Lyle J. Braun

Bovine Viral Diarrhea Virus Multiorgan Infection in Two White-Tailed Deer in Southeastern South Dakota

The susceptibility of wild ruminants, especially cervids, to bovine viral diarrhea virus (BVDV) has remained an enigma. Two white-tailed deer (Odocoileus virginianus) were submitted to the Animal Disease Research and Diagnostic Laboratory (ADRDL) in the fall of 2003 by the South Dakota Game Fish and Parks for chronic wasting disease (CWD) testing. Both animals were CWD negative. The animals were necropsied and histopathology, viral antigen detection, and virus isolation were performed. A noncytopathic (NCP) BVDV was isolated from the lungs and several other tissues of both animals. Formalin-fixed ear notches from both animals were positive for BVDV antigen by immunohistochemistry. The BVDV isolates were typed with the use of polymerase chain reaction in 5′ untranslated region (UTR) and one isolate was typed a Type 2a and the other a Type 1b. Future field surveys to determine the incidence of BVDV along with experimental studies to determine if white-tailed deer fawns can be persistently infected with BVDV are needed.

Detection of bovine herpesvirus-1 in peripheral blood mononuclear cells eight months postinfection.

Peripheral blood mononuclear cells (PBMCs) from 5 calves (3 controls and 2 vaccinates) used in a bovine herpesvirus 1 (BHV-1) vaccine study with a BHV-1 Cooper strain challenge were collected 6 months after challenge. The PBMCs from the control animals were positive by immunofluorescence for the BHV-1 glycoprotein D (gD) while the vaccinates were negative. The PBMC samples from 4 of the 5 animals were examined for BHV-1 DNA by polymerase chain reaction (PCR) and for gD immunofluorescence at 8 months after challenge. The BHV-1 DNA and viral antigen were detected in PBMC samples at 8 months postinfection, but no virus was isolated.

Comparison of the breadth and complexity of bovine viral diarrhea (BVDV) populations circulating in 34 persistently infected cattle generated in one outbreak.

Exposure to bovine viral diarrhea viruses (BVDV) results in acute and persistent infections. Persistent infections result from in utero exposure during the first trimester of gestation. Clinical presentation, in persistently infected cattle (PI), is highly variable. The reasons for this variation is largely unknown. The BVDV circulating in PI exist as quasispecies (swarms of individual viruses). An outbreak resulting in 34 PI cattle presented an opportunity to compare a large number of PI׳s. Methods were developed to compare the circulating viral populations within PI animals. It was found that PI animals generated in the same outbreak carry circulating viral populations that differ widely in size and diversity. Further, it was demonstrated that variation in PI viral populations could be used as a quantifiable phenotype. This observation makes it possible to test the correlation of this phenotype to other phenotypes such as growth rate, congenital defects, viral shed and cytokine expression.

Bovine viral diarrhea virus outbreak in a beef cow herd in South Dakota.

CASE DESCRIPTION 136 pregnant beef cows were purchased in the fall of 2003. The following spring, 128 cows calved as expected; 8 cows were believed to have aborted with the fetuses unavailable for evaluation. Of the 128 calves born, 8 died within 2 weeks after birth and 9 were born with congenital abnormalities. CLINICAL FINDINGS Cows and their calves were evaluated for bovine viral diarrhea virus (BVDV) infection. Forty-four of 120 calves, but 0 cows, tested positive for BVDV antigen by immunohistochemical staining of ear notch specimens. TREATMENT AND OUTCOME Five BVDV test-positive calves died shortly after weaning, and the remaining 39 BVDV test-positive calves were moved to an isolated feedlot and retested for BVDV at 5 to 6 months of age; 36 had positive results, which indicated that they were persistently infected (PI) with BVDV, whereas 3 had negative results, which indicated that they were transiently infected with BVDV at the time of the first test. All PI calves were infected with the same BVDV type 2a strain. As yearlings, 17 of the 36 PI calves died peracutely with lesions consistent with mucosal disease, 6 died without gross lesions, and 2 were euthanized because of chronic ill thrift. The remaining 11 PI calves appeared healthy and were sold for slaughter. Screening of the following years calf crop for BVDV by use of immunohistochemical staining of ear-notch specimens yielded negative results for all calves. CLINICAL RELEVANCE Introduction of BVDV into a naïve cow herd resulted in a loss of 44% of the calf crop subsequent to reproductive loss, poor thrift, and mucosal disease.

Both cytopathic and non-cytopathic bovine viral diarrhea virus (BVDV) induced autophagy at a similar rate

Autophagy is a cellular process that maintains cellular homeostasis by the proteolytic recycling of cytoplasm. Autophagy occurs at basal levels in almost all cells. It is upregulated in cellular stress including starvation, oxidative stress or during infection. Several viruses including flavivirus have developed strategies to subvert or use autophagy for their efficient replication. Bovine viral diarrhea virus (BVDV) is a member of the Flaviviridae family and the pestivirus virus group. BVDV is responsible for significant economic loss in cattle industry worldwide. A unique characteristic of BVDV is the well-characterized genetic changes that can result in two different phenotypes (biotypes) in cell culture: cytopathic (cp) or non-cytopathic (ncp) effects. The ncp viruses are the most prevalent and important for clinical disease. This study was carried out to determine the effect of different BVDV phenotypes using the virus pair, cp TGAC and ncp TGAN in autophagy induction, as well as to investigate the role of autophagy in BVDV induced cytopathic effect. RESULTS showed that both biotypes (cp and ncp) of BVDV induced autophagy in immortal Madin-Darby bovine kidney (MDBK) cell line as well as primary bovine turbinate (Bt) cells following infection. There was no significant difference between cp or ncp strains of BVDV in autophagosome formation (p<0.05) in either MDBK or Bt cells. The autophagy inhibiting drug, 3-methyladenine (3MA) significantly reduced autophagy (p<0.05) as well as viral replication. While autophagy inducing drug rapamycin significantly enhanced autophagy as well as viral replication. The co-localization study using, BVDV NS5A, Erns and E1 proteins with autophagy marker, light chain-3 (LC3) revealed that BVDV replication was associated with autophagosomes. This study revealed that both cp and ncp strains of BVDV induced autophagy at similar level and used autophagy machinery for their replication.

Fetal Hepatic Response to Bovine Viral Diarrhea Virus Infection in Utero

Non-cytopathic bovine viral diarrhea virus (ncp BVDV) can cause persistent infection (PI) in animals infected in utero during early gestation. PI animals shed the virus for life and are the major source of the virus in herds. The mechanism responsible for BVDV immune tolerance in the PI fetus is unknown. We assessed the impact of BVDV infection on the fetal liver. Dams were inoculated with ncp BVDV at gestational day 75. Fetal liver samples were collected at necropsy, 7 and 14 days post-maternal-BVDV inoculation. BVDV antigen was not detected in the liver at gestational day 82 (7 days post-maternal inoculation). However, at 14 days post-maternal inoculation, BVDV was detected by immunohistochemistry in fetal Kupffer cells. Flow cytometry analysis showed a higher percentage of hepatic immune cells expressed MHC I and MHC II in BVDV-infected fetal liver (as compared to uninfected controls). Immunofluorescence was used to identify Kupffer cells, which were positive for BVDV antigen, near populations of CD3+ lymphocytes. The identification of BVDV in the fetal liver Kupffer cells at 14 days post inoculation is interesting in the context of establishment of tolerance in persistent infection. These data indicate the presence of a hepatic immune response to fetal infection.

BVDV Npro protein mediates the BVDV induced immunosuppression through interaction with cellular S100A9 protein

Abstract The innate immune response is a vital part of the bodys antiviral defense system. The innate immune response is initiated by various receptor interactions, including danger associated molecular patterns (DAMPs). The S100A9 is a member of the DAMPs protein family and, is released by activated phagocytic cells such as neutrophils, monocytes, macrophages or endothelial cells, and S100A9 induces its effect through TLR4/MyD88 pathway. Bovine viral diarrhea virus (BVDV) is one of the major devastating disease in the cattle industry worldwide. It shows its effect through immunosuppression and develops persistent infection in calves born from infected cows. The current study revealed that BVDV potentially induced immunosuppression by the interaction of BVDV Npro protein with cellular S100A9 protein. The Inhibition of S100A9 protein expression by small interfering RNA (siRNA) enhanced the virus replication in infected cells. Overexpression of bovine S100A9 enhanced the ncpBVDV2a 1373 mediated Type-I interferon production. A co-immunoprecipitation experiment demonstrated a strong interaction between ncp BVDV2a 1373 Npro protein and cellular S100A9 protein. This suggested that BVDV Npro reduced the S100A9 protein availability/activity in infected cells, resulting in reduced Type-I interferon production. A further study of S100A9-BVDV interaction will be need for better understanding of BVDV pathophysiology.

Evaluation of Responses to Vaccination of Angus Cattle for Four Viruses that Contribute to Bovine Respiratory Disease Complex

Initial antibody titers are maternally-derived from colostrum, then decay with age. Change in antibody titer levels were compared between four viruses contributing to the Bovine Respiratory Disease Complex (BRDC), and evaluation of response to vaccination indicated that antibody production will not occur when high levels of maternal antibodies are present. The maternal antibodies were found to decay with calf age for each of the four viruses, which allowed for the estimation of a maximum circulating titer level under which a positive antibody response to vaccination could occur. Phenotypic correlations were calculated between the antibody titers for the four viruses across multiple time points. Results indicate a difference in the response to vaccination between the four virus antigens