Upregulation of interferon-alpha gene in bovine embryos produced in vitro in response to experimental infection with noncytophatic bovine-viral-diarrhea virus.

In-vitro fertilization is a routine livestock-breeding technique widely used around the world. Several studies have reported the interaction of bovine viral-diarrhea virus (BVDV) with gametes and in-vitro-produced (IVP) bovine embryos. Since, gene expression in BVDV-infected IVP bovine embryos is scarcely addressed. The aim of this work was to evaluate the differential expression of genes involved in immune and inflammatory response. Groups of 20-25 embryos on Day 6 (morula stage) were exposed (infected) or not (control) to an NCP-BVDV strain in SOF medium. After 24 h, embryos that reached expanded blastocyst stage were washed. Total RNA of each embryo group was extracted to determine the transcription levels of 9 specific transcripts related with antiviral and inflammatory response by SYBR Green real time quantitative (RT-qPCR). Culture media and an aliquot of the last embryos wash on Day 7 were analyzed by titration and virus isolation, respectively. A conventional PCR confirmed BVDV presence in IVP embryos. A significantly higher expression of interferon-α was observed in blastocysts exposed to NCP-BVDV compared to the controls (p < 0.05). In this study, the upregulation of INFα and TLR7 genes involved in inflammatory and immune response in BVDV-infected IVP bovine embryos is a new finding in this field. This differential expression suggest that embryonic cells could function in a manner like immune cells by recognizing and responding early to interaction with viral pathogens. These results provide new insights into the action of BVDV on the complex molecular pathways controlling bovine early embryonic development.

Genomic evolution of bovine viral diarrhea virus based on complete genome and individual gene analyses.

Bovine viral diarrhea virus (BVDV) genome consists of a single-stranded, positive-sense RNA with high genetic diversity. In the last years, significant progress has been achieved in BVDV knowledge evolution through phylodynamic analysis based on the partial 5'UTR sequences, whereas a few studies have used other genes or the complete coding sequence (CDS). However, no research has evaluated and compared BVDV evolutionary history based on the complete genome (CG), CDS, and individual genes. In this study, phylodynamic analyses were carried out with BVDV-1 (Pestivirus A) and BVDV-2 (Pestivirus B) CG sequences available on the GenBank database and each genomic region: CDS, UTRs, and individual genes. In comparison to the CG, the estimations for both BVDV species varied according to the dataset used, pointing out the importance of considering the analyzed genomic region when concluding. This study may provide new insight into BVDV evolution history while highlighting the need to increase the available BVDV CG sequences to perform more comprehensive phylodynamic studies in the future.

Temporal and geographic dynamics of bovine viral diarrhea virus in American countries.

Bovine viral diarrhea virus (BVDV) is a worldwide distributed pathogen of livestock classified into three species, BVDV-1 (Pestivirus A), BVDV-2 (Pestivirus B), and HoBi-like pestivirus (HoBiPeV; Pestivirus H). Despite being considered endemic in several regions of the Americas, the spatiotemporal distribution of BVDV is scarcely known. This study aimed to reconstruct the population dynamics of BVDV in American countries. The analyses performed with the partial 5´UTR gene showed that BVDV-1 and -2 would have started their diversification in the 1670s and 1790s in the United States, whereas HoBiPeV probably emerged in the 1980s in Brazil. No evident geographic clustering was observed in the Bayesian trees, which may indicate that multiple introductions events would have occurred following the first introduction. This study provides new insights into BVDV dynamics, although further analyses including sequences from other American countries and continents will help to expand the knowledge of BVDV evolution and transmission.

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

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

Frequency of bovine viral diarrhea virus (BVDV) in Argentinean bovine herds and comparison of diagnostic tests for BVDV detection in bovine serum samples: a preliminary study.

Bovine viral diarrhea (BVD) is a major worldwide disease with negative economic impact on cattle production. Successful control programs of BVD require the identification and culling of persistently infected (PI) animals with bovine viral diarrhea virus (BVDV). A variety of diagnostic tests are available to detect BVDV, but no comparison has been performed among those tests in Argentina. Sera collected from 2864 cattle, belonging to 55 herds from three Argentinean provinces, were analyzed by nested RT-PCR (RT-nPCR) to detect BVDV for diagnostic purposes. Additionally, this study evaluated the agreement of the RT-nPCR along with virus isolation, antigen-capture ELISA, and real-time RT-PCR for BVDV detection in archived bovine serum samples (n = 90). The RT-nPCR was useful for BVDV detection in pooled and individual serum samples. BVDV was detected in 1% (29/2864) of the cattle and in 20% (11/55) of the herds. The proportion of BVDV-positive sera was not statistically different among the tests. In addition, comparisons showed high agreement levels, with the highest values between both RT-PCR protocols. The frequency of BVDV infection at individual and herd level was lower than the reported values worldwide. Since follow-up testing was not performed, the frequency of PI cattle was unknown. Also, this study demonstrated that the four diagnostic tests can be used reliably for BVDV identification in individual serum samples. Further epidemiologically designed studies that address prevalence, risk factors, and economic impact of BVDV in Argentina will be necessary to implement effective control programs.

Detection methods and characterization of bovine viral diarrhea virus in aborted fetuses and neonatal calves over a 22-year period.

Detection of bovine viral diarrhea virus (BVDV) in aborted fetus samples is often difficult due to tissue autolysis and inappropriate sampling. Studies assessing different methods for BVDV identification in fetal specimens are scarce. The present study evaluated the agreement between different diagnostic techniques to detect BVDV infections in specimens from a large number of bovine aborted fetuses and neonatal deaths over a period of 22 years. Additionally, genetic, serological, and pathological analyses were conducted in order to characterize BVDV strains of fetal origin. Samples from 95 selected cases from 1997 to 2018 were analyzed by antigen-capture ELISA (AgELISA), nested RT-PCR (RT-nPCR), and real-time RT-PCR (RT-qPCR). In addition, amplification and sequencing of the 5'UTR region were performed for phylogenetic purposes. Virus neutralization tests against the BVDV-1a, BVDV-1b, and BVDV-2b subtypes were conducted on 60 fetal fluids of the selected cases. Furthermore, the frequency and severity of histopathological lesions were evaluated in BVDV-positive cases. This study demonstrated that RT-nPCR and RT-qPCR were more suitable than AgELISA for BVDV detection in fetal specimens. However, the agreement between the two RT-PCR methods was moderate. The BVDV-1b subtype was more frequently detected than the BVDV-1a and BVDV-2b subtypes. Neutralizing antibodies to any of the three subtypes evaluated were present in 94% of the fetal fluids. Microscopically, half of the BVDV-positive cases showed a mild non-suppurative inflammatory response. These results emphasize the need to consider different methods for a diagnostic approach of BVDV associated to reproductive losses.

Interaction of bovine viral diarrhea virus with bovine cumulus-oocyte complex during IVM: Detection in permissive cells.

Structural changes in the zona pellucida (ZP) of bovine oocytes seem to modulate their interaction with various viral agents, facilitating the viral infection in in vitro production systems. To evaluate the susceptibility of bovine oocytes to noncytopathogenic bovine viral diarrhea virus (ncp-BVDV), cumulus-oocyte complexes were exposed to 10(7) ​tissue culture-infective doses (TCID50)/mL of an ncp-BVDV strain during IVM (in vitro maturation). After that, cumulus cells and the ZP were removed by hyaluronidase and pronase treatment, respectively, and the percentages of oocytes with polar body were analyzed as a sign of nuclear maturation. After passage through cell culture, the virus was isolated from granulosa cells, ZP-free mature oocytes, and ZP-intact mature oocytes. These results were confirmed by reverse transcription-polymerase chain reaction. After consecutive washes, the virus remained associated with ZP-free oocytes, maintaining its replication and infectivity in permissive cells. Based on these findings, it is concluded that the classical viral isolation procedure has a predictive value to detect BVDV associated with ZP-free oocytes and that it was novelty demonstrated that both washing and trypsin treatment of oocytes were ineffective to remove BVDV infection.