Refinement of the equine influenza model in the natural host: A meta-analysis to determine the benefits of individual nebulisation for experimental infection and vaccine evaluation in the face of decreased strain pathogenicity.

Equine Influenza (EI) is an important respiratory disease of horses caused by H3N8 equine influenza viruses (EIV). Vaccination is a key strategy to prevent or control this disease. However, EIV undergoes continuous antigenic drift and whilst numerous EI vaccines are commercially available worldwide, an accurate evaluation of their efficacy is frequently required through clinical trials conducted in the natural host. Room nebulisation is one of the chosen methods to challenge horses during EI vaccine studies. A potential decreased pathogenicity observed with recent Florida Clade 2 (FC2) EIV isolates have increased the heterogeneity of the clinical response and virus shedding measured after infection by room nebulisation, which reduced the statistical power of studies. Our objectives were to compare clinical and virological parameters following experimental infection with several different EIV strains and to confirm that individual nebulisation is a model refinement that prevents an increase of the number of animals per group. This study is a retrospective comparison and meta-analysis of clinical and virological results collected from 9 independent EIV infection studies in the natural host. Naïve Welsh mountain ponies were experimentally infected by room or individual nebulisation with FC2 EIV strains, including A/equine/Richmond/1/07 (R/07), A/equine/East Renfrewshire/11 (ER/11), A/equine/Cambremer/1/2012 (C/12) and A/equine/Northamptonshire/1/13 (N/1/13). The retrospective meta-analysis confirmed a decreased pathogenicity of the EIV ER/11 and C/12 strains when compared with R/07. Experimental infection by individual nebulisation improved the clinical and virological parameters induced by recent FC2 strains, when compared with conventional room nebulisation. In conclusion, individual nebulisation offers a better control of the challenge dose administered and a greater homogeneity of the response measured in control animals. This in turn, helps maintain the number of animals per group to the minimum necessary required to obtain meaningful results in vaccine efficacy studies, which adheres to the 3Rs (Replacement, Reduction and Refinement) principles.

Chimeric viruses containing the N-terminal ectodomains of GP5 and M proteins of porcine reproductive and respiratory syndrome virus do not change the cellular tropism of equine arteritis virus.

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are members of family Arteriviridae; they are highly species specific and differ significantly in cellular tropism in cultured cells. In this study we examined the role of the two major envelope proteins (GP5 and M) of EAV and PRRSV in determining their cellular tropism. We generated three viable EAV/PRRSV chimeric viruses by swapping the N-terminal ectodomains of these two proteins from PRRSV IA1107 strain into an infectious cDNA clone of EAV (rMLVB4/5 GP5ecto, rMLVB4/5/6 Mecto and rMLVB4/5/6 GP5&Mecto). The three chimeric viruses could only infect EAV susceptible cell lines but not PRRSV susceptible cells in culture. Therefore, these data unequivocally demonstrate that the ectodomains of GP5 and M are not the major determinants of cellular tropism, further supporting the recent findings that the minor envelope proteins are the critical proteins in mediating cellular tropism (Tian et al., 2012).

Development and characterization of an infectious cDNA clone of the modified live virus vaccine strain of equine arteritis virus.

A stable full-length cDNA clone of the modified live virus (MLV) vaccine strain of equine arteritis virus (EAV) was developed. RNA transcripts generated from this plasmid (pEAVrMLV) were infectious upon transfection into mammalian cells, and the resultant recombinant virus (rMLV) had 100% nucleotide identity to the parental MLV vaccine strain of EAV. A single silent nucleotide substitution was introduced into the nucleocapsid gene (pEAVrMLVB), enabling the cloned vaccine virus (rMLVB) to be distinguished from parental MLV vaccine as well as other field and laboratory strains of EAV by using an allelic discrimination real-time reverse transcription (RT)-PCR assay. In vitro studies revealed that the cloned vaccine virus rMLVB and the parental MLV vaccine virus had identical growth kinetics and plaque morphologies in equine endothelial cells. In vivo studies confirmed that the cloned vaccine virus was very safe and induced high titers of neutralizing antibodies against EAV in experimentally immunized horses. When challenged with the heterologous EAV KY84 strain, the rMLVB vaccine virus protected immunized horses in regard to reducing the magnitude and duration of viremia and virus shedding but did not suppress the development of signs of EVA, although these were reduced in clinical severity. The vaccine clone pEAVrMLVB could be further manipulated to improve the vaccine efficacy as well as to develop a marker vaccine for serological differentiation of EAV naturally infected from vaccinated animals.