Rapid visual detection of porcine reproductive and respiratory syndrome virus via recombinase polymerase amplification combined with a lateral flow dipstick.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically devastating infectious diseases in the global swine industry. A rapid and sensitive on-site detection method for PRRS virus (PRRSV) is critically important for diagnosing PRRS. In this study, we established a method that combines reverse transcription recombinase polymerase amplification (RT-RPA) with a lateral flow dipstick (LFD) for detecting North American PRRSV (PRRSV-2). The primers and probe were designed based on the conserved region of all complete PRRSV-2 genomic sequences available in China (n = 512) from 1996 to 2020. The detection limit of the assay was 5.6 × 10-1 median tissue culture infection dose (TCID50) per reaction within 30 min at 42 °C, which was more sensitive than that of reverse transcription polymerase chain reaction (RT-PCR) (5.6 TCID50 per reaction). The assay was highly specific for the epidemic lineages of PRRSV-2 in China and did not cross-react with pseudorabies virus, porcine circovirus 2, classical swine fever virus, or porcine epidemic diarrhea virus. The assay performance was evaluated by testing 179 samples and comparing the results with those of quantitative RT-PCR (RT-qPCR). The results showed that the detection coincidence rate of RT-RPA and RT-qPCR was 100% when the cycle threshold values of RT-qPCR were < 32. The assay provides a new alternative for simple and reliable detection of PRRSV-2 and has great potential for application in the field.

Recombination of Porcine Reproductive and Respiratory Syndrome Virus: Features, Possible Mechanisms, and Future Directions.

Recombination is a pervasive phenomenon in RNA viruses and an important strategy for accelerating the evolution of RNA virus populations. Recombination in the porcine reproductive and respiratory syndrome virus (PRRSV) was first reported in 1999, and many case reports have been published in recent years. In this review, all the existing reports on PRRSV recombination events were collected, and the genotypes, parental strains, and locations of the recombination breakpoints have been summarized and analyzed. The results showed that the recombination pattern constantly changes; whether inter- or intra-lineage recombination, the recombination hotspots vary in different recombination patterns. The virulence of recombinant PRRSVs was higher than that of the parental strains, and the emergence of virulence reversion was caused by recombination after using MLV vaccines. This could be attributed to the enhanced adaptability of recombinant PRRSV for entry and replication, facilitating their rapid propagation. The aim of this paper was to identify common features of recombinant PRRSV strains, reduce the recombination risk, and provide a foundation for future research into the mechanism of PRRSV recombination.

Antigenicity, epitope mapping, and intracellular distribution of the NSP7α protein of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that causes huge economic losses to the global pig industry. Nonstructural protein 7α (NSP7α) of PRRSV is highly conserved among different lineages of PRRSV and could be a potential target for the development of detection methods. In this study, NSP7α was expressed in prokaryote (Escherichia coli) and purified. An NSP7α-ab-ELISA detection method was established, the NSP7α-ab-ELISA has 93.1 % coincidence rate with IDEXX PRRS X3 ab test kit. NSP7α antibody was detected in pig serum by ELISA 14 days following PRRSV infection. Three monoclonal antibodies (4H9, 3F2, and C10) against NSP7α prepared by a hybridoma technique were used for epitope mapping by indirect immunofluorescence. The 4H9, 3F2, and C10 antibodies all recognized the C-terminal 72-149 amino acid region of NSP7α. 4H9 reacted with amino acids 135-143, but 3F2 and C10 did not react with any truncated polypeptide. In addition, by using the monoclonal antibodies, NSP7α was localized solely in the cytoplasm, while the N protein was distributed in the cytoplasm and nucleus. The collective findings of the antigenicity and epitope of NSP7α will be helpful for understanding the antigenicity of NSP7α and developing PRRSV diagnostic methods.

Multivalent nanobody-based sandwich enzyme-linked immunosorbent assay for sensitive detection of porcine reproductive and respiratory syndrome virus.

The pandemic of the porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses and continues to threaten the swine industry worldwide. Nucleocapsid protein (N protein) is the primary antigen of PRRSV for development of sensitive diagnostic assays. Two high affinity nanobodies against N protein, Nb12 and Nb35, were selected and employed to develop a sandwich ELISA. Further we improved the ELISA method to obtain greater sensitivity, a trivalent nanobody (3 × Nb35) and a bivalent nanobody-HRP fusion protein (2 × Nb12-HRP) were expressed and used. This modified ELISA was found to have high sensitivity for detecting PRRSV, with a detection limit of 10 TCID50/ml (median tissue culture infectious dose), which was approximately 200-fold greater than the single-copy nanobody-based sandwich ELISA. The developed assay shows high specificity and can detect almost all circulating lineages of PRRSV-2 in China. This study provides suggestions for reforming nanobodies and for the further development of multivalent nanobody-based ELISAs for other various viruses.

Analysis of Recombinant Characteristics Based on 949 PRRSV-2 Genomic Sequences Obtained from 1991 to 2021 Shows That Viral Multiplication Ability Contributes to Dominant Recombination.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases affecting the pig-raising industry. The PRRS virus (PRRSV) has high genetic diversity, partly owing to viral recombination. Some individual recombinant type 2 PRRSV (PRRSV-2) strains have been detected; however, the sequence composition characteristics of recombination hot spots and potential driving forces for recombinant PRRSV-2 are still unreported. Therefore, all available genomic sequences of PRRSV-2 (n = 949, including 29 genomes sequenced in this study) from 11 countries from 1991 to 2021 were collected and analyzed. The results revealed that the dominant major recombinant parent has been converted from lineage 3 (L3) to L1 since 2012. The recombination hot spots were located at nucleotides (nt) 7900 to 8200 (in NSP9, encoding viral RNA-dependent RNA polymerase) and nt 12500 to nt 13300 (in ORF2-ORF4, mean ORF2 to ORF4); no AU-rich characteristics were found in the recombination hot spots. Based on infectious clones of L1 and L8 PRRSV-2, recombinant PRRSVs were generated by switching complete or partial NSP9 (harboring the recombination hot spot). The results showed that recombinant PRRSVs based on the L1 backbone, but not the L8 backbone, acquired a higher replication capacity in pig primary alveolar macrophages. These findings will help to understand the reason behind the dominance of L1-based recombination in PRRSV-2 strains and provide new clues for an in-depth study of the recombination mechanism of PRRSV-2. IMPORTANCE Recombination is an important driver of the genetic shifts that are tightly linked to the evolution of RNA viruses. Viral recombination contributes substantially to the emergence of new variants, alterations in virulence, and pathogenesis. PRRSV is genetically diverse, partly because of extensive recombination. In this study, we analyzed interlineage recombination based on available genomic sequences of PRRSV-2 from 1991 to 2021. The study revealed the temporal and geographical distribution of recombinant PRRSVs and the recombination hot spot's location and showed that artificially constructed recombinant PRRSVs (harboring a high-frequency region) had more viral genomic copies than their parental virus, indicating that dominant recombination was shaped by a tendency to benefit viral replication. This finding will enrich our understanding of PRRSV recombination and provide new clues for an in-depth study of the recombination mechanism.

Recombinant characteristics, pathogenicity, and viral shedding of a novel PRRSV variant derived from twice inter-lineage recombination.

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is a significant threat to the global pig industry. In this study, a novel recombinant PRRSV, SD043, was isolated from a pig farm experiencing disease in 2019. Phylogenetic analysis revealed that SD043 belonged to lineage 1 of PRRSV-2 while recombination analyses revealed that it is a recombinant virus from lineage 1 and lineage 8 strains. Based on further analysis, SD043 underwent recombination twice. Pathogenicity studies revealed that SD043 causes mild clinical symptoms, thymus atrophy, and severe histopathological lesions in the lungs. Notably, virus shedding in SD043-infected piglets was detectable at 10 days post-inoculation with a high viral load in the respiratory or digestive tract, indicating that the recombinant PRRSV appears to shed higher numbers of virus. Furthermore, genomic surveillance based on all available PRRSVs circulating in Shandong province revealed an increasing increase in recombinant PRRSV since 2015, with the recombinant pattern (between lineages 1 and 8) being the same as that of SD043. These findings enable a better understanding of the process of twice recombination and virus shedding of recombinant PRRSV and can strengthen the prevention and control of the PRRSV epidemic.

Recent Advances in Porcine Reproductive and Respiratory Syndrome Virus NADC30-Like Research in China: Molecular Characterization, Pathogenicity, and Control.

The name porcine reproductive and respiratory syndrome virus (PRRSV) NADC30-like was first coined in 2015. It originated from the NADC30 strain that was introduced into China by importing breeding pigs and has since undergone mutations or recombination, resulting in variant viruses. Following widespread outbreaks in China in recent years, these NADC30-like strains have presented major health challenges in swine production systems. Outcomes induced by PRRSV NADC30-like infection are highly variable, ranging from inapparent to severe, depending on the recombination between NADC30 and field PRRSV strains prevalent in swine farms. Vaccines and strict biosecurity measures have been explored to fight this disease; however, current PRRSV commercially modified-live virus vaccines (MLVs) have the potential to revert to virulence and only provide limited or no cross-protection efficacy against NADC30-like strains. PRRSVs will remain an ongoing challenge to the swine industry until safe and effective vaccines or antiviral reagents are developed.

Identification and assessment of pathogenicity of a naturally reassorted infectious bursal disease virus from Henan, China.

Infectious bursal disease virus (IBDV) is still a vital etiological agent in poultry farms. IBDV outbreaks occasionally occur due to the presence of very virulent, reassortment or variant strains. Vaccine immunization has played crucial roles in IBD control for decades. However, survival pressure of IBDV from the vaccine immunization also increases the reassortments of circulating viruses. In this study, an IBDV strain was isolated from several broiler farms in Henan Province, central part of China, and named IBDV HN strain. Based on the results of RT-PCR, sequencing and phylogenic analyses of VP1 and VP2 genes, the IBDV HN strain is a novel reassortment strain in the Henan region. Segment A of this strain appears to originate from the very virulent IBDV strain, while segment B comes from the other field reassortment strains. This may be the result of natural reassortant of virus circulating in the field. About 60% (6/10) of experimentally infected specific pathogen-free chickens died after 3 to 5 d post-infection with typical symptom and pathological lesions. The IBDV HN strain was prone to horizontal transmission, which poses a serious threat to the chicken industry. Further investigation on the prevalence, virulence, and evolution of HN strain IBDV will provide a foundation for the prevention and control of the disease in this region.