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 is currently available on the full-length genome of PRRSV. We hypothesized that whole genome sequencing (WGS) of PRRSV genome would allow a better epidemiological monitoring compared to ORF5 gene sequencing. PRRSV PCR positive sera, oral fluids 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 RT-qPCR 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 an MiSeq Illumina sequencer. Ninety-two 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 the ORF1a were found in most wildtype (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 sera 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 compared to ORF5 classification method. Overall, WGS of PRRSV enables a better strains classification and/or interpretation of results in 9.10% of clinical samples compared to ORF5 sequencing, as well as allowing interesting research avenues.

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.

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.

INTRODUCTION: IgG₄-related disease is a rare autoimmune syndrome that causes fibrosis of glands, most commonly of the pancreas and biliary tract1,3. The overall prevalence is unknown, with an estimated less than 1:100,000 of the general population typically found in middle-aged males2,3. Diagnosis is made with IgG₄ levels at least 2x the upper limit of normal and dense lymphoplasmacytic infiltrate, however, it may be seen with normal IgG₄ levels6. Although rare, it can present similar to GI malignancies and should be considered in the differential for patients with autoimmune disease1,3,4,5. Here we present one of those unusual cases. CASE DESCRIPTION/METHODS: A 65-year-old lady with PMH of DM2 and Sjogren's Syndrome (SS) who presented to the hospital with intractable nausea, vomiting, and failure to thrive for over 1 month. The physical exam, labs that included CMP, LFTs, and serology was unremarkable; previous EGD revealed diffuse thickening of the gastric submucosa. Biopsy via open gastrectomy revealed chronic inflammation with multiple IgG₄ plasma cells and no signs of lymphoma, suspicious for IgG₄-RD. After the partial gastrectomy, serum Na levels were 162, which improved with DDAVP and was later confirmed to be diabetes insipidus (DI). Further workup and imaging were negative and baseline IgG and IgG₄ levels were 1178 and 61, respectively. Treatment included steroid therapy and DDAVP which improved her symptoms and preceded an uneventful hospital course. DISCUSSION: IgG₄-RD is a disease with numerous presentations based on the organs involved. It presents as a GI-specific illness due to its affinity for the pancreas and biliary tract1,2,3. As seen in our patient, although rare, it may also affect the stomach, form mass lesions and cause symptomatology similar to a GI malignancy3,5. In patients with autoimmune diseases, where GI malignancy is considered, it is reasonable to include IgG₄-RD in the differential. IgG₄-RD may mimic symptoms of SS, and therefore case-patients with SS should also be considered for IgG₄-RD. This patient also experienced DI which may be a manifestation of IgG₄-RD, as the syndrome may affect the pituitary4. This case is one of the few instances reported to have pituitary involvement after the onset of multiorgan involvement4. Like other autoimmune disorders, IgG₄-RD is responsive to steroid therapy or rituximab. Therefore, a timely diagnosis can help slow down or prevent symptomatic manifestations, as seen in the case. Clinicians should be aware of this clinical presentation.