- Email: [email protected]
Development, validation and evaluation of added diagnostic value of a q(RT)-PCR for the detection of genotype A strains of small ruminant lentiviruses
Accepted Manuscript Title: Development, validation and evaluation of added diagnostic value of a q(RT)-PCR for the detection of genotype A strains of small ruminant lentiviruses Author: Nick De Regge Brigitte Cay PII: DOI: Reference:
S0166-0934(13)00388-1 http://dx.doi.org/doi:10.1016/j.jviromet.2013.09.001 VIRMET 12306
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
22-2-2013 28-8-2013 3-9-2013
Please cite this article as: De Regge, N., Cay, B., Development, validation and evaluation of added diagnostic value of a q(RT)-PCR for the detection of genotype A strains of small ruminant lentiviruses., Journal of Virological Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.09.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Development, validation and evaluation of added diagnostic value of a q(RT)-PCR for the
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detection of genotype A strains of small ruminant lentiviruses.
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Nick De Regge, Brigitte Cay
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CODA-CERVA, Groeselenberg 99, 1180 Brussel, Belgium
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Nick De Regge: [email protected]
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Brigitte Cay: [email protected]
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Corresponding author:
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Nick De Regge
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Groeselenberg 99
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1180 Brussel
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Belgium
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Phone: 0032 (0)2 379 05 80
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Fax: 0032 (0)2 379 06 70
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Email: [email protected] 1 Page 1 of 30
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Summary
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Small ruminant lentiviruses (SRLV) infect sheep and goats. Diagnosis of SRLV infection
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mostly relies on serological testing but more recently, also PCR is regarded as a useful
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complementary tool in SRLV diagnosis. The goal of this study was to develop and validate a
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quantitative PCR capable to detect a broad range of SRLV strains from genotype A, including
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strains circulating in Belgium.
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The developed q(RT)-PCR targets a region of the gag gene and showed to be highly sensitive
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and specific with a limit of detection of 6 DNA and 40 RNA copies/reaction respectively.
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SRLV sequences could be detected in lung samples and leukocytes pellets. The q(RT)-PCR
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identified SRLV positive animals in Belgian sheep flocks, but also SRLV isolates and
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samples from Scotland, The Netherlands, Spain, Portugal, UK, Iceland, Finland and USA
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were found positive. Samples known to contain ‘CAEV like’ SRLV from France and Spain
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were not identified as positive. Combined serological and PCR analysis of a limited number
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(n=35) of Belgian sheep underlined the usefulness of the described PCR as a complementary
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diagnostic tool since 3 seronegative animals were found positive by the PCR.
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In conclusion, the validated q(RT)-PCR shows excellent analytical characteristics and is
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capable to detect SRLV strains belonging to genotype A from various countries.
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Keywords: small ruminant lentiviruses; diagnosis; q(RT)-PCR; validation; control program
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1. Introduction
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Small ruminant lentiviruses (SRLV), including Maedi-Visna virus (MVV) and Caprine
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arthritis-encephalitis virus (CAEV), form a viral continuum of strains which can infect sheep
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and goats (Leroux et al., 2010). They cause a chronic inflammatory degenerative disease
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sometimes associated with clinical signs such as neurological disorders, pneumonia, dyspnea,
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arthritis, mastitis and weight lose leading to economic losses, trade limitations and a negative
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impact on animal welfare (reviewed in Blacklaws, 2012; Peterhans et al., 2004). Vertical
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transmission through ingestion of infected colostrum or milk seems the main route of virus
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transmission, but horizontal transmission via respiratory secretions between animals that are
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in close contact has also been described (reviewed in Blacklaws et al., 2004; McNeilly et al.,
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2008; Peterhans et al., 2004).
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SRLV belong to the family of the Retroviridae and are ss(+) RNA viruses with a tropism for
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cells of the monocyte/macrophage lineage and dendritic cells, but also other cell types can
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become infected in tissues (Bolea et al., 2006; Gendelman et al., 1985, 1986; Lerondelle et al.,
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1999; Ryan et al., 2000; Zink et al., 1990). For virus replication, as other lentiviruses, they
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integrate as a provirus in the DNA of the host cell genome. By consequence, infection of bone
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marrow stem cells or precursor cells allows for continuous production of infected cells and a
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persistent life-long infection of the animal (Gendelman et al., 1985; Grossi et al., 2005).
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Partial genetic characterization of SRLV strains from many different countries and subsequent
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phylogenetic analysis classifies SRLV strains for the moment into five different groups, from
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A to E. Group A, B and E are further subdivided in subgroups. Group A consist of MVV like
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genotypes, group B of CAEV like genotypes, while the other three groups harbor genotypes
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from more restricted geographical regions (Bertolotti et al., 2011; Giammarioli et al., 2011;
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Gjerset et al., 2006; Glaria et al., 2009; Grego et al., 2007; Olech et al., 2012; Pisoni et al.,
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2010; Reina et al., 2006; Reina et al., 2010; Shah et al., 2004).
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Since no adequate prophylactic tools are on the market, many countries rely on control
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programs to decrease SRLV prevalence (Peterhans et al., 2004; Reina et al., 2009). Good
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diagnostic tools are of crucial importance for the detection of SRLV infected animals.
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Serological tests as agar gel immunodiffusion (AGID) and ELISA are the most frequently
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used assays to identify anti-SRLV antibodies in positive animals (reviewed by de Andres et
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al., 2005; Hermann-Hoesing, 2010; Ramirez et al., 2013). The long time between infection
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and seroconversion, the existence of non-responders, fluctuating antibody titers throughout
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the animal’s life, and the antigenic differences between circulating strains and test antigens
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used can however interfere with the sensitivity of the serological tests (Cardinaux et al., 2013;
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de Andrés et al., 2013; Peterhans et al., 2004; Ramirez et al., 2013). More recently, also (RT)-
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PCR and quantitative (RT)-PCR assays have been developed that are able to detect the viral
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RNA genome, proviral sequences and viral RNA transcripts (reviewed by Hermann-Hoesling,
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2010). PCR based diagnosis of SRLV infected animals can however be hampered by the low
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proviral load during a latent state of infection and the large genetic variation between strains
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(Peterhans et al., 2004). Studies comparing the sensitivity of PCRs and serological tests have
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shown that PCR can sometimes identify SRLV positive animals that tested negative in
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serology, thereby indicating that both tests should be seen as complementary tests in SRLV
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diagnosis (reviewed by Ramirez et al., 2013).
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Most of the described q(RT)-PCR assays are developed for the detection of a specific SRLV
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strain and mostly no broad evaluation of their capacity to detect strains circulating in different
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geographical regions has been done (Brajon et al., 2012; Carrozza et al., 2010; Gudmundsson
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et al., 2003; Herrmann-Hoesling et al., 2007; Zhang et al., 2000). An exception is the qPCR
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developed by Brinkhof et al. (2008) that is described to detect several SRLV strains. This
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PCR uses the SybrGreen technology, thereby partly circumventing the problem of high
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genetic variation. It requires however a melting curve analysis to confirm specific
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amplification what is not always straightforward seen the probability that variable SRLV
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sequences will denature at different temperatures.
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In Belgium, a voluntary control program for SRLV exists and certification of SRLV negative
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flocks depends on serological results obtained by AGID (Maeditect, VLA scientific) and
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ELISA (Elitest, Hyphen) testing. In this study the development and validation of a sensitive
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q(RT)-PCR assay capable to detect SRLV strains belonging to genotype A is described. This
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test can in the future be implemented in the Belgian control program as a complementary or
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confirmatory test besides serology to help identifying additional ‘Maedi Visna like’ SRLV
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infected animals or to help defining the status of ELISA positive animals that cannot be
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confirmed in AGID. This qPCR could also be of interest to other countries since the
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validation demonstrates that the PCR is capable to detect SRLV strains belonging to genotype
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A originating from different geographical regions.
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2. Material and Methods
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2.1. Ethical statement
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All samples used in this study were collected by veterinarians in accordance with existing
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legislation on animal welfare.
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2.2. Clinical specimens and virus isolates
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Whole blood and organ samples from 79 Belgian sheep belonging to flocks that are certified
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as SRLV negative were collected at the slaughterhouse. Whole blood from 35 sheep
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belonging to 5 Belgian sheep flocks and 16 goats belonging to 4 Belgian goat flocks in which 5 Page 5 of 30
SRLV seropositive animals were present was collected by the farm veterinarian and sent to
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CODA-CERVA. Another SRLV seropositive Belgian sheep (6612) was housed and
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euthanized at the animal facilities of CODA-CERVA and lung samples were collected.
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Other samples of defined SRLV positive status (by serology or PCR) were sent to CODA-
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CERVA including whole blood samples from 31 sheep belonging to 4 Scottish sheep flocks; a
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lung sample from a Finnish sheep (H S2L 2006 Fin); SRLV virus isolates WLC1, P1OLV,
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EV-1, 1514 and 2 Dutch isolates (ZVV-1050, MVV-7); and DNA extracts from organs or
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PBMC originating from The Netherlands (positive control, 5282652786000016, 11 (20890),
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12 (302739)), Portugal (3951, 5834-09), France (623, 663, 683, 684, 685, 760, 761, 762, 763,
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766) and Spain (697, 258, 496).
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2.3. SRLV serology
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The serological status for SRLV of Belgian sheep used in this study was determined by the
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use of the Elitest ELISA (Hyphen, Neuville-sur-Oise, France) and the Maeditect AGID test
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(VLA scientific, London, United Kingdom) following manufacturer’s instructions.
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2.4. Preparation of control DNA and RNA transcripts from strain 1514 background
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A plasmid containing a part of the SRLV strain 1514 gag sequence and the corresponding
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RNA transcripts were produced using standard cloning and in vitro transcription protocols.
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After RNA extraction from a virus stock of strain 1514 and cDNA synthesis, a DNA fragment
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corresponding to 524 base pairs (bp) was produced by classical PCR using the Seq 1 and Seq
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2 primer described in table 1. The fragment was cloned into the pCR 2.1-TOPO cloning
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vector (Life Technologies, Ghent, Belgium) and subsequently transformed into competent
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Escherichia coli TOP10F’ cells and multiplied. Purified plasmids were linearised with BamHI
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and in vitro transcribed (TranscriptAid T7 High Yield Transcription Kit, Thermo Scientific,
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Aalst, Belgium). Remaining plasmid DNA was eliminated by a Turbo DNA free treatment
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(Life Technologies, Ghent, Belgium) and the RNA was purified using the RNeasy Mini kit
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(Qiagen, Hilden, Germany). The produced plasmid and RNA transcripts were quantified
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using a Nanodrop device (Thermofisher Scientific, Aalst, Belgium) and the copy number was
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calculated based on the predicted molecular weight of either the plasmid or RNA transcripts.
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Aliquots of the DNA plasmid and RNA transcripts were stored at -80 °C.
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2.5. Sample preparation, DNA and RNA extraction and reverse transcription
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Whole blood samples were used to prepare leucocyte pellets by adding 1,5 ml blood to 8,5 ml
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hemolysis buffer (16.6 g NH4Cl, 2.0 g NaHCO3, 0.185g diNa EDTA per l H2O; pH 7.4
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(Mignon et al., 1992)). After 15 min of incubation, the samples were centrifuged for 15 min at
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1000g and after elimination of the supernatans, the pellet was resuspended in 100 µl of
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phosphate buffered saline (PBS). Lung samples were homogenized by adding ± 0.5 g of tissue
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to 1 ml PBS and 10 to 15 1mm silicon carbid beads (Biospec Products, Bartlesville, OK,
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USA), followed by high speed shaking (2 min, 25 Hz) in a TissueLyser (Qiagen, Hilden,
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Germany).
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Extraction of DNA and total RNA from leucocyte pellets, lung homogenates and virus
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isolates was respectively done by the QIAamp DNA minikit (Qiagen, Hilden, Germany) and
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the RNeasy Mini kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. DNA
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and RNA were respectively eluted in 100 and 50 µl H2O.
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Total RNA was converted to cDNA using the M-MLV reverse transcriptase system (Life
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Technologies, Ghent, Belgium). For each reaction, a mix of 4 µl 5x first strand buffer, 2 µl
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0.1M DTT, 1 µl 10nM dNTP mix (Roche, Basel, Switzerland), 2 µl 1x hexanucleotide mix
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(Roche, Basel, Switzerland), 1 µl M-MLV RT and 10 µl RNA was prepared and incubated at
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37°C for 45min, followed by inactivation at 95°C for 10min.
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2.6. Real time PCR assay
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All DNA and cDNA samples were analysed for the presence of SRLV by using the FastStart
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TaqMan Probe Master kit (Roche, Basel, Switzerland) following manufacturer’s instructions
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with primers and probe directed against a part of the gag gene of SRLV. To check the DNA or
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RNA extraction, reverse transcription and amplification reaction, each sample was also tested
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for the presence of -actin in a separate reaction (forward primer: 5’-
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cagcacaatgaagatcaagatcatc-3’; reverse primer: 5’-cggactcatcgtactcctgctt-3’; probe: HEX-5’-
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tcgctgtccaccttccagcagatgt-3’-BHQ1 (Toussaint et al., 2007)). Briefly, a master mix consisting
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of 5 µl RNase-free water, 10 µl 2x Faststart Taqman probe master buffer, 1 µl forward primer
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(10µM), 1 µl reverse primer (10µm) and 1 µl probe (4µM) for one reaction was prepared and
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2 µl DNA or cDNA template was added. For amplification, the following temperature profile
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was used: 10 min at 95°C, followed by 45 cycles of 15s at 95°C and 45s at 60°C. All qRT-
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PCRs were done on a LightCycler 480 Real-Time PCR system (Roche, Basel, Switzerland).
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In all q(RT)-PCR analyses performed, negative extraction controls and negative and positive
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amplification controls were included and tests were only validated when all controls were
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satisfactory.
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2.7. Construction of standard curve and parameters used for evaluation of q(RT)-PCR
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performance
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For the construction of the DNA standard curve, the constructed plasmid containing part of
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the 1514 strain gag sequence was 10 fold serially diluted in a DNA extract obtained from an
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SRLV negative lung and all dilutions were assayed in the qPCR with input material ranging
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from 3x107 to 3x10-2 copies/µl. Same was done for RNA whereby the RNA transcripts were
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10 fold serially diluted in an RNA extract obtained from an SRLV negative lung. Input RNA
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transcripts ranging from 4x107 to 4x10-2 RNA copies/µl were reverse transcribed, followed by
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analysis in the qPCR. For both DNA and RNA, three independent replicates were run, mean
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values of each dilution were calculated and a standard curve was constructed by plotting the
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Ct values against the log of the input DNA or RNA copy number. These standard curves were
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used to evaluate the q(RT)-PCR performance based on dynamic range (the range of
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respectively template DNA and RNA copies/µl for which accurate CT values were obtained),
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r2 value (a statistical measure indicating how well the linear regression curve fits the real data
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points; an r² of 1.0 indicates that the regression line perfectly fits the data), efficiency (%)
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(efficiency was calculated using the following formula: Efficiency = -1+10(-1/slope)) and
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sensitivity (limit of detection (LOD) was determined as the lowest concentration for which
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3/3 replicates produced a positive result).
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2.8. Sequencing of a gag gene fragment of SRLV strain 6612 and amplified fragments
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obtained by the developed q(RT)-PCR
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For sequencing of a gag gene fragment of SRLV strain 6612, PCR amplification using
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primers targeting a part of the gag gene of SRLV1514 strain (Seq1 and Seq 2 primers in Table
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1) was performed on cDNA prepared from the lung of the Belgian sheep 6612 using the
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FastStart PCR Master kit from Roche (Basel, Switzerland) following manufacturer’s
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instruction. An initial denaturation for 5 min at 95°C was followed by 40 cycles of 1 min at
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95°C, 1 min at 55°C and 1 min at 72°C, and a final extention at 72°C for 10 min. PCR
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products were visualised on a 1% agarose gel and bands of the correct size were cut from the
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gel and purified using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany).
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Approximately 5 ng of PCR product was used as template for sequencing using the forward
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and reverse primer mentioned above and 3 additional primers (Seq 3, Seq 4 and Seq 5 primer
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in Table 1) with the BigDye Terminator v3.1 sequencing kit (Life Technologies, Ghent,
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Belgium). Sequencing reactions were purified using the BigDye Xterminator reagent (Life
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Technologies, Ghent, Belgium) and ran on an ABI3130 Genetic Analyser (Life Technologies,
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Ghent, Belgium).
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For sequencing of the fragments obtained by the developed q(RT)-PCR, the realtime reaction
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product was separated on an 0.6% agarose gel and a similar protocol as described above was
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followed using 2 ng of PCR product and the forward and reverse primer of the developed
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q(RT)-PCR (Table 1) as sequencing primers.
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3. Results
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3.1. Partial gag gene sequencing of Belgian SRLV strain 6612, sequence alignment and
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primer/probe selection
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The lung of a Belgian serological SRLV positive sheep 6612 was found positive for the
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presence of SRLV by the classical PCR described by Celer et al. (2000). Using primers
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homologous to the gag gene sequence of SRLV strain 1514, a part of the gag gene from
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SRLV strain 6612 was amplified and sequenced (Genbank accession number: KC560792). A
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similarity search in the NCBI nucleotide database using BLAST showed that considerable
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similarity (between 80 and 90%) existed with the EV-1, 1514, SA-OMVV, 83.7, WLC-1 and
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P1OLV SRLV strain sequences in the database. These strains have been classified before as
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strains belonging to genotype A, based on phylogenetic analysis of ± 1.8 kb sequences of the
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gag-pol region and ± 1.2 kb sequences of the pol region (Shah et al., 2004). Clearly less
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similarity was found after alignment of the 6612 sequence with corresponding sequences of
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SRLV strains of genotype B, C and E.
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Since the high genetic variability in this genomic region did not allow to find conserved
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sequences when all genotypes were taken into account, primer express software was used to
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select primers (Eurogentec, Seraing, Belgium) and a minor groove binding (MGB) probe
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(Life Technologies, Ghent, Belgium) in the conserved regions of genotype A strains (Table
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1). Figure 1 shows the similarity between the selected primers and probe sequences with
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corresponding sequences of SRLV strains of genotype A and the heterogeneity with SRLV
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strains belonging to the other genotypes.
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3.2. Analytical q(RT)-PCR performance
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After production of control DNA and RNA from strain 1514 background, standard curves
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were constructed and used to determine the analytical q(RT)-PCR performance.
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For DNA, the limit of detection of the real time PCR was 3 plasmid copies/µl (corresponding
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to 6 copies/reaction). The typical standard curve amplification plot and linear regression
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analysis (Fig.2a) showed an amplification efficiency of 91% and a dynamic range of detection
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between 3x107 and 3x100 copies/µl. For RNA, the detection limit was 40 copies/µl
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(corresponding to 40 copies/reaction) (Fig. 2b). An amplification efficiency of 92% and a
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linear range of detection between 4x107 and 4x101 copies/µl were obtained.
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3.3. Matrix effect
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Since the q(RT)-PCR on plasmid DNA and RNA transcripts showed promising results
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regarding the limit of detection and PCR efficiency, its capability to detect SRLV in different
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biological samples was evaluated in a next step. Again, this was done for SRLV detection on
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both DNA and RNA level.
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The lung of the Belgian sheep 6612 (Ct=29.3) and a leucocyte pellet prepared from whole
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blood of a Scottish sheep (Ct=29.8) tested positive in the q(RT)-PCR under validation. These
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samples were used to study the impact of sample material on SRLV detection after DNA
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extraction. A homogenate of the positive lung was 10 fold serially diluted in a homogenate of
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an SRLV negative lung. Same was done for the blood by diluting it in whole blood of an
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SRLV negative animal. After sample preparation and DNA extraction, all dilutions were
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tested in the qPCR. This resulted (Fig.3a) in linear assays over several dilutions and the slopes
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(respectively -3.3 and -3.38 for lung and blood), and therefore the PCR efficiency, were
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similar to the slope found for the DNA standard curve (-3.56).
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To study the impact of sample material on SRLV detection upon RNA extraction, the same
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experiment with the lung material of animal 6612 as described above was repeated with the
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difference that now RNA was extracted from the dilution series, followed by reverse
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transcription and PCR analysis. Furthermore, a homogenate of an SRLV negative lung and a
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whole blood sample of an SRLV negative sheep were spiked with SRLV strain WLC-1 (104.8
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TCID50/ml homogenate or whole blood). These spiked samples were then respectively 10
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fold serially diluted in a homogenate of a negative lung or in whole blood from a negative
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sheep, followed by sample preparation, RNA extraction, reverse transcription and qPCR
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analysis. Figure 3b shows that SRLV could be detected in all biological samples and that the
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qPCR was linear over several dilutions. Also here, the slopes of all dilution series (-3.29 for
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lung pos in lung neg; -3.54 for WLC-1 in lung neg; -3.29 for WLC-1 in blood neg) were
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similar to the slope of the RNA standard curve (-3.54).
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These results indicate that the PCR assay is capable to detect SRLV DNA and RNA
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sequences in different biological samples and that the DNA and RNA standard curves (Fig.2)
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can be used for the calculation of the (pro)viral load in the different samples.
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3.4. Specificity
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To test the diagnostic specificity, 79 leucocyte pellets prepared from whole blood samples
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from AGID and ELISA negative sheep from SRLV free certified Belgian farms were tested
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using the developed qPCR after DNA extraction. All samples were found negative, showing a
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100% concordance with the serological results. Also 2 organ samples known to be PCR
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positive for equine infectious anemia virus, a lentivirus of horses, were tested and were both
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negative, giving a preliminary idea of the analytical specificity of the PCR.
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3.5. Diagnostic sensitivity
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The results described above show that the PCR is capable to detect SRLV sequences present
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in the lung of Belgian sheep 6612 and in the blood of a Scottish serological positive sheep. To
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get further insight in the range of SRLV strains that can be detected by the PCR, reference
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laboratories and research institutes of several countries were contacted and asked to provide
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us with SRLV strains or material from SRLV positive animals. Most laboratories responded
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positively to our demand and provided us with virus isolates, organ or blood samples, or DNA
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extracts from organs or blood from defined SRLV positive animals (by serology or PCR).
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Table 2 gives an overview of the received samples and the obtained PCR results.
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All isolates and samples containing SRLV strains known to belong to genotype A tested
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positive in the qPCR. Also several samples containing genetically non-characterized SRLV
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strains were found positive. The specificity of the amplification products of all q(RT)-PCR
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positive samples mentioned in table 2 was confirmed by sequencing. All amplicons showed
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similarity with sequences of the gag region of SRLV strains when a similarity search was
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done with the NCBI database using BLAST (data not shown).
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The samples that remained negative were a sample containing group B SRLV strain 496 from
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Spain (Glaria et al., 2009), 10 samples containing ‘CAEV like’ strains from France (Leroux et
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al., 1995) and samples from 4 serological SRLV positive Belgian goat flocks. The SRLV
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positive status of these samples was confirmed by the use of the classical PCR assay
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described by Leroux et al. (1995), except for samples from 2 out of 4 Belgian goat flocks
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(data not shown).
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3.6. Testing of Belgian field samples
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To get an idea of the concordance between serological tests and the developed qPCR for
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SRLV detection in sheep and the usefulness of implementing the qPCR in the Belgian SRLV
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control program, whole blood samples of 35 animals from 5 different farms in which at least
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one serological positive sheep was detected were analyzed by the qPCR while the
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corresponding serum samples were tested in AGID and ELISA. Cross table 3a shows that
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there is a positive concordance of 87% (13/15) between AGID and qPCR results. Four
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samples were found positive in the qPCR while they were negative in the AGID test. The
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positive concordance between the ELISA and qPCR test was 61% (14/23), while 3 samples
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positive in the qPCR were negative in the ELISA (table 3b). Taken together, 3 out of 35
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samples were positive in the qPCR but negative in both serological tests. On the other hand, 2
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AGID and 9 ELISA positive samples were not confirmed in the qPCR, indicating that not all
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seropositive animals are detected by the qPCR.
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4. Discussion
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A combination of aspects related to the SRLV replication cycle, their genetic variability and
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the slow and sometimes hampered humoral immune response to these viruses makes it not
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always straightforward to correctly identify the SRLV infection status of an animal (de
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Andres et al., 2013; Peterhans et al., 2004; Ramirez et al., 2013). In Belgium, the voluntary
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SRLV control program relies on serological testing using ELISA and AGID. The discrepancy
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that is sometimes observed between both test results raises questions about the true infection
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status of an animal and the related measures to apply since these have important economic
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impact for the farmers. To assist in the determination of the SRLV infection status of
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suspected sheep, a q(RT)-PCR capable to detect SRLV strains from genotype A circulating in
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Belgian herds was developed.
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The validation showed that the developed q(RT)-PCR is capable to detect SRLV in different
348
biological samples and that it can be used to detect SRLV sequences on both DNA and RNA
349
levels. This latter property can be important since depending on the moment of infection
350
(acute or latent), the sample received, and the context of the analysis (diagnosis, in vivo or in
351
vitro experimental infection), it can be preferable to search for proviral DNA, the viral RNA
352
genome or viral RNA transcripts (since hexanucleotides were used during reverse
353
transcription, no differentiation between both RNA molecules was made in this study).
354
Furthermore, the assay turned out to be sensitive with a limit of detection of 3 copies/µl DNA
355
extract and 40 copies/µl RNA extract. This LOD is similar to sensitivities that were
356
previously reported for other q(RT)-PCR assays for the detection of SRLV (Carroza et al.,
357
2010; Gudmundsson et al., 2003). Since positive results were obtained for blood and organ
358
samples of sheep without clinical symptoms in this study, it seems reasonable to assume that
359
the sensitivity of the test is satisfactory to detect SRLV from genotype A in most infected
360
animals. Follow up of SRLV positive animals over time will have to show if the PCR is able
361
to detect the virus during the latent phase of infection, the moment where the lowest provirus
362
quantities are present.
363
The analysis of the diagnostic sensitivity has shown that the PCR is capable to detect SRLV
364
strains circulating in Belgium, as well as strains from several other countries. The fact that the
365
primer/probe selection was done using conserved regions within the gag gene of SRLV strains
366
belonging to the phylogenetic group A and that all received samples containing genotype A
367
sequences tested positive in the PCR makes it reasonable to assume that this PCR is especially
368
useful to detect SRLV strains belonging to this genotype (‘Maedi-Visna like strains’).
369
Although uncertain, probably also the non-characterized samples from Portugal, Finland and
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15 Page 15 of 30
Scotland that tested positive contain genotype A SRLV strains since mainly strains belonging
371
to this genotype have been isolated in these countries (Barros et al., 2004; Laamanen et al.,
372
2007; Sargan et al., 1991). Further indications that the PCR detects probably SRLV strains
373
belonging to genotype A are found in the fact that all ‘CAEV like’ strains tested were not
374
identified as positive. The observation that seropositive samples from Belgian goats were
375
found positive in the PCR described by Leroux et al. (1995) but tested negative in the newly
376
developed q(RT)-PCR indicates that probably strains belonging to different genotypes
377
circulate in Belgium. The sequences used for primer selection by Leroux et al. (1995) and the
378
widespread distribution of genotype B strains in goats (Ramirez et al., 2013) suggest that the
379
Belgian goats are probably infected by strains of genotype B. It will be a future challenge to
380
identify these strains and to adapt the PCR accordingly so they can also be detected. However,
381
surveillance on both species is highly recommended since SRLV strains are able to cross
382
species barriers between sheep and goats (Leroux et al, 2010).
383
When interpreting results obtained with this q(RT)-PCR, it should be kept in mind that this
384
PCR is directed to a part of the gag region that falls out of the genomic region that is used for
385
the phylogenetic classification (Shah et al., 2004) and that strains positive in this PCR should
386
not automatically be regarded as strains belonging to group A. Further sequencing is required
387
before such phylogenetic classification of the strain can be done.
388
In the limited number of serological SRLV positive Belgian sheep flocks tested till now,
389
always one or more qPCR positive animals were identified, supporting its capability to detect
390
SRLV strains circulating in Belgian sheep. The comparison between serological test results
391
and qPCR results on samples from Belgian sheep showed that a high positive concordance
392
existed between AGID results and the qPCR, but that this was considerably lower between the
393
ELISA and qPCR results. Care should however be taken in the interpretation of these data
394
since they represent only a very limited sample size (n=35) which moreover was not selected
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16 Page 16 of 30
randomly, since it contains samples from SRLV free certified flocks that tested unexpectedly
396
positive in ELISA what could not be confirmed by other tests. More samples from a random
397
sampling need to be tested before making definite statements about the concordance of the
398
qPCR with serological tests. What is however interesting is that 3 out of 35 animals were
399
positive in the qPCR while they were negative in both ELISA and AGID. This indicates that
400
the qPCR can be a useful complementary tool in a control program to identify animals that
401
would not be detected if only serological testing is done. These animals would pose a risk for
402
transmission of the virus to the rest of the herd if they remain in the flock. Since the validation
403
demonstrated that the q(RT)-PCR is capable to detect SRLV strains originating from different
404
geographical regions, it could for the same reason also be of interest to diagnostic and
405
research laboratories in other countries. It should however also be emphasized that several
406
ELISA and AGID positive samples were not confirmed in the qPCR. These could be
407
explained as false positive reactions in serology, but they most likely represent animals in
408
which the viral load was too low or that were infected by a strain that is not recognized by the
409
PCR. This further supports the view that PCR and serology have to be considered as
410
complementary tests in SRLV diagnosis.
412 413
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5. Conclusion
414
The q(RT)-PCR developed in this study targets a region of the gag gene of SRLV. During
415
validation, the PCR showed to have excellent q(RT)-PCR characteristics. It is capable to
416
detect SRLV proviral DNA and RNA in blood and lung and showed to be highly sensitive
417
and specific. Furthermore, several SRLV isolates and positive samples, most probably ‘Maedi
418
Visna like’, from Belgium, Scotland, The Netherlands, Spain, Portugal, UK, Iceland, Finland
419
and USA were identified as positive. This q(RT)-PCR could therefore be used as a useful
17 Page 17 of 30
420
diagnostic tool to detect genotype A strains of SRLV, and it can also be an interesting tool for
421
research purposes.
422
6. Acknowledgements
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We thank Virginie Colasse, Sarah Matuyia, and Thibault De Maesschalck for the excellent
426
technical assistance during the qPCR validation. We thank Guido Bertels for his help in the
427
collection of samples from Belgian sheep. We particularly thank Hans Kramps, Gerard
428
Wellenberg, Beatriz Amorena, Barbara Blacklaws, Brian Hosie, Miguel Fevereiro, Liisa
429
Sihvonen, Valgerdur Andresdottir and Caroline Leroux for their willingness to provide us
430
with SRLV positive samples that were used in this validation.
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7. References
433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456
Barros, S.C., Ramos, F., Duarte, M., Fagulha, T., Cruz, B., Fevereiro, M., 2004. Genomic characterization of a slow/low Maedi Visna virus. Virus Genes 29, 199-210.
te
d
432
Ac ce p
Bertolotti, L., Mazzei, M., Puggioni, G., Carrozza, M.L., Dei Giudici, S., Muz, D., Juganaru, M., Patta, C., Tolari, F., Rosati, S., 2011. Characterization of new small ruminant lentivirus subtype B3 suggests animal trade within the Mediterranean Basin. J. Gen. Virol. 92, 1923– 1929. Blacklaws B.A., 2012. Small ruminant lentiviruses: Immunopathogenesis of visna-maedi and caprine arthritis and encephalitis virus. Comp. Immunol. Microbiol. Infect. Dis. 35, 259-269. Blacklaws, B.A., Berriatua, E., Torsteinsdottir, S., Watt, N.J., de Andres, D., Klein, D., Harkiss, G.D., 2004. Transmission of small ruminant lentiviruses. Vet. Microbiol. 101, 199– 208. Bolea, R., Monleon, E., Carrasco, L., Vargas, A., de Andrés, D., Amorena, B., Badiola, J.J., Lujan, L., 2006. Maedi-visna virus infection of ovine mammary epithelial cells. Vet. Res. 37, 133-44. Brinkhof, J.M., van Maanen, C., Wigger, R., Peterson, K., Houwers, D.J., 2008. Specific detection of small ruminant lentiviral nucleic acid sequences located in the proviral long terminal repeat and leader-gag regions using real-time polymerase chain reaction. J. Virol. Methods 147, 338-344.
18 Page 18 of 30
Cardinaux, L., Zahno, M.L., Deubelbeiss, M., Zanoni, R., Vogt, H.R., Bertoni, G., 2013. Virological and phylogenetic characterization of attenuated small ruminant lentivirus isolates eluding efficient serological detection. Vet. Microbiol. 162, 572-581.
ip t
Carrozza, M.L., Mazzei, M., Bandecchi, P., Fraisier, C., Pérez, M., Suzan-Monti, M., de Andrés, D., Amorena, B., Rosati, S., Andrésdottir, V., Lujan, L., Pepin, M., Blacklaws, B., Tolari, F., Harkiss, G.D., 2010. Development and comparison of strain specific gag and pol real-time PCR assays for the detection of Visna/maedi virus. J. Virol. Methods 165, 161-167.
cr
Celer, V. Jr., Celer, V., Nejekla, E., Bertoni, G., Peterhans, E., Zanoni, R.G., 2000. The detection of proviral DNA by semi-nested polymerase chain reaction and phylogenetic analysis of Czech maedi-visna isolates based on gag gene sequences. J. Vet. Med. B Infect. Dis. Vet. Public Health 47, 203–215.
us
de Andres, D., Klein, D., Watt, N.J., Berriatua, E., Torsteinsdottir, S., Blacklaws, B.A., Harkiss, G.D., 2005. Diagnostic tests for small ruminant lentiviruses. Vet. Microbiol. 107, 49–62.
M
an
de Andres, X., Ramirez, H., Bertolotti, L., San Roman, B., Glaria, I., Crespo, H., Jauregui, P., Minguijon, E., Juste, R., Leginagoikoa, I., Perez, M., Lujan, L., Badiola, J.J., Polledo, L., Garcia-Marin, J.F., Riezu, J.I., Borras-Cuesta, F., de Andres, D., Rosati, S., Reina, R., Amorena, B., 2013. An insight into a combination of ELISA strategies to diagnose small ruminant lentivirus infections. Vet. Immunol. Immunopathol. 152, 277-288.
te
d
Gendelman, H.E., Narayan, O., Kennedy-Stoskopf, S., Kennedy, P.G., Ghotbi, Z., Clements, J.E., Stanley, J., Pezeshkpour, G., 1986. Tropism of sheep lentiviruses for monocytes: susceptibility to infection andvirus gene expression increase during maturation of monocytes to macrophages. J. Virol. 58, 67-74. Gendelman, H.E., Narayan, O., Molineaux, S., Clements, J.E., Ghotbi, Z., 1985. Slow, persistent replication of lentiviruses: role of tissue macrophages and macrophage precursors in bone marrow. Proc. Natl. Acad. Sci. USA 82, 7086–7090.
Ac ce p
457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505
Giammarioli, M., Bazzucchi, M., Puggioni, G., Brajon, G., Dei Giudici, S., Taccori, F., Feliziani, F., De Mia, G.M., 2011. Phylogenetic analysis of small ruminant lentivirus (SRLV) in Italian flocks reveals the existence of novel genetic subtypes. Virus Genes 43, 380–384. Gjerset, B., Storset, A.K., Rimstad, E., 2006. Genetic diversity of small-ruminant lentiviruses: characterization of Norwegian isolates of caprine arthritis encephalitis virus. J. Gen. Virol., 87, 573–580. Glaria, I., Reina, R., Crespo, H., de Andrés, X., Ramírez, H., Biescas, E., Pérez, M.M., Badiola, J., Luján, L., Amorena, B., de Andrés, D., 2009. Phylogenetic analysis of SRLV sequences from an arthritic sheep outbreak demonstrates the introduction of CAEV-like viruses among Spanish sheep. Vet. Microbiol. 138, 156-162. Glaria, I., Reina, R., Ramírez, H., de Andrés, X., Crespo, H., Jauregui, P., Salazar, E., Lujan, L., Pérez, M.M., Benavides, J., Pérez, V., Polledo, L., Garcia-Marin, J.F., Riezu, J.I., Borras, F., Amorena, B., de Andrés, D., 2012. Visna/Maedi virus genetic characterization and
19 Page 19 of 30
cr
ip t
Grossi, P., Giudice, C., Bertoletti, I., Cioccarelli, G., Brocchi, E., Cammarata, G., Gelmetti, D., 2005. Immunohistochemical detection of the p27 capsid protein of caprine arthritis– encephalitis virus (CAEV) in bone-marrow cells of seropositive goats. J. Comp. Pathol., 133, 197–200.
us
Gudmundsson, B., Bjarnadottir, H., Kristjansdottir, S., Jonsson, J.J., 2003. Quantitative assays for maedi-visna virus genetic sequences and mRNA's based on RT-PCR with real-time FRET measurements. Virology, 307, 135–142.
an
Herrmann-Hoesing L.M., 2010. Diagnostic assays used to control small ruminant lentiviruses. J. Vet. Diagn. Invest., 22, 843-55.
M
Herrmann-Hoesing, L.M., White, S.N., Lewis, G.S., Mousel, M.R., Knowles, D.P., 2007. Development and validation of an ovine progressive pneumonia virus quantitative PCR. Clin. Vaccine Immunol., 14, 1274–1278. Laamanen, I., Jakava-Viljanen, M., Sihvonen, L., 2007. Genetic characterization of maedivisna virus (MVV) detected in Finland. Vet. Microbiol. 122, 357-365.
d
528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554
Grego, E., Bertolotti, L., Quasso, A., Profiti, M., Lacerenza, D., Muz, D., Rosati, S., 2007. Genetic characterization of small ruminant lentiviruses in Italian mixed flocks: evidence for a novel genotype circulating in a local goat population. J. Gen. Virol., 88, 3423–3427.
Lerondelle, C., Godet, M., Mornex, J.F., 1999. Infection of primary cultures of mammary epithelial cells by small ruminant lentiviruses. Vet. Res. 30, 467-74.
te
517 518 519 520 521 522 523 524 525 526 527
serological diagnosis of infection in sheep from a neurological outbreak. Vet. Microbiol., 155, 137-146.
Leroux, C., Cruz, J.C.M., Mornex, J.F., 2010. SRLVs: A Genetic Continuum of Lentiviral Species in Sheep and Goats with Cumulative Evidence of Cross Species Transmission. Current HIV Research, 8, 94-100.
Ac ce p
506 507 508 509 510 511 512 513 514 515 516
Leroux, C., Vuillermoz, S., Mornex, J.F., Greenland, T., 1995. Genomic heterogeneity in the pol region of ovine lentiviruses obtained from bronchoalveolar cells of infected sheep from France. J. Gen. Virol. 76, 1533-1537. McNeilly, T.N., Baker, A., Brown, J.K., Collie, D., Maclachlan, G., Rhind, S.M., Harkiss, G.D., 2008. Role of alveolar macrophages in respiratory transmission of visna/maedi virus. J. Virol. 82, 1526–1536. Mignon, B., Waxweiler, S., Thiry, E., Boulanger, D., Dubuisson, J., Pastoret, P.P., 1992. Epidemiological evaluation of a monoclonal ELISA detecting bovine viral diarrhoea pestivirus antigens in field blood samples of persistently infected cattle. J. Virol. Methods, 40, 85-94. Olech, M., Rachid, A., Croisé, B., Kuźmak, J., Valas, S., 2012. Genetic and antigenic characterization of small ruminant lentiviruses circulating in Poland. Virus Res. 163, 528-536. 20 Page 20 of 30
Peterhans, E., Greenland, T., Badiola, J., Harkiss, G., Bertoni, G., Amorena, B., Eliaszewicz, M., Juste, R.A., Krassnig, R., Lafont, J.P., Lenihan, P., Petursson, G., Pritchard, G., Thorley, J., Vitu, C., Mornex, J.F., Pepin, M., 2004. Routes of transmission and consequences of small ruminant lentiviruses (SRLVs) infection and eradication schemes. Vet. Res. 35, 257-274.
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Pisoni, G., Bertoni, G., Manarolla, G., Vogt, H.R., Scaccabarozzi, L., Locatelli, C., Moroni, P., 2010. Genetic analysis of small ruminant lentiviruses following lactogenic transmission. Virology 407, 91–99.
cr
Ramirez, H., Reina, R., Amorena, B., de Andres, D., Martinez, H.A., 2013. Small ruminant lentiviruses : genetic variability, tropism and diagnosis. Viruses 5, 1175-1207.
us
Reina, R., Berriatua, E., Luján, L., Juste, R., Sánchez, A., de Andrés, D., Amorena, B., 2009. Prevention strategies against small ruminant lentiviruses: an update. Vet. J. 182, 31-37.
an
Reina, R., Bertolotti, L., Dei Giudici, S., Puggioni, G., Ponti, N., Profiti, M., Patta, C., Rosati, S., 2010. Small ruminant lentivirus genotype E is widespread in Sarda goat. Vet. Microbiol., 144, 24–31.
M
Reina, R., Mora, M.I., Glaria, I., Garcia, I., Solano, C., Lujan, L., Badiola, J.J., Contreras, A., Berriatua, E., Juste, R., Mamoun, R.Z., Rolland, M., Amorena, B., de Andres, D., 2006. Molecular characterization and phylogenetic study of maedi visna and caprine arthritis encephalitis viral sequences in sheep and goats from Spain. Virus Res. 121, 189–198.
d
Ryan, S., Tiley, L., McConnel, I., Blacklaws, B., 2000. Infection of dendrictic cells by the maedi-visna lentivirus. J. Virol. 74, 10096-10103.
te
Sargan, D.R., Bennet, E.D., Cousens, C., Roby, D.J., Blacklaws, B.A., Dalziel, R.G., Watt, N.J., McConnell, I., 1991. Nucleotide sequence of EV1, a British isolate of maedi-visna virus. J. Gen. Virol. 72, 1893-1903.
Ac ce p
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Shah, C., Böni, J., Huder, J.B., Vogt, H.R., Mühlherr, J., Zanoni, R., Miserez, R., Lutz, H., Schüpbach, J., 2004. Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade. Virology 319, 12–26. Toussaint, J.F., Sailleau, C., Breard, E., Zientara, S., De Clerq, K., 2007. Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments. J. Virol. Methods 140, 115-123. Zhang, Z., Watt, N.J., Hopkins, J., Harkiss, G., Woodall, C.J., 2000. Quantitative analysis of maedi-visna virus DNA load in peripheral blood monocytes and alveolar macrophages. J. Virol. Methods 86, 13–20. Zink, M.C., Yager, J.A., Myers, J.D., 1990. Pathogenesis of caprine arthritis encephalitis virus. Cellular localization of viral transcripts in tissues of infected goats. Am. J. Pathol. 136, 843-854.
603 21 Page 21 of 30
604
Tables
605
Table 1. Overview of developed primers and probe for the q(RT)-PCR detection of SRLV and
607
sequencing of the gag fragment of SRLV strain 6612.
608
Nucleotide sequence (5’-3’)
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caa gag caa cac tgg taa gg cta ccg cct tcc aac ttc tc tag aga cat ggc gaa gca agg tgc aag gag gca aat tct ct cat taa gca agc cat tgt gg
613 614 615 616 617
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612
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611
d
609 610
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tag aga cat ggc gaa gca agg gcc cat aga cag ttc cct tc 6-FAM - tac ccc gag ctc aa - MGBNFQ
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q(RT)-PCR forward reverse MGB-probe 6612 sequencing seq primer 1 seq primer 2 seq primer 3 seq primer 4 seq primer 5
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606
618 619 620 621 622 22 Page 22 of 30
623
Table 2. Overview of qPCR results for SRLV isolates and defined SRLV positive samples
624
from various countries. genotype
qPCR result
a
pos pos
P1OLV
isolate
A
U.K.
EV-1
isolate
Ab
The Netherlands
ZVV 1050
isolate
n.d.
pos
MVV-7
isolate
n.d.
pos
Iceland
1514
isolate
Ab
pos
USA
WLC-1
isolate
n.d.
pos
Belgium
35 sheep from 5 seropositive sheep flocks
whole blood
n.d.
pos
16 goats from 4 seropositive goat flocks
whole blood
n.d.
neg
sheep 6612
lung
n.d.
pos
DNA extract
n.d.
pos
528265278600016
DNA extract
n.d.
pos
11(20890)
DNA extract
n.d.
pos
12 (302739)
DNA extract
n.d.
pos
3951
DNA extract
n.d.
pos
5834-09
DNA extract
n.d.
pos
H S2L 2006 Fin
lung
n.d.
pos
pos contr
Portugal
Ac ce p
Finland
te
M
The Netherlands
cr
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Portugal
us
Field samples
sample
an
Virus isolates
strain/identification
d
origin
Scotland
31 sheep from 4 seropositive sheep flocks
whole blood
n.d.
pos
Spain
697
DNA extract
Ac
pos
258
DNA extract
n.d.
pos
France
d
496
DNA extract
B
10 SRLV pos sheep
DNA extract
CAEV likee
neg neg
625
n.d.= not determined; Barros et al., 2004; Shah et al., 2004; Glaria et al., 2012; Glaria et al., 2009; Leroux et
626
al., 1995
a
b
c
d
e
627 628 629
23 Page 23 of 30
630
Table 3. Comparison between serological (AGID (A) and ELISA (B)) and qPCR SRLV
631
diagnosis of 35 Belgian sheep from 5 different flocks.
B ELISA
pos neg
15 20 35
qPCR pos neg 14 9 3 9 17 18
23 12 35
cr
pos neg
us
AGID
qPCR pos neg 13 2 4 16 17 18
an
A
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632
633
M
634
638 639 640 641 642 643
te
637
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644 645 646 647 648 24 Page 24 of 30
649
Figure legends
650
Figure 1. Alignment of the developed primer and probe sequences with corresponding
652
sequences of selected SRLV strains.
653
The sequences of the developed primers and probe were aligned with corresponding regions
654
of the Belgian strain 6612 and SRLV sequences of genotype A (1514 (NC_001452.1), EV1
655
(S51392.1), P1OLV (AF476938.1), WLC1.3 (GQ255434.1), 83.7 (GQ255410.1), SA-OMVV
656
(NC_001511.1)), B (CAEV Co (M33677)), C (1GA-NOR (AF322109)) and E (Roccaverano
657
(EU293537)).
us
cr
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651
an
658
Figure 2. (A) DNA and (B) RNA standard curve for detection of SRLV. A plasmid containing
660
a part of the gag gene sequence of the 1514 SRLV strain and corresponding RNA transcripts
661
were serially diluted in respectively a DNA and RNA extract from a SRLV negative sheep
662
lung. Each dilution was tested by the developed q(RT)-PCR using FastStart TaqMan Probe
663
Master kit (Roche). A standard curve was constructed by plotting the mean CT values of three
664
independent replicates against the log of the DNA and RNA copy number in each dilution.
d
te
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665
M
659
666
Figure 3. Matrix effect. A lung homogenate and whole blood of an SRLV positive sheep were
667
10 fold serially diluted in respectively a lung homogenate and whole blood of an SRLV
668
negative sheep followed by sample preparation and DNA extraction (A). The SRLV WLC1
669
isolate was spiked and subsequently 10 fold serially diluted in respectively a lung homogenate
670
and blood of an SRLV negative sheep. Also a lung homogenate of an SRLV positive sheep
671
was 10 fold serially diluted in a lung homogenate of an SRLV negative sheep. For all
672
dilutions, RNA extraction and reverse transcription was performed after sample preparation
25 Page 25 of 30
673
(B). All DNA extracts and cDNA samples were tested in the developed q(RT)-PCR using the
674
FastStart TaqMan Probe Master kit (Roche).
675
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676
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677
26 Page 26 of 30
Highlights
678
A q(RT)-PCR detecting genotype A SRLV strains was developed and validated.
679
It has a limit of detection of 6 DNA and 40 RNA copies/reaction.
680
It detects SRLV RNA and proviral DNA in lungs and leukocyte pellets.
681
It detects SRLV strains circulating in Belgium and various other countries.
682
The complementary value of PCR based diagnosis besides serology is evidenced.
cr
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677
Ac ce p
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M
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683
27 Page 27 of 30
Ac
ce
pt
ed
M
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Figure(s)
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Figure 2
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Figure 3
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S0166-0934(13)00388-1 http://dx.doi.org/doi:10.1016/j.jviromet.2013.09.001 VIRMET 12306
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
22-2-2013 28-8-2013 3-9-2013
Please cite this article as: De Regge, N., Cay, B., Development, validation and evaluation of added diagnostic value of a q(RT)-PCR for the detection of genotype A strains of small ruminant lentiviruses., Journal of Virological Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.09.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Development, validation and evaluation of added diagnostic value of a q(RT)-PCR for the
2
detection of genotype A strains of small ruminant lentiviruses.
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Nick De Regge, Brigitte Cay
5
CODA-CERVA, Groeselenberg 99, 1180 Brussel, Belgium
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Nick De Regge: [email protected]
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Brigitte Cay: [email protected]
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Corresponding author:
15
Nick De Regge
16
Groeselenberg 99
17
1180 Brussel
18
Belgium
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Phone: 0032 (0)2 379 05 80
20
Fax: 0032 (0)2 379 06 70
21
Email: [email protected] 1 Page 1 of 30
22
Summary
23
Small ruminant lentiviruses (SRLV) infect sheep and goats. Diagnosis of SRLV infection
25
mostly relies on serological testing but more recently, also PCR is regarded as a useful
26
complementary tool in SRLV diagnosis. The goal of this study was to develop and validate a
27
quantitative PCR capable to detect a broad range of SRLV strains from genotype A, including
28
strains circulating in Belgium.
29
The developed q(RT)-PCR targets a region of the gag gene and showed to be highly sensitive
30
and specific with a limit of detection of 6 DNA and 40 RNA copies/reaction respectively.
31
SRLV sequences could be detected in lung samples and leukocytes pellets. The q(RT)-PCR
32
identified SRLV positive animals in Belgian sheep flocks, but also SRLV isolates and
33
samples from Scotland, The Netherlands, Spain, Portugal, UK, Iceland, Finland and USA
34
were found positive. Samples known to contain ‘CAEV like’ SRLV from France and Spain
35
were not identified as positive. Combined serological and PCR analysis of a limited number
36
(n=35) of Belgian sheep underlined the usefulness of the described PCR as a complementary
37
diagnostic tool since 3 seronegative animals were found positive by the PCR.
38
In conclusion, the validated q(RT)-PCR shows excellent analytical characteristics and is
39
capable to detect SRLV strains belonging to genotype A from various countries.
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Keywords: small ruminant lentiviruses; diagnosis; q(RT)-PCR; validation; control program
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1. Introduction
47
Small ruminant lentiviruses (SRLV), including Maedi-Visna virus (MVV) and Caprine
49
arthritis-encephalitis virus (CAEV), form a viral continuum of strains which can infect sheep
50
and goats (Leroux et al., 2010). They cause a chronic inflammatory degenerative disease
51
sometimes associated with clinical signs such as neurological disorders, pneumonia, dyspnea,
52
arthritis, mastitis and weight lose leading to economic losses, trade limitations and a negative
53
impact on animal welfare (reviewed in Blacklaws, 2012; Peterhans et al., 2004). Vertical
54
transmission through ingestion of infected colostrum or milk seems the main route of virus
55
transmission, but horizontal transmission via respiratory secretions between animals that are
56
in close contact has also been described (reviewed in Blacklaws et al., 2004; McNeilly et al.,
57
2008; Peterhans et al., 2004).
58
SRLV belong to the family of the Retroviridae and are ss(+) RNA viruses with a tropism for
59
cells of the monocyte/macrophage lineage and dendritic cells, but also other cell types can
60
become infected in tissues (Bolea et al., 2006; Gendelman et al., 1985, 1986; Lerondelle et al.,
61
1999; Ryan et al., 2000; Zink et al., 1990). For virus replication, as other lentiviruses, they
62
integrate as a provirus in the DNA of the host cell genome. By consequence, infection of bone
63
marrow stem cells or precursor cells allows for continuous production of infected cells and a
64
persistent life-long infection of the animal (Gendelman et al., 1985; Grossi et al., 2005).
65
Partial genetic characterization of SRLV strains from many different countries and subsequent
66
phylogenetic analysis classifies SRLV strains for the moment into five different groups, from
67
A to E. Group A, B and E are further subdivided in subgroups. Group A consist of MVV like
68
genotypes, group B of CAEV like genotypes, while the other three groups harbor genotypes
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from more restricted geographical regions (Bertolotti et al., 2011; Giammarioli et al., 2011;
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Gjerset et al., 2006; Glaria et al., 2009; Grego et al., 2007; Olech et al., 2012; Pisoni et al.,
71
2010; Reina et al., 2006; Reina et al., 2010; Shah et al., 2004).
72
Since no adequate prophylactic tools are on the market, many countries rely on control
73
programs to decrease SRLV prevalence (Peterhans et al., 2004; Reina et al., 2009). Good
74
diagnostic tools are of crucial importance for the detection of SRLV infected animals.
75
Serological tests as agar gel immunodiffusion (AGID) and ELISA are the most frequently
76
used assays to identify anti-SRLV antibodies in positive animals (reviewed by de Andres et
77
al., 2005; Hermann-Hoesing, 2010; Ramirez et al., 2013). The long time between infection
78
and seroconversion, the existence of non-responders, fluctuating antibody titers throughout
79
the animal’s life, and the antigenic differences between circulating strains and test antigens
80
used can however interfere with the sensitivity of the serological tests (Cardinaux et al., 2013;
81
de Andrés et al., 2013; Peterhans et al., 2004; Ramirez et al., 2013). More recently, also (RT)-
82
PCR and quantitative (RT)-PCR assays have been developed that are able to detect the viral
83
RNA genome, proviral sequences and viral RNA transcripts (reviewed by Hermann-Hoesling,
84
2010). PCR based diagnosis of SRLV infected animals can however be hampered by the low
85
proviral load during a latent state of infection and the large genetic variation between strains
86
(Peterhans et al., 2004). Studies comparing the sensitivity of PCRs and serological tests have
87
shown that PCR can sometimes identify SRLV positive animals that tested negative in
88
serology, thereby indicating that both tests should be seen as complementary tests in SRLV
89
diagnosis (reviewed by Ramirez et al., 2013).
90
Most of the described q(RT)-PCR assays are developed for the detection of a specific SRLV
91
strain and mostly no broad evaluation of their capacity to detect strains circulating in different
92
geographical regions has been done (Brajon et al., 2012; Carrozza et al., 2010; Gudmundsson
93
et al., 2003; Herrmann-Hoesling et al., 2007; Zhang et al., 2000). An exception is the qPCR
94
developed by Brinkhof et al. (2008) that is described to detect several SRLV strains. This
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PCR uses the SybrGreen technology, thereby partly circumventing the problem of high
96
genetic variation. It requires however a melting curve analysis to confirm specific
97
amplification what is not always straightforward seen the probability that variable SRLV
98
sequences will denature at different temperatures.
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In Belgium, a voluntary control program for SRLV exists and certification of SRLV negative
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flocks depends on serological results obtained by AGID (Maeditect, VLA scientific) and
101
ELISA (Elitest, Hyphen) testing. In this study the development and validation of a sensitive
102
q(RT)-PCR assay capable to detect SRLV strains belonging to genotype A is described. This
103
test can in the future be implemented in the Belgian control program as a complementary or
104
confirmatory test besides serology to help identifying additional ‘Maedi Visna like’ SRLV
105
infected animals or to help defining the status of ELISA positive animals that cannot be
106
confirmed in AGID. This qPCR could also be of interest to other countries since the
107
validation demonstrates that the PCR is capable to detect SRLV strains belonging to genotype
108
A originating from different geographical regions.
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2. Material and Methods
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2.1. Ethical statement
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All samples used in this study were collected by veterinarians in accordance with existing
114
legislation on animal welfare.
115 116
2.2. Clinical specimens and virus isolates
117
Whole blood and organ samples from 79 Belgian sheep belonging to flocks that are certified
118
as SRLV negative were collected at the slaughterhouse. Whole blood from 35 sheep
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belonging to 5 Belgian sheep flocks and 16 goats belonging to 4 Belgian goat flocks in which 5 Page 5 of 30
SRLV seropositive animals were present was collected by the farm veterinarian and sent to
121
CODA-CERVA. Another SRLV seropositive Belgian sheep (6612) was housed and
122
euthanized at the animal facilities of CODA-CERVA and lung samples were collected.
123
Other samples of defined SRLV positive status (by serology or PCR) were sent to CODA-
124
CERVA including whole blood samples from 31 sheep belonging to 4 Scottish sheep flocks; a
125
lung sample from a Finnish sheep (H S2L 2006 Fin); SRLV virus isolates WLC1, P1OLV,
126
EV-1, 1514 and 2 Dutch isolates (ZVV-1050, MVV-7); and DNA extracts from organs or
127
PBMC originating from The Netherlands (positive control, 5282652786000016, 11 (20890),
128
12 (302739)), Portugal (3951, 5834-09), France (623, 663, 683, 684, 685, 760, 761, 762, 763,
129
766) and Spain (697, 258, 496).
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2.3. SRLV serology
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The serological status for SRLV of Belgian sheep used in this study was determined by the
133
use of the Elitest ELISA (Hyphen, Neuville-sur-Oise, France) and the Maeditect AGID test
134
(VLA scientific, London, United Kingdom) following manufacturer’s instructions.
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2.4. Preparation of control DNA and RNA transcripts from strain 1514 background
137
A plasmid containing a part of the SRLV strain 1514 gag sequence and the corresponding
138
RNA transcripts were produced using standard cloning and in vitro transcription protocols.
139
After RNA extraction from a virus stock of strain 1514 and cDNA synthesis, a DNA fragment
140
corresponding to 524 base pairs (bp) was produced by classical PCR using the Seq 1 and Seq
141
2 primer described in table 1. The fragment was cloned into the pCR 2.1-TOPO cloning
142
vector (Life Technologies, Ghent, Belgium) and subsequently transformed into competent
143
Escherichia coli TOP10F’ cells and multiplied. Purified plasmids were linearised with BamHI
144
and in vitro transcribed (TranscriptAid T7 High Yield Transcription Kit, Thermo Scientific,
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Aalst, Belgium). Remaining plasmid DNA was eliminated by a Turbo DNA free treatment
146
(Life Technologies, Ghent, Belgium) and the RNA was purified using the RNeasy Mini kit
147
(Qiagen, Hilden, Germany). The produced plasmid and RNA transcripts were quantified
148
using a Nanodrop device (Thermofisher Scientific, Aalst, Belgium) and the copy number was
149
calculated based on the predicted molecular weight of either the plasmid or RNA transcripts.
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Aliquots of the DNA plasmid and RNA transcripts were stored at -80 °C.
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2.5. Sample preparation, DNA and RNA extraction and reverse transcription
153
Whole blood samples were used to prepare leucocyte pellets by adding 1,5 ml blood to 8,5 ml
154
hemolysis buffer (16.6 g NH4Cl, 2.0 g NaHCO3, 0.185g diNa EDTA per l H2O; pH 7.4
155
(Mignon et al., 1992)). After 15 min of incubation, the samples were centrifuged for 15 min at
156
1000g and after elimination of the supernatans, the pellet was resuspended in 100 µl of
157
phosphate buffered saline (PBS). Lung samples were homogenized by adding ± 0.5 g of tissue
158
to 1 ml PBS and 10 to 15 1mm silicon carbid beads (Biospec Products, Bartlesville, OK,
159
USA), followed by high speed shaking (2 min, 25 Hz) in a TissueLyser (Qiagen, Hilden,
160
Germany).
161
Extraction of DNA and total RNA from leucocyte pellets, lung homogenates and virus
162
isolates was respectively done by the QIAamp DNA minikit (Qiagen, Hilden, Germany) and
163
the RNeasy Mini kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. DNA
164
and RNA were respectively eluted in 100 and 50 µl H2O.
165
Total RNA was converted to cDNA using the M-MLV reverse transcriptase system (Life
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Technologies, Ghent, Belgium). For each reaction, a mix of 4 µl 5x first strand buffer, 2 µl
167
0.1M DTT, 1 µl 10nM dNTP mix (Roche, Basel, Switzerland), 2 µl 1x hexanucleotide mix
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(Roche, Basel, Switzerland), 1 µl M-MLV RT and 10 µl RNA was prepared and incubated at
169
37°C for 45min, followed by inactivation at 95°C for 10min.
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2.6. Real time PCR assay
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All DNA and cDNA samples were analysed for the presence of SRLV by using the FastStart
173
TaqMan Probe Master kit (Roche, Basel, Switzerland) following manufacturer’s instructions
174
with primers and probe directed against a part of the gag gene of SRLV. To check the DNA or
175
RNA extraction, reverse transcription and amplification reaction, each sample was also tested
176
for the presence of -actin in a separate reaction (forward primer: 5’-
177
cagcacaatgaagatcaagatcatc-3’; reverse primer: 5’-cggactcatcgtactcctgctt-3’; probe: HEX-5’-
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tcgctgtccaccttccagcagatgt-3’-BHQ1 (Toussaint et al., 2007)). Briefly, a master mix consisting
179
of 5 µl RNase-free water, 10 µl 2x Faststart Taqman probe master buffer, 1 µl forward primer
180
(10µM), 1 µl reverse primer (10µm) and 1 µl probe (4µM) for one reaction was prepared and
181
2 µl DNA or cDNA template was added. For amplification, the following temperature profile
182
was used: 10 min at 95°C, followed by 45 cycles of 15s at 95°C and 45s at 60°C. All qRT-
183
PCRs were done on a LightCycler 480 Real-Time PCR system (Roche, Basel, Switzerland).
184
In all q(RT)-PCR analyses performed, negative extraction controls and negative and positive
185
amplification controls were included and tests were only validated when all controls were
186
satisfactory.
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2.7. Construction of standard curve and parameters used for evaluation of q(RT)-PCR
189
performance
190
For the construction of the DNA standard curve, the constructed plasmid containing part of
191
the 1514 strain gag sequence was 10 fold serially diluted in a DNA extract obtained from an
192
SRLV negative lung and all dilutions were assayed in the qPCR with input material ranging
193
from 3x107 to 3x10-2 copies/µl. Same was done for RNA whereby the RNA transcripts were
194
10 fold serially diluted in an RNA extract obtained from an SRLV negative lung. Input RNA
8 Page 8 of 30
transcripts ranging from 4x107 to 4x10-2 RNA copies/µl were reverse transcribed, followed by
196
analysis in the qPCR. For both DNA and RNA, three independent replicates were run, mean
197
values of each dilution were calculated and a standard curve was constructed by plotting the
198
Ct values against the log of the input DNA or RNA copy number. These standard curves were
199
used to evaluate the q(RT)-PCR performance based on dynamic range (the range of
200
respectively template DNA and RNA copies/µl for which accurate CT values were obtained),
201
r2 value (a statistical measure indicating how well the linear regression curve fits the real data
202
points; an r² of 1.0 indicates that the regression line perfectly fits the data), efficiency (%)
203
(efficiency was calculated using the following formula: Efficiency = -1+10(-1/slope)) and
204
sensitivity (limit of detection (LOD) was determined as the lowest concentration for which
205
3/3 replicates produced a positive result).
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2.8. Sequencing of a gag gene fragment of SRLV strain 6612 and amplified fragments
208
obtained by the developed q(RT)-PCR
209
For sequencing of a gag gene fragment of SRLV strain 6612, PCR amplification using
210
primers targeting a part of the gag gene of SRLV1514 strain (Seq1 and Seq 2 primers in Table
211
1) was performed on cDNA prepared from the lung of the Belgian sheep 6612 using the
212
FastStart PCR Master kit from Roche (Basel, Switzerland) following manufacturer’s
213
instruction. An initial denaturation for 5 min at 95°C was followed by 40 cycles of 1 min at
214
95°C, 1 min at 55°C and 1 min at 72°C, and a final extention at 72°C for 10 min. PCR
215
products were visualised on a 1% agarose gel and bands of the correct size were cut from the
216
gel and purified using QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany).
217
Approximately 5 ng of PCR product was used as template for sequencing using the forward
218
and reverse primer mentioned above and 3 additional primers (Seq 3, Seq 4 and Seq 5 primer
219
in Table 1) with the BigDye Terminator v3.1 sequencing kit (Life Technologies, Ghent,
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Belgium). Sequencing reactions were purified using the BigDye Xterminator reagent (Life
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Technologies, Ghent, Belgium) and ran on an ABI3130 Genetic Analyser (Life Technologies,
222
Ghent, Belgium).
223
For sequencing of the fragments obtained by the developed q(RT)-PCR, the realtime reaction
224
product was separated on an 0.6% agarose gel and a similar protocol as described above was
225
followed using 2 ng of PCR product and the forward and reverse primer of the developed
226
q(RT)-PCR (Table 1) as sequencing primers.
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3. Results
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3.1. Partial gag gene sequencing of Belgian SRLV strain 6612, sequence alignment and
231
primer/probe selection
232
The lung of a Belgian serological SRLV positive sheep 6612 was found positive for the
233
presence of SRLV by the classical PCR described by Celer et al. (2000). Using primers
234
homologous to the gag gene sequence of SRLV strain 1514, a part of the gag gene from
235
SRLV strain 6612 was amplified and sequenced (Genbank accession number: KC560792). A
236
similarity search in the NCBI nucleotide database using BLAST showed that considerable
237
similarity (between 80 and 90%) existed with the EV-1, 1514, SA-OMVV, 83.7, WLC-1 and
238
P1OLV SRLV strain sequences in the database. These strains have been classified before as
239
strains belonging to genotype A, based on phylogenetic analysis of ± 1.8 kb sequences of the
240
gag-pol region and ± 1.2 kb sequences of the pol region (Shah et al., 2004). Clearly less
241
similarity was found after alignment of the 6612 sequence with corresponding sequences of
242
SRLV strains of genotype B, C and E.
243
Since the high genetic variability in this genomic region did not allow to find conserved
244
sequences when all genotypes were taken into account, primer express software was used to
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select primers (Eurogentec, Seraing, Belgium) and a minor groove binding (MGB) probe
246
(Life Technologies, Ghent, Belgium) in the conserved regions of genotype A strains (Table
247
1). Figure 1 shows the similarity between the selected primers and probe sequences with
248
corresponding sequences of SRLV strains of genotype A and the heterogeneity with SRLV
249
strains belonging to the other genotypes.
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3.2. Analytical q(RT)-PCR performance
252
After production of control DNA and RNA from strain 1514 background, standard curves
253
were constructed and used to determine the analytical q(RT)-PCR performance.
254
For DNA, the limit of detection of the real time PCR was 3 plasmid copies/µl (corresponding
255
to 6 copies/reaction). The typical standard curve amplification plot and linear regression
256
analysis (Fig.2a) showed an amplification efficiency of 91% and a dynamic range of detection
257
between 3x107 and 3x100 copies/µl. For RNA, the detection limit was 40 copies/µl
258
(corresponding to 40 copies/reaction) (Fig. 2b). An amplification efficiency of 92% and a
259
linear range of detection between 4x107 and 4x101 copies/µl were obtained.
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3.3. Matrix effect
262
Since the q(RT)-PCR on plasmid DNA and RNA transcripts showed promising results
263
regarding the limit of detection and PCR efficiency, its capability to detect SRLV in different
264
biological samples was evaluated in a next step. Again, this was done for SRLV detection on
265
both DNA and RNA level.
266
The lung of the Belgian sheep 6612 (Ct=29.3) and a leucocyte pellet prepared from whole
267
blood of a Scottish sheep (Ct=29.8) tested positive in the q(RT)-PCR under validation. These
268
samples were used to study the impact of sample material on SRLV detection after DNA
269
extraction. A homogenate of the positive lung was 10 fold serially diluted in a homogenate of
11 Page 11 of 30
an SRLV negative lung. Same was done for the blood by diluting it in whole blood of an
271
SRLV negative animal. After sample preparation and DNA extraction, all dilutions were
272
tested in the qPCR. This resulted (Fig.3a) in linear assays over several dilutions and the slopes
273
(respectively -3.3 and -3.38 for lung and blood), and therefore the PCR efficiency, were
274
similar to the slope found for the DNA standard curve (-3.56).
275
To study the impact of sample material on SRLV detection upon RNA extraction, the same
276
experiment with the lung material of animal 6612 as described above was repeated with the
277
difference that now RNA was extracted from the dilution series, followed by reverse
278
transcription and PCR analysis. Furthermore, a homogenate of an SRLV negative lung and a
279
whole blood sample of an SRLV negative sheep were spiked with SRLV strain WLC-1 (104.8
280
TCID50/ml homogenate or whole blood). These spiked samples were then respectively 10
281
fold serially diluted in a homogenate of a negative lung or in whole blood from a negative
282
sheep, followed by sample preparation, RNA extraction, reverse transcription and qPCR
283
analysis. Figure 3b shows that SRLV could be detected in all biological samples and that the
284
qPCR was linear over several dilutions. Also here, the slopes of all dilution series (-3.29 for
285
lung pos in lung neg; -3.54 for WLC-1 in lung neg; -3.29 for WLC-1 in blood neg) were
286
similar to the slope of the RNA standard curve (-3.54).
287
These results indicate that the PCR assay is capable to detect SRLV DNA and RNA
288
sequences in different biological samples and that the DNA and RNA standard curves (Fig.2)
289
can be used for the calculation of the (pro)viral load in the different samples.
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3.4. Specificity
292
To test the diagnostic specificity, 79 leucocyte pellets prepared from whole blood samples
293
from AGID and ELISA negative sheep from SRLV free certified Belgian farms were tested
294
using the developed qPCR after DNA extraction. All samples were found negative, showing a
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100% concordance with the serological results. Also 2 organ samples known to be PCR
296
positive for equine infectious anemia virus, a lentivirus of horses, were tested and were both
297
negative, giving a preliminary idea of the analytical specificity of the PCR.
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3.5. Diagnostic sensitivity
300
The results described above show that the PCR is capable to detect SRLV sequences present
301
in the lung of Belgian sheep 6612 and in the blood of a Scottish serological positive sheep. To
302
get further insight in the range of SRLV strains that can be detected by the PCR, reference
303
laboratories and research institutes of several countries were contacted and asked to provide
304
us with SRLV strains or material from SRLV positive animals. Most laboratories responded
305
positively to our demand and provided us with virus isolates, organ or blood samples, or DNA
306
extracts from organs or blood from defined SRLV positive animals (by serology or PCR).
307
Table 2 gives an overview of the received samples and the obtained PCR results.
308
All isolates and samples containing SRLV strains known to belong to genotype A tested
309
positive in the qPCR. Also several samples containing genetically non-characterized SRLV
310
strains were found positive. The specificity of the amplification products of all q(RT)-PCR
311
positive samples mentioned in table 2 was confirmed by sequencing. All amplicons showed
312
similarity with sequences of the gag region of SRLV strains when a similarity search was
313
done with the NCBI database using BLAST (data not shown).
314
The samples that remained negative were a sample containing group B SRLV strain 496 from
315
Spain (Glaria et al., 2009), 10 samples containing ‘CAEV like’ strains from France (Leroux et
316
al., 1995) and samples from 4 serological SRLV positive Belgian goat flocks. The SRLV
317
positive status of these samples was confirmed by the use of the classical PCR assay
318
described by Leroux et al. (1995), except for samples from 2 out of 4 Belgian goat flocks
319
(data not shown).
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3.6. Testing of Belgian field samples
322
To get an idea of the concordance between serological tests and the developed qPCR for
323
SRLV detection in sheep and the usefulness of implementing the qPCR in the Belgian SRLV
324
control program, whole blood samples of 35 animals from 5 different farms in which at least
325
one serological positive sheep was detected were analyzed by the qPCR while the
326
corresponding serum samples were tested in AGID and ELISA. Cross table 3a shows that
327
there is a positive concordance of 87% (13/15) between AGID and qPCR results. Four
328
samples were found positive in the qPCR while they were negative in the AGID test. The
329
positive concordance between the ELISA and qPCR test was 61% (14/23), while 3 samples
330
positive in the qPCR were negative in the ELISA (table 3b). Taken together, 3 out of 35
331
samples were positive in the qPCR but negative in both serological tests. On the other hand, 2
332
AGID and 9 ELISA positive samples were not confirmed in the qPCR, indicating that not all
333
seropositive animals are detected by the qPCR.
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4. Discussion
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A combination of aspects related to the SRLV replication cycle, their genetic variability and
338
the slow and sometimes hampered humoral immune response to these viruses makes it not
339
always straightforward to correctly identify the SRLV infection status of an animal (de
340
Andres et al., 2013; Peterhans et al., 2004; Ramirez et al., 2013). In Belgium, the voluntary
341
SRLV control program relies on serological testing using ELISA and AGID. The discrepancy
342
that is sometimes observed between both test results raises questions about the true infection
343
status of an animal and the related measures to apply since these have important economic
344
impact for the farmers. To assist in the determination of the SRLV infection status of
14 Page 14 of 30
suspected sheep, a q(RT)-PCR capable to detect SRLV strains from genotype A circulating in
346
Belgian herds was developed.
347
The validation showed that the developed q(RT)-PCR is capable to detect SRLV in different
348
biological samples and that it can be used to detect SRLV sequences on both DNA and RNA
349
levels. This latter property can be important since depending on the moment of infection
350
(acute or latent), the sample received, and the context of the analysis (diagnosis, in vivo or in
351
vitro experimental infection), it can be preferable to search for proviral DNA, the viral RNA
352
genome or viral RNA transcripts (since hexanucleotides were used during reverse
353
transcription, no differentiation between both RNA molecules was made in this study).
354
Furthermore, the assay turned out to be sensitive with a limit of detection of 3 copies/µl DNA
355
extract and 40 copies/µl RNA extract. This LOD is similar to sensitivities that were
356
previously reported for other q(RT)-PCR assays for the detection of SRLV (Carroza et al.,
357
2010; Gudmundsson et al., 2003). Since positive results were obtained for blood and organ
358
samples of sheep without clinical symptoms in this study, it seems reasonable to assume that
359
the sensitivity of the test is satisfactory to detect SRLV from genotype A in most infected
360
animals. Follow up of SRLV positive animals over time will have to show if the PCR is able
361
to detect the virus during the latent phase of infection, the moment where the lowest provirus
362
quantities are present.
363
The analysis of the diagnostic sensitivity has shown that the PCR is capable to detect SRLV
364
strains circulating in Belgium, as well as strains from several other countries. The fact that the
365
primer/probe selection was done using conserved regions within the gag gene of SRLV strains
366
belonging to the phylogenetic group A and that all received samples containing genotype A
367
sequences tested positive in the PCR makes it reasonable to assume that this PCR is especially
368
useful to detect SRLV strains belonging to this genotype (‘Maedi-Visna like strains’).
369
Although uncertain, probably also the non-characterized samples from Portugal, Finland and
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Scotland that tested positive contain genotype A SRLV strains since mainly strains belonging
371
to this genotype have been isolated in these countries (Barros et al., 2004; Laamanen et al.,
372
2007; Sargan et al., 1991). Further indications that the PCR detects probably SRLV strains
373
belonging to genotype A are found in the fact that all ‘CAEV like’ strains tested were not
374
identified as positive. The observation that seropositive samples from Belgian goats were
375
found positive in the PCR described by Leroux et al. (1995) but tested negative in the newly
376
developed q(RT)-PCR indicates that probably strains belonging to different genotypes
377
circulate in Belgium. The sequences used for primer selection by Leroux et al. (1995) and the
378
widespread distribution of genotype B strains in goats (Ramirez et al., 2013) suggest that the
379
Belgian goats are probably infected by strains of genotype B. It will be a future challenge to
380
identify these strains and to adapt the PCR accordingly so they can also be detected. However,
381
surveillance on both species is highly recommended since SRLV strains are able to cross
382
species barriers between sheep and goats (Leroux et al, 2010).
383
When interpreting results obtained with this q(RT)-PCR, it should be kept in mind that this
384
PCR is directed to a part of the gag region that falls out of the genomic region that is used for
385
the phylogenetic classification (Shah et al., 2004) and that strains positive in this PCR should
386
not automatically be regarded as strains belonging to group A. Further sequencing is required
387
before such phylogenetic classification of the strain can be done.
388
In the limited number of serological SRLV positive Belgian sheep flocks tested till now,
389
always one or more qPCR positive animals were identified, supporting its capability to detect
390
SRLV strains circulating in Belgian sheep. The comparison between serological test results
391
and qPCR results on samples from Belgian sheep showed that a high positive concordance
392
existed between AGID results and the qPCR, but that this was considerably lower between the
393
ELISA and qPCR results. Care should however be taken in the interpretation of these data
394
since they represent only a very limited sample size (n=35) which moreover was not selected
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16 Page 16 of 30
randomly, since it contains samples from SRLV free certified flocks that tested unexpectedly
396
positive in ELISA what could not be confirmed by other tests. More samples from a random
397
sampling need to be tested before making definite statements about the concordance of the
398
qPCR with serological tests. What is however interesting is that 3 out of 35 animals were
399
positive in the qPCR while they were negative in both ELISA and AGID. This indicates that
400
the qPCR can be a useful complementary tool in a control program to identify animals that
401
would not be detected if only serological testing is done. These animals would pose a risk for
402
transmission of the virus to the rest of the herd if they remain in the flock. Since the validation
403
demonstrated that the q(RT)-PCR is capable to detect SRLV strains originating from different
404
geographical regions, it could for the same reason also be of interest to diagnostic and
405
research laboratories in other countries. It should however also be emphasized that several
406
ELISA and AGID positive samples were not confirmed in the qPCR. These could be
407
explained as false positive reactions in serology, but they most likely represent animals in
408
which the viral load was too low or that were infected by a strain that is not recognized by the
409
PCR. This further supports the view that PCR and serology have to be considered as
410
complementary tests in SRLV diagnosis.
412 413
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5. Conclusion
414
The q(RT)-PCR developed in this study targets a region of the gag gene of SRLV. During
415
validation, the PCR showed to have excellent q(RT)-PCR characteristics. It is capable to
416
detect SRLV proviral DNA and RNA in blood and lung and showed to be highly sensitive
417
and specific. Furthermore, several SRLV isolates and positive samples, most probably ‘Maedi
418
Visna like’, from Belgium, Scotland, The Netherlands, Spain, Portugal, UK, Iceland, Finland
419
and USA were identified as positive. This q(RT)-PCR could therefore be used as a useful
17 Page 17 of 30
420
diagnostic tool to detect genotype A strains of SRLV, and it can also be an interesting tool for
421
research purposes.
422
6. Acknowledgements
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We thank Virginie Colasse, Sarah Matuyia, and Thibault De Maesschalck for the excellent
426
technical assistance during the qPCR validation. We thank Guido Bertels for his help in the
427
collection of samples from Belgian sheep. We particularly thank Hans Kramps, Gerard
428
Wellenberg, Beatriz Amorena, Barbara Blacklaws, Brian Hosie, Miguel Fevereiro, Liisa
429
Sihvonen, Valgerdur Andresdottir and Caroline Leroux for their willingness to provide us
430
with SRLV positive samples that were used in this validation.
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7. References
433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456
Barros, S.C., Ramos, F., Duarte, M., Fagulha, T., Cruz, B., Fevereiro, M., 2004. Genomic characterization of a slow/low Maedi Visna virus. Virus Genes 29, 199-210.
te
d
432
Ac ce p
Bertolotti, L., Mazzei, M., Puggioni, G., Carrozza, M.L., Dei Giudici, S., Muz, D., Juganaru, M., Patta, C., Tolari, F., Rosati, S., 2011. Characterization of new small ruminant lentivirus subtype B3 suggests animal trade within the Mediterranean Basin. J. Gen. Virol. 92, 1923– 1929. Blacklaws B.A., 2012. Small ruminant lentiviruses: Immunopathogenesis of visna-maedi and caprine arthritis and encephalitis virus. Comp. Immunol. Microbiol. Infect. Dis. 35, 259-269. Blacklaws, B.A., Berriatua, E., Torsteinsdottir, S., Watt, N.J., de Andres, D., Klein, D., Harkiss, G.D., 2004. Transmission of small ruminant lentiviruses. Vet. Microbiol. 101, 199– 208. Bolea, R., Monleon, E., Carrasco, L., Vargas, A., de Andrés, D., Amorena, B., Badiola, J.J., Lujan, L., 2006. Maedi-visna virus infection of ovine mammary epithelial cells. Vet. Res. 37, 133-44. Brinkhof, J.M., van Maanen, C., Wigger, R., Peterson, K., Houwers, D.J., 2008. Specific detection of small ruminant lentiviral nucleic acid sequences located in the proviral long terminal repeat and leader-gag regions using real-time polymerase chain reaction. J. Virol. Methods 147, 338-344.
18 Page 18 of 30
Cardinaux, L., Zahno, M.L., Deubelbeiss, M., Zanoni, R., Vogt, H.R., Bertoni, G., 2013. Virological and phylogenetic characterization of attenuated small ruminant lentivirus isolates eluding efficient serological detection. Vet. Microbiol. 162, 572-581.
ip t
Carrozza, M.L., Mazzei, M., Bandecchi, P., Fraisier, C., Pérez, M., Suzan-Monti, M., de Andrés, D., Amorena, B., Rosati, S., Andrésdottir, V., Lujan, L., Pepin, M., Blacklaws, B., Tolari, F., Harkiss, G.D., 2010. Development and comparison of strain specific gag and pol real-time PCR assays for the detection of Visna/maedi virus. J. Virol. Methods 165, 161-167.
cr
Celer, V. Jr., Celer, V., Nejekla, E., Bertoni, G., Peterhans, E., Zanoni, R.G., 2000. The detection of proviral DNA by semi-nested polymerase chain reaction and phylogenetic analysis of Czech maedi-visna isolates based on gag gene sequences. J. Vet. Med. B Infect. Dis. Vet. Public Health 47, 203–215.
us
de Andres, D., Klein, D., Watt, N.J., Berriatua, E., Torsteinsdottir, S., Blacklaws, B.A., Harkiss, G.D., 2005. Diagnostic tests for small ruminant lentiviruses. Vet. Microbiol. 107, 49–62.
M
an
de Andres, X., Ramirez, H., Bertolotti, L., San Roman, B., Glaria, I., Crespo, H., Jauregui, P., Minguijon, E., Juste, R., Leginagoikoa, I., Perez, M., Lujan, L., Badiola, J.J., Polledo, L., Garcia-Marin, J.F., Riezu, J.I., Borras-Cuesta, F., de Andres, D., Rosati, S., Reina, R., Amorena, B., 2013. An insight into a combination of ELISA strategies to diagnose small ruminant lentivirus infections. Vet. Immunol. Immunopathol. 152, 277-288.
te
d
Gendelman, H.E., Narayan, O., Kennedy-Stoskopf, S., Kennedy, P.G., Ghotbi, Z., Clements, J.E., Stanley, J., Pezeshkpour, G., 1986. Tropism of sheep lentiviruses for monocytes: susceptibility to infection andvirus gene expression increase during maturation of monocytes to macrophages. J. Virol. 58, 67-74. Gendelman, H.E., Narayan, O., Molineaux, S., Clements, J.E., Ghotbi, Z., 1985. Slow, persistent replication of lentiviruses: role of tissue macrophages and macrophage precursors in bone marrow. Proc. Natl. Acad. Sci. USA 82, 7086–7090.
Ac ce p
457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505
Giammarioli, M., Bazzucchi, M., Puggioni, G., Brajon, G., Dei Giudici, S., Taccori, F., Feliziani, F., De Mia, G.M., 2011. Phylogenetic analysis of small ruminant lentivirus (SRLV) in Italian flocks reveals the existence of novel genetic subtypes. Virus Genes 43, 380–384. Gjerset, B., Storset, A.K., Rimstad, E., 2006. Genetic diversity of small-ruminant lentiviruses: characterization of Norwegian isolates of caprine arthritis encephalitis virus. J. Gen. Virol., 87, 573–580. Glaria, I., Reina, R., Crespo, H., de Andrés, X., Ramírez, H., Biescas, E., Pérez, M.M., Badiola, J., Luján, L., Amorena, B., de Andrés, D., 2009. Phylogenetic analysis of SRLV sequences from an arthritic sheep outbreak demonstrates the introduction of CAEV-like viruses among Spanish sheep. Vet. Microbiol. 138, 156-162. Glaria, I., Reina, R., Ramírez, H., de Andrés, X., Crespo, H., Jauregui, P., Salazar, E., Lujan, L., Pérez, M.M., Benavides, J., Pérez, V., Polledo, L., Garcia-Marin, J.F., Riezu, J.I., Borras, F., Amorena, B., de Andrés, D., 2012. Visna/Maedi virus genetic characterization and
19 Page 19 of 30
cr
ip t
Grossi, P., Giudice, C., Bertoletti, I., Cioccarelli, G., Brocchi, E., Cammarata, G., Gelmetti, D., 2005. Immunohistochemical detection of the p27 capsid protein of caprine arthritis– encephalitis virus (CAEV) in bone-marrow cells of seropositive goats. J. Comp. Pathol., 133, 197–200.
us
Gudmundsson, B., Bjarnadottir, H., Kristjansdottir, S., Jonsson, J.J., 2003. Quantitative assays for maedi-visna virus genetic sequences and mRNA's based on RT-PCR with real-time FRET measurements. Virology, 307, 135–142.
an
Herrmann-Hoesing L.M., 2010. Diagnostic assays used to control small ruminant lentiviruses. J. Vet. Diagn. Invest., 22, 843-55.
M
Herrmann-Hoesing, L.M., White, S.N., Lewis, G.S., Mousel, M.R., Knowles, D.P., 2007. Development and validation of an ovine progressive pneumonia virus quantitative PCR. Clin. Vaccine Immunol., 14, 1274–1278. Laamanen, I., Jakava-Viljanen, M., Sihvonen, L., 2007. Genetic characterization of maedivisna virus (MVV) detected in Finland. Vet. Microbiol. 122, 357-365.
d
528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554
Grego, E., Bertolotti, L., Quasso, A., Profiti, M., Lacerenza, D., Muz, D., Rosati, S., 2007. Genetic characterization of small ruminant lentiviruses in Italian mixed flocks: evidence for a novel genotype circulating in a local goat population. J. Gen. Virol., 88, 3423–3427.
Lerondelle, C., Godet, M., Mornex, J.F., 1999. Infection of primary cultures of mammary epithelial cells by small ruminant lentiviruses. Vet. Res. 30, 467-74.
te
517 518 519 520 521 522 523 524 525 526 527
serological diagnosis of infection in sheep from a neurological outbreak. Vet. Microbiol., 155, 137-146.
Leroux, C., Cruz, J.C.M., Mornex, J.F., 2010. SRLVs: A Genetic Continuum of Lentiviral Species in Sheep and Goats with Cumulative Evidence of Cross Species Transmission. Current HIV Research, 8, 94-100.
Ac ce p
506 507 508 509 510 511 512 513 514 515 516
Leroux, C., Vuillermoz, S., Mornex, J.F., Greenland, T., 1995. Genomic heterogeneity in the pol region of ovine lentiviruses obtained from bronchoalveolar cells of infected sheep from France. J. Gen. Virol. 76, 1533-1537. McNeilly, T.N., Baker, A., Brown, J.K., Collie, D., Maclachlan, G., Rhind, S.M., Harkiss, G.D., 2008. Role of alveolar macrophages in respiratory transmission of visna/maedi virus. J. Virol. 82, 1526–1536. Mignon, B., Waxweiler, S., Thiry, E., Boulanger, D., Dubuisson, J., Pastoret, P.P., 1992. Epidemiological evaluation of a monoclonal ELISA detecting bovine viral diarrhoea pestivirus antigens in field blood samples of persistently infected cattle. J. Virol. Methods, 40, 85-94. Olech, M., Rachid, A., Croisé, B., Kuźmak, J., Valas, S., 2012. Genetic and antigenic characterization of small ruminant lentiviruses circulating in Poland. Virus Res. 163, 528-536. 20 Page 20 of 30
Peterhans, E., Greenland, T., Badiola, J., Harkiss, G., Bertoni, G., Amorena, B., Eliaszewicz, M., Juste, R.A., Krassnig, R., Lafont, J.P., Lenihan, P., Petursson, G., Pritchard, G., Thorley, J., Vitu, C., Mornex, J.F., Pepin, M., 2004. Routes of transmission and consequences of small ruminant lentiviruses (SRLVs) infection and eradication schemes. Vet. Res. 35, 257-274.
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Pisoni, G., Bertoni, G., Manarolla, G., Vogt, H.R., Scaccabarozzi, L., Locatelli, C., Moroni, P., 2010. Genetic analysis of small ruminant lentiviruses following lactogenic transmission. Virology 407, 91–99.
cr
Ramirez, H., Reina, R., Amorena, B., de Andres, D., Martinez, H.A., 2013. Small ruminant lentiviruses : genetic variability, tropism and diagnosis. Viruses 5, 1175-1207.
us
Reina, R., Berriatua, E., Luján, L., Juste, R., Sánchez, A., de Andrés, D., Amorena, B., 2009. Prevention strategies against small ruminant lentiviruses: an update. Vet. J. 182, 31-37.
an
Reina, R., Bertolotti, L., Dei Giudici, S., Puggioni, G., Ponti, N., Profiti, M., Patta, C., Rosati, S., 2010. Small ruminant lentivirus genotype E is widespread in Sarda goat. Vet. Microbiol., 144, 24–31.
M
Reina, R., Mora, M.I., Glaria, I., Garcia, I., Solano, C., Lujan, L., Badiola, J.J., Contreras, A., Berriatua, E., Juste, R., Mamoun, R.Z., Rolland, M., Amorena, B., de Andres, D., 2006. Molecular characterization and phylogenetic study of maedi visna and caprine arthritis encephalitis viral sequences in sheep and goats from Spain. Virus Res. 121, 189–198.
d
Ryan, S., Tiley, L., McConnel, I., Blacklaws, B., 2000. Infection of dendrictic cells by the maedi-visna lentivirus. J. Virol. 74, 10096-10103.
te
Sargan, D.R., Bennet, E.D., Cousens, C., Roby, D.J., Blacklaws, B.A., Dalziel, R.G., Watt, N.J., McConnell, I., 1991. Nucleotide sequence of EV1, a British isolate of maedi-visna virus. J. Gen. Virol. 72, 1893-1903.
Ac ce p
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Shah, C., Böni, J., Huder, J.B., Vogt, H.R., Mühlherr, J., Zanoni, R., Miserez, R., Lutz, H., Schüpbach, J., 2004. Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade. Virology 319, 12–26. Toussaint, J.F., Sailleau, C., Breard, E., Zientara, S., De Clerq, K., 2007. Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments. J. Virol. Methods 140, 115-123. Zhang, Z., Watt, N.J., Hopkins, J., Harkiss, G., Woodall, C.J., 2000. Quantitative analysis of maedi-visna virus DNA load in peripheral blood monocytes and alveolar macrophages. J. Virol. Methods 86, 13–20. Zink, M.C., Yager, J.A., Myers, J.D., 1990. Pathogenesis of caprine arthritis encephalitis virus. Cellular localization of viral transcripts in tissues of infected goats. Am. J. Pathol. 136, 843-854.
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604
Tables
605
Table 1. Overview of developed primers and probe for the q(RT)-PCR detection of SRLV and
607
sequencing of the gag fragment of SRLV strain 6612.
608
Nucleotide sequence (5’-3’)
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caa gag caa cac tgg taa gg cta ccg cct tcc aac ttc tc tag aga cat ggc gaa gca agg tgc aag gag gca aat tct ct cat taa gca agc cat tgt gg
613 614 615 616 617
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609 610
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tag aga cat ggc gaa gca agg gcc cat aga cag ttc cct tc 6-FAM - tac ccc gag ctc aa - MGBNFQ
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q(RT)-PCR forward reverse MGB-probe 6612 sequencing seq primer 1 seq primer 2 seq primer 3 seq primer 4 seq primer 5
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618 619 620 621 622 22 Page 22 of 30
623
Table 2. Overview of qPCR results for SRLV isolates and defined SRLV positive samples
624
from various countries. genotype
qPCR result
a
pos pos
P1OLV
isolate
A
U.K.
EV-1
isolate
Ab
The Netherlands
ZVV 1050
isolate
n.d.
pos
MVV-7
isolate
n.d.
pos
Iceland
1514
isolate
Ab
pos
USA
WLC-1
isolate
n.d.
pos
Belgium
35 sheep from 5 seropositive sheep flocks
whole blood
n.d.
pos
16 goats from 4 seropositive goat flocks
whole blood
n.d.
neg
sheep 6612
lung
n.d.
pos
DNA extract
n.d.
pos
528265278600016
DNA extract
n.d.
pos
11(20890)
DNA extract
n.d.
pos
12 (302739)
DNA extract
n.d.
pos
3951
DNA extract
n.d.
pos
5834-09
DNA extract
n.d.
pos
H S2L 2006 Fin
lung
n.d.
pos
pos contr
Portugal
Ac ce p
Finland
te
M
The Netherlands
cr
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Portugal
us
Field samples
sample
an
Virus isolates
strain/identification
d
origin
Scotland
31 sheep from 4 seropositive sheep flocks
whole blood
n.d.
pos
Spain
697
DNA extract
Ac
pos
258
DNA extract
n.d.
pos
France
d
496
DNA extract
B
10 SRLV pos sheep
DNA extract
CAEV likee
neg neg
625
n.d.= not determined; Barros et al., 2004; Shah et al., 2004; Glaria et al., 2012; Glaria et al., 2009; Leroux et
626
al., 1995
a
b
c
d
e
627 628 629
23 Page 23 of 30
630
Table 3. Comparison between serological (AGID (A) and ELISA (B)) and qPCR SRLV
631
diagnosis of 35 Belgian sheep from 5 different flocks.
B ELISA
pos neg
15 20 35
qPCR pos neg 14 9 3 9 17 18
23 12 35
cr
pos neg
us
AGID
qPCR pos neg 13 2 4 16 17 18
an
A
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632
633
M
634
638 639 640 641 642 643
te
637
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635
644 645 646 647 648 24 Page 24 of 30
649
Figure legends
650
Figure 1. Alignment of the developed primer and probe sequences with corresponding
652
sequences of selected SRLV strains.
653
The sequences of the developed primers and probe were aligned with corresponding regions
654
of the Belgian strain 6612 and SRLV sequences of genotype A (1514 (NC_001452.1), EV1
655
(S51392.1), P1OLV (AF476938.1), WLC1.3 (GQ255434.1), 83.7 (GQ255410.1), SA-OMVV
656
(NC_001511.1)), B (CAEV Co (M33677)), C (1GA-NOR (AF322109)) and E (Roccaverano
657
(EU293537)).
us
cr
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651
an
658
Figure 2. (A) DNA and (B) RNA standard curve for detection of SRLV. A plasmid containing
660
a part of the gag gene sequence of the 1514 SRLV strain and corresponding RNA transcripts
661
were serially diluted in respectively a DNA and RNA extract from a SRLV negative sheep
662
lung. Each dilution was tested by the developed q(RT)-PCR using FastStart TaqMan Probe
663
Master kit (Roche). A standard curve was constructed by plotting the mean CT values of three
664
independent replicates against the log of the DNA and RNA copy number in each dilution.
d
te
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665
M
659
666
Figure 3. Matrix effect. A lung homogenate and whole blood of an SRLV positive sheep were
667
10 fold serially diluted in respectively a lung homogenate and whole blood of an SRLV
668
negative sheep followed by sample preparation and DNA extraction (A). The SRLV WLC1
669
isolate was spiked and subsequently 10 fold serially diluted in respectively a lung homogenate
670
and blood of an SRLV negative sheep. Also a lung homogenate of an SRLV positive sheep
671
was 10 fold serially diluted in a lung homogenate of an SRLV negative sheep. For all
672
dilutions, RNA extraction and reverse transcription was performed after sample preparation
25 Page 25 of 30
673
(B). All DNA extracts and cDNA samples were tested in the developed q(RT)-PCR using the
674
FastStart TaqMan Probe Master kit (Roche).
675
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676
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677
26 Page 26 of 30
Highlights
678
A q(RT)-PCR detecting genotype A SRLV strains was developed and validated.
679
It has a limit of detection of 6 DNA and 40 RNA copies/reaction.
680
It detects SRLV RNA and proviral DNA in lungs and leukocyte pellets.
681
It detects SRLV strains circulating in Belgium and various other countries.
682
The complementary value of PCR based diagnosis besides serology is evidenced.
cr
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683
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Figure(s)
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Figure 2
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Figure 3
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