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Genetic heterogeneity of bovine viral diarrhoea virus (BVDV) isolates from Turkey: Identification of a new subgroup in BVDV-1
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Veterinary Microbiology 130 (2008) 258–267 www.elsevier.com/locate/vetmic
Genetic heterogeneity of bovine viral diarrhoea virus (BVDV) isolates from Turkey: Identification of a new subgroup in BVDV-1 Kadir Yes¸ilbag˘a,*, Christine Fo¨rsterb, Barbara Bank-Wolfb, Zeki Yılmazc, Feray Alkand, Aykut Ozkuld, Ibrahim Burgud, Sibilina Cedillo Rosalesb, Heinz-Ju¨rgen Thielb, Matthias Ko¨nigb a
Uludag˘ University, Faculty of Veterinary Medicine, Department of Microbiology, Virology, TR-16059 Go¨ru¨kle, Bursa, Turkey b Institute of Virology (Faculty of Veterinary Medicine), Justus-Liebig-University Giessen, Frankfurter Str. 107, D-35392, Giessen – Germany c Uludag˘ University Faculty of Veterinary Medicine, Department of Internal Medicine, TR-16059 Go¨ru¨kle, Bursa, Turkey d Ankara University Faculty of Veterinary Medicine, Department of Virology, Ankara, Turkey Received 9 November 2007; received in revised form 26 January 2008; accepted 31 January 2008
Abstract Genetic heterogeneity of Turkish ruminant pestiviruses was investigated by phylogenetic analysis of complete Npro encoding nucleotide sequences. A total of 30 virus isolates obtained from 15 provinces around the country between 1997 and 2005 were included in the phylogenetic analysis. Virus isolates mostly originated from cattle with one isolate from sheep. The bovine isolates all belonged to BVDV-1, the sheep isolate to BVDV-2. Fifteen isolates formed a new subgroup within BVDV-1, tentatively named BVDV-1l. The remaining bovine isolates were typed as BVDV-1a (n = 4), BVDV-1b (n = 4), BVDV-1d (n = 3), BVDV-1f (n = 2) and BVDV-1h (n = 1). The isolates allocated to BVDV-1l originated from various geographical regions in different years. There was no correlation between genetic grouping and locations where isolates were obtained. Viruses originating from one farm in most cases belonged to the same subgroup (n = 5). This study indicates that the newly detected subgroup BVDV-1l is predominant and widespread in Turkey. Moreover, an ovine virus isolate was identified as the first member of BVDV-2 reported in Turkey. A serological survey using samples from western Turkey indicated that BVDV-2 is also present in cattle. # 2008 Elsevier B.V. All rights reserved. Keywords: BVDV-1; BVDV-2; Genetic heterogeneity; RT-PCR; Serological survey; Turkey
1. Introduction * Corresponding author at: Uludag˘ University, Faculty of Veterinary Medicine, Virology Section, Go¨ru¨kle Campus, 16059 Bursa, Turkey. Tel.: +90 224 2941295; fax: +90 224 2941202. E-mail address: [email protected] (Y. Kadir).
Bovine viral diarrhoea virus (BVDV) leads to various clinical problems with significant economic impact. The spectrum of disease manifestations
0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2008.01.016
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comprises acute haemorrhagic syndrome, persistent infection, mucosal disease (MD), as well as diseases of the respiratory tract and/or reproductive systems including abortion. Two virus species, namely BVDV-1 and BVDV-2 have been recognized (Fauquet et al., 2005). For both species cytopathogenic (cp) and non-cytopathogenic (ncp) virus strains are distinguished according to their growth characteristics in cultured cells. In adult cattle BVDV infection generally is inapparent. Diaplacental infection of foetuses with a ncp BVDV strain during first trimester of gestation may result in the birth of immunotolerant persistently infected (PI) calves which shed the virus during their life. While cattle serve as natural host, BVDV-1 and BVDV-2 strains may also infect sheep, goats, wild ruminants and pigs (Becher et al., 1997). BVDV-2 was first reported in USA and Canada (Corapi et al., 1989; Pellerin et al., 1994; Ridpath et al., 1994). After these first reports, BVDV-2 has been detected in South America, Japan and many European countries (Becher et al., 1995; Wolfmeyer et al., 1997; Sakoda et al., 1999; Flores et al., 2000; Couvreur et al., 2002; Drew et al., 2002; Barros et al., 2006). BVD viruses are classified in the genus Pestivirus of the family Flaviviridae together with classical swine fever virus (CSFV) and border disease viruses (BDV) (Fauquet et al., 2005). The genome of BVDV is a single stranded RNA of 12.3 kb with positive polarity and a single open reading frame (ORF) flanked by untranslated regions (UTR) at 50 and 30 ends. The polyprotein is cleaved co- and posttranslationally into structural (C, Erns, E1, E2) and nonstructural proteins (Npro, p7, NS2-3, NS4A, NS4B,
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NS5A, NS5B). Genetic and antigenic differences between BVDV-1 and BVDV-2 viruses have been previously described (Pellerin et al., 1994; AvalosRamirez et al., 2001). Genetic typing of BVDV isolates is often based on comparison of nucleotide sequences of 5’UTR, Npro or E2 coding regions. It was demonstrated that analyses based on entire Npro and E2 gene sequences are supported by high bootstrap values (Becher et al., 1997). Analyses of virus strains from different regions of the world revealed at least 12 BVDV-1 subgroups (Vilcek et al., 2001, 2004). Knowledge about genetic diversity of BVDV strains and their geographic distribution is important for laboratory diagnosis and disease control programs. In Turkey occurrence of BVDV infections is known since 1964. The serological prevalence has been reported to range between 24.8% and 64.7%. In intensively managed dairy herds the average rate of PI animals is lower than 0.25% (Burgu et al., 1999, 2003). This study describes phylogenetic analyses of BVDV isolates from Turkey that revealed a new genetic cluster in BVDV-1 as well as the presence of BVDV-2.
2. Materials and methods 2.1. Sera, viruses and cell line For serological screening a total of 722 serum samples from cattle were collected in 7 provinces in western Turkey. From the same area 582 blood samples were obtained for virus isolation (Table 1).
Table 1 Distribution of serum samples according to provinces located in the western part of Turkey Region
Number of serum samples tested
Seropositive for BVDV
Seropositive for BVDV-2
Number of blood samplesa
Bursa Balıkesir Bilecik Yalova Izmir Edirne/Kırklareli Mug˘la Afyon
258 166 15 15 101 139 28 –
101 81 5 3 42 79 20 –
6 7 1 – 1 27 – –
148 140 15 15 101 80 28 55
Total
722
331 (45.8%)
a
(39.1) (48.7) (33.3) (20.0) (41.5) (56.8) (71.4)
(2.3) (4.2) (6.6) (0.9) (19.4)
42 (5.8%)
582
For virus isolation 582 blood samples were collected from the animals that also served for serological analyses. From the buffy coat samples four BVDV isolates were obtained.
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All the animals were from dairy herds, not vaccinated against BVDV and older than 1 year. A total of 45 pestivirus strains isolated from 17 different farms in Turkey was analysed in this study. Among them 41 isolates had been obtained from different geographical regions of the country during field screening studies between 1997 and 2000 (Burgu et al., 1999, 2003). The remaining 4 pestivirus strains were isolated from the buffy coat samples (see above) obtained during this study in 2005 (Fig. 1). Forty-four isolates came from cattle and one isolate was of sheep origin (TR-15). Virus isolates labelled TR-1 to TR-45 were propagated on Madin-Darby bovine kidney (MDBK) cells tested for endogenous pestivirus infection by immunoperoxidase monolayer assay ¨ zkul et al., 2002). Viruses were isolated (IPMA) (O from buffy coat serum and lung samples by long-term co-cultivation and identified by IPMA. Bovine isolates came from PI cattle (n = 10) including one case of MD and from cattle either transiently infected or not tested for persistent viremia (n = 35). Besides the virus from the MD case all isolates were non-cytopathogenic. All tested viruses were 3rd to 4th passage level, since original samples were not available. Cell cultures and foetal bovine sera used during these passages were strictly tested for pestivirus contamination and antiviral antibodies. For neutralization assays cytopathogenic virus strains NADL (BVDV-1a) and Gi-2 (BVDV-2a) were used.
2.2. Serum neutralization test (SNT) All 722 serum samples were screened for neutralizing antibodies against BVDV-1 (NADL) and BVDV-2 (Gi-2). Two-fold dilutions of serum samples were incubated with an equal volume of virus suspension (100TCID50) for 1 h followed by addition of 50 ml MDBK cell suspension (300,000 cells/ml). Results were evaluated under inverted microscope after 4–5 days of incubation at 37 8C in a 5% CO2 atmosphere. The highest dilution of a serum showing complete inhibition of viral infectivity was taken as neutralizing antibody titre. 2.3. RNA isolation and RT-PCR Viral RNA was isolated from infected MDBK cells using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. For quick differentiation of BVDV-2 isolates from other pestiviruses a discriminating RT-PCR was applied to 29 isolates (TR-1 to TR-29) as previously described (Becher et al., 1997; Cedillo Rosales, 2004). Panpestivirus primers p380_reverse (50 -AAC TCC ATG TGC CAT GTA GAG-30 ) and p100_forward (50 CAT GCC CWY AGT AGG ACT AGC-30 ) were combined with 3 additional sense primers (S200a: 50 CAC TCC ATC AGT YGA GGA G-30 ; S200b: 50 CGC TCT GGC AAC AAG AGA G-30 and S200c: 50 -
Fig. 1. Geographical origins in Turkey of BVD virus isolates characterized in this study. Numbers of isolates taken from a defined region are shown in brackets. Total of four isolates from Edirne, Bursa and Izmir provinces have been isolated during this study (2005), while other 41 isolates had been obtained between 1997 and 2000 Turkey.
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CGC TCT GGC AAC AAG AGA G-30 ) designed to detect various BVDV-2 strains in this test. Reverse transcription was performed using primer p380_reverse for 30 min at 45 8C followed by 30 cycles of denaturation at 94 8C for 30 s, annealing at 55 8C for 30 s and elongation at 72 8C for 30 s. For selective detection of BVDV-1 strains an additional RT-PCR using primers p380_forward in combination with anti-sense primers pBVDV1ra (50 GCC CYT TGC TGT TAC CCG T-30 ) and pBVDV1rb (50 -GTC CTY TGC TAT TGC CTA A-30 ) was performed on all 29 isolates (Cedillo Rosales, 2004). 2.4. Sequencing and phylogenetic analysis Thirty virus isolates (TR-1 to TR-29 and TR-38) were analysed by nucleic acid sequencing. A fragment encompassing 1.3 kb of the viral genome covering part of 50 UTR, Npro, C and part of Erns gene was amplified by RT-PCR as previously described (Becher et al., 1997). In the test, primers p100 (50 -CAT GCC CWY AGT AGG ACT AGC-30 ) and p1400R (50 -CCA GTT GCA CCA ACC ATG-30 ) were employed. Amplified cDNA fragments were cloned using the TOPO TA cloning kit (Invitrogen) and sequenced using the Thermo Sequenase Kit (Amersham Pharmacia Biotech) in a DNA sequencing device Li-COR 4000L (MWG Biotech). For each of the 30 isolates three independent cDNA clones were sequenced and data analysed using the computer programme HUSAR (DKFZ, Heidelberg, Germany). Pairwise genetic distances for the complete Npro encoding regions were calculated using the Kimura 2 parameter method (Kimura, 1980). Phylogenetic trees were created using the neighbor-joining method (Saitou and Nei, 1987). Bootstrap analyses of 1000 replicates ensured statistical significance of the tree branching (Felsenstein, 1985). Genbank accession numbers are listed in Table 2. 2.5. Indirect immunoperoxidase monolayer assay (IIPMA) Seventeen pestivirus isolates (TR-30 to TR-45 and TR-15) were characterized by IIPMA using different mAbs (Yesilbag and Burgu, 2003). For that purpose, MDBK cell cultures were prepared in 24-well plates and infected with approximately 100 TCID50 of the
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respective isolates (MOI 0.01). Following 72 h of incubation at 37 8C in a 5% CO2 atmosphere, the monolayers were washed with 1:3 PBS and heat fixed at 80 8C for 1 h. Different mAb’s including SCR-4 which was shown to be specific for BVDV-2a strains (Cedillo Rosales, 2004) were added to each well in 200 ml volume and incubated at room temperature for 1 h. After washing biotin labelled anti-mouse secondary antibody and streptavidin-HRPO conjugate were used to demonstrate mAb binding. Both steps were applied as 1 h incubation at room temperature. Substrate solution was prepared as 2 mg AEC in 0.3 ml DMF, 4.7 ml Na-Acetate buffer (pH 5.5) and 0.05% H2O2. After 20 min of incubation positive results were evaluated in inverted phase-contrast microscope as brown-reddish cytoplasmic staining.
3. Results 3.1. Survey for BVDV in turkey A total of 722 serum samples from Turkish cattle collected during 2004 and 2005 in western Turkey were tested for neutralizing antibodies (nAb) against BVDV1 and BVDV-2 (Table 1). Taken together 331 samples (45.8%) reacted positive against either test virus. BVDV strains induce neutralizing antibodies which show a significant degree of cross neutralization between the virus species BVDV-1 and BVDV-2. When comparing neutralizing antibody titres against the two test viruses BVDV-1 NADL and BVDV-2 Gi2, a cut-off was set in order to identify animals that had been infected with BVDV-2 on a serological basis. Animals with nAb titres at least 4-fold higher against BVDV-2 than against BVDV-1 were considered as candidates for a BVDV-2 infection. Using this cut-off 5.8% of the samples had significantly higher BVDV-2 specific antibody titres. This result indicated the presence of BVDV-2 in cattle in Turkey. To demonstrate directly the presence of BVDV-2 in Turkey virus isolates had to be investigated. As part of 1997–2000 survey 41 virus isolates from cattle and sheep were obtained; 4 additional pestiviruses were isolated from blood samples in 2005 as part of this study. The 45 virus isolates were tested using different techniques in order to discriminate between BVDV-1 and BVDV-2.
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Table 2 Turkish BVDV isolates analysed in this study Isolate
Province of origina
Year of isolation
Sample
Case b
Biotype
Genetic subgroupc
Genbank accession nos.
TR-1 TR-2 TR-3 TR-4 TR-5 TR-6 TR-7 TR-8 TR-9 TR-10 TR-11 TR-12 TR-13 TR-14 TR-15 TR-16 TR-17 TR-18 TR-19 TR-20 TR-21 TR-22 TR-23 TR-24 TR-25 TR-26 TR-27 TR-28 TR-29 TR-38
Mug˘la Malatya Sivas Samsun-1 Samsun-1 Samsun-1 Samsun-1 Mug˘la Aksaray Aksaray Sanlıurfa Kayseri Kayseri Samsun-2 Amasya Ankara Adana Adana Mug˘la Mug˘la Samsun-2 Samsun-2 Konya Mug˘la Mug˘la Edirne-1 Edirne-2 Izmir Bursa Eskis¸ehir
1999 1999 1998 1999 1999 1999 1999 1999 1998 1998 1999 2000 2000 1997 1999 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 2005 2005 2005 2005 1999
Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Lung Blood Blood Blood Blood Blood Blood Blood Serum Serum Blood Blood Blood
PI PI T T T T T T T T T T T T n.d. PI T MD PI PI PI PI PI T T n.d. n.d. n.d. n.d. PI
ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp cp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp
BVDV-1l BVDV-1a BVDV-1a BVDV-1l BVDV-1l BVDV-1l BVDV-1l BVDV-1l BVDV-1b BVDV-1b BVDV-1d BVDV-1b BVDV-1b BVDV-1d BVDV-2b BVDV-1l BVDV-1a BVDV-1a BVDV-1l BVDV-1l BVDV-1l BVDV-1l BVDV-1h BVDV-1l BVDV-1l BVDV-1f BVDV-1l BVDV-1d BVDV-1l BVDV-1f
EU163950 EU163951 EU163952 EU163953 EU163954 EU163955 EU163956 EU163957 EU163958 EU163959 EU163960 EU163961 EU163962 EU163963 EU163979 EU163964 EU163965 EU163966 EU163967 EU163968 EU163969 EU163970 EU163971 EU163972 EU163973 EU163974 EU163975 EU163976 EU163977 EU163978
a
Location names labeled with a number (like Samsun-1) indicate more than one farm in the same province from which isolates have been obtained. b PI: persistently infected; T: transient viremia; MD: mucosal disease case; n.d.: persistent viremia was not determined; cp: cytopathogenic; ncp: non-cytopathogenic. c The new subgroup in BVDV-1 is shown as BVDV-1l.
3.2. Discrimination by RT-PCR The first 29 virus isolates (TR-1 to TR-29) were tested using RT-PCR. An RT-PCR assay designed to discriminate BVDV-2 from other pestiviruses was employed that had been used to type numerous virus isolates in the past (Cedillo Rosales, 2004). With this system positive signals indicating pestivirus infection were obtained for all 29 isolates (data not shown). A second band indicative for the presence of BVDV-2 was not observed in any case. In order to confirm this result and for positive differentiation of BVDV-1 a second RT-PCR test specific for BVDV-1 isolates was
employed. With one exception all virus isolates showed the expected positive reaction in this test which confirmed the identity of the bovine virus isolates as members of BVDV-1. The sole sample that failed to react in the BVDV-1 RT-PCR was of ovine origin (TR-15). 3.3. IIPMA screening for BVDV-2 Discrimination of BVDV-1 and BVDV-2 can also be performed using virus species specific antibodies. Accordingly an IIPMA was performed using the remaining 16 virus isolates (TR-30 to TR-45) and
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TR-15 which could not be discriminated by RT-PCR. A mAb (SCR-4) prepared against the E2 protein of BVDV-2 strain 890 is particularly suited for this purpose (Cedillo Rosales, 2004). Using more than 30 pestivirus strains from different virus species mAb SCR-4 was previously shown to recognize the major viral membrane glycoprotein E2 of members of BVDV-2a the predominant BVDV-2 subgroup in Europe but not of other pestiviruses including BVDV1 (Cedillo Rosales, 2004). As expected all 17 virus isolates were recognized using mAbs against the highly conserved nonstructural protein NS2/3. In contrast only two virus isolates, TR-15 isolated from sheep and TR-38, reacted with mAb SCR-4 in the IIPMA (data not shown). 3.4. Determination of the Npro coding sequence and phylogenetic analyses In order to confirm and expand the results of the RT-PCR and IIPMA tests the complete Npro coding region of the 29 virus isolates employed in the RTPCR assays (TR-1 to TR-29) and the additional putative BVDV-2 isolate (TR-38) identified by immunoperoxidase screening was amplified and sequenced. Obtained nucleic acid sequences were identical in length (504 bp). The newly determined sequences were compared to sequences from Genbank. The analysis comprised all pestivirus species and representatives of numerous subgroups. The phylogenetic analyses showed that all virus isolates from cattle belonged to BVDV-1 (Fig. 2, Table 2). Turkish BVDV-1 isolates were distributed into various subgroups (Fig. 2). Interestingly, half of the new virus isolates (n = 15) allocated in a single new subgroup. This new subgroup of BVDV-1 was tentatively named as BVDV-1l (Fig. 2, Table 2). The remaining isolates were grouped into BVDV-1a (n = 4), BVDV-1b (n = 4), BVDV-1d (n = 3), BVDV-1f (n = 2) and BVDV-1h (n = 1). There was no clear correlation between the years of virus isolation, the geographic origin and the phylogenetic allocation. Accordingly isolates taken from different provinces far from each other belonged to the same subgroups (BVDV-1a, 1b, 1d and 1l). In contrast virus isolates obtained from one farm in different years were mostly placed within the same genetic cluster.
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The only virus isolate from sheep (TR-15) investigated in this study gave no clear result when the discriminating RT-PCR and the BVDV-1 assay were evaluated. This could indicate the presence of a pestivirus not belonging to either BVDV species; such isolates have been described previously (Becher et al., 1999). However, the phylogenetic analyses based on the complete Npro encoding sequence demonstrated affiliation of the ovine isolate to subgroup BVDV-2b (Fig. 2). Using a mAb specific for BVDV-2a (SCR-4) one additional virus isolate (TR-38) of bovine origin was originally typed as BVDV-2. The discriminating RTPCR however gave no evidence for BVDV-2 in the respective sample. Sequencing of the Npro coding region followed by phylogentic analysis grouped the questionable isolate into subgroup BVDV-1f. MAb SCR-4 has been shown to react exclusively with the E2 protein from members of BVDV-2a. The unexpected binding of the mAb to a BVDV-1 isolate requires further investigation including cloning and sequencing of the E2 coding region of TR38. A comparison with E2 sequences from BVDV-1 and BVDV-2 strains may explain the reactivity of the mAb.
4. Discussion Serological studies in cattle demonstrated a wide distribution of antibodies against pestiviruses around the country indicating that BVDV infections are common in Turkey (Alkan et al., 1997; Burgu et al., 2003). The rate of persistently infected animals in intensively managed dairy herds is however lower than 0.25% (Burgu et al., 1999). In our study the serological prevalence of antibodies against BVDV was relatively low (45.8%) when compared to other studies (Alkan et al., 1997; Burgu et al., 1999). Interestingly, we found in the serological survey clear evidence for the presence of BVDV-2 infections. Thus, far BVDV-2 infections had not been recorded in Turkey. Consequently we chose to investigate available virus isolates more closely by detection of viral genomes as well as viral antigens. RT-PCR typing of 29 isolates demonstrated only BVDV-1 with one ambiguous result for an ovine isolate indicated the presence of a pestivirus but not of
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Fig. 2. Phylogenetic tree of Turkish BVDV isolates generated according to nucleotide sequences of Npro region comprising 504 bp in length. Accession nos. of reference strains are as follows: NADL (M31182), Paplitz (AY735488), Deer-NZ1 (U80903), 519 (AF144464), Antilope-G1 (AY735482), Osloss (M96687), CP7 (V63479), RIT (AF144465), S10 (AY735489), S14-1 (AY735490), 721 (AF144463), W-AU (AF287290), A-AU (AF287283), L-AU (AF287287), 2313-UK (AF287279), Deer-GB1 (U80902), G-AU (AF287285), Giraffe-1 (AF144617), Hobi (AY735486), Chamois-1 (AY738083), 137-4 (L05402), X818 (AF037405), Gifhorn (AY163653), Reindeer-1 (AF144618), 466 (AY163650), Alfort-T (J04358), C-Strain (Z46258), Soldan (AY735495), Gi-4 (AF144468), 28508-5 (AF145968), 890 (U18059), 659 (AY735480), Suwa-CH (AY894998), Gi-7 (EU178816, submitted within this study).
BVDV. Under natural conditions sheep can be infected by different pestivirus species including BVDV-1, BVDV-2 and BDV. Unexpectedly phylogenetic analyses based on the complete Npro encoding sequence showed that the ovine isolate allocated within subgroup b of BVDV-2. The fact that the isolate was not correctly identified by the BVDV-2 specific primers underlines the need for continuous refinement of assay systems. Members of BVDV-2b so far only have been found in Middle- and South-America while BVDV-2 isolates from Europe belonged to BVDV-2a (Cedillo Rosales, 2004). It has not been possible until date to identify the origin of BVDV-2 in Turkey. It appears likely that import of either living animals or biological products is responsible for the introduction of the virus to Turkish farms. For example, the virus could have been brought into Turkey by fetal bovine serum (FBS) used to propagate culture cells. FBS from
the Americas is frequently used in cell cultures. In several independent cases it has been strongly suggested that pestiviruses originate from contaminated FBS including strain ‘‘Hobi’’ (Schirrmeier et al., 2004) and members of BVDV-2b (Becher et al., 1999; Cedillo Rosales, 2004). Cell cultures and FBS used in this study are regularly tested for pestivirus contamination. Nevertheless, the presence of a minimal dose of infectious virus in a batch of FBS cannot be excluded. The discrepancy between the results of the serological survey and the virus isolation is remarkable. From serological data we would have expected a higher percentage of BVDV-2 isolates in our samples. Since we can exclude a selection towards BVDV-1 in our methodology the reason for the observed bias probably is associated with the sampling procedure. The sera used in this study were collected from 7 provinces in western Turkey and may not be
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representative for the entire country. Isolates for genetical typing came from all over Turkey with only 5 isolates originating from 3 western provinces (Bursa, ˙Izmir and Edirne) included in the serological survey. It will be interesting to analyse more isolates from these region to confirm serological results. Further investigations are needed to determine the extent of the presence of BVDV-2b in Turkey. Within the panel of 16 additional isolates analysed by mAbs one further candidate for BVDV-2 was identified. RT-PCR typing based on sequences from the 5’UTR or the Npro region failed to demonstrate the presence of BVDV-2. Based on the entire Npro sequence the isolate was finally grouped into BVDV-1f. The initial miss-typing of the isolate can be explained as follows: either the epitope recognized by mAb SCR-4 is present in the E2 protein of TR-38 or the virus isolate consists of a mixture of virus strains. The reaction pattern of mAb SCR-4 was tested with more than 30 different strains representing all pestivirus species and numerous subgroups including BVDV-1f. Nevertheless, cross reactivity between individual isolates of different species and/or subgroups cannot be excluded. Alternatively TR-38 was generated by recombination between members of BVDV-1f and BVDV-2. In order to discriminate between both possibilities we are currently analyzing the E2 protein of TR-38. In addition biological cloning of the virus isolate will be performed. BVDV-2 infections have previously been demonstrated in various European countries including Germany, France, Italy, United Kingdom, Belgium, Portugal, Slovakia and Austria (Wolfmeyer et al., 1997; Pratelli et al., 2001; Vilcek et al., 2002, 2003; Couvreur et al., 2002; Drew et al., 2002; Barros et al., 2006), while they have not yet been detected in Ireland, Slovenia, Spain and Scandinavian countries. In marked contrast to the situation in the Americas, in Europe only a low amount of BVDV infections are due to BVDV-2. As expected the majority of virus isolates characterized during this study belonged to BVDV-1. Within the latter species there is a marked heterogeneity with more than 11 subgroups that can be distinguished by sequence comparison. According to our phylogenetic studies Turkish BVDV-1 isolates belong to 6 genetic clusters, five of which (BVDV-1a, 1b, 1d, 1f, 1h) have previously been described (Vilcek et al., 2001). Interestingly, half of the isolates formed a
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new subgroup tentatively labelled BVDV-1l. It is important to mention that BVDV-1l strains had been collected in different years and from a number of locations throughout Turkey. Recently, Vilcek et al. (2004) reported that 2 Swiss BVDV isolates belong to a novel 12th genetic group BVDV-1k. Sequence data used in that study were from the 5’UTR while Npro encoding sequences are only available for one strain of this group (Stalder et al., 2005). This latter strain was shown to group separately from the new BVDV-1l subgroup. In addition pairwise comparison between members of BVDV-1k and isolates from the new subgroup BVDV-1l on the basis of sequences from the 5’UTR revealed a sequence homology of less than 88%; this indicates that 1k and 1l are indeed separate subgroups (data not shown). Different methods for subtyping of BVDV isolates were used in this study. All test systems had been validated using a variety of different virus strains. Nevertheless, individual methods failed to correctly identify BVDV species. Unambiguous results were obtained finally by elaborate nucleic acid sequencing and phylogenetic analyses. Taken together the results of this study actually demonstrate that the significant heterogeneity of pestivirus isolates limits the reliability of fast typing assays. Further studies and constant refinement are required to improve test systems. Distinct patterns of geographical distribution have been reported for BVDV subgroups. BVDV-1a and 1b are predominant subgroups in Ireland, the United Kingdom, Spain, India, and the Americas (Graham et al., 2001; Vilcek et al., 2001; Arias et al., 2003; Mishra et al., 2004; Fulton et al., 2005; Pizarro-Lucero et al., 2006). In contrast BVDV-1d and 1f dominate in some European countries (Toplak et al., 2004; Uttenthal et al., 2005). BVDV-1c originally thought to be unique to Germany was possibly transferred to Spain by animal export (Arias et al., 2003). BVDV-1c also predominates in Australia (Mahony et al., 2005). Another subgroup, BVDV-1g, is restricted to an area of middle Europe (Toplak et al., 2004). Our results show that the main subgroups of BVDV-1 circulating in Europe namely BVDV-1a, 1b, 1d, 1f and 1h can also be found in Turkey. Detection of a variety of genetic subgroups in Turkey may be related to animal import or import of biological products from different
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countries during the last decades. Surprisingly a so far unknown subgroup of BVDV-1 was predominant in Turkey. This finding has implications for disease control programs. Serological cross reactions between BVDV-1l and other subgroups have not been determined so far. However, significant differences in serological response against different BVDV subgroups have been described (Pizarro-Lucero et al., 2006). Available vaccines against BVD/MD are mainly based on BVDV-1a strains. Although an official control program for BVDV is not yet applied in Turkey, inactivated vaccines are used in many farms. Whether the predominance of BVDV-1l strains in Turkey reduces the effectiveness of current vaccination campaigns remains to be determined. This study on the genetic characterisation of Turkish BVDV strains demonstrates the presence of a new subgroup (BVDV-1l) that is predominant in Turkey. Currently, there are not enough data on epidemiology and distribution of BVDV in Front Asian, Middle Eastern and Balkan countries. A remarkably different genetic pattern is reported in India where all BVDV strains analysed were BVDV1b (Mishra et al., 2004). Hence, it will be valuable to continue to characterize more isolates from different regions of Turkey as well as from neighbouring countries to clarify epidemiological patterns in that area. These attempts will also help to understand global epidemiology of BVDV.
Acknowledgment This work was supported by the Research Fund of the University of Uludag˘, Project no: V-2004/33.
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Veterinary Microbiology 130 (2008) 258–267 www.elsevier.com/locate/vetmic
Genetic heterogeneity of bovine viral diarrhoea virus (BVDV) isolates from Turkey: Identification of a new subgroup in BVDV-1 Kadir Yes¸ilbag˘a,*, Christine Fo¨rsterb, Barbara Bank-Wolfb, Zeki Yılmazc, Feray Alkand, Aykut Ozkuld, Ibrahim Burgud, Sibilina Cedillo Rosalesb, Heinz-Ju¨rgen Thielb, Matthias Ko¨nigb a
Uludag˘ University, Faculty of Veterinary Medicine, Department of Microbiology, Virology, TR-16059 Go¨ru¨kle, Bursa, Turkey b Institute of Virology (Faculty of Veterinary Medicine), Justus-Liebig-University Giessen, Frankfurter Str. 107, D-35392, Giessen – Germany c Uludag˘ University Faculty of Veterinary Medicine, Department of Internal Medicine, TR-16059 Go¨ru¨kle, Bursa, Turkey d Ankara University Faculty of Veterinary Medicine, Department of Virology, Ankara, Turkey Received 9 November 2007; received in revised form 26 January 2008; accepted 31 January 2008
Abstract Genetic heterogeneity of Turkish ruminant pestiviruses was investigated by phylogenetic analysis of complete Npro encoding nucleotide sequences. A total of 30 virus isolates obtained from 15 provinces around the country between 1997 and 2005 were included in the phylogenetic analysis. Virus isolates mostly originated from cattle with one isolate from sheep. The bovine isolates all belonged to BVDV-1, the sheep isolate to BVDV-2. Fifteen isolates formed a new subgroup within BVDV-1, tentatively named BVDV-1l. The remaining bovine isolates were typed as BVDV-1a (n = 4), BVDV-1b (n = 4), BVDV-1d (n = 3), BVDV-1f (n = 2) and BVDV-1h (n = 1). The isolates allocated to BVDV-1l originated from various geographical regions in different years. There was no correlation between genetic grouping and locations where isolates were obtained. Viruses originating from one farm in most cases belonged to the same subgroup (n = 5). This study indicates that the newly detected subgroup BVDV-1l is predominant and widespread in Turkey. Moreover, an ovine virus isolate was identified as the first member of BVDV-2 reported in Turkey. A serological survey using samples from western Turkey indicated that BVDV-2 is also present in cattle. # 2008 Elsevier B.V. All rights reserved. Keywords: BVDV-1; BVDV-2; Genetic heterogeneity; RT-PCR; Serological survey; Turkey
1. Introduction * Corresponding author at: Uludag˘ University, Faculty of Veterinary Medicine, Virology Section, Go¨ru¨kle Campus, 16059 Bursa, Turkey. Tel.: +90 224 2941295; fax: +90 224 2941202. E-mail address: [email protected] (Y. Kadir).
Bovine viral diarrhoea virus (BVDV) leads to various clinical problems with significant economic impact. The spectrum of disease manifestations
0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2008.01.016
Y. Kadir et al. / Veterinary Microbiology 130 (2008) 258–267
comprises acute haemorrhagic syndrome, persistent infection, mucosal disease (MD), as well as diseases of the respiratory tract and/or reproductive systems including abortion. Two virus species, namely BVDV-1 and BVDV-2 have been recognized (Fauquet et al., 2005). For both species cytopathogenic (cp) and non-cytopathogenic (ncp) virus strains are distinguished according to their growth characteristics in cultured cells. In adult cattle BVDV infection generally is inapparent. Diaplacental infection of foetuses with a ncp BVDV strain during first trimester of gestation may result in the birth of immunotolerant persistently infected (PI) calves which shed the virus during their life. While cattle serve as natural host, BVDV-1 and BVDV-2 strains may also infect sheep, goats, wild ruminants and pigs (Becher et al., 1997). BVDV-2 was first reported in USA and Canada (Corapi et al., 1989; Pellerin et al., 1994; Ridpath et al., 1994). After these first reports, BVDV-2 has been detected in South America, Japan and many European countries (Becher et al., 1995; Wolfmeyer et al., 1997; Sakoda et al., 1999; Flores et al., 2000; Couvreur et al., 2002; Drew et al., 2002; Barros et al., 2006). BVD viruses are classified in the genus Pestivirus of the family Flaviviridae together with classical swine fever virus (CSFV) and border disease viruses (BDV) (Fauquet et al., 2005). The genome of BVDV is a single stranded RNA of 12.3 kb with positive polarity and a single open reading frame (ORF) flanked by untranslated regions (UTR) at 50 and 30 ends. The polyprotein is cleaved co- and posttranslationally into structural (C, Erns, E1, E2) and nonstructural proteins (Npro, p7, NS2-3, NS4A, NS4B,
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NS5A, NS5B). Genetic and antigenic differences between BVDV-1 and BVDV-2 viruses have been previously described (Pellerin et al., 1994; AvalosRamirez et al., 2001). Genetic typing of BVDV isolates is often based on comparison of nucleotide sequences of 5’UTR, Npro or E2 coding regions. It was demonstrated that analyses based on entire Npro and E2 gene sequences are supported by high bootstrap values (Becher et al., 1997). Analyses of virus strains from different regions of the world revealed at least 12 BVDV-1 subgroups (Vilcek et al., 2001, 2004). Knowledge about genetic diversity of BVDV strains and their geographic distribution is important for laboratory diagnosis and disease control programs. In Turkey occurrence of BVDV infections is known since 1964. The serological prevalence has been reported to range between 24.8% and 64.7%. In intensively managed dairy herds the average rate of PI animals is lower than 0.25% (Burgu et al., 1999, 2003). This study describes phylogenetic analyses of BVDV isolates from Turkey that revealed a new genetic cluster in BVDV-1 as well as the presence of BVDV-2.
2. Materials and methods 2.1. Sera, viruses and cell line For serological screening a total of 722 serum samples from cattle were collected in 7 provinces in western Turkey. From the same area 582 blood samples were obtained for virus isolation (Table 1).
Table 1 Distribution of serum samples according to provinces located in the western part of Turkey Region
Number of serum samples tested
Seropositive for BVDV
Seropositive for BVDV-2
Number of blood samplesa
Bursa Balıkesir Bilecik Yalova Izmir Edirne/Kırklareli Mug˘la Afyon
258 166 15 15 101 139 28 –
101 81 5 3 42 79 20 –
6 7 1 – 1 27 – –
148 140 15 15 101 80 28 55
Total
722
331 (45.8%)
a
(39.1) (48.7) (33.3) (20.0) (41.5) (56.8) (71.4)
(2.3) (4.2) (6.6) (0.9) (19.4)
42 (5.8%)
582
For virus isolation 582 blood samples were collected from the animals that also served for serological analyses. From the buffy coat samples four BVDV isolates were obtained.
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All the animals were from dairy herds, not vaccinated against BVDV and older than 1 year. A total of 45 pestivirus strains isolated from 17 different farms in Turkey was analysed in this study. Among them 41 isolates had been obtained from different geographical regions of the country during field screening studies between 1997 and 2000 (Burgu et al., 1999, 2003). The remaining 4 pestivirus strains were isolated from the buffy coat samples (see above) obtained during this study in 2005 (Fig. 1). Forty-four isolates came from cattle and one isolate was of sheep origin (TR-15). Virus isolates labelled TR-1 to TR-45 were propagated on Madin-Darby bovine kidney (MDBK) cells tested for endogenous pestivirus infection by immunoperoxidase monolayer assay ¨ zkul et al., 2002). Viruses were isolated (IPMA) (O from buffy coat serum and lung samples by long-term co-cultivation and identified by IPMA. Bovine isolates came from PI cattle (n = 10) including one case of MD and from cattle either transiently infected or not tested for persistent viremia (n = 35). Besides the virus from the MD case all isolates were non-cytopathogenic. All tested viruses were 3rd to 4th passage level, since original samples were not available. Cell cultures and foetal bovine sera used during these passages were strictly tested for pestivirus contamination and antiviral antibodies. For neutralization assays cytopathogenic virus strains NADL (BVDV-1a) and Gi-2 (BVDV-2a) were used.
2.2. Serum neutralization test (SNT) All 722 serum samples were screened for neutralizing antibodies against BVDV-1 (NADL) and BVDV-2 (Gi-2). Two-fold dilutions of serum samples were incubated with an equal volume of virus suspension (100TCID50) for 1 h followed by addition of 50 ml MDBK cell suspension (300,000 cells/ml). Results were evaluated under inverted microscope after 4–5 days of incubation at 37 8C in a 5% CO2 atmosphere. The highest dilution of a serum showing complete inhibition of viral infectivity was taken as neutralizing antibody titre. 2.3. RNA isolation and RT-PCR Viral RNA was isolated from infected MDBK cells using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. For quick differentiation of BVDV-2 isolates from other pestiviruses a discriminating RT-PCR was applied to 29 isolates (TR-1 to TR-29) as previously described (Becher et al., 1997; Cedillo Rosales, 2004). Panpestivirus primers p380_reverse (50 -AAC TCC ATG TGC CAT GTA GAG-30 ) and p100_forward (50 CAT GCC CWY AGT AGG ACT AGC-30 ) were combined with 3 additional sense primers (S200a: 50 CAC TCC ATC AGT YGA GGA G-30 ; S200b: 50 CGC TCT GGC AAC AAG AGA G-30 and S200c: 50 -
Fig. 1. Geographical origins in Turkey of BVD virus isolates characterized in this study. Numbers of isolates taken from a defined region are shown in brackets. Total of four isolates from Edirne, Bursa and Izmir provinces have been isolated during this study (2005), while other 41 isolates had been obtained between 1997 and 2000 Turkey.
Y. Kadir et al. / Veterinary Microbiology 130 (2008) 258–267
CGC TCT GGC AAC AAG AGA G-30 ) designed to detect various BVDV-2 strains in this test. Reverse transcription was performed using primer p380_reverse for 30 min at 45 8C followed by 30 cycles of denaturation at 94 8C for 30 s, annealing at 55 8C for 30 s and elongation at 72 8C for 30 s. For selective detection of BVDV-1 strains an additional RT-PCR using primers p380_forward in combination with anti-sense primers pBVDV1ra (50 GCC CYT TGC TGT TAC CCG T-30 ) and pBVDV1rb (50 -GTC CTY TGC TAT TGC CTA A-30 ) was performed on all 29 isolates (Cedillo Rosales, 2004). 2.4. Sequencing and phylogenetic analysis Thirty virus isolates (TR-1 to TR-29 and TR-38) were analysed by nucleic acid sequencing. A fragment encompassing 1.3 kb of the viral genome covering part of 50 UTR, Npro, C and part of Erns gene was amplified by RT-PCR as previously described (Becher et al., 1997). In the test, primers p100 (50 -CAT GCC CWY AGT AGG ACT AGC-30 ) and p1400R (50 -CCA GTT GCA CCA ACC ATG-30 ) were employed. Amplified cDNA fragments were cloned using the TOPO TA cloning kit (Invitrogen) and sequenced using the Thermo Sequenase Kit (Amersham Pharmacia Biotech) in a DNA sequencing device Li-COR 4000L (MWG Biotech). For each of the 30 isolates three independent cDNA clones were sequenced and data analysed using the computer programme HUSAR (DKFZ, Heidelberg, Germany). Pairwise genetic distances for the complete Npro encoding regions were calculated using the Kimura 2 parameter method (Kimura, 1980). Phylogenetic trees were created using the neighbor-joining method (Saitou and Nei, 1987). Bootstrap analyses of 1000 replicates ensured statistical significance of the tree branching (Felsenstein, 1985). Genbank accession numbers are listed in Table 2. 2.5. Indirect immunoperoxidase monolayer assay (IIPMA) Seventeen pestivirus isolates (TR-30 to TR-45 and TR-15) were characterized by IIPMA using different mAbs (Yesilbag and Burgu, 2003). For that purpose, MDBK cell cultures were prepared in 24-well plates and infected with approximately 100 TCID50 of the
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respective isolates (MOI 0.01). Following 72 h of incubation at 37 8C in a 5% CO2 atmosphere, the monolayers were washed with 1:3 PBS and heat fixed at 80 8C for 1 h. Different mAb’s including SCR-4 which was shown to be specific for BVDV-2a strains (Cedillo Rosales, 2004) were added to each well in 200 ml volume and incubated at room temperature for 1 h. After washing biotin labelled anti-mouse secondary antibody and streptavidin-HRPO conjugate were used to demonstrate mAb binding. Both steps were applied as 1 h incubation at room temperature. Substrate solution was prepared as 2 mg AEC in 0.3 ml DMF, 4.7 ml Na-Acetate buffer (pH 5.5) and 0.05% H2O2. After 20 min of incubation positive results were evaluated in inverted phase-contrast microscope as brown-reddish cytoplasmic staining.
3. Results 3.1. Survey for BVDV in turkey A total of 722 serum samples from Turkish cattle collected during 2004 and 2005 in western Turkey were tested for neutralizing antibodies (nAb) against BVDV1 and BVDV-2 (Table 1). Taken together 331 samples (45.8%) reacted positive against either test virus. BVDV strains induce neutralizing antibodies which show a significant degree of cross neutralization between the virus species BVDV-1 and BVDV-2. When comparing neutralizing antibody titres against the two test viruses BVDV-1 NADL and BVDV-2 Gi2, a cut-off was set in order to identify animals that had been infected with BVDV-2 on a serological basis. Animals with nAb titres at least 4-fold higher against BVDV-2 than against BVDV-1 were considered as candidates for a BVDV-2 infection. Using this cut-off 5.8% of the samples had significantly higher BVDV-2 specific antibody titres. This result indicated the presence of BVDV-2 in cattle in Turkey. To demonstrate directly the presence of BVDV-2 in Turkey virus isolates had to be investigated. As part of 1997–2000 survey 41 virus isolates from cattle and sheep were obtained; 4 additional pestiviruses were isolated from blood samples in 2005 as part of this study. The 45 virus isolates were tested using different techniques in order to discriminate between BVDV-1 and BVDV-2.
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Table 2 Turkish BVDV isolates analysed in this study Isolate
Province of origina
Year of isolation
Sample
Case b
Biotype
Genetic subgroupc
Genbank accession nos.
TR-1 TR-2 TR-3 TR-4 TR-5 TR-6 TR-7 TR-8 TR-9 TR-10 TR-11 TR-12 TR-13 TR-14 TR-15 TR-16 TR-17 TR-18 TR-19 TR-20 TR-21 TR-22 TR-23 TR-24 TR-25 TR-26 TR-27 TR-28 TR-29 TR-38
Mug˘la Malatya Sivas Samsun-1 Samsun-1 Samsun-1 Samsun-1 Mug˘la Aksaray Aksaray Sanlıurfa Kayseri Kayseri Samsun-2 Amasya Ankara Adana Adana Mug˘la Mug˘la Samsun-2 Samsun-2 Konya Mug˘la Mug˘la Edirne-1 Edirne-2 Izmir Bursa Eskis¸ehir
1999 1999 1998 1999 1999 1999 1999 1999 1998 1998 1999 2000 2000 1997 1999 1997 1997 1997 1997 1997 1997 1997 1997 1997 1997 2005 2005 2005 2005 1999
Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Blood Lung Blood Blood Blood Blood Blood Blood Blood Serum Serum Blood Blood Blood
PI PI T T T T T T T T T T T T n.d. PI T MD PI PI PI PI PI T T n.d. n.d. n.d. n.d. PI
ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp cp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp ncp
BVDV-1l BVDV-1a BVDV-1a BVDV-1l BVDV-1l BVDV-1l BVDV-1l BVDV-1l BVDV-1b BVDV-1b BVDV-1d BVDV-1b BVDV-1b BVDV-1d BVDV-2b BVDV-1l BVDV-1a BVDV-1a BVDV-1l BVDV-1l BVDV-1l BVDV-1l BVDV-1h BVDV-1l BVDV-1l BVDV-1f BVDV-1l BVDV-1d BVDV-1l BVDV-1f
EU163950 EU163951 EU163952 EU163953 EU163954 EU163955 EU163956 EU163957 EU163958 EU163959 EU163960 EU163961 EU163962 EU163963 EU163979 EU163964 EU163965 EU163966 EU163967 EU163968 EU163969 EU163970 EU163971 EU163972 EU163973 EU163974 EU163975 EU163976 EU163977 EU163978
a
Location names labeled with a number (like Samsun-1) indicate more than one farm in the same province from which isolates have been obtained. b PI: persistently infected; T: transient viremia; MD: mucosal disease case; n.d.: persistent viremia was not determined; cp: cytopathogenic; ncp: non-cytopathogenic. c The new subgroup in BVDV-1 is shown as BVDV-1l.
3.2. Discrimination by RT-PCR The first 29 virus isolates (TR-1 to TR-29) were tested using RT-PCR. An RT-PCR assay designed to discriminate BVDV-2 from other pestiviruses was employed that had been used to type numerous virus isolates in the past (Cedillo Rosales, 2004). With this system positive signals indicating pestivirus infection were obtained for all 29 isolates (data not shown). A second band indicative for the presence of BVDV-2 was not observed in any case. In order to confirm this result and for positive differentiation of BVDV-1 a second RT-PCR test specific for BVDV-1 isolates was
employed. With one exception all virus isolates showed the expected positive reaction in this test which confirmed the identity of the bovine virus isolates as members of BVDV-1. The sole sample that failed to react in the BVDV-1 RT-PCR was of ovine origin (TR-15). 3.3. IIPMA screening for BVDV-2 Discrimination of BVDV-1 and BVDV-2 can also be performed using virus species specific antibodies. Accordingly an IIPMA was performed using the remaining 16 virus isolates (TR-30 to TR-45) and
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TR-15 which could not be discriminated by RT-PCR. A mAb (SCR-4) prepared against the E2 protein of BVDV-2 strain 890 is particularly suited for this purpose (Cedillo Rosales, 2004). Using more than 30 pestivirus strains from different virus species mAb SCR-4 was previously shown to recognize the major viral membrane glycoprotein E2 of members of BVDV-2a the predominant BVDV-2 subgroup in Europe but not of other pestiviruses including BVDV1 (Cedillo Rosales, 2004). As expected all 17 virus isolates were recognized using mAbs against the highly conserved nonstructural protein NS2/3. In contrast only two virus isolates, TR-15 isolated from sheep and TR-38, reacted with mAb SCR-4 in the IIPMA (data not shown). 3.4. Determination of the Npro coding sequence and phylogenetic analyses In order to confirm and expand the results of the RT-PCR and IIPMA tests the complete Npro coding region of the 29 virus isolates employed in the RTPCR assays (TR-1 to TR-29) and the additional putative BVDV-2 isolate (TR-38) identified by immunoperoxidase screening was amplified and sequenced. Obtained nucleic acid sequences were identical in length (504 bp). The newly determined sequences were compared to sequences from Genbank. The analysis comprised all pestivirus species and representatives of numerous subgroups. The phylogenetic analyses showed that all virus isolates from cattle belonged to BVDV-1 (Fig. 2, Table 2). Turkish BVDV-1 isolates were distributed into various subgroups (Fig. 2). Interestingly, half of the new virus isolates (n = 15) allocated in a single new subgroup. This new subgroup of BVDV-1 was tentatively named as BVDV-1l (Fig. 2, Table 2). The remaining isolates were grouped into BVDV-1a (n = 4), BVDV-1b (n = 4), BVDV-1d (n = 3), BVDV-1f (n = 2) and BVDV-1h (n = 1). There was no clear correlation between the years of virus isolation, the geographic origin and the phylogenetic allocation. Accordingly isolates taken from different provinces far from each other belonged to the same subgroups (BVDV-1a, 1b, 1d and 1l). In contrast virus isolates obtained from one farm in different years were mostly placed within the same genetic cluster.
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The only virus isolate from sheep (TR-15) investigated in this study gave no clear result when the discriminating RT-PCR and the BVDV-1 assay were evaluated. This could indicate the presence of a pestivirus not belonging to either BVDV species; such isolates have been described previously (Becher et al., 1999). However, the phylogenetic analyses based on the complete Npro encoding sequence demonstrated affiliation of the ovine isolate to subgroup BVDV-2b (Fig. 2). Using a mAb specific for BVDV-2a (SCR-4) one additional virus isolate (TR-38) of bovine origin was originally typed as BVDV-2. The discriminating RTPCR however gave no evidence for BVDV-2 in the respective sample. Sequencing of the Npro coding region followed by phylogentic analysis grouped the questionable isolate into subgroup BVDV-1f. MAb SCR-4 has been shown to react exclusively with the E2 protein from members of BVDV-2a. The unexpected binding of the mAb to a BVDV-1 isolate requires further investigation including cloning and sequencing of the E2 coding region of TR38. A comparison with E2 sequences from BVDV-1 and BVDV-2 strains may explain the reactivity of the mAb.
4. Discussion Serological studies in cattle demonstrated a wide distribution of antibodies against pestiviruses around the country indicating that BVDV infections are common in Turkey (Alkan et al., 1997; Burgu et al., 2003). The rate of persistently infected animals in intensively managed dairy herds is however lower than 0.25% (Burgu et al., 1999). In our study the serological prevalence of antibodies against BVDV was relatively low (45.8%) when compared to other studies (Alkan et al., 1997; Burgu et al., 1999). Interestingly, we found in the serological survey clear evidence for the presence of BVDV-2 infections. Thus, far BVDV-2 infections had not been recorded in Turkey. Consequently we chose to investigate available virus isolates more closely by detection of viral genomes as well as viral antigens. RT-PCR typing of 29 isolates demonstrated only BVDV-1 with one ambiguous result for an ovine isolate indicated the presence of a pestivirus but not of
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Fig. 2. Phylogenetic tree of Turkish BVDV isolates generated according to nucleotide sequences of Npro region comprising 504 bp in length. Accession nos. of reference strains are as follows: NADL (M31182), Paplitz (AY735488), Deer-NZ1 (U80903), 519 (AF144464), Antilope-G1 (AY735482), Osloss (M96687), CP7 (V63479), RIT (AF144465), S10 (AY735489), S14-1 (AY735490), 721 (AF144463), W-AU (AF287290), A-AU (AF287283), L-AU (AF287287), 2313-UK (AF287279), Deer-GB1 (U80902), G-AU (AF287285), Giraffe-1 (AF144617), Hobi (AY735486), Chamois-1 (AY738083), 137-4 (L05402), X818 (AF037405), Gifhorn (AY163653), Reindeer-1 (AF144618), 466 (AY163650), Alfort-T (J04358), C-Strain (Z46258), Soldan (AY735495), Gi-4 (AF144468), 28508-5 (AF145968), 890 (U18059), 659 (AY735480), Suwa-CH (AY894998), Gi-7 (EU178816, submitted within this study).
BVDV. Under natural conditions sheep can be infected by different pestivirus species including BVDV-1, BVDV-2 and BDV. Unexpectedly phylogenetic analyses based on the complete Npro encoding sequence showed that the ovine isolate allocated within subgroup b of BVDV-2. The fact that the isolate was not correctly identified by the BVDV-2 specific primers underlines the need for continuous refinement of assay systems. Members of BVDV-2b so far only have been found in Middle- and South-America while BVDV-2 isolates from Europe belonged to BVDV-2a (Cedillo Rosales, 2004). It has not been possible until date to identify the origin of BVDV-2 in Turkey. It appears likely that import of either living animals or biological products is responsible for the introduction of the virus to Turkish farms. For example, the virus could have been brought into Turkey by fetal bovine serum (FBS) used to propagate culture cells. FBS from
the Americas is frequently used in cell cultures. In several independent cases it has been strongly suggested that pestiviruses originate from contaminated FBS including strain ‘‘Hobi’’ (Schirrmeier et al., 2004) and members of BVDV-2b (Becher et al., 1999; Cedillo Rosales, 2004). Cell cultures and FBS used in this study are regularly tested for pestivirus contamination. Nevertheless, the presence of a minimal dose of infectious virus in a batch of FBS cannot be excluded. The discrepancy between the results of the serological survey and the virus isolation is remarkable. From serological data we would have expected a higher percentage of BVDV-2 isolates in our samples. Since we can exclude a selection towards BVDV-1 in our methodology the reason for the observed bias probably is associated with the sampling procedure. The sera used in this study were collected from 7 provinces in western Turkey and may not be
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representative for the entire country. Isolates for genetical typing came from all over Turkey with only 5 isolates originating from 3 western provinces (Bursa, ˙Izmir and Edirne) included in the serological survey. It will be interesting to analyse more isolates from these region to confirm serological results. Further investigations are needed to determine the extent of the presence of BVDV-2b in Turkey. Within the panel of 16 additional isolates analysed by mAbs one further candidate for BVDV-2 was identified. RT-PCR typing based on sequences from the 5’UTR or the Npro region failed to demonstrate the presence of BVDV-2. Based on the entire Npro sequence the isolate was finally grouped into BVDV-1f. The initial miss-typing of the isolate can be explained as follows: either the epitope recognized by mAb SCR-4 is present in the E2 protein of TR-38 or the virus isolate consists of a mixture of virus strains. The reaction pattern of mAb SCR-4 was tested with more than 30 different strains representing all pestivirus species and numerous subgroups including BVDV-1f. Nevertheless, cross reactivity between individual isolates of different species and/or subgroups cannot be excluded. Alternatively TR-38 was generated by recombination between members of BVDV-1f and BVDV-2. In order to discriminate between both possibilities we are currently analyzing the E2 protein of TR-38. In addition biological cloning of the virus isolate will be performed. BVDV-2 infections have previously been demonstrated in various European countries including Germany, France, Italy, United Kingdom, Belgium, Portugal, Slovakia and Austria (Wolfmeyer et al., 1997; Pratelli et al., 2001; Vilcek et al., 2002, 2003; Couvreur et al., 2002; Drew et al., 2002; Barros et al., 2006), while they have not yet been detected in Ireland, Slovenia, Spain and Scandinavian countries. In marked contrast to the situation in the Americas, in Europe only a low amount of BVDV infections are due to BVDV-2. As expected the majority of virus isolates characterized during this study belonged to BVDV-1. Within the latter species there is a marked heterogeneity with more than 11 subgroups that can be distinguished by sequence comparison. According to our phylogenetic studies Turkish BVDV-1 isolates belong to 6 genetic clusters, five of which (BVDV-1a, 1b, 1d, 1f, 1h) have previously been described (Vilcek et al., 2001). Interestingly, half of the isolates formed a
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new subgroup tentatively labelled BVDV-1l. It is important to mention that BVDV-1l strains had been collected in different years and from a number of locations throughout Turkey. Recently, Vilcek et al. (2004) reported that 2 Swiss BVDV isolates belong to a novel 12th genetic group BVDV-1k. Sequence data used in that study were from the 5’UTR while Npro encoding sequences are only available for one strain of this group (Stalder et al., 2005). This latter strain was shown to group separately from the new BVDV-1l subgroup. In addition pairwise comparison between members of BVDV-1k and isolates from the new subgroup BVDV-1l on the basis of sequences from the 5’UTR revealed a sequence homology of less than 88%; this indicates that 1k and 1l are indeed separate subgroups (data not shown). Different methods for subtyping of BVDV isolates were used in this study. All test systems had been validated using a variety of different virus strains. Nevertheless, individual methods failed to correctly identify BVDV species. Unambiguous results were obtained finally by elaborate nucleic acid sequencing and phylogenetic analyses. Taken together the results of this study actually demonstrate that the significant heterogeneity of pestivirus isolates limits the reliability of fast typing assays. Further studies and constant refinement are required to improve test systems. Distinct patterns of geographical distribution have been reported for BVDV subgroups. BVDV-1a and 1b are predominant subgroups in Ireland, the United Kingdom, Spain, India, and the Americas (Graham et al., 2001; Vilcek et al., 2001; Arias et al., 2003; Mishra et al., 2004; Fulton et al., 2005; Pizarro-Lucero et al., 2006). In contrast BVDV-1d and 1f dominate in some European countries (Toplak et al., 2004; Uttenthal et al., 2005). BVDV-1c originally thought to be unique to Germany was possibly transferred to Spain by animal export (Arias et al., 2003). BVDV-1c also predominates in Australia (Mahony et al., 2005). Another subgroup, BVDV-1g, is restricted to an area of middle Europe (Toplak et al., 2004). Our results show that the main subgroups of BVDV-1 circulating in Europe namely BVDV-1a, 1b, 1d, 1f and 1h can also be found in Turkey. Detection of a variety of genetic subgroups in Turkey may be related to animal import or import of biological products from different
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countries during the last decades. Surprisingly a so far unknown subgroup of BVDV-1 was predominant in Turkey. This finding has implications for disease control programs. Serological cross reactions between BVDV-1l and other subgroups have not been determined so far. However, significant differences in serological response against different BVDV subgroups have been described (Pizarro-Lucero et al., 2006). Available vaccines against BVD/MD are mainly based on BVDV-1a strains. Although an official control program for BVDV is not yet applied in Turkey, inactivated vaccines are used in many farms. Whether the predominance of BVDV-1l strains in Turkey reduces the effectiveness of current vaccination campaigns remains to be determined. This study on the genetic characterisation of Turkish BVDV strains demonstrates the presence of a new subgroup (BVDV-1l) that is predominant in Turkey. Currently, there are not enough data on epidemiology and distribution of BVDV in Front Asian, Middle Eastern and Balkan countries. A remarkably different genetic pattern is reported in India where all BVDV strains analysed were BVDV1b (Mishra et al., 2004). Hence, it will be valuable to continue to characterize more isolates from different regions of Turkey as well as from neighbouring countries to clarify epidemiological patterns in that area. These attempts will also help to understand global epidemiology of BVDV.
Acknowledgment This work was supported by the Research Fund of the University of Uludag˘, Project no: V-2004/33.
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