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The rapid molecular subtyping and pathotyping of avian influenza viruses
Journal of Virological Methods 156 (2009) 157–161
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Short communication
The rapid molecular subtyping and pathotyping of avian influenza viruses A. Yacoub a,∗ , I. Kiss a , S. Zohari a , M. Hakhverdyan a , G. Czifra a , N. Mohamed b , P. Gyarmati a , J. Blomberg b , S. Belák a a The National Veterinary Institute and The Swedish University of Agricultural Sciences, OIE Collaborating Centre for the Biotechnology-Based Diagnosis of Infectious Diseases in Veterinary Medicine, Ulls väg 2B, SE 751 89 Uppsala, Sweden b Section of Virology, Department of Medical Sciences, Uppsala University at Academic Hospital, SE 751 85 Uppsala, Sweden
a b s t r a c t Article history: Received 23 June 2008 Received in revised form 3 October 2008 Accepted 13 October 2008 Available online 6 December 2008 Keywords: Avian influenza Subtype Pathotype Real-time PCR SYBR Green
Highly conserved nucleotide stretches flanking the cleavage site of the haemagglutinin (HA) gene of influenza type A viruses were utilised for generating PCR amplicons from a broad range of avian influenza viruses (AIV) in a one-step real-time SYBR Green RT-PCR assay. The nucleotide sequencing of the amplified PCR products simultaneously reveals both the HA subtype and the pathotype of the AIV isolates, as we demonstrated in case of H5 subtype viruses. The specificity of the assay was confirmed by investigating 66 strains of AIV and nine heterologous pathogens, including influenza B, C and various avian pathogenic viruses. This assay enables a general HA subtype identification and pathotype determination of AIV isolates providing a useful alternative tool for avian influenza diagnosis. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Influenza viruses cause endemic, epidemic, and pandemic diseases among a wide variety of birds and mammals. The causative viral agents belong to the Orthomyxoviridae family and are divided into types A, B and C on the basis of antigenic differences in the matrix and nucleoproteins (Liu, 2006). Influenza type A viruses are classified into subtypes according to the antigenic differences of the two surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA). So far 16 HA and 9 NA subtypes have been identified (Wallensten, 2006). Avian influenza viruses (AIV) belong to influenza type A and are responsible for enormous economic losses in the poultry industry and present a potential threat of zoonotic disease to human populations (Capua and Marangon, 2006). Wild birds are the natural reservoirs for the virus, which is distributed mainly by the movement of migratory waterfowl (Olsen et al., 2006). The virus genome consists of eight segments of negative stranded RNA that code for the viral polymerase complex (PB1, PB2 and PA), the HA and NA, the nucleoprotein (NP), the matrix proteins (M1 and M2), and the non-structural proteins (NS1 and NEP) (Wallensten, 2006). The HA protein is synthesized initially as a single polypep-
∗ Corresponding author at: Department of Virology, SVA, Ulls väg 2B, SE 751 89 Uppsala, Sweden. E-mail address: [email protected] (A. Yacoub). 0166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2008.10.019
tide precursor (HA0) which is cleaved into two subunits HA1 and HA2. This cleavage is necessary for virus infectivity. The occurrence of certain specific amino acids at the cleavage site is indicative for the highly pathogenic avian influenza viruses (HPAIV). While low pathogenic avian influenza viruses (LPAIV) generally lack this marker (Horimoto and Kawaoka, 1994), this criterion is not absolute (Lee et al., 2005). Highly pathogenic H5 and H7 subtypes are virulent to birds (Alexander, 2000) and some strains of these subtypes have crossed the species barrier and cause serious disease in mammalian species, including humans. Numerous outbreaks of HPAIV in avian and in mammalian species have been reported in recent years in several African, Asian and European countries. These occurrences are tracked on the web site of the OIE (Office International des Epizooties; World Organisation for Animal Health; http://www.oie.int/eng/en index.htm). The prevailing theory is that HPAI variants evolve from subtypes of LPAIV in domestic poultry by mutation, therefore, regardless of pathogenicity all AIV infections of poultry caused by H5 and H7 subtypes have been classified as notifiable to OIE (OIE, 2004). In epizootic situations, there is a clear need for the rapid detection of influenza viruses and for the prompt discrimination between LPAIV and HPAIV. The traditional diagnosis of avian influenza is based on virus isolation in embryonated chicken eggs, followed by subtype determination, which is based on HA and NA inhibition (HI and NI) assays (OIE, 2004). The complex diagnosis of influenza outbreaks, including the HA typing and the pathotyping takes considerable time. In
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principle, the laboratory has to perform several PCR procedures, followed by sequencing and chicken pathogenicity tests. First, in general, a wide-range PCR is performed, targeting the matrix or the nucleoprotein genes, in order to detect a broad spectrum of variants of the virus (Fouchier et al., 2000; Lee et al., 2001; Spackman et al., 2002; Kiss et al., 2006). If these assays yield positive results, further PCR procedures are required to perform the HA typing. In case of H5 or H7 subtypes a pathotyping in ensuing tests including the sequence analysis over the cleavage site and experimental animal infections are to be done (see standards of the OIE). Considering the complexity, labour and time involved in the standard procedures, a simpler, more rapid diagnostic procedure for subtyping and pathotyping AIV is desirable. Thus, the assay developed in this study provides a simple diagnostic system based on PCR amplification and sequencing for the rapid and simultaneous determination of AIV subtypes and pathotypes. The one-step SYBR Green real-time RT-PCR protocol was optimised for rapid running conditions, allowing positive samples to be characterized in a timely fashion. The allantoic fluids containing the AIV isolates and the heterologous pathogens used in the study are listed in Table 1 . The HPAIV H5N1 virus isolates were collected during the 2006 influenza outbreaks in Sweden (Zohari et al., 2008) and were handled and identified according to the relevant EU legislation (Council Directive 2005/94/EC), or received from Dr. Ilaria Capua, Istituto Zooprofilattico Sperimentale delle Venezia [IZSVE], Padova, Italy. Rare AIV subtypes as well as heterologous pathogens used in the specificity testing, originated from the archive pathogen collection of the OIE Collaborating Centre or from partner institutions (IZSVE, Italy, and Veterinary Laboratories Agency, Weybridge, UK). Virus isolation in embryonated eggs was performed in a Biosafety Level 3 (BSL-3) laboratory at the National Veterinary Institute (SVA) as described by Zohari et al. (2008). The subtypes of the virus isolates were determined by conventional HI and NI assays (OIE, 2004), using subtype specific antisera (Schild et al., 1980). RNA was extracted from the allantoic fluids of the infected embryonated eggs with the Magnatrix 8000 extraction robot (Magnetic Biosolutions, Sweden) and MagAttract Virus Mini M48 kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. RNA was recovered in 70 l of nuclease-free water and either used immediately or stored at −80 ◦ C. All control viruses underwent the same extraction procedure. A BLAST search in GenBank with an Influenza A HA consensus sequence resulted in an alignment with 1862 members covering most haemagglutinin types. It was processed by the ConSort© program (J. Blomberg, description available in Mohamed et al., 2006), which identifies conserved genomic sequences suitable for primer and probe design. Primers HA FW (5 –3 ): RAGTAMYGGRAAYTTYATWGCYCC and HA RV (5 –3 ): TGRTGRTANCCRTACCANCC were selected to target a fragment of approximately 340 base pairs (between positions 776-1116 of the respective sequence of A/chicken/Burkina Faso/1347-16/2006(H5N1), GenBank reference No. EU277833.1). A one-step real-time RT-PCR was optimised by using the iScriptTM One-Step RT-PCR kit with SYBR Green (Bio-Rad, Hercules, USA) for amplification in a 25 l reaction mixture containing 1× SYBR Green RT-PCR mix, 0.7 M of each primer, 0.5 l of the iScriptTM reverse transcriptase and 3 l of RNA. The reactions were performed on a Rotor-Gene 3000 real-time PCR instrument (Corbett Research, Mortlake, Australia). Reverse transcription was carried out at 50 ◦ C for 15 min, followed by the inactivation of iScript reverse transcriptase at 95 ◦ C for 3 min. Cycling consisted of the following three steps (40 cycles): denaturation at 95 ◦ C for 10 s, annealing at 51 ◦ C for 20 s, extension at 70 ◦ C for 20 s. Different concentrations of forward and reverse primers (0.1–1.3 M) and
Table 1 Avian influenza virus strains and heterologous pathogens investigated by SYBR Green RT-PCR in specificity test. AIV strains with GenBank accession numbers where available
Subtype
HA RT-PCR results
A/Mallard/Swe/S90768/05 A/Mallard/Swe/S90586/05 A/Mallard/Swe/S90780/05 A/Mallard/Swe/S90568/03—EU057728 A/Mallard/Swe/S90772/05 A/Mallard/Swe/S90805/05 A/Mallard/Swe/S90391/05—EU057721 A/Mallard/Swe/S90406/05 A/Mallard/Swe/S90586/05 A/Mallard/Swe/S90800/05 A/Mallard/Swe/S90822/05 A/Mallard/Swe/S90770/03 A/Mallard/Swe/S90780/03 A/Mallard/Swe/S90807/03 A/TuftedDuck/Swe/V526/06 A/Goosander/Swe/V539/06 A/Greater Scaup/Swe/V543/06 A/TuftedDuck/Swe/V599/06 A/Eurasian Eagel Owel/Swe/V618/06* A/Buzzard/Swe/V651/06 A/Mallard/It/3401/05/Inactivated A/Tufted Duck/Swe/V685/06* A/Tufted Duck/Swe/V686/06* A/Tufted Duck/Swe/V788/06—EU057725 A/Tufted Duck/Swe/V789/06* A/Greater Scaup/Swe/V791/06 A/Smew/Swe/V820/06* A/MuteSwan/Swe/V827/06* A/CanadaGoose/Swe/V828/06* A/Mink/Swe/V907/06* A/CanadaGoose/Swe/V978/06* A/Tufted Duck/Swe/V998/06 A/Tufted Duck/Swe/V1027 A/Herring Gull/Swe/V1116/06 A/Eurasian Eagel Owel/Swe/V1218/06 A/Chicken/It/Matrico/L7/04/97—EU057722* A/ost/Den/72420/96 A/Tk/It/90302/05 A/Mallard/Swe/1174/05—EU057727* A/Mallard/Swe/S90436/05* A/teal/Eng/7394-2805/06 A/Chicken/It/9097/97 A/Mallard/Swe/S8012/03 A/Mallard/Swe/S90360/05 A/Mallard/Swe/S90412/05 A/Mallard/Swe/S90418/05 A/Turkey/It/Matrico/L5/04/95—EU057723 A/Mallard/Swe/S90597/05 A/Mallard/Swe/S90598/05 A/Mallard/Swe/S90735/03—EU057726 Unknown/Swe/05 A/Chicken/It/1994—EU057724 A/Mallard/Swe/S90515/05 A/Mallard/Swe/S90457/05 A/Whistling Swan/31/3/88 A/Mallard/Swe/S90229/03 A/Mallard/Swe/S90739/05 A/Mallard/Swe/S90792/05 A/Mallard/Swe/S90812/05 A/Mallard/Swe/S907494/05 A/Mallard/Swe/S90820/03 A/Black-headed gull/Swe/S90005 A/Gull/Maryland/704/77/Inactivated A/Mallarad/Gurjev/263/82/Inactivated A/Shear Water/Aut/2576/79/Inactivated A/Gull/Den/68110/02/Inactivated
H1N1 H1N1 H1N1 H2N3 H2N3 H2N3 H3N8 H3N8 H3N8 H4N3 H4N3 H4N6 H4N6 H4N6 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N2 H5N2 H5N2 H5N3 H5N3 H5N3 H5N9 H6N2 H6N8 H6N8 H6N8 H7N1 H7N7 H7N7 H7N7 H8N4 H9N2 H9N2 H10N4 H10N4 H10N8 H11N9 H11N9 H11N9 H12N5 H12N5 H13N3 H13N6 H14N5 H15N9 H16N3
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + − − + − + − + + + + − − −
A. Yacoub et al. / Journal of Virological Methods 156 (2009) 157–161 Table 1 (Continued ) Heterologous pathogens
HA RT-PCR results
Influenza B-B/Russia/69 (ATCC # VR-790) Influenza B- and B/Lee/40 (ATCC # VR-101) Influenza C-(C/Yamagata/13/98/640) Infectious bronchitis virus (IBV) M41 vaccine strain AHS, Deventer, NL Infectious bronchitis virus (IBV) 4/91/AHS, Deventer, NL Newcastle Disease Virus LaSota vaccine strain/AHS, Deventer, NL Newcastle Disease Virus velogenic field isolate SE-981/Sweden/97 Avian metapneumovirus CVO3 (86004) Rhone Merieux Lyon/France Infectious laryngotracheitis virus reference strain (ATCC# N-71851) AHS, Deventer, NL
− − − − − − − − −
annealing temperatures (49, 51, 53 and 55 ◦ C) were tested during optimization. For sensitivity testing the areas of the HA gene that cover the cleavage site (HA1/HA2) were amplified for three subtypes H5N1, H5N3 and H7N7. The PCR products were purified by using the QIAquick PCR purification kit (Qiagen, Hilden, Germany), cloned into pCR® II-TOPO® Vector, by using TOPO TA Cloning® kit (Invitrogen) following the manufacturer’s instructions. The plasmid mini-preparations were confirmed by PCR. The plasmids were linearised with EcoRV followed by in vitro transcription using MEGA script SP6 kit (Ambion, Cambridgeshire, UK). Transcripts were treated with DNase for 30 min. Complementary RNA (cRNA) was purified by phenol/chloroform (Sigma) and precipitated in isopropanol (Sigma). The pellet was resuspended in 50 l of nuclease-free water and quantified on a NanoDrop spectrophotometer (NanoDrop Technologies, Inc., Wilmington, USA). The copy numbers of the cRNA standard were calculated and the RNA was diluted serially in TE buffer containing 20 ng/l of RNA carrier (Qiagen, Hilden Germany). The cRNA standards were used to assess
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sensitivity of the real-time PCR assay. The sensitivity of the assay was investigated by testing 10-fold serial dilutions of cRNA (109 to 0.1 copies) originating from H5N3, H5N1 and H7N7 subtypes of AIV. All dilutions were tested in triplicates. In order to determine the subtype and pathotype of the detected viruses, the PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Sequence reaction was performed using the Big Dye terminators Reaction Kit v.3.1 (Applied Biosystems, Foster City, California, USA). After precipitation, the products were sequenced on the ABI 3100 genetic analyzer (Applied Biosystems). The obtained sequence data were analyzed by the Lasergene DNASTAR (DNASTAR Inc., Madison, WI) and by the CLC Combined Workbench 3.0.2. (CLC bio A/S, Aarhus, Denmark) program packages. A representative set of sequences was submitted to GenBank (see Table 1). The ConSort© analysis of the aligned sequences revealed that the HA1/HA2 cleavage site was surrounded by three conserved stretches (data not shown). The primers were designed to target the two outer stretches resulting in the amplification of an approximately 340mer segment (Fig. 1). The sequence information contained in this portion of the HA gene enables HA subtype identification and pathotyping as well. Out of the 66 tested influenza virus samples, representing different subtypes, 59 samples gave positive results in the real-time PCR assay and yielded the expected 340mer product. The samples that were not amplified belonged either to HA subtype 10 and 11 or were -propiolactone-inactivated viruses (and of HA subtypes 14, 15, and 16), wherein positive results were obtained only with samples having higher HI titer. No dual infections were revealed by the routine isolation protocol regarding the samples tested by the typing assay. In case of dual (multiple) infection, presumably the most predominant variant would be characterized by the assay. However, this is largely dependent on the quality of the sequencing process whether or not the sequence data would indicate the presence of more strains in the sample. No amplification was observed with the heterologous pathogens, as shown in Table 1. The standard curve obtained from the ampli-
Fig. 1. Degree of conservation in a portion of the influenza A haemagglutinin segment. Graphical output from ConSort© , based on 1862 haemagglutinin sequences, of most haemagglutinin types, collected from GenBank. Positions of the forward and reverse primers, and the cleavage site, are shown.
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Fig. 2. Standard curve of the real-time RT-PCR assay generated from a 10-fold serial dilution series of H5N1 cRNA solution.
fication of the cRNA dilution series displayed a linear relationship between Ct values and cRNA copy numbers (Fig. 2) with a correlation coefficient (R2 value) of 0.99. The reaction efficiency was 0.89; as calculated by comparative quantitation analysis by the software of the Rotor-Gene 3000 instrument. The quantitation analysis showed that the system is capable of detecting approximately100 copies of in vitro transcribed RNA molecules. The intra-assay reproducibility was evaluated by standard deviation of the Ct values of each replicate. A standard deviation between 0.03 and 0.26 Ct was observed in the standard curve. The BLAST search confirmed the results of the traditional HA subtyping in each case. Amplimer sequences of eight strains were submitted to GenBank (Table 1). Furthermore, the analysis of the nucleotide sequences revealed that the GC content of the highly pathogenic H5 subtype is lower by 4–5% than that of the low pathogenic counterparts (data not shown). This feature was expected to affect the melting peaks of the corresponding PCR products. In consequence, melting peaks for the HP H5N1 (82.05 ± 0.09 ◦ C) could be differentiated from those concerning the LP H5 subtype viruses (83 ± 0.07 ◦ C) by more than 0.7 ◦ C at a 95% confidence level (p < 0.05) according to a Student’s t-test analysis (Fig. 3). The possibility of exploring the same approach regarding the H7 subtypes was considered as well. However, the GC content of the HA amplicons of the different pathotypes was not significant (45.27% versus 45.43% as an average regarding the analyzed nucleotide sequences of 27 HP and 60 LP pathotype viruses, respectively, originating from this study and the GenBank, data not shown). The corresponding predicted melting temperatures reflected this finding (83.45 and 83.54 ◦ C as an average for the HP and LP H7 subtype viruses, respectively), which precludes differentiation on this ground. The OIE-approved molecular technology used for influenza diagnosis is based on TaqMan® real-time PCR methods (Spackman
Fig. 3. Demonstration of the nucleotide composition difference of the amplified HA PCR products between highly pathogenic (HP) and low pathogenic (LP) H5 subtype avian influenza viruses by the melting curve analysis of the PCR product of the HA gene covering the cleavage site. The left panel represents nine different samples of highly pathogenic H5N1 and the right panel represents the low pathogenic subtypes, two samples of H5N3 and one sample H5N2.
et al., 2002), which provide important, but limited information about the biological features of the viruses. Thus, due to the importance of influenza in general and avian influenza in particular, there are a number of different novel approaches gaining ground in influenza diagnostics. One approach is pyrosequencing, where pathogenicity is determined by receptor binding and glycosylation sequences, as well as the phylogenetic traits of H5N1 influenza viruses (Pourmand et al., 2006). Straightforward amplification/sequencing of the polymerase complex of influenza viruses provides further important insight into the genetic determinants of virulence (Salomon et al., 2006). The full-length amplification of the genomes, followed by sequencing, provides comprehensive information on influenza A viruses (Chan et al., 2006). However, these methods require extensive customized optimisations on an individual basis and their general application has, at least in our hands, proved to be rather difficult (see also Li et al., 2007). The method described in this paper provides a versatile and robust tool for the quick identification of AIV HA subtypes and, importantly, the determination of the pathotype upon nucleotide sequencing, as demonstrated with H5N1 HPAIVs. The SYBR Green dye intercalates into double-stranded DNA in a non-specific manner and it provides this assay the advantage of being less affected by mutations of the targeted viral sequences than other real-time PCR assays, particularly when compared to the probe-based methods (Pham et al., 2005). The utility of the application of this dye is further supported by recent reports describing that influenza A detection based on SYBR Green chemistry had a higher performance than a specific probe-based system (Karlsson et al., 2007) and than the virus isolation method (Ong et al., 2007). Such robustness is an important requirement for a method developed with the aim of broad range detection. The inner conserved region may be useful for future developments like a probe-based assay, and as a target for pyrosequencing primers. An assay conceptually similar to the one presented herein was published by Phipps et al. (2004) that also uses a single set of primers for generating PCR amplicons from a wide range of influenza A viruses. However, those primers target the HA2 portion of the HA gene and thus, would not provide information on the pathotype of the isolate in question. The probe-based H5N1 pathotyping protocol published by Hoffmann et al. (2006) clearly identifies and characterizes the Qinghai-like H5N1 HPAIVs, but not the others. A further study, related to the subject, evaluated large number of clinical samples, but it was based on using four pairs of primers targeting different regions of the HA gene in separate reactions (Wang et al., 2008). The discrimination between H5N1 HP and LP AIVs based on melting curve analysis has been demonstrated by Payungporn et al. (2006), but their assay targeted specifically H5 subtype viruses. Nevertheless, the sequence variability within the cleavage site region makes melting curve analysis of limited use for distinguishing virulence of AIV variants.
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Therefore, melting point differences should be evaluated in that context, for e.g., in case of viruses isolated within an outbreak it might have an indicative value of the pathotype, but not in case of viruses originating from different outbreaks. In summary, the assay described is able to determine a wide range of HA subtypes of AIV and to identify the pathotypes (HPAIV or LPAIV), within 6 h upon virus isolation. The assay identified and characterized all those subtypes that present the highest risk to devastate avian populations today and which are considered as potential sources of a pandemic. In view of its broad detection range, simplicity and rapidity, this assay has the potential for wide application in influenza diagnosis. It can be adapted and optimised to direct testing of field samples obtained from various hosts in different scenarios of the disease outbreaks. This new assay was tested and optimised on a wide range of viruses of allantoic fluid origin and was effective as a tool for the simple and rapid characterizing of these AIV isolates. Future development, such as testing and optimizing on other samples types, could expand the range of application of this new method. Acknowledgements This work was supported by a grant from the 6th Framework Programme of the European Union (project SSPE-CT-2004513645, http://www.labonsite.com), 7th Framework European Union project FLUTEST (SSPE-CT-2007-044429), Formas influenza project Dnr 221-2006-2169, Formas research grants 2003-1059 and 2004-2698, the EU Network of Excellence, EPIZONE (Contract No FOOD-CT-2006-016236) and by the R&D budget of the National Veterinary Institute (SVA), Uppsala, Sweden. Influenza virus samples were kindly provided by Dr. Ana Moreno Martin, Isituto Zooprofilattico Sperimentale Lombardia ed Emilia Romagna Brescia Italy and Dr. Marek Slomka, Veterinary Laboratories Agency, Weybridge, UK. Thanks are due to Dr. Peter Thorén for practical advice during the work and to Dr. Mikael Leijon for his support in sequence analyses. Dr. Neil LeBlanc is thanked for correcting the manuscript linguistically. References Alexander, D.J., 2000. A review of avian influenza in different bird species. Vet. Microbiol. 74, 3–13. Capua, I., Marangon, S., 2006. Control of avian influenza in poultry. Emerg. Infect. Dis. 12, 1319–1324. Chan, C.H., Lin, K.L., Chan, Y., Wang, Y.L., Chi, Y.T., Tu, H.L., Shieh, H.K., Liu, W.T., 2006. Amplification of the entire genome of influenza A virus H1N1 and H3N2 subtypes by reverse-transcription polymerase chain reaction. J. Virol. Methods 136, 38–43. Fouchier, R.A., Bestebroer, T.M., Herfst, S., Van Der Kemp, L., Rimmelzwaan, G.F., Osterhaus, A.D., 2000. Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J. Clin. Microbiol. 38, 4096–4101. Hoffmann, B., Harder, T., Starick, E., Depner, K., Werner, O., Beer, M., 2006. Rapid and highly sensitive pathotyping of avian influenza A H5N1 virus by using real-time reverse transcription-PCR. J. Clin. Microbiol. 45, 600–603.
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Short communication
The rapid molecular subtyping and pathotyping of avian influenza viruses A. Yacoub a,∗ , I. Kiss a , S. Zohari a , M. Hakhverdyan a , G. Czifra a , N. Mohamed b , P. Gyarmati a , J. Blomberg b , S. Belák a a The National Veterinary Institute and The Swedish University of Agricultural Sciences, OIE Collaborating Centre for the Biotechnology-Based Diagnosis of Infectious Diseases in Veterinary Medicine, Ulls väg 2B, SE 751 89 Uppsala, Sweden b Section of Virology, Department of Medical Sciences, Uppsala University at Academic Hospital, SE 751 85 Uppsala, Sweden
a b s t r a c t Article history: Received 23 June 2008 Received in revised form 3 October 2008 Accepted 13 October 2008 Available online 6 December 2008 Keywords: Avian influenza Subtype Pathotype Real-time PCR SYBR Green
Highly conserved nucleotide stretches flanking the cleavage site of the haemagglutinin (HA) gene of influenza type A viruses were utilised for generating PCR amplicons from a broad range of avian influenza viruses (AIV) in a one-step real-time SYBR Green RT-PCR assay. The nucleotide sequencing of the amplified PCR products simultaneously reveals both the HA subtype and the pathotype of the AIV isolates, as we demonstrated in case of H5 subtype viruses. The specificity of the assay was confirmed by investigating 66 strains of AIV and nine heterologous pathogens, including influenza B, C and various avian pathogenic viruses. This assay enables a general HA subtype identification and pathotype determination of AIV isolates providing a useful alternative tool for avian influenza diagnosis. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Influenza viruses cause endemic, epidemic, and pandemic diseases among a wide variety of birds and mammals. The causative viral agents belong to the Orthomyxoviridae family and are divided into types A, B and C on the basis of antigenic differences in the matrix and nucleoproteins (Liu, 2006). Influenza type A viruses are classified into subtypes according to the antigenic differences of the two surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA). So far 16 HA and 9 NA subtypes have been identified (Wallensten, 2006). Avian influenza viruses (AIV) belong to influenza type A and are responsible for enormous economic losses in the poultry industry and present a potential threat of zoonotic disease to human populations (Capua and Marangon, 2006). Wild birds are the natural reservoirs for the virus, which is distributed mainly by the movement of migratory waterfowl (Olsen et al., 2006). The virus genome consists of eight segments of negative stranded RNA that code for the viral polymerase complex (PB1, PB2 and PA), the HA and NA, the nucleoprotein (NP), the matrix proteins (M1 and M2), and the non-structural proteins (NS1 and NEP) (Wallensten, 2006). The HA protein is synthesized initially as a single polypep-
∗ Corresponding author at: Department of Virology, SVA, Ulls väg 2B, SE 751 89 Uppsala, Sweden. E-mail address: [email protected] (A. Yacoub). 0166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2008.10.019
tide precursor (HA0) which is cleaved into two subunits HA1 and HA2. This cleavage is necessary for virus infectivity. The occurrence of certain specific amino acids at the cleavage site is indicative for the highly pathogenic avian influenza viruses (HPAIV). While low pathogenic avian influenza viruses (LPAIV) generally lack this marker (Horimoto and Kawaoka, 1994), this criterion is not absolute (Lee et al., 2005). Highly pathogenic H5 and H7 subtypes are virulent to birds (Alexander, 2000) and some strains of these subtypes have crossed the species barrier and cause serious disease in mammalian species, including humans. Numerous outbreaks of HPAIV in avian and in mammalian species have been reported in recent years in several African, Asian and European countries. These occurrences are tracked on the web site of the OIE (Office International des Epizooties; World Organisation for Animal Health; http://www.oie.int/eng/en index.htm). The prevailing theory is that HPAI variants evolve from subtypes of LPAIV in domestic poultry by mutation, therefore, regardless of pathogenicity all AIV infections of poultry caused by H5 and H7 subtypes have been classified as notifiable to OIE (OIE, 2004). In epizootic situations, there is a clear need for the rapid detection of influenza viruses and for the prompt discrimination between LPAIV and HPAIV. The traditional diagnosis of avian influenza is based on virus isolation in embryonated chicken eggs, followed by subtype determination, which is based on HA and NA inhibition (HI and NI) assays (OIE, 2004). The complex diagnosis of influenza outbreaks, including the HA typing and the pathotyping takes considerable time. In
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principle, the laboratory has to perform several PCR procedures, followed by sequencing and chicken pathogenicity tests. First, in general, a wide-range PCR is performed, targeting the matrix or the nucleoprotein genes, in order to detect a broad spectrum of variants of the virus (Fouchier et al., 2000; Lee et al., 2001; Spackman et al., 2002; Kiss et al., 2006). If these assays yield positive results, further PCR procedures are required to perform the HA typing. In case of H5 or H7 subtypes a pathotyping in ensuing tests including the sequence analysis over the cleavage site and experimental animal infections are to be done (see standards of the OIE). Considering the complexity, labour and time involved in the standard procedures, a simpler, more rapid diagnostic procedure for subtyping and pathotyping AIV is desirable. Thus, the assay developed in this study provides a simple diagnostic system based on PCR amplification and sequencing for the rapid and simultaneous determination of AIV subtypes and pathotypes. The one-step SYBR Green real-time RT-PCR protocol was optimised for rapid running conditions, allowing positive samples to be characterized in a timely fashion. The allantoic fluids containing the AIV isolates and the heterologous pathogens used in the study are listed in Table 1 . The HPAIV H5N1 virus isolates were collected during the 2006 influenza outbreaks in Sweden (Zohari et al., 2008) and were handled and identified according to the relevant EU legislation (Council Directive 2005/94/EC), or received from Dr. Ilaria Capua, Istituto Zooprofilattico Sperimentale delle Venezia [IZSVE], Padova, Italy. Rare AIV subtypes as well as heterologous pathogens used in the specificity testing, originated from the archive pathogen collection of the OIE Collaborating Centre or from partner institutions (IZSVE, Italy, and Veterinary Laboratories Agency, Weybridge, UK). Virus isolation in embryonated eggs was performed in a Biosafety Level 3 (BSL-3) laboratory at the National Veterinary Institute (SVA) as described by Zohari et al. (2008). The subtypes of the virus isolates were determined by conventional HI and NI assays (OIE, 2004), using subtype specific antisera (Schild et al., 1980). RNA was extracted from the allantoic fluids of the infected embryonated eggs with the Magnatrix 8000 extraction robot (Magnetic Biosolutions, Sweden) and MagAttract Virus Mini M48 kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. RNA was recovered in 70 l of nuclease-free water and either used immediately or stored at −80 ◦ C. All control viruses underwent the same extraction procedure. A BLAST search in GenBank with an Influenza A HA consensus sequence resulted in an alignment with 1862 members covering most haemagglutinin types. It was processed by the ConSort© program (J. Blomberg, description available in Mohamed et al., 2006), which identifies conserved genomic sequences suitable for primer and probe design. Primers HA FW (5 –3 ): RAGTAMYGGRAAYTTYATWGCYCC and HA RV (5 –3 ): TGRTGRTANCCRTACCANCC were selected to target a fragment of approximately 340 base pairs (between positions 776-1116 of the respective sequence of A/chicken/Burkina Faso/1347-16/2006(H5N1), GenBank reference No. EU277833.1). A one-step real-time RT-PCR was optimised by using the iScriptTM One-Step RT-PCR kit with SYBR Green (Bio-Rad, Hercules, USA) for amplification in a 25 l reaction mixture containing 1× SYBR Green RT-PCR mix, 0.7 M of each primer, 0.5 l of the iScriptTM reverse transcriptase and 3 l of RNA. The reactions were performed on a Rotor-Gene 3000 real-time PCR instrument (Corbett Research, Mortlake, Australia). Reverse transcription was carried out at 50 ◦ C for 15 min, followed by the inactivation of iScript reverse transcriptase at 95 ◦ C for 3 min. Cycling consisted of the following three steps (40 cycles): denaturation at 95 ◦ C for 10 s, annealing at 51 ◦ C for 20 s, extension at 70 ◦ C for 20 s. Different concentrations of forward and reverse primers (0.1–1.3 M) and
Table 1 Avian influenza virus strains and heterologous pathogens investigated by SYBR Green RT-PCR in specificity test. AIV strains with GenBank accession numbers where available
Subtype
HA RT-PCR results
A/Mallard/Swe/S90768/05 A/Mallard/Swe/S90586/05 A/Mallard/Swe/S90780/05 A/Mallard/Swe/S90568/03—EU057728 A/Mallard/Swe/S90772/05 A/Mallard/Swe/S90805/05 A/Mallard/Swe/S90391/05—EU057721 A/Mallard/Swe/S90406/05 A/Mallard/Swe/S90586/05 A/Mallard/Swe/S90800/05 A/Mallard/Swe/S90822/05 A/Mallard/Swe/S90770/03 A/Mallard/Swe/S90780/03 A/Mallard/Swe/S90807/03 A/TuftedDuck/Swe/V526/06 A/Goosander/Swe/V539/06 A/Greater Scaup/Swe/V543/06 A/TuftedDuck/Swe/V599/06 A/Eurasian Eagel Owel/Swe/V618/06* A/Buzzard/Swe/V651/06 A/Mallard/It/3401/05/Inactivated A/Tufted Duck/Swe/V685/06* A/Tufted Duck/Swe/V686/06* A/Tufted Duck/Swe/V788/06—EU057725 A/Tufted Duck/Swe/V789/06* A/Greater Scaup/Swe/V791/06 A/Smew/Swe/V820/06* A/MuteSwan/Swe/V827/06* A/CanadaGoose/Swe/V828/06* A/Mink/Swe/V907/06* A/CanadaGoose/Swe/V978/06* A/Tufted Duck/Swe/V998/06 A/Tufted Duck/Swe/V1027 A/Herring Gull/Swe/V1116/06 A/Eurasian Eagel Owel/Swe/V1218/06 A/Chicken/It/Matrico/L7/04/97—EU057722* A/ost/Den/72420/96 A/Tk/It/90302/05 A/Mallard/Swe/1174/05—EU057727* A/Mallard/Swe/S90436/05* A/teal/Eng/7394-2805/06 A/Chicken/It/9097/97 A/Mallard/Swe/S8012/03 A/Mallard/Swe/S90360/05 A/Mallard/Swe/S90412/05 A/Mallard/Swe/S90418/05 A/Turkey/It/Matrico/L5/04/95—EU057723 A/Mallard/Swe/S90597/05 A/Mallard/Swe/S90598/05 A/Mallard/Swe/S90735/03—EU057726 Unknown/Swe/05 A/Chicken/It/1994—EU057724 A/Mallard/Swe/S90515/05 A/Mallard/Swe/S90457/05 A/Whistling Swan/31/3/88 A/Mallard/Swe/S90229/03 A/Mallard/Swe/S90739/05 A/Mallard/Swe/S90792/05 A/Mallard/Swe/S90812/05 A/Mallard/Swe/S907494/05 A/Mallard/Swe/S90820/03 A/Black-headed gull/Swe/S90005 A/Gull/Maryland/704/77/Inactivated A/Mallarad/Gurjev/263/82/Inactivated A/Shear Water/Aut/2576/79/Inactivated A/Gull/Den/68110/02/Inactivated
H1N1 H1N1 H1N1 H2N3 H2N3 H2N3 H3N8 H3N8 H3N8 H4N3 H4N3 H4N6 H4N6 H4N6 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N1 H5N2 H5N2 H5N2 H5N3 H5N3 H5N3 H5N9 H6N2 H6N8 H6N8 H6N8 H7N1 H7N7 H7N7 H7N7 H8N4 H9N2 H9N2 H10N4 H10N4 H10N8 H11N9 H11N9 H11N9 H12N5 H12N5 H13N3 H13N6 H14N5 H15N9 H16N3
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + − − + − + − + + + + − − −
A. Yacoub et al. / Journal of Virological Methods 156 (2009) 157–161 Table 1 (Continued ) Heterologous pathogens
HA RT-PCR results
Influenza B-B/Russia/69 (ATCC # VR-790) Influenza B- and B/Lee/40 (ATCC # VR-101) Influenza C-(C/Yamagata/13/98/640) Infectious bronchitis virus (IBV) M41 vaccine strain AHS, Deventer, NL Infectious bronchitis virus (IBV) 4/91/AHS, Deventer, NL Newcastle Disease Virus LaSota vaccine strain/AHS, Deventer, NL Newcastle Disease Virus velogenic field isolate SE-981/Sweden/97 Avian metapneumovirus CVO3 (86004) Rhone Merieux Lyon/France Infectious laryngotracheitis virus reference strain (ATCC# N-71851) AHS, Deventer, NL
− − − − − − − − −
annealing temperatures (49, 51, 53 and 55 ◦ C) were tested during optimization. For sensitivity testing the areas of the HA gene that cover the cleavage site (HA1/HA2) were amplified for three subtypes H5N1, H5N3 and H7N7. The PCR products were purified by using the QIAquick PCR purification kit (Qiagen, Hilden, Germany), cloned into pCR® II-TOPO® Vector, by using TOPO TA Cloning® kit (Invitrogen) following the manufacturer’s instructions. The plasmid mini-preparations were confirmed by PCR. The plasmids were linearised with EcoRV followed by in vitro transcription using MEGA script SP6 kit (Ambion, Cambridgeshire, UK). Transcripts were treated with DNase for 30 min. Complementary RNA (cRNA) was purified by phenol/chloroform (Sigma) and precipitated in isopropanol (Sigma). The pellet was resuspended in 50 l of nuclease-free water and quantified on a NanoDrop spectrophotometer (NanoDrop Technologies, Inc., Wilmington, USA). The copy numbers of the cRNA standard were calculated and the RNA was diluted serially in TE buffer containing 20 ng/l of RNA carrier (Qiagen, Hilden Germany). The cRNA standards were used to assess
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sensitivity of the real-time PCR assay. The sensitivity of the assay was investigated by testing 10-fold serial dilutions of cRNA (109 to 0.1 copies) originating from H5N3, H5N1 and H7N7 subtypes of AIV. All dilutions were tested in triplicates. In order to determine the subtype and pathotype of the detected viruses, the PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Sequence reaction was performed using the Big Dye terminators Reaction Kit v.3.1 (Applied Biosystems, Foster City, California, USA). After precipitation, the products were sequenced on the ABI 3100 genetic analyzer (Applied Biosystems). The obtained sequence data were analyzed by the Lasergene DNASTAR (DNASTAR Inc., Madison, WI) and by the CLC Combined Workbench 3.0.2. (CLC bio A/S, Aarhus, Denmark) program packages. A representative set of sequences was submitted to GenBank (see Table 1). The ConSort© analysis of the aligned sequences revealed that the HA1/HA2 cleavage site was surrounded by three conserved stretches (data not shown). The primers were designed to target the two outer stretches resulting in the amplification of an approximately 340mer segment (Fig. 1). The sequence information contained in this portion of the HA gene enables HA subtype identification and pathotyping as well. Out of the 66 tested influenza virus samples, representing different subtypes, 59 samples gave positive results in the real-time PCR assay and yielded the expected 340mer product. The samples that were not amplified belonged either to HA subtype 10 and 11 or were -propiolactone-inactivated viruses (and of HA subtypes 14, 15, and 16), wherein positive results were obtained only with samples having higher HI titer. No dual infections were revealed by the routine isolation protocol regarding the samples tested by the typing assay. In case of dual (multiple) infection, presumably the most predominant variant would be characterized by the assay. However, this is largely dependent on the quality of the sequencing process whether or not the sequence data would indicate the presence of more strains in the sample. No amplification was observed with the heterologous pathogens, as shown in Table 1. The standard curve obtained from the ampli-
Fig. 1. Degree of conservation in a portion of the influenza A haemagglutinin segment. Graphical output from ConSort© , based on 1862 haemagglutinin sequences, of most haemagglutinin types, collected from GenBank. Positions of the forward and reverse primers, and the cleavage site, are shown.
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Fig. 2. Standard curve of the real-time RT-PCR assay generated from a 10-fold serial dilution series of H5N1 cRNA solution.
fication of the cRNA dilution series displayed a linear relationship between Ct values and cRNA copy numbers (Fig. 2) with a correlation coefficient (R2 value) of 0.99. The reaction efficiency was 0.89; as calculated by comparative quantitation analysis by the software of the Rotor-Gene 3000 instrument. The quantitation analysis showed that the system is capable of detecting approximately100 copies of in vitro transcribed RNA molecules. The intra-assay reproducibility was evaluated by standard deviation of the Ct values of each replicate. A standard deviation between 0.03 and 0.26 Ct was observed in the standard curve. The BLAST search confirmed the results of the traditional HA subtyping in each case. Amplimer sequences of eight strains were submitted to GenBank (Table 1). Furthermore, the analysis of the nucleotide sequences revealed that the GC content of the highly pathogenic H5 subtype is lower by 4–5% than that of the low pathogenic counterparts (data not shown). This feature was expected to affect the melting peaks of the corresponding PCR products. In consequence, melting peaks for the HP H5N1 (82.05 ± 0.09 ◦ C) could be differentiated from those concerning the LP H5 subtype viruses (83 ± 0.07 ◦ C) by more than 0.7 ◦ C at a 95% confidence level (p < 0.05) according to a Student’s t-test analysis (Fig. 3). The possibility of exploring the same approach regarding the H7 subtypes was considered as well. However, the GC content of the HA amplicons of the different pathotypes was not significant (45.27% versus 45.43% as an average regarding the analyzed nucleotide sequences of 27 HP and 60 LP pathotype viruses, respectively, originating from this study and the GenBank, data not shown). The corresponding predicted melting temperatures reflected this finding (83.45 and 83.54 ◦ C as an average for the HP and LP H7 subtype viruses, respectively), which precludes differentiation on this ground. The OIE-approved molecular technology used for influenza diagnosis is based on TaqMan® real-time PCR methods (Spackman
Fig. 3. Demonstration of the nucleotide composition difference of the amplified HA PCR products between highly pathogenic (HP) and low pathogenic (LP) H5 subtype avian influenza viruses by the melting curve analysis of the PCR product of the HA gene covering the cleavage site. The left panel represents nine different samples of highly pathogenic H5N1 and the right panel represents the low pathogenic subtypes, two samples of H5N3 and one sample H5N2.
et al., 2002), which provide important, but limited information about the biological features of the viruses. Thus, due to the importance of influenza in general and avian influenza in particular, there are a number of different novel approaches gaining ground in influenza diagnostics. One approach is pyrosequencing, where pathogenicity is determined by receptor binding and glycosylation sequences, as well as the phylogenetic traits of H5N1 influenza viruses (Pourmand et al., 2006). Straightforward amplification/sequencing of the polymerase complex of influenza viruses provides further important insight into the genetic determinants of virulence (Salomon et al., 2006). The full-length amplification of the genomes, followed by sequencing, provides comprehensive information on influenza A viruses (Chan et al., 2006). However, these methods require extensive customized optimisations on an individual basis and their general application has, at least in our hands, proved to be rather difficult (see also Li et al., 2007). The method described in this paper provides a versatile and robust tool for the quick identification of AIV HA subtypes and, importantly, the determination of the pathotype upon nucleotide sequencing, as demonstrated with H5N1 HPAIVs. The SYBR Green dye intercalates into double-stranded DNA in a non-specific manner and it provides this assay the advantage of being less affected by mutations of the targeted viral sequences than other real-time PCR assays, particularly when compared to the probe-based methods (Pham et al., 2005). The utility of the application of this dye is further supported by recent reports describing that influenza A detection based on SYBR Green chemistry had a higher performance than a specific probe-based system (Karlsson et al., 2007) and than the virus isolation method (Ong et al., 2007). Such robustness is an important requirement for a method developed with the aim of broad range detection. The inner conserved region may be useful for future developments like a probe-based assay, and as a target for pyrosequencing primers. An assay conceptually similar to the one presented herein was published by Phipps et al. (2004) that also uses a single set of primers for generating PCR amplicons from a wide range of influenza A viruses. However, those primers target the HA2 portion of the HA gene and thus, would not provide information on the pathotype of the isolate in question. The probe-based H5N1 pathotyping protocol published by Hoffmann et al. (2006) clearly identifies and characterizes the Qinghai-like H5N1 HPAIVs, but not the others. A further study, related to the subject, evaluated large number of clinical samples, but it was based on using four pairs of primers targeting different regions of the HA gene in separate reactions (Wang et al., 2008). The discrimination between H5N1 HP and LP AIVs based on melting curve analysis has been demonstrated by Payungporn et al. (2006), but their assay targeted specifically H5 subtype viruses. Nevertheless, the sequence variability within the cleavage site region makes melting curve analysis of limited use for distinguishing virulence of AIV variants.
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Therefore, melting point differences should be evaluated in that context, for e.g., in case of viruses isolated within an outbreak it might have an indicative value of the pathotype, but not in case of viruses originating from different outbreaks. In summary, the assay described is able to determine a wide range of HA subtypes of AIV and to identify the pathotypes (HPAIV or LPAIV), within 6 h upon virus isolation. The assay identified and characterized all those subtypes that present the highest risk to devastate avian populations today and which are considered as potential sources of a pandemic. In view of its broad detection range, simplicity and rapidity, this assay has the potential for wide application in influenza diagnosis. It can be adapted and optimised to direct testing of field samples obtained from various hosts in different scenarios of the disease outbreaks. This new assay was tested and optimised on a wide range of viruses of allantoic fluid origin and was effective as a tool for the simple and rapid characterizing of these AIV isolates. Future development, such as testing and optimizing on other samples types, could expand the range of application of this new method. Acknowledgements This work was supported by a grant from the 6th Framework Programme of the European Union (project SSPE-CT-2004513645, http://www.labonsite.com), 7th Framework European Union project FLUTEST (SSPE-CT-2007-044429), Formas influenza project Dnr 221-2006-2169, Formas research grants 2003-1059 and 2004-2698, the EU Network of Excellence, EPIZONE (Contract No FOOD-CT-2006-016236) and by the R&D budget of the National Veterinary Institute (SVA), Uppsala, Sweden. Influenza virus samples were kindly provided by Dr. Ana Moreno Martin, Isituto Zooprofilattico Sperimentale Lombardia ed Emilia Romagna Brescia Italy and Dr. Marek Slomka, Veterinary Laboratories Agency, Weybridge, UK. Thanks are due to Dr. Peter Thorén for practical advice during the work and to Dr. Mikael Leijon for his support in sequence analyses. Dr. Neil LeBlanc is thanked for correcting the manuscript linguistically. References Alexander, D.J., 2000. A review of avian influenza in different bird species. Vet. Microbiol. 74, 3–13. Capua, I., Marangon, S., 2006. Control of avian influenza in poultry. Emerg. Infect. Dis. 12, 1319–1324. Chan, C.H., Lin, K.L., Chan, Y., Wang, Y.L., Chi, Y.T., Tu, H.L., Shieh, H.K., Liu, W.T., 2006. Amplification of the entire genome of influenza A virus H1N1 and H3N2 subtypes by reverse-transcription polymerase chain reaction. J. Virol. Methods 136, 38–43. Fouchier, R.A., Bestebroer, T.M., Herfst, S., Van Der Kemp, L., Rimmelzwaan, G.F., Osterhaus, A.D., 2000. Detection of influenza A viruses from different species by PCR amplification of conserved sequences in the matrix gene. J. Clin. Microbiol. 38, 4096–4101. Hoffmann, B., Harder, T., Starick, E., Depner, K., Werner, O., Beer, M., 2006. Rapid and highly sensitive pathotyping of avian influenza A H5N1 virus by using real-time reverse transcription-PCR. J. Clin. Microbiol. 45, 600–603.
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