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Pseudorabies virus (PRV) is related to alphaherpesvirus and varicellovirus. pUL16 is a conserved protein in all herpesviruses, and studies have shown that UL16 can interact with the viral proteins pUL11, pUL49, pUL21, gD, and gE. In this study, we found that pUL16 interacted with the viral capsid protein VP26, which could not translocate into the nucleus itself but did appear in the nucleus. We further determined whether pUL16 assists the translocation of VP26 into the nucleus. We found that pUL16 interacted with VP26 with or without viral proteins, and since VP26 itself did not contain a nuclear location signal, we concluded that pUL16 assisted the translocation of VP26 into the nucleus. Deletion of UL16 and UL35 significantly reduced the 50 % tissue culture infective dose, virulence, attachment, and internalization of PRV in cells. These results show that the interaction between pUL16 and VP26 influences the growth and virulence of pseudorabies virus. Our research is the first study to show that pUL16 interacts with VP26, which may explain the targeting site of UL16 and viral capsids. It is also the first to show that UL16 assists the transport of other viral proteins to organelles. Previous researches on pUL16 usually emphasized its interaction with pUL11, pUL21, and gE, and sometimes commented on pUL49 and gD. Our research focuses on the novel interaction between pUL16 and VP26, thereby enriching the studies on herpesviruses and possibly providing different directions for researchers.

The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to be highly heterogeneous and variable among PRRSV strains and some sequences in the middle region of the nsp2 are not essential to viral replication. Recent studies have attempted to insert foreign genes in the nsp2 nonessential regions but the foreign genes were not stably expressed by recombinant viruses in vitro. In the present study, we first constructed an infectious cDNA clone with deletion of 75 nucleotides (25 amino acids) in the nsp2 region (rHuN4-F112-Δ508-532) of the attenuated vaccine virus HuN4-F112 derived from a highly pathogenic PRRSV HuN4 and then inserted a gene fragment encoding a immunodominant B-cell epitope (49 amino acids) of Newcastle disease virus (NDV) nucleoprotein (NP) in-frame into the deletion site. The viable recombinant virus was rescued from the full-length cDNA infectious clone in vitro. The engineered viruses rescued from the cDNA clone indicated that the deletions of 75 nucleotides and insertion of NDV NP gene in the nsp2 region did not affect viral replication; they had similar growth kinetics to its parental virus. The inserting gene could be expressed consistently when the recombinant virus was passaged up to twenty times in cell cultures as determined by immunofluorescence assay (IFA) and genomic sequencing. To investigate the potential application of the NDV NP gene-inserted PRRSV as a marker vaccine, piglets were immunized with the recombinant virus and then challenged with lethal dose of highly pathogenic PRRSV. The immunized piglets produced specific antibodies against both the NDV NP and PRRSV, and lacked antibodies against the deleted 25aa nsp2 epitope. After challenge, all immunized piglets were protected from clinical disease or death, while all piglets in control group died (5/5) by ten days post challenge. The results of the present study indicated that the recombinant PRRSV (rHuN4-F112-Δ508-532) could be used as a potential marker vaccine against PRRS.

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.

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.

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.

Pseudorabies virus (PRV) causes Pseudorabies (PR), an economically important disease in domestic swine. PR outbreaks on pig farms caused by PRV variant strains in Bartha-K61-vaccinated pigs have resulted in considerable economic losses in China since 2011. In this study, the pathogenicity of the PRV variant JS-2012 strain to pigs was investigated by experimentally inoculating piglets of different ages in comparison with a classic virulent PRV SC strain. The JS-2012 strain caused an earlier onset of clinical signs and higher mortality in 15, 30, and 60-day-old pigs, as compared with a classic virulent PRV SC strain. The Bartha-K61 vaccination provided complete protection against challenge with classical virulent PRV, but only partial protection against challenge with the JS-2012 strain in piglets. In conclusion, the increased virulence of the PRV variant may have partly contributed to the PR outbreak in China.

Three porcine reproductive and respiratory syndrome viruses (PRRSV), NT1, NT2, and NT3, were isolated from three dying piglets from a single pig farm in Jiangsu Province, China. Whole genome sequencing revealed that the three isolates share the highest homology with JXA1-P80, an attenuated vaccine strain developed by serial passage of highly pathogenic PRRSV JXA1 in MARC-145 cells. More than ten amino acids residues in ORF1a, ORF1b, GP4, and GP5 that were thought to be unique to JXA1 attenuated on MARC-145 cells were each found in the corresponding locations of NT1, NT2, and NT3. In virulence assays, piglets infected with NT1, NT2, or NT3 exhibited clinical signs of disease, including high fever, anorexia, and respiratory distress, leading to the death of the majority of the piglets within two weeks. Collectively, these data indicate that NT1, NT2, and NT3 are highly pathogenic PRRSVs and they are likely to be revertants of the vaccine strain JXA1-P80.

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 (P0.05), 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.