An Importance of Long-Term Clinical Analysis to Accurately Diagnose Calves Persistently and Acutely Infected by Bovine Viral Diarrhea Virus 2.

Bovine viral diarrhea virus (BVDV) infection results in a wide variety of clinical manifestations and is a pathogen that is able to cause huge economic losses in the cattle industry worldwide. It is important to identify cattle that are persistently infected (PI) by BVDV within the herd as early as possible because PI animals are the main reservoir of the virus. In contrast, cattle who are acutely infected (AI) with BVDV show various clinical signs, but most cattle show either mild symptoms or are asymptomatic. In general, AI and PI animals can be distinguished by repeat testing within an interval of at least 21 days. However, we found a rare case of a BVDV2-infected AI animal with long-term viral presence, making it indistinguishable from PI through two tests within an interval of 21 days. As a result, we diagnosed one infected animal as AI after 35 days from the initial sample collection via multiple analyses. Our findings recommend performing an additional test using samples that have been collected after 14-21 days from the second sample collection in cases where it is difficult to accurately differentiate an AI diagnosis from a PI diagnosis after only two tests. Additionally, our analysis exhibits that monitoring the number of copies of viruses with similar genomes in the sera by means of quantitative real-time RT-PCR through several sample collections periods might be useful to distinguish AI from PI. Furthermore, our data suggest that the AI animals with a long-term viral presence who show test results similar to those of PI animals might be the result of a coincidental combination of various factors that are present in cattle fields. These findings provide useful information that can be used to improve the diagnosis of BVDV in the field.

Do modified live virus vaccines against bovine viral diarrhea induce fetal cross-protection against HoBi-like Pestivirus?

Bovine Pestivirus heterogeneity is a major challenge for vaccines against bovine viral diarrhea (BVD). In breeding herds, fetal protection is a high priority issue. To some degree, fetal infections in vaccinated heifers have been attributed to the antigenic diversity of bovine Pestiviruses. The purpose of this study was to assess fetal protection against a divergent bovine Pestivirus (Hobi-like Pestivirus, HoBiPeV) with a commercially available modified live vaccine (MLV) claiming fetal protection against BVDV 1 and BVDV 2 up to one year after the first inoculation. Five vaccinated and four unvaccinated heifers were challenged by intranasal inoculation with the HoBiPeV Italy-1/10-1 strain between 82 and 89 days after insemination, i.e. between 4 and 6 months after vaccination. At challenge, neutralizing antibody titers to HoBiPeV in vaccinated heifers were low or even undetectable. Of the four unvaccinated heifers, one control animal aborted (fetus not available) and the remaining three gave birth to HoBiPeV positive calves. Among the heifers of the vaccinated group, one aborted the fetus in the sixth month of pregnancy, which tested Pestivirus negative, while three others gave birth to healthy, HoBiPeV negative calves; the remaining heifer delivered one HoBiPeV positive calf. The results suggest that the BVDV vaccine might be able to elicit a partial fetal protection against HobiPeV, even in absence of a strong specific antibody response.

The identification of a B-cell epitope in bovine viral diarrhea virus (BVDV) core protein based on a mimotope obtained from a phage-displayed peptide library.

Bovine pestivirus A and B, previously known as bovine viral diarrhea virus (BVDV)-1 and 2, respectively, are important pathogens of cattle worldwide, which causes significant economic losses. B-cell epitopes in BVDV glycoprotein E2 and nonstructural protein NS2/3 have been extensively identified. In this study, we screened a 12-mer phage display peptide library using commercial goat anti-BVDV serum, and identified a mimotope "LTPHKHHKHLHA" referred to as P3. With sequence alignment, a putative B-cell epitope "77ESRKKLEKALLA88" termed as P3-BVDV1/2 residing in BVDV core protein was identified. The synthesized peptides of both P3 and P3-BVDV1/2 show strong reactivity with BVDV serum in immune blot assay. Immunization of mice with these individual peptides leads to the production of antibody that cannot neutralize virus infectivity. Thus for the first time we identified a B-cell epitope, "77ESRKKLEKALLA88", in BVDV core protein. Interestingly, the epitope was highly conserved in Pestivirus A, B, C, D, as well as emerging Pestivirus E and I, but highly variable in Pestiviruses H, G, F, and J, as well as unclassified Pestivirus originated from non-ruminant animals. Whether this putative B-cell epitope is implicated in pestivirus pathogenesis or evolution needs further investigations once large numbers of isolates are available in the future.

Detection and genotyping of bovine viral diarrhea virus found contaminating commercial veterinary vaccines, cell lines, and fetal bovine serum lots originating in Mexico.

In this communication, we report the presence of RNA of bovine viral diarrhea virus (BVDV) as a contaminant of different biological products used in Mexico for veterinary vaccine production. For this purpose, six batches of monovalent vaccines, eight cell line batches used for vaccine production, and 10 fetal bovine serum lots (FBS) commercially available in Mexico from different suppliers were tested by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was detected in 62.5% of the samples analyzed. Phylogenetic analysis revealed the presence of the subgenotypes BVDV-1a, 1b, and BVDV-2a in the tested samples. Collectively, these findings indicate that contamination by BVDV RNA occurs in commercial vaccines and reagents used in research and production of biological products. The ramifications of this contamination are discussed.

An outbreak of abortions, stillbirths and malformations in a Spanish sheep flock associated with a bovine viral diarrhoea virus 2-contaminated orf vaccine.

Bovine viral diarrhoea virus (BVDV) is a pestivirus that affects both cattle and sheep, causing an array of clinical signs, which include abortions and malformations in the offspring. Manufacturing of modified live virus (MLV) vaccines often includes the use of bovine-derived products, which implies a risk of contamination with viable BVDV. Recently, the circulation of a specific strain of BVDV 2b among Spanish sheep flocks, associated with outbreaks of abortions and malformations, and whose origin was not determined, has been observed. On February 2018, a MLV orf vaccine was applied to a 1,600 highly prolific sheep flock in the Northeast of Spain that included 550 pregnant ewes. In May 2018, during the lambing season, an unusual high rate (72.7%) of abortions, stillbirths, congenital malformations and neurological signs in the offspring was observed. It was estimated that about 1,000 lambs were lost. Three 1- to 3-day-old affected lambs and a sealed vial of the applied vaccine were studied. Lambs showed variable degrees of central nervous system malformations and presence of pestiviral antigen in the brain. Molecular studies demonstrated the presence of exactly the same BVDV 2b in the tissues of the three lambs and in the orf vaccine, thus pointing to a pestivirus contamination in the applied vaccine as the cause of the outbreak. Interestingly, sequencing at the 5'-untranslated region-(UTR) of the contaminating virus showed a complete match with the virus described in the previously reported outbreaks in Spain, thus indicating that the same contaminated vaccine could have also played a role in those cases. This communication provides a clear example of the effects of the application of this contaminated product in a sheep flock. The information presented here can be of interest in putative future cases of suspected circulation of this or other BVDV strains in ruminants.

Interferon lambda protects cattle against bovine viral diarrhea virus infection.

Interferon lambda (IFN-λ) plays an important role in inducing an antiviral state in mucosal surfaces and has been used as an effective biotherapeutic against several viral diseases. Here we performed a proof of concept study on the activity of a biologically active recombinant bovine IFN-λ (rIFN-λ) produced in eukaryotic cells against Bovine Viral Diarrhea Virus (BVDV) in cattle. We first confirmed the lack of toxicity of different concentrations of rIFN-λ in bovine peripheral blood cells and the safety of its subcutaneous application in calves in doses up to 12 IU/kg. The antiviral activity of the rIFN-λ against BVDV was assessed in calves that were inoculated with 6 IU/kg of rIFN-λ (n = 4) or mock-treated (n = 2) two days before and after challenge with a BVDV type-2 non-cytopathic strain. Mock-treated animals developed respiratory disease, shedded the virus from 4 to 7 days post-infection (dpi) and had viremia between 4 and 14 dpi. Conversely, calves treated with rIFN-λ did not develop clinical symptoms. The virus was not found in nasal secretions or sera. Only one animal had a positive viral RNA detection in serum at 7 dpi. All infected animals treated with rIFN-λ increased systemic type-I IFNs levels at 4 dpi. The antiviral treatment induced an earlier onset of the anti-BVDV neutralizing antibodies. Altogether, these results constitute the proof-of-principle of bovine IFN-λ as an antiviral biotherapeutic to protect cattle against the clinical disease caused by BVDV.

Bovine Pestivirus Heterogeneity and Its Potential Impact on Vaccination and Diagnosis.

Bovine Pestiviruses A and B, formerly known as bovine viral diarrhoea viruses (BVDV)-1 and 2, respectively, are important pathogens of cattle worldwide, responsible for significant economic losses. Bovine viral diarrhoea control programmes are in effect in several high-income countries but less so in low- and middle-income countries where bovine pestiviruses are not considered in disease control programmes. However, bovine pestiviruses are genetically and antigenically diverse, which affects the efficiency of the control programmes. The emergence of atypical ruminant pestiviruses (Pestivirus H or BVDV-3) from various parts of the world and the detection of Pestivirus D (border disease virus) in cattle highlights the challenge that pestiviruses continue to pose to control measures including the development of vaccines with improved cross-protective potential and enhanced diagnostics. This review examines the effect of bovine pestivirus diversity and emergence of atypical pestiviruses in disease control by vaccination and diagnosis.

An Assessment of Secondary Clinical Disease, Milk Production and Quality, and the Impact on Reproduction in Holstein Heifers and Cows from a Single Large Commercial Herd Persistently Infected with Bovine Viral Diarrhea Virus Type 2.

The aim of this study was to evaluate secondary clinical disease, milk production efficiency and reproductive performance of heifers and cows persistently infected (PI) with bovine viral diarrhea virus type 2 (BVDV type 2). PI animals (n = 25) were identified using an antigen capture ELISA of ear notch samples. They were distributed into three age groups: ≤ 12 (n = 8), 13 to 24 (n = 6) and 25 to 34 (n = 11) months old. A control group of BVDV antigen ELISA negative female cattle that were age matched to the PI animals was utilized from the same herd. The PI group had a 1.29 higher odds ratio for diarrhea than controls (p = 0.001, IC95% = 1.032-1.623) and 1.615 greater chance of developing bovine respiratory disease (BRD) (p = 0.012, IC95% = 1.155-2.259). The age at first insemination (p = 0.012) and number of insemination attempts required to establish the first pregnancy (p = 0.016) were both higher for PI than controls. Milk production was higher for control cows than PI cows during most of the sampling periods. Somatic cell counts (SCC) were higher in PI cows than the controls at all sampling points across lactation (p ≤ 0.042). PI cattle had a higher incidence of disease, produced less milk, a higher SCC, and poorer reproductive performance than control cattle in this study.

Evaluation of protection against bovine viral diarrhea virus type 2 after vaccination of the calves with bovine viral diarrhea virus type 1 combo inactivated vaccine / Avaliação da proteção contra o vírus da diarreia viral bovina tipo 2 após vacinação de bezerros com vacina combinada inativada do vírus da diarreia viral bovina tipo 1

This study was designed to evaluate the extent of the protection for bovine viral diarrhea virus type 2 (BVDV-2) infection, afforded by vaccination with a combo inactivated vaccine, which contains bovine viral diarrhea virus type 1 (BVDV-1) and infectious bovine rhinotracheitis virus (IBRV). Five 3-4-month-old calves were intramuscularly vaccinated with a single dose of the combo vaccine and boosted with same dose three weeks after the first vaccination, with five mock immunized calves serving as a control group. Twenty-one days after the second vaccination, all calves were challenged with BVDV-2 SX08 strain by spray into nostril. The unvaccinated animals developed typical clinical signs of high rectal temperature, diarrhoea with erosions and a dramatic drop in leukocyte counts. These signs occured markedly less in all vaccinated animals, the rectal temperature, leukopenia and virarmia of which, were significantly less than the mock immunized calves. It can be concluded that vaccination with the combo inactivated vaccine affords cross-protection against clinical effects of a challenge-infection with BVDV-2 SX08 strain, although it was part protection.(AU)

Este estudo foi desenvolvido para avaliar a extensão da proteção contra a infecção pelo vírus da diarréia viral bovina tipo 2 (BVDV-2) através da vacinação com uma vacina combinada inativada contendo o vírus da diarréia viral bovina tipo 1 (BVDV-1) e vírus da rinotraqueíte de bovinos infecciosos (IBRV). Cinco bezerros com 3 a 4 meses de idade foram vacinados via intramuscular com uma dose única da vacina combinada e reforçados com a mesma dose três semanas após a primeira vacinação, com cinco bezerros imunizados em simulação servindo como grupo controle. Vinte e um dias após a segunda vacinação, todos os bezerros foram desafiados com a cepa BVDV-2 SX08 por spray na narina. Os animais não vacinados desenvolveram sinais clínicos típicos, como alta temperatura retal, diarréia com erosões e queda drástica na contagem de leucócitos. Estes sinais tiveram ocorrência significativamente menor em todos os animais vacinados, cuja temperatura retal, leucopenia e virarmia eram significativamente menores do que os bezerros simulados. É possível concluir que a vacinação com a vacina combinada inativada proporciona proteção cruzada contra os efeitos clínicos de uma infecção provocada pela cepa BVDV-2 SX08, embora tenha sido parcialmente protegida.(AU)

Diseases associated with bovine viral diarrhea virus subtypes 1a and 2b in beef and dairy cattle in Uruguay.

Bovine viral diarrhea virus (BVDV, Pestivirus) causes significant economic losses to the livestock industry worldwide. Although serological surveys show that BVDV exposure is widespread in cattle in Uruguay, BVDV-associated diseases are greatly underreported. The aim of this work is to describe the epidemiological, clinical, pathological, and virological findings from spontaneous outbreaks of BVDV-associated diseases in cattle in Uruguay. Diagnostic investigations were performed during 6 spontaneous disease outbreaks on beef and dairy cattle farms in the departments of Colonia, Rio Negro, and Soriano between November 2016 and April 2018. Carcasses of 8 naturally deceased cattle from these outbreaks were necropsied and subjected to histological examination and immunohistochemistry to detect BVDV antigen in the tissues. Reverse transcription real-time PCR and genomic sequencing were also performed to identify BVDV at the species and subtype levels. Other ancillary diagnostic tests, including bacterial cultures, were performed on a case-by-case basis to rule in/out differential diagnoses based on initial clinicopathological presumptive diagnoses. BVDV-associated conditions that were diagnosed in the 8 cases included mucosal disease, transient postnatal BVDV infections associated with digestive/septicemic salmonellosis by Salmonella serovar typhimurium, Histophilus somni bronchopneumonia, urinary tract coinfections with Escherichia coli and Streptococcus sp., enteric coinfection with coccidia, and transplacental fetal infections and abortions with Neospora caninum coinfection. BVDV-1a and BVDV-2b were each identified in four of the eight cases. We conclude that BVDV-1a and BVDV-2b contribute significantly to disease and mortality in cattle in Uruguay. Future research should estimate the economic impact of BVDV in the Uruguayan livestock sector.

An immunomodulatory feed additive enhances in vitro viral vaccine recall antigen responses in dairy heifers.

Enhancing immunological responses to vaccination is an important goal in many herd health management systems. OmniGen-AF®(OG) is an immunomodulatory feed additive that has been shown to enhance innate immune function in ruminants and its effects on adaptive immunity require additional study. The objective of this study was to evaluate post-vaccine antibody titers and circulating cellular memory development in heifers fed OG and administered a commercially available modified-live bovine respiratory disease (BRD) vaccine. Twenty-four Holstein heifers were assigned to one of two diets for 170 days: Control TMR (CON; n = 11), or TMR plus OG (TRT; 9 g/100 kg BW/day; n = 13). Samples for hematology, serology, and cellular assays were collected on D-110, 0, 21, 42, and 60 of the trial. Heifers were administered two priming doses of a modified-live BRD vaccine, with a third dose given on D0. There were no significant differences in total WBC and absolute number or the percentage of circulating lymphocytes, monocytes, neutrophils, RBC, or platelets on D-110 through D21. On D42 and D60, CON had significantly higher numbers of lymphocytes. On D0, mean serum neutralizing (SN) titer to BHV-1 was significantly higher for CON compared to TRT. SN titers were not significantly different between CON and TRT at any other time point for BHV-1, BVDV type 1, or BVDV type 2. TRT mounted a significantly stronger recall proliferative response to 0.5 multiplicity of infection (MOI) of BHV-1, BVDV type 1 and BVDV type 2 on D42 and D60; 0.25 MOI of BVDV type 1 on D21 and D42; and 0.25 MOI BVDV type 2 on D42 compared to CON. IL-4 production induced by 0.5 and 1.0 MOI BHV-1 (D42 and D60); 0.25 MOI of BVDV type 1 (D21); and 0.25 and 0.5 MOI of BVDV type 2 (D60) were significantly higher for TRT than CON. IL-17 production induced by 0.25 MOI of BVDV type 1 was significantly higher on D60 for TRT compared to CON. IFN-gamma and IL-10 were not significantly different between treatments. These data indicate feeding OG has a beneficial effect on responses to vaccine antigens in Holstein dairy heifers.

Transcriptome analysis reveals differential immune related genes expression in bovine viral diarrhea virus-2 infected goat peripheral blood mononuclear cells (PBMCs).

BACKGROUND: Bovine viral diarrhea virus (BVDV) is an economically important viral pathogen of domestic and wild ruminants. Apart from cattle, small ruminants (goats and sheep) are also the susceptible hosts for BVDV. BVDV infection could interfere both of the innate and adaptive immunity of the host, while the genes and mechanisms responsible for these effects have not yet been fully understood. Peripheral blood mononuclear cells (PBMCs) play a pivotal role in the immune responses to viral infection, and these cells were the target of BVDV infection. In the present study, the transcriptome of goat peripheral blood mononuclear cells (PBMCs) infected with BVDV-2 was explored by using RNA-Seq technology. RESULTS: Goat PBMCs were successfully infected by BVDV-2, as determined by RT-PCR and quantitative real-time RT-PCR (qRT-PCR). RNA-Seq analysis results at 12 h post-infection (hpi) revealed 499 differentially expressed genes (DEGs, fold-change ≥ ± 2, p < 0.05) between infected and mock-infected PBMCs. Of these genes, 97 were up-regulated and the remaining 352 genes were down-regulated. The identified DEGs were found to be significantly enriched for locomotion/ localization, immune response, inflammatory response, defense response, regulation of cytokine production, etc., under GO enrichment analysis. Cytokine-cytokine receptor interaction, TNF signaling pathway, chemokine signaling pathway, etc., were found to be significantly enriched in KEGG pathway database. Protein-protein interaction (PPI) network analysis indicated most of the DEGs related to innate or adaptive immune responses, inflammatory response, and cytokine/chemokine-mediated signaling pathway. TNF, IL-6, IL-10, IL-12B, GM-CSF, ICAM1, EDN1, CCL5, CCL20, CXCL10, CCL2, MAPK11, MAPK13, CSF1R and LRRK1 were located in the core of the network and highly connected with other DGEs. CONCLUSIONS: BVDV-2 infection of goat PBMCs causes the transcription changes of a series of DEGs related to host immune responses, including inflammation, defense response, cell locomotion, cytokine/chemokine-mediated signaling, etc. The results will be useful for exploring and further understanding the host responses to BVDV-2 infection in goats.

Experimental inoculation of gilts with bovine viral diarrhea virus 2 (BVDV-2) does not induce transplacental infection.

Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus and can cause reproductive problems in cattle. However, there is still a lack of research to clarify its pathogenicity in different gestational periods of sows and its effects in neonates. In this study, 12 gilts divided into groups (G) were experimentally inoculated with the strain BVDV-2 (SV-253) oronasally at a dose of 106·85 TCID50; one group was inoculated 30 days before insemination (G0; n = 2), three groups were inoculated during gestation (first (G1; n = 2), second (G2; n = 3), third (G3; n = 3)), and a fourth was the control group (G4; n = 2). Samples of blood and nasal swabs from the gilts were collected every three days until delivery for a virus neutralization (VN) test, qRT-PCR, and blood count. On the day of delivery, 40% of the neonates were euthanized to obtain tissue and blood samples at necropsy for histopathology and qRT-PCR. The sows were seroconverted between 12 and 33 days after inoculation, and the virus was detected in the blood between 3 and 12 days and on the nasal swab between 6 and 24 days in the G0, G1, G2 and G3 sows but was not detected in piglet tissues, and no significant alterations were found through histopathology. The mean and standard deviation of the mean cycles (Cq) from blood (Cq = 34.87 ± 0.60) and nasal swab (Cq = 34.61 ± 0.87) samples were between 107 and 490 TCID50/ml. Transient infection was demonstrated with a low viral load, but transplacental infection was not possible in gilts.

Recombinant E2 protein enhances protective efficacy of inactivated bovine viral diarrhea virus 2 vaccine in a goat model.

BACKGROUND: Inactivated and subunit bovine viral diarrhea virus (BVDV) vaccines have shown limited protective efficacy. This study aimed to evaluate the effectiveness of a vaccine containing both inactivated BVDV (iBVDV) and baculovirus-expressed recombinant E2 (rE2), an important BVDV antigen with strongly neutralizing epitopes. RESULTS: Four groups of goats were immunized twice with one of four vaccine preparations: 1) iBVDV+rE2, 2) rE2, 3) iBVDV, and 4) saline, and challenged with BVDV. For goats vaccinated with the iBVDV+rE2 vaccine, no viremia was observed after challenge, and clinical signs, pyrexia, and leukopenia were reduced compared to the saline group. In contrast, for goats vaccinated with either iBVDV or rE2 alone, viremia was still detectable. CONCLUSION: The combination of iBVDV and rE2 elicited stronger protective immune responses against BVDV than iBVDV or rE2 alone.

The Occurrence of a Commercial Npro and Erns Double Mutant BVDV-1 Live-Vaccine Strain in Newborn Calves.

The major source for the spread of bovine viral diarrhea virus (BVDV) are in-utero infected, immunotolerant, persistently infected (PI) animals since they shed enormous amounts of viruses throughout their lives. During the sequence-based virus typing of diagnostic ear notch samples performed in the context of the obligatory German BVDV eradication program, the commercial Npro and Erns double mutant BVDV-1 live-vaccine strain KE-9 was detected in seven newborn calves; their mothers were immunized in the first trimester of gestation. Six calves either succumbed or were culled immediately, but the one remaining animal was closely monitored for six months. The viral RNA was detected in the skin sample taken in its first and fifth week of life, but the virus could not be isolated. Further skin biopsies that were taken at monthly intervals as well as every serum and urine sample, nasal, oral, and rectal swabs taken weekly tested BVDV negative. However, neutralizing titers against BVDV-1 remained at a consistently high level. To further control for virus shedding, a BVDV antibody and antigen negative calf was co-housed which remained negative throughout the study. The missing viremia, a lack of excretion of infectious virus and negative follow-up skin samples combined with consistently high antibody titers speak against the induction of the classical persistent infection by vaccination with recombinant KE-9 during gestation. We, therefore, suggest that the epidemiological impact of the RNA/antigen positivity for an extended period in the skin is very low. The detection of live-vaccine viruses in skin biopsies mainly represents a diagnostic issue in countries that implemented ear notch-based control programs; and KE9-specific RT-PCRs or sequence analysis can be used to identify these animals and avoid culling measures.

Serological survey for antibodies against pestiviruses in Wyoming domestic sheep.

Pestiviruses including Bovine viral diarrhea virus type 1 (BVDV-1), BVDV-2 and Border disease virus (BDV) have been reported in both sheep and cattle populations, together with the HoBi-like, an emerging group of pestiviruses. Pestivirus control programs in the United States have focused on the control of BVDV-1 and 2. The incidence of pestivirus infection in sheep in the United States and the risk of transmission between cattle and sheep populations are unknown. The aim of this study was to perform serological surveillance for pestivirus exposure in sheep from an important sheep producing state in the Unites States, Wyoming. For this, sera from 500 sheep, collected across the state of Wyoming (US) in 2015-2016, were examined by comparative virus neutralization assay against four species/proposed species of pestiviruses: BVDV-1, BVDV-2, BDV and HoBi-like virus. Rates of exposure varied between geographic regions within the state. The overall pestivirus prevalence of antibodies was 5.6%. Antibodies were most frequently detected against BVDV-1 (4%), and the highest antibody titers were also against BVDV-1. Data from this study highlights understanding of the dynamics of sheep pestivirus exposure, consideration of reference strains used for VN assays, transmission patterns, and potential vaccination history should be taken into account in implementation of control measures against pestiviruses in sheep and for successful BVDV control programs in cattle.

Evaluation of bovine viral diarrhea virus transmission potential to naïve calves by direct and indirect exposure routes.

Bovine viral diarrhea viruses (BVDV) can cause both acute and persistent infections in cattle. Exposure to BVDV persistently infected (PI) animals results in transmission of the virus to a naïve animal which causes a transient acute infection. While it is known that direct exposure to PI animals is a highly efficient means of transmission, less information is available regarding the potential for transmission from acutely infected either by direct or indirect exposure to naïve animals. Therefore, the objective of this study was to evaluate the potential for spread of the virus from calves acutely infected, with typical virulence field viruses know to have minimal shedding and viremia, to naïve contact animals either by direct or indirect exposure. To accomplish this objective, two BVDV isolates belonging to two species of BVDV, type 1 and type 2, were used to inoculate calves. Subsequently on day 2 post-infection, naïve calves were exposed to inoculated calves, either directly or indirectly, over a period of two weeks. All calves were evaluated for the presence of virus in blood samples and nasal swabs, pyrexia, lymphopenia and seroconversion. BVDV was isolated from inoculated calves but not from any of the direct and indirect contact animals or from control calves. Similarly, pyrexia and lymphopenia were observed in the inoculated calves, but not in contact and control calves. Only the inoculated calves seroconverted by day 38 of the study indicating that no transmission had occurred to the naïve contact calves. This data would suggest that there may be an infectious dose needed for transmission of virus for typical virulent isolates.

Comparison of reproductive protection against bovine viral diarrhea virus provided by multivalent viral vaccines containing inactivated fractions of bovine viral diarrhea virus 1 and 2.

Bovine viral diarrhea virus (BVDV) is an important viral cause of reproductive disease, immune suppression and clinical disease in cattle. The objective of this study was to compare reproductive protection in cattle against the impacts of bovine viral diarrhea virus (BVDV) provided by three different multivalent vaccines containing inactivated BVDV. BVDV negative beef heifers and cows (n = 122) were randomly assigned to one of four groups. Groups A-C (n = 34/group) received two pre-breeding doses of one of three commercially available multivalent vaccines containing inactivated fractions of BVDV 1 and BVDV 2, and Group D (n = 20) served as negative control and received two doses of saline prior to breeding. Animals were bred, and following pregnancy diagnosis, 110 cattle [Group A (n = 31); Group B (n = 32); Group C (n = 31); Group D (n = 16)] were subjected to a 28-day exposure to cattle persistently infected (PI) with BVDV (1a, 1b and 2a). Of the 110 pregnancies, 6 pregnancies resulted in fetal resorption with no material for testing. From the resultant 104 pregnancies, BVDV transplacental infections were demonstrated in 73 pregnancies. The BVDV fetal infection rate (FI) was calculated at 13/30 (43%) for Group A cows, 27/29 (93%) for Group B cows, 18/30 (60%) for Group C cows, and 15/15 (100%) for Group D cows. Statistical differences were observed between groups with respect to post-vaccination antibody titers, presence and duration of viremia in pregnant cattle, and fetal infection rates in offspring from BVDV-exposed cows. Group A vaccination resulted in significant protection against BVDV infection as compared to all other groups based upon outcome measurements, while Group B vaccination did not differ in protection against BVDV infection from control Group D. Ability of inactivated BVDV vaccines to provide protection against BVDV fetal infection varies significantly among commercially available products; however, in this challenge model, the inactivated vaccines provided unacceptable levels of BVDV FI protection.

Npro His49 and Erns Lys412 mutations in pig bovine viral diarrhea virus type 2 synergistically enhance the cellular antiviral response.

Type I interferons are major components of the innate immune response of hosts, and accordingly, many viruses have evolved mechanisms to modulate the host response during infection. Bovine viral diarrhea virus (BVDV) nonstructural protein Npro and structural protein Erns play important roles in inhibiting type I interferon. The aim of this study was to explore the epistatic effects of amino acid mutations in Npro and Erns in porcine ST cells to characterize the immune response induced by BVDV-2. Plasmids with mutant amino acids His49 (H49), Glu22 (E22) in Npro, and His300 (H300), Lys412 (K412) in Erns which had been changed to Alanine (A) had similar effects on type I interferon production in MDBK and ST cells, but resulted in much greater ISG15, OAS, and Mx production in ST cells. The rescued vASH/NproH49ErnsK412 virus showed the best efficiency with respect to modulating antiviral cytokines, indicating that the amino acids Npro H49 and Erns K412 had highly synergistic effects in abolishing the ability to inhibit type I interferon. These findings have importance practical implications owing to the increasing prevalence of BVDV infections, including persistent infections, in domestic pigs.

Antigenic diversity of Brazilian isolates of HoBi-like pestiviruses.

Hobi-like viruses comprise an unclassified group of bovine pestiviruses related to bovine viral diarrhea virus 1 (BVDV-1) and 2 (BVDV-2). These viruses were originally identified in fetal bovine serum from Brazilian origin and, subsequently, isolated from diseased animals in several countries. Herein we performed an antigenic characterization of eight Brazilian HoBi-like viruses isolated from persistently infected (PI) animals and from gastroenteric disease (2007-2015). Phylogenetic analysis based on the 5' unstranslated region (UTR) clustered these viruses with other HoBi-like viruses from European and Asiatic origin. Monoclonal antibody (MAb) binding indicated variability in the Hobi-like virus glycoprotein E2 and significant differences from the homologous BVDV-1 and BVDV-2 glycoprotein. Analysis of antigenic relatedness based on virus-neutralizing titers using virus-specific antisera revealed that HoBi-like viruses are antigenically very different from BVDV-1 and, to a lesser extent, from BVDV-2. Cross-neutralizing assays between pairs of HoBi-like viruses and their respective antisera indicated the existence of antigenic variability among these viruses, even for viruses isolated from the same herd in different occasions. Moreover, the identification of a HoBi-like isolate with low antigenic similarity with the other isolates indicates the potential existence of antigenic subgroups among HoBi-like virus isolates. Finally, sera of lambs immunized with commercial BVDV vaccines showed low or undetectable neutralizing activity against HoBi-like isolates. These results indicate significant antigenic differences between BVDV genotypes and Brazilian HoBi-like viruses and the existence of antigenic variability within this atypical group of pestiviruses. These findings extend the knowledge about the antigenic diversity of HoBi-like viruses and reinforce the need for their inclusion in current BVDV vaccines.