Host Immune Response Modulation in Avian Coronavirus Infection: Tracheal Transcriptome Profiling In Vitro and In Vivo.

Infectious bronchitis virus (IBV) is a highly contagious Gammacoronavirus causing moderate to severe respiratory infection in chickens. Understanding the initial antiviral response in the respiratory mucosa is crucial for controlling viral spread. We aimed to characterize the impact of IBV Delmarva (DMV)/1639 and IBV Massachusetts (Mass) 41 at the primary site of infection, namely, in chicken tracheal epithelial cells (cTECs) in vitro and the trachea in vivo. We hypothesized that some elements of the induced antiviral responses are distinct in both infection models. We inoculated cTECs and infected young specific pathogen-free (SPF) chickens with IBV DMV/1639 or IBV Mass41, along with mock-inoculated controls, and studied the transcriptome using RNA-sequencing (RNA-seq) at 3 and 18 h post-infection (hpi) for cTECs and at 4 and 11 days post-infection (dpi) in the trachea. We showed that IBV DMV/1639 and IBV Mass41 replicate in cTECs in vitro and the trachea in vivo, inducing host mRNA expression profiles that are strain- and time-dependent. We demonstrated the different gene expression patterns between in vitro and in vivo tracheal IBV infection. Ultimately, characterizing host-pathogen interactions with various IBV strains reveals potential mechanisms for inducing and modulating the immune response during IBV infection in the chicken trachea.

DUSP1 mRNA modulation during porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus co-infection regulates viruses replication.

The effects of porcine circovirus type 2b (PCV2b) and porcine reproductive and respiratory syndrome virus (PRRSV) co-infection in epithelial cells of the swine respiratory tract is unknown. In the present study, the newborn pig trachea cell line NPTr-CD163, which is permissive to both viruses, was persistently infected with PCV2b and then with PRRSV. Viral replication, cell viability, cytokines' mRNA expression, and modulation of cellular genes expression were evaluated in infected cells. In NPTr-CD163 co-infection model, PCV2b replication was enhanced while PRRSV replication was suppressed. Cell viability was significantly decreased during PCV2b single infection and co-infection compared to mock-infected and PRRSV single infected cells. However, no difference was observed in cell viability between PCV2b and PCV2b/PRRSV infected cells. The IL6, IL8 and IL10 mRNA expression was significantly higher in co-infected cells compared to PCV2b and PRRSV single infected cells. Moreover, the IFN-α/ß expression was significantly reduced in co-infected cells compared to PCV2b infected cells whereas it remained higher compared to PRRSV infected cells. The differential gene expression analysis revealed that the mRNA expression level of the cellular gene DUSP1 was significantly higher in all PRRSV infection models compared to PCV2b single infected cells. Knockdown of DUSP1 expression in co-infected cells significantly reduced PCV2b replication, suggesting a role for DUSP1 in PCV2b/PRRSV pathogenesis.

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, 3'mRNA 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. RNA-sequencing 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.

Comparative full genome sequence analysis of wild-type and chicken embryo origin vaccine-like infectious laryngotracheitis virus field isolates from Canada.

Infectious laryngotracheitis (ILT), caused by infectious laryngotracheitis virus (ILTV), occurs sporadically in poultry flocks in Canada. Live attenuated chicken embryo origin (CEO) vaccines are being used routinely to prevent and control ILTV infections. However, ILT outbreaks still occur since vaccine strains could revert to virulence in the field. In this study, 7 Canadian ILTV isolates linked to ILT outbreaks across different time in Eastern Canada (Ontario; ON and Quebec; QC) were whole genome sequenced. Phylogenetic analysis confirmed the close relationship between the ON isolates and the CEO vaccines, whereas the QC isolates clustered with strains previously known as CEO revertant and wild-type ILTVs. Recombination network analysis of ILTV sequences revealed clear evidence of historical recombination between ILTV strains circulating in Canada and other geographical regions. The comparison of ON CEO clustered and QC CEO revertant clustered isolates with the LT Blen® CEO vaccine reference sequence showed amino acid differences in 5 and 12 open reading frames (ORFs), respectively. Similar analysis revealed amino acid differences in 32 ORFs in QC wild-type isolates. Compared to all CEO vaccine strains in the public domain, the QC wild-type isolates showed 15 unique mutational sites leading to amino acid changes in 13 ORFs. Our outcomes add to the knowledge of the molecular mechanisms behind ILTV genetic variance and provide genetic markers between wild-type and vaccine strains.

Phylogenetic analysis of porcine circovirus 3 circulating in Canadian pigs.

INTRODUCTION: Porcine circovirus 3 (PCV3) has been detected in pigs worldwide and associated with several clinical signs. METHODS: To investigate the genetic diversity of PCV3 strains circulating in Canada, 44 PCV3 positive samples from Saskatchewan (2/44), Manitoba (2/44), Quebec (4/44), Alberta (11/44) and Ontario (25/44) submitted to diagnostic laboratories in Canada between 2019 and 2021 were sequenced and analyzed. RESULTS: Phylogenetic analysis of capsid genes showed that all of the 44 Canadian strains classified into PCV3a and segregated into seven lineages with common amino acid changes observed at A24V, R27K, N56D, T77S, Q98R, L150I (F) and R168K positions. CONCLUSION: Future studies are required to determine whether the polymorphisms in capsid proteins, as revealed in this study, could be associated with differences in the pathogenicity or antigenicity of PCV3 strains. This is the first phylogenetic analysis of PCV3 strains among different provinces in Canada.

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.

Untargeted and targeted metabolomics reveal that adenosine nucleotides released in Actinobacillus pleuropneumoniae supernatant inhibit porcine reproductive and respiratory syndrome virus replication.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viruses in the swine industry and causes major economic losses. To date, there has not been an effective antiviral treatment for the disease. We have shown in previous studies that culture supernatant of Actinobacillus pleuropneumoniae (App), the causative agent of porcine pleuropneumonia, possesses antiviral activity in vitro against PRRSV, and we have clearly established that the antiviral activity was mediated by small molecular weight (i.e., <1 kDa), heat resistant metabolites present in the App supernatant ultrafiltrates. However, the identity of those metabolites remains unknown. The objective of the current study was to identify the active metabolites using untargeted and targeted mass spectrometry-based metabolomics and test their respective antiviral activity against PRRSV in the Jude Porcine Lung Epithelial Cell Line (SJPL). The results presented reveal very significant antiviral activity of App supernatant ultrafiltrates against PRRSV in SJPL cells. Consequently, we identified and quantified several adenosine nucleotide metabolites present in App supernatant ultrafiltrates using mass spectrometry-based metabolomics, and the concentrations detected were very high. SJPL cells infected with PRRSV and treated with 2'-adenosine monophosphate (2-AMP), 3'-adenosine monophosphate (3-AMP) or 5'-adenosine monophosphate (5-AMP) significantly reduced PRRSV infection. Interestingly, many antiviral drugs or prodrugs are adenosine analogs, and the mechanism of action was previously elucidated. Currently marketed nucleoside analog drugs could potentially be used to treat PRRSV infection.

IN SITU HYBRIDIZATION AND VIRUS CHARACTERIZATION OF SKUNK ADENOVIRUS IN NORTH AMERICAN WILDLIFE REVEALS MULTISYSTEMIC INFECTIONS IN A BROAD RANGE OF HOSTS.

Skunk adenovirus-1 (SkAdV-1) has been reported infecting several North American wildlife species; however, lesions associated with disease have not yet been completely characterized, particularly in porcupines. We describe and characterize the tissue distribution and lesions associated with SkAdV-1 infection in 24 wildlife diagnostic cases submitted between 2015 and 2020, including 16 North American porcupines (Erethizon dorsatum), three striped skunks (Mephitis mephitis), and five raccoons (Procyon lotor), which constitute a new host species. The most common lesion in all species was severe necrotizing bronchopneumonia with (n=12) or without (n=10) interstitial involvement. Intranuclear inclusion bodies were common in respiratory epithelium (n=21) and less often in renal tubular (n=6) and biliary epithelium (n=1). Several cases (n=4) had secondary bacterial infections, including Bordetella bronchiseptica, Pasteurella multocida, and Streptococcus zooepidemicus. In situ hybridization in porcupine (n=6), raccoon (n=1), and skunk (n=1) revealed SkAdV-1 DNA in multiple tissue types, including lung, trachea, turbinates, liver, kidney, lymph node, and brain, and multiple cell types including epithelial, endothelial, and mesothelial cells. These findings were consistent across species. Comparison of viral genomes from a porcupine and a raccoon with that originally isolated from a skunk demonstrated DNA point mutations affecting several viral genes, including the fiber protein gene. Our findings show the spectrum of disease associated with SkAdV-1 infection in a broad host range of wildlife species.

First report and genomic characterization of a bovine-like coronavirus causing enteric infection in an odd-toed non-ruminant species (Indonesian tapir, Acrocodia indica) during an outbreak of winter dysentery in a zoo.

Bovine coronavirus (BCoV) is associated with three distinct clinical syndromes in cattle that is, neonatal diarrhoea, haemorrhagic diarrhoea in adults (the so-called winter dysentery syndrome, WD) and respiratory infections in cattle of different ages. In addition, bovine-like CoVs have been detected in various species including domestic and wild ruminants. However, bovine-like CoVs have not been reported so far in odd-toed ungulates. We describe an outbreak of WD associated with a bovine-like CoV affecting several captive wild ungulates, including Indonesian tapirs (Acrocodia indica) an odd-toed ungulate species (Perissodactyla) which, with even-toed ungulates species (Artiodactyla) form the clade Euungulata. Genomic characterization of the CoV revealed that it was closely related to BCoVs previously reported in America. This case illustrates the adaptability of bovine-like CoVs to new species and the necessity of continued surveillance of bovine-like CoVs in various species.

Evaluation of Recombinant Herpesvirus of Turkey Laryngotracheitis (rHVT-LT) Vaccine against Genotype VI Canadian Wild-Type Infectious Laryngotracheitis Virus (ILTV) Infection.

In Alberta, infectious laryngotracheitis virus (ILTV) infection is endemic in backyard poultry flocks; however, outbreaks are only sporadically observed in commercial flocks. In addition to ILTV vaccine revertant strains, wild-type strains are among the most common causes of infectious laryngotracheitis (ILT). Given the surge in live attenuated vaccine-related outbreaks, the goal of this study was to assess the efficacy of a recombinant herpesvirus of turkey (rHVT-LT) vaccine against a genotype VI Canadian wild-type ILTV infection. One-day-old specific pathogen-free (SPF) White Leghorn chickens were vaccinated with the rHVT-LT vaccine or mock vaccinated. At three weeks of age, half of the vaccinated and the mock-vaccinated animals were challenged. Throughout the experiment, weights were recorded, and feather tips, cloacal and oropharyngeal swabs were collected for ILTV genome quantification. Blood was collected to isolate peripheral blood mononuclear cells (PBMC) and quantify CD4+ and CD8+ T cells. At 14 dpi, the chickens were euthanized, and respiratory tissues were collected to quantify genome loads and histological examination. Results showed that the vaccine failed to decrease the clinical signs at 6 days post-infection. However, it was able to significantly reduce ILTV shedding through the oropharyngeal route. Overall, rHVT-LT produced a partial protection against genotype VI ILTV infection.

The administration of diets contaminated with low to intermediate doses of deoxynivalenol and supplemented with antioxidants and binding agents slightly affects the growth, antioxidant status, and vaccine response in weanling pigs.

This study aimed to evaluate the impact of grading levels of deoxynivalenol (DON) in the diet of weaned pigs, as well as the effects of a supplementation with antioxidants (AOX), hydrated sodium calcium aluminosilicates (HSCAS), and their combination on the growth, AOX status, and immune and vaccine responses against the porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2). At weaning, 336 piglets were allocated to six dietary treatments according to a randomized complete block design. Treatments were as follows: basal diet (CTRL); basal diet containing DON at 1.2 mg/kg (DON1.2); basal diet containing DON at 2.4 mg/kg (DON2.4); DON2.4 diet + a mix of AOX which included vitamins A and E at 20,000 IU and 200 IU/kg feed respectively, selenized yeast at 0.3 mg/kg, and a grape seed extracts at 100 mg/kg feed (DON2.4 + AOX); DON2.4 diet + the mix of AOX and the modified HSCAS mentioned above (DON2.4 + AOX + HSCAS); DON2.4 + AOX + HSCAS. Pigs were vaccinated against PRRSV and PCV2 at 7 d; on 0, 14, and 35 d, growth performance was recorded, and blood samples were collected in order to evaluate the oxidative status, inflammatory blood markers, lymphocyte blastogenic response, and vaccine antibody response. Increasing intake of DON resulted in a quadratic effect at 35 d in the lymphocyte proliferative response to concanavalin A and PCV2 as well as in the anti-PRRSV antibody response, whereas the catalase activity decreased in DON2.4 pigs compared with the CTRL and DON1.2 groups (P ≤ 0.05). Compared with the DON2.4 diet, the AOX supplementation slightly reduced gain to feed ratio (P = 0.026) and increased the ferric reducing ability of plasma as well as α-tocopherol concentration (P < 0.05), whereas the association of AOX + HSCAS increased the anti-PRRSV IgG (P < 0.05). Furthermore, the HSCAS supplement reduced haptoglobin levels in serum at 14 d compared with the DON2.4 group; however, its concentration decreased in all the experimental treatments from 14 to 35 d and particularly in the DON2.4 + AOX pigs, whereas a different trend was evidenced in the DON2.4 + HSCAS group, where over the same period haptoglobin concentration increased (P < 0.05). Overall, our results show that the addition of AOX and HSCAS in the diet may alleviate the negative effects due to DON contamination on the AOX status and immune response of vaccinated weanling pigs.

Comparison of Primary Virus Isolation in Pulmonary Alveolar Macrophages and Four Different Continuous Cell Lines for Type 1 and Type 2 Porcine Reproductive and Respiratory Syndrome Virus.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) has a highly restricted cellular tropism. In vivo, the virus primarily infects tissue-specific macrophages in the nose, lungs, tonsils, and pharyngeal lymphoid tissues. In vitro however, the MARC-145 cell line is one of the few PRRSV susceptible cell lines that are routinely used for in vitro propagation. Previously, several PRRSV non-permissive cell lines were shown to become susceptible to PRRSV infection upon expression of recombinant entry receptors (e.g., PK15Sn-CD163, PK15S10-CD163). In the present study, we examined the suitability of different cell lines as a possible replacement of primary pulmonary alveolar macrophages (PAM) cells for isolation and growth of PRRSV. The susceptibility of four different cell lines (PK15Sn-CD163, PK15S10-CD163, MARC-145, and MARC-145Sn) for the primary isolation of PRRSV from PCR positive sera (both PRRSV1 and PRRSV2) was compared with that of PAM. To find possible correlations between the cell tropism and the viral genotype, 54 field samples were sequenced, and amino acid residues potentially associated with the cell tropism were identified. Regarding the virus titers obtained with the five different cell types, PAM gave the highest mean virus titers followed by PK15Sn-CD163, PK15S10-CD163, MARC-145Sn, and MARC-145. The titers in PK15Sn-CD163 and PK15S10-CD163 cells were significantly correlated with virus titers in PAM for both PRRSV1 (p < 0.001) and PRRSV2 (p < 0.001) compared with MARC-145Sn (PRRSV1: p = 0.22 and PRRSV2: p = 0.03) and MARC-145 (PRRSV1: p = 0.04 and PRRSV2: p = 0.12). Further, a possible correlation between cell tropism and viral genotype was assessed using PRRSV whole genome sequences in a Genome-Wide-Association Study (GWAS). The structural protein residues GP2:187L and N:28R within PRRSV2 sequences were associated with their growth in MARC-145. The GP5:78I residue for PRRSV2 and the Nsp11:155F residue for PRRSV1 was linked to a higher replication on PAM. In conclusion, PK15Sn-CD163 and PK15S10-CD163 cells are phenotypically closely related to the in vivo target macrophages and are more suitable for virus isolation and titration than MARC-145/MARC-145Sn cells. The residues of PRRSV proteins that are potentially related with cell tropism will be further investigated in the future.

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.(Traduit par les auteurs).

Epidemiological study of Coxiella burnetii in dairy cattle and small ruminants in Québec, Canada.

The bacterium Coxiella burnetii (C. burnetii) can infect a wide range of animals, most notably ruminants where it causes mainly asymptomatic infections and, when clinical, it is associated with reproductive disorders such as abortion. It is also the etiological agent of Q fever in humans, a zoonosis of increasingly important public health concern. A cross-sectional study was performed to estimate the apparent prevalence and spatial distribution of C. burnetii positivity in dairy cattle and small ruminant herds of two regions of Québec, Canada, and identify potential risk factors associated with positivity at animal and herd levels. In dairy cattle herds, individual fecal samples and repeated bulk tank milk samples (BTM) were collected. In small ruminant herds, serum and feces were sampled in individual animals. ELISA analyses were performed on serum and BTM samples. Real-time quantitative PCR (qPCR) was done on fecal and BTM samples. An animal was considered C. burnetii-positive when at least one sample was revealed positive by ELISA and/or qPCR, while a herd was considered C. burnetii-positive when at least one animal inside that herd was revealed positive. None of the 155 cows had a qPCR-positive fecal sample, whereas 37.2 % (95 % CI = 25.3-49.1) of the 341 sheep and 49.2 % (95 % CI = 25.6-72.7) of the 75 goats were C. burnetii-positive. The apparent prevalence of C. burnetii-positive herds was 47.3 % (95 % CI = 35.6-59.3) in dairy cattle herds (n = 74), 69.6 % (95 % CI = 47.1-86.8) in sheep flocks (n = 23) and 66.7 % (95 % CI = 22.3-95.7) in goat herds (n = 6). No spatial cluster of positive herds was detected. At the individual level, the only significant association with positivity in multivariable regressions was higher parity number in small ruminants. At the herd level, the use of calving group pen, the distance to the closest positive bovine herd, and small ruminant herd density in a 5 km radius were associated with dairy cattle herd positivity, whereas small ruminant herds with more than 100 animals and with a dog on the farm had greater odds of C. burnetii positivity. Our study shows that the infection is frequent on dairy cattle and small ruminant herds from the two studied regions and that some farm and animal characteristics might influence the transmission dynamics of the C. burnetii infection.

Pathogenic and Transmission Potential of Wildtype and Chicken Embryo Origin (CEO) Vaccine Revertant Infectious Laryngotracheitis Virus.

Infectious laryngotracheitis (ILT) is an infectious upper respiratory tract disease that impacts the poultry industry worldwide. ILT is caused by an alphaherpesvirus commonly referred to as infectious laryngotracheitis virus (ILTV). Vaccination with live attenuated vaccines is practiced regularly for the control of ILT. However, extensive and improper use of live attenuated vaccines is related to vaccine viruses reverting to virulence. An increase in mortality and pathogenicity has been attributed to these vaccine revertant viruses. Recent studies characterized Canadian ILTV strains originating from ILT outbreaks as related to live attenuated vaccine virus revertants. However, information is scarce on the pathogenicity and transmission potential of these Canadian isolates. Hence, in this study, the pathogenicity and transmission potential of two wildtype ILTVs and a chicken embryo origin (CEO) vaccine revertant ILTV of Canadian origin were evaluated. To this end, 3-week-old specific pathogen-free chickens were experimentally infected with each of the ILTV isolates and compared to uninfected controls. Additionally, naïve chickens were exposed to the experimentally infected chickens to mimic naturally occurring infection. Pathogenicity of each of these ILTV isolates was evaluated by the severity of clinical signs, weight loss, mortality, and lesions observed at the necropsy. The transmission potential was evaluated by quantification of ILTV genome loads in oropharyngeal and cloacal swabs and tissue samples of the experimentally infected and contact-exposed chickens, as well as in the capacity to produce ILT in contact-exposed chickens. We observed that the CEO vaccine revertant ILTV isolate induced severe disease in comparison to the two wildtype ILTV isolates used in this study. According to ILTV genome load data, CEO vaccine revertant ILTV isolate was successfully transmitted to naïve contact-exposed chickens in comparison to the tested wildtype ILTV isolates. Overall, the Canadian origin CEO vaccine revertant ILTV isolate possesses higher virulence, and dissemination potential, when compared to the wildtype ILTV isolates used in this study. These findings have serious implications in ILT control in chickens.

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.

Prevalence of shedding and antibody to Coxiella burnetii in post-partum dairy cows and its association with reproductive tract diseases and performance: A pilot study.

The bacterium Coxiella burnetii has been associated with reproduction disorders in dairy cattle. A cross-sectional study was conducted in Québec, Canada, to estimate the prevalence of C. burnetii in dairy cows from C. burnetii RT-PCR-positive and/or ELISA-positive herds. As a secondary objective, the associations between C. burnetii-positivity and three reproductive outcomes (purulent vaginal discharge, cytological endometritis, and success at first service) were assessed. A total of 202 post-parturient dairy cows from nine herds were sampled at 35 ± 7 days in milk. Vaginal mucus and composite milk were collected from each cow and screened for the presence of C. burnetii by real-time PCR (RT-PCR) and ELISA, respectively. Purulent vaginal discharge and cytological endometritis were evaluated using a Metricheck device and a modified cytobrush, respectively. The first insemination postpartum was done following an ovulation synchronization protocol around 70 days in milk, and success at first service was recorded. Multilevel logistic regressions adjusted for parity were used to model purulent vaginal discharge, cytological endometritis and success at first service according to C. burnetii cow status. All 202 RT-PCR-assayed vaginal samples were C. burnetii-negative. A positive result for anti-C. burnetii antibodies detection in composite milk was obtained in 25/202 samples and a doubtful result in 4/202 samples. After adjustment for sampling weights, the 202 ELISA-assayed composite milk samples gave an estimated overall prevalence of C. burnetii positive cows of 12.9 % (CI = 6.1-19.6 %) and of doubtful cows of 1.4 % (CI = 0.0-3.3 %). The proportion of ELISA-positive cows was lower in first parity (0%) compared to second (17.1 %) or third parity cows (20.0 %). The associations between ELISA positivity and reproductive outcomes were not statistically significant, perhaps due to the limited sample size, but could be used as pilot estimate for large-scale studies investigating the impact of C. burnetii infection on reproduction disorders in dairy cattle.

Analysis of Whole-Genome Sequences of Infectious laryngotracheitis Virus Isolates from Poultry Flocks in Canada: Evidence of Recombination.

Infectious laryngotracheitis virus (ILTV) is a herpes virus that causes an acute respiratory disease of poultry known as infectious laryngotracheitis (ILT). Chicken embryo origin (CEO) and tissue culture origin (TCO) live attenuated vaccines are routinely used for the control of ILT. However, vaccine virus is known to revert to virulence, and it has been recently shown that ILT field viral strains can undergo recombination with vaccinal ILTV and such recombinant ILT viruses possess greater transmission and pathogenicity potential. Based on complete or partial genes of the ILTV genome, few studies genotyped ILTV strains circulating in Canada, and so far, information is scarce on whole-genome sequencing or the presence of recombination in Canadian ILTV isolates. The objective of this study was to genetically characterize the 14 ILTV isolates that originated from three provinces in Canada (Alberta, British Columbia and Quebec). To this end, a phylogenetic analysis of 50 ILTV complete genome sequences, including 14 sequences of Canadian origin, was carried out. Additional phylogenetic analysis of the unique long, unique short and inverted repeat regions of the ILTV genome was also performed. We observed that 71%, 21% and 7% of the ILTV isolates were categorized as CEO revertant, wild-type and TCO vaccine-related, respectively. The sequences were also analyzed for potential recombination events, which included evidence in the British Columbia ILTV isolate. This event involved two ILTV vaccine (CEO) strains as parental strains. Recombination analysis also identified that one ILTV isolate from Alberta as a potential parental strain for a United States origin ILTV isolate. The positions of the possible recombination breakpoints were identified. These results indicate that the ILTV wild-type strains can recombine with vaccinal strains complicating vaccine-mediated control of ILT. Further studies on the pathogenicity of these ILTV strains, including the recombinant ILTV isolate are currently ongoing.

Chicken Astrovirus (CAstV) Molecular Studies Reveal Evidence of Multiple Past Recombination Events in Sequences Originated from Clinical Samples of White Chick Syndrome (WCS) in Western Canada.

In this study, we aimed to molecularly characterize 14 whole genome sequences of chicken astrovirus (CAstV) isolated from samples obtained from white chick syndrome (WCS) outbreaks in Western Canada during the period of 2014-2019. Genome sequence comparisons showed all these sequences correspond to the novel Biv group from which no confirmed representatives were published in GenBank. Molecular recombination analyses using recombination detection software (i.e., RDP5 and SimPlot) and phylogenetic analyses suggest multiple past recombination events in open reading frame (ORF)1a, ORF1b, and ORF2. Our findings suggest that recombination events and the accumulation of point mutations may have contributed to the substantial genetic variation observed in CAstV and evidenced by the current seven antigenic sub-clusters hitherto described. This is the first paper that describes recombination events in CAstV following analysis of complete CAstV sequences originated in Canada.