Genetic heterogeneity of bovine viral diarrhoea virus (BVDV) isolates from Turkey: identification of a new subgroup in BVDV-1

Genetic heterogeneity of Turkish ruminant pestiviruses was investigated by phylogenetic analysis of complete N(pro) 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.     

Heterogeneity of ruminant pestiviruses: academic interest or important basis for the development of vaccines and diagnostics?

Pestiviruses cause economically important diseases of farm animals. Members of the Pestiviruses are bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, classical swine fever virus (CSFV) and border disease virus (BDV). Phylogenetic analyses based on the entire nucleic acid sequence encoding the Npro allow a statistically significant segregation of established species and of subgroups within the species. BVDV-1 strains isolated in Germany can be associated with at least five different subgroups. In contrast all BVDV-2 isolates detected in Germany so far are closely related, belonging to one subgroup. A group of virus isolates from sheep and zoo animals is clearly different from established pestivirus species and can be designated as BDV-2. Antigenetic relatedness of pestiviruses was studied using defined virus isolates and antisera in cross-neutralization assays. Six antigenic groups were distinguished corresponding to the genetic clusters BVDV-1, BVDV-2, CSFV, BDV-1, BDV-2 and Giraffe-1. A significant antigenic difference was also observed between members of subgroups 1a and 1b of BVDV-1. Studies on the genetic and antigenic heterogeneity of pestiviruses are important for the development of new vaccines, diagnostic tests and for eradication programs.     

Genetic diversity of BVDV: consequences for classification and molecular epidemiology

Genetic typing of bovine viral diarrhoea virus (BVDV) is important for the precise classification of viruses as well as for the development of molecular epidemiology. BVDV isolates were usually typed based on comparison of genomic sequences from the 5'-untranslated region (5'-UTR), N(pro) and E2 region. Recently we have identified 11 genetic groups (subgenotypes) of BVDV-1. Our further experiments confirmed a new subgenotype, BVDV-1k, isolated from cattle in Switzerland. BVDV isolates from India were typed as BVDV-1b whereas BVDV-1c is a predominant subgenotype in Australia. The results of genetic typing of BVDV indicate that distribution of subgenotypes has no relationship to the geographic origin of viral isolates.     

Genetic and antigenic characterization of bovine viral diarrhea viruses isolated from cattle in Hokkaido,Japan

In our previous study, we genetically analyzed bovine viral diarrhea viruses (BVDVs) isolated from 2000 to 2006 in Japan and reported that subgenotype 1b viruses were predominant. In the present study, 766 BVDVs isolated from 2006 to 2014 in Hokkaido, Japan, were genetically analyzed to understand recent epidemics. Phylogenetic analysis based on nucleotide sequences of the 5′-untranslated region of viral genome revealed that 766 isolates were classified as genotype 1 (BVDV-1; 544 isolates) and genotype 2 (BVDV-2; 222). BVDV-1 isolates were further divided into BVDV-1a (93), 1b (371) and 1c (80) subgenotypes, and all BVDV-2 isolates were grouped into BVDV-2a subgenotype (222). Further comparative analysis was performed with BVDV-1a, 1b and 2a viruses isolated from 2001 to 2014. Phylogenetic analysis based on nucleotide sequences of the viral glycoprotein E2 gene, a major target of neutralizing antibodies, revealed that BVDV-1a, 1b and 2a isolates were further classified into several clusters. Cross-neutralization tests showed that BVDV-1b isolates were antigenically different from BVDV-1a isolates, and almost BVDV-1a, 1b and 2a isolates were antigenically similar among each subgenotype and each E2 cluster. Taken together, BVDV-1b viruses are still predominant, and BVDV-2a viruses have increased recently in Hokkaido, Japan. Field isolates of BVDV-1a, 1b and 2a show genetic diversity on the E2 gene with antigenic conservation among each subgenotype during the last 14 years.     

Genetic diversity of international bovine viral diarrhoea virus (BVDV) isolates: identification of a new BVDV-1 genetic group

In the last decade, several studies were performed to characterise bovine viral diarrhoea virus (BVDV) isolates and define genetic groups by genotyping. Much data is now available from GenBank, predominantly sequences from the 5' untranslated region (5'-UTR). In order to find out whether genetic grouping of isolates from different countries could be harmonised, 22 new isolates from five countries were analysed in combination with published sequences. Eighteen of these isolates were typed as BVDV genotype 1 (BVDV-1), and one isolate from Argentina and three isolates from Brazil were typed as BVDV-2. BVDV-1 isolates were clustered into five previously defined genetic groups: BVDV-1a, b, d, e and f. Two isolates from Finland and one from Egypt formed a group which was tentatively labelled as BVDV-1j, since statistical support was low. By using a fragment of the Npro gene for typing, we found that these isolates fall into the same group as a deer strain, and are statistically significant. Some Swiss BVDV strains taken from GenBank were found in a new genetic group which was designated as BVDV-1k. The BVDV-2 isolates included in this study seemed to fall into two genetic groups.     

Endemic infections of bovine viral diarrhea virus genotypes 1b and 2a isolated from cattle in Japan between 2014 and 2020

Bovine viral diarrhea virus (BVDV) is a causative agent of bovine viral diarrhea. In Japan, a previous study reported that subgenotype 1b viruses were predominant until 2014. Because there is little information regarding the recent epidemiological status of BVDV circulating in Japan, we performed genetic characterization of 909 BVDV isolates obtained between 2014 and 2020. We found that 657 and 252 isolates were classified as BVDV-1 and BVDV-2, respectively, and that they were further subdivided into 1a (35 isolates, 3.9%), 1b (588, 64.7%), 1c (34, 3.7%), and 2a (252, 27.7%). Phylogenetic analysis using entire E2 coding sequence revealed that a major domestic cluster in Japan among BVDV-1b and 2a viruses were unchanged from a previous study conducted from 2006 to 2014. These results provide updated information concerning the epidemic strain of BVDV in Japan, which would be helpful for appropriate vaccine selection.     

甘肃、青海和宁夏地区牛病毒性腹泻病毒Erns基因的变异分析

分析2013—2019年中国西北部分省区不同基因亚型牛病毒性腹泻病毒（BVDV）抗原基因E^rns的分子特征,了解其遗传演化规律。从甘肃、青海、宁夏规模化牛场送检的疑似牛病毒性腹泻发病牛150份EDTA抗凝血提取总RNA,利用RT-PCR扩增病毒基因组E^rns-E1区,克隆测序后比对,构建系统进化树进行遗传演化关系分析。利用牛肾细胞MDBK对检出的不同基因亚型BVDV进行分离,并鉴定其生物型。RT-PCR扩增结果表明,BVDV总体阳性率为37.33%,其中甘肃省、青海省、宁夏回族自治区BVDV阳性率分别为37.68%、35.71%、40.00%。获得56份E^rns-E1 DNA,克隆测序获得33条不同的E^rns序列,长度均为681 bp,分析表明流行株分属10个BVDV基因亚型：BVDV-1a （2株）、BVDV-1b （5株）、BVDV-1c （1株）、BVDV-1d （3株）、BVDV-1m （11株）、BVDV-1o （1株）、BVDV-1p （4株）、BVDV-1q （4株）、BVDV-1v （1株）、BVDV-2a （1株）。分离获得BVDV-1a亚型、BVDV-1b亚型、BVDV-1v亚型、BVDV-2a亚型分离株各1株,BVDV-1 d亚型分离株2株,均为非致细胞病变型。各亚型株间E^rns基因核苷酸相似性以BVDV-1a～1d经典亚型株（79.8%～85.9%）或1m～1q及1v新亚型株（81.0%～87.3%）较高,以BVDV-1 m和BVDV-1p流行株亚型间相似性最高（87.3%）。各亚型株E^rns基因编码蛋白的RNA酶活性位点以及双链RNA作用基序（₁₃₉KKGK₁₄₂）保守,但E^rns第26位糖基化位点（26 NRSL）在1m～1q、1v亚型株移位（24 NVSR）。首次以E^rns核苷酸序列构建系统进化树,结果显示1m～1q及1v等亚型BVDV株在进化上关系较为密切。本研究首次选用E^rns靶标基因对甘肃、青海、宁夏部分省区牛源BVDV株进行同源性及系统进化分析,发现10个基因亚型流行株,以1m亚型株最为普遍,1m～1q及1v等亚型株亲缘关系密切。     

Genetic Heterogeneity of Bovine Viral Diarrhoea Virus In Italy

The genetic characteristics, of 38 field isolates of bovine viral diarrhoea virus (BVDV) collected in 1999 from sick or healthy and persistently infected cattle of dairy farms situated in northern Italy, were investigated. A partial 5-untranslated region (5-UTR) sequence of each isolate was determined and a phylogenetic analysis was performed. All the isolates were classified as belonging to the BVDV-1 genotype and could be assigned to different BVDV-1 groups, namely BVDV-1b (n = 20), BVDV-1d (n = 6) and BVDV-1e (n = 10). Two remaining isolates could be classified as BVDV-1f and BVDV-1h, respectively. These results provided evidence for genetic heterogeneity of BVDV in Italy, and contribute to a better knowledge of the circulation of BVDV strains, and to their classification.     

Genetic diversity of ruminant <Emphasis Type="Italic">Pestivirus</Emphasis> strains collected in Northern Ireland between 1999 and 2011 and the role of live ruminant imports

Background

The genus pestivirus within the family Flaviviridae includes bovine viral diarrhoea virus (BVDV) types 1 and 2, border disease virus (BDV) and classical swine fever virus. The two recognised genotypes of BVDV are divided into subtypes based on phylogenetic analysis, namely a-p for BVDV-1 and a-c for BVDV-2.

Methods

Three studies were conducted to investigate the phylogenetic diversity of pestiviruses present in Northern Ireland. Firstly, pestiviruses in 152 serum samples that had previously tested positive for BVDV between 1999 and 2008 were genotyped with a RT-PCR assay. Secondly, the genetic heterogeneity of pestiviruses from 91 serum samples collected between 2008 and 2011 was investigated by phylogenetic analysis of a 288 base pair portion of the 5’ untranslated region (UTR). Finally, blood samples from 839 bovine and 4,437 ovine animals imported in 2010 and 2011 were tested for pestiviral RNA. Analysis of animal movement data alongside the phylogenetic analysis of the strains was carried out to identify any links between isolates and animal movement.

Results

No BVDV-2 strains were detected. All of the 152 samples in the first study were genotyped as BVDV-1. Phylogenetic analysis indicated that the predominant subtype circulating was BVDV-1a (86 samples out of 91). The remaining five samples clustered close to reference strains in subtype BVDV-1b. Out of the imported animals, 18 bovine samples tested positive and 8 inconclusive (Ct ≥36), while all ovine samples were negative. Eight sequences were obtained and were defined as BVDV-1b. Analysis of movement data between herds failed to find links between herds where BVDV-1b was detected.

Conclusion

Given that only BVDV-1a was detected in samples collected between 1968 and 1999, this study suggests that at least one new subtype has been introduced to Northern Ireland between 1999 and 2011 and highlights the potential for importation of cattle to introduce new strains.

     

In vitro permissivity of bovine peripheral blood mononuclear cells to bovine viral diarrhoea virus is dependent on the animal specific immune status

The in vitro permissivity to infection with homologous and heterologous bovine viral diarrhoea virus (BVDV) strains of bovine peripheral blood mononuclear cells (PBMCs) from eight na?ve and eight BVDV-1b immune animals was studied. Four reference strains (BVDV-1a NADL, BVDV-1b NY-1, BVDV-2 125 and BVDV-2 890) were selected, based on genotype, prevalence and biotype. Virus neutralizing antibody titres were determined at bleeding and the viral loads were measured in PBMCs by end point titration in cell culture and by real-time PCR. PBMCs from both na?ve and immune animals became infected by all BVDV strains tested, although virus titres were lower for immune heifers than na?ve ones; the differences were significant for NADL (P<0.05) and 890 (P<0.001) strains. The in vitro model used in this study showed that PBMCs from immune animals are susceptible to re-infection with both homologous and heterologous BVDV strains, albeit at a lower extent than na?ve cattle.     

Genetic and pathobiological characterization of bovine viral diarrhea viruses recently isolated from cattle in Japan

The 475 strains of bovine viral diarrhea virus (BVDV) isolated from cattle in 12 prefectures of Japan in the last 7 years were phylogenetically classified as BVDV-1 or BVDV-2 on the basis of the nucleotide sequence of the 5'-untranslated region. BVDV-1 strains were further subtyped as 1a (101 strains), 1b (163), 1c (128), 1j (3), and So CP/75-like (1), and all of the 79 BVDV-2 strains belonged to subtype 2a. These 2a BVDVs contain two isolates that had high nucleotide identities with those of highly pathogenic BVDV-2 strains reported in North America (Pellerin et al., 1994). However, acute infection with severe mortality like North American outbreak was not observed and most of the present BVDV-2 strains were isolated from persistently infected (PI) cattle showing mild or no clinical sign. Moreover, it was revealed that 61.5% of the 39 PI cattle with cytopathogenic BVDVs did not show typical mucosal disease and 54.6% of the 405 PI animals only with non-cytopathogenic BVDVs were apparently healthy. The present results indicate that the prevention of the infection with an appropriate vaccine and active surveillance covering healthy cattle are required for the control of BVD.     

Reactivity and prevalence of neutralizing antibodies against Japanese strains of bovine viral diarrhea virus subgenotypes

Bovine viral diarrhea virus (BVDV) field isolates show genetic and antigenic diversity. At least 14 subgenotypes of BVDV-1 and 4 of BVDV-2 have been identified in Artiodactyla worldwide. Of these, 6 subgenotypes of BVDV-1 and 1 of BVDV-2 have been isolated in Japan. Previously, we reported that each subgenotype virus expresses different antigenic characteristics. Here we investigated the reactivity of neutralizing antibodies against representative strains of Japanese BVDV subgenotypes using sera from 266 beef cattle to estimate the prevalence of this epidemic virus among cattle in Japan. Antibody titers at concentrations at least 4-fold higher than antibodies against other subgenotype viruses were considered subgenotype specific. Subgenotype-specific antibodies were detected from 117 (80.7%) of 145 sera samples (69.7% against BVDV-1a, 1.4% against BVDV-1b, 8.3% against BVDV-1c, and 1.4% against BVDV-2a). The results suggest that neutralization tests are useful in estimating currently epidemic subgenotypes of BVDV in the field.     

Variation in pestivirus growth in testicle primary cell culture is more dependent on the individual cell donor than cattle breed

The causes of bovine respiratory disease complex (BRDC) are multifactorial and include infection with both viral and bacterial pathogens. Host factors are also involved as different breeds of cattle appear to have different susceptibilities to BRDC. Infection with bovine pestiviruses, including bovine viral diarrhea virus 1 (BVDV1), BVDV2 and ‘HoBi’-like viruses, is linked to the development of BRDC. The aim of the present study was to compare the growth of different bovine pestiviruses in primary testicle cell cultures obtained from taurine, indicine and mixed taurine and indicine cattle breeds. Primary cells strains, derived from testicular tissue, were generated from three animals from each breed. Bovine pestivirus strains used were from BVDV-1a, BVDV-1b, BVDV-2a and ‘HoBi’-like virus. Growth was compared by determining virus titers after one passage in primary cells. All tests were run in triplicate. Virus titers were determined by endpoint dilution and RT-qPCR. Statistical analysis was performed using one way analysis of variance (ANOVA) followed by the Tukey’s Multiple Comparison Test (P?0.05). Significant differences in virus growth did not correlate with cattle breed. However, significant differences were observed between cells derived from different individuals regardless of breed. Variation in the replication of virus in primary cell strains may reflect a genetic predisposition that favors virus replication.     

The prevalent genotypes of bovine viral diarrhea virus in Japan, Germany and the United States of America

Genotypes and subgenotypes of bovine viral diarrhea virus (BVDV) field isolates from Japan, Germany and the United States of America (USA) were identified, and the prevalent pattern of BVDV in individual countries was estimated genetically. Subgenotypes were determined based on phylogenetic analyses of nucleotide sequences of a part of the E2-coding gene of BVDV. Forty-five, 61 and 56 BVDV strains were isolated from naturally infected cattle in Japan, Germany and USA, respectively, between 1980 and 2003. The most prevalent BVDV in these three countries was BVDV-1b. The second most prevalent BVDV strains were 1a, 1d and BVDV-2 in Japan, Germany and USA, respectively. The most prevalent subgenotype 1b in each country constructed individual small clusters in the subgenotype 1b branch in the phylogenetic tree. Although cattle and/or cattle products were moving among the three countries as part of international trade, the distribution of BVDV in the field in each country showed long-standing individual patterns.     

High prevalence of bovine viral diarrhea virus 1 in Chinese swine herds

Nested RT-PCR was used to investigate bovine viral diarrhea virus in 511 specimens collected from Chinese pigs exhibiting clinical symptoms between 2007 and 2010. Of these, 137 samples were BVDV-positive and the BVDV prevalence rate was 23.1% (9/39) in 2007, 27.7% (44/159) in 2008, 33.6% (34/101) in 2009, and 23.6% (50/212) in 2010. Twenty of 137 BVDV-positive samples were used for further genetic analysis of the 5'-UTR. Phylogenetic analysis revealed that they were BVDV-1 and subtyped into BVDV-1a, BVDV-1b, BVDV-1m, BVDV-1o and an unknown subgenotype. This study showed that BVDVs were highly prevalent in Chinese pig herds and appropriate measures should be taken to control BVDV prevalence in pig herds.