Epigenetic Modifications of White Blood Cell DNA Caused by Transient Fetal Infection with Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) infections cause USD 1.5-2 billion in losses annually. Maternal BVDV after 150 days of gestation causes transient fetal infection (TI) in which the fetal immune response clears the virus. The impact of fetal TI BVDV infections on postnatal growth and white blood cell (WBC) methylome as an index of epigenetic modifications was examined by inoculating pregnant heifers with noncytopathic type 2 BVDV or media (sham-inoculated controls) on Day 175 of gestation to generate TI (n = 11) and control heifer calves (n = 12). Fetal infection in TI calves was confirmed by virus-neutralizing antibody titers at birth and control calves were seronegative. Both control and TI calves were negative for BVDV RNA in WBCs by RT-PCR. The mean weight of the TI calves was less than that of the controls (p < 0.05). DNA methyl seq analysis of WBC DNA demonstrated 2349 differentially methylated cytosines (p ≤ 0.05) including 1277 hypomethylated cytosines, 1072 hypermethylated cytosines, 84 differentially methylated regions based on CpGs in promoters, and 89 DMRs in islands of TI WBC DNA compared to controls. Fetal BVDV infection during late gestation resulted in epigenomic modifications predicted to affect fetal development and immune pathways, suggesting potential consequences for postnatal growth and health of TI cattle.

Uncommon bovine viral diarrhea virus subtype 1e associated with abortions in cattle in southern Brazil.

We characterized bovine viral diarrhea virus (BVDV)-related abortions in cattle and identified the species and subgenotypes in the state of Santa Catarina, southern Brazil. Our RT-PCR assay was positive for BVDV in 5 fetuses from different farms; however, 3 of the 5 fetuses were also PCR-positive for Neospora caninum. In the 5 BVDV-positive fetuses, gross lesions included fetal mummification (1), hepatomegaly (1), subcutaneous edema (1), and perirenal edema (1). Predominant histologic lesions included epicarditis and mild-to-moderate lymphoplasmacytic myocarditis (5), mild multifocal lymphoplasmacytic interlobular pneumonia (4), nephrosis associated with moderate multifocal interstitial nephritis (1), moderate multifocal lymphoplasmacytic necrotic hepatitis (1), and mild multifocal lymphoplasmacytic meningitis (1). The amplification products from the Pestivirus 5'UTR region of 4 of the 5 fetuses had 96.3-100% similarity between fetal strains and reference strains. The samples were distributed into 2 branches of the phylogenetic tree; strains UDESC:01, UDESC:02, and UDESC:05 clustered in the BVDV-1e branch, uncommon in the Americas, and strain UDESC:04 clustered in the BVDV-2b branch. The three 1e strains had 96.9-97.4% similarity.

Immune Responses to Influenza D Virus in Calves Previously Infected with Bovine Viral Diarrhea Virus 2.

Bovine viral diarrhea virus (BVDV) induces immunosuppression and thymus depletion in calves. This study explores the impact of prior BVDV-2 exposure on the subsequent immune response to influenza D virus (IDV). Twenty 3-week-old calves were divided into four groups. Calves in G1 and G3 were mock-treated on day 0, while calves in G2 and G4 received BVDV. Calves in G1 (mock) and G2 (BVDV) were necropsied on day 13 post-infection. IDV was inoculated on day 21 in G3 calves (mock + IDV) and G4 (BVDV + IDV) and necropsy was conducted on day 42. Pre-exposed BVDV calves exhibited prolonged and increased IDV shedding in nasal secretions. An approximate 50% reduction in the thymus was observed in acutely infected BVDV calves (G2) compared to controls (G1). On day 42, thymus depletion was observed in two calves in G4, while three had normal weight. BVDV-2-exposed calves had impaired CD8 T cell proliferation after IDV recall stimulation, and the α/ß T cell impairment was particularly evident in those with persistent thymic atrophy. Conversely, no difference in antibody levels against IDV was noted. BVDV-induced thymus depletion varied from transient to persistent. Persistent thymus atrophy was correlated with weaker T cell proliferation, suggesting correlation between persistent thymus atrophy and impaired T cell immune response to subsequent infections.

Molecular detection and characterization of bovine viral diarrhea virus type 2 and bluetongue virus 9 in forest flies (Hippobosca equina) collected from livestock in southern Kazakhstan.

Keds are hematophagous ectoparasites of animals belonging to the family Hippoboscidae (Diptera: Hippoboscoidea). Because of their importance as vectors of some pathogens of medical and veterinary importance, they have received special attention. There are numerous studies demonstrating the presence of various parasites and pathogenic bacteria in keds. At the same time, there are very few reports on ked-related viruses. The aim of this study was to perform a molecular survey of viral pathogens in the forest fly (Hippobosca equina) from southern Kazakhstan. In this study, 104H. equina were collected from livestock in Turkistan oblast (southern region of Kazakhstan), which has the largest concentration of livestock in the country. Insect homogenates were screened by PCR for pestiviruses, orbiviruses, flaviviruses, orthobunyaviruses, phleboviruses, orthopoxviruses, capripoxviruses, parapoxviruses, and asfiviruses. The causative agents of two livestock diseases, bovine viral diarrhea virus (BVDV) (3/104; 2.88%; 95% confidence interval (CI): 0.6-8.2%) and bluetongue virus (BTV) (1/104; 0.96%; 95% CI: 0.02-5.24%), were identified and subjected to further analysis. The BTV strain was isolated and all ten genomic RNA segments were sequenced using the Sanger technique. The isolated BTV strain showed >99.6% identity in all genomic segments with the BTV-9 strains belonging to the 'western' topotype. Partial analysis of the 5'-untranslated region demonstrated that both BVDV strains are closely related to Pestivirus B. Flaviviruses, phleboviruses, orthobunyaviruses, poxviruses, and asfiviruses were not detected. This is the first report describing BVDV type 2 in Kazakhstan. The study also confirms the presence of BTV serotype 9 in southern Kazakhstan. The data presented here can help improve preventive measures to control the spread of viral diseases in livestock by using forest flies as an object of epidemiological studies. However, further studies are needed to investigate the vector capacity of H. equina and its suitability for xenodiagnosis of veterinary relevant pathogens.

Novel genomic targets for proper subtyping of bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2.

Whole-genome phylogenetic analysis, the most suitable strategy for subtyping bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2, is not feasible for many laboratories. Consequently, BVDV isolates/strains have been frequently subtyped based on analysis of single genomic regions, mainly the 5' untranslated region (UTR). This approach, however, may lead to inaccurate and/or poorly statistically supported viral classification. Herein, we describe novel primer sets whose amplicons may be easily sequenced and used for BVDV subtyping. Initially, genomic regions previously described as the most suitable targets for BVDV subtyping were analyzed for design of high-coverage primers. The putative amplicons were analyzed in silico for their suitability to reproduce the phylogenetic classification of 118 BVDV-1 and 88 BVDV-2 complete/near-complete genomes (CNCGs) (GenBank). This analysis was also performed considering the region amplifiable by primers HCV90-368, 324-326 and BP189-389 (5'UTR), which have been used for BVDV diagnosis and/or classification. After confirming the agreement between the analyses of our primers' amplicon versus the CNCGs, we optimized the RT-PCRs and evaluated their performance for amplification of BVDV isolates/strains (n = 35 for BVDV-1; n = 33 for BVDV-2). Among the potential targets for BVDV subtyping, we designed high-coverage primers for NS3-NS4A (BVDV-1) (526 bp amplicon) and NS5B (BVDV-2) (728 bp). The classification based on these regions fully reproduced the subtyping of all CNCGs. On the other hand, subtyping based on the putative amplicons from primers HCV90-368, 324-326 and BP189-389 showed disagreements in relation the CNCG analysis. The NS3-NS4A and NS5B primers also allowed the amplification of all BVDV isolates/strains tested. Finally, we suggest the use of these primers in future phylogenetic and epidemiological studies of BVDVs.

Molecular and serological survey of bovine viral diarrhea virus infection in cattle in Kazakhstan.

The aim of this study was to estimate the occurrence of bovine viral diarrhea virus (BVDV) infection and to assess the population immunity in cattle vaccinated against BVDV in different regions of Kazakhstan. Cattle samples were collected in 12 oblasts (43 districts) of Kazakhstan. A total of 2477 cattle from 114 herds and 21 Bukhara deer (Cervus elaphus bactrianus) were examined by ELISA and conventional RT-PCR. Univariate and multivariate logistic regression analysis was performed to identify risk factors associated with BVDV infection in the country. In total, antibodies against BVDV were found in 79.3% (1965/2477) of all the animals and 92.1% (105/114) of all the herds examined. Seroprevalence in unvaccinated and vaccinated animals was 48.6% (447/920) and 98.7% (1391/1410), respectively. Seroprevalence in deer was 19.1% (4/21). The BVDV RNA was detected in six unvaccinated cattle (0.2%). Sequence analysis of the 5'-untranslated region demonstrated that four of the detected strains belonged to BVDV-1 and two strains to BVDV-2. Regression analysis revealed that age, production type, housing method, farm size, and geographic location were risk factors for BVDV infection in cattle in Kazakhstan. The present data confirm circulation of BVDV-1 and BVDV-2 in Kazakhstan and highlight the need to improve strategies for prevention and control of BVDV infection in the country.

Evaluation of the stress-reducing effect of trace mineral injection in beef calves.

BACKGROUND: Little is known about the effects of trace mineral supplementation on the stress response in beef calves. OBJECTIVES: To investigate the effect of injectable trace mineral supplementation (ITM) on the stress response in beef calves exposed to different types of stress. ANIMALS: Thirty weaned Angus and Angus crossbred calves. METHODS: The enrolled calves were randomly assigned to 2 groups: ITM, 15 calves received modified-live virus vaccine (MLV) and ITM SC and 15 calves received MLV and saline SC (CONT). The calves were exposed to 3 types of stress: the stress of MLV vaccination (d0), nasal aerosol with bovine viral diarrhea virus-2 (BVDV-2) challenge (d5), and liver biopsy (d26). The calves' body weights and health status were monitored. Leukocyte counts, serum cortisol concentration ([cort]), BVDV-2 serum neutralizing antibodies (SNA), and percentages of CD4+ , CD8+ , WC1+ , and CD25+ T-lymphocytes were measured. RESULTS: Serum cortisol concentration ([cort]) showed strong associations with the percentage of CD8+ (rs = .50), BVDV2-SNA (rs = -.43), and WC1CD25+ (rs = .41) cells, and rectal temperature (rs = .40). The highest [cort] was reported 3 days after aerosol BVDV-2 challenge. Serum [cort] was decreased in ITM-treated calves 3 days post-BVDV-2 challenge, compared with CONT calves, with an average decrease of 18.5 ng/µL (95% confidence interval [CI], -6.07 to -31.3). The ITM-treated calves were heavier and healthier (P < .01) than the CONT calves. CONCLUSIONS AND CLINICAL IMPORTANCE: Trace mineral supplementation appears to have stress mitigation effects in beef cattle that may reflect positively on growth and health performance. Viral exposure is associated with a high degree of stress, which is considered a major welfare concern.

Comparative efficacy of modified-live and inactivated vaccines in boosting disease-sparing responses to bovine viral diarrhea virus challenge in neonatally mucosally primed weanling beef calves.

Objective: This study compares immune and clinical responses of bovine viral diarrhea virus (BVDV)-maternal antibody (MatAb)-positive beef calves primed with intranasal modified-live virus vaccine (MLV) and differentially boosted with a systemic MLV or an inactivated vaccine (KV). Animal: Eighteen commercial Black Angus steers. Procedure: Calves were mucosally primed at ~24 h of age with an MLV and boosted by injection of a MLV (IN-MLV) or inactivated vaccine (IN-KV) at an average age of 54 d. Challenge occurred at weaning with a virulent non-cytopathic BVDV-2 strain, 24515. Results: Clinically, the IN-KV group had a longer duration of fever, leukopenia, and viremia, whereas the IN-MLV group had greater BVDV Types-1 and -2 heterospecific antibody responses. Conclusion: Altogether, these data indicated that systemic MLV boosting resulted in a more robust protection to BVDV Type-2 challenge at weaning. Clinical relevance: Mucosal prime-boosting of neonatal calves provided protection against BVDV Type-2 challenge at weaning.

Efficacité comparative des vaccins vivants modifiés et inactivés pour stimuler les réponses épargnant la maladie à la provocation par le virus de la diarrhée virale bovine chez des veaux de boucherie sevrés sensibilisés par voie mucosale en période néo-natale. Objectif: Cette étude compare les réponses immunitaires et cliniques des veaux de boucherie positifs au virus de la diarrhée virale bovine (BVDV) dus aux anticorps maternels (MatAb), sensibilisés avec un vaccin intranasal à virus vivant modifié (MLV) et différentiellement stimulés avec un vaccin MLV systémique ou un vaccin inactivé (KV). Animal: Dix-huit bouvillons commerciaux Black Angus. Procédure: Les veaux ont été sensibilisés par voie mucosale à environ 24 h d'âge avec un MLV et ont reçu un rappel par injection d'un MLV (IN-MLV) ou d'un vaccin inactivé (IN-KV) à un âge moyen de 54 jours. L'épreuve a eu lieu au sevrage avec une souche virulente non cytopathique de BVDV-2, 24515. Résultats: Cliniquement, le groupe IN-KV présentait une durée plus longue de fièvre, de leucopénie et de virémie, tandis que le groupe IN-MLV présentait des réponses en anticorps hétérospécifiques BVDV de types 1 et 2 plus importantes. Conclusion: Dans l'ensemble, ces données ont indiqué que le renforcement par le vaccin MLV systémique entraînait une protection plus robuste contre la provocation par le BVDV de type 2 au sevrage. Pertinence clinique: La stimulation mucosale des veaux nouveau-nés a fourni une protection contre la provocation par le BVDV de type 2 au sevrage.(Traduit par Dr Serge Messier).

HoBi-like Pestivirus Is Highly Prevalent in Cattle Herds in the Amazon Region (Northern Brazil).

Pestiviruses are globally distributed and cause substantial economic losses to the cattle industry. In Brazil, the country with the world's largest cattle population, pestivirus infections are well described in some regions, such as in the south, where a high frequency of BVDV-2 is described and contrasts with the high prevalence of HoBi-like pestivirus (HoBiPeV) in the northeast. However, there is a lack of information about pestiviruses in the Amazon Region, in northern Brazil, with a cattle population estimated at 55.7 million head, which has a significant impact on the international livestock market. Therefore, this study investigated the seroprevalence and genetic variability of ruminant pestiviruses in 944 bovine serum samples from four states in northern Brazil: Pará (PA), Amapá (AP), Roraima (RR), and Amazonas (AM). Our results showed that 45.4% of the samples were seropositive (19.8% for BVDV-1, 14.1% for BVDV-2, and 20.9% for HoBiPeV). All samples were tested by RT-qPCR, and three were positive and classified as HoBiPeV in a phylogenetic analysis. These serological and molecular results contrast with those from other regions of the world, suggesting that the northern Brazilian states have a high prevalence of all bovine pestiviruses including HoBiPeV.

Evaluation of the Mucosal Immunity Effect of Bovine Viral Diarrhea Virus Subunit Vaccine E2Fc and E2Ft.

Classified as a class B infectious disease by the World Organization for Animal Health (OIE), bovine viral diarrhea/mucosal disease is an acute, highly contagious disease caused by the bovine viral diarrhea virus (BVDV). Sporadic endemics of BVDV often lead to huge economic losses to the dairy and beef industries. To shed light on the prevention and control of BVDV, we developed two novel subunit vaccines by expressing bovine viral diarrhea virus E2 fusion recombinant proteins (E2Fc and E2Ft) through suspended HEK293 cells. We also evaluated the immune effects of the vaccines. The results showed that both subunit vaccines induced an intense mucosal immune response in calves. Mechanistically, E2Fc bonded to the Fc γ receptor (FcγRI) on antigen-presenting cells (APCs) and promoted IgA secretion, leading to a stronger T-cell immune response (Th1 type). The neutralizing antibody titer stimulated by the mucosal-immunized E2Fc subunit vaccine reached 1:64, which was higher than that of the E2Ft subunit vaccine and that of the intramuscular inactivated vaccine. The two novel subunit vaccines for mucosal immunity developed in this study, E2Fc and E2Ft, can be further used as new strategies to control BVDV by enhancing cellular and humoral immunity.

Non-cytopathic bovine viral diarrhoea virus 2 induces autophagy to enhance its replication.

BACKGROUND: Bovine viral diarrhoea virus (BVDV) is an important viral pathogen that has an economic impact on the livestock industry worldwide. Autophagy is one of the earliest cell-autonomous defence mechanisms against microbial invasion, and many types of viruses can induce autophagy by infecting host cells. OBJECTIVES: The aim of this study was to identify the role of autophagy in the pathogenesis of non-cytopathic (ncp) BVDV2 infection. METHODS: Madin-Darby bovine kidney (MDBK) cells were treated with ncp BVDV2, rapamycin, or 3-methyladenine (MA) and ncp BVDV2 and then incubated at 37°C for 24 h. Cells were harvested, and the effects of autophagy were determined by transmission electron microscopy (TEM), confocal laser microscopy, western blotting and qRT-PCR. Apoptotic analysis was also performed using western blotting and flow cytometry. RESULTS: In ncp BVDV2-infected MDBK cells, more autophagosomes were observed by TEM, and the number of microtubule-associated protein 1 light chain 3B (LC3B) with green fluorescent protein puncta was also increased. The ncp BVDV2-infected cells showed significantly enhanced conversion of LC3-I to LC3-II, as well as upregulation of autophagy-related proteins, including ATG5 and Beclin 1, and substantial degradation of p62/SQSTM1. These results are similar to those induced by rapamycin, an autophagy inducer. E2 protein expression, which is associated with viral replication, increased over time in ncp BVDV2-infected cells. Inhibition of autophagy by 3-MA in ncp BVDV2-infected MDBK cells downregulated the expressions of LC3-II, ATG5 and Beclin 1 and prevented the degradation of p62/SQSTM1. Moreover, the expressions of phosphorylated Akt and procaspase-3 were significantly increased in ncp BVDV2-infected cells. In addition, the mRNA level of protein kinase R (PKR) was significantly reduced in ncp BVDV2-infected cells. CONCLUSIONS: Our results demonstrate that ncp BVDV2 infection induced autophagy in MDBK cells via anti-apoptosis and PKR suppression. Therefore, autophagy may play a role in establishing persistent infection caused by ncp BVDV.

bta-miR-2904 inhibits bovine viral diarrhea virus replication by targeting viral-infection-induced autophagy via ATG13.

MicroRNAs (miRNAs) are endogenous small and noncoding RNA molecules (18-25 nt) that can regulate expression of their target genes post-transcriptionally. Previously, using high-throughput sequencing data obtained on a Solexa platform, we found that Bos taurus bta-miR-2904 (miR-2904) was significantly upregulated in Madin-Darby bovine kidney (MDBK) cells infected with bovine viral diarrhea virus (BVDV) strain NADL at 2, 6, and 18 h postinfection (hpi) compared to uninfected MDBK cells. Moreover, miR-2904 overexpression significantly reduced BVDV replication. However, the mechanism by which miR-2904 inhibits viral replication remains unclear. In this study, we used electron microscopy, laser confocal microscopy, dual-luciferase reporter analysis, real-time PCR, and Western blot assays to investigate the effect of the miR-2904 expression on BVDV NADL replication and virus-infection-induced autophagy. The results indicate that miR-2904 inhibits autophagy of MDBK cells by targeting autophagy-related gene 13 (ATG13), and overexpression of miR-2904 inhibited the replication of BVDV NADL.

Clinical status and endoscopy of the upper respiratory tract of dairy calves infected with Bovine viral diarrhea virus 2 and Bovine herpes virus 1 after vaccination and trace minerals injection.

The objective was to compare clinical protection [evaluated through health scoring, endoscopy score of the upper respiratory tract (URT-ES), leukocyte count, viremia, and virus shedding in nasal secretions] following Bovine viral diarrhea virus 2 (BVDV2) and Bovine herpes virus 1 (BHV1) challenge among calves submitted to modified-live virus (MLV) booster vaccination (either intranasal or subcutaneous) concurrent with injectable trace minerals (ITM) or saline. Forty-eight dairy calves received an MLV intranasal (IN) vaccine containing BHV1, BRSV, and BPI3V and subcutaneous (SC) ITM (Se, Cu, Zn & Mn; ITM, n = 24) or saline (SAL, n = 24). Ten weeks later, calves received a second dose of ITM, or saline, according to previous groups and were randomly assigned to receive the same IN vaccine [ITM-IN (n = 12), SAL-IN (n = 12)] or a SC MLV vaccine containing BHV1, BRSV, BPI3V, BVDV1 & 2 [ITM-SC (n = 12), SAL-SC (n = 12)]. Additionally, 12 calves did not receive vaccine or treatment and served as a control group (UNVAC, n = 12). Forty-nine days after booster, calves were challenged with BVDV2; and seven days later with BHV1. Health scores indicated disease in UNVAC on days 6, 10 and 12 compared to the vaccinated groups. Unvaccinated calves had the highest URT-ES after BHV1 challenge. Calves that received SC booster had lower URT-ES after BHV1 challenge than UNVAC calves. Calves in ITM-IN had significantly lower URT-ES after BHV1 infection than SAL-IN and UNVAC calves. In conclusion, IN or SC MLV vaccination was similarly effective in protecting calves from BVDV2 + BHV1 challenges (reducing clinical and endoscopy scores, preventing leukopenia, and viremia), compared to unvaccinated calves. Endoscopic evaluation of the URT allowed visualization of the inflammation and damage at multiple depths in the URT caused by a serial BVDV2 + BHV1 challenge. Calves that received SC vaccination had significantly lower URT-ES after BHV1 challenge than the UNVAC calves. Administration of ITM concurrent with IN vaccination was associated with reduced URT inflammation after BVDV2 + BHV1 challenge.

A new (old) bovine viral diarrhea virus 2 subtype: BVDV-2e.

Bovine pestiviruses are members of the species Pestivirus A (bovine viral diarrhea virus 1, BVDV-1), Pestivirus B (BVDV-2) or Pestivirus H (HoBiPeV). To date, BVDV-2 isolates/strains have been classified into three subtypes (a-c) by phylogenetic analysis, and an additional subtype (d) has been proposed based on 5' untranslated region (UTR) secondary structures. In a previous study, we identified some BVDV-2 sequences in the GenBank database that could not be classified as subtype a, b or c by phylogenetic analysis of their genomes, UTRs or individual genes. Here, we performed a detailed study of these sequences and assessed whether they might represent a distinct BVDV-2 subtype. Initially, we collected 85 BVDV-2 complete/near-complete genomes (CNCGs) from GenBank and performed a "proof of equivalence" between phylogenetic analyses based on CNCGs and open reading frames (ORFs), which showed that ORFs may be reliably used as a reference target for BVDV-2 phylogeny, allowing us to increase our dataset to 139 sequences. Among these, we found seven sequences that could not be classified as BVDV-2a-c. The same was observed in the phylogenetic analysis of CNCGs and viral genes. In addition, the seven non-BVDV-2a-c sequences formed a distinct cluster in all phylogenetic trees, which we propose to term BVDV-2e. BVDV-2e also showed 44 amino acid changes compared to BVDV-2a-c, 20 of which are in well-defined positions. Importantly, an additional phylogenetic analysis including BVDV-2d and a pairwise comparison of BVDV-2e and BVDV-2d sequences also supported the difference between these subtypes. Finally, we propose the recognition of BVDV-2e as a distinct BVDV-2 subtype and encourage its inclusion in future phylogenetic analyses to understand its distribution and evolution.

Research advances on interferon (IFN) response during BVDV infection.

Bovine viral diarrhea virus (BVDV) is an important pathogen responsible for significant economic loss to cattle. BVDV infection in pregnant cattle leads to fetal infection and reproductive losses, including early embryonic death, abortion, and stillbirth. Importantly, vaccinated heifers could not provide fetal protection against BVDV. It can be divided into two genotypes (BVDV-1 and BVDV-2) and two biotypes (cytopathic (CP) and non-cytopathic (NCP)). Infection with NCP-BVDV during gestation, the fetus becomes persistently infected (PI) and sheds BVDV throughout life, serving as the main source of infection for other cattle. BVDV potentially induces immunosuppression and aggravates bovine respiratory disease (BRD). Accordingly, BVDV infection results in a heterogeneous range of clinical signs and immune responses. Interferon (IFN) plays a vital role by mediating the innate immune response against antiviral infection through the Janus Kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. BVDV infection can reportedly exert variable degrees of influence on IFN response. Interestingly, reports have suggested that IFN can exert a significant inhibitory effect on various viruses. Human IFN-α was used to restrain BVDV in vitro. In this article, we summarized the latest researches on IFN response during BVDV infection.

Use of multivariate analysis to evaluate antigenic relationships between US BVDV vaccine strains and non-US genetically divergent isolates.

Bovine viral diarrhea virus (BVDV) comprises two species, BVDV-1 and BVDV-2. But given the genetic diversity among pestiviruses, at least 22 subgenotypes are described for BVDV-1 and 3-4 for BVDV-2. Genetic characterization is generally accomplished through complete or partial sequencing and phylogeny, but it is not a reliable method to define antigenic relationships. The traditional method for evaluating antigenic relationships between pestivirus isolates is the virus neutralization (VN) assay, but interpretation of the data to define antigenic relatedness can be difficult to discern for BVDV isolates within the same BVDV species. Data from this study utilized a multivariate analysis for visualization of VN results to analyze the antigenic relationships between US vaccine strains and field isolates from Switzerland, Italy, Brazil, and the UK. Polyclonal sera were generated against six BVDV strains currently contained in vaccine formulations, and each serum was used in VNs to measure the titers against seven vaccine strains (including the six homologous strains) and 23 BVDV field isolates. Principal component analysis (PCA) was performed using VN titers, and results were interpreted from PCA clustering within the PCA dendrogram and scatter plot. The results demonstrated clustering patterns among various isolates suggesting antigenic relatedness. As expected, the BVDV-1 and BVDV-2 isolates did not cluster together and had the greatest spatial distribution. Notably, a number of clusters representing antigenically related BVDV-1 subgroups contain isolates of different subgenotypes. The multivariate analysis may be a method to better characterize antigenic relationships among BVDV isolates that belong to the same BVDV species and do not have distinct antigenic differences. This might be an invaluable tool to ameliorate the composition of current vaccines, which might well be important for the success of any BVDV control program that includes vaccination in its scheme.

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.

Th17 cell differentiation induced by cytopathogenic biotype BVDV-2 in bovine PBLCs.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is a major pathogen that causes bovine viral diarrhea/mucosal disease (BVD-MD), which has become a global infectious disease due to its wide spread and the lack of effective treatment. The process of BVDV infection is complex. Once infected, host immune cells are activated and modulated. As a major immune cell, peripheral blood lymphocyte cells (PBLCs) are the primary target of BVDV. In order to further understand the mechanism of BVDV- host interaction, the expression profiles of host lymphocytes mRNAs associated with BVDV infection were investigated by transcriptomic sequencing analysis. RESULTS: The transcriptomic sequencing analysis was performed on bovine PBLCs infected with CP BVDV-2 GS2018 after 12 h of infection. Gene expression profiling demonstrated that 1052 genes were differentially expressed in GS2018 infected PBLCs compared with the control group. Of these genes, 485 genes were up-regulated and 567 were down-regulated. The 19 differential expressed genes (DEGs) were selected for validation using quantitative real-time PCR and the results were consistent with the results of RNA-Seq. Gene ontology enrichment and KEGG pathway analysis showed that 1052 DEGs were significantly enriched in 16 pathways, including cytokine-cytokine receptor interaction, IL17, PI3K-Akt, MAPK and TNF signaling pathway. PPI network analysis showed that IL17A, IFN-γ and TNF-α interacted with various proteins and may play crucial roles in BVDV-2 infection. Of note, we confirmed that GS2018 induced Th17 cell differentiation in PBLCs and persistently increased the expression levels of IL17A. In turn, the replication of GS2018 was inhibited by IL17A. CONCLUSION: In this study, the transcription changes of DEGs related to host immune responses in bovine PBLCs were caused by CP BVDV-2 infection. In particular, the effector molecules IL17A of Th17 cells were significantly up-regulated, which inhibited viral replication. These results will contribute to exploration and further understanding of the host immune response mechanism and interaction between host and BVDV-2.

Genomic diversity and phylodynamic of bovine viral diarrhea virus in Argentina.

Bovine viral diarrhea virus (BVDV) is an important pathogen of ruminants worldwide and is characterized by high genetic diversity and a wide range of clinical presentations. In Argentina, several studies have evaluated the genetic diversity of BVDV but no phylodynamic study has been published yet. In this study, a comprehensive compilation and update of Argentinean BVDV sequences were performed, and the evolutionary history of BVDV was characterized by phylodynamic analyses based on the 5´UTR. Although BVDV-1b and BVDV-1a were the most frequent subtypes, novel subtypes for Argentina, 1e and 1i, were identified. The phylodynamic analysis suggested that BVDV started its diversification in the mid-1650s with an exponential increase in viral diversity since the late 1990s, possibly related to the livestock expansion and intensification in the country. Evolutionary rate in the 5´UTR was faster for BVDV-1a than for BVDV-1b, and both subtypes presented an endemic nature according to the demographic reconstructions. The current study contributes to clarify the evolutionary history of BVDV in the main cattle region of the country and provides useful information about the epidemiology and future development of diagnostic and control tools in Argentina.

Cattle connection: molecular epidemiology of BVDV outbreaks via rapid nanopore whole-genome sequencing of clinical samples.

BACKGROUND: As a global ruminant pathogen, bovine viral diarrhea virus (BVDV) is responsible for the disease Bovine Viral Diarrhea with a variety of clinical presentations and severe economic losses worldwide. Classified within the Pestivirus genus, the species Pestivirus A and B (syn. BVDV-1, BVDV-2) are genetically differentiated into 21 BVDV-1 and four BVDV-2 subtypes. Commonly, the 5' untranslated region and the Npro protein are utilized for subtyping. However, the genetic variability of BVDV leads to limitations in former studies analyzing genome fragments in comparison to a full-genome evaluation. RESULTS: To enable rapid and accessible whole-genome sequencing of both BVDV-1 and BVDV-2 strains, nanopore sequencing of twelve representative BVDV samples was performed on amplicons derived through a tiling PCR procedure. Covering a multitude of subtypes (1b, 1d, 1f, 2a, 2c), sample matrices (plasma, EDTA blood and ear notch), viral loads (Cq-values 19-32) and species (cattle and sheep), ten of the twelve samples produced whole genomes, with two low titre samples presenting 96 % genome coverage. CONCLUSIONS: Further phylogenetic analysis of the novel sequences emphasizes the necessity of whole-genome sequencing to identify novel strains and supplement lacking sequence information in public repositories. The proposed amplicon-based sequencing protocol allows rapid, inexpensive and accessible obtainment of complete BVDV genomes.