Neuropilin-1 Facilitates Pseudorabies Virus Replication and Viral Glycoprotein B Promotes Its Degradation in a Furin-Dependent Manner.

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 (Δfurin PRV) and evaluated its pathogenesis; in vivo, we found that Δfurin 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, Δfurin 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.

Host BAG3 Is Degraded by Pseudorabies Virus pUL56 C-Terminal 181L-185L and Plays a Negative Regulation Role during Viral Lytic Infection.

Bcl2-associated athanogene (BAG) 3, which is a chaperone-mediated selective autophagy protein, plays a pivotal role in modulating the life cycle of a wide variety of viruses. Both positive and negative modulations of viruses by BAG3 were reported. However, the effects of BAG3 on pseudorabies virus (PRV) remain unknown. To investigate whether BAG3 could modulate the PRV life cycle during a lytic infection, we first identified PRV protein UL56 (pUL56) as a novel BAG3 interactor by co-immunoprecipitation and co-localization analyses. The overexpression of pUL56 induced a significant degradation of BAG3 at protein level via the lysosome pathway. The C-terminal mutations of 181L/A, 185L/A, or 181L/A-185L/A in pUL56 resulted in a deficiency in pUL56-induced BAG3 degradation. In addition, the pUL56 C-terminal mutants that lost Golgi retention abrogated pUL56-induced BAG3 degradation, which indicates a Golgi retention-dependent manner. Strikingly, BAG3 was not observed to be degraded in either wild-type or UL56-deleted PRV infected cells as compared to mock infected ones, whereas the additional two adjacent BAG3 cleaved products were found in the infected cells in a species-specific manner. Overexpression of BAG3 significantly suppressed PRV proliferation, while knockdown of BAG3 resulted in increased viral yields in HEK293T cells. Thus, these data indicated a negative regulation role of BAG3 during PRV lytic infection. Collectively, our findings revealed a novel molecular mechanism on host protein degradation induced by PRV pUL56. Moreover, we identified BAG3 as a host restricted protein during PRV lytic infection in cells.

Pseudorabies Virus UL24 Abrogates Tumor Necrosis Factor Alpha-Induced NF-κB Activation by Degrading P65.

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.

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.

The gene expression profile of porcine alveolar macrophages infected with a highly pathogenic porcine reproductive and respiratory syndrome virus indicates overstimulation of the innate immune system by the virus.

Since the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) variant emerged in 2006, it has caused death in more than 20 million pigs in China and other Southeast Asian countries, making it the most destructive swine pathogen currently in existence. To characterize the cellular responses to HP-PRRSV infection, the gene expression profile of porcine alveolar macrophage (PAM) cells, the primary target cells of PRRSV, was analyzed in HP-PRRSV-infected and uninfected PAMs by suppression subtractive hybridization. After confirmation by Southern blot, genes that were differentially expressed in the HP-PRRSV-infected and uninfected PAMs were sequenced and annotated. Genes that were upregulated mainly in HP-PRRSV-infected PAM cells were related to immunity and cell signaling. Among the differentially expressed genes, Mx1 and HSP70 protein expression was confirmed by western blotting, and IL-8 expression was confirmed by ELISA. In PAM cells isolated from HP-PRRSV-infected piglets, the differential expression of 21 genes, including IL-16, TGF-beta type 1 receptor, epidermal growth factor, MHC-I SLA, Toll-like receptor, hepatoma-derived growth factor, FTH1, and MHC-II SLA-DRB1, was confirmed by real-time PCR. To our knowledge, this is the first study to demonstrate differential gene expression between HP-PRRSV-infected and uninfected PAMs in vivo. The results indicate that HP-PRRSV infection excessively stimulates genes involved in the innate immune response, including proinflammatory cytokines and chemokines.

Pseudorabies virus variant in Bartha-K61-vaccinated pigs, China, 2012.

The widely used pseudorabies virus (PRV) Bartha-K61 vaccine has played a key role in the eradication of PRV. Since late 2011, however, a disease characterized by neurologic symptoms and a high number of deaths among newborn piglets has occurred among Bartha-K61-vaccinated pigs on many farms in China. Clinical samples from pigs on 15 farms in 6 provinces were examined. The PRV gE gene was detectable by PCR in all samples, and sequence analysis of the gE gene showed that all isolates belonged to a relatively independent cluster and contained 2 amino acid insertions. A PRV (named HeN1) was isolated and caused transitional fever in pigs. In protection assays, Bartha-K61 vaccine provided 100% protection against lethal challenge with SC (a classical PRV) but only 50% protection against 4 challenges with strain HeN1. The findings suggest that Bartha-K61 vaccine does not provide effective protection against PRV HeN1 infection.

Highly pathogenic porcine reproductive and respiratory syndrome virus GP5 B antigenic region is not a neutralizing antigenic region.

In 2006, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) caused great economic losses emerged in China and continues to be a threat for the pig industry. B antigenic region (AR) ((37)SHL/FQLIYNL(45)) of GP5 was considered to be a major linear neutralizing AR in PRRSV classical strains. However, peptide-purified antibodies against this AR did not neutralize PRRSV in a recent report. Compared with classical PRRSV, one amino acid mutation (L/F(39)→ I(39)) was found in B AR of HP-PRRSV. To study the ability of B AR of HP-PRRSV to induce neutralizing antibody (NA) in vitro and in vivo, rabbit antisera against B AR with and without the mutation and pig hyperimmune sera with high titer of NAs against HP-PRRSV were prepared. Immunofluorescence assays (IFA) showed that the two rabbit antisera both had reactivity to classical PRRSV CH-1a and HP-PRRSV HuN4 with no observable difference in IFA titer. However, antisera did not have neutralizing activity against classical PRRSV CH-1a and HP-PRRSV HuN4. No correlation was observed between the levels of anti-B AR peptide antibodies and NAs in pig hyperimmune sera that were detected by indirect ELISA and virus neutralization, respectively. B AR peptide-specific serum antibodies had no neutralizing activity and, GST-B fusion protein could not inhibit neutralization of NAs in pig hyperimmune sera. Based on these findings, we conclude that B AR of HP-PRRSV is not a neutralizing AR of HP-PRRSV GP5.

Comparative genomic analysis of five pairs of virulent parental/attenuated vaccine strains of PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of porcine reproductive and respiratory syndrome, is responsible for serious disease in pigs resulting in substantial economic losses in the porcine industry. An attenuated vaccine strain, HuN4-F112, was obtained by passaging virulent PRRSV strain HuN4 on Marc-145 cells (for 112 passages), and the full-genomic sequence was determined. To understand the molecular basis of attenuation of PRRSV, we compared and analyzed the genomic sequences of HuN4/HuN4-F112, together with those of other four virulent parental/attenuated vaccine strains. Among the 19 PRRSV proteins, two (NSP6 and NSP8) were highly conserved, without any mutations and considered irrelative to attenuation. The mutation rates of envelope-associated structural proteins were obviously higher than those of most non-structural proteins. It is interesting that the gene of the smallest structural protein, E protein, had the highest mutation rate among all of the structural genes analyzed, and also harbored a highly variable region. Our results indicate that determinants of PRRSV attenuation are multigenic products of both non-structural and structural genes. To our knowledge, this is the first report showing that the envelope-associated structural proteins (including E and GP2-GP5 proteins) may play a significant role. These findings contribute towards our understanding of PRRSV attenuation and will provide an important clue for further study.

Isolation of serotype 2 porcine teschovirus in China: evidence of natural recombination.

Porcine teschovirus (PTV), the pathogen of porcine polioencephalomyelitis, is a member of the family Picornaviridae. In this study, a new PTV strain (designated as JF613) was isolated from pigs in China. It was confirmed by the specific CPE on susceptible cells, RT-PCR and nucleotide sequencing. Analysis of its amino acids sequence of complete polyprotein indicated that the isolate belongs to serotype 2. Genetic recombination is a well-known phenomenon for picornavirus which has been demonstrated in many other members of the family, but it remains so far unclear whether recombination occurs in PTV. To detect possible recombination events, 30 sequences of complete coding regions of PTV strains accessible in GenBank were examined. Putative recombinant sequence was identified with the use of SimPlot program. The result showed that the genomic sequence of our isolate exhibited highest similarities with strains of serotypes 2 and 5, respectively, in two crossover regions, suggesting the recombination event in PTV. Then the mosaic structure of viral genome was confirmed by bootscanning and genetic algorithm for recombination detection (GARD). This represents the first PTV-2 isolate in China. Furthermore, our study provided the first evidence of natural recombination in PTV and indicated that homologous recombination may be a driving force in PTV evolution.

Porcine reproductive and respiratory syndrome virus attachment is mediated by the N-terminal domain of the sialoadhesin receptor.

Sialoadhesin (Sn) is an important receptor for viral attachment and internalization of porcine reproductive and respiratory syndrome virus (PRRSV) to porcine alveolar macrophages (PAM). To investigate whether the N-terminal domain of Sn is sufficient and/or necessary for PRRSV attachment, we constructed a series of truncated fragments of porcine Sn and expressed these in the non-permissive PK15 cell line. The first 150 amino acids comprising the entire first domain of the Sn N-terminal region was necessary for PRRSV binding to cells, and the N-terminal domain alone was sufficient for virus attachment. The attachment of PRRSV to PAM cells was inhibited by polyclonal anti-serum against the N-terminal region of porcine Sn in a dose-dependent manner. The present study demonstrates that the first domain at the N-terminus of Sn mediates PRRSV attachment to PAM cells and contributes to better understanding the interaction between PRRSV and its host cells.

Genetic diversity of the ORF5 gene of porcine reproductive and respiratory syndrome virus isolates in China from 2006 to 2008.

Since April 2006, swine herds have experienced the outbreaks of a highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China. To explore the possible mechanism of the emergence of the highly pathogenic PRRS and more fully understand the extent of genetic diversity of PRRSV in China, we analyzed the ORF5 gene sequences of 159 representative PRRSV isolates in 16 provinces from 2006 to 2008. Sequence and phylogenetic analyses showed that all these 159 isolates belonged to the North American genotype and were further divided into six subgenotypes; 140 of 159 isolates were closely related to the highly pathogenic PRRSV with 98.5-100% nucleotide and 98.3-100% amino acid sequence identities and belonged to Subgenotype I; and 3, 8, 4, 3, 1 of 159 isolates were part of Subgenotypes II-VI, respectively. Amino acid analysis of the GP5 protein revealed that all the isolates in Subgenotypes I-III were found to be highly variable in the primary neutralizing epitope; most of the isolates in Subgenotypes I and IV had more glycosylation sites than those in Subgenotypes II, III, V and VI; and 1, 5, and 9 unique amino acid mutations were observed in Subgenotypes I, IV and VI, respectively. In conclusion, our study provides the evidence of coexistence of six different subgenotype isolates in pigs in China from 2006 to 2008, and emphasizes the importance of reinforcing PRRSV surveillance, especially after the emergence of highly pathogenic PRRS in China.

Monoclonal antibodies and conserved antigenic epitopes in the C terminus of GP5 protein of the North American type porcine reproductive and respiratory syndrome virus.

Glycoprotein 5 (GP5) is the major glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the gene encoding rtGP5, lacking signal peptide sequence, was expressed as GST-fusion protein in E. coli. Fifteen monoclonal antibodies (MAbs) against rtGP5 were developed and used to probe a series of GP5 peptides by ELISA, in which two MAbs specifically recognized the epitope GP5EP3 (146-156aa), four recognized GP5EP5 (164-180aa) and nine recognized GP5EP7 (192-200aa). After precise analysis by sequential deletion of the terminal amino acid residues, the three minimal epitopes (R(152)LYRWR(156), E(169)GHLIDLKRV(178) and Q(196)WGRL(200)) were determined, which were highly conserved among the North American type isolates, with the exception of one amino acid mutation (L(200) to P(200)). Mutational analysis showed that the mutant (Q(196)WGRP(200)) could be recognized by four of nine anti-GP5EP7 MAbs, indicating Q(196)WGRP(200) was also one minimal epitope. Western blot analysis showed that GP5EP5 and GP5EP7 (L(200) or P(200)) could be recognized by PRRSV-positive sera of CH-1a and/or BJ-4, suggesting GP5EP5 and GP5EP7 (L(200) or P(200)) were antigenic epitopes in the PRRSV-infected pigs. MAbs against GP5EP3, GP5EP5, and GP5EP7 could react with MARC-145 cells infected with the North American type isolates from China in IFA. However, very interestingly, when the highly pathogenic PRRSV, represented by HUN4, was passaged in MARC-145 cells, MAbs against GP5EP7 did not react with HUN4-F20-HUN4-F112 (20-112th passage virus), where Q(196)WGRL(200) had mutated to R(196)WGRL(200). Due to no mutations observed in GP5EP3 and GP5EP5, MAbs against GP5EP3 and GP5EP5 could recognize HUN4-F20-HUN4-F112. All the results herein might deepen the understanding of the antigen structure of in the C terminus of GP5 and facilitate the development of diagnostic antigens of the North American type PRRSV in China.

An attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) protects piglets against HP-PRRS.

Porcine infections with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) cause significant morbidity and mortality and currently there are no effective vaccines for disease prevention. An attenuated strain, HuN4-F112, was obtained by passaging the HP-PRRSV HuN4 on Marc-145 cells (112th-passage). PRRSV-free pigs were inoculated intramuscularly with HuN4-F112 (10(2.0), 10(3.0), 10(4.0), 10(5.0) and 10(6.0) TCID(50) for groups 1-5, respectively). The groups 3-5 could resist the lethal challenge and did not show any obvious changes in body temperature nor clinical signs throughout the experiment, the pathological lesions were milder and the gained weight at a greater rate (P<0.05), compared to group 1 and control. Sequence analysis of the HuN4 passages showed a conserved epitope in GP5 protein was mutated ((196)QWGRL/P(200)-->(196)RWGRL/P(200)), as a result the monoclonal antibody could not recognize the HuN4-F112 any more. These results suggested that the HuN4-F112 could protect piglets from lethal challenge and might be a candidate vaccine against the HP-PRRSV.

A recombinant plasmid co-expressing swine ubiquitin and the GP5 encoding-gene of porcine reproductive and respiratory syndrome virus induces protective immunity in piglets.

We transfected a recombinant plasmid that co-expressed swine ubiquitin and a codon optimized GP5 encoding-gene of porcine reproductive and respiratory syndrome virus (PRRSV), designated pCA-U-optiGP5, as well as the plasmid pCA-optiGP5 encoding codon optimized GP5, and the plasmid pCA-GP5 expressing wild-type pGP5 into 293T cells. Expression of GP5 was measured by indirect immunofluorescence (IIF) assay and Western blot and found to be considerably higher in response to pCA-U-optiGP5 than the wild-type vector. GP5 protein was rapidly degraded in pCA-U-optiGP5-transfected 293T cells. The proteasome inhibitor, MG-132, however, successfully inhibited degradation. Immunogenicity of the three constructs was examined by measuring GP5-specific antibody production, lymphocyte proliferation, cytotoxic T lymphocyte (CTL) responses, and cytokine secretion in intramuscularly immunized pigs. Three weeks after the last inoculation, all animals were challenged intranasally with 2-ml 10(5)TCID(50)/ml PRRSV CH-1a. DNA immunization with pCA-optiGP5 produced a higher level of GP5-specific antibody than immunization with pCA-GP5, and the humoral response remained undetectable in the pCA-U-optiGP5 group. However, the fusion DNA had a significantly enhanced stimulation index (SI) and induced a stronger Th1 type cellular immune response than the single gene DNA, suggesting that ubiquitin conjugation improved the cellular but not the humoral immune response. Four of six pigs in the pCA-U-optiGP5 group, three of six in the pCA-optiGP5 group, and two of six in the pCA-GP5 group were devoid of visible pathological changes that were present in other vaccinated and control animals after challenge. Viral replication and distribution in the blood and tissues was lower in the pCA-U-optiGP5 vaccinated group than the other groups, suggesting that codon optimization, along with the development of rapidly processed antigen, represents a novel strategy to increase the immune efficacy of DNA vaccines against PRRSV.

Long terminal repeat sequences from virulent and attenuated equine infectious anemia virus demonstrate distinct promoter activities.

In the early 1970s, the Chinese Equine Infectious Anemia Virus (EIAV) vaccine, EIAV(DLA), was developed through successive passages of a wild-type virulent virus (EIAV(L)) in donkeys in vivo and then in donkey macrophages in vitro. EIAV attenuation and cell tropism adaptation are associated with changes in both envelope and long terminal repeat (LTR). However, specific LTR changes during Chinese EIAV attenuation have not been demonstrated. In this study, we compared LTR sequences from both virulent and attenuated EIAV strains and documented the diversities of LTR sequence from in vivo and in vitro infections. We found that EIAV LTRs of virulent strains were homologous, while EIAV vaccine have variable LTRs. Interestingly, experimental inoculation of EIAV(DLA) into a horse resulted in a restriction of the LTR variation. Furthermore, LTRs from EIAV(DLA) showed higher Tat transactivated activity than LTRs from virulent strains. By using chimeric clones of wild-type LTR and vaccine LTR, the main difference of activity was mapped to the changes of R region, rather than U3 region.