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Porcine reproductive and respiratory syndrome virus (PRRSV) has seriously affected the viability of swine industries worldwide, and effective measures to control PRRSV are urgently required. Understanding the mechanisms of action of antiviral proteins is crucial for developing antiviral strategies. Interferon-induced bone marrow stromal cell antigen 2 (BST2) can inhibit the replication of various viruses via different pathways. However, little is known about the effects of BST2 on PRRSV. Therefore, this study aimed to evaluate whether the interferon-induced BST2 can inhibit PRRSV replication. We used western blotting and RT-qPCR techniques to analyze the effect of BST2 overexpression and knockdown on PRRSV replication. Overexpression of BST2 inhibited the replication of PRRSV, whereas knockdown of BST2 by small interfering RNA promoted PRRSV replication. Additionally, the expression of BST2 was upregulated during the early phase of PRRSV infection in porcine alveolar macrophages. Analysis of PRRSV proteins showed that BST2 restricted the expression of several non-structural viral proteins. BST2 downregulated the expression of Nsp12 through a proteasome-dependent pathway and downregulated the expression and transcription of E protein. These findings demonstrate the potential of BST2 as a critical regulator of PRRSV replication., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Porcine reproductive and respiratory syndrome (PRRS) is an important viral disease, causing significant economic losses to the swine industry worldwide. Atypical cases caused by highly pathogenic PRRS virus (HP-PRRSV) emerged in 2006 in China. The vaccine strain HuN4-F112 has been developed from the wild-type HP-PRRSV HuN4 through repeated passages on MARC-145 cells. However, the mechanisms of attenuation have yet to be defined. Previous studies have shown that the vaccine strain HuN4-F112 could not effectively replicate in porcine alveolar macrophages (PAMs). In the present study, a series of chimeric and mutant PRRSVs were constructed to investigate regions associated with the virus attenuation. Firstly, the corresponding genome regions (ORF1a, ORF1b and ORFs 2-7) were exchanged between two infectious clones of HuN4 and HuN4-F112, and then the influence of small regions in ORF1a and ORF2-7 was evaluated, then influence of specific amino acids on NSP2 was tested. NSP2 was determined to be the key gene that regulated infection efficiency on PAMs, and amino acids at 893 and 979 of NSP2 were the key amino acids. The results of in vivo study indicated that NSP2 was not only important for infection efficiency in vitro, but also influenced the virulence, which was indicated by the results of survival rate, temperature, viremia, lung score and tissue score.

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Highly pathogenic porcine reproductive and respiratory syndrome virus PRRSV (HP-PRRSV) was one of the most devastating diseases of the pig industry, among various strategies, vaccination was one of the most useful tools for PRRS control. Attenuated live vaccine was used worldwide, however, the genetic basis of HP-PRRSV virulence change during attenuation remain to be determined. Here, to identify virulence associated regions of HP-PRRSV during attenuation in vitro, six full-length infectious cDNA clones with interchanges of 5'UTR + ORF1a, ORF1b, and ORF2-7 + 3'UTR regions between HP-PRRSV strain HuN4-F5 and its attenuated vaccine strain HuN4-F112 were generated, and chimeric viruses were rescued. Piglets were inoculated with chimeric viruses and their parental viruses, and rectal temperature were recorded daily, and serum were collected for future experiments. Our results showed that ORF1a played an important role on virus replication, cytokine response and lung damage, the exchange of ORF1b and ORF2-7 in different backbone led to different exhibition on virus replication in vivo/vitro and cytokine response. Among 9 PRRSV attenuated series, consistent amino acid changes during PRRSV attenuation were found in NSP4, NSP9, GP2, E, GP3 and GP4. Our study provides a fundamental data for the investigation of PRRSV attenuation, the different results of the virulence change among different studies indicated that different mechanisms might be used during PRRSV virulence enhancement in vivo and attenuation in vitro.

Currently, various porcine reproductive and respiratory syndrome virus (PRRSV) variants emerged worldwide with different genetic characteristics and pathogenicity, increasing the difficulty of PRRS control. In this study, a PRRSV strain named HBap4-2018 was isolated from swine herds suffering severe respiratory disease with high morbidity in Hebei Province of China in 2018. The genome of HBap4-2018 is 15,003 nucleotides in length, and compared with NADC30-like PRRSV, nsp2 of HBap4-2018 has an additional continuous deletion of five amino acids. Phylogenetic analysis based on complete genome and ORF5 showed that HBap4-2018 belonged to lineage 8 of PRRSV-2, which was characterized by highly variable genome. However, HBap4-2018 was classified into lineage 1 based on phylogenetic analysis of nsp2, sharing higher amino acid homology (85.3%-85.5%) with NADC30-like PRRSV. Further analysis suggested that HBap4-2018 was a novel natural recombinant PRRSV with three recombinant fragments in the genome, of which highly pathogenic PRRSV (HP-PRRSV) served as the major parental strains, while NADC30-like PRRSV served as the minor parental strains. Five recombination break points were identified in nsp2, nsp3, nsp5, nsp9 and ORF6, respectively, presenting a novel recombinant pattern in the genome. Piglets inoculated with HBap4-2018 presented typical clinical signs with a mortality rate of 60%. High levels of viremia and obvious macroscopic and histopathological lesions in the lungs were observed, revealing the high pathogenicity of HBap4-2018 in piglets., (© 2021. Wuhan Institute of Virology, CAS.)

Porcine reproductive and respiratory syndrome (PRRS) is one of the major drivers of economic loss in the swine industry worldwide. In commercial pig production, vaccination is the first option in an attempt to control infectious diseases. Pigs are therefore often immunized with different vaccines, and almost all of them are delivered via the intramuscular (IM) route. However, the IM injection may result in physical damage, stress reactions, and is labor demanding. An alternative route is urgently needed to reduce the disadvantages of conventional vaccination. In this study, a needle-free intradermal (ID) delivery system was evaluated for delivering a live PRRS vaccine as compared with the traditional needle-syringe method. Fifty-two 4-week-old piglets were divided into six groups: piglets in groups A-C were immunized using ID delivery system with 10 4 , 10 5 and 10 6 TCID 50 of PRRS candidate vaccine strain rHN-NP49, respectively; piglets in group D were immunized IM with 10 5 TCID 50 of rHN-NP49; and group E and F were used as challenge and control groups, respectively. At 28 days post vaccination, piglets in group A to E were challenged with a lethal dose of highly-pathogenic PRRSV. Similar results were found in viremia and antibody response among the ID and IM groups during the immunization stage. After challenge, similar results were found in average body weight gain, viral shedding, serum viral load, and clinical score among the immunization groups, with a higher protection ratio in the ID group compared with IM group with the same immunization dose. These results demonstrated that the ID delivery system could provide similar or even better protection compared with IM route, and could be an effective route for PRRS vaccination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Classical swine fever virus (CSFV) and Porcine reproductive and respiratory syndrome virus (PRRSV) are both important pathogens which seriously harm the economic swine industry worldwide. We have previously demonstrated that rPRRSV-E2 is a promising live, virus-vectored vaccine that provides 100% protection against highly pathogenic PRRSV (HP-PRRSV) and CSFV. Here, we evaluated the duration of immunity (DOI) of the vaccine strain, rPRRSV-E2. Vaccine or cell culture medium was administered to piglets at 4 weeks of age. All immunized piglets developed high levels of antibodies, which could maintain for up to 23 weeks, against PRRSV and CSFV. All immunized pigs were well protected from the challenge of HP-PRRSV or CSFV at 20 weeks and 24 weeks post vaccination. The vaccine protection rate was still 100% at 24 weeks after immunization. The immune efficacy results showed that the immune duration of rPRRSV-E2 could be up to 5 months., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Currently live attenuated porcine reproductive and respiratory syndrome (PRRS) and classical swine fever (CSF) vaccines are widely used in Chinese swine herds. However, the mutual effects of vaccination procedures and severe stress caused by successive vaccinations harm piglets and make it difficult to stimulate robust and effective immune responses. In our previous study, a recombinant PRRS virus (PRRSV) vectored vaccine candidate rPRRSV-E2, which expresses CSF virus (CSFV) E2 protein, has been demonstrated being able to protect piglets against lethal challenge of highly-pathogenic (HP)-PRRSV and CSFV. In this study, we determine whether preexisting maternally derived antibodies (MDA) interfere with the immune efficacy of rPRRSV-E2. 8 experimental groups of piglets, with or without PRRSV MDAs or CSFV MDAs were immunized with a single dose of 10 5 TCID 50 rPRRSV-E2 or DMEM and challenged with HP-PRRSV or CSFV. Clinical characteristics, PRRSV- or CSFV-specific antibodies, viremia and pathological changes were monitored, examined and analyzed. The results showed that rPRRSV-E2-vaccinated piglets, either with or without MDAs directed against PRRSV or CSFV were completely protected from the lethal challenge of HP-PRRSV or CSFV. These results demonstrate that the MDAs do not interfere with the immune efficacy of rPRRSV-E2, which indicates that rPRRSV-E2 could have great significance in the effective prevention and control of HP-PRRSV and CSFV., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Notch signaling, an evolutionarily conserved signal pathway has emerged as a key signal pathway to regulate host immune response but the contribution of Notch signaling to immune response in pigs remains unknown. Infection of porcine alveolar macrophages (PAM) with porcine reproductive and respiratory syndrome virus (PRRSV) triggers expression of Jagged1 mRNA, suggesting that Notch signaling might play a role in the immune response to PRRSV infection. To further explore it, we examined the expression profile of Notch molecules in PAM following a highly pathogenic PRRSV (HP-PRRSV) strain infection. We demonstrated that HP-PRRSV infection resulted in the induction of Notch ligands (Jagged1, Dll3, Dll4), the transcription factor RBP-J, and the target gene Hes1, consistent with activation of Notch signaling. Next, using DAPT treatment and the knockdown of RBP-J illustrated that inhibition of activation of Notch signaling attenuated induction of the inflammatory cytokines (TNF-α and IL-1β) instead of viral replication in PAM during HP-PRRSV infection. Furthermore, the knockdown of Jagged1, the most induced ligand not only inhibited activation of Notch signaling, but also reduced the expression of inflammatory cytokines without any influence in viral replication. Moreover, our data revealed that several signaling including NF-κB, MAPK and Notch signaling contributed to the induction of Jagged1 in PAM during HP-PRRSV infection. In summary, these findings reveal that Notch as an important signaling pathway could contribute to the regulation of inflammatory response induced by HP-PRRSV infection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Different strains of porcine reproductive and respiratory syndrome virus (PRRSV) have emerged and circulated in different regions of mainland China since 1996, particularly after 2006. In 2012, NADC30-like PRRSV was first isolated in Henan Province. By 2016, it had spread to most provinces in China. In the present study, the whole genomes (excluding the poly(A) tails) of 13 newly emerged NADC30-like PRRSV strains were sequenced and analyzed. Furthermore, the pathogenicity of SD53-1603, one of the 13 PRRSV strains, was assessed. Phylogenetic analysis showed that these 13 newly emerged NADC30-like PRRSV strains, together with some reference strains, formed a new subgroup (subgroup 5), characterized by a predicted 131-amino-acid deletion in the nonstructural protein (NSP) 2. However, low levels of whole-genome similarity and a wide variety of recombination patterns complicated the classification of the NADC30-like PRRSV isolates. Interestingly, almost all of the recombination breakpoints found in these 13 PRRSV isolates and other NADC30-like PRRSV isolates occurred in genes encoding NSPs and/or minor structural proteins. In addition, piglets infected with the newly emerged NADC30-like strain SD53-1603 displayed clear clinical respiratory symptoms and underwent typical pathological changes. The findings may be useful for elucidating the characteristics and epidemic status of NADC30-like PRRSV in China.

Porcine galectin-3 (GAL3) is a 29-kDa protein encoded by a single gene, LGALS3, located on chromosome 1. Here, using a yeast two-hybrid screen of a cDNA library from porcine alveolar macrophage cells (PAMs), we report for the first time that GAL3 interacts with nonstructural protein 12 (Nsp12) of the porcine reproductive and respiratory syndrome virus (PRRSV). Although extensive research has focused on porcine reproductive and respiratory syndrome (PRRS), little is known about the pathogen and host interactions involving individual nonstructural viral proteins, especially Nsp12. Here, we showed that GAL3 interacted with viral Nsp12 following co-transfection of HEK293 cells with GAL3- and Nsp12-expressing plasmids. Additionally, we observed that PPRSV infection led to reduced GAL3 levels during the late phase of infection in both MARC-145 cells and PAMs. Importantly, GAL3 overexpression significantly suppressed the replication of both type 1 and 2 PRRSV strains, whereas knockout of endogenous LGALS3 in MARC-145 cells significantly increased viral titer and expression of the nucleocapsid protein. These results strongly support a direct inhibitory effect of GAL3 on PRRSV replication, which might contribute to an overall antiviral effect. Furthermore, our findings provide insights into the molecular basis of the role Nsp12 plays in PRRSV pathogenesis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Porcine reproductive and respiratory syndrome (PRRS) and classical swine fever (CSF) are economically significant diseases that affect the swine industry worldwide. However, the current vaccination strategy, which uses two single live attenuated vaccines, can result in interference for each other. In addition, the universally used CSFV vaccine C-strain does not allow for differentiation of infected and vaccinated animals. In this study, rPRRSV-E2, PRRS virus (PRRSV) expressing CSF virus (CSFV) E2, was constructed by reverse genetics. The E2 gene of CSFV was inserted between ORF1b and ORF2 in the genome of the PRRS vaccine virus, HuN4-F112. A copy of transcriptional regulatory sequence 6 was inserted at the 3' terminal of the exogenous gene to produce CSFV E2 as a unique subgenomic mRNA transcript. The rPRRSV-E2 was stable for at least 25 serial cell passages. Single-shot intramuscular immunization of rPRRSV-E2 into pigs induced PRRSV-specific and CSFV-specific antibodies and fully protected pigs from lethal challenge with highly-pathogenic PRRSV and CSFV. These results demonstrate that a novel strategy for recombinant PRRSV production is effective, and suggest that rPRRSV-E2 is a promising live, virus-vectored vaccine against PRRS and a marker vaccine against CSF., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Significant differences exist between the highly pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) and its attenuated pathogenic (AP) strain in the ability to infect host cells. The mechanisms by which different virulent strains invade host cells remain relatively unknown. In this study, pulmonary alveolar macrophages (PAMs) are infected with HP-PRRSV (HuN4) and AP-PRRSV (HuN4-F112) for 24 h, then harvested and subjected to label-free quantitative MS. A total of 2849 proteins are identified, including 95 that are differentially expressed. Among them, 26 proteins are located on the membrane. The most differentially expressed proteins are involved in response to stimulus, metabolic process, and immune system process, which mainly have the function of binding and catalytic activity. Cluster of differentiation CD163, vimentin (VIM), and nmII as well as detected proteins are assessed together by string analysis, which elucidated a potentially different infection mechanism. According to the function annotations, PRRSV with different virulence may mainly differ in immunology, inflammation, immune evasion as well as cell apoptosis. This is the first attempt to explore the differential characteristics between HP-PRRSV and its attenuated PRRSV infected PAMs focusing on membrane proteins which will be of great help to further understand the different infective mechanisms of HP-PRRSV and AP-PRRSV., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Currently, porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viral pathogens in swine in most countries, especially China. Two PRRSV attenuated live vaccine strains (HuN4-F112 and CH-1R) are currently widely used in China. Our previous study showed that HuN4-F112, but not CH-1R, induced high anti-nucleocapsid (N) antibody and neutralizing antibody (NA) titers. Additionally, sera from HuN4-F112 inoculated pigs induced low cross neutralization of CH-1R., Methods: In the present study, 6 chimeric viruses through exchanging 5' untranslated region (UTR) + open reading frame (ORF)1a, ORF1b, and ORF2-7 + 3'UTR between HuN4-F112 and CH-1R were constructed and rescued based on the infectious clones of rHuN4-F112 and rCH-1R. The characteristics of these viruses were investigated in vitro and vivo., Results: All the three fragments, 5'UTR + ORF1a, ORF1b, and ORF2-7 + 3'UTR, could affect the replication efficiencies of rHuN4-F112 and rCH-1R in vitro. Additionally, both 5'UTR + ORF1a and ORF2-7 + 3'UTR affected the anti-N antibody and NA responses targeting rHuN4-F112 and rCH-1R in piglets., Conclusions: The 5'UTR + ORF1a region of HuN4-F112 played a key role in inducing NAs in piglets. Furthermore, we confirmed for the first time that ORF1a contains a neutralization region. This study provides important information that can be used for further study of the generation of anti-PRRSV NAs.

Porcine Reproductive and Respiratory Syndrome (PRRS), which is caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection, has caused substantial economic losses for the global swine industry. To date, there are limited commercially available measures to control the spread of PRRSV. The available vaccines are unstable and there is no anti-PRRSV therapeutic available. Therefore, this study designed a novel recombinant antiviral protein that included a novel polypeptide that binds to the PRRSV polymerase (p9), the transduction ability of the HIV TAT, and the nucleotide degradation activity of interferon stimulated gene 20 (ISG20). The recombinant proteins TAT-p9-ISG20 and p9-ISG20 were expressed in MARC-145 cells by transient transfection and then tested for antiviral activity and entry efficiency. The p9-ISG20 construct had greater antiviral activity than either p9 alone (1.37-fold) or ISG20 alone (1.9-fold). Addition of the HIV TAT protein increased the entry efficiency of p9-ISG20 by 1.57-fold, which was associated with increased anti-viral activity (1.52-fold) compared to P9-ISG20. In summary, TAT-p9-ISG20 achieved a synergistic effect by combining three different antiviral proteins and may be a novel PRRSV therapeutic platform., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which has important impacts on the pig industry. PRRSV infection results in disruption of the swine leukocyte antigen class I (SLA-I) antigen presentation pathway. In this study, highly pathogenic PRRSV (HP-PRRSV) infection inhibited transcription of the β2-microglobulin (β2M) gene ( B2M ) and reduced cellular levels of β2M, which forms a heterotrimeric complex with the SLA-I heavy chain and a variable peptide and plays a critical role in SLA-I antigen presentation. HP-PRRSV nonstructural protein 4 (Nsp4) was involved in the downregulation of β2M expression. Exogenous expression of Nsp4 downregulated β2M expression at both the mRNA and the protein level and reduced SLA-I expression on the cell surface. Nsp4 bound to the porcine B2M promoter and inhibited its transcriptional activity. Domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter were identified as essential for the interaction between Nsp4 and B2M These findings demonstrate a novel mechanism whereby HP-PRRSV may modulate the SLA-I antigen presentation pathway and provide new insights into the functions of HP-PRRSV Nsp4. IMPORTANCE PRRSV modulates the host response by disrupting the SLA-I antigen presentation pathway. We show that HP-PRRSV downregulates SLA-I expression on the cell surface via transcriptional inhibition of B2M expression by viral Nsp4. The interaction between domain III of Nsp4 and the enhancer PAM element of the porcine B2M promoter is essential for inhibiting B2M transcription. These observations reveal a novel mechanism whereby HP-PRRSV may modulate SLA-I antigen presentation and provide new insights into the functions of viral Nsp4., (Copyright © 2017 American Society for Microbiology.)

Porcine reproductive and respiratory syndrome virus (PRRSV) has a condensed single-stranded positive-sense RNA genome that contains several overlapping regions. The transcription regulatory sequence (TRS) is the important cis-acting element participating in PRRSV discontinuous transcription process. Based on reverse genetic system of type 2 highly pathogenic PRRSV cell-passage attenuated strain pHuN4-F112, firefly luciferase or Renilla luciferase genes were inserted between ORF1b and ORF2. An extra TRS6 was embedded behind the foreign luciferase genes. pA-Fluc and pA-Rluc were constructed and successfully rescued in MARC-145 cells. The phenotypical characteristics of the progeny virus were indistinguishable from those of vHuN4-F112 and were genetically stable for at least 25 cell passages. Mutant virus-infected cells were lysed at different time points to assess luciferase activities and measure foreign gene expression levels. The results showed identical variations in the luciferase activities of the recombinants in MARC-145 cells, indicating that they were suitable for monitoring viral propagation in PRRSV-permissive cell cultures. They were also used to infect pulmonary alveolar macrophages, which yielded similar variations in luciferase activities. Therefore, vA-Fluc and vA-Rluc present powerful new tools to monitor PRRSV propagation in both passaged and target cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The sterile alpha motif and HD domain 1 (SAMHD1) protein is a novel innate immunity restriction factor that inhibits HIV-1 infection in myeloid cells. Here, we cloned the full-length SAMHD1 complementary DNA (cDNA) from porcine peripheral blood lymphocytes. The porcine SAMHD1 cDNA was of 3951 bp with an open reading frame of 1884 bp, encoding a polypeptide of 627 amino acids. Porcine SAMHD1 mRNA was detected in all swine tissues examined, with the higher expression in the tonsil, lung, liver, and lymph node tissues. The SAMHD1 protein was localized to the nucleus. Overexpression of SAMHD1 blocked the proliferation of HuN4, a highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV), in MARC-145 cells, by inhibiting the synthesis of the HuN4 complement RNA. The antiviral effects of the simian SAMHD1 protein were nearly equivalent to those of porcine SAMHD1 in the HuN4-infected MARC-145 cells. Phosphorylation analysis of SAMHD1 showed that overexpressed SAMHD1 protein was in primarily an unphosphorylated state. SAMHD1 overexpression increased the transcript abundance of IFN-stimulated genes ISG15 and ISG56. The mRNA levels of SAMHD1 and ISGs were significantly increased in porcine alveolar macrophages infected with HP-PRRSV. SAMHD1 protein level was also elevated, and the protein was not phosphorylated during infection. Collectively, our data indicate that SAMHDI inhibits HP-PRRSV proliferation through inhibiting the replication of HP-PRRSV. SAMHD1 might be the protein participating in the IFN signaling and is thus an important immunoregulatory protein in innate immunity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The factors that determine the transcription-regulating sequence (TRS) activity of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unclear. In this study, the effect of mutagenesis of conserved C nucleotides at positions 5 and 6 in the leader TRS (TRS-L) and/or canonical body TRS7 (TRS-B7) on the synthesis of subgenomic (sg) mRNA and virus infectivity was investigated in the context of a type 2 PRRSV infectious cDNA clone. The results showed that a double C mutation in the leader TRS completely abolished sg mRNAs synthesis and virus infectivity, but a single C mutation did not. A single C or double C mutation in TRS-B7.1 or/and TRS-B7.2 impaired or abolished the corresponding sg mRNA synthesis. Introduction of identical mutations in the leader and body TRSs partially restored sg mRNA7.1 and/or sg mRNA7.2 transcription, indicating that the base-pairing interaction between sense TRS-L and cTRS-B is a crucial factor influencing sg mRNA synthesis. Analysis of the mRNA leader-body junctions of mutants provided evidence for a mechanism of discontinuous minus-strand transcription. This study also showed that mutational inactivation of TRS-B7.1 or TRS-B7.2 did not affect the production of infectious progeny virus, and the sg mRNA formed from each of them could express N protein. However, TRS-B7.1 plays more important roles than TRS-B7.2 in maintaining the growth characteristic of type 2 PRRSV. These results provide more insight into the molecular mechanism of genome expression and subgenomic mRNA transcription of PRRSV.

Phosphatidylinositol-3-kinase (PI3K)/Akt is an important cellular pathway that has been shown to participate in various replication steps of multiple viruses. In the present study, we compared the phosphorylation status of Akt during infection of MARC-145 cells and porcine alveolar macrophages (PAMs) with highly pathogenic PRRSV (HP-PRRSV) strain HuN4. We observed that biphasic activation of Akt was induced in at both the early stage (5, 15 and 30 min postinfection) and the late stage (12 and 24 h postinfection) of HP-PRRSV infection of MARC-145 cells, while an early-phase activation of Akt was found exclusively in virus-infected PAMs in vitro. Analysis with the PI3K-specific inhibitor LY294002 confirmed that PI3K acted as the upstream activator for the virus-induced activation of Akt. UV-irradiation-inactivated virus still induced the early event in PAMs but not in MARC-145 cells, suggesting that different mechanisms are employed for the early-stage induction of phosphorylated Akt within different cell cultures. We further demonstrated that FoxO1 and Bad, which serve as downstream targets of Akt, were phosphorylated in virus-infected MARC-145 cells. Moreover, the suppression of phosphorylated Akt with LY294002 significantly inhibited the virus-induced cytopathic effect (CPE) on MARC-145 cells, but it had a negligible effect on virus propagation. Collectively, our data provide new evidence of a novel role for the PI3K/Akt pathway in PRRSV infection of MARC-145 cells.

SYBR Green coupled to melting curve analysis has been suggested to detect RNA viruses showing high genomic variability. Here, a SYBR Green-based real-time RT-PCR assay was developed for simultaneous detection and differentiation of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and classical type 2 PRRSV (C-PRRSV). The different strains were identified by their distinctive melting temperatures: 82.98 ± 0.25 °C and 85.95 ± 0.24 °C for HP-PRRSVs or 82.74 ± 0.26 °C for C-PRRSVs. Specificity was tested using nine other viral and bacterial pathogens of swine. The detection limit was 1 TCID(50) for HP- or C-PRRSV. Furthermore, the detection results for samples from an animal trial with HP- or C-PRRSV infections showed that the SYBR Green-based real-time RT-PCR was more sensitive than the conventional RT-PCR. Additionally, an analysis of 319 field samples from North China, Central China and Northeast China showed that HP- and C-PRRSVs co-circulated in pig herds. Thus, the SYBR Green-based real-time RT-PCR, which can be performed within one hour, is a rapid, sensitive and low-cost diagnostic tool for rapid differential detection and routine surveillance of HP- and classical type 2 PRRSVs in China.

Since 2006, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has become the major pathogen attributed to the prevalent porcine reproductive and respiratory syndrome (PRRS) in China. The present study aims to identify serum proteins modified in response to infection of HuN4, a HP-PRRSV strain isolated from a farm in 2006. 2-D DIGE analysis allowed for the detection of 19 differentially expressed protein spots, of which 18 were identified by MALDI-TOF/TOF MS. These 18 spots represented for a total of 9 proteins (6 up-regulated and 3 down-regulated), most of which belonged to the acute phase proteins in swine and showed a trend of regression in the late phase of the experiment. One of a series of AGP spots was identified for the first time to be decreased in acute phase of PRRSV infection in swine. But the whole level of the protein in the serum did not show significant changes by Western blot. The rising tendency of Hp was confirmed by Western blot and ELISA. These altered proteins were probably involved in the inflammatory process triggered by HuN4 and in alleviating the oxidative damage occurring in the process. In summary, these results may provide new insights into understanding the mechanisms of HP-PRRSV infection., (Copyright © 2012. Published by Elsevier B.V.)

It has been proposed that the N-linked glycan of the minor proteins of porcine reproductive and respiratory syndrome virus (PRRSV) is important for the production of infectious virus. In this study, we showed that N-linked glycosylation of GP2 is not essential for virus viability and none of the individual glycosylation sites in GP3 has a vital effect on the production of infectious virus. Moreover, mutations of single and double glycosylation sites in GP4 are not critically important for infectious virus recovery, triple and quadruple mutations are lethal. The bimolecular fluorescence complementation (BiFC) analysis also showed that GP4, but might be not GP2, is involved in interaction with cellular receptor CD163 and that glycosylation of GP4 might not play a vital role in the interaction with CD163. The study further revealed that none of the N-glycosylation sites in the minor proteins is critical for the susceptibility of mutants to neutralizing antibody., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Nowadays, PRRS has become one of the most economically important infectious diseases of pig worldwide. To better characterize and understand the molecular basis of PRRSV virulence determinants, it would be important to develop the infectious cDNA clones. In this regard, HuN4-F112, a live-attenuated North-American-type PRRSV vaccine strain, could serve as an excellent model., Results: In the study, genomic sequence of HuN4-F112, an attenuated vaccine virus derived from the highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) HuN4 strain, was determined and its full-length cDNA was cloned. Capped RNA was transcribed in vitro from the cDNA clone and transfected into BHK-21 cells. The supernatant from transfected monolayers were serially passaged in Marc-145 cells. The rescued virus exhibited a similar growth pattern to its parental virus in Marc-145 cells with peak titers at 48 h post-infection., Conclusion: In conclusion, we rescued virus from an infectious cDNA clone of attenuated vaccine. It is possible in the future that a new attenuated PRRSV vaccine with broader specificity and good immunogenicity can be designed in vitro via an infectious cDNA clone platform coupled with validated information on virulence determinants.