Construction and manipulation of a full-length infectious bacterial artificial chromosome clone of equine herpesvirus type 3 (EHV-3).

Equine herpesvirus type 3 (EHV-3) is the causal agent of equine coital exanthema, a disease characterized by pox-like lesions on the penis of stallions and the vulva of mares. Although the complete genomic sequence of EHV-3 has been recently made available, its genomic content remains poorly characterized and the molecular mechanisms of disease development not yet elucidated. In an attempt to facilitate genetic manipulation of EHV-3, we describe here the construction of a full-length infectious bacterial artificial chromosome (BAC) clone of EHV-3. Mini-F vector sequences were inserted into the intergenic region between ORF19 and ORF20 (UL41 and UL40, respectively) of EHV-3 strain C175 by homologous recombination in equine dermal cells (NBL-6). DNA of the resulting recombinant virus was electroporated into E. coli and a full-length EHV-3 BAC clone was recovered. Virus reconstituted after transfection of the EHV-3 BAC into NBL-6 cells showed growth properties in vitro that were indistinguishable from those of the parental virus. To assess the feasibility of mutagenesis of the cloned EHV-3 genome, recombinant viruses targeting the glycoprotein E (gE) gene were generated using Red recombination in E. coli and in vitro growth properties of the recombinant viruses were evaluated. We first repaired the gE (ORF74) coding region, since the parental virus used for BAC cloning specifies a truncated version of the gene, and then created gE-tagged and gE-null versions of the virus. Our results demonstrated that: (i) EHV-3 can be efficiently cloned as a BAC allowing easy manipulation of its genome; (ii) gE is dispensable for EHV-3 growth in vitro and is expressed as a product of approximately 110-kDa in infected cells; (iii) viruses having a deletion compromising gE expression or with a truncation of the cytoplasmic and transmembrane domains are significantly compromised with regard cell-to-cell spread. The cloning of EHV-3 as a BAC simplifies future studies to identify the role of its coding genes in viral pathogenesis and host immune responses.

Epidemiological and virological findings during multiple outbreaks of equine influenza in South America in 2012.

BACKGROUND: In 2012, equine influenza (EI) virus was confirmed as the cause of outbreaks of respiratory disease in horses throughout South America. In Uruguay and Argentina, hundreds of vaccinated thoroughbred horses in training and racing facilities were clinically affected. OBJECTIVE: To characterise the EI viruses detected during the outbreak in Uruguay and Argentina. METHODS: Virus was detected in nasopharyngeal swabs by a pan-reactive influenza type A real-time RT-PCR. The nucleotide sequence of the HA1 gene was determined and analysed phylogenetically using mega 5 software. Amino acid sequences alignments were constructed and virus was antigenically characterised with specific ferret antisera. Paired serum samples were tested by haemagglutination inhibition and single radial haemolysis. RESULTS: The diagnosis of EIV was confirmed by real-time RT-PCR, virus isolation and serological testing. The phylogenetic analysis of HA1 gene sequences of 18 EI viruses indicated that all of them belong to clade 1 of the Florida sublineage of the American lineage and are closely related to viruses isolated in the United States in 2012. The HA1 of viruses identified in horses in racing facilities in Maroñas, Uruguay, and in Palermo, Argentina, displayed 100% amino acid sequence identity and were identical to that of a virus isolated in Dubai in 2012, from vaccinated endurance horses recently imported from Uruguay. CONCLUSIONS: The surveillance data reported illustrate the international spread of EI viruses and support the recommendations of the OIE expert surveillance panel to include viruses of the Florida sublineage in vaccines.

Complete genome sequence of equid herpesvirus 3.

Equid herpesvirus 3 (EHV-3) is a member of the subfamily Alphaherpesvirinae that causes equine coital exanthema. Here, we report the first complete genome sequence of EHV-3. The 151,601-nt genome encodes 76 distinct genes like other equine alphaherpesviruses, but genetically, EHV-3 is significantly more divergent.

West Nile virus isolation from equines in Argentina, 2006.

West Nile virus (WNV) was isolated from the brains of 3 horses that died from encephalitis in February 2006. The horses were from different farms in central Argentina and had not traveled outside the country. This is the first isolation of WNV in South America.