Metagenomic Approach Reveals the Second Subtype of PRRSV-1 in a Pathogen Spectrum during a Clinical Outbreak with High Mortality in Western Siberia, Russia.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a significant economic impact on pig farming worldwide by causing reproductive problems and affecting the respiratory systems of swine. In Eastern Europe, PRRSV-1 strains are characterized by high genetic variability, and pathogenicity differs among all known subtypes. This case study describes the detection of a wide pathogen spectrum, including the second subtype PRRSV-1, with a high mortality rate among nursery piglets (23.8%). This study was conducted at a farrow-to-finish farm in the Western Siberia region of Russia. Clinical symptoms included apathy, sneezing, and an elevation in body temperature, and during the autopsy, degenerative lesions in different tissues were observed. Moreover, 1.5 percent of the affected animals displayed clinical signs of the central nervous system and were characterized by polyserositis. Nasal swabs from diseased piglets and various tissue swabs from deceased animals were studied. For diagnostics, the nanopore sequencing method was applied. All the samples tested positive for PRRSV, and a more detailed analysis defined it as a second subtype of PRRSV-1. The results, along with the clinical picture, showed a complex disease etiology with the dominant role of PRRSV-1 and were informative about the high pathogenicity of the subtype in question under field conditions.

Whole-Genome Analysis of Porcine Circovirus Type 2 in Russia.

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVAD) that bring about significant economic losses in the pig industry all over the world. The aim of this study was to investigate the genetic diversity of PCV2 in Russia and characterize the available complete genome sequences. PCV2 DNA was detected at all investigated farms located in different regions of Russia. Whole-genome analysis demonstrated that the majority of PCV2 strains belonged to genotype PCV2d (12 out of 14), while PCV2a and PCV2b were only detected at 2 farms (one at each). Further analysis revealed that all antibody recognition sites in Russian PCV2 strains were different from the corresponding epitopes in a PCV2a vaccine strain, suggesting that PCV2a-based vaccines may only provide limited protection against these strains. PCV2d strains could be grouped into 3 distinct lines which shared 98.7-100% identity within open reading frame 2 (ORF2). It is the first study reporting the genetic diversity of PCV2 strains in Russia. Our data indicated that, similarly to China, Europe, and USA, PCV2a and PCV2b have largely been replaced by PCV2d.

Development of a Potential Penside Colorimetric LAMP Assay Using Neutral Red for Detection of African Swine Fever Virus.

African swine fever (ASF) has caused huge economic losses to the swine industry worldwide. Since there is no commercial ASF vaccine available, an early diagnosis is extremely important to prevent and control the disease. In this study, ASF virus (ASFV) capsid protein-encoding gene (p72) was selected and used to design primers for establishing a one-step visual loop-mediated isothermal amplification (LAMP) assay with neutral red, a pH-sensitive dye, as the color shift indicator. Neutral red exhibited a sharp contrast of color change from faint orange (negative) to pink (positive) during LAMP for detection of ASFV. The designed primer set targeting highly conserved region of the p72 gene was highly specific to ASFV and showed no cross-reactivity with other swine viruses. The detection limit for the one-step visual LAMP developed was 10 copies/reaction based on the recombinant plasmid containing the p72 gene of ASFV. More importantly, the developed one-step visual LAMP showed high consistency with the results of the real-time polymerase chain reaction (qPCR) method recommended by World Organization for Animal Health (OIE). Furthermore, the results demonstrate that the colorimetric detection with this LAMP assay could be directly applied for the whole blood and serum samples without requiring genome extraction. Based on our results, the developed one-step visual LAMP assay is a promising penside diagnostic tool for development of early and cost-effective ASF monitoring program that would greatly contribute to the prevention and control of ASF.

An Outbreak of a Respiratory Disorder at a Russian Swine Farm Associated with the Co-Circulation of PRRSV1 and PRRSV2.

We conducted a cross-sectional study to identify the major respiratory pathogen responsible for an outbreak of respiratory disease at a swine farm in West Siberia in 2019. We discovered that the peak of morbidity and mortality coincided with a high level of porcine reproductive and respiratory syndrome virus (PRRSV) 1 and 2-related viremia. Based on longer PRRSV2 viremia, the dominant role of PRRSV2 over PRRSV1 in the outbreak was assumed. Phylogenetic analysis revealed that the PRRSV1 strain belonged to sub-genotype 2-one of the predominant groups of genotype 1 PRRSVs in Russia. A partial open reading frame 7 sequence of the PRRSV2 isolate demonstrated a high identity with modified live vaccine-related strains from Denmark (93%) and wild-type VR2332 (92%). We identified the first instance of PRRSV1/PRRSV2 mixed infection in Russia. This finding indicates that further field investigations are needed to access PRRSV2 epidemiology in eastern Europe.

Full-genome analysis and pathogenicity of a genetically distinct Russian PRRSV-1 Tyu16 strain.

Porcine reproductive and respiratory syndrome virus-1 (PRRSV-1) strains from Eastern Europe have a high diversity. All three known subtypes (1, 2, 3) of PRRSV-1 have been detected in Russia. There are two different groups of viruses belonging to the subtype 1: pan-European subtype 1 strains, and insufficiently studied Russian strains. The main objective of this study was to characterize the full genomic structure of the atypical Tyu16 strain of the Russian group subtype 1 PRRSV-1 and to assess its pathogenicity. Complete sequencing of the Tyu16 strain revealed that it did not belong to any existing subtype. Comparison of the whole genome sequence of the Tyu16 strain with that of PRRSV-1 prototype strains revealed 78.1 % (subtype 1 Lelystad), 78.1 % (subtype 2 WestSib13) and 77.7 % (subtype 3 Lena) nucleotide identity level, respectively. The coding sequence of different parts of the Tyu16 strain genome demonstrated a varying percentage identity to the different reference PRRSV-1 strains, which may indicate recombination events in its evolutionary history. We assume that among PRRSV-1 isolates, the Tyu16 is the closest relative to the common ancestor of PRRSV-1 and PRRSV-2. Low pathogenicity of the Tyu16 was demonstrated by experimental infection of 70-day-old piglets. Infected animals showed fever not exceeding 7 days, dyspnea in two out of five pigs and reduced weight gain. The virus shedding was undetectable and viremia was at low level.

Novel Mammalian orthorubulavirus 5 Discovered as Accidental Cell Culture Contaminant.

A distinct Russian Mammalian orthorubulavirus 5 (PIV5) was detected in cell culture exhibiting cytopathic effect and hypothesized to be contaminated by a scientist with respiratory symptoms. The identification of the divergent strain indicated a lack of knowledge on the diversity of PIV5 strains and calls for surveillance of global PIV5 strains.

First detection and full genome sequence of porcine circovirus type 3 in Russia.

Porcine circovirus type 3 (PCV3) was firstly detected in 2016 in USA. Later PCV3 was discovered in Asia, Europe, and South America. The present investigation demonstrates for the first time the circulation of PCV3 among pigs in Russia. The viruses were detected at two geographically distant unrelated commercial farms with records of reproductive failure (abortions, stillbirth), porcine dermatitis, and nephropathy syndrome (PDNS). The two farms were located in the region of Smolensk (western part of Russia) and the region of Tyumen (West Siberia, Russia). We investigated samples collected from pigs of different ages. We performed PCR for the PCV3 DNA detection. The DNA of PCV3 was detected in serum, kidney, heart, spleen, pleural effusion, and peritoneal cavity fluid samples. Two viral genomes were sequenced and the corresponding strains were named PCV3-RU/SM17 (the strain from Smolensk region) and PCV3-RU/TY17 (the strain from Tyumen region). The full genome sequences of both strains were 2000 nucleotides in length and contained at least two ORFs, encoding the Cap and Rep proteins. Full sequence alignment revealed a 99.3% identity between the PCV3-RU/SM17 and PCV3-RU/TY17 strains. Molecular analysis showed that the two strains from Russia are highly homologous to viruses identified in other countries, with a 98.5-99.6% homology for PCV3-RU/TY17 and 97.9-99.0 for PCV3-RU/SM17. The PCV3-RU/SM17 and PCV3-RU/TY17 strains were found to form a monophyletic group in a phylogenetic tree based on PCV3 complete genome sequences.

Genome Characterization of a Pathogenic Porcine Rotavirus B Strain Identified in Buryat Republic, Russia in 2015.

An outbreak of enteric disease of unknown etiology with 60% morbidity and 8% mortality in weaning piglets occurred in November 2015 on a farm in Buryat Republic, Russia. Metagenomic sequencing revealed the presence of rotavirus B in feces from diseased piglets while no other pathogens were identified. Clinical disease was reproduced in experimentally infected piglets, yielding the 11 RVB gene segments for strain Buryat15, with an RVB genotype constellation of G12-P[4]-I13-R4-C4-M4-A8-N10-T4-E4-H7. This genotype constellation has also been identified in the United States. While the Buryat15 VP7 protein lacked unique amino acid differences in the predicted neutralizing epitopes compared to the previously published swine RVB G12 strains, this report of RVB in Russian swine increases our epidemiological knowledge on the global prevalence and genetic diversity of RVB.

Genetic and pathogenic characterization of a Russian subtype 2 PRRSV-1 isolate.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure and respiratory problems. Data about the virulence and pathogenicity of subtype 2 PRRSV-1 strains are limited. The main purposes of this investigation were to characterize the full genome sequence of the subtype 2 PRRSV-1 WestSib13 strain and to compare the pathogenicity with that of the subtype 1 PRRSV-1 Lelystad strain. Comparison of the whole genome sequence of the WestSib13 strain with that of PRRSV-1 prototype strains revealed a 76.2% (subtype 1 Lelystad virus) and 79.0% (subtype 3 Lena virus) identity, respectively The virulence and pathogenicity of the European subtype 2 PRRSV strain WestSib13 and the European subtype 1 PRRSV strain Lelystad were compared in 3-week-old piglets upon inoculation of 105.4 TCID50 of virus. Non-infected animals (control group) as well as animals infected with the Lelystad strain were clinically healthy until 14days post challenge. In contrast, animals infected with the WestSib13 strain demonstrated dyspnea starting at 3days post-inoculation (dpi). All piglets in this group died between 5 and 8 dpi. During that period, fever was not observed in WestSib13-infected animals. Viremia was detected in animals from both infected groups starting from 2 dpi. Viral loads in serum and lungs upon euthanasia were significantly higher (3 log10) in the WestSib13-infected than in the LV-infected animals. Taken together, this study provides the full genome sequence and the unusual virological and clinical outcome (high level viremia without fever) of the novel WestSib13 strain.