The porcine reproductive and respiratory syndrome virus (PRRSV) is a highly significant infectious disease that poses a substantial threat to the global pig industry. In recent years, the NADC30-like strain has gradually emerged as prevalent in China, causing a profound impact on the country\'s pig farming industry. Therefore, it is important to conduct an in-depth study on the characteristics and gene functions of the NADC30-like strain. An infectious cDNA clone is an indispensable tool for investigating the functions of viral genes. In this current study, we successfully isolated a NADC30-like strain and constructed its full-length infectious cDNA clone. The utilization of this clone will facilitate our investigation into the viral replication, pathogenesis, and immune response associated with the PRRSV NADC30-like strain.

China has the largest pig herd in the world which accounts for more than 50% of the global pig population. Over the past three decades, the porcine reproductive and respiratory syndrome virus (PRRSV) has caused significant economic loss to the Chinese swine industry. Currently, the prevalent PRRSV strains in the field are extremely complicated, and the NADC30-like strains, NADC34-like strains, and novel recombinant viruses have become a great concern to PRRS control in China. In this study, a novel NADC30-like PRRSV, named GS2022, was isolated from the lung of a dead pig collected from a farm that experienced a PRRS outbreak. The complete genome of GS2022 shares the highest identity with the NADC30 strain and contains a discontinuous deletion of 131 aa in nsp2. Novel deletion and insertion have been identified in ORF7 and 3\'UTR. Recombination analysis revealed that the GS2022 is a potential recombinant of NADC30-like and JXA1-like strains. Both inter-lineage and intra-lineage recombination events were predicted to be involved in the generation of the GS2022. An infectious cDNA clone of GS2022 was assembled to generate the isogenic GS2022 (rGS2022). The growth kinetics of rGS2022 were almost identical to those of GS2022. The pathogenicity of the GS2022 and rGS2022 was evaluated using a nursery piglet model. In the infection groups, the piglets exhibited mild clinical symptoms, including short periods of fever and respiratory diseases. Both gross lesions and histopathological lesions were observed in the lungs and lymph nodes of the infected piglets. Therefore, we reported a novel recombinant NADC30-like PRRSV strain with moderate pathogenicity in piglets. These results provide new information on the genomic characteristics and pathogenicity of the NADC30-like PRRSV in China.

Since it was first reported in 2013, the NADC30-like PRRSV has been epidemic in China. Hubei Province is known as China\'s key hog-exporting region. To understand the prevalence and genetic variation of PRRSV, herein, we detected and analyzed 317 lung tissue samples from pigs with respiratory disease in Hubei Province, and demonstrated that the NADC30-like strain was the second-most predominant strain during 2017-2018, following the highly pathogenic PRRSV (HP-PRRSV). Additionally, we isolated a new NADC30-like PRRSV strain, named CHN-HB-2018, which could be stably passaged in Marc-145 cells. Genetic characterization analysis showed that compared with the NADC30 strain, the CHN-HB-2018 strain had several amino acid variations in glycoprotein (GP) 3, GP5, and nonstructural protein 2 (NSP2). Moreover, the CHN-HB-2018 strain showed a unique 5-amino acid (aa) deletion in NSP2, which has not previously been reported. Gene recombination analysis identified the CHN-HB-2018 strain as a potentially recombinant PRRSV of the NADC30-like strain and HP-PRRSV. Animal experiments indicated that the CHN-HB-2018 strain has a mild pathogenicity, with no mortality and only mild fever observed in piglets. This study contributes to defining the evolutionary characteristics of PRRSV and its molecular epidemiology in Hubei Province, and provides a potential candidate strain for PRRSV vaccine development.

The porcine reproductive and respiratory syndrome virus (PRRSV) has significantly impacted the global pork industry for over three decades. Its high mutation rates and frequent recombination greatly intensifies its epidemic and threat. To explore the fidelity characterization of Chinese highly pathogenic PRRSV JXwn06 and the NADC30-like strain CHsx1401, self-recombination and mutation in PAMs, MARC-145 cells, and pigs were assessed. In vitro, CHsx1401 displayed a higher frequency of recombination junctions and a greater diversity of junction types than JXwn06. In vivo, CHsx1401 exhibited fewer junction types yet maintained a higher junction frequency. Notably, JXwn06 showed more accumulation of mutations. To pinpoint the genomic regions influencing their fidelity, chimeric viruses were constructed, with the exchanged nsp9-10 regions between JXwn06 and CHsx1401. The SJn9n10 strain, which incorporates JXwn06\'s nsp9-10 into the CHsx1401 genome, demonstrated reduced sensitivity to nucleotide analogs compared to CHsx1401. Conversely, compared with JXwn06, the JSn9n10 strain showed increased sensitivity to these inhibitors. The swapped nsp9-10 also influences the junction frequency and accumulated mutations as their donor strains. The results indicate a propensity for different types of genetic variations between these two strains and further highlight the nsp9-10 region as a critical determinant of their fidelity.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains one of the major threats to swine industry, resulting in huge economic losses worldwide. Currently, PRRSV has diversified into multiple lineages with characteristics of extensive recombination in China. In this research, three virus strains were isolated and four virus whole genome sequences were generated and analyzed from clinical samples collected in Gansu province of China in 2023. The four virus strains were designated GSTS4-2023, GSLX2-2023, GSFEI2-2023 and GSBY4-2023. Phylogenetic analysis based on ORF5 sequences showed that GSTS4-2023, GSLX2-2023, GSFEI2-2023 and GSBY4-2023 shared 91.7, 91.2, 93.2 and 92.9% homology with NADC30 strain respectively, and belonged to lineage 1 of PRRSV-2. In addition, one amino acid deletion was observed at position 33 in ORF5 of GSTS4-2023, GSLX2-2023 and GSFEI2-2023. Moreover, amino acid alignment of the four strains showed a typical discontinuous 131-amino acid (aa) deletion in NSP2 for NADC30-like virus strains. Recombination analysis revealed that all four strains originated from NADC30 (lineage 1), with their minor parents coming from JXA1-like strains (lineage 8), VR-2332-like strains (lineage5) and QYYZ-like strains (lineage3). Finally, the three isolated virus strains, GSTS4-2023, GSLX2-2023 and GSFEI2-2023 showed relatively low levels of replication in cell culture. Our findings provide important implications for the field epidemiology of PRRSV.

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.

Recently, the emergence of a NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV), which causes a large number of abortions in swine herds, has raised great concern in China. In this study, a PRRSV variant strain, PRRSV/CN/FJGD01/2021, evolved from recombination between NADC30-like, NADC34-like, and JXA1-like viruses was isolated in Fujian province in 2021, and its pathogenicity in piglets was examined. Animal experiments demonstrated that PRRSV/CN/FJGD01/2021 infection could induce 100% morbidity and cause higher viremia, a persistently higher fever (>40°C for 14 consecutive days), significant weight loss, and severe histopathological lung lesions compared to the NADC30-like FJZ03 strain and NADC34-like FJ0908 strain in piglets. The PRRSV/CN/FJGD01/2021 strain displayed higher pathogenicity than the FJZ03 and FJ0908 strains, but lower pathogenicity than the Chinese highly pathogenic (HP)-PRRSVs in piglets. Moreover, the Ingelvac PRRS modified live vaccine (MLV) provides incomplete cross-protection against heterologous PRRSV/CN/FJGD01/2021 in piglets. Our findings contribute to the understanding of the current epidemic situation of NADC34-like PRRSV in China. IMPORTANCE The pathogenicity of NADC34-like PRRSV has broad variations in virulence. Importantly, NADC34-like PRRSV has undergone complex recombination with local strains since it first emerged in 2017 in China. However, the pathogenicity of the recombinant NADC34-like virus was rarely experimentally evaluated in pigs. In this study, a novel PRRSV strain, PRRSV/CN/FJGD01/2021, was isolated from sows enduring a high-abortion-rate (20%) period in China in 2021. Notably, phylogenetic and recombination analyses revealed that PRRSV/CN/FJGD01/2021 is a recombinant virus from NADC30-, NADC34-, and JXA1-like isolates. PRRSV/CN/FJGD01/2021 was shown to cause higher virus load, persistent fever, significant weight loss, moderate respiratory clinical signs, and severe histopathological lung lesions in piglets. PRRSV/CN/FJGD01/2021 exhibited higher pathogenicity than NADC30-like FJZ03 and NADC34-like FJ0908, but lower than Chinese HP-PRRSVs for piglets. These data indicated that PRRSV/CN/FJGD01/2021 has intermediate virulence for piglets. Furthermore, the Ingelvac PRRS MLV could partly provide protective efficacy against PRRSV/CN/FJGD01/2021 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.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic loss to China\'s swine industry. Currently, a novel type 2 PRRSV, called the NADC30-like strain, is epidemic in numerous provinces of China. In this study, a NADC30-Like PRRSV strain was isolated in primary alveolar macrophage (PAM) cells from fecal samples collected from a local pig farm, which suffered severe diarrhea. A pathogenicity comparison study was conducted in 6-week-old piglets by inoculating highly pathogenic HP-PRRSV and NADC30-Like PRRSV isolates. RT-qPCR revealed detection of NADC30-Like PRRSV but not the HP-PRRSV in the intestine. PRRSV infection-related lesions were observed in the intestine were further confirmed by histopathological and immunohistochemical examination (IHC). In addition, severe virus infections were also detected by RT-qPCR. Based on clinical observation and pathogenicity experiments, we confirmed that NADC30-Like PRRSV gained more tissue tropism, especially in the small intestine. This may be the one reason explaining why NADC-Like 30 PRRSV become a major epidemic strain in China since the first outbreak in 2013.

The diversity of porcine reproductive and respiratory syndrome virus (PRRSV) in China is increasing rapidly along with mutation and recombination. Recombination could occur between inter- and intra-lineage of PRRSV, which accelerated the complexity of pathogenicity and cell tropism of the recombinant strain. In the present study, a novel PRRSV strain named HN-YL1711 was isolated from a pig farm suffering from severe respiratory difficulty in Henan province, China. The whole genomic sequence analysis indicated that the genome of HN-YL1711 was 15018 nt. It shared 86%, 87.3%, 88.1%, 91.1%, 84.2%, and 84.1% nucleotide similarities with PRRSVs VR2332, CH1a, JXA1, NADC30, QYYZ, and GM2, respectively. Based on phylogenetic analysis of Nsp2, ORF5 and complete genomes, HN-YL1711 was classified into lineage 1 of PRRSV. However, seven genomic break points were detected in recombination analysis, which indicated that the HN-YL1711 originated from multiple recombination among NADC30-like (major parent, lineage 1), JXA1-like (minor parent, lineage 8), and QYYZ-like (minor parent, lineage 3) PRRSV. Porcine alveolar macrophages (PAMs), 3D4/21-CD163 and MARC-145 cells were used to explore the viral adaptation of HN-YL1711. The results indicated that it could infect the PAMs but failed to infect MARC-145 cells. Challenge experiments showed that HN-YL1711 exhibits intermediate virulence in pigs, compared with HP-PRRSV JXA1 and LP-PRRSV CH1a. Taken together, our findings suggest that recombination remains an important factor in PRRSV evolution and that recombination further complicates the cell tropism and pathogenicity of PRRSV.