42 research outputs found
Whole genome sequencing of porcine reproductive and respiratory syndrome virus 2 (PRRSV) from field clinical samples improves the genomic surveillance of the virus
Porcine reproductive and respiratory syndrome virus (PRRSV) is a major
economic concern worldwide. There are currently large data sets available about the
ORF5 gene of the virus, with thousands of sequences available, but little data are
currently available on the full-length genome of PRRSV. We hypothesized that
whole-genome sequencing (WGS) of the PRRSV genome would allow better epidemiological monitoring than ORF5 gene sequencing. PRRSV PCR-positive serum, oral
fluid, and tissue clinical samples submitted to the diagnostic laboratory for routine
surveillance or diagnosis of PRRSV infection in Québec, Canada, swine herds were
used. The PRRSV reverse transcription-quantitative PCR Cq values of the processed
samples varied between 11.5 and 34.34. PRRSV strain genomes were isolated using a
poly (A)-tail method and were sequenced with a MiSeq Illumina sequencer. Ninetytwo full-length PRRSV genomes were obtained from 88 clinical samples out of 132
tested samples, resulting in a PRRSV WGS success rate of 66.67%. Three important
deletions in ORF1a were found in most wild-type (i.e., not vaccine-like) strains. The
importance of these deletions remains undetermined. Two different full-length
PRRSV genomes were found in four different samples (three serum samples and one
pool of tissues), suggesting a 4.55% PRRSV strain coinfection prevalence in swine.
Moreover, six PRRSV whole genomes (6.52% of PRRSV strains) were found to cluster
differently than they did under the ORF5 classification method. Overall, WGS of
PRRSV enables better strain classification and/or interpretation of results in 9.10% of
clinical samples than ORF5 sequencing, as well as allowing interesting research avenues
Coding-complete genome sequence of a Falcon aviadenovirus A strain associated with necrotizing hepatitis in an American Kestrel (Falco sparverius)
A necropsy was performed on an American kestrel (Falco sparverius) with necrotizing hepatitis associated with inclusion bodies, suggesting an adenovirus infection. A next-generation sequencing assay was conducted on the liver, and the coding-complete genome sequence of a Falcon aviadenovirus A strain was revealed
Porcine reproductive and respiratory syndrome virus whole-genome sequencing efficacy with field clinical samples using a poly(A)-tail viral genome purification method
The genomic surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) is based on sequencing of the ORF5 gene of the virus, which covers only 4% of the entire viral genome. It is expected that PRRSV whole-genome sequencing (WGS) will improve PRRSV genomic data and allow better understanding of clinical discrepancies observed in the field when using ORF5 sequencing. Our main objective was to implement an efficient method for WGS of PRRSV from clinical samples. The viral genome was purified using a poly(A)-tail viral genome purification method and sequenced using Illumina technology. We tested 149 PRRSV-positive samples: 80 sera, 33 lungs, 33 pools of tissues, 2 oral fluids, and 1 processing fluid (i.e., castration liquid). Overall, WGS of 67.1% of PRRSV-positive cases was successful. The viral load, in particular for tissues, had a major impact on the PRRSV WGS success rate. Serum was the most efficient type of sample to conduct PRRSV WGS poly(A)-tail assays, with a success rate of 76.3%, and this result can be explained by improved sequencing reads dispersion matching throughout the entire viral genome. WGS was unsuccessful for all pools of tissue and lung samples with Cq values > 26.5, whereas it could still be successful with sera at Cq ≤ 34.1. Evaluation of results of highly qualified personnel confirmed that laboratory skills could affect PRRSV WGS efficiency. Oral fluid samples seem very promising and merit further investigation because, with only 2 samples of low viral load (Cq = 28.8, 32.8), PRRSV WGS was successful
Dual infections of CD163 expressing NPTr epithelial cells with influenza A virus and PRRSV
In the pig, respiratory co-infections involving various pathogens are far more frequent than single infections.
Amongst respiratory viruses, swine influenza type A virus (swIAV) and porcine reproductive and respiratory
syndrome virus (PRRSV) are frequently associated. Previously, we performed co-infections with swIAV and
PRRSV in porcine alveolar macrophages (PAM) and precision cut lung slices (PCLS). With these two approaches
it was practically impossible to have co-infections of the same cells as the main target cell of swIAV is the
epithelial cell while the main target of PRRSV is the PAM. This constraint makes the study of interference
between the two viruses difficult at the cellular level. In the current report, an epithelial cell line expressing,
CD163, the main receptor of PRRSV was generated. This cell line receptive for both viruses was used to assess the
interference between the two viruses. Results showed that swIAV as well as PRRSV, even if they interacted
differently with the modified epithelial cells, were clearly interfering with each other regarding their replication
when they infected a same cell with consequences within the cellular antiviral response. Our modified cell line,
receptive to both viruses, can be used as a tool to assess interference between swIAV and PRRSV in a same cell as
it probably happens in the porcine host
Persistence of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in bacterial biofilms
The aim of this pilot project was to investigate
association of viruses with bacterial
biofilms. Our preliminary data indicate that
important viral pathogens of swine, namely,
porcine reproductive and respiratory syndrome
virus and porcine circovirus type 2,
can associate with and persist within bacterial
biofilms for several days
Porcine circovirus modulates swine influenza virus replication in pig tracheal epithelial cells and porcine alveolar macrophages
The pathogenesis of porcine circovirus type 2b (PCV2b) and swine influenza A virus
(SwIV) during co-infection in swine respiratory cells is poorly understood. To elucidate the impact
of PCV2b/SwIV co-infection, newborn porcine tracheal epithelial cells (NPTr) and immortalized
porcine alveolar macrophages (iPAM 3D4/21) were co-infected with PCV2b and SwIV (H1N1 or
H3N2 genotype). Viral replication, cell viability and cytokine mRNA expression were determined and
compared between single-infected and co-infected cells. Finally, 30mRNA sequencing was performed
to identify the modulation of gene expression and cellular pathways in co-infected cells. It was found
that PCV2b significantly decreased or improved SwIV replication in co-infected NPTr and iPAM
3D4/21 cells, respectively, compared to single-infected cells. Interestingly, PCV2b/SwIV co-infection
synergistically up-regulated IFN expression in NPTr cells, whereas in iPAM 3D4/21 cells, PCV2b
impaired the SwIV IFN induced response, both correlating with SwIV replication modulation. RNAsequencing analyses revealed that the modulation of gene expression and enriched cellular pathways
during PCV2b/SwIV H1N1 co-infection is regulated in a cell-type-dependent manner. This study
revealed different outcomes of PCV2b/SwIV co-infection in porcine epithelial cells and macrophages
and provides new insights on porcine viral co-infections pathogenesis
In vivo effect of deoxynivalenol (DON) naturally contaminated feed on porcine reproductive and respiratory syndrome virus (PRRSV) infection
Deoxynivalenol (DON), also known as vomitoxin, is the most prevalent type B trichothecene mycotoxin worldwide. Pigs show a great sensitivity to DON, and because of the high proportion of grains in their diets, they are frequently exposed to this mycotoxin. The objective of this study was to determine the impact of DON naturally contaminated feed on porcine reproductive and respiratory syndrome virus (PRRSV) infection, the most important porcine viral pathogen in swine. Experimental infections were performed with 30 animals. Piglets were randomly divided into three groups of 10 animals based on DON content of diets (0, 2.5 and 3.5 mg/kg DON). All experimental groups were further divided into subgroups of 6 pigs and were inoculated with PRRSV. The remaining pigs (control) were sham-inoculated with PBS. Pigs were daily monitored for temperature, weight and clinical signs for 21 days. Blood samples were collected and tested for PRRSV RNA and for virus specific antibodies. Results of PRRSV infection showed that ingestion of diet highly contaminated with DON greatly increases the effect of PRRSV infection on weight gain, lung lesions and mortality, without increasing significantly viral replication, for which the tendency is rather directed toward a decrease of replication. These results suggest that PRRSV infection could exacerbate anorectic effect of DON, when ingested in large doses. Results also demonstrate a DON negative effect on PRRSV-specific humoral responses. This study demonstrate that high concentrations of DON naturally contaminated feed decreased the immune response against PRRSV and influenced the course of PRRSV infection in pigs
In vitro effect of deoxynivalenol (DON) mycotoxin on porcine reproductive and respiratory syndrome virus replication
Deoxynivalenol (DON) is a mycotoxin produced by Fusarium spp. Among monogastric farm animals,
swine are the most susceptible to DON as it markedly reduces feed intake and decreases weight gain.
DON has also been shown to increase susceptibility to viral infections; therefore the objective of this
study was to investigate in vitro impact of DON on porcine reproductive and respiratory syndrome virus
(PRRSV). Permissive cells were infected or not with PRRSV and were treated with increasing concentrations
of DON. Cell survival and mortality were evaluated by determining the number of viable cells with a
tetrazolium compound and by measuring lactate dehydrogenase (LDH) release, respectively. Virus titration
and antiviral cytokines mRNA expression were evaluated by quantitative PCR. DON significantly
affected the survival of noninfected cells in a dose dependent manner. However, DON concentrations
between 140 and 280 significantly increased the survival of cells infected with PRRSV. These concentrations
significantly decreased PRRSV replication by inducing a pro-inflammatory cytokines environment
and an early activation of apoptosis, which in turn seem to interrupt viral replication. For the first time,
this study showed that DON had significant effects on the survival of PRRSV infected cells and on virus
replication, in a dose dependent manner
Identification of a novel herpesvirus associated with a penile proliferative lesion in a beluga (Delphinapterus leucas)
The carcass of an adult male beluga (Delphinapterus leucas) was found beach cast in 2008 on the shore of the St. Lawrence Estuary at Rivière-Ouelle, Quebec, Canada. The carcass was transported to the Faculté de médecine vétérinaire of the Université de Montréal for postmortem examination. Aspiration pneumonia was the probable cause of death. Necropsy revealed a focal papilloma-like penile lesion, characterized by focal mucosal thickening with disorganization of the epithelial layers and lymphoplasmacytic infiltration. A pan-herpesvirus nested PCR assay on frozen tissue from the penile lesion was positive. The PCR product sequencing revealed a partial herpesvirus DNA polymerase (DPOL) gene sequence of 600 nucleotides. Its nearest nucleotide identity was with the partial DPOL gene of an alphaherpesvirus, bovine herpesvirus 5 (79.5% identity). It also shared high identity with several other marine mammal herpesviruses (50.2 to 77.3% identity). This new herpesvirus was tentatively named beluga whale herpesvirus (BWHV). Virus isolation was unsuccessful. The pathogenic potential of BWHV is unknown, but the evaluation of archived tissues suggests that the virus is endemic in the St. Lawrence Estuary beluga population
Whole genome sequencing of methicillin-resistant and methicillin-sensitive Staphylococcus aureus isolated from 4 horses in a veterinary teaching hospital and its ambulatory service
Genomic characterization was conducted on 2 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from 2 horses
hospitalized during an overlapping period of time and 2 methicillin-sensitive S. aureus (MSSA) strains isolated from 2 distinct
horses. Phylogenetic proximity was traced and the genotypic and phenotypic characteristics of the antimicrobial resistance of
the strains were compared.
Whole genome sequencing of MRSA strains for this report was similar but differed from whole genome sequencing of MSSA
strains. The MRSA strains were closely related, belonging to sequence type (ST) 612, spa type t1257, and SCCmec type IVd2B. The
MSSA strains were also closely related, belonging to ST1660, spa type t3043, and having no detectable staphylococcal cassette
chromosome mec elements. All MSRA and MSSA strains were Panton-Valentine leukocidin negative. There were discrepancies
in the genotypic analysis and the antimicrobial susceptibility testing (phenotypic analysis) of MRSA strains for rifampin,
trimethoprim-sulfamethoxazole, gentamicin, amikacin, and enrofloxacin.La caractérisation génomique a été effectuée sur deux souches de Staphylococcus aureus résistantes à la méticilline (SARM) isolées de
deux chevaux hospitalisés sur une période de chevauchement, et de deux S. aureus sensibles à la méticilline (SASM) isolés de deux chevaux
distincts. Leur proximité phylogénétique a été retracée. Les caractéristiques génotypiques et phénotypiques de la résistance aux antimicrobiens
de ces souches ont été comparées.
Le séquençage complet du génome des souches de SARM pour ce rapport était similaire, mais différent du séquençage complet du génome
des souches de SASM. Les souches de SARM étaient étroitement apparentées, appartenant à la séquence type (ST) 612, au spa type t1257
et au SCCmec type IVd2B. Les souches MSSA étaient étroitement apparentées appartenant au ST1660, spa type t3043 et aucun élément de
la cassette contenant le gène mec n’a été détecté. Toutes les souches MSRA et MSSA étaient négatives pour la leucocidine Panton-Valentine.
Il y avait des divergences entre l’analyse génotypique et les tests de sensibilité aux antimicrobiens (phénotype) des souches de SARM pour
la rifampicine, le triméthoprime-sulfaméthoxazole, la gentamicine, l’amikacine et l’enrofloxacine