Molecular Characterization of the First Bovine Herpesvirus 4 (BoHV-4) Strains Isolated from In Vitro Bovine Embryos production in Argentina

Abstract

Bovine herpesvirus 4 (BoHV-4) is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as "starting material" for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these strains circulate in the herds.

Fig 1. Cytopathic effect induced by BoHV-4 strains from bovine ovary batches on MDBK cells.

Cytopathic effect associated with BoHV-4 infections. Light microscope (10X).

Fig 2. Phylogenetic analysis of the BoHV-4 strains studied here.

(A) Obtained from glycoprotein B gene sequence. (B) Obtained from thymidine kinase gene sequence. Branch lengths are proportional to genetic distances (the scale bar units are substitutions per aligned position). Numbers close to nodes correspond to bootstrap supports (n = 100); non-significant bootstrap supports are not shown. The trees were midpoint rooted.

Fig 3. Bayesian Evidence for the BoHV-4 sequences studied here.

(A) Mirrored maximum clade credibility trees. (B) Times to most recent common ancestor (tMRCA) posterior densities, medians, and 95% HPD intervals of the studied strains are shown. Branch lengths are proportional to median node heights. The links connecting terminal nodes are colored according to which genotype the corresponding strains belong to (tan Genotype 1; light blue Genotype 2). The link connecting terminals corresponding to strain 08_404 are dashed and gray colored to indicate uncertainty in genotype assignment. Ov strains isolated from ovaries (granulosa cells and oocyte_h3); Gt1 Genotype 1; gB glycoprotein B; TK thymidine kinase.