Effect of bovine viral diarrhea virus on subsequent infectivity of bovine gammaherpesvirus 4 in endometrial cells in primary culture: An in vitro model of viral co-infection.

Bovine viral diarrhea virus (BVDV) and bovine gammaherpesvirus 4 (BoHV-4) infect the uterus of cattle, being responsible for huge economic losses. Most of the pathogenesis of BoHV-4 in the bovine reproductive tract has been elucidated by conducting tests on primary cultures. Thus, it is important to have optimal in vitro conditions, avoiding the presence of other pathogens that can alter the results. BVDV is one of the most frequent viral contaminants of cell cultures. Considering that non-cytopathic (NCP) BVDV biotype can generate persistently infected (PI) cattle, which are the major source for virus transmission in susceptible herds, it is important to check products derived from cattle that are intended to be used in research laboratories. The aim of this work was to evaluate how the natural infection of bovine endometrial cells (BEC) with a NCP BVDV strain (BEC + BVDV) affects BoHV-4 replication. We have demonstrated a delay in BoHV-4 gene expression and a decrease in viral load in the extracellular environment in BEC + BDVD cells compared to BEC (BVDV-free) cells. These results confirm that replication of BoHV-4 in BEC primary cultures is affected by previous infection with BVDV. This finding highlights the importance of ruling out BVDV infection in bovine primary cell cultures to avoid biological interference or misinterpretation of results at the time of performing in vitro studies with BoHV-4.

Gene expression and in vitro replication of bovine gammaherpesvirus type 4.

In vitro cell cultures are widely used models for dissecting cellular and molecular mechanisms that lead to certain physiological conditions and diseases. The pathogenesis of BoHV-4 in the bovine reproductive tract has been studied by conducting tests on primary cultures. However, many questions remain to be answered about the role of BoHV-4 in endometrial cells. The aim of this study was to compare the replication and gene expression of BoHV-4 in cell lines and bovine reproductive tract primary cells as an in vitro model for the study of this virus. We demonstrated that BoHV-4 strains differ in their in vitro growth kinetics and gene expression but have the same cell type preference. Our results demonstrate that BoHV-4 replicates preferentially in bovine endometrial cells (BEC). However, its replication capacity extends to various cell types, since all cells that were tested were permissive to BoHV-4 infection. The highest virus titers were obtained in BEC cells. Nevertheless, virus replication efficiency could not be fully predicted from the mRNA expression profiles. This implies that there are multiple cell-type-dependent factors and strain properties that determine the level of BoHV-4 replication. The results of this study provide relevant information about the in vitro behavior of two field isolates of BoHV-4 in different cell cultures. These findings may be useful for the design of future in vitro experiments to obtain reliable results not only about the pathogenic role of BoHV-4 in the bovine female reproductive tract but also in the development of efficient antiviral strategies.

Molecular and antigenic characterization of bovine Coronavirus circulating in Argentinean cattle during 1994-2010.

Bovine coronavirus (BCoV) is an important viral pathogen associated with neonatal calf diarrhea. Our aim was to investigate the incidence of BCoV in diarrhea outbreaks in beef and dairy herds from Argentina during 1994-2010. A total of 5.365 fecal samples from diarrheic calves were screened for BCoV diagnosis by ELISA. The virus was detected in 1.71% (92/5365) of the samples corresponding to 5.95% (63/1058) of the diarrhea cases in 239 beef and 324 dairy farms. The detection rate of BCoV was significantly higher in dairy than in beef herds: 12.13% (29/239) vs. 4.32% (14/324) respectively. Phylogenetic analysis of the hypervariable S1 region of seven representative samples (from different husbandry systems, farm locations and years of sampling) indicated that BCoV strains circulating in Argentinean beef and dairy herds formed a cluster distinct from other geographical regions. Interestingly, Argentinean strains are distantly related (at both the nucleotide and amino acid levels) with the Mebus historic reference BCoV strain included in the vaccines currently available in Argentina. However, Mebus-induced antibodies were capable of neutralizing the BCoV Arg95, a field strain adapted to grow in vitro, and vice versa, indicating that both strains belong to the same CoV serotype reported in cattle. This work represents the first large survey describing BCoV circulation in Argentinean cattle.

Phylogenetic analyses of typical bovine rotavirus genotypes G6, G10, P[5] and P[11] circulating in Argentinean beef and dairy herds.

Group A rotavirus (RVA) is one of the main causes of neonatal calf diarrhea worldwide. RVA strains affecting Argentinean cattle mainly possess combinations of the G6, G10, P[5] and P[11] genotypes. To determine RVA diversity among Argentinean cattle, representative bovine RVA strains detected in diarrheic calves were selected from a survey conducted during 1997-2009. The survey covered the main livestock regions of the country from dairy and beef herds. Different phylogenetic approaches were used to investigate the genetic evolution of RVA strains belonging to the prevalent genotypes. The nucleotide phylogenetic tree showed that all genotypes studied could be divided into several lineages. Argentinean bovine RVA strains were distributed across multiple lineages and most of them were distinct from the lineage containing the vaccine strains. Only the aminoacid phylogenetic tree of G6 RVA strains maintained the same lineages as observed at the nucleotide level, whereas a different clustering pattern was observed for the aminoacid phylogenetic trees of G10, P[5] and P[11] suggesting that the strains are more closely related at the aminoacid level than G6 strains. Association between P[5] and G6(IV), prevalent in beef herd, and between P[11] and G6(III) or G10 (VI and V), prevalent in dairy herds, were found. In addition, Argentinean G6(III), G10, P[5] and P[11] bovine RVA strains grouped together with human strains, highlighting their potential for zoonotic transmission. Phylogenetic studies of RVA circulating in animals raised for consumption and in close contact with humans, such as cattle, contribute to a better understanding of the epidemiology of the RVA infection and evolution.