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

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

MARCH8 inhibits pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in the trans-Golgi network.

The membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, has broad-spectrum antiviral activity. However, some viruses hijack MARCH8 to promote virus replication, highlighting its dual role in the viral lifecycle. Most studies on MARCH8 have focused on RNA viruses, leaving its role in DNA viruses largely unexplored. Pseudorabies virus (PRV) is a large DNA virus that poses a potential threat to humans. In this study, we found that MARCH8 inhibited PRV replication at the cell-to-cell fusion stage. Interestingly, our findings proved that MARCH8 blocks gB cleavage by recruiting furin but this activity does not inhibit viral infection in vitro. Furthermore, we confirmed that MARCH8 inhibits cell-to-cell fusion independent of its E3 ubiquitin ligase activity but dependent on the interaction with the cell-to-cell fusion complex (gB, gD, gH, and gL). Finally, we discovered that the distribution of the cell-to-cell fusion complex is significantly altered and trapped within the trans-Golgi network. Overall, our results indicate that human MARCH8 acts as a potent antiviral host factor against PRV via trapping the cell-to-cell fusion complex in the trans-Golgi network.

MARCH1 and MARCH2 inhibit pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in trans-Golgi network.

The membrane-associated RING-CH (MARCH) family of proteins are members of the E3 ubiquitin ligase family and are essential for a variety of biological functions. Currently, MARCH proteins are discovered to execute antiviral functions by directly triggering viral protein degradation or blocking the furin cleavage of viral class I fusion proteins. Here, we report a novel antiviral mechanism of MARCH1 and MARCH2 (MARCH1/2) in the replication of Pseudorabies virus (PRV), a member of the Herpesviridae family. We discovered MARCH1/2 restrict PRV replication at the cell-to-cell fusion step. Furthermore, MARCH1/2 block gB cleavage, and this is dependent on their E3 ligase activity. Interestingly, the blocking of gB cleavage by MARCH1/2 does not contribute to their antiviral activity in vitro. We discovered that MARCH1/2 are associated with the cell-to-cell fusion complex of gB, gD, gH, and gL and trap these viral proteins in the trans-Golgi network (TGN) rather than degrading them. Overall, we conclude that MARCH1/2 inhibit PRV by trapping the viral cell-to-cell fusion complex in TGN.

Minor envelope proteins from GP2a to GP4 contribute to the spread pattern and yield of type 2 PRRSV in MARC-145 cells.

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.

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

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

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

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

Characterization of Two Immunodominant Antigenic Peptides in NSP2 of PRRSV-2 and Generation of a Marker PRRSV Strain Based on the Peptides.

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.

Long-Term Genome Monitoring Retraces the Evolution of Novel Emerging Porcine Reproductive and Respiratory Syndrome Viruses.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes tremendous economic losses to the swine industry worldwide. In China, novel PRRSVs have frequently emerged in recent years, but the evolutionary relationship among these viruses has remained unclear. In the present study, a 4-year PRRSV genome-monitoring study was performed on samples from a pig farm. We observed that NADC30-like PRRSVs with higher mutation rates replaced HP-PRRSVs as the epidemic strains. We monitored the variation in the same PRRSV strain evolved in a pig herd over 2 years and observed that the low genomic similarity of NADC30-like PRRSVs results from rapid mutation. We also showed that recombination events between NADC30-like and QYYZ-like PRRSVs resulted in the complex recombination patterns of PRRSVs, which have formed gradually over time. Furthermore, recombination of the same strain can occur at different locations and increase the diversity of recombination events. Overall, these findings interpret the evolutionary patterns of novel and emerging PRRSVs, information that is crucial for PRRSV control.

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

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

Molecular and Cellular Mechanisms for PRRSV Pathogenesis and Host Response to Infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused tremendous amounts of economic losses to the swine industry for more than three decades, but its control is still unsatisfactory. A significant amount of information is available for host cell-virus interactions during infection, and it is evident that PRRSV has evolved to equip various strategies to disrupt the host antiviral system and provide favorable conditions for survival. The current study reviews viral strategies for modulations of cellular processes including innate immunity, apoptosis, microRNAs, inflammatory cytokines, and other cellular pathways.

Single Virus Tracking with Quantum Dots Packaged into Enveloped Viruses Using CRISPR.

Labeling viruses with high-photoluminescence quantum dots (QDs) for single virus tracking provides a visual tool to aid our understanding of viral infection mechanisms. However, efficiently labeling internal viral components without modifying the viral envelope and capsid remains a challenge, and existing strategies are not applicable to most viruses. Here, we have devised a strategy using the clustered regularly interspaced short palindromic repeats (CRISPR) imaging system to label the nucleic acids of Pseudorabies virus (PRV) with QDs. In this strategy, QDs were conjugated to viral nucleic acids with the help of nuclease-deactivated Cas9/gRNA complexes in the nuclei of living cells and then packaged into PRV during virion assembly. The processes of PRV-QD adsorption, cytoplasmic transport along microtubules, and nuclear entry were monitored in real time in both Vero and HeLa cells, demonstrating the utility and efficiency of the strategy in the study of viral infection.

Phylogenetics, Genomic Recombination, and NSP2 Polymorphic Patterns of Porcine Reproductive and Respiratory Syndrome Virus in China and the United States in 2014-2018.

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.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine diseases. However, the phylogenetic and genomic recombination properties of the PRRS virus (PRRSV) have not been completely elucidated. In this study, we systematically compared differences in the lineage distribution, recombination, NSP2 polymorphisms, and evolutionary dynamics between North American (NA)-type PRRSVs in China and in the United States. Strikingly, we found high frequency of interlineage recombination hot spots in nonstructural protein 9 (NSP9) and in the GP2 to GP3 region. Also, intralineage recombination hot spots were scattered across the genome between Chinese and US strains. Furthermore, we proposed novel methods based on NSP2 indel patterns for the classification of PRRSVs. Evolutionary dynamics analysis revealed that NADC30-like PRRSVs are undergoing a decrease in population genetic diversity, suggesting that a dominant population may occur and cause an outbreak. Our findings offer important insights into the recombination of PRRSVs and suggest the need for coordinated international epidemiological investigations.

Genetic diversity of porcine reproductive and respiratory syndrome virus 1 in the United States of America from 2010 to 2018.

Porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) was first detected in the United States of America (USA) in 1999, several strains were also recognized soon later, and these isolates are typically called North American (NA) PRRSV-1. However, few reports have characterized PRRSV-1 viruses in the USA. We explored the genetic characteristics and diversity of PRRSV-1 viruses circulating in the USA. PRRSV-1 PCR-positive samples collected from seven states in 2010-2018 (n = 27) were subjected to next-generation sequencing. The 27 PRRSV-1 viruses had 88.4-91.3% nucleotide identity to the PRRSV-1 Lelystad-virus strain (the type 1 prototype strain) and 87.4-89.8% to the previously reported NA PRRSV-1 viruses. Individual proteins had several unique genetic characteristics and only one of the 27 tested samples had the characteristic 17-amino acid (aa) deletion in Nsp2, a genetic marker of NA PRRSV-1 viruses described previously. Fourteen isolates displayed a 3-aa C-terminal truncation in the highly conserved Nsp12 gene; 16 samples had a 21- or 18-aa C-terminal truncation in GP3 gene; and one was observed with a 1-aa deletion at the overlapping region of GP3 and GP4. In addition, the GP5 protein in most isolates, excluding one exception, demonstrated similar genetic variation as other reported NA PRRSV-1 isolates. All tested isolates clustered within subtype 1 together with other available NA PRRSV-1 viruses. Collectively, our results provide up-to-date information on PRRSV-1 viruses circulating in the USA in the past 9 years although the number of PRRSV-1 isolates included in this study is limited. These PRRSV-1 viruses have undergone gradual genetic variation and exhibited some previously undescribed genetic characteristics and diversity, which complicates the diagnosis and control of NA PRRSV-1.

Two novel recombinant porcine reproductive and respiratory syndrome viruses belong to sublineage 3.5 originating from sublineage 3.2.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an agent of porcine reproductive and respiratory syndrome (PRRS), which causes substantial economic losses to the swine industry. PRRSV displays rapid variation, and five lineages coexist in mainland China. Lineage 3 PRRSVs emerged in mainland China in 2005 and prevailed in southern China after 2010. In the present study, two lineage 3 PRRSV strains, which are named SD110-1608 and SDWH27-1710, were isolated from northern China in 2017. To explore the characteristics and origins of the two strains, we divided lineage 3 into five sublineages (3.1-3.5) based on 146 open reading frame (ORF) 5 sequences. Both strains and the strains isolated from mainland China were classified into sublineage 3.5. Lineage 3 PRRSVs isolated from Taiwan and Hong Kong were classified into sublineages 3.1-3.3 and sublineage 3.4, respectively. Recombination analysis revealed that SD110-1608 and SDWH27-1710 were derived from recombination of QYYZ (major parent strain) and JXA1 (minor parent strain). Sequence alignment showed that SD110-1608 and SDWH27-1710 shared a 36-aa insertion in Nsp2 with QYYZ isolated from Guangdong Province in 2010. Based on the evolutionary relationship among GP2a, GP3, GP4, GP5 and N proteins between sublineages 3.2 (FJ-1) and 3.5 (FJFS), we speculated that sublineage 3.5 (mainland China) originated from sublineage 3.2 (Taiwan, China). This study provides important information regarding the classification and transmission of lineage 3 PRRSVs.

Correction to: Annexin A2 binds to vimentin and contributes to porcine reproductive and respiratory syndrome virus multiplication.

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'.

Adaptions of field PRRSVs in Marc-145 cells were determined by variations in the minor envelope proteins GP2a-GP3.

The recent rapid evolution of PRRSVs has resulted in certain biological characteristic changes, such as the fact that an increasing number of field PRRSVs can be isolated from PAMs but not from Marc-145 cells. In this study, we first isolated Marc-145-unadaptive field PRRSV strains from PAMs; sequence analysis showed that these PRRSVs belong to the HP-PRRSV (lineage 8) branch or NADC30-Like (lineage 1) branch. We further found major variations in ORF2-4 regions. To explore the viral adaptation mechanisms in detail, we constructed a full-length cDNA clone of MY-376, a Marc-145-unadaptive PRRSV. Construction of serially chimeric viruses of HuN4-F112 (a Marc-145-adaptive strain) and MY-376 demonstrated that variation in the minor envelope protein (GP2a and GP3) complex is a main determinant of PRRSV tropism for Marc-145 cells.

Annexin A2 binds to vimentin and contributes to porcine reproductive and respiratory syndrome virus multiplication.

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.

Porcine alveolar macrophage CD163 abundance is a pivotal switch for porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a problematic virus that is difficult to control. The principal target cells for PRRSV infection are porcine alveolar macrophages (PAMs). Increasing evidence has demonstrated that CD163 is the determinant receptor for PRRSV infection. However, the relationship between CD163 abundance and PRRSV infection is unclear. In this study, we first generated primary immortalized PAMs (iPAMs) using SV40 large T antigen and demonstrated that CD163 expression is suppressed by the alternative splicing of mRNA in iPAMs. Two forms of CD163 transcripts were discovered, and most iPAMs expressed a short-form CD163 transcript that lacked from scavenger receptor cysteine-rich tandem repeat 1 (SRCR1) to SRCR5 of the functional domain. More importantly, using flow cytometric cell sorting technology, we isolated CD163-positive single-cell-derived clones with varying CD163 abundances to investigate the relationship between CD163 abundance and PRRSV infection. For the first time, we showed that cells with low CD163 abundance (approximately 20%) do not initiate PRRSV infection, while cells with moderate CD163 abundance display limited infection. PRRSV initiated efficient infection only in cells with high CD163 abundances. Our results demonstrate that CD163 abundance is a pivotal switch for PRRSV replication.

CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.

Several groups have used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) for DNA virus editing. In most cases, one single-guide RNA (sgRNA) is used, which produces inconsistencies in gene editing. In this study, we used a swine herpesvirus, pseudorabies virus, as a model to systematically explore the application of CRISPR/Cas9 in DNA virus editing. In our current report, we demonstrated that cotransfection of 2 sgRNAs and a viral genome resulted in significantly better knockout efficiency than the transfection-infection-based approach. This method could result in 100% knockout of ≤3500 bp of viral nonessential large fragments. Furthermore, knockin efficiency was significantly improved by using 2 sgRNAs and was also correlated with the number of background viruses. We also demonstrated that the background viruses were all 2-sgRNA-mediated knockout mutants. Finally, this study demonstrated that the efficacy of gene knockin is determined by the replicative kinetics of background viruses. We propose that CRISPR/Cas9 coupled with 2 sgRNAs creates a powerful tool for DNA virus editing and offers great potential for future applications.-Tang, Y.-D., Guo, J.-C., Wang, T.-Y., Zhao, K., Liu, J.-T., Gao, J.-C., Tian, Z.-J., An, T.-Q., Cai, X.-H. CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.