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Abstract Background A cost-effective Escherichia coli expression system has gained popularity for producing virus-like particle (VLP) vaccines. However, the challenge lies in balancing the endotoxin residue and removal costs, as residual endotoxins can cause inflammatory reactions in the body. Results In this study, porcine parvovirus virus-like particles (PPV-VLPs) were successfully assembled from Decreased Endotoxic BL21 (BL21-DeE), and the effect of structural changes in the lipid A of BL21 on endotoxin activity, immunogenicity, and safety was investigated. The lipopolysaccharide purified from BL21-DeE produced lower IL-6 and TNF-α than that from wild-type BL21 (BL21-W) in both RAW264.7 cells and BALB/c mice. Additionally, mice immunized with PPV-VLP derived form BL21-DeE (BL21-DeE-VLP) showed significantly lower production of inflammatory factors and a smaller increase in body temperature within 3 h than those immunized with VLP from BL21-W (BL21-W-VLP) and endotoxin-removed VLP (ReE-VLP). Moreover, mice in the BL21-DeE-VLP immunized group had similar levels of serum antibodies as those in the BL21-W-VLP group but significantly higher levels than those in the ReE-VLP group. Furthermore, the liver, lungs, and kidneys showed no pathological damage compared with the BL21-W-VLP group. Conclusion Overall, this study proposes a method for producing VLP with high immunogenicity and minimal endotoxin activity without chemical or physical endotoxin removal methods. This method could address the issue of endotoxin residues in the VLP and provide production benefits.

Abstract Viral infections continue to pose a significant threat to the health of both humans and animals. Currently, live attenuated vaccines (LAVs) remain the most efficacious and widely utilized tool for combating viral infections. Conventional LAVs involve the adaptation of virulent viruses to novel hosts, cell cultures, or suboptimal environments, resulting in a reduction in pathogenicity while retaining immunogenicity. This process entails directed evolution of the virus to enhance its replication efficiency under these modified conditions. In this review, the development of traditional animal‐adapted and cold‐adapted LAVs is specially discussed. Additionally, the factors that contribute to virus attenuation from a viral lifecycle perspective are summarized. Finally, we propose future directions for next‐generation LAVs.

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.

In China, porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are widely used. These vaccines, which contain inactivated and live attenuated vaccines (LAVs), are produced by MARC-145 cells derived from the monkey kidney cell line. However, some PRRSV strains in MARC-145 cells have a low yield. Here, we used two type 2 PRRSV strains (CH-1R and HuN4) to identify the genes responsible for virus yield in MARC-145 cells. Our findings indicate that the two viruses have different spread patterns, which ultimately determine their yield. By replacing the viral envelope genes with a reverse genetics system, we discovered that the minor envelope proteins, from GP2a to GP4, play a crucial role in determining the spread pattern and yield of type 2 PRRSV in MARC-145 cells. The cell-free transmission pattern of type 2 PRRSV appears to be more efficient than the cell-to-cell transmission pattern. Overall, these findings suggest that GP2a to GP4 contributes to the spread pattern and yield of type 2 PRRSV.

In recent years, an increasing number of emerging and remerging virus outbreaks have occurred and the rapid development of vaccines against these viruses has been crucial. Controlling the replication of premature termination codon (PTC)-containing viruses is a promising approach to generate live but replication-defective viruses that can be used for potent vaccines. Here, we used anticodon-engineered transfer RNAs (ACE-tRNAs) as powerful precision switches to control the replication of PTC-containing viruses. We showed that ACE-tRNAs display higher potency of reading through PTCs than genetic code expansion (GCE) technology. Interestingly, ACE-tRNA has a site preference that may influence its read-through efficacy. We further attempted to use ACE-tRNAs as a novel viral vaccine platform. Using a human immunodeficiency virus type 1 (HIV-1) pseudotyped virus as an RNA virus model, we found that ACE-tRNAs display high potency for read-through viral PTCs and precisely control their production. Pseudorabies virus (PRV), a herpesvirus, was used as a DNA virus model. We found that ACE-tRNAs display high potency for reading through viral PTCs and precisely controlling PTC-containing virus replication. In addition, PTC-engineered PRV completely attenuated and lost virulence in mice in vivo, and immunization with PRV containing a PTC elicited a robust immune response and provided complete protection against wild-type PRV challenge. Overall, replication-controllable PTC-containing viruses based on ACE-tRNAs provide a new strategy to rapidly attenuate virus infection and prime robust immune responses. This technology can be used as a platform for rapidly developing viral vaccines in the future.

ABSTRACT 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.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused serious economic losses to the pig industry worldwide. During the continuous monitoring of PRRSV, a new PRRSV strain type with novel characteristics was first identified in three different regions of Shandong Province. These strains presented a novel deletion pattern (1 + 8 + 1) in the NSP2 region and belonged to a new branch in sublineage 8.7 based on the ORF5 gene phylogenetic tree. To further study the genomic characteristics of the new-branch PRRSV, we selected a sample from each of the three farms for whole-genome sequencing and sequence analysis. Based on the phylogenetic analysis of the whole genome, these strains formed a new independent branch in sublineage 8.7, which showed a close relationship with HP-PRRSV and intermediate PRRSV according to nucleotide and amino acid homology but displayed a completely different deletion pattern in NSP2. Recombinant analysis showed that these strains presented similar recombination patterns, all of which involved recombination with QYYZ in the ORF3 region. Furthermore, we found that the new-branch PRRSV retained highly consistent nucleotides at positions 117–120 (AGTA) of a quite conserved motif in the 3’-UTR; showed similar deletion patterns in the 5’-UTR, 3’-UTR and NSP2; retained characteristics consistent with intermediate PRRSV and exhibited a gradual evolution trend. The above results showed that the new-branch PRRSV strains may have the same origin and be similar to HP-PPRSV also evolved from intermediate PRRSV, but are distinct strains that evolved simultaneously with HP-PRRSV. They persist in some parts of China through rapid evolution, recombine with other strains and have the potential to become epidemic strains. The monitoring and biological characteristics of these strains should be further studied.

Porcine reproductive and respiratory syndrome (PRRS) is a widespread disease with great economic importance in the pig industry. Although vaccines against the PRRS virus (PRRSV) have been employed for more than 20 years, differentiating infected from vaccinated animals remains challenging. In this study, all 907 non-structural protein 2 (NSP2) full-length sequences of PRRSV-2 available from GenBank were aligned. Two peptides, at positions 562–627 (m1B) and 749–813 (m2B) of NSP2, were selected, and their potential for use in differential diagnosis was assessed. Both m1B and m2B were recognized by PRRSV-positive pig serum in peptide-coated enzyme-linked immunosorbent assays. Further epitope identification yielded five overlapping short peptides for the immunodominant regions of m1B and m2B. Using the infectious clone of PRRSV HuN4-F112 as a template, the deletion mutants, rHuN4-F112-m1B, rHuN4-F112-m2B, and rHuN4-F112-C5-m1B-m2B, were generated and successfully rescued in Marc-145 cells. Growth kinetics revealed that the deletion of m1B and m2B did not significantly affect virus replication. Hence, m1B and m2B show potential as molecular markers for developing a PRRSV vaccine.

In recent years, Seneca Valley virus (SVV) as a newly identified pathogen of porcine vesicular disease spread quickly and has posed a potential threat to the swine industry in several countries resulting in economic losses. Considering the evolution of SVV, attention should be given to controlling SVV epidemics. So far there are no commercial vaccines or drugs available to combat SVV. Therefore, development of strategies for preventing and controlling SVV infection should be taken into account. In the current study, we evaluated whether the CRISPR-Cas13d system could be used as a powerful tool against SVV infection. Besides, selected crRNAs showed different capacity against SVV infection. Our study suggests the CRISPR-Cas13d system significantly inhibited SVV replication and exhibited potent anti-SVV activity. This knowledge may provide a novel alternative strategy to control epidemics of SVV in the future.

The aim of this study was to investigate the antimicrobial resistance profiles and genotypes of Streptococcus suis in Heilongjiang Province, China. A total of 29 S. suis were isolated from 332 samples collected from 6 pig farms. The results showed that serotypes 2, 4 and 9 were prevalent, and all the clinical isolates were resistant to at least two antibacterial drugs. The most resisted drugs were macrolides, and the least resisted drugs were fluoroquinolones. Resistant genes ermB and aph (3′)-IIIa were highly distributed among the isolates, with the detection rates of 79.31% and 75.86%. The formation of biofilm could be observed in all the isolated S. suis, among which D-1, LL-1 and LL-3 strains formed stronger biofilm structure than other strains. The results indicate that S. suis in Heilongjiang Province presents a multi-drug resistance to commonly used antimicrobial drugs, which was caused by the same target gene, the dissemination of drug resistance genes, and bacterial biofilm.

Abstract PCV3 capsid protein (Cap) is an important antigen for diagnosis and vaccine development. To achieve high-level expression of recombinant PCV3 Cap in Escherichia coli (E. coli), the gene of wild-type entire Cap (wt-eCap) was amplified from clinical samples, and three optimized entire Cap (opti-eCap) and one optimized Cap deleted nuclear location signal (NLS) (opti-dCap) gene fragments encoding the same amino acid sequence with wt-eCap were synthesized based on the codon bias of E. coli. Those gene fragments were inserted into the pET30a expression vector. One recombinant strain with the highest expressed soluble eCap from four entire Cap (one wt-eCap and three opti-eCap) and one recombinant strain expressed opti-dCap were selected for further purification. The purified eCap and dCap were identified by transmission electron microscopy (TEM), a large number of round hollow particles with a diameter of 10 nm virus-like particles (VLPs) were observed in eCap, whereas irregular aggregation of proteins observed in dCap. After formation the VLPs were applied as a coating antigen to establish an indirect ELISA (I-ELISA) for detection of PCV3-specific antibody in swine serum. 373 clinical swine serum samples from China collected in 2019 were tested utilizing the VLP-based I-ELISA method under optimized conditions. To the best of our knowledge, this is the first report of self-assembly into VLPs of PCV3 recombinant Cap. Our results demonstrated that the VLP-based I-ELISA will be a valuable tool for detecting the presence of PCV3 antibodies in serum samples and will facilitate screening of large numbers of swine serum for clinical purposes.

Innate immunity is the front line for antiviral immune responses and bridges adaptive immunity against viral infections. However, various viruses have evolved many strategies to evade host innate immunity. A typical virus is the porcine reproductive and respiratory syndrome virus (PRRSV), one of the most globally devastating viruses threatening the swine industry worldwide. PRRSV engages several strategies to evade the porcine innate immune responses. This review focus on the underlying mechanisms employed by PRRSV to evade pattern recognition receptors signaling pathways, type I interferon (IFN-α/β) receptor (IFNAR)-JAK-STAT signaling pathway, and interferon-stimulated genes. Deciphering the antiviral immune evasion mechanisms by PRRSV will enhance our understanding of PRRSV’s pathogenesis and help us to develop more effective methods to control and eliminate PRRSV.

ABSTRACTAs one of many nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV), nonstructural protein 12 (Nsp12) has received relatively little attention, and its role in virus replication, if any, is essentially unknown. By the application of reverse genetic manipulation of an infectious PRRSV clone, the current study is the first to demonstrate that Nsp12 is a key component of PRRSV replication. In addition, the biochemical properties of Nsp12 were evaluated, revealing that Nsp12 forms dimers when exposed to oxidative conditions. Furthermore, we systemically analyzed the function of Nsp12 in PRRSV RNA synthesis using a strand-specific PCR method. To our surprise, Nsp12 was not found to be involved in minus-strand genomic RNA (-gRNA) synthesis; importantly, our results indicate that Nsp12 is involved in the synthesis of both plus- and minus-strand subgenomic mRNAs (+sgmRNA and -sgmRNA). Finally, we found that the combination of cysteine 35 and cysteine 79 in Nsp12 is required for sgmRNA synthesis. To our knowledge, we are the first to report the biological role of Nsp12 in the PRRSV lifecycle, and we conclude that Nsp12 is involved in the synthesis of both + sgRNA and -sgRNA.

Despite many efforts and diverse approaches, developing an effective herpesvirus vaccine remains a great challenge. Traditional inactivated and live-attenuated vaccines always raise efficacy or safety concerns. This study used Pseudorabies virus (PRV), a swine herpes virus, as a model. We attempted to develop a live but replication-incompetent PRV by genetic code expansion (GCE) technology. Premature termination codon (PTC) harboring PRV was successfully rescued in the presence of orthogonal system MbpylRS/tRNAPyl pair and unnatural amino acids (UAA). However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, we failed to establish a stable transgenic cell line containing orthogonal translation machinery for PTC virus replication, and we demonstrated that orthogonal tRNAPyl is a key limiting factor. This study is the first to demonstrate that orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC engineered virus replication. To our knowledge, this is the first reported PTC herpesvirus generated by GCE technology. Our work provides a proof-of-concept for generating UAAs-controlled PRV-PTC virus, which can be used as a safe and effective vaccine.

Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) is an important globally distributed and highly contagious pathogen that has restricted cell tropism in vivo and in vitro. In the present study, we found that annexin A2 (ANXA2) is upregulated expressed in porcine alveolar macrophages infected with PRRSV. Additionally, PRRSV replication was significantly suppressed after reducing ANXA2 expression in Marc-145 cells using siRNA. Bioinformatics analysis indicated that ANXA2 may be relevant to vimentin, a cellular cytoskeleton component that is thought to be involved in the infectivity and replication of PRRSV. Co-immunoprecipitation assays and confocal analysis confirmed that ANXA2 interacts with vimentin, with further experiments indicating that the B domain (109–174 aa) of ANXA2 contributes to this interaction. Importantly, neither ANXA2 nor vimentin alone could bind to PRRSV and only in the presence of ANXA2 could vimentin interact with the N protein of PRRSV. No binding to the GP2, GP3, GP5, nor M proteins of PRRSV was observed. In conclusion, ANXA2 can interact with vimentin and enhance PRRSV growth. This contributes to the regulation of PRRSV replication in infected cells and may have implications for the future antiviral strategies.

Abstract There is currently a pandemic of pseudorabies virus (PRV) variant strains in China. Despite extensive research on PRV variant strains in the past two years, few studies have investigated PRV pathogenicity-related genes. To determine which gene(s) is/are linked to PRV virulence, ten putative virulence genes were knocked out using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 technology. The pathogenicity of these mutants was evaluated in a mouse model. Our results demonstrated that of the ten tested genes, the thymidine kinase (TK) and glycoprotein M (gM) knockout mutants displayed significantly reduced virulence. However, mutants of other putative virulence genes, such as glycoprotein E (gE), glycoprotein I (gI), Us2, Us9, Us3, glycoprotein G (gG), glycoprotein N (gN) and early protein 0 (EP0), did not exhibit significantly reduced virulence compared to that of the wild-type PRV. To our knowledge, this study is the first to compare virulence genes from the current pandemic PRV variant strain. This study will provide a valuable reference for scientists to design effective live attenuated vaccines in the future.

The transcription factor NF-κB plays a critical role in diverse biological processes. The NF-κB pathway can be activated by incoming pathogens and then stimulates both innate and adaptive immunity. However, many viruses have evolved corresponding strategies to balance NF-κB activation to benefit their replication. Pseudorabies virus (PRV) is an economically important pathogen that belongs to the alphaherpesvirus group. There is little information about PRV infection and NF-κB regulation. This study demonstrates for the first time that the UL24 protein could abrogate tumor necrosis factor alpha (TNF-α)-mediated NF-κB activation. An overexpression assay indicated that UL24 inhibits this pathway at or downstream of P65. Furthermore, co-immunoprecipitation analysis demonstrated that UL24 selectively interacts with P65. We demonstrated that UL24 could significantly degrade P65 by the proteasome pathway. For the first time, PRV UL24 was shown to play an important role in NF-κB evasion during PRV infection. This study expands our understanding that PRV can utilize its encoded protein UL24 to evade NF-κB signaling.

To develop a modified live vaccine (MLV) against porcine reproductive and respiratory syndrome virus (PRRSV), virulent CH-1a strain was attenuated by serial passages up to 130 passage (P130) in Marc-145 cells. The virulence and immune efficacy of the attenuated CH-1a were evaluated in pigs. The results showed that animals inoculated with P130 did not develop any clinical sign of the disease, but produced rapid and effective humoral immune responses against PRRSV challenge, indicating that attenuated CH-1a P130 is the candidate as the effective vaccine against PRRSV. To define the potential mutations in the attenuated CH-1a genome, we sequenced and analyzed the ORF5 gene of CH-1a strain of different passages (P39, P55, P65, P70, P85, P100, P115, P120, P125, and P130) and found that three mutations (C5Y, H38Q and L146Q) which may be related with the attenuation of CH-1a. In addition, we also found a unique restriction enzyme site (TspEI) in the ORF5 gene of attenuated CH-1a, which can be used as a genetic marker to distinguish original and attenuated CH-1a.

In the original publication of this article [1], the author found the brand of vimentin antibody was wrong in Fig. 3. The legend of Fig. 3, ‘mouse anti-vimentin mAb (Cell Signaling Technology) at 4 °C overnight’ should be ‘mouse anti-vimentin mAb (Sigma-Aldrich) at 4 °C overnight’.

A Pseudorabies virus (PRV) variant has emerged in China since 2011 that is not protected by commercial vaccines, and has not been well studied. The PRV genome is large and difficult to manipulate, but it is feasible to use clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. However, identification of single guide RNA (sgRNA) through screening is critical to the CRISPR/Cas9 system, and is traditionally time and labor intensive, and not suitable for rapid and high throughput screening of effective PRV sgRNAs. In this study, we developed a recombinant PRV strain expressing firefly luciferase and enhanced green fluorescent protein (EGFP) as a reporter virus for PRV-specific sgRNA screens and rapid evaluation of antiviral compounds. Luciferase activity was apparent as soon as 4 h after infection and was stably expressed through 10 passages. In a proof of the principle screen, we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods. Using the reporter virus, we also identified PRV variants lacking US3, US2, and US9 gene function, and showed anti-PRV activity for chloroquine. Our results suggest that the reporter PRV strain will be a useful tool for basic virology studies, and for developing PRV control and prevention measures.

A 2- to 4-year-old uncastrated male Siberian tiger (Panthera tigris altica) bred in a local wild animal park presented with generalized clinical signs including abdominal pain, fever, lethargy, and anorexia, along with subcutaneous nodules along the trunk. The patient subsequently died of chronic, progressive dyspnea despite 45 days of antibiotic treatment. At necropsy, mesenteric fat inflammation and multiple subcutaneous, peritoneal, and intraabdominal nodules were observed. The lungs demonstrated congestion and heavy coagulation, and necrotic foci were observed on the cut surface. Histopathologically, the nodules were identified as granulomatous fatty tissue with numerous lymphocytes, infiltration with lipid-laden macrophages, and fibrosis. These changes were also noted in the lung. The etiology of this condition remains undetermined. [ABSTRACT FROM AUTHOR]

Bacterial artificial chromosomes (BACs) are powerful tools for the manipulation of the large genomes of DNA viruses, such as herpesviruses. However, the methods currently used to construct the recombinant viruses, an important intermediate link in the generation of BACs, involve the laborious process of multiple plaque purifications. Moreover, some fastidious viruses may be lost or damaged during these processes, making it impossible to generate BACs from these large-genome DNA viruses. Here, we introduce the CRISPR/Cas9 as a site-specific gene knock-in instrument that promotes the homologs recombination of a linearized transfer vector and the Pseudorabies virus genome through double incisions. The efficiency of recombination is as high as 86%. To our knowledge, this is the highest efficiency ever reported for Pseudorabies virus recombination. We also demonstrate that the positions and distances of the CRISPR/Cas9 single guide RNAs from the homology arms correlate with the efficiency of homologous recombination. Our work show a simple and fast cloning method of BACs with large genome inserted by greatly enhancing the HR efficiencies through CRISPR/Cas9-mediated homology-directed repair mechanism, and this method could be of helpful for manipulating large DNA viruses, and will provide a successful model for insertion of large DNA fragments into other viruses. [ABSTRACT FROM AUTHOR]

Pseudorabies virus (PRV), which is extremely infectious and can infect numerous mammals, has a risk of spillover into humans. Virus–host interactions determine viral entry and spreading. Here, we showed that neuropilin-1 (NRP1) significantly potentiates PRV infection. Mechanistically, NRP1 promoted PRV attachment and entry, and enhanced cell-to-cell fusion mediated by viral glycoprotein B (gB), gD, gH, and gL. Furthermore, through in vitro coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays, NRP1 was found to physically interact with gB, gD, and gH, and these interactions were C-end Rule (CendR) motif independent, in contrast to currently known viruses. Remarkably, we illustrated that the viral protein gB promotes NRP1 degradation via a lysosome-dependent pathway. We further demonstrate that gB promotes NRP1 degradation in a furin-cleavage-dependent manner. Interestingly, in this study, we generated gB furin cleavage site (FCS)-knockout PRV (Dfurin PRV) and evaluated its pathogenesis; in vivo, we found that Dfurin PRV virulence was significantly attenuated in mice. Together, our findings demonstrated that NRP1 is an important host factor for PRV and that NRP1 may be a potential target for antiviral intervention. IMPORTANCE Recent studies have shown accelerated PRV cross-species spillover and that PRV poses a potential threat to humans. PRV infection in humans always manifests as a high fever, tonic-clonic seizures, and encephalitis. Therefore, understanding the interaction between PRV and host factors may contribute to the development of new antiviral strategies against PRV. NRP1 has been demonstrated to be a receptor for several viruses that harbor CendR, including SARS-CoV-2. However, the relationships between NRP1 and PRV are poorly understood. Here, we found that NRP1 significantly potentiated PRV infection by promoting PRV attachment and enhanced cell-to-cell fusion. For the first time, we demonstrated that gB promotes NRP1 degradation via a lysosome-dependent pathway. Last, in vivo, Dfurin PRV virulence was significantly attenuated in mice. Therefore, NRP1 is an important host factor for PRV, and NRP1 may be a potential target for antiviral drug development. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV), an important pathogen that affects the pig industry, is a highly genetically diverse RNA virus. However, the phylogenetic and genomic recombination properties of this virus have not been completely elucidated. In this study, comparative analyses of all available genomic sequences of North American (NA)-type PRRSVs (n = 355, including 138 PRRSV genomes sequenced in this study) in China and the United States during 2014–2018 revealed a high frequency of interlineage recombination hot spots in nonstructural protein 9 (NSP9) and the GP2 to GP3 regions. Lineage 1 (L1) PRRSV was found to be susceptible to recombination among PRRSVs both in China and the United States. The recombinant major parent between the 1991–2013 data and the 2014–2018 data showed a trend from complex to simple. The major recombination pattern changed from an L8 to L1 backbone during 2014–2018 for Chinese PRRSVs, whereas L1 was always the major backbone for US PRRSVs. Intralineage recombination hot spots were not as concentrated as interlineage recombination hot spots. In the two main clades with differential diversity in L1, NADC30-like PRRSVs are undergoing a decrease in population genetic diversity, NADC34-like PRRSVs have been relatively stable in population genetic diversity for years. Systematic analyses of insertion and deletion (indel) polymorphisms of NSP2 divided PRRSVs into 25 patterns, which could generate novel references for the classification of PRRSVs. The results of this study contribute to a deeper understanding of the recombination of PRRSVs and indicate the need for coordinated epidemiological investigations among countries. [ABSTRACT FROM AUTHOR]

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) possesses greater replicative capacity and pathogenicity than classical PRRSV. However, the factors that lead to enhanced replication and pathogenicity remain unclear. In our study, an alignment of all available full-length sequences of North American-type PRRSVs (n = 204) revealed two consistent amino acid mutations that differed between HP-PRRSV and classical PRRSV and were located at positions 519 and 544 in nonstructural protein 9. Next, a series of mutant viruses with either single or double amino acid replacements were generated from HP-PRRSV HuN4 and classical PRRSV CH-1a infectious cDNA clones. Deletion of either of the amino acids led to a complete loss of virus viability. In both Marc-145 and porcine alveolar macrophages, the replicative efficiencies of mutant viruses based on HuN4 were reduced compared to the parent, whereas the replication level of CH-1aderived mutant viruses was increased. Plaque growth assays showed clear differences between mutant and parental viruses. In infected piglets, the pathogenicity of HuN4-derived mutant viruses, assessed through clinical symptoms, viral load in sera, histopathology examination, and thymus atrophy, was reduced. Our results indicate that the amino acids at positions 519 and 544 in NSP9 are involved in the replication efficiency of HP-PRRSV and contribute to enhanced pathogenicity. This study is the first to identify specific amino acids involved in PRRSV replication or pathogenicity. These findings will contribute to understanding the molecular mechanisms of PRRSV replication and pathogenicity, leading to better therapeutic and prognostic options to combat the virus. IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a significant threat to the global pig industry. Highly pathogenic PRRSV (HP-PRRSV) first emerged in China in 2006 and has subsequently spread across Asia, causing considerable damage to local economies. HP-PRRSV strains possess a greater replication capacity and higher pathogenicity than classical PRRSV strains, although the mechanisms that underlie these characteristics are unclear. In the present study, we identified two mutations in HP-PRRSV strains that distinguish them from classical PRRSV strains. Further experiments that swapped the two mutations in an HP-PRRSV strain and a classical PRRSV strain demonstrated that they are involved in the replication efficiency of the virus and its virulence. Our findings have important implications for understanding the molecular mechanisms of PRRSV replication and pathogenicity and also provide new avenues of research for the study of other viruses. [ABSTRACT FROM AUTHOR]

In China, a majority of the highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRSV) strains were seeded by the 2006 outbreak. However, the most recently emerged (2013-2014) HP-PRRSV strain has a very different genetic background. It is a NADC30-like PRRSV strain recently introduced from North America that has undergone genetic exchange with the classic HP-PRRSV strains in China. Subsequent isolation and characterization of this variant suggest high pathogenicity, so it merits special attention in control and vaccine strategies. [ABSTRACT FROM AUTHOR]