Yifeng Jiang

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.

Genomic characterization of emergent pseudorabies virus in China reveals marked sequence divergence: Evidence for the existence of two major genotypes

Recently pseudorabies outbreaks have occurred in many vaccinated farms in China. To identify genetic characteristics of pseudorabies virus (PRV) strains, we obtained the genomic sequences of PRV strains HeN1 and JS, which were compared to 4 PRV genomes and 729 partial gene sequences. PRV strains isolated in China showed marked sequence divergence compared to European and American strains. Phylogenetic analysis revealed that for the first time PRV can be divided into 2 distinct clusters, with Chinese strains being genotype II and PRVs isolated from other countries being genotype I. Restriction fragment length polymorphism analysis confirmed differences between HeN1 and Bartha strains, as did the presence of unique insertion/deletion polymorphisms and microsatellites. This divergence between the two genotypes may have been generated from long-term, independent evolution, which could also explain the low efficacy of the Bartha vaccine in protecting pigs infected with genotype II PRV.

Host miR-26a suppresses replication of porcine reproductive and respiratory syndrome virus by upregulating type I interferons.

Abstract MicroRNAs (miRNAs) play important roles in viral infections, especially by modulating the expression of cellular factors essential to viral replication or the host innate immune response to infection. To identify host miRNAs important to controlling porcine reproductive and respiratory syndrome virus (PRRSV) infection, we screened 15 miRNAs that were previously implicated in innate immunity or antiviral functions. Over-expression of the miR-26 family strongly inhibited PRRSV replication in vitro, as shown by virus titer assays, Western blotting, and qRT-PCR assays. MiR-26a inhibited the replication of both type 1 and type 2 PRRSV strains. Mutating the seed region of miR-26 restored viral titers. Luciferase reporters showed that miR-26a does not target the PRRSV genome directly but instead affects the expression of type I interferon and the IFN-stimulated genes MX1 and ISG15 during PRRSV infection. These results demonstrate the important role of miR-26a in modulating PRRSV infection and also support the possibility of using host miR-26a to achieve RNAi-mediated antiviral therapeutic strategies.

Identification of immunodominant T-cell epitopes in membrane protein of highly pathogenic porcine reproductive and respiratory syndrome virus.

The development of cell-mediated immunity has been known extremely important in clearing porcine reproductive and respiratory syndrome virus (PRRSV) in infected pigs. However, the PRRS immunology regarding the interaction of T-cells and PRRSV proteins is poorly understood. To identify the T-cell immunodominant epitopes on the membrane (M) protein of PRRSV, a series of 31 overlapping pentadecapeptides covering the entire M protein were designed and synthesized. These peptides were screened by ELIspot analysis for their capabilities to elicit interferon-gamma (IFN-γ) responses in the peripheral blood mononuclear cells (PBMCs), which were collected from pigs immunized with attenuated PRRSV HuN4-F112 strain and challenged with highly pathogenic HuN4 strain. After three rounds of screening, 4 peptides (M3, M6, M8 and M12) were shown to elicit high expression of IFN-γ. The stimulation of high IFN-γ transcription in PBMCs by these 4 peptides was further confirmed in real-time PCR. The sequence alignment revealed that the epitope represented by peptide M6 was fully conserved in all of examined 42 North American genotype II PRRSV isolates and the epitopes represented by peptides M3, M8 and M12 showed 2-4 amino acid replacements. The finding of 4 T-cell immunodominant epitopes in the M protein of PRRSV will be beneficial to the understanding of the development of cell-mediated immunity.

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.

Generation of an infectious clone of HuN4-F112, an attenuated live vaccine strain of porcine reproductive and respiratory syndrome virus

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

Emergence of a Pseudorabies virus variant with increased virulence to piglets

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.

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.

Cellular miR-130b inhibits replication of porcine reproductive and respiratory syndrome virus in vitro and in vivo.

MicroRNAs (miRNAs) can impact viral infections by binding to sequences with partial complementarity on viral RNA transcripts, usually resulting in the repression of virus replication. In the present study, we identified a potential binding site for miR-130 in the 5′ untranslated region (bps 155-162) of the porcine reproductive and respiratory syndrome virus (PRRSV) genome. We found that the delivery of multiple miR-130 family mimics, especially miR-130b, resulted in inhibition of PRRSV replication in vitro. miR-130 was effective in inhibiting the replication of multiple type 2 PRRSV strains, but not against vSHE, a classical type 1 strain. miR-130 over-expression did not induce IFN-α or TNF-α expression in either uninfected or PRRSV-infected porcine alveolar macrophages. Results from luciferase reporter assays indicated that miR-130 directly targeted the PRRSV 5′ UTR. Intranasal inoculation of piglets with miR-130b exhibited antiviral activity in vivo and partially protected piglets from an otherwise lethal challenge with HP-PRRSV strain vJX143. Overall, these results demonstrate the importance of the miR-130 family in modulating PRRSV replication and also provide a scientific basis for using cellular miRNAs in anti-PRRSV therapies.

Stable expression of foreign gene in nonessential region of nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus: applications for marker vaccine design.

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.