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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 BPI

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.

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 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.

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.

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, 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. Electronic supplementary material The online version of this article (10.1007/s42770-019-00170-7) contains supplementary material, which is available to authorized users.

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 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.

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.

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

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.

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

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

Bovine viral diarrhea virus (BVDV) is an important pathogen causally associated with morbidity and mortality, and production losses in both suckling and weaned beef calves. Vaccination for protection against disease caused by BVDV is challenging because of the inhibitory effect of maternal antibodies; however, it is most convenient for beef producers to vaccinate calves before moving herds to summer pastures. We compared modified live and inactivated vaccines used for priming and boosting beef calves and found that neither type of vaccine results in strong antibody responses in the face of maternal antibodies. These data are generally consistent with previous studies and suggest that alternative protocols using early mucosal delivery followed by parenteral boosting should be examined to improve vaccine efficacy.

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&ndash, 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 ERNS in three of the four isolates tested. In the fourth isolate, the exchange of a cysteine at position 441 in the ERNS resulted in a loss of ERNS 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&ndash, receptor interaction.

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 Npro-Erns 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.

Made available in DSpace on 2018-12-11T17:38:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-11-01 Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus and can cause reproductive problems in cattle. However, there is still a lack of research to clarify its pathogenicity in different gestational periods of sows and its effects in neonates. In this study, 12 gilts divided into groups (G) were experimentally inoculated with the strain BVDV-2 (SV-253) oronasally at a dose of 106·85 TCID50; one group was inoculated 30 days before insemination (G0; n = 2), three groups were inoculated during gestation (first (G1; n = 2), second (G2; n = 3), third (G3; n = 3)), and a fourth was the control group (G4; n = 2). Samples of blood and nasal swabs from the gilts were collected every three days until delivery for a virus neutralization (VN) test, qRT-PCR, and blood count. On the day of delivery, 40% of the neonates were euthanized to obtain tissue and blood samples at necropsy for histopathology and qRT-PCR. The sows were seroconverted between 12 and 33 days after inoculation, and the virus was detected in the blood between 3 and 12 days and on the nasal swab between 6 and 24 days in the G0, G1, G2 and G3 sows but was not detected in piglet tissues, and no significant alterations were found through histopathology. The mean and standard deviation of the mean cycles (Cq) from blood (Cq = 34.87 ± 0.60) and nasal swab (Cq = 34.61 ± 0.87) samples were between 107 and 490 TCID50/ml. Transient infection was demonstrated with a low viral load, but transplacental infection was not possible in gilts. School of Agricultural and Veterinarian Sciences (FCAV) São Paulo State University (Unesp), Via de Acesso Prof. Paulo Donato Castellane s/n Biological Institute of São Paulo, Av. Conselheiro Rodrigues Alves, 1252 - Vila Mariana School of Agricultural and Veterinarian Sciences (FCAV) São Paulo State University (Unesp), Via de Acesso Prof. Paulo Donato Castellane s/n

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.

The objective was to evaluate the effects of injectable trace minerals (ITM) concurrent with modified-live virus (MLV) vaccination on protection from bovine viral diarrhea virus (BVDV) infection in dairy calves. In a previous study (Palomares et al., 2016), thirty dairy calves received two doses of a MLV vaccine subcutaneously (SC), concurrently with ITM (n = 15) or saline (n = 15), SC. Five months later, 20 of these calves received ITM (G1, n = 10) or saline (G2, n = 10) according to their previous groups and were challenged intranasally with BVDV2. Five unvaccinated calves were also challenged with BVDV2 (G3). Blood samples were collected on days 0 (BVDV challenge), 3, 5, 6, 7, 8, 9, 11, 14, 18, 21, 32 and 61 for leukocyte count, virus isolation and BVDV serum neutralizing antibodies (SNA). Mild-moderate clinical signs were observed in G3 after BVDV challenge. Group 1 showed lower sum health score and nasal score on d5 and fecal score on d8 compared to G2. Rectal temperature and leukocyte counts were not different between G1 and G2. In contrast, G3 calves had significant leukopenia and lymphopenia from d3 to d7 (P .05) and higher rectal temperatures on d6 to d8, compared to values on d0 (P .05). All unvaccinated calves became viremic, while viremia was not detected in G1 or G2. Average daily gain was not different between vaccinated groups, however, only G1 calves had significantly greater (P = .04) ADG compared to non-vaccinated calves during the first 14 days post challenge. Vaccinated calves treated or not with ITM were protected from BVDV2 infection five months post-vaccination.

Made available in DSpace on 2018-12-11T17:37:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-07-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 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. São Paulo State University (Unesp) School of Agricultural and Veterinarian Sciences (FCAV), Via de Acesso Prof. Paulo Donato Castellane s/n Biological Institute of São Paulo, Av. Conselheiro Rodrigues Alves, 1252 - Vila Mariana São Paulo State University (Unesp) School of Agricultural and Veterinarian Sciences (FCAV), Via de Acesso Prof. Paulo Donato Castellane s/n FAPESP: 2016/21421-2 CNPq: 409435/2016-3

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.

Naive pregnant cattle exposed to pestiviruses between 40-125 days of gestation can give birth to persistently infected (PI) calves. Clinical presentation and survivability, in PI cattle, is highly variable even with the same pestivirus strain whereas the clinical presentation in acute infections is more uniform with severity of symptoms being primarily a function of virulence of the infecting virus. The aim of this study was to compare thymic depletion, as measured by comparing the area of the thymic cortex to the medulla (corticomedullary ratio), in acute and persistent infections of the same pestivirus isolate. The same general trends were observed with each pestivirus isolate. Thymic depletion was observed in both acutely and persistently infected calves. The average thymic depletion observed in acutely infected calves was greater than that in age matched PI calves. PI calves, regardless of infecting virus, revealed a greater variability in amount of depletion compared to acutely infected calves. A trend was observed between survivability and depletion of the thymus, with PI calves surviving less than 5 weeks having lower corticomedullary ratios and greater depletion. This is the first study to compare PI and acutely infected calves with the same isolates as well as to evaluate PI calves based on survivability. Further, this study identified a quantifiable phenotype associated with potential survivability.

Bovine viral diarrhea (BVD) is one of the most economically important diseases of cattle. With its very high prevalence, cattle kept on pastures become a source of the virus for the wildlife which, due to their susceptibility, then easily can serve as a source for re-infections of cattle. Therefore, we investigated the BVDV infection in Serbian wild boar and assessed the role of wild boar in BVDV epidemiology including possible spreading to domestic species. This study was based on examination of 50 spleen samples which were collected from wild boars located in Eastern Serbia during the hunting season 2016/2017. BVDV genome was detected in 4 of 50 samples (8%). Phylogenetic analysis based on 5'UTR revealed that BVDV strains from wild boars shared 100% identity. Belonging to the BVDV 1f subgenotype, the most common in cattle, we showed that BVDV infections of wild boar occurred as a result of either direct or indirect contact with domestic animals. Therefore, the occurrence of infectious disease in wildlife emphasizes the need to study the pathogens shared by wildlife and domestic animals by investigating the incidence of pathogens and disease patterns of those populations.

This study was designed to determine whether congenital persistent infection occurs in piglets from gilts experimentally inoculated with bovine viral diarrhea virus type 2 (BVDV-2). Six pregnant gilts were divided into 2 groups, infected (n = 4), and control (n = 2). The gilts were inoculated at 45 days gestation. Piglets were assessed for 35 days following birth with nasal swab and blood sample collections every 72 hours. Reverse transcriptase-polymerase chain reaction (RT-PCR) tests were performed for direct diagnosis of virus in blood and nasal swabs, and virus neutralization was used for antibody detection. Transplacental transmission of BVDV-2 did not occur. Piglets were born free of the virus and did not shed BVDV during the experimental period.

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.

Strategies to improve the onset of protective immunity induced by vaccination against respiratory pathogens may have a significant impact on health of newly received beef calves. The objective was to determine if the use of injectable trace minerals (ITM; Se, Zn, Cu, and Mn) concurrent with a modified-live virus (MLV) vaccine enhances the immune response and onset of protection in beef calves challenged with BVDV2 five days after vaccination. Forty-five calves were randomly assigned to one of three groups (15/group): VAC + ITM, received MLV-vaccine and ITM (Multimin®90) subcutaneously (SC); VAC + SAL, received the same vaccine and saline SC; or UNVAC, unvaccinated. Five days after vaccination (d.0), calves were challenged with BVDV2 strain 890. Health status was evaluated and blood samples were collected for leukocyte counts, BVDV1 and 2 serum neutralizing antibodies (SNA), BVDV-PCR, and percentage of CD4+, CD8+, WC1+ and CD25+ T-cells. VAC + ITM had lower health scores than UNVAC (d.8 and 9). VAC + ITM had higher BVDV1 & 2 SNA titers than VAC + SAL and UNVAC on d.21 and 28. Lymphocyte counts decreased in UNVAC but not in VAC + ITM or VAC + SAL (d.3 to 11). CD4+ T-cells significantly decreased in UNVAC and VAC + SAL (d.3). VAC + ITM had higher percentage of CD4+ T-cells than UNVAC (d.3 and 7). VAC + ITM had lower percentage of activated CD4+ and CD8+ T-cells than UNVAC (d.7). In summary, vaccination induced a rapid protection against BVDV2 infection. Administration of ITM was associated with increased SNA response to BVDV1 & 2, enhanced health status, mitigation of CD4+ T-cells decrease, and reduction of T-cell activation in calves challenged with BVDV2 five days after immunization. These results support the strategic use of ITM concurrent with vaccination, especially when a rapid protection is needed in newly received beef calves.

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.

Four species of ruminant pestivirus are currently circulating in the United States: Bovine viral diarrhea virus 1 and 2 (BVDV-1, -2; predominant host: cattle), Border disease virus (BDV; predominant host: sheep), and pronghorn virus (sporadically detected in wild ruminants). A third bovine pestivirus called HoBi-like virus has been detected in cattle and water buffalo in South America, Asia, and Europe. To date, no isolations of HoBi-like viruses from U.S. cattle have been reported. To assess exposure, 2,000 cattle sera, collected between 2014 and 2015 as part of the U.S. brucellosis surveillance program, were tested for antibodies against BVDV-1, BVDV-2, and HoBi-like viruses. In addition, RNA was extracted and tested by reverse transcription–polymerase chain reaction for the presence of pestiviruses; all samples tested negative. The percent of VN-positive samples was 91.3% for BVDV-1, 89.3% for BVDV-2, and 84.9% for HoBi-like viruses. Because the 3 bovine pestiviruses are antigenically cross-reactive, the comparative level of antibody against each pestivirus species was determined. Based on comparative titers, samples were segregated into 6 categories: no titers (7.6%), titers clearly higher against BVDV-1 (22.2%), titers substantially higher against BVDV-2 (9.1%), BVDV-1 and BVDV-2 titers equivalent but substantially higher than HoBi titers (25.7%), titers substantially higher against HoBi-like viruses (0%), and equivocal (35.4%). Titers tended to be higher against BVDV-1 than BVDV-2. However, the overall percentage of animals with titers below levels considered protective against acute bovine pestivirus infection were ~11% for BVDV-1, 12% BVDV-2, and 18% for HoBi-like virus.

Subclinical illness associated with infection is thought to reduce performance and increase production costs in feedlot cattle, but underlying components remain largely unidentified. Vaccination is frequently used in feedlot settings but producers lack metrics that evaluate the effectiveness of vaccination programs. The goal of this study was to determine if levels of serum neutralizing antibody titers were predictive of levels of vaccine protection in a commercial setting. During this four-year study, Angus-Nellore steers housed in a production feedlot setting were assigned to 1 of 3 vaccine treatments: killed vaccine (kV), modified live virus (MLV) vaccine, or no vaccine (control), and were challenged with a noncytopathic 1b field strain of bovine viral diarrhea virus. Rectal temperature and levels of circulating lymphocytes and platelets were monitored following challenge. While no animals were diagnosed as clinically ill with respiratory disease, indicators of disease (pyrexia, lymphopenia, and thrombocytopenia) were observed. The MLV treatment elicited higher antibody titers to the vaccination than the kV, and calves in the MLV treatment had higher mean titers at challenge. The year that elicited the highest antibody response to the vaccination and the year with the lowest frequency of phenotypic responses to the challenge were not concurrent. The MLV treatment had the highest proportion, 34.68%, of animals that were protected against the challenge regardless of the pre-challenge antibody titer and had the fewest number of lymphopenia cases in response to the challenge. Both vaccine treatments mitigated thrombocytopenia when compared to the control treatment, and the MLV treatment reduced lymphopenia; however, these symptoms were not completely eliminated in vaccinated animals. Pyrexia was present in 40.11% of the animals, but no difference in the frequency of cases between treatments was observed. Pre-challenge vaccination response was not indicative of the level of protection nor was anamnestic antibody response correlated with health status.

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.

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.

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

Bovine 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.

Bovine viral diarrhea virus (BVDV), a prominent viral pathogen worldwide, causes substantial economic losses in the cattle industry. BVDV comprises two recognized species, BVDV-1 and BVDV-2, and at least 21 subtypes (1a–1u) for BVDV-1 and four subtypes (2a–2d) for BVDV-2 based on its 5′-untranslated region. This study aimed at investigating the prevalence and genetic analysis of BVDV in calf feces in the Republic of Korea (ROK). We collected fecal samples from 635 pre-weaned native Korean calves aged 1–60 days, regardless of diarrhea, and subjected them to RT-PCR and phylogenetic analysis. Thirty-five (5.5%) of the 635 samples were positive for BVDV infection. BVDV was detected in 20, 10, and 5 calves aged 1–20 days, 21–40 days, and 41–60 days, respectively. BVDV was the most frequent in 17 normal feces, followed by 16 diarrheic feces, and 2 hemorrhagic feces. Phylogenetic analysis revealed that 25 samples belonged to BVDV-1b; 1 sample, BVDV-1c; and 9 samples, BVDV-2a. Moreover, the BVDV-1b and BVDV-2a isolates showed genetic variations. BVDV-1b was detected in diarrheic, hemorrhagic, and normal fecal samples. Thus, BVDV-1b is the most prevalent in calves and causes enteric disease with differing severity. BVDV-1c was newly identified in diarrheic calves. Further studies are warranted to elucidate the pathogenesis of BVDV-1c infection and its clinical manifestations. Our results indicate that effective vaccines and control programs against BVDV are required in the ROK.

Current methods for evaluating bovine viral diarrhea virus (BVDV) vaccination response typically rely on measurement of humoral responses as determined by virus neutralizing antibody titers (VNT) against BVDV. While VNT are correlated with increased protection, research has also shown that cell mediated immunity (CMI) is an important component of a protective response against BVDV. For example, improved protection against BVDV by modified-live viral (MLV) vaccines as compared to killed vaccines is thought to be due to better CMI induced by the MLV. The goal of this work was to evaluate the cell mediated response in vaccinated calves using a novel PrimeFlow RNA assay that incorporates cell surface marker staining with intracellular RNA expression of cytokines and viral RNA detection. Results from this study evaluating mRNA for IFN-γ and IL-2 at 24 h post-BVDV stimulation are similar to previous studies in which IFN-γ was detected in the CD4+ and CD8+ T cell population. However, a novel observation was the detection of IFN-γ mRNA in the NK cell population in vaccinated animals. The NK cell population contributed a significant portion of the IFN-γ produced. This study also demonstrated a decrease in the frequency and amount of BVDV in PBMCs, harvested from vaccinated calves and exposed to BVDV in vitro. Collectively data from this study highlights the association between an increase in IFN-γ and a decreased infection rate of isolated PBMC's, based on the frequency and amount of BVDV positive cells following in vitro exposure. This new method combines not only the ability to evaluate cellular responses, but also the ability to understand potential antiviral properties associated with cellular responses. This is the first assay to describe and simultaneously measure CMI responses and intracellular viral RNA quantity as a method to evaluate protective responses associated with vaccination.

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.

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.

Background Bovine viral diarrhea virus (BVDV) causes significant economic losses worldwide in the cattle industry through decrease in productive performance and immunosuppression of animals in herds. Recent studies conducted by our group showed that mice can be infected with BVDV-1 by the oral route. The purpose of this study was to assess the clinical signs, hematological changes, histopathological lesions in lymphoid tissues, and the distribution of the viral antigen after oral inoculation with a Korean noncytopathic (ncp) BVDV-2 field isolate in mice. Methods Mice were orally administered a low or high dose of BVDV-2; blood and tissue samples were collected on days 2, 5, and 9 postinfection (pi). We monitored clinical signs, hematological changes, histopathological lesions, and tissue distribution of a viral antigen by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) and then compared these parameters with those in ncp BVDV-1 infections. Results None of the infected mice developed any clinical signs of the illness. Significant thrombocytopenia was found in both low- and high-dose-inoculated mice on day 2 pi. Leukopenia was apparent only in low-dose-inoculated mice on day 2 pi, whereas lymphopenia was not observed in any ncp BVDV-2-infected animal. Viral RNA was found in the spleen in of low- and high-dose-inoculated mice by RT-PCR. According to the results of IHC, the viral antigen was consistently detected in lymphocytes of bone marrow and spleen and less frequently in bronchus-associated lymphoid tissue (BALT), mesenteric lymph nodes, and Peyer’s patches. Despite the antigen detection in BALT and mesenteric lymph nodes, histopathological lesions were not observed in these tissues. Lympholysis, infiltration by inflammatory cells, and increased numbers of megakaryocytes were seen in Peyer’s patches, spleens, and bone marrow, respectively. In contrast to ncp BVDV-1 infection, lympholysis was found in the spleen of ncp BVDV-2-infected mice. These histopathological lesions were more severe in high-dose-inoculated mice than in low-dose-inoculated mice. Conclusions Our results provide insight into the pathogenesis of ncp BVDV-2 infection in mice. Collectively, these results highlight significant differences in pathogenesis between ncp BVDV-1 and ncp BVDV-2 infections in a murine model.

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

OBJECTIVE To determine the effect of a commercially available multivalent killed virus vaccine on serum neutralizing (SN) and colostrum neutralizing (CN) antibodies against bovine herpesvirus (BHV) type 1 and bovine viral diarrhea virus (BVDV) types 1 and 2 in pregnant dairy cattle. ANIMALS 49 Holstein dairy cattle. PROCEDURES 25 cattle were vaccinated (IM injection) at least 60 days prior to calving (ie, at the end of the lactation period or according to the expected calving date for heifers) and again 5 weeks later. The remaining 24 cattle were not vaccinated (control group). Titers of SN antibodies were measured at the 5-week time point. Titers of SN and CN antibodies were measured at parturition. RESULTS 5 weeks after initial vaccination, titers of SN antibodies against BHV-1 and BVDV types 1 and 2 were 1:512, 1:128, and 1:2,048, respectively, in vaccinates and 1:64, 1:128, and 1:64, respectively, in unvaccinated controls. Equivalent SN antibody titers at parturition were 1:256, 1:64, and 1:512, respectively, in vaccinates and 1:128, 1:128, and 1:64, respectively, in controls. Median titers of CN antibodies against BHV-1 and BVDV types 1 and 2 were 1:1,280, 1:10,240, and 1:20,480, respectively, in vaccinates and 1:80, 1:1,280, and 1:2,560, respectively, in controls. CONCLUSIONS AND CLINICAL RELEVANCE Titers of antibodies against viral respiratory pathogens were significantly enhanced in both serum (BHV-1 and BVDV type 2) and colostrum (BHV-1 and BVDV types 1 and 2) in cattle receiving a killed virus vaccine (with no adverse reactions) before parturition. To maximize protection of bovine neonates, this method of vaccination should be considered.

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 (Npro ) 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.

The aim of this work was to investigate the effect of pre-infection with bovine viral diarrhoea virus (BVDV) on thymus immune cells from calves challenged with bovine herpesvirus 1 (BHV-1). Twelve Friesian calves, aged 8 to 9 months, were inoculated with non-cytopathic BVDV-1. Ten of them were subsequently challenged with BHV-1 and euthanized in batches of two at 1, 2, 4, 7 or 14 dpi with BHV-1. The other two calves were euthanized prior to the second inoculation and were used as BVDV-infected controls. A further 10 calves were inoculated solely with BHV-1 and euthanized at the same time points. Two calves were not inoculated with any agent and were used as negative controls. Quantitative changes in immune cells were evaluated with immunohistochemical methods to compare coinfected calves and calves challenged only with BHV-1. The results of this study pointed out BVDV as responsible for the thymic lesions observed in the experiment as well as for the majority of immunopathologic changes, including a downregulation of Foxp3 lymphocytes and TGFβ, which reverted as BVDV was cleared, and an overexpression of medullary CD8+ T cells. However, despite not inducing evident lesions in the thymus, BHV-1 seemed to prompt some immune alterations. Collectively, these data contribute to the knowledge on the immunopathologic alterations of the thymus during BVDV infections, and its importance in the development of secondary infections., This work was supported by grants from the Junta de Andalucía‐FEDER (project P09‐AGR‐4671). F. Romero‐Palomo was financially supported by a predoctoral grant associated with the aforementioned project.

Aims The aim of the study was the development of cross-priming amplification for ubiquitous detection of bovine viral diarrhoea virus (BVDV) species 1 and 2. Methods and Results Three and five specific primers, respectively, for the detection of BVDV-1 and BVDV-2, were designed on the basis of the sequences of the 5′UTR region. Incubation temperature and reaction time were determined. The optimal incubation conditions using water bath were 63°C for 75 min. Reverse transcription step (RT) was not required. The results were visualized under UV-light as a bright yellow fluorescence in positive samples. Additional method for results interpretation was agarose gel electrophoresis. Positive samples showed the presence of ladder-like banding patterns, formed by harpin-like cross-priming amplification (CPA) products. Sensitivity of CPA was compared with conventional RT-PCR and real-time RT-PCR. The CPA detection limit was 3500 copies for BVDV-1 and 80000 copies for BVDV-2 per reaction. For RT-PCR it was 350 and 80 copies for BVDV-1 and BVDV-2, respectively, and for real-time RT-PCR it was 35 copies for BVDV-1 and 80 copies for BVDV-2. The sensitivity of the developed method is sufficient to detect persistently infected (PI) animals. Positive results were found in 24 of 25 BVDV isolates belonging to species 1 and 2. Additionally, one false-negative result for BVDV-2 was detected. There were no false-positive results in negative samples and in the negative control. Both sets of primers used for the detection of BVDV-1 and BVDV-2 were not able to detect atypical pestiviruses. CPA positive results were confirmed by RT-PCR and real-time RT-PCR. Conclusions CPA is a rapid method for the detection of BVDV-1 and BVDV-2 in field samples from PI animals. Significance and Impact of Study This is the first report on the application of the CPA method for the detection of BVDV.

Infection of water buffaloes (Bubalus bubalis) with bovine viral diarrhea viruses (BVDV) has been confirmed in several studies by serological and molecular techniques. In order to determine the presence of persistently infected animals and circulating species and subtypes of BVDV we conducted this study on a buffalo herd, whose habitat was shared with bovine cattle (Bossp.). Our serological results showed a high level of positivity for BVDV-1 and BVDV-2 within the buffalo herd. The molecular analyses of blood samples in serologically negative animals revealed the presence of viral nucleic acid, confirming the existence of persistent infection in the buffaloes. Cloning and sequencing of the 5′ UTR of some of these samples revealed the presence of naturally mix-infected buffaloes with at least two different subtypes (1a and 1b), and also with both BVDV species (BVDV-1 and BVDV-2).

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

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

Although vaccination is an effective measure in reducing the risk of bovine respiratory disease complex (BRDC) in cattle, BRDC losses remain significant. Increasing the efficacy of vaccination depends on elucidating the protective immune response to different antigens included in vaccines, determining the best timing for vaccination, and understanding the impact of the age of the calf on vaccination. This study measured the serum antibodies present in calves following vaccination against 4 viruses commonly associated with BRDC: bovine viral diarrhea virus type 1 and 2 (BVDV1 and BVDV2), bovine respiratory syncytial virus (BRSV), and bovine herpesvirus 1 (BHV1). Serum antibody titers were measured in more than 1,600 calves at 3-wk intervals starting at the time of the first vaccination. This first vaccination occurred at weaning for approximately half of the individuals and 3 wk before weaning for the other half. Dam age (years), time of weaning (initial vaccination or booster vaccination), and age of calf within year–season (days within year–season) classification all were found to have a significant effect on measured traits such as the initial titer and overall response. An increased initial titer was negatively correlated with each response trait (initial, booster, and overall response). Calves that were weaned at initial vaccination had greater overall antibody response to BVDV1 and BVDV2 compared with calves weaned 3 wk before initial vaccination. In contrast, calves given their initial vaccination 3 wk before weaning had greater overall antibody response to BRSV and BHV1 compared with calves that were vaccinated at weaning. Furthermore, the circulating antibody titer at which each virus needed to be below for an individual calf to positively respond to vaccination was determined (log2 titer of 0.38 for BVDV1, 1.5 for BVDV2, 3.88 for BRSV, and 1.5 for BHV1). This information can be used to improve vaccination protocols to allow for a greater response rate of individuals to vaccination and, hopefully, improved protection.

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.

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