Rabbit hemorrhagic disease virus 2 (RHDV2) is a newly emerging Lagovirus belonging to the family Caliciviridae. After its first discovery in 2010 in France, this highly pathogenic virus rapidly spread to neighboring countries and has become the dominant strain, replacing the classical RHDV1 strains. RHDV2 was first reported in North America in 2016 in Mont-Joli, Quebec, Canada and it was reported again in 2018 and 2019 on Vancouver, Island and the southeast mainland of British Columbia (BC). The whole genome sequence of the RHDV2 Quebec isolate resembled the RHDV-N11 isolate from Navarra, Spain identified in 2011 with 97% identity. The epidemiological investigation involved three hobby farms and one personal residence. In December and February 2018, high mortality was reported in first a private feral rabbit refuge and then, a large colony of feral rabbits on the Vancouver Island University Campus, Nanaimo, BC. The virus responsible showed only 93% identity to the Quebec RHDV2 isolate at the nucleotide level. Additional cases of RHDV2 on Vancouver Island and on the BC mainland affecting feral, captive domestic and commercial rabbits were reported subsequently. Vaccination was recommended to control the outbreak and an inactivated bivalent vaccine was made available to the private veterinary practices. In June 2019 an isolated RHDV2 outbreak was reported in an apartment building in Vancouver, BC. This virus showed only 97% identity to the RHDV2 isolate responsible for the BC outbreak in 2018 at the nucleotide level suggesting that it was an independent incursion. In October 2020, there are reports of partial recovery of the feral population in Nanaimo and to date there are no confirmed deaths of native rabbit species in BC.

Pseudorabies virus (PRV) causes Aujeszky’s disease or pseudorabies (PR), which is characterized by fatal encephalitis in newborn piglets, respiratory infection in growing and fattening pigs, and reproductive failures in pregnant sows. It establishes a lifelong latent infection in the peripheral nervous system followed by subsequent intermittent shedding of infectious virus. Since 2011, highly virulent PRV strains that are genetically different from the classic PRV strains surfaced in pig herds in China. Availability of a highly sensitive and specific polymerase chain reaction (PCR)-based diagnostic assay for rapid differential detection of PRV variants is critical to prevent huge economic losses to the U.S. and Canadian pork industries if these strains enter North America and cause an outbreak. Here we describe the development and evaluation of a single-tube triplex real-time-PCR assay for differential detection of variant strains of PRV. The assay targets the intergenic region between the US2 and US6 genes in the PRV genome, is highly sensitive and specific, and it did not detect other non-target viruses, including related herpesviruses. The clinical specificity and sensitivity of the assay was evaluated using whole blood, serum, tissue and swab samples collected from known negative and experimentally inoculated pigs with either classical (Bristol) or variant (JS-2012 and HeN1) PRV strains. The targeted genomic region of this assay is also deleted in commonly used PRV gE-deleted marker vaccines, and therefore, the triplex assay did not detect viral DNA extracted from two commercial vaccine strains Bartha K-61 and Bucharest. This single-tube triplex assay can be used for routine diagnostics and epidemiological studies for detection and differentiation of classical strains from variant strains of PRV, and as a differentiation of infected and vaccinated animals (DIVA) assay when PRV gE- deletion mutant marker vaccines are used.

African swine fever (ASF) continues to cause outbreaks throughout regions of Africa, Europe and Asia. The disease can cause severe morbidity and mortality resulting in serious economic losses. Since there is no vaccine available to control ASF, early detection is critical to contain and control the disease. The aim of this study was to develop a novel real-time PCR assay based on highly conserved ASFV gene E183L (p54). The limit of detection of the assay, VNUA-54 real-time PCR, was 2.63 copies/reaction and 2 Log10 HAD50/ml. The VNUA-54 real-time PCR was able to detect fifteen different ASFV reference strains representing p72 genotypes I, II and V. The assay was specific and did not amplify other swine viruses including CSF, FMD, PRRS, and PED. The diagnostic sensitivity of the real-time PCR assay was evaluated using 187 field clinical specimens collected from swine farms located in different provinces in Vietnam. The VNUA-54 real-time PCR assay is an additional tool for ASF diagnostics and can be used in combination with other p72 based ASFV real-time PCR assays as a rapid confirmatory assay.