NADC30-like Porcine Reproductive And Respiratory Syndrome Virus Research Articles

Isolation and pathogenicity comparison of two novel natural recombinant porcine reproductive and respiratory syndrome viruses with different recombination patterns in Southwest China.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses in the swine industry. Frequent mutations and recombinations account for PRRSV immune evasion and the emergence of novel strains. In this study, we isolated and characterized two novel PRRSV-2 strains from Southwest China exhibiting distinct recombination patterns. They were designated SCABTC-202305 and SCABTC-202309. Phylogenetic results indicated that SCABTC-202305 was classified as lineage 8, and SCABTC-202309 was classified as lineage 1.8. Amino acid mutation analysis identified unique amino acid substitutions and deletions in ORF5 and Nsp2 genes. The results of the recombination analysis revealed that SCABTC-202305 is a recombinant with JXA1 as the major parental strain and NADC30 as the minor parental strain. At the same time, SCABTC-202309 is identified as a recombinant with NADC30 as the major parental strain and JXA1 as the minor parental strain. In this study, we infected piglets with SCABTC-202305, SCABTC-202309, or mock inoculum (control) to study the pathogenicity of these isolates. Although both isolated strains were pathogenic, SCABTC-202305-infected piglets exhibited more severe clinical signs and higher mortality, viral load, and antibody response than SCABTC-202309-infected piglets. SCABTC-202305 also caused more extensive lung lesions based on histopathology. Our findings suggest that the divergent pathogenicity observed between the two novel PRRSV isolates may be attributed to variations in the genetic information encoded by specific genomic regions. Elucidating the genetic determinants governing PRRSV virulence and transmissibility will inform efforts to control this devastating swine pathogen.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) is one of the most critical pathogens impacting the global swine industry. Frequent mutations and recombinations have made the control of PRRSV increasingly difficult. Following the NADC30-like PRRSV pandemic, recombination events involving PRRSV strains have further increased. We isolated two novel field PRRSV recombinant strains, SCABTC-202305 and SCABTC-202309, exhibiting different recombination patterns and compared their pathogenicity in animal experiments. The isolates caused higher viral loads, persistent fever, marked weight loss, moderate respiratory clinical signs, and severe histopathologic lung lesions in piglets. Elucidating correlations between recombinant regions and pathogenicity in these isolates can inform epidemiologic tracking of emerging strains and investigations into viral adaptive mechanisms underlying PRRSV immunity evasion. Our findings underscore the importance of continued genomic surveillance to curb this economically damaging pathogen.

Novel characterization of NADC30-like and NADC34-like PRRSV strains in China: epidemiological status and pathogenicity analysis of L1A variants1

NADC34-like Porcine reproductive and respiratory syndrome virus (PRRSV), which first appeared in China in 2017, is currently one of the main epidemic strains in China. In this study, we found that a new variant of NADC34-like PRRSV evolved, named the L1A variant. The phylogenetics, epidemic status, and pathogenicity of the LA variants were subsequently comprehensively evaluated. Based on the results of the ORF5 phylogenetic analysis, the L1A variants were classified as NADC34-like PPRSV. All the strains had the same discontinuous 131-aa deletion in the NSP2 region (similar to that in the NADC30). Recombination analysis revealed that the L1A variants were recombinant viruses that contained an NADC30-like PRRSV skeleton, a nonstructural protein-encoding gene region obtained in part from JXA1-like PRRSV and a ORF2-ORF6 gene region partly obtained from NADC34-like PRRSV and that exhibited similar recombination patterns. We successfully isolated the L1A variant TZJ2756 from PAMs and Marc-145 cells. In animal experiments, TZJ2756 exhibited moderate pathogenicity in piglets, causing obvious clinical symptoms, namely, persistent fever, significantlyreduced body weight, interstitial edema and severe interstitial pneumonia in the lungs, and prolonged high-load viremia. L1A variants have been detected in at least 12 provinces in China and share many similar epidemiological characteristics with the American L1C variant. This research will enhance our understanding of the prevalence of L1A variants and furnish valuable data for the ongoing monitoring of NADC34-like PRRSV in China.

Isolation, identification, recombination analysis and pathogenicity experiment of a PRRSV recombinant strain in Sichuan Province, China.

Since 2013, the porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2), lineage 1.8 (NADC30-like PRRSV) has emerged and become widely prevalent in China. The NADC30-like PRRSV poses significant challenges for disease control, primarily because of its propensity for frequent mutations and recombinations. We successfully isolated and identified a NADC30-like strain, designated SCCD22, in Chengdu, Sichuan Province, China. We meticulously examined the genetic recombination properties and evaluated its pathogenicity in 28-day-old piglets. SCCD22 showed 93.02% nucleotide homology with the NADC30 PRRSV strain, and its non-structural protein 2 coding region showed the same 131 amino acid deletion pattern as that seen in NADC30. Furthermore, we identified two recombination events in SCCD22: one in the NSP2 region (1,028-3,290 nt), where it was highly similar to the JXA1-like strain GZ106; and another in the NSP10 ~ 12 region (9,985-12,279 nt), closely resembling the NADC30-like strain CY2-1604. Piglets infected with SCCD22 exhibited clinical symptoms such as elevated body temperature, prolonged fever, reduced appetite, and roughened fur. Postmortem examinations underscored the typical lung pathology associated with PRRSV, indicating that the lungs were the primary affected organs. Furthermore, extended viral shedding accompanied by progressive viremia was observed in the serum and nasal excretions of infected piglets. In summary, this study reports a domestic PRRSV recombination strain in the Sichuan Province that can provide critical insights into preventing and controlling PRRSV in this region.

Pathogenicity Studies of NADC34-like Porcine Reproductive and Respiratory Syndrome Virus LNSY-GY and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus GXGG-8011 in Piglets.

The porcine reproductive and respiratory syndrome virus (PRRSV) has caused significant economic losses to the swine industry. The U.S., China, and Peru have reported NADC30-like or NADC34-like PRRSV-infected piglets, which have been identified as the cause of a significant number of abortions in clinics. Although the pathogenicity of NADC30-like PRRSV and NADC34-like PRRSV in piglets exhibits significant variability globally, studies on their pathogenicity in China are limited. In this study, the animal experiments showed that within 8-14 days post-infection, both piglets infected with NADC30-like PRRSV GXGG-8011 and those infected with NADC34-like PRRSV LNSY-GY exhibited significant weight loss compared to the control piglets. Additionally, the viremia of the LNSY-GY persisted for 28 days, while the viremia of piglets infected with the GXGG-8011 lasted for 17 days. Similarly, the duration of viral shedding through the fecal-oral route after the LNSY-GY infection was longer than that observed after the GXGG-8011 infection. Furthermore, post-infection, both the LNSY-GY and GXGG-8011 led to pronounced histopathological lesions in the lungs of piglets, including interstitial pneumonia and notable viral colonization. However, the antibody production in the LNSY-GY-infected group occurred earlier than that in the GXGG-8011-infected group. Our research findings indicate that LNSY-GY is a mildly pathogenic strain in piglets, whereas we speculate that the GXGG-8011 might be a highly pathogenic strain.

Characterization of Rongchang piglets after infection with type 2 porcine reproductive and respiratory syndrome virus strains differing in pathogenicity.

Porcine reproductive and respiratory syndrome virus (PRRSV) affects the production and health of pigs and causes severe economic losses to the swine industry worldwide. Different pig breeds have been reported to have different levels of susceptibility to PRRSV, and different PRRSV strains may also influence the infectivity and pathogenicity of the virus. In this study, the susceptibility of Rongchang pigs (a prominent local pig breed in China) to PRRSV infection was thoroughly investigated. Rongchang piglets were exposed to two PRRSV strains: HuN4 (highly pathogenic PRRSV) and SD53-1603 (moderately virulent NADC30-like PRRSV). We observed that Rongchang pigs infected with HuN4 displayed significant clinical manifestations, including fever, reduced body weight, and interstitial pneumonia lesions. Routine blood tests revealed that HuN4-infected pigs exhibited slightly decreased levels of red blood cells, hemoglobin, reticulocytes, and a notable increase in monocytes than control pigs. Additionally, the Rongchang pigs exhibiting severe clinical signs presented a higher neutrophil-to-lymphocyte ratio and a lower lymphocyte-to-monocyte ratio. In contrast, SD53-1603 infection did not cause considerable harm to Rongchang pigs, only resulting in slightly elevated leukocytes and lymphocytes. Furthermore, these two PRRSV strains elicited divergent cytokine responses, such that SD53-1603 infection induced higher levels of TNF-α and IFN-γ, whereas HuN4 infection upregulated IL-1β. These dissimilarities in clinical symptoms, pathological changes, viremia, cytokine expression, and routine blood indices between HuN4 and SD53-1603 infections are critical in understanding the mechanisms of PRRSV infection and developing rational prevention and control strategies against PRRSV.

Protective efficacy of a candidate live attenuated vaccine derived from the SD-R strain of lineage 1 porcine reproductive and respiratory syndrome virus against a lethal challenge with HP-PRRSV HuN4 in piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen endangering the swine industry worldwide. Our previous study confirmed that the live attenuated vaccine SD-R strain could provide broad cross-protection against homologous and heterologous NADC30-like viruses. However, it is unclear whether SD-R can provide protection for piglets against HP-PRRSV. In the present study, 4-week-old PRRSV-free piglets were vaccinated intramuscularly with 106.2 TCID50 of SD-R and then challenged intramuscularly (2 mL) and intranasally (2 mL) with HP-PRRSV HuN4 (1 × 105.0 TCID50/mL). The results showed that two of five piglets died in the HuN4 challenge group and five of five immunized piglets survived in the immune challenge group. Five piglets in the HuN4 challenge group had a fever for 6-12 days and showed obvious clinical symptoms, but 3 of 5 piglets had a fever for only 1 day at 5 days post inoculation, and only 1 of the 5 piglets in the immune challenge group had a cough. Compared to challenged control piglets, vaccinated piglets had significantly improved daily weight gain and obviously alleviated pathological lesions. Furthermore, piglets immunized with SD-R had reduced thymus atrophy caused by HP-PRRSV HuN4. These results demonstrate that the lineage 1 PRRSV attenuated vaccine SD-R strain provides protection against lethal HP-PRRSV HuN4 challenge. As such, it is a promising vaccine candidate for use against PRRSV infection. IMPORTANCE Both highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and NADC30-like PRRSV have caused tremendous economic losses to the Chinese pig industry. In this study, a good challenge model was established to evaluate the protection afforded by the candidate SD-R vaccine against infection with a representative HP-PRRSV strain (HuN4). The control piglets in the challenge experiment displayed obvious clinical symptoms of PRRSV infection, with a mortality rate up to 40%. In contrast, all the piglets in the vaccinated challenged group survived, and only some pigs had transient fever. The daily gain of SD-R immunized group piglets was significantly increased, and the pathological changes were significantly reduced. In addition, the viral replication levels in the serum of the immunized group were significantly lower than those of the challenged control group. The live attenuated vaccine SD-R strain can provide protection against HP-PRRSV challenge, indicating that the SD-R strain is a promising vaccine candidate for use in the swine industry.

Epidemiological investigation and pathogenicity of porcine reproductive and respiratory syndrome virus in Sichuan, China.

Porcine reproductive and respiratory syndrome virus type 2 (PRRSV-2) lineage 8 was first detected in mainland China in 2006 and has since rapidly spread to become the primary epidemic strain in the country. In this study, samples such as lung tissue, hilar lymph nodes, abortion fetuses, and blood were collected from large-scale pig farms across 11 prefecture-level cities in Sichuan province between 2019 and 2020 for antigen detection and PRRS virus isolation. The antigen detection results indicated that the positive rate of HP-PRRSV (JXA1-Like strain) was 44.74% (51/114), NADC30-Like PRRSV was 17.54% (20/114), and classical PRRSV (VR2332-Like strain) was 37.72% (43/114). The predominant strain was HP-PRRSV. Positive samples were further inoculated into Marc-145 cells for virus isolation and identification, leading to the isolation of a new JXA1-Like PRRSV strain named SCSN2020. The strain was characterized by RT-qPCR, indirect immunofluorescence assay (IFA), plaque purification, electron microscopy, and whole genome sequencing. The total length of the viral genome was determined to be approximately 15,374 bp. A comparison of the SCSN2020 genome with VR2332 revealed that both strains had the same discontinuous 30-amino acid deletion on the Nsp2 gene. ORF5 genotyping classified the SCSN2020 strain as sublineage 8.7, with a whole genome sequence identity of 99.34% with JXA1. Furthermore, we evaluated the pathogenicity of the SCSN2020 strain in 28-day-old piglets and observed persistent fever from day 4 to day 10, weight loss started on day 7, dyspnea and severe lung lesions began started on day 14. The results of this study highlight the current PRRSV epidemic situation in Sichuan province and provide a scientific reference for subsequent prevention and control measures.

Genomic similarity and antibody-dependent enhancement of immune serum potentially affect the protective efficacy of commercial MLV vaccines against NADC30-like PRRSV

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant diseases affecting the pig industry worldwide. The PRRSV mutation rate is the highest among the RNA viruses. To date, NADC30-like PRRSV and highly pathogenic PRRSV (HP-PRRSV) are the dominant epidemic strains in China; however, commercial vaccines do not always provide sufficient cross-protection, and the reasons for insufficient protection are unclear. This study isolated a wild-type NADC30-like PRRSV, SX-YL1806, from Shaanxi Province. Vaccination challenge experiments in piglets showed that commercial modified live virus (MLV) vaccines provided good protection against HP-PRRSV. However, it could not provide sufficient protection against the novel strain SX-YL1806. To explore the reasons for this phenomenon, we compared the genomic homology between the MLV strain and HP-PRRSV or NADC30-like PRRSV and found that the MLV strain had a lower genome similarity with NADC30-like PRRSV. Serum neutralization assay showed that MLV-immune serum slightly promoted the homologous HP-PRRSV replication and significantly promoted the heterologous NADC30-like PRRSV strain replication in vitro, suggesting that antibody-dependent enhancement (ADE) might also play a role in decreasing MLV protective efficacy. These findings expand our understanding of the potential factors affecting the protective effect of PRRSV MLV vaccines against the NADC30-like strains.

Protective Efficacy of a Candidate Live-Attenuated Vaccine Derived from the SD-R Strain against NADC34-like Porcine Reproductive and Respiratory Syndrome Virus.

NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV) strains were first detected in China in 2017 and became major circulating strains in 2021. Our previous study showed that the live-attenuated vaccine candidate SD-R strain could provide broad cross-protection against different NADC30-like PRRSVs (sublineage 1.8). However, the protective effect of SD-R against NADC34-like PRRSV is unclear. Here, a novel NADC34-like PRRSV, LNTZJ1341-2012, was isolated from a pig farm experiencing disease in 2020. Sequence analysis revealed that LNTZJ1341-2012 belonged to PRRSV-2 sublineage 1.5, exhibited the same Nsp2 amino-acid deletion characteristics as IA/2014/NADC34, and had not recombined with other strains. Additionally, a good challenge model was established to evaluate the protection afforded by the candidate SD-R vaccine against infection with a representative NADC34-like strain (LNTZJ1341-2012). The control piglets in the challenge experiment displayed clinical signs typical of PRRSV infection, including transient fever, high viremia, mild clinical symptoms, and histopathological changes in the lungs and submaxillary lymph nodes. In contrast, SD-R vaccination significantly reduced serum and lung tissue viral loads, and vaccinated piglets did not show any clinical symptoms or histopathological changes. Our results demonstrated that LNTZJ1341-2012 is a mildly virulent NADC34-like PRRSV and that the live-attenuated vaccine SD-R can prevent the onset of clinical signs upon challenge with the NADC34-like PRRSV LNTZJ1341-2012 strain, indicating that SD-R is a promising vaccine candidate for the swine industry.

Genetic and pathogenic characterization of an NADC-34-like isolate of porcine reproductive and respiratory syndrome virus.

In this study, an NADC34-like strain of porcine reproductive and respiratory syndrome virus (PRRSV), YC-2020, was isolated from a pig farm in Yuncheng, Shanxi Province, China. Phylogenetic and molecular evolutionary analysis showed that the genome sequence of YC-2020 was very similar to those of NADC34-like PRRSV strains in the ORF2-7 region. However, it was more closely related to NADC30-like PRRSV and highly pathogenic (HP) PRRSV in the NSP2 and NSP3-9 coding regions, respectively, suggesting that recombination had occurred between viruses belonging to lineages 1 and 8. Piglets infected with YC-2020 exhibited mild clinical signs, but they had severe histopathological lesions in their lungs. These findings reveal novel genetic and pathogenic features of this isolate.

Isolation, identification, and pathogenicity of a NADC30-like porcine reproductive and respiratory disorder syndrome virus strain affecting sow production.

Since it was first reported in 1987, porcine reproductive and respiratory syndrome virus (PRRSV) has caused several economic crises worldwide. The current prevalence of PRRSV NADC30-like stains causing clinical disease outbreaks in Chain is highly concerning. Immunization against and the prevention of this infection are burdensome for farming organizations as the pathogen frequently mutates and undergoes recombination. Herein, the genetic characterization of a NADC30-like strain (termed BL2019) isolated from a farm in Guangdong Province, China, was analyzed and its pathogenicity for piglets and sows was assessed. Results revealed that BL2019 exhibits a nucleotide homology of 93.7% with NADC30 PRRSV and its NSP2 coding region demonstrates the same 131aa deletion pattern as that of NADC30 and NADC30-like. Furthermore, we identified two recombination breakpoints located nt5804 of the NSP5-coding region and nt6478 of NSP2-coding region, the gene fragment between the two breakpoints showed higher homology to the TJ strain(a representative strain of highly pathogenic PRRSV) compared to the NADC30 strain. In addition, BL2019 infection in piglets caused fever lasting for 1 week, moderate respiratory clinical signs and obvious visual and microscopic lung lesions; infection in gestating sows affected their feed intake and increased body temperature, abortion rates, number of weak fetuses, and other undesirable phenomena. Therefore, we report a NADC30-like PRRSV strain with partial recombination and a representative strain of HP-PRRSV, strain TJ, that can provide early warning and support for PRRS immune prevention and control.

A potent CD8 T-cell response may be associated with partial cross-protection conferred by an attenuated Chinese HP-PRRSV vaccine against NADC30-like PRRSV challenge.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating pathogens to the global swine industry. Many commercial PRRSV vaccines, originally designed to provide homologous protection, have shown partial protection against heterologous strains. However, the protective immune mechanisms mediated by these PRRSV vaccines are not fully understood. In this study, we investigated the factors responsible for partial protection conferred by an attenuated Chinese HP-PRRSV vaccine (TJM-F92) against heterologous NADC30-like PRRSV. By analysing peripheral T-cell responses induced by the TJM-F92 vaccine and local and systemic memory responses following challenge with NADC30-like PRRSV (SD17-38 strains) as well as neutralizing antibody response, we found that the TJM-F92 vaccine induced a significant expansion of CD8 T cells but not CD4 T cells or γδ T cells. The expanded CD8 T cells exhibited a phenotype of effector memory T cells and secreted IFN-γ upon restimulation with SD17-38 strains in vitro. In addition, only CD8 T cells in the prior immunized pigs rapidly expanded in the blood and spleen after heterologous challenge, with higher magnitude, compared to the unvaccinated pigs, showing a remarkable memory response. In contrast, no obvious humoral immune response was enhanced in the vaccinated and challenged pigs, and no heterologous neutralizing antibodies were detected throughout the experiment. Our results suggested that CD8 T cells elicited by the TJM-F92 vaccine may be responsible for partial heterologous protection against NADC30-like PRRSV strains and potentially recognize the conserved antigens among PRRSV strains.

Allicin Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection In Vitro and Alleviates Inflammatory Responses.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogens to the swine industry worldwide over the past three decades. No approved effective antiviral drug is available to control this virus. The antiviral effects of allicin (diallyl thiosulfinate) on many human and animal viruses have been documented. However, the antiviral effect of allicin on PRRSV infection remains unknown. In this study, we found that allicin exhibited an inhibitory effect on HP-PRRSV and NADC30-like PRRSV in a dose-dependent manner by interfering with viral entry, replication, and assembly. Furthermore, allicin alleviated the expression of pro-inflammatory cytokines (IFN-β, IL-6, and TNFα) induced by PRRSV infection. The pro-inflammatory signaling pathways, TNF signaling pathway and MAPK signaling pathway, up-regulated by PRRSV infection were restored by allicin treatment. Taken together, these results demonstrate that allicin has antiviral activity against PRRSV and ameliorates inflammatory responses induced by PRRSV infection, suggesting that allicin is a promising drug candidate for anti-PRRSV therapy in vivo.

Systematic Identification and Comparison of the Expressed Profiles of Exosomal MiRNAs in Pigs Infected with NADC30-like PRRSV Strain.

Exosomes are biological vesicles secreted and released by cells that act as mediators of intercellular communication and play a unique role in virus infection, antigen presentation, and suppression/promotion of body immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most damaging pathogens in the pig industry and can cause reproductive disorders in sows, respiratory diseases in pigs, reduced growth performance, and other diseases leading to pig mortality. In this study, we used the PRRSV NADC30-like CHsx1401 strain to artificially infect 42-day-old pigs and isolate serum exosomes. Based on high-throughput sequencing technology, 305 miRNAs were identified in serum exosomes before and after infection, among which 33 miRNAs were significantly differentially expressed between groups (13 relatively upregulated and 20 relatively downregulated). Sequence conservation analysis of the CHsx1401 genome identified 8 conserved regions, of which a total of 16 differentially expressed (DE) miRNAs were predicted to bind to the conserved region closest to the 3' UTR of the CHsx1401 genome, including 5 DE miRNAs capable of binding to the CHsx1401 3' UTR (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, ssc-miR-6529). Further analysis revealed that the target genes of differentially expressed miRNAs were widely involved in exosomal function-related and innate immunity-related signaling pathways, and 18 DE miRNAs (ssc-miR-4331-3p, ssc-miR-744, ssc-miR-320, ssc-miR-10b, ssc-miR-124a, ssc-miR-128, etc.) associated with PRRSV infection and immunity were screened as potential functional molecules involved in the regulation of PRRSV virus infection by exosomes.

Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) type 1 (European genotype) and PRRSV type 2 (North American genotype) are prevalent all over the world. Nowadays, the North American genotype PRRSV (NA-PRRSV) has been widely circulating in China and has caused huge economic losses to the pig industry. In recent years, classical PRRSV (C-PRRSV), highly pathogenic PRRSV (HP-PRRSV), and NADC30-like PRRSV (NL-PRRSV) have been the most common circulating strains in China. In order to accurately differentiate the circulating strains of NA-PRRSV, three pairs of specific primers and corresponding probes were designed for the Nsp2 region of C-PRRSV, HP-PRRSV, and NL-PRRSV. After optimizing the annealing temperature, primer concentration, and probe concentration, a multiplex real-time quantitative RT-PCR (qRT-PCR) and a multiplex Crystal digital RT-PCR (cdRT-PCR) for the differential detection of C-PRRSV, HP-PRRSV, and NL-PRRSV were developed. The results showed that the two assays illustrated high sensitivity, with a limit of detection (LOD) of 3.20 × 100 copies/μL for the multiplex qRT-PCR and 3.20 × 10-1 copies/μL for the multiplex cdRT-PCR. Both assays specifically detected the targeted viruses, without cross-reaction with other swine viruses, and indicated excellent repeatability, with coefficients of variation (CVs) of less than 1.26% for the multiplex qRT-PCR and 2.68% for the multiplex cdRT-PCR. Then, a total of 320 clinical samples were used to evaluate the application of these assays, and the positive rates of C-PRRSV, HP-PRRSV, and NL-PRRSV by the multiplex qRT-PCR were 1.88%, 21.56%, and 9.69%, respectively, while the positive rates by the multiplex cdRT-PCR were 2.19%, 25.31%, and 11.56%, respectively. The high sensitivity, strong specificity, excellent repeatability, and reliability of these assays indicate that they could provide useful tools for the simultaneous and differential detection of the circulating strains of C-PRRSV, HP-PRRSV, and NL-PRRSV in the field.

Molecular Characterization of the Nsp2 and ORF5s of PRRSV Strains in Sichuan China during 2012–2020

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that poses a serious threat to the global pig industry. Sichuan Province is one of the largest pig breeding provinces in China. There is a lack of reports on the continuous surveillance and systematic analysis of prevalent strains of PRRSV in Sichuan Province in recent years. To fill this gap, a total of 539 samples were collected from 13 breeding regions in Sichuan during 2012-2020. The detection result showed that the positive rate of PRRSV was 52.32% (282/539). The ORF5s and Nsp2 were obtained and further analyzed, with Chinese reference strains downloaded from the GenBank. Phylogenetic analysis showed that the PRRSV strains sequenced in this study belonged to PRRSV-1 and PRRSV-2 (lineage 1, 3, 5 and 8). In total, 168 PRRSV-2 strains were selected for ORF5 analyses, and these strains were classified into sub-lineage 8.7 (HP-PRRSV), sub-lineage 5.1 (classical PRRSV), sub-lineage 1.8 (NADC30-like), sub-lineage 1.5 (NADC34-like) and sub-lineage 3.5 (QYYZ-like), accounting for 60.71% (102/168), 11.31% (19/168), 18.45% (31/168), 2.97% (5/168) and 6.55% (11/168) of the total analyzed strains, respectively. The Nsp2 of identified PRRSV strains exhibited a nucleotide identity of 44.5-100%, and an amino acid identity of 46.82-100%. The ORF5 of the identified PRRSV strains exhibited a nucleotide identity of 81.3-100%, and an amino acid identity of 78.5-100%. A sequence analysis of ORF5 revealed that the mutation sites of GP5 were mainly concentrated in HVR1 and HVR2 and the virulence sites. In summary, the HP-PRRSV, NADC30-like PRRSV, Classic-PRRSV, QYYZ-like PRRSV, NADC34-like PRRSV and PRRSV-1 strains exist simultaneously in pigs in Sichuan. NADC30-like PRRSV was gradually becoming the most prevalent genotype currently in Sichuan province. This study suggested that PRRSV strains in Sichuan were undergoing genomic divergence.

CD163-Expressing Porcine Macrophages Support NADC30-like and NADC34-like PRRSV Infections.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a strict cell tropism. In addition to the primary alveolar macrophages, PRRSV is strictly cytotropic to African green monkey kidney cells, such as MARC-145 cells; however, MARC-145 cells are not infected by most NADC30-like and NADC34-like PRRSV strains. The essential scavenger receptor CD163 has been proved to mediate productive infection of PRRSV in various non-permissive cell lines. In this study, we systematically tested the porcine CD163 stably expressing 3D4/21 cells for infections with various PRRSV strains. The results showed that the porcine CD163-expressing macrophages support the infections of PRRSV2 of lineages 1, 5, and 8, as evidenced by Western blotting, immunofluorescence assay, quantitative PCR, and virus titration assay. Considering the current prevalence of NADC30-like and NADC34-like PRRSV2 of lineage 1 in China, the CD163-expressing macrophages are very useful for PRRSV research and disease management.

Comparison of pathogenicity of different subgenotype porcine reproductive and respiratory syndrome viruses isolated in China

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen causing huge economic losses to the swine industry worldwide, because of its rapid evolution and variation in genomes. It is necessary to monitor the newly emerging epidemic strains and compare their virulence diversity in swine. In this study, two strains of PRRSV2 were isolated from clinical samples by using primary porcine alveolar macrophage (PAMs) cells and designated as SD1805 and FJ1805, respectively. They were unable to grow in MARC-145 cells. Full-length genomes sequence analysis revealed that SD1805 strain has the molecular characterization of NADC30 strain from American, and belongs to the branch of NADC30-like PRRSV (NL-PRRSV) (lineagea 1). FJ1805 isolate came from the inter-subgenotype recombinant of NADC30 strain with a highly pathogenic PRRSV (HP-PRRSV) strain HUN4. Moreover, it belongs to a lineagea 3 represented by the Chinese isolate QYYZ. Pathogenicity analysis showed that SD1805 strain had similar mortality and viral loads in lungs to HP-PRRSV BB0907 strain. FJ1805 strain showed milder pathogenicity compared to NL-PRRSV strain FJ1402 that was previously isolated with Marc-145 cells. It provides evidence of the circulation of the different subgenotype PRRSV strains in China with variations in cell adaption and pathogenic abilities.

Lineage 1 Porcine Reproductive and Respiratory Syndrome Virus Attenuated Live Vaccine Provides Broad Cross-Protection against Homologous and Heterologous NADC30-Like Virus Challenge in Piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that endangers the swine industry worldwide. Recently, lineage 1 PRRSVs, especially NADC30-like PRRSVs, have become the major endemic strains in many pig-breeding countries. Since 2016, NADC30-like PRRSV has become the predominant strain in China. Unfortunately, current commercial vaccines cannot provide sufficient protection against this strain. Here, an attenuated lineage 1 PRRSV strain, named SD-R, was obtained by passaging an NADC30-like PRRSV strain SD in Marc-145 cells for 125 passages. Four-week-old PRRSV-free piglets were vaccinated intramuscularly with 105.0TCID50 SD-R and then challenged intramuscularly (2 mL) and intranasally (2 mL) with homologous NADC30-like PRRSV SD (1 × 105.0TCID50/mL) and heterologous NADC30-like PRRSV HLJWK108-1711 (1 × 105.0TCID50/mL). The results showed that antibodies against specific PRRSVs in 5 of 5 immunized piglets were positive after a 14-day post-vaccination and did not develop fever or clinical diseases after NADC30-like PRRSV challenges. Additionally, compared with challenge control piglets, vaccinated piglets gained significantly more weight and showed much milder pathological lesions. Furthermore, the viral replication levels of the immunized group were significantly lower than those of the challenge control group. These results demonstrate that lineage 1 PRRSV SD-R is a good candidate for an efficacious vaccine, providing complete clinical protection for piglets against NADC30-like PRRSVs.

A rescued NADC30-like virus by reverse genetic manipulation exhibits moderate virulence and a promising application perspective

NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV), which is highly homologous to the NADC30 strain isolated in the United States. The NADC30-like PRRSV was first reported in 2014 in China, where it spread and gradually caused an epidemic. Currently, growing research has shown that NADC30-like strains have greater propensity to recombine with other PRRSV strains, particularly the PPRSV vaccine virus used clinically, making the prevention and control of PRRSV highly complex. To carry out an in-depth molecular biology and virulence analysis, a full-length infectious clone of the NADC30-like strain was successfully constructed and rescued by reverse genetic manipulation. The rescued virus, rZJqz, was indistinguishable from its parental virus, ZJqz21, based on virological characteristics. Further animal experiments demonstrated that rZJqz retained similar pathogenicity and induced the typical clinical symptoms and viral shedding observed in the ZJqz21 challenge model. Together, these results provide a useful tool for further study of the biological characteristics and pathogenicity of NADC30-like strains. Moreover, these findings also provide a solid foundation for studying the recombination of different PRRSVs and developing new and effective universal vaccines in the future.