Impact of alignment algorithm on the estimation of pairwise genetic similarity of porcine reproductive and respiratory syndrome virus (PRRSV).

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a major threat to the swine industry. It is caused by the PRRS virus (PRRSV). Determination and comparison of the nucleotide sequences of PRRSV strains provides useful information in support of control initiatives or epidemiological studies on transmission patterns. The alignment of sequences is the first step in analyzing sequence data, with multiple algorithms being available, but little is known on the impact of this methodological choice. Here, a study was conducted to evaluate the impact of different alignment algorithms on the resulting aligned sequence dataset and on practical issues when applied to a large field database of PRRSV open reading frame (ORF) 5 sequences collected in Quebec, Canada, from 2010 to 2014. Five multiple sequence alignment programs were compared: Clustal W, Clustal Omega, Muscle, T-Coffee and MAFFT. RESULTS: The resulting alignments showed very similar results in terms of average pairwise genetic similarity, proportion of pairwise comparisons having ≥97.5% genetic similarity and sum of pairs (SP) score, except for T-Coffee where increased length of aligned datasets as well as limitation to handle large datasets were observed. CONCLUSIONS: Based on efficiency at minimizing the number of gaps in different dataset sizes with default open gap values as well as the capability to handle a large number of sequences in a timely manner, the use of Clustal Omega might be recommended for the management of PRRSV extensive database for both research and surveillance purposes.

The DataHarmonizer: a tool for faster data harmonization, validation, aggregation and analysis of pathogen genomics contextual information.

Pathogen genomics is a critical tool for public health surveillance, infection control, outbreak investigations as well as research. In order to make use of pathogen genomics data, they must be interpreted using contextual data (metadata). Contextual data include sample metadata, laboratory methods, patient demographics, clinical outcomes and epidemiological information. However, the variability in how contextual information is captured by different authorities and how it is encoded in different databases poses challenges for data interpretation, integration and their use/re-use. The DataHarmonizer is a template-driven spreadsheet application for harmonizing, validating and transforming genomics contextual data into submission-ready formats for public or private repositories. The tool's web browser-based JavaScript environment enables validation and its offline functionality and local installation increases data security. The DataHarmonizer was developed to address the data sharing needs that arose during the COVID-19 pandemic, and was used by members of the Canadian COVID Genomics Network (CanCOGeN) to harmonize SARS-CoV-2 contextual data for national surveillance and for public repository submission. In order to support coordination of international surveillance efforts, we have partnered with the Public Health Alliance for Genomic Epidemiology to also provide a template conforming to its SARS-CoV-2 contextual data specification for use worldwide. Templates are also being developed for One Health and foodborne pathogens. Overall, the DataHarmonizer tool improves the effectiveness and fidelity of contextual data capture as well as its subsequent usability. Harmonization of contextual information across authorities, platforms and systems globally improves interoperability and reusability of data for concerted public health and research initiatives to fight the current pandemic and future public health emergencies. While initially developed for the COVID-19 pandemic, its expansion to other data management applications and pathogens is already underway.

Oral immunization with F4 fimbriae and CpG formulated with carboxymethyl starch enhances F4-specific mucosal immune response and modulates Th1 and Th2 cytokines in weaned pigs.

PURPOSE: F4 fimbriae are a potential candidate for an oral subunit vaccine for prevention of post-weaning diarrhea in swine due to infection with F4-positive enterotoxigenic Escherichia coli. However, large quantities of F4 fimbriae are required to induce a specific antibody response. The aim of the present study was to evaluate the effect of supplementation of F4 fimbriae with Cytosine-phosphate-Guanosine-oligodeoxynucleotide (CpG-A D19) or with complete cholera toxin (CT) as adjuvants on the F4-specific antibody response and cytokine production in weaned pigs following oral administration of F4 fimbrial antigen formulated with Carboxymethyl Starch (CMS). METHODS: Oral dosage forms of F4 fimbriae alone or supplemented with CpG-A D19 or with CT were formulated with CMS as monolithic tablets, obtained by direct compression, and administered to weaned pigs. Blood and faecal samples were collected to determine the systemic and mucosal immune status of animals at various times until necropsy. During necropsy, contents of the jejunum and ileum were collected for determination of mucosal F4 specific antibodies. Segments of jejunum and ileum were also used to measure mRNA cytokine production. RESULTS: The presence of CpG in the formulation of the fimbriae significantly increased F4-specific immunoglobulin (Ig) IgM and IgG levels in intestinal secretions, and enhanced Th1 (Interferon-gamma / IFN-γ, Tumour Necrosis Factor-alpha / TNF-α, Interleukin-12p40 / IL-12p40, IL-1ß) and Th2 (IL-4, IL-6) cytokine production in intestinal tissues. Supplementation with CT did not result in induction of F4-specific antibodies in secretions, although a significant Th1 response (IFN-α, IFN-γ, IL-18) was detected in tissues. Neither F4-specific systemic antibodies, nor intestinally secreted IgA were detected throughout the immunization trial for all groups. CONCLUSIONS: CpG-A D19 appeared to be a promising adjuvant for an oral F4 subunit vaccine formulated with CMS excipient as monolithic tablets. This matrix afforded gastro-protection and delivered the F4 fimbriae at their intestinal sites.

SARS-CoV-2 Omicron Spike recognition by plasma from individuals receiving BNT162b2 mRNA vaccination with a 16-week interval between doses.

Continuous emergence of SARS-CoV-2 variants of concern (VOCs) is fueling the COVID-19 pandemic. Omicron (B.1.1.529) rapidly spread worldwide. The large number of mutations in its Spike raise concerns about a major antigenic drift that could significantly decrease vaccine efficacy and infection-induced immunity. A long interval between BNT162b2 mRNA doses elicits antibodies that efficiently recognize Spikes from different VOCs. Here, we evaluate the recognition of Omicron Spike by plasma from a cohort of SARS-CoV-2 naive and previously infected individuals who received their BNT162b2 mRNA vaccine 16 weeks apart. Omicron Spike is recognized less efficiently than D614G, Alpha, Beta, Gamma, and Delta Spikes. We compare with plasma activity from participants receiving a short (4 weeks) interval regimen. Plasma from individuals of the long-interval cohort recognize and neutralize better the Omicron Spike compared with those who received a short interval. Whether this difference confers any clinical benefit against Omicron remains unknown.

Positioning Quebec ORF5 sequences of porcine reproductive and respiratory syndrome virus (PRRSV) within Canada and worldwide diversity.

Sequencing of ORF5 gene is widely used and considered essential for diagnostics and control of porcine reproductive and respiratory syndrome (PRRS) in Canada. The objective of this study was to position Quebec ORF5 sequences of PRRS virus within Canada and worldwide diversity. Overall, 76.8% of the 5204 sequences gathered from Quebec (n = 5031), Ontario (n = 151) and Manitoba (n = 18) were classified into one of 34 genetic clusters defined as groupings including ≥15 sequences and having ≥70% rapid bootstrap support value from a maximum likelihood (ML)-phylogeny. Following the addition of PRRSV 2 international reference dataset from Shi et al. (2010), the most predominant lineages in our dataset were wild-type 1 and vaccine-like 5.1 (MLV) and 8.9 (ATP). No strains or only a very few (1 or 2) were assigned to lineages 1.3-1.5, 3, 4, 5.2, 6, 7 or 9. Most wild-type clusters (97%) detected in a dataset from Canada did not include any sequence from the international reference dataset. It might reflect recent subpopulations that were absent at the time of Shi's publication. As an example, cluster #25 first appeared in 2007, but since then had expanded considerably and is now the most prevalent wild-type cluster found in Quebec. A total of 117 RFLP patterns were identified and those were poorly correlated with genetic clusters based on phylogeny. Factors modulating PRRSV diversity such as pig movement that occurred within and between provinces should be further investigated in a perspective of disease control.

Porcine reproductive and respiratory syndrome virus: web-based interactive tools to support surveillance and control initiatives.

BACKGROUND: Control of porcine reproductive and respiratory syndrome (PRRS) represents a tremendous challenge. The trend is now toward managing the disease collectively. In Quebec, area and regional control and elimination (ARC&E) initiatives started in 2011; diagnostic testing, including ORF5 sequencing, and sharing of information among stakeholders are largely promoted. At the provincial level, a data-sharing agreement was signed by Quebec swine practitioners allowing PRRS virus (PRRSV) sequences to be transferred to a database maintained by the Laboratoire d'épidémiologie et de médecine porcine (LEMP-DB). Several interactive tools were developed and are available to veterinarians to allow comparison of PRRSV ORF5 sequences within ARC&E projects or provincially while managing confidentiality issues. RESULTS: Between January 1st 2010 and December 31st 2018, 4346 PRRSV ORF5 sequences were gathered into the LEMP-DB, involving 1254 sites and 43 practicing veterinarians. Approximately 34% of the submissions were from ARC&E projects. Using a novel web-based sequence comparison tool, each veterinarian has access to information on his/her client sequences and can compare each sequence with 1) commercial vaccine strains, 2) historical samples from the same site, and 3) all sequences submitted to the database over the last 4 years. Newly introduced PRRSV into breeding herds can be monitored using a new sequence comparison tool based on comparison of sequences at the provincial level. Each month, graphs providing the number of introductions per month and the yearly cumulative are updated. Between August 1st 2014 and December 31st 2018, 233 introductions were detected on 180 different breeding sites. Following a data-sharing agreement, veterinarians involved in ARC&E projects have access to an interactive mapping tool to locate pig sites, compare sequence similarity between participating sites and visualize the results on the map. CONCLUSIONS: The structure developed in Quebec to collect, analyse and share sequencing data was efficient to provide useful information to the swine industry at both provincial and regional levels while dealing with confidentiality issues.

Evaluating an automated clustering approach in a perspective of ongoing surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) field strains.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a major economic impact on the swine industry. The important genetic diversity needs to be considered for disease management. In this regard, information on the circulating endemic strains and their dispersal patterns through ongoing surveillance is beneficial. The objective of this project was to classify Quebec PRRSV ORF5 sequences in genetic clusters and evaluate stability of clustering results over a three-year period using an in-house automated clustering system. Phylogeny based on maximum likelihood (ML) was first inferred on 3661 sequences collected in 1998-2013 (Run 1). Then, sequences collected between January 2014 and September 2016 were sequentially added into 11 consecutive runs, each one covering a three-month period. For each run, detection of clusters, which were defined as groups of ≥15 sequences having a≥70% rapid bootstrap support (RBS) value, was automated in Python. Cluster stability was described for each cluster and run based on the number of sequences, RBS value, maximum pairwise distance and agreement in sequence assignment to a specific cluster. First and last run identified 29 and 33 clusters, respectively. In the last run, about 77% of the sequences were classified by the system. Most clusters were stable through time, with sequences attributed to one cluster in Run 1 staying in the same cluster for the 11 remaining runs. However, some initial groups were further subdivided into subgroups with time, which is important for monitoring since one specific wild-type cluster increased from 0% in 2007 to 45% of all sequences in 2016. This automated classification system will be integrated into ongoing surveillance activities, to facilitate communication and decision-making for stakeholders of the swine industry.

The spread of type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in North America: a phylogeographic approach.

The emergence and spread of Type 2 Porcine Reproductive and Respiratory Syndrome virus (Type 2 PRRSV) in North America is heavily influenced by the multiple site production system used in the hog industry. However, it is unclear how anthropogenic factors such has this have shaped the current spatial distribution of PRRSV genotypes. We employed Bayesian phylogeographic analyses of 7040 ORF5 sequences to reveal the recent geographical spread of Type 2 PRRSV in North America. The directions and intensities in our inferred virus traffic network closely mirror the hog transportation. Most notably, we reveal multiple viral introductions from Canada into the United States causing a major shift in virus genetic composition in the Midwest USA that went unnoticed by the regular surveillance and field epidemiological studies. Overall, these findings provide important insights into the dynamics of Type 2 PRRSV evolution and spread that will facilitate programs for control and prevention.

Porcine reproductive and respiratory syndrome virus diversity of Eastern Canada swine herds in a large sequence dataset reveals two hypervariable regions under positive selection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to be genetically highly variable, but knowledge of sequence diversity from Eastern Canada and its degree of genetic plasticity in or near the principal neutralizing epitope (PNE) in association with evolutionary selective pressure is limited. The purposes of our study were to investigate the extent of strain diversity, the existing glycotypes and the amino acid sites under selective evolutionary pressure in its encoded protein, GP5, for a dataset of 1301 sequences (1998-2009). This was addressed by partitioning and clustering into subgenotypes a large number of open reading frame 5 sequences from the province of Quebec and analyzing the content of these subgenotypes. The overall pairwise diversity was 12% and was comparable to what has been reported around the world. The mean diversity for sequences within subgenotypes was around 7%. No marked variations in subgenotype emergence could be observed through time. Thirty-eight GP5 glycotype patterns were observed which included a newly identified site at position N57 which was already present in 1998. These patterns possessed one to six N-glycosylation sites in total and could be located in eight different positions. No obvious grouping of glycotypes could be established in relation to subgenotypes. Positions N44 and N51 were confirmed to be fixed N-glycosylation positions, whereas other positions where found to be shifting and located in or near hypervariable regions (HVRs) 1 and 2. Both HVRs were under selective evolutionary pressure in half of all subgenotypes including vaccine-like groups. Conversely, the PNE flanked by both HVRs was well conserved among most subgenotypes demonstrating potential molecular constraint in a probable viral binding region. The analysis of this dataset increased knowledge of evolutionary change inferred from genetic data, more specifically regarding the implications of both HVRs in PRRSV diversity.