Comparative susceptibility of eastern cottontails and New Zealand white rabbits to classical rabbit haemorrhagic disease virus (RHDV) and RHDV2

Rabbit haemorrhagic disease virus (RHDV) is associated with high morbidity and mortality in the European rabbit (Oryctolagus cuniculus). In 2010, a genetically distinct RHDV named RHDV2 emerged in Europe and spread to many other regions, including North America in 2016. Prior to this study it was unknown if eastern cottontails (ECT(s); Sylvilagus floridanus), one of the most common wild lagomorphs in the United States, were susceptible to RHDV2. In this study, 10 wild-caught ECTs and 10 New Zealand white rabbits (NZWR(s); O. cuniculus) were each inoculated orally with either RHDV (RHDVa/GI.1a; n = 5 per species) or RHDV2 (a recombinant GI.1bP-GI.2; n = 5 per species) and monitored for the development of disease. Three of the five ECTs that were infected with RHDV2 developed disease consistent with RHD and died at 4 and 6 days post-inoculation (DPI). The RHDV major capsid protein/antigen (VP60) was detected in the livers of three ECTs infected with RHDV2, but none was detected in the ECTs infected with RHDV. Additionally, RHD viral RNA was detected in the liver, spleen, intestine and blood of ECTs infected with RHDV2, but not in the ECTs infected with RHDV. RHD viral RNA was detected in urine, oral swabs and rectal swabs in at least two of five ECTs infected with RHDV2. One ECT inoculated with RHDV2 seroconverted and developed a high antibody titre by the end of the experimental period (21 DPI). ECTs inoculated with the classic RHDV did not seroconvert. In comparison, NZWRs inoculated with RHDV2 exhibited high mortality (five of five) at 2 DPI and four of five NZWRs inoculated with RHDV either died or were euthanized at 2 DPI indicating both of these viruses were highly pathogenic to this species. This experiment indicates that ECTs are susceptible to RHDV2 and can shed viral RNA, thereby suggesting this species could be involved in the epidemiology of this virus

Real-Time Reverse Transcription PCR Assay for Detection of Senecavirus A in Swine Vesicular Diagnostic Specimens.

Senecavirus A (SV-A), formerly, Seneca Valley virus (SVV), has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD), a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS), that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88%) were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18%) or without (6%) vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates

RT-qPCR linearity and sensitivity.

<p>Serial dilutions of standard control and SV-A positive tissue RNA produced linear standard amplification plot up to extinction with y-slopes of (-3.68 and -3.51, respectively) demonstrated >90% RT-qPCR efficiency with r² > 0.99.</p

DNA doubling efficiency.

<p>Linear amplification of circular plasmid DNA was between copy numbers of 4 x 10⁰ and 4 x 10⁶, and generated a standard curve with calculated efficiency of 86% from y-slope (-3.72) with correlation coefficient (R²) of 0.9993.</p

Sequence alignment of qRT-PCR viral isolates to SVV-001 and qRT-PCR primers.

<p>Five viral isolates were confirmed positive by qRT-PCR and the genome was sequenced. The amplification region was aligned with the qRT-PCR SV-A primers used in the assay, and to the reference sequence SVV-001: NC_011349.1 (NCBI).</p

Melting curve for SV-A qPCR on the Step One Plus.

<p>Standard plasmid DNA containing SV-A gene fragment was used as the template for amplification in a SYBR green based qPCR reaction. RT-qPCR product had a melting temperature of ~78°C.</p

RT-qPCR data with amplicons visualized by DNA gel electrophoresis.

<p>Five diagnostic RNA samples with three controls were analyzed by RT-qPCR on the SmartCyclerII. (a) Ct and melt curves are displayed. (b) All products were subjected to high resolution gel electrophoresis to verify amplification of specific products of ~90bp. Control samples included a negative extraction (NEC), a negative template (NTC) and a positive amplification (PAC) control. (c) Ct values and melting temperature data is summarized.</p

Diagnosis of \u3ci\u3ePorcine teschovirus\u3c/i\u3e encephalomyelitis in the Republic of Haiti

In February and March 2009, approximately 1,500 backyard pigs of variable age became sick, and approximately 700 of them died or were euthanized in the Lower Artibonite Valley and the Lower Plateau of the Republic of Haiti. The main clinical sign was posterior ataxia followed by paresis and/or paralysis on the second or third day of illness. No gross lesions were observed at postmortem examinations. The morbidity and mortality were approximately 60% and 40%, respectively. Diagnostic samples (whole blood, brain, tonsil, lymph nodes, spleen, and lung) were negative for Classical swine fever virus and African swine fever virus. Porcine teschovirus type 1 was detected by reverse transcription polymerase chain reactions in brain samples. Results of virus isolation, electron microscopy of virus particles, histopathological analysis on brain tissues, nucleic acid sequencing, and phylogenetic analysis of the viral isolate supported the diagnosis of teschovirus encephalomyelitis. The outbreak of the disease in Haiti is the first appearance of the severe form of teschovirus encephalomyelitis in the Americas. This disease poses a potential threat to the swine industries in other Caribbean countries, as well as to Central and North American countries

A partial deletion in non-structural protein 3A can attenuate foot-and-mouth disease virus in cattle

AbstractThe role of non-structural protein 3A of foot-and-mouth disease virus (FMDV) on the virulence in cattle has received significant attention. Particularly, a characteristic 10–20 amino acid deletion has been implicated as responsible for virus attenuation in cattle: a 10 amino acid deletion in the naturally occurring, porcinophilic FMDV O1 Taiwanese strain, and an approximately 20 amino acid deletion found in egg-adapted derivatives of FMDV serotypes O1 and C3. Previous reports using chimeric viruses linked the presence of these deletions to an attenuated phenotype in cattle although results were not conclusive. We report here the construction of a FMDV O1Campos variant differing exclusively from the highly virulent parental virus in a 20 amino acid deletion between 3A residues 87–106, and its characterization in vitro and in vivo. We describe a direct link between a deletion in the FMDV 3A protein and disease attenuation in cattle