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Bovine viral diarrhoea (BVD) is one of the most economically damaging livestock enzootic diseases in the world. BVD aetiological agents are three pestiviruses (BVDV-1, -2 and HoBi-like pestivirus), which exhibit high genetic diversity and complex transmission cycles. This considerably hampers the management of the disease, which is why eradication plans have been implemented in several countries. In France, a national plan has been in place since 2019. Our understanding of its impact on the distribution of BVDV genotypes is limited by the availability of French genetic data. Here, we conducted a molecular epidemiology study to refine our knowledge of BVDV genetic diversity in France, characterise its international relationships, and analyse national spatio-temporal genotypic distribution. We collated 1037 BVDV-positive samples throughout France between 2011 and 2023, with a greater sampling effort in two major cattle production areas. We developed a high-throughput sequencing protocol which we used to complete the 5'UTR genotyping of this collection. We show that two main BVDV-1 genotypes, 1e and 1b, account for 88% of genotyped sequences. We also identified seven other BVDV-1 genotypes occurring at low frequencies and three BVDV-2 samples (genotype 2c). Phylogenetic analyses indicate different worldwide distribution patterns between the two main BVDV-1 genotypes. Their relative frequencies present no major changes in France since the 1990s and few variations at the national scale. We also found some degree of local spatial structuring in western France. Overall, our results demonstrate the potential of large-scale sequence-based surveillance to monitor changes in the epidemiological situation of enzootic diseases., (© 2024. The Author(s).)

Bovine viral diarrhea virus (BVDV) is a widespread pathogen of cattle and other mammals that causes major economic losses in the livestock industry. N4-TSC and 6NO 2 -TSC are two thiosemicarbazones derived from 1-indanone that exhibit anti-BVDV activity in vitro. These compounds selectively inhibit BVDV and are effective against both cytopathic and non-cytopathic BVDV-1 and BVDV-2 strains. We confirmed that N4-TSC acts at the onset of viral RNA synthesis, as previously reported for 6NO 2 -TSC. Moreover, resistance selection and characterization showed that N4-TSC R mutants were highly resistant to N4-TSC but remained susceptible to 6NO 2 -TSC. In contrast, 6NO 2 -TSC R mutants were resistant to both compounds. Additionally, mutations N264D and A392E were found in the viral RNA-dependent RNA polymerase (RdRp) of N4-TSC R mutants, whereas I261 M was found in 6NO 2 -TSC R mutants. These mutations lay in a hydrophobic pocket within the fingertips region of BVDV RdRp that has been described as a "hot spot" for BVDV non-nucleoside inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (Published by Elsevier B.V.)

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.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bovine viral diarrhea virus-1 (BVDV-1, Pestivirus A) and BVDV-2 (Pestivirus B) have been clustered into 21 and 4 subtypes, respectively. This genetic diversity, in addition to the lack of consensus on which genomic region to use for BVDV subtyping, has resulted in conflicting classifications depending on the target analyzed. Here, we investigated which genes or UTRs would reproduce the phylogeny obtained by complete genome (CG) analyses. The study was carried out with 91 (BVDV-1) and 85 (BVDV-2) CG available on GenBank database. The viruses were subtyped by analyzing their CG, as well as their individual genes and UTRs (complete 3' and 5'UTRs, and partial 5'UTR); and the phylogeny results were compared to each other. The sequences were aligned using the ClustalW multiple method (BioEdit Alignment Editor software, v.7.0.5.3) and the phylogenetic analyses were performed by the Maximum Likelihood method (MEGA-X software, v.10.2.4), with 1000 bootstrap replicates. The best analysis model for each gene/UTR was defined using the jModelTest software. The geodesic distance between the CG (reference) and individual genes/UTRs trees was also calculated (TreeCmp software, v.2.0). In general, 3'UTR-based analyses, followed by 5'UTR, presented the least reliable subtyping results. Regarding BVDV-1, phylogeny based on C, E rns , E1, E2, p7, NS2, NS3, NS4B, NS5A and NS5B was consistent with that of CG. In contrast, analyses performed with individual BVDV-2 genes showed at least one different clustering from the phylogeny based on the CG. After analyzing the geodesic distance between the CG and genes/UTRs trees, we observed that NS4B (for BVDV-1) and NS5A (BVDV-2) presented the closest topology and edge length to the CG analyses. Finally, comparing the phylogeny performed with the CG and the genes/UTRs, as well as the geodesic distance between them, we understand that NS4B and NS5A represent the most suitable targets for BVDV-1 and -2 subtyping, respectively, and may be considered in future phylogenetic studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 " 77 ESRKKLEKALLA 88 " 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, " 77 ESRKKLEKALLA 88 ", 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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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 N pro 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., (© 2021. The Author(s).)

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.

Bovine viral diarrhea virus (BVDV) is a viral pathogen associated with serious problems in the cattle industry. Cattle persistently infected (PI) with BVDV are mild or asymptomatic; however, they become a source of BVDV transmission to other cattle. Hence, it is important to rapidly identify and remove the PI animals from cattle herds. Whereas cattle acutely infected (AI) with BVDV have various symptoms, yet they generally recover within 3 weeks. However, there is a paucity of information concerning clinical characteristics of AI cattle. Further accumulation of information would be required to accurately diagnose AI cattle with BVDV. Here, we attempted to obtain valuable information via various analyses using a case report of BVD outbreak that occurred for approximately four months in Iwate Prefecture in 2017. Using eight calves and multiple tests (real-time RT-PCR, virus isolation, enzyme-linked immunosorbent assay, and virus neutralization assay) over 6 weeks, we diagnosed the continuous BVD outbreak as an acute infection and not a persistent one. Additionally, we revealed that the sporadic case was caused by low pathogenic BVDV2 via BVDV genotyping and phylogenetic analysis. The data suggest that BVDV2 AI animals might also be a source of transmission to susceptible calves; hence, it might persist for a long period owing to multiple AI animals. These findings provide useful information to diagnose AI and PI cattle with BVDV in the field.

Bovine viral diarrhea virus (BVDV) is comprised of two species, BVDV-1 and BVDV-2, but given the genetic diversity among pestiviruses, at least 21 subgenotypes are described for BVDV-1 and 4 for BVDV-2. Genetic characterization can be achieved through complete or partial sequencing and phylogeny, but antigenic characterization can be difficult to determine due to the antigenic diversity and cross-neutralization that exists among isolates. The traditional method for evaluating antigenic relationships between pestivirus isolates is the virus neutralization (VN) assay, but interpretation of the data to determine antigenic difference can be unclear. Data from this study utilized a multivariate analysis for visualization of VN results to analyze the antigenic relationships between vaccine strains and multiple field isolates. Polyclonal sera were generated against 6 BVDV strains currently contained in vaccine formulations, and each serum was used in VN's to measure the neutralizing antibody titers against 15 BVDV field isolates characterized as prevalent and divergent subgenotypes in the USA. Principal component analysis (PCA) were performed on the VN assay datasets, and results were interpreted from PCA clustering within the PCA dendrogram and scatter plot. The results demonstrated clustering patterns among isolates suggestive of antigenic differences. While expected, the BVDV-1 and BVDV-2 isolates did not cluster together and had the greatest spatial distribution. In addition, other BVDV isolates had distinct spatial patterns suggesting antigenically divergent isolates. This analysis provides an alternative and more efficient means to analyze large VN datasets to visualize antigenic relationships between pestivirus isolates. This analysis could be beneficial for vaccine development and evaluation of efficacy, since most vaccines cannot fully protect animals from the broad range diversity of BVDV viruses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Bovine viral diarrhea virus (BVDV) entry into a host cell is mediated by the interaction of the viral glycoprotein E2 with the cellular transmembrane CD46 receptor. In this study, we generated a stable Madin-Darby Bovine Kidney (MDBK) CD46-knockout cell line to study the ability of different pestivirus A and B species (BVDV-1 and -2) to escape CD46-dependent cell entry. Four different BVDV-1/2 isolates showed a clearly reduced infection rate after inoculation of the knockout cells. However, after further passaging starting from the remaining virus foci on the knockout cell line, all tested virus isolates were able to escape CD46-dependency and grew despite the lack of the entry receptor. Whole-genome sequencing of the escape-isolates suggests that the genetic basis for the observed shift in infectivity is an amino acid substitution of an uncharged (glycine/asparagine) for a charged amino acid (arginine/lysine) at position 479 in the E RNS in three of the four isolates tested. In the fourth isolate, the exchange of a cysteine at position 441 in the E RNS resulted in a loss of E RNS dimerization that is likely to influence viral cell-to-cell spread. In general, the CD46-knockout cell line is a useful tool to analyze the role of CD46 for pestivirus replication and the virus-receptor interaction.

Infectious bovine viral diarrhea virus (BVDV) cDNA clones have been used for the expression of classical swine fever virus (CSFV) genes for immune prevention and control. However, can it be used for the expression of an allogenetic fragment? To answer this question, a BVDV chimeric virus expressing the spike (S) antigen fragment of porcine epidemic diarrhea virus (PEDV) was constructed. Antigen S 499-602 was inserted into pig-derived BVDV-2 infectious cDNA clone pASH28 in tandem by overlapping PCR, located between the seventh and eighth amino acids at the N-terminus of the capsid (C) protein of BVDV. Indirect immunofluorescence assay confirmed that the chimeric virus vASH-dS312 containing double S 499-602 sequences was successfully assembled, which could react with the monoclonal antibody (MAb) against BVDV E2 and PEDV S proteins. Further western blot analysis confirmed that the exogenous S 499-602 double protein could be stably expressed. Next, the chimeric virus vASH-dS312 was administered to BALB/C mice either orally or by intramuscular injection. The immunized mice were healthy and showed no signs of toxicity. IgG against BVDV and PEDV antibodies could be detected in the mice administered vASH-dS312 by intramuscular injection, which had neutralization activity against BVDV and PEDV. Thus, this study reported a new insertion site in the BVDV infectious cDNA clone that could successfully express an allogenetic antigen., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

Bovine viral diarrhea virus (BVDV) infections in pigs may result in transient leukopenia, chronic gastroenteritis, septicemia, and hemorrhagic lesions. Both classical swine fever virus (CSF) and the atypical porcine pestivirus (APPV) are shed in the semen of infected boars. Because these viruses share conserved regions and present antigenic similarity, they may not be the only species belonging to the genus Pestivirus that can be shed in the semen of infected pigs. The objective of this study was to evaluate the testicular and epididymal changes, seminal parameters, and viral shedding in the reproductive tract of boars experimentally inoculated with noncytopathic BVDV-2. Six males were selected, and samples of blood, semen, and preputial swabs were collected every four days until the 52nd day after inoculation. The samples were tested for the presence of viral RNA by RT-PCR. An aliquot of whole blood was used to perform hematological analyses, which showed a significant reduction in monocyte counts and a significant increase in lymphocyte counts when comparing the pre- and postinoculation periods. The neutralizing antibody titers were determined by the virus neutralization test. None of the animals presented clinical signs or worsening of the seminal parameters that were evaluated. Moreover, BVDV-2 shedding by the reproductive route was not observed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bovine viral diarrhea virus (BVDV) is one of the most important pathogens of cattle worldwide. BVDV-1 is widely distributed in Italy, while BVDV-2 has been detected occasionally. BVDV can be classified in two biotypes, cytopathic (CP) or noncytopathic (NCP). The characteristic of the virus is linked with the infection of a pregnant dam with a NCP strain: due to viral establishment before maturation of the fetal immune system the calf remains persistently infected (PI) and immunotolerant to the infecting BVDV strain. Thanks to their immunotolerance, PI animals represent a unique model to study the viral distribution and compartmentalization in absence of immunoresponse in vivo. In the present study, NGS sequencing was used to characterize the BVDV2 viral strain infecting a PI calf and to describe the viral quasispecies in tissues. Even if the consensus sequences obtained by all the samples were highly similar, quasispecies was described evaluating the presence and the frequency of variants among all the sequencing reads in each tissue. The results suggest a high heterogeneity of the infecting viral strain suggesting viral compartmentalization. The quasispecies analysis highlights the complex dynamics of viral population structure and can increase the knowledge about viral evolution in BVDV-2 persistently infected animals.

Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Bovine viral diarrhea virus (BVDV) is a pathogen associated with loss of meat, milk, and reproductive performance in cattle across the world. There have been two types of BVDV identified worldwide: BVDV-1 and BVDV-2. However, a new type of BVDV, named HoBi-like pestivirus (BVDV-3), has been identified recently. BVDV presence in Turkey has been reported since the 1990s, but a mandatory vaccination program has not been implemented in Turkey so far. In serological studies conducted in Turkey for BVDV, reported seropositivity has been 50% on average. The aim of this study is to determine the genetic diversity of BVDV in blood and abortion materials from bovine in eastern Turkey. The presence of the virus was determined by antigen ELISA test. As a result of the phylogenetic analysis of 5'UTR, Npro and E2 genomic regions of the BVDV (n = 28), BVDV-1 (n = 25) was identified as the dominant type. In addition, BVDV-2 (n = 2) and BVDV-3 (n = 1) were determined which is the first report of HoBi-like pestivirus in Turkey. Although BVDV-1l (n = 19) was detected as the predominant sub-type of BVDV-1, 1a (n = 2), 1b (n = 1), 1c (n = 1), and 1d (n = 2) were also identified. In 2 samples, the BVDV-2 type detected was the 2a sub-type. In this study, it is emphasized that BVDV can be present in the abort materials as an agent and that it should be examined in the herd screening. In addition, it is understood that molecular epidemiological studies should continue for determining the genetic diversity of the viruses and that such studies should be carried out on the country basis. Necessary diagnostic programs should be developed for animals, which are imported or buying from other barns, and protection and control measures should be taken. The increase of reports on BVDV heterogeneity in Turkey and worldwide gets up related to the occurrence and spread of new BVDV types or variants, with potential implications for animal health and disease control.

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.

Bovine pestiviruses, e.g., bovine viral diarrhea virus types 1 (BVDV-1 or Pestivirus A), BVDV-2 (Pestivirus B), and HoBi-like pestiviruses (HoBiPeV or Pestivirus H), have been shown to circulate in Brazilian cattle in varied proportions. In this study, we identified genetically pestiviruses circulating in beef cattle in Rio Grande do Sul, the southern most Brazilian state. Screening of serum of 15.584 beef calves destined to be export by an antigen capture ELISA and, subsequently, by reverse-transcription polymerase chain reaction (RT-PCR), revealed 135 containing pestivirus RNA. Genetic typing of these viruses based on nucleotide sequencing and phylogenetic analysis of the 5' untranslated region (5' UTR) of the viral genome allowed for the identification of 90 different viruses, being 38 BVDV-1 (42.2%), 31 BVDV-2 (34.4%), and 21 HoBiPeV (23.4%). Among BVDV-1, only subtypes BVDV-1a (n = 28, 31.1%) and BVDV-1b (n = 10, 11.1%) were identified. All 31 BVDV-2 isolates belonged to BVDV-2b subtype and the 21 HoBiPeV viruses clustered to subgroup 3a. Thus, this study provides an approximate genetic profile of pestiviruses circulating in beef cattle in a traditional Brazilian beef cattle-raising state.

We examined the circulating BVDV species and genotypes among cattle herds from Northeast, Southeast, and Midwest regions in Brazil. A total of 77 animals tested positive through standard PCR. Phylogenetic analyses revealed the presence of BVDV-1a, highlighting the need for better surveillance strategies to prevent BVDV spread in the country.

Bovine viral diarrhea virus (BVDV), a ubiquitous pathogen of cattle, causes subclinical to severe acute disease. Two species of BVDV are recognized, BVDV1 and BVDV2 with BVDV1 divided into at least 21 subgenotypes and BVDV2 into 3-4 subgenotypes, most commonly using sequences from the 5' untranslated region (5' UTR). We report genomic sequencing of 8 BVDV2 isolates that did not segregate into the 2a subgenotype; but represented two additional BVDV2 subgenotypes. One BVDV2 subgenotype was previously recognized only in Asia. The other seven viruses fell into a second subgenotype that was first reported in Brazil and the U.S. in 2002. Neutralization assays using antiserum raised against vaccine strain BVDV2a 296c revealed varying degrees of neutralization of genetically diverse BVDV2 isolates. Neutralization titers decreased from 1.8 to more than a four log(2) decrease. This study illustrated the considerable genetic and antigenic diversity in BVDV2 circulating in the U.S., (Published by Elsevier Inc.)

Bovine viral diarrhea viruses (BVDV) cause acute and persistent infections. Acute infection results in generalized immunosuppression characterized by a decrease in circulating lymphocytes as a result of depletion of CD4+ and CD8 + T cell populations. Persistent infection with BVDV is the result of immune tolerance and is generally not associated with lymphocytopenia. The health outcome of persistently infected (PI) calves varies widely; some die of mucosal disease, some succumb to ill thrift and others appear normal and survive to adulthood. Detection of BVDV at the single lymphoid cell level is important to the study of subpopulations of peripheral blood mononuclear cells (PBMC) during BVDV infections, however there are few methods available for the detection and quantification of BVDV at this level. To circumvent this difficulty, a novel flow cytometry-based PrimeFlow RNA assay using in-situ detection of BVDV was developed. This assay was used to evaluate differences in viral distribution within subpopulations of PBMC over time in PI calves carrying one of two different species of BVDV (type 1 and type 2). Calves were sampled at 3 different time points approximately one month apart. During the course of the study, a subset of the calves died from ill thrift. Mucosal disease was not indicated in any of the deaths. Using RNA probes specific for the BVDV N pro -E rns coding regions for each respective virus, BVDV RNA was detected in all PBMC of PI that appeared clinically healthy. Calves that succumbed to ill thrift were found to have no or little virus in T cells. The clearance of virus from T cells suggests a breakdown in immune tolerance in these calves. This is the first report of a pattern observed in the viral load in the T cell subpopulations and survival in PI calves., (Published by Elsevier B.V.)

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 10 6 · 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Bovine Viral Diarrhea Virus causes significant economic losses in cattle. BVDV has high genomic diversity, with two species, BVDV-1 and BVDV-2, and at least twenty-one subgenotypes for BVDV-1 and four subgenotypes for BVDV-2. Vaccines are important tools to reduce the economic losses caused by this virus. However, vaccine strains must correspond to the antigenic profile of the viruses present in the region where the vaccine is applied. A restricted phylogenetic study with 14 viruses isolated from cattle between 1993 and 2001 showed that the genetic profile of BVDV in Chile consisted of viruses of both species and sub-genotypes 1a, 1b, 1c (currently 1j) and 2a. To determine more accurately the genetic profile of BVDV in Chile, in this study a larger number of viruses obtained from bovines between 2003 and 2007 were typed., Results: The study was performed using partial sequences from the 5' noncoding region (5'UTR) and E2 coding region of the viral genome of thirty-five Chilean viruses isolated from geographic regions that have 84.6% of the Chilean cattle. All tested viruses belonged to species BVDV-1. Eighteen viruses belonged to BVDV-1j subgenotype (51.4%), twelve belonged to BVDV-1b (34.3%) and five belonged to BVDV-1a (14.3%). The Chilean BVDV-1j viruses showed low genetic diversity, both among themselves and with the BVDV-1j present in other regions of the world. This could be explained by a relatively recent introduction of this viral subgenotype in cattle, which agrees with its low geographical distribution worldwide. Otherwise, Chilean BVDV-1b viruses grouped into a single cluster, different even than the viruses present in Argentina and Brazil, countries geographically close to Chile, a process of local evolution that could generate antigenic differences between the Chilean viruses and the viruses used as vaccine strains., Conclusions: The high presence of viruses of the BVDV-1j subgenotype, which show major antigenic differences with BVDV-1a and BVDV-1b subgenotypes used in the commercial vaccines, suggest that BVDV-1j viruses could be an emergent subgenotype of BVDV in cattle in South America and suggest evaluating an update of the vaccines used in Chile.

Background: Bovine viral diarrhea virus (BVDV) is prevalent in Korean indigenous cattle, leading to substantial economic losses. This study was conducted to investigate the occurrence of BVDV. In 2016, a total of 143 blood samples were collected from asymptomatic Korean indigenous calves younger than 3-months of age from six different farms in the Republic of Korea (ROK)., Results: Eighty-seven calves (60.8%, 87/143) were tested positive for BVDV as evaluated by RT-PCR analysis. Phylogenetic analysis based on the 5'-untranslated region was used to classify these cases into three subtypes: BVDV-1b, BVDV-1o, and BVDV-2a. These results showed that BVDV-1b was the predominant subtype, while 2 samples clustered with BVDV-2a. Interestingly, one sample formed a separate group as a potentially new subtype, BVDV-1o. To our knowledge, this is the first report of BVDV-1o infection in Korean native calves. The BVDV-1o subtype identified in this study was closely related to cattle isolates obtained from Japan, indicating that this subtype is a new introduction to the ROK., Conclusions: This study provides useful information for carrying out epidemiological surveys of BVDV in the ROK and developing a vaccine for future use in the ROK, particularly for the first detection of BVDV-1o in Korean indigenous calves. Further studies are required to investigate the prevalence and pathogenicity of this BVDV-1o subtype.

Congenital tremor in pigs involves several etiologies, including pestivirus, which may cause neurological injuries in different animal species. To evaluate whether bovine viral diarrhea virus (BVDV), an important pestivirus, is one of the etiological agents of congenital tremor in swine, gilts and the fetuses were challenged at 45 days of gestation with BVDV-2. Four pregnant gilts were inoculated oronasally, four gilts underwent fetal intrauterine inoculation, and two gilts constituted the control group. Antibody titers were determined by virus neutralization (VN), and viral RNA was detected by RT-PCR. Blood samples were collected from all gilts and piglets born to obtain whole blood and serum for analysis. One third of the neonates were euthanized at three days old, and samples of the encephalon, brain stem and spinal cord were collected for anatomopathological evaluation and viral RNA detection. The piglets that remained alive were clinically evaluated every day, and blood sampling was performed regularly for 35 days. The piglets from gilts in both inoculation treatment groups showed no clinical neurological signs and were born with no viral RNA in their blood and organs. Piglets born from oronasally inoculated gilts did not present antibodies against BVDV-2 at birth, although they were acquired by passive maternal transfer. In contrast, intrauterine-inoculated piglets were born with high antibody titers (80 to 640) against the agent, which remained high until the end of the experimental period. Microscopically, no noticeable changes were observed. Macroscopically, 29.5% of the total piglets euthanized, from both inoculation groups, were born with a low cerebellar:brain ratio. Nevertheless, some piglets had a high cerebellar:brain ratio, indicating the need for standardizing this value. Thus, it was concluded that BVDV is not an etiological agent for congenital swine tremor., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study, the systemic analyses of nucleotide, codon and amino acid usages for E2 gene of bovine viral diarrhea virus (BVDV) were carried out for estimating its genetic features. The nucleotide usage pattern at the first codon position was strongly influenced by the overall nucleotide composition, while the nucleotide usage patterns at the second and third codon positions seemed to have little link to the overall nucleotide composition. The result indicated that the mutation pressure from nucleotide composition constraint was not the single evolutionary force for genetic features of BVDV E2 gene. Just 18 out of 59 synonymous codons were similar with synonymous codon usage patterns for E2 gene between BVDV1 and BVDV2, while all synonymous codons which contain CpG dinucleotides were selected at the low level by E2 gene, suggesting that this gene suppressed the usages of codons containing CpG dinucleotides to regulate E2 gene replicate and transcript efficiently and avoid immune response from infected hosts. Amino acid usage patterns of E2 protein were generally different between BVDV1 and BVDV2. The patterns of synonymous codon and amino acid usages for E2 gene might be caused by the equilibrium of evolutionary forces from virus and host. Our work gave new investigations into the role of host origin in the formations of synonymous codon/amino acid usages and the evolutionary trend of BVDV E2 gene. The genetic characteristics that codon/amino acid usages of E2 gene adapted to the internal environment of individual animals might assist in understanding the changes of genetics and antigenicity for newly emerging BVDV., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Fifty-three cattle of unknown serologic status that were not persistently infected (PI) with bovine viral diarrhea virus (BVDV) were commingled with 10 cattle that were PI with different strains of BVDV, and were monitored for an extended commingle period using a reverse-transcription real-time PCR (RT-rtPCR) BVDV assay on various sample types. Transient infections with BVDV were also assessed by virus isolation, virus neutralization (VN) assays, and direct buffy coat 5'-UTR sequencing. Infections were demonstrated in all cattle by RT-rtPCR; however, the detection rate was dependent on the type of sample. Buffy coat samples demonstrated a significantly greater number of positive results ( p ≤ 0.05) than either serum or nasal swab samples. Presence of elevated BVDV VN titers at the onset inversely correlated with the number of test days positive that an individual would be identified by RT-rtPCR from buffy coat samples, and directly correlated with the average Ct values accumulated over all RT-rtPCR test days from buffy coat samples. Both single and mixed genotype/subgenotype/strain infections were detected in individual cattle by direct sample 5'-UTR sequencing. A BVDV-2a strain from a PI animal was found to be the predominant strain infecting 64% of all non-PI cattle; BVDV-1b strains originating from 3 PI cattle were never detected in non-PI cattle. Although direct sample 5'-UTR sequencing was capable of demonstrating mixed BVDV infections, identifying all strains suspected was not always efficient or possible.

The ruminant pestiviral species BVDV-1, BVDV-2 and BDV, along with the putative species HoBi-like, may cause substantial economic losses in cattle, sheep and goats. Brazil's large size, variable biomes and wide range of ruminant animal production within different geographic regions suggest that the presence and prevalence of ruminant pestivirus may differ by regions within Brazil. This study investigated the genetic diversity of ruminant pestiviruses and determined the frequency of active infections within two states of the Northeast Region of Brazil, Maranhão and Rio Grande do Norte. Serum samples from 16,621 cattle and 2,672 small ruminants from 569 different herds residing in this region were tested by RT-PCR followed by DNA sequencing. Seventeen positive cattle were detected (0.1%) from fifteen different herds (2.64%). All isolates were classified as HoBi-like pestiviruses based on phylogenetic analysis. All small ruminant samples tested negative. The findings presented herein suggest that the Northeast Region of Brazil has a uniquely high prevalence of HoBi-like viruses. The increasing reports of HoBi-like viruses detected in cattle in the field suggest that natural infection with these viruses may be more widespread than previously thought. The identification of HoBi-like viruses as the most prevalent type of ruminant pestivirus circulating in the Northeast Region of Brazil indicates the need for both continued monitoring and determination of the extent of economic losses associated with HoBi-like virus infections. In addition, it must be taken into account in the choice of diagnostic tests and in vaccine formulations., (© 2017 Blackwell Verlag GmbH.)

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 N pro and structural protein E rns play important roles in inhibiting type I interferon. The aim of this study was to explore the epistatic effects of amino acid mutations in N pro and E rns in porcine ST cells to characterize the immune response induced by BVDV-2. Plasmids with mutant amino acids His49 (H49), Glu22 (E22) in N pro , and His300 (H300), Lys412 (K412) in E rns 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/N pro H49E rns K412 virus showed the best efficiency with respect to modulating antiviral cytokines, indicating that the amino acids N pro H49 and E rns 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.

Background: Bovine viral diarrhea virus (BVDV) types 1 and 2 are members of the Pestivirus genus of the Flaviviridae family. This genus also includes the HoBi-like virus, tentatively classified as BVDV type 3. BVDV-1 is widely distributed in Italy despite the extensive use of BVDV-1-based vaccines, while BVDV-2 and HoBi-like Pestivirus have been detected occasionally. Monitoring the occurrence of sporadic or atypical pestiviruses is a useful approach to evaluate the need for additional vaccine strains that can be used in BVDV control programs., Results: In this study we developed a multiwell antibody ELISA based on the recombinant E2 protein of the three bovine pestiviruses. We evaluated the assay's applicability for surveillance purposes using pooled milk samples, each prepared from a maximum of 35 lactating cows and collected from 176 dairy herds. As expected, the majority of the pooled samples reacted to a greater extent against the BVDV-1 E2 antigen. All three milk pools from a single farm reacted to the BVDV-2 antigen, however. Further analysis using spot tests, antigen detection, and sequence analysis of the 5'-UTR region confirmed the presence of five persistently infected calves carrying a BVDV-2a strain., Conclusions: This study highlights for the first time that sporadic circulation of BVDV-2 can be predicted by immunoenzymatic methods in the absence of specific vaccination.

The ability of ruminant pestivirus including bovine viral diarrhoea virus (BVDV) and the related emerging pestivirus, HoBi-like virus, to establish persistent infection (PI) following foetal infection is central to keeping these viruses in circulation. Non-PI dams carrying BVDV PI calves develop high levels of immunity due constantly viral exposure. A study to determine whether the immunity developed following the generation of a BVDV PI is enough to prevent HoBi-like virus infection of a subsequent foetus was performed. This study consisted of nine pregnant cows, four had birthed BVDV-1 PI calves in a previous pregnancy, three cows had birthed BVDV-2 PIs and two had birthed pestivirus negative calves. From this, six pregnant cows were challenged with HoBi-like virus about day 85 of gestation (four BVDV-1 and two BVDV-2 cows) and three non-challenged cows (negative control). At the day of challenge, the serum neutralizing titres against the homologous BVDV strains of the first inoculation ranged from 1148 to 5793. At day 6 post-challenge, HoBi-like RNA was detected in the serum of all four BVDV-1 cows but not in the two BVDV-2 cows. The foetuses harvested from five of the exposed dams (three BVDV-1 and two BVDV-2 cows) at day 30 post-challenge were positive for HoBi-like virus RNA. The sixth cow, BVDV-1 cow #541, while pregnant at the time of exposure, had no foetus 30 days after exposure. Foetuses from HoBi-like virus exposed dams were significantly smaller and lighter than control foetuses. HoBi-like RNA was detected in samples of all challenged foetuses. The identification of viral RNA in the serum of 4 cows at day 6 post-challenge, as well viral RNA detection in all foetuses 30 days post-inoculation, indicates that the foetuses of dams with high antibodies titres against BVDV-1 or BVDV-2 would not be protected from challenge with a HoBi-like virus., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

Bovine viral diarrhea virus (BVDV) infection induces immunosuppression and in conjunction with bovine respiratory syncytial virus (BRSV) contributes to the bovine respiratory disease complex. Bovine turbinate cells were single or co-infected with type 2 BVDV wild-type (BVDV2-wt), its dysfunctional N pro mutant (BVDV2-E), and/or BRSV. BVDV2-E significantly up-regulated PKR, IRF-7, TBK-1, IRF-3, and IFN-β mRNAs based on real-time Q-RT-PCR. BRSV-infected cells expressed significantly up-regulated PKR, IRF-3, IRF-7, and IFN-β mRNAs, whereas BVDV2-wt, but not BVDV2-E, abolished this up-regulation in co-infection. No significant differences were observed in MAVS, NF-κB, and PIN-1 mRNAs. A dual-luciferase reporter assay showed that BVDV2-wt significantly increased NF-κB activity compared to BVDV2-E, while BVDV2-E significantly increased IFN-β activity compared to BVDV2-wt. The BRSV titer and RNA levels significantly increased in cells co-infected with BRSV/BVDV2-wt compared to cells co-infected with BRSV/BVDV2-E or infected with BRSV alone. This data supports the synergistic action of BVDV2-wt and BRSV inhibition of IFN-1., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pestiviruses of cattle include bovine viral diarrhoea 1 (BVDV-1) and 2 (BVDV-2) plus an emerging group, named HoBi-like pestivirus. In the present paper, the results of an epidemiological survey for pestiviruses circulating in cattle in southern Italy are presented. Molecular assays carried out on a total of 924 bovine samples detected 74 BVDV strains, including 73 BVDV-1 and 1 BVDV-2 viruses. Phylogenetic analysis carried out on partial 5'UTR and N pro sequences revealed the presence of 6 different subtypes of BVDV-1 and a single BVDV-2c strain. BVDV-1 displayed a high level of genetic heterogeneity, which can have both prophylactic and diagnostic implications. In addition, the detection of BVDV-2c highlights the need for a continuous surveillance for the emergence of new pestivirus strains in cattle farms in southern Italy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bovine viral diarrhea virus (BVDV) infects cattle populations worldwide, causing significant economic losses though its impact on animal health. Previous studies have reported the prevalence of BVDV species and subgenotypes in cattle from the United States and Canada. We investigated the genetic diversity of BVDV strains detected in bovine serum samples from 6 different Mexican regions. Sixty-two BVDV isolates from Mexico were genetically typed based on comparison of sequences from the 5' untranslated region (5'-UTR) of the viral genome. Phylogenetic reconstruction indicated that 60 of the samples belonged to the BVDV-1 genotype and 2 to the BVDV-2 genotype. Comparison of partial 5'-UTR sequences clustered 49 samples within BVDV-1c, 8 samples within BVDV-1a, 3 samples within BVDV-1b, and 2 samples clustered with the BVDV-2a subgenotypes. Our study, combined with information previously published on BVDV field strain diversity in the United States and Canada, benefits the development of effective detection assays, vaccines, and control programs for North America.

Pestivirus infections in ruminants result in significant economic losses worldwide. The aetiological agents are three species from the genus Pestivirus, family Flaviviridae, including bovine viral diarrhoea virus type 1 (BVDV-1), BVDV-2, border disease virus (BDV), and an atypical pestivirus named HoBi-like pestivirus. In this study, eighty-nine pestivirus isolates that were collected in Brazil between 1995 and 2014 and that originated from either cattle, fetal bovine serum (FBS) or as cell culture contaminants were genotyped based on a comparison of gene sequences from their 5' untranslated regions (5'UTR), N-terminal autoprotease (N pro ) and envelope glycoprotein 2 (E2). Of these isolates, 53.9% of the sequences were genotyped as BVDV-1, 33.7% as BVDV-2 and 12.4% as HoBi-like pestivirus. The prevalence of subgenotypes within the species was as follows: BVDV-1a (35.9%), BVDV-2b (31.4%), BVDV-1b (10.1%), BVDV-1d (6.7%), BVDV-2c (2.2%) and BVDV-1e (1.1%). BVDV-2c and BVDV-1e were detected for the first time in Brazil. This study revealed extensive genetic diversity among Brazilian pestivirus isolates, and the combination of pestiviruses that was detected is unique to Brazil. This information may serve as a foundation for designing and evaluating diagnostic tools and in the development of more effective vaccines; therefore, it may potentially contribute to pestivirus control and eradication., (© 2015 Blackwell Verlag GmbH.)

In this study, we constructed for the first time a full-length cDNA clone of pig-original bovine viral diarrhea virus 2 (BVDV-2) strain SH-28, modified the cDNA clone (pASH28) for mutant pASHΔN pro and derived virus strain vASHΔN pro by deleting the genomic region encoding the N pro polypeptide, and examined significance of protein N pro for antiviral responses in vitro. Data showed that N pro -deletion mutant virus vASHΔN pro led to significant overexpression of oligo adenylate synthetase (OAS), myxovirus-resistant protein 1 (Mx1), and ubiquitin-like protein 15 (ISG15). Data also revealed that overexpression of N pro , but not NS2 and NS3 proteins, resulted in significant down-regulation of OAS, Mx1, and ISG15 production (p ≤ 0.05) in bovine cells as well as porcine cells transfected with N pro recombinant eukaryotic expression plasmids. N pro (but not NS2 and NS3) was also found to inhibit poly(IC) from inducing production of type I interferon (IFN-I). These results indicated that protein N pro may play multiple roles in regulating antiviral response in host cells interfered by pig BVDV-2 strain, and provided useful information to understand better the mechanism of BVDV-2 persistent infection in pigs.

Bovine viral diarrhea virus (BVDV) plays a key role in bovine respiratory disease complex, which can lead to pneumonia, diarrhea and death of calves. Current vaccines are not very effective due, in part, to immunosuppressive traits and failure to induce broad protection. There are diverse BVDV strains and thus, current vaccines contain representative genotype 1 and 2 viruses (BVDV-1 & 2) to broaden coverage. BVDV modified live virus (MLV) vaccines are superior to killed virus vaccines, but they are susceptible to neutralization and complement-mediated destruction triggered by passively acquired antibodies, thus limiting their efficacy. We generated three novel mosaic polypeptide chimeras, designated NproE2123; NS231; and NS232, which incorporate protective determinants that are highly conserved among BVDV-1a, 1b, and BVDV-2 genotypes. In addition, strain-specific protective antigens from disparate BVDV strains were included to broaden coverage. We confirmed that adenovirus constructs expressing these antigens were strongly recognized by monoclonal antibodies, polyclonal sera, and IFN-γ-secreting T cells generated against diverse BVDV strains. In a proof-of-concept efficacy study, the multi-antigen proto-type vaccine induced higher, but not significantly different, IFN-γ spot forming cells and T-cell proliferation compared to a commercial MLV vaccine. In regards to the humoral response, the prototype vaccine induced higher BVDV-1 specific neutralizing antibody titers, whereas the MLV vaccine induced higher BVDV-2 specific neutralizing antibody titers. Following BVDV type 2a (1373) challenge, calves immunized with the proto-type or the MLV vaccine had lower clinical scores compared to naïve controls. These results support the hypothesis that a broadly protective subunit vaccine can be generated using mosaic polypeptides that incorporate rationally selected and validated protective determinants from diverse BVDV strains. Furthermore, regarding biosafety of using a live vector in cattle, we showed that recombinant human adenovirus-5 was cleared within one week following intradermal inoculation., Competing Interests: The authors have declared that no competing interests exist.

Bovine viral diarrhea virus (BVDV) is classified into 2 genotypes, BVDV-1 and BVDV-2, each of which contains distinct subtypes with genetic and antigenic variation. To effectively control BVDV by vaccination, it is important to know which subtypes of the virus are circulating and how their prevalence is changing over time. Accordingly, the purpose of our study was to estimate the current prevalence and diversity of BVDV subtypes from persistently infected (PI) beef calves in the central United States. Phylogenetic analysis of the 5'-UTR (5' untranslated region) for 119 virus strains revealed that a majority (82%) belonged to genotype 1b, and the remaining strains were distributed between genotypes 1a (9%) and 2 (8%); however, BVDV-2 subtypes could not be confidently resolved. Therefore, to better define the variability of U.S. BVDV isolates and further investigate the division of BVDV-2 isolates into subtypes, complete genome sequences were obtained for these isolates as well as representatives of BVDV-1a and -1b. Phylogenetic analyses of the complete coding sequence provided more conclusive genetic classification and revealed that U.S. BVDV-2 isolates belong to at least 3 distinct genetic groups that are statistically supported by both complete and individual coding gene analyses. These results show that a more complex set of BVDV-2 subtypes has been circulating in this region than was previously thought., (© 2016 The Author(s).)

Bovine viral diarrhea virus (BVDV) is classified into two species, namely, Bovine viral diarrhea virus 1 and Bovine viral diarrhea virus 2, and affects cattle worldwide, resulting in significant economic loss. The prevalence of BVDV-1 and BVDV-2 infections and its genotypes in Mongolian animals has not been studied. In this study, we surveyed BVDV infection in dairy cattle and yaks from Bornuur and Bulgan counties by RT-PCR, and the average infection rate in the sampling sites was 15.8 % and 20.0 %, respectively. In addition, molecular features of the 5'-UTR region of the BVDV genome in Mongolian cattle and yaks were identified as belonging to the subtypes BVDV-1a and BVDV-2a, respectively. Determining the prevalence, geographical distribution, and molecular diversity of BVDV-1 and BVDV-2 in various host species in Mongolia is important for further studies and process control programs.

Bovine viral diarrhea virus (BVDV) is one of the most important viral pathogens of livestock and causes substantial economic losses to the livestock industry worldwide. BVDV is not necessarily species specific and is known to infect domesticated and wild ruminants. In the present study, BVDV infection was identified in two Saanen goats from one farm, and two different viral subtypes were found, BVDV-1a and BVDV-2a. Each isolate was closely related to cattle isolates identified in the Republic of Korea. The two sequences obtained in this study were not consistent with border disease virus (BDV). The incidence of BVDV in this farm apparently occurred in the absence of contact with cattle and may be associated with grazing. This study demonstrates that BVDV infection may be possible to transmit among goats without exposure to cattle. Therefore, this result indicates that Saanen goats may act as natural reservoirs for BVDV. This is the first report of BVDV-1a infection in a Saanen goat.