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.

Bovine herpes virus type-4 infection among postpartum dairy cows in California: risk factors and phylogenetic analysis

“…Detection of BoVH-4 and other co-infecting viruses including bovine viral diarrhoea virus (BVDV) [18], bovine herpes virus-1 (BoHV-1), bovine herpes virus-2 (BoHV-2) and parapox virus (PPV) in uterine/vaginal samples was confirmed by real-time (RT) PCR at the CAHFS laboratory, Davis. The assay targeted three open reading frames [19] of the viral genome (ORF 3 – encoding thymidine kinase, ORF 8 – encoding glycoprotein B and ORF 22 – encoding glycoprotein H). Fifty microlitres of swab fluid or cell culture was extracted (Max 96 Viral RNA Isolation kit (AM 1836-5), Life Technologies, Carlsbad, CA, USA) to prepare samples for RT PCR testing following the recommendations of the manufacturer.…”

“…Detection of BoVH-4 and other co-infecting viruses including bovine viral diarrhoea virus (BVDV) [18], bovine herpes virus-1 (BoHV-1), bovine herpes virus-2 (BoHV-2) and parapox virus (PPV) in uterine/vaginal samples was confirmed by real-time (RT) PCR at the CAHFS laboratory, Davis. The assay targeted three open reading frames [19]…”

“…Whereas TK genotype 2 isolates were more closely related to MGA514 (EU244699) strain than DN 599 and Movar. On the other hand, neither of TK genotype 1 and TK genotype 2 were closely related to 07-435 (JQ838046) and 09-227(JQ838056)the two genetically analogous strains, described from Argentina [19]. Figure 2 presents phylogenetic analysis of BoHV-4 field isolates based on sequencing of gB coding for ORF-3.…”

Comparative analysis of replicative properties of phylogenetically divergent, Argentinean BoHV-4 strains in cell lines from different origins

“…Genotype 1 comprises Movar-like strains (European group), Genotype 2 includes DN599-like strains (American group) and Genotype 3 corresponds to a novel genotype group [26]. Thereafter, the virus was isolated from nasal swabs, brain tissue, granulosa cells, oocytes [28] and bovine semen [29,30].…”

Tissular Distribution of Argentinean Strains of Bovine Herpesvirus Type 4 (BoHV-4) in Experimentally-Infected Calves

“…In Argentina, this viral agent was virtually unknown until its detection and identification in 2007 in samples of cervical-vaginal mucus (CVM) from aborted cows (Verna et al 2008), by the Specialized Veterinary Diagnostic Service of INTA Balcarce. Thereafter, the virus was isolated from nasal swabs, lungs, granulosa cells, oocytes (González Altamiranda et al 2015) and bovine semen (Lomónaco et al 2009, Morán et al 2013). To date, more than 50 isolates have been obtained in our country, mainly associated with abortions in bovine females.…”