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.     

Differences in virulence between two noncytopathic bovine viral diarrhea viruses in calves.

A noncytopathic bovine viral diarrhea virus (BVDV), BVDV-890, isolated from a yearling heifer that died with extensive internal hemorrhages, was compared for virulence in calves with noncytopathic BVDV-TGAN, isolated from an apparently healthy persistently infected calf. After challenge exposure with BVDV-890, nonimmune calves (n = 7) developed fever > 40 C, diarrhea, leukopenia, lymphopenia, neutropenia, and thrombocytopenia. Most calves (n = 6) died or were euthanatized by 19 days after challenge exposure. Challenge exposure with BVDV-890 did not induce disease in 2 calves that had congenital persistent infection with BVDV or in 3 calves that had neutralizing antibody titer > 4 against BVDV-890. After challenge exposure with BVDV-TGAN, nonimmune calves (n = 7) developed fever > 40 C and, rarely, diarrhea or lymphopenia. All of those calves survived challenge exposure. The average maximal titer of BVDV-890 isolated from serum was 1,000 times that of BVDV-TGAN. In calves infected with BVDV-890, the average maximal percentages of lymphocytes and platelets associated with virus were greater than those found in calves infected with BVDV-TGAN. Additional findings of epidemiologic significance were prolonged shedding of virus and delayed production of viral-neutralizing antibody in 1 calf challenge-exposed with BVDV-890. Also, after production of neutralizing antibody, mutant virus that was refractory to neutralization was isolated from calves challenge-exposed with BVDV-TGAN.     

Antigenic variability in bovine viral diarrhea virus (BVDV) isolates from alpaca (Vicugna pacos), llama (Lama glama) and bovines in Chile

Llamas and alpacas are domesticated South American camelids (SACs) important to ancestral population in the Altiplano region, and to different communities where they have been introduced worldwide. These ungulates have shown to be susceptible to several livestock viral pathogens such as members of the Pestivirus genus and mainly to bovine viral diarrhea virus (BVDV). Seventeen Chilean BVDV isolates were analyzed by serum cross neutralization with samples obtained from five llama, six alpacas, three bovines, plus three reference strains belonging to different subgroups and genotypes. The objective was to describe antigenic differences and similarities among them. Antigenic comparison showed significant differences between different subgroups. Consequently, antigenic similarities were observed among isolates belonging to the same subgroup and also between isolates from different animal species belonging the same subgroup. Among the analyzed samples, one pair of 1b subgroup isolates showed significant antigenic differences. On the other hand, one pair of isolates from different subgroups (1b and 1j) shared antigenic similarities indicating antigenic relatedness. This study shows for the first time the presence of antigenic differences within BVDV 1b subgroup and antigenic similarities within 1j subgroup isolates, demonstrating that genetic differences within BVDV subgroups do not necessary corresponds to differences on antigenicity.     

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.     

Outbreak of acute bovine viral diarrhea in Brazilian beef cattle: clinicopathological findings and molecular characterization of a wild-type BVDV strain subtype 1b

When first described in 1946, bovine viral diarrhea (BVD) was characterized as an acute transmissible disease associated with severe leucopenia, high fever, depression, diarrhea, gastrointestinal erosions, and hemorrhages. Recently the severe acute form has been related only to some hypervirulent BVDV-2 strains. This article reports the detection of BVDV-1b associated with an acute and fatal outbreak of BVD in a Brazilian beef cattle herd. Depression, anorexia, watery diarrhea, sialorrhea, and weakness were observed in six steers. One of these animals was evaluated for laboratorial, clinical, and pathological alterations. Laboratory findings were non-specific; clinically, the animal was weak, with dehydration and erosive oral lesions. Pathological alterations were predominant at the tongue, esophagus, and rumen. A RT-PCR assay using primers to partially amplify the 5′ untranslated region (5′UTR) of the BVDV genome was performed and identified BVDV in all clinical samples analyzed. Phylogenetic analysis of BVDV derived from lymph node revealed that this strain was clustered within the BVDV subtype 1b. This differentiating was only possible to be performed by molecular characterization since both clinical presentation and pathologic findings were similar to BVDV-2 infection.     

Co-existence of genetically and antigenically diverse bovine viral diarrhoea viruses in an endemic situation

Bovine viral diarrhoea virus (BVDV) is an important cattle pathogen that causes acute or persistent infections. These are associated with immunotolerance to the viral strain persisting in animals that became infected early in their intrauterine development. To this date, the epidemiology of BVD in Switzerland runs virtually undisturbed by control measures such as restrictions on animal traffic or vaccination. Here, we analysed the viral genetics of 169 Swiss isolates and carried out crossed serum neutralisation tests to assess the antigenic spectrum of BVDV strains present in the cattle population. Besides confirming the presence of BVDV type 1 subgroups b, e, h and k, a single "orphan" BVDV-1 virus was detected that does not belong to any known BVDV-1 subgroup. No BVDV type 2 viruses were detected, suggesting that they are rare or not present in the cattle population. Antigenic comparison revealed significant differences between the different subgroups, with anti-1k immune serum having up to tenfold lower neutralising activity against 1b, 1e and 1h subgroup viruses, which however may still suffice to protect 1k-immune animals against superinfection by viruses of those other subgroups. Serum from routinely vaccinated animals revealed generally low titres but good cross-neutralisation. A geographic information system revealed that the viruses of the different subgroups are distributed in an apparently randomised fashion in the cattle population. This geographic distribution pattern may reflect peculiarities of the management practice in the Swiss cattle industry that, especially through annual transhumance of up to 25% of the entire population in the alpine region, tend to optimise the spread of BVDV.     

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。     

Evaluation of a modified live virus type-1a bovine viral diarrhea virus vaccine (Singer strain) against a type-2 (strain 890) challenge

Both type-1 and type-2 bovine viral diarrhea virus (BVDV) infections are responsible for major losses in the cattle industry. However, several commercial BVDV vaccines contain only a type-1 strain. A vaccine trial was conducted to evaluate the efficacy of BVDV type-1 (Singer strain; BVDV-1) vaccine for protecting calves challenged with virulent BVDV type-2 (890 strain; BVDV-2). Thirty-eight BVDV-negative calves were randomly allocated to four groups. One group was treated with a modified live virus (MLV) BVDV-1 vaccine by i.m. injection and another group was treated with the same vaccine by s.c. injection. Two groups served as nonvaccinated controls (one i.m. and one s.c.). Twenty-eight days following vaccination, the calves were challenged with BVDV-2 and monitored for 21 days. Clinical scores and body temperatures of vaccinated calves were significantly (P<.05) lower than for controls on several days, and peak differences occurred 8 days after challenge. The control calves had significantly (P<.05) lower leukocyte counts 3 through 8 days after challenge; leukocyte counts for vaccinated animals did not decline significantly from prechallenge levels. There were no differences in protection between the i.m. and s.c. routes of vaccination. The study demonstrated satisfactory cross protection of the BVDV-1 vaccine against BVDV-2 challenge.     

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.     

Multiple diagnostic tests to identify cattle with Bovine viral diarrhea virus and duration of positive test results in persistently infected cattle

Several tests for Bovine viral diarrhea virus (BVDV) were applied to samples collected monthly from December 20, 2005, through November 27, 2006 (day 0 to day 342) from 12 persistently infected (PI) cattle with BVDV subtypes found in US cattle: BVDV-1a, BVDV-1b, and BVDV-2a. The samples included clotted blood for serum, nasal swabs, and fresh and formalin-fixed ear notches. The tests were as follows: titration of infectious virus in serum and nasal swabs; antigen-capture (AC) enzyme-linked immunosorbent assay (ELISA), or ACE, on serum, nasal swabs, and fresh ear notches; gel-based polymerase chain reaction (PCR) testing of serum, nasal swabs, and fresh ear notches; immunohistochemical (IHC) testing of formalin-fixed ear notches; and serologic testing for BVDV antibodies in serum. Of the 12 animals starting the study, 3 died with mucosal disease. The ACE and IHC tests on ear notches had positive results throughout the study, as did the ACE and PCR tests on serum. There was detectable virus in nasal swabs from all the cattle throughout the study except for a few samples that were toxic to cell cultures. The serum had a virus titer ≥ log₁₀ 1.60 in all samples from all the cattle except for 3 collections from 1 animal. Although there were several equivocal results, the PCR test most often had positive results. The BVDV antibodies were due to vaccination or exposure to heterologous strains and did not appear to interfere with any BVDV test. These findings illustrate that PI cattle may be identified by several tests, but differentiation of PI cattle from cattle with acute BVDV infection requires additional testing, especially of blood samples and nasal swabs positive on initial testing. Also, calves PI with BVDV are continual shedders of infectious virus, as shown by the infectivity of nasal swabs over the 11-mo study.     

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.     

猪源牛病毒性腹泻病毒的流行初探

根据牛病毒性腹泻病毒(BVDV)代表株NADL的NS5B基因序列,设计合成2对引物进行套式PCR,检测从浙江、安徽、湖南、江西、广西、辽宁等地采集的43份猪病料中BVDV的流行情况,结果有7份病料可以扩增出360bp的特异性条带,阳性率为16.3%。为追踪BVDV在猪体中的流行来源,我们又对细胞培养用国产及进口犊牛血清进行了检测,结果也能扩增到目的条带。对这些病料和血清中扩增出的目的条带进行克隆、测序,并用DNASTAR软件分析,结果表明这些目的片段均为BVDV序列,这些不同BVDV序列可以分为两个亚群,ZJ133、HN386、LN247、NCS-J属于BVDV-Ⅰa亚型,ZJ114、AH138、JX60、GX96、NCS-G、DZ属于BVDV-Ⅰb亚型。这些毒株与BVDVI型间的同源性为80.3%-98.6%,与BVDV-Ⅱ型的同源性为72.2%-74.4%。本文研究结果说明我国猪群中BVDV感染情况已经比较严重,应该予以重视。     

Sequence-optimised E2 constructs from BVDV-1b and BVDV-2 for DNA immunisation in cattle

We report DNA immunisation experiments in cattle using plasmid constructs that encoded glycoprotein E2 from bovine viral diarrhoea virus (BVDV)-1 (E2.1) and BVDV-2 (E2.2). The coding sequences were optimised for efficient expression in mammalian cells. A modified leader peptide sequence from protein gD of BoHV1 was inserted upstream of the E2 coding sequences for efficient membrane export of the proteins. Recombinant E2 were efficiently expressed in COS7 cells and they presented the native viral epitopes as judged by differential recognition by antisera from cattle infected with BVDV-1 or BVDV-2. Inoculation of pooled plasmid DNA in young cattle elicited antibodies capable of neutralising viral strains representing the major circulating BVDV genotypes.     

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.

     

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.     

First report of BVDV circulation in sheep in Argentina

Pestiviruses are capable of infecting a wide range of animals within the order Artyodactila. Currently, the genus Pestivirus includes Bovine Viral Diarrhea Virus 1 (BVDV-1) and 2 (BVDV-2), Border Disease Virus (BDV), and Classical Swine Fever Virus (CSFV). BVDV-1, BVDV-2 and BDV are able to cross species barrier to infect a wide range of hosts, whereas CSFV is restricted to domestic pigs and wild boars. In Argentina, 70% of cattle are seropositive to BVDV. Although there were some serological studies in llamas, alpacas and buffaloes, no reports existed about the circulation of BVDV in sheep in Argentina. Based on these, 54 blood samples of healthy ovines were analysed by serology. The results showed that 46.3% of the analysed sheep were seropositive to BVDV-1, 13% to BVDV-2 and 20.4% for both BVDV-1 and BVDV-2. The molecular analysis confirmed the presence of BVDV-1a in some samples.     

牛病毒性腹泻病毒BVDV-JL分离株全基因序列测定与分析

为了解中国农业科学院特产研究所分子生物学重点实验室前期分离的牛病毒性腹泻病毒BVDV-JL毒株完整的基因序列信息,本试验对BVDV-JL F6代毒株进行了完整基因序列测定。利用反转录—聚合酶链式反应(RT-PCR)方法分段扩增了BVDV-JL分离毒株的7段cDNA片段,分别克隆于pMD18-T载体并进行测序。BVDV-JL株基因组序列全长为12276 bp,编码3901个氨基酸。基因组两端为非编码区5'UTR和3'UTR。基因比对及进化树分析结果表明BVDV-JL与BVDV CP7同源性最高,核苷酸同源性为93.2%,归类于BVDV-1b2基因亚型。BVDV-JL是第1个在中国报道的BVDV-1b2亚型毒株。了解BVDV-JL完整基因序列有利于中国BVDV流行病学调查。