[Show abstract][Hide abstract] ABSTRACT: Background
Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be an important problem for the swine industry. Inactivated vaccines and modified-live virus vaccines are widely used in the field; however, the efficacy of these PRRSV vaccines is suboptimal due to poor immunogenicity. Granulocyte¿macrophage colony stimulating factor (GM-CSF) has been extensively used as an effective genetic and protein adjuvant to enhance the efficiencies vaccines expressing tumor or pathogen antigens. The purpose of this study was to determine if GM-CSF could increase the efficiency of PRRSV vaccine.Methods
The GM-CSF gene was inserted in the HuN4-F112 vaccine strain by overlap PCR. The expression of GM-CSF by the recombinant virus was confirmed with methods of indirect immunofluorescent assay (IFA) and Western blotting. The stability of recombinant virus was assessed by cDNA sequence and IFA after 20 passages. To detect the biological activity of GM-CSF expressed by the recombinant virus, bone marrow-derived dendritic cells (BMDCs) were isolated and co-cultured with the recombinant virus or parental virus and the surface phenotypes of BMDCs were examined by flow cytometric analysis. The cytokines secreted by BMDCs infected with PRRSV,or treated with LPS, GM-CSF or medium alone were evaluated by ProcartaPlexTM Multiplex Immunoassays and qRT-PCR.ResultsA novel modified-live PRRSV vaccine strain expressing GM-CSF (rHuN4-GM-CSF) was successfully constructed and rescued. The GM-CSF protein was stable expressed in recombinant virus-infected cells after 20 passages. Analysis of virus replication kinetics showed that the novel vaccine strain expressing GM-CSF had a similar replication rate as the parental virus. In vitro studies showed that infection of porcine BMDCs with rHuN4-GM-CSF resulted in increased surface expression of MHCI+, MHCII¿+¿and CD80/86+ that was dependent on virus expressed GM-CSF. The expression of representative cytokines was significantly up-regulated when BMDCs were incubated with the recombinant GM-CSF expressing virus.Conclusions
Our results indicated that the expression of GM-CSF during infection with a vaccine strain could enhance the activation of BMDCs and increase cytokine response, which is expected to result in higher immune responses and may improve vaccine efficacy against PRRSV infection.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: The sterile alpha motif and HD domain 1 (SAMHD1) protein is an novel innate immunity restriction factor that inhibits HIV-1 infection in myeloid cells. Here, we cloned the full-length SAMHD1 complementary DNA (cDNA) from porcine peripheral blood lymphocytes. The porcine SAMHD1 cDNA was of 3951 bp with an open reading frame of 1884 bp, encoding a polypeptide of 627 amino acids. Porcine SAMHD1 mRNA was detected in all swine tissues examined, with the higher expression in the tonsil, lung, liver, and lymph node tissues. The SAMHD1 protein was localized to the nucleus. Overexpression of SAMHD1 blocked the proliferation of HuN4, a highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV), in MARC-145 cells, by inhibiting the synthesis of the HuN4 complement RNA. The antiviral effects of the simian SAMHD1 protein were nearly equivalent to those of porcine SAMHD1 in the HuN4-infected MARC-145 cells. Phosphorylation analysis of SAMHD1 showed that overexpressed SAMHD1 protein was in primarily an unphosphorylated state. SAMHD1 overexpression increased the transcript abundance of IFN-stimulated genes ISG15 and ISG56. The mRNA levels of SAMHD1 and ISGs were significantly increased in porcine alveolar macrophages infected with HP-PRRSV. SAMHD1 protein level was also elevated, and the protein was not phosphorylated during infection. Collectively, our data indicate that SAMHDI inhibits HP-PRRSV proliferation through inhibiting the replication of HP-PRRSV. SAMHD1 might be the protein participating in the IFN signaling and is thus an important immunoregulatory protein in innate immunity.
[Show abstract][Hide abstract] ABSTRACT: The factors that determine the transcription-regulating sequence (TRS) activity of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unclear. In this study, the effect of mutagenesis of conserved C nucleotides at positions 5 and 6 in the leader TRS (TRS-L) and/or canonical body TRS7 (TRS-B7) on the synthesis of subgenomic (sg) mRNA and virus infectivity was investigated in the context of a type 2 PRRSV infectious cDNA clone. The results showed that a double C mutation in the leader TRS completely abolished sg mRNAs synthesis and virus infectivity, but a single C mutation did not. A single C or double C mutation in TRS-B7.1 or/and TRS-B7.2 impaired or abolished the corresponding sg mRNA synthesis. Introduction of identical mutations in the leader and body TRSs partially restored sg mRNA7.1 and/or sg mRNA7.2 transcription, indicating that the base-pairing interaction between sense TRS-L and cTRS-B is a crucial factor influencing sg mRNA synthesis. Analysis of the mRNA leader-body junctions of mutants provided evidence for a mechanism of discontinuous minus-strand transcription. This study also showed that mutational inactivation of TRS-B7.1 or TRS-B7.2 did not affect the production of infectious progeny virus, and the sg mRNA formed from each of them could express N protein. However, TRS-B7.1 plays more important roles than TRS-B7.2 in maintaining the growth characteristic of type 2 PRRSV. These results provide more insight into the molecular mechanism of genome expression and subgenomic mRNA transcription of PRRSV.
Archives of Virology 02/2014; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that is causing massive economic loss in the Chinese duck industry. To obtain a live vaccine candidate against the disease, the DTMUV isolate FX2010 was passaged serially in chicken embryo fibroblasts (CEFs). Characterization of FX2010-180P revealed that it was unable to replicate efficiently in chicken embryonated eggs, nor intranasally infect mice or shelducks at high doses of 5.5 log10 tissue culture infectious doses (TCID50). FX2010-180P did not induce clinical symptoms, or pathological lesions in ducks at a dose of 5.5 log10 TCID50. The attenuation of FX2010-180P was due to 19 amino acid changes and 15 synonymous mutations. Importantly, FX2010-180P elicited good immune responses in ducks inoculated at low doses (3.5 log10 TCID50) and provided complete protection against challenge with a virulent strain. These results indicate that FX2010-180P is a promising candidate live vaccine for prevention of duck Tembusu viral disease.
Virology 01/2014; s 450–451:233–242. · 3.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vaccination is considered as the most effective preventive method to control influenza. The hallmark of influenza virus is the remarkable variability of its major surface glycoproteins, HA and NA, which allows the virus to evade existing anti-influenza immunity in the target population. So it is necessary to develop a novel vaccine to control animal influenza virus. Also we know that the ectodomain of influenza matrix protein 2 (M2e) is highly conserved in animal influenza A viruses, so a vaccine based on the M2e could avoid several drawbacks of the traditional vaccines. In this study we designed a novel tetra-branched multiple antigenic peptide (MAP) based vaccine, which was constructed by fusing four copies of M2e to one copy of foreign T helper (Th) cell epitope, and then investigated its immune responses.
Our results show that the M2e-MAP induced strong M2e-specific IgG antibody,which responses following 2 doses immunization in the presence of Freunds' adjuvant. M2e-MAP vaccination limited viral replication substantially. Also it could attenuate histopathological damage in the lungs of challenged mice and counteracted weight loss. M2e-MAP-based vaccine protected immunized mice against the lethal challenge with PR8 virus.
Based on these findings, M2e-MAP-based vaccine seemed to provide useful information for the research of M2e-based influenza vaccine. Also it show huge potential to study vaccines for other similarly viruses.
[Show abstract][Hide abstract] ABSTRACT: Goose parvovirus (GPV), Newcastle disease virus (NDV), goose herpesvirus (GHV) and goose adenovirus (GAV) are considered collectively to be four of the most important and widespread viruses of geese. Because all of these viruses cause similar pathological changes, histological differentiation among these viruses is difficult. A reliable, specific and sensitive multiplex PCR (mPCR) assay was developed for the combined detection of GPV, NDV, GHV and GAV in clinical samples of geese. Using the mPCR technique, single infections with GPV (28/76;36.8%), NDV (9/76;11.8%), GHV (3/76;3.9%) and GAV (12/76;15.8%) were identified in the samples; co-infections with GAV and either GPV or NDV (31.6%; 24/76) were also identified with this approach. The results for all of the samples tested were the same in both the uPCR and mPCR systems. The mPCR approach is considered to be useful for routine molecular diagnosis and epidemiological applications in geese.
Journal of virological methods 03/2013; · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 3' untranslated region (3' UTR), including the poly (A) tail, reportedly plays an important role in arterivirus replication, but the roles of the cis-acting elements present in the 3' UTR of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unknown. In the present study, PCR-based mutagenic analysis was conducted on the 3' UTR of PRRSV infectious full-length cDNA clone pAPRRS to investigate the structure and function of the conserved terminal nucleotides between the poly (A) tail and the 3' UTR region. Our findings indicated that the conservation of the primary sequence of the 3' terminal nucleotides, rather than the surrounding secondary structure, was vital for viral replication and infectivity. Four nucleotides (nt) (5'-(15517)AAUU(15520)-3') at the 3' proximal end of the 3' UTR and the dinucleotide 5'-AU-3' exerted an important regulatory effect on viral viability. Of the five 3'-terminal nucleotides of the 3' UTR (5'-(15503)AACCA(15507)-3'), at least three, including the last dinucleotide (5'-CA-3'), were essential for maintaining viral infectivity. Taken together, the 3'-terminal conserved sequence plays a critical role in PRRSV replication and may function as a contact site for specific assembly of the replication complex.
Archives of Virology 03/2013; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 5' untranslated region (UTR) is believed to be vital for the replication of porcine reproductive and respiratory syndrome virus (PRRSV), yet its functional mechanism remains largely unknown. In this study, to define the cis-acting elements for viral replication and infectivity, The 5' UTR swapping chimeric clones pTLV8 and pSHSP5 were constructed based on two different genotypes full-length infectious cDNA clone pAPRRS and pSHE backbones. Between them, vTLV8 could be rescued from pTLV8 and had similar virological properties to vAPRRS, including phenotypic characteristic and RNA synthesis level. However, pSHSP5 exhibited no evidence of infectivity. Taken together, the results presented here demonstrate that only the 5' UTR of type 1 PRRSV did not affect the infectivity and replication of type 2 PRRSV in vitro. The 5' UTR of type 2 PRRSV could be functionally replaced by its counterpart from type 1.
[Show abstract][Hide abstract] ABSTRACT: Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis in Asia and domestic pigs are its amplifying hosts. In the present study, the full genomic sequences of two JEV strains HEN0701 and SH0601 isolated from pigs in China were determined and compared with other 12 JEV strains deposited in GenBank. These two strains shared 88.8% of nucleotide sequence and 97.9% of deduced amino acid sequence. HEN0701 had high nucleotide sequence identity and high amino acid sequence identity with the GI strains, and SH0601 had high nucleotide sequence identity and high amino acid sequence identity with GIII strains at both the gene level and full genome. Further phylogenetic analysis showed that HEN0701 belonged to JEV genotype I (GI) and SH0601 to GIII. Analysis of codon usage showed there were a few differences in nucleotide composition and codon usage for open reading frames between GI strains and GIII strains.
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylinositol-3-kinase (PI3K)/Akt is an important cellular pathway that has been shown to participate in various replication steps of multiple viruses. In the present study, we compared the phosphorylation status of Akt during infection of MARC-145 cells and porcine alveolar macrophages (PAMs) with highly pathogenic PRRSV (HP-PRRSV) strain HuN4. We observed that biphasic activation of Akt was induced in at both the early stage (5, 15 and 30 min postinfection) and the late stage (12 and 24 h postinfection) of HP-PRRSV infection of MARC-145 cells, while an early-phase activation of Akt was found exclusively in virus-infected PAMs in vitro. Analysis with the PI3K-specific inhibitor LY294002 confirmed that PI3K acted as the upstream activator for the virus-induced activation of Akt. UV-irradiation-inactivated virus still induced the early event in PAMs but not in MARC-145 cells, suggesting that different mechanisms are employed for the early-stage induction of phosphorylated Akt within different cell cultures. We further demonstrated that FoxO1 and Bad, which serve as downstream targets of Akt, were phosphorylated in virus-infected MARC-145 cells. Moreover, the suppression of phosphorylated Akt with LY294002 significantly inhibited the virus-induced cytopathic effect (CPE) on MARC-145 cells, but it had a negligible effect on virus propagation. Collectively, our data provide new evidence of a novel role for the PI3K/Akt pathway in PRRSV infection of MARC-145 cells.
Archives of Virology 02/2013; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human guanylate-binding protein 1 (hGBP1) is an interferon-inducible protein involved in the host immune response against viral infection. In response to infection by influenza A virus (IAV), hGBP1 transcript and protein were significantly upregulated. Overexpression of hGBP1 inhibited IAV replication in a dose-dependent manner in vitro. The lysine residue at position 51 (K51) of hGBP1 was essential for inhibition of IAV replication. Mutation of K51 resulted in an hGBP1 that was unable to inhibit IAV replication. The viral nonstructural protein 1 (NS1) was found to interact directly with hGBP1. K51 of hGBP1 and a region between residues 123 and 144 in NS1 were demonstrated to be essential for the interaction between NS1 and hGBP1. Binding of NS1 to hGBP1 resulted in a significant reduction in both GTPase activity and the anti-IAV activity of hGBP1. These findings indicated that hGBP1 contributed to the host immune response against IAV replication and that hGBP1-mediated antiviral activity was antagonized by NS1 via binding to hGBP1.
PLoS ONE 01/2013; 8(2):e55920. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It has been shown that ORF5a protein in EAV is important but not essential for virus infectivity. In this study, we found that RNA changes in the overlapping region (1-104 nucleotide, nt) between ORF5 and ORF5a introduced by codon-optimized GP5 was lethal for virus viability, suggesting that the nt changes or amino acid (aa) mutations in the GP5 or ORF5a protein did not permit the production of infectious virus. Furthermore, inactivation of ORF5a expression in the context of type 1 (pSHE) and type 2 (pAJXM and pAPRRS) full-length PRRSV cDNA clones was lethal for the production of infectious virus, while viable PRRSV could be recovered by expressing ORF5a protein in trans, suggesting that ORF5a protein was essential for virus viability. Finally, ORF5a protein could be putatively extended to 63 aas by inactivation of the downstream stop codon candidates, thereby demonstrating that the C-terminus of ORF5a may be variable.
[Show abstract][Hide abstract] ABSTRACT: Astroviruses have been widely described in mammalian and avian species. Here, we report a complete genome sequence of a novel porcine astrovirus (PoAstV) isolated from a porcine fecal sample in China. The genome consists of 6,611 nucleotides, excluding the 3' poly(A) tail, and has two open reading frames (ORFs). ORF1 maps between nucleotide positions 19 and 4211 and encodes a 1,396-amino-acid (aa) polyprotein precursor consisting of nonstructural protein and putative RNA-dependent RNA polymerase, and ORF2 maps between nucleotide positions 4202 and 6531 and encodes a 775-aa polyprotein which is a capsid precursor protein. The genome sequence of the virus was distinct enough from those of the known PoAstVs to be considered a novel sequence. Phylogenetic analysis based on the predicted amino acid sequence of the complete capsid region showed that this strain may be a novel porcine astrovirus.
Journal of Virology 12/2012; 86(24):13820-1. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using mutation PCR, we cloned the target gene containing 421-480nt (141-160aa) and 598-639nt (200-213aa) of VP1 gene of foot and mouth disease virus (FMDV) into the deleted region (508-532aa) of Nsp2 gene of a highly pathogenic porcine reproductive and respiratory syndrome virus derived vaccine strain (HuN4-F112) that was used as vector. The recombinant cDNA was in vitro transcribed followed by transfection of BHK-21 cells for 36 h. Then, the supernatant of the cell culture was continuously seeded to monolayer of MARC-145 cells for recovery of the recombinant virus. CPE was obviously visible after a couple of passages in the seeded MARC-145, and the rescued virus (designated as rPRRSV-F112-O/VP1ep) was identified by Mlu I digestion, sequencing and immunofluorescence assay. Meanwhile, expression of inserted FMDV epitopes was also detected by indirect immunofluorescence assay with polyclonal antibodies against VP1 protein of FMDV. The analysis of biological characteristics shows that the titer of the rescued recombinant PRRSV (TCID50 = -log10(-6.75)/0.1 mL) was similar to its direct parental virus rHuN4-F112-delta508-532, but higher than rHuN4-F112.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 12/2012; 28(12):1431-40.
[Show abstract][Hide abstract] ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) is renowned for its genetic, antigenic, and pathogenic heterogeneity. As a consequence, highly pathogenic PRRSV (HP-PRRSV) has emerged and caused tremendous economic losses in the swine industry. In this study, a Chinese HP-PRRSV JX143 isolate was serially passaged in MARC-145 cells up to 100 times. We found that phenotypic changes involved with the cell adaptation process of PRRSV JX143 were characterized by higher titers, faster growth kinetics, and larger plaque sizes as the passage number increased compared with the parental virus. We found that the virulence of the JX143 strain in piglets was decreased greatly at passage 100 (JXM100). The attenuated strain JXM100 could protect piglets from lethal challenge by HP-PRRSV JX143. Genome-wide analysis showed that JXM100 contained a continuous 264 nucleotide (88 amino acids; 88 aa) deletion in the nsp2 region and 75 random nucleotide mutations throughout the genome. The nucleotide changes that arose during MARC-145 passaging of HP-PRRSV JX143 provide a potential molecular basis for the observed cell-adapted phenotype in MARC-145 cells and the attenuated phenotype in vivo. We also showed that pigs inoculated with JXM100 with an 88 aa deletion (del88) in nsp2 elicited a strong antibody response against the N protein but they did not develop antibody against the del88, whereas strong reactivity was observed in sera obtained from piglets infected with JX143 using the same del88-based ELISA. This suggests that del88 can be used as a genetic marker for serologically differentiating JXM100 from JX143.
[Show abstract][Hide abstract] ABSTRACT: Human parechoviruses (HPeVs) belonging to the family Picornaviridae are widely spread pathogens among young children. We report the complete genome sequence of a novel HPeV isolated from the stool sample of a hospitalized child with diarrhea in China. The genome consists of 7,305 nucleotides, excluding the 3' poly(A) tail, and has an open reading frame that maps between nucleotide positions 675 and 7217 and encodes a 2,180-amino-acid polyprotein. The genome sequence of the virus was sufficiently distinct from the 8 known HPeV types. Phylogenetic analysis based on the complete genome indicated that the HPeV strain represents a new genotype.
Journal of Virology 11/2012; 86(21):11945-6. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular adjuvants were used to augment the amplitude of the immune response in many studies recently. Ubiquitin (ub), the peptide binding truncated C-terminal portion of heat shock protein 70 (hsp70c) and interleukin-2 (IL-2) are widely investigated adjuvants which have been proved to be efficient. In our study, we compared the enhancing ability of these three adjuvants based on DNA vaccination using the porcine circovirus type 2 ORF2 (capsid) gene in mice. The results of lymphocyte proliferation assay, flow cytometric analysis (FCM), antibody titer and cytokine production showed that ub conjugated plasmid induced a stronger Th1 type cellular immune response and an observably higher level of Cap-specific serum immunoglobulin G antibody compared with hsp70c or IL-2 conjugated plasmids during the period of post-immunization. Meanwhile, the ub conjugation vaccinated group elicited stronger speciﬁc immunity against PCV2 challenge than the others during most of the time of post-challenge. Thus, these data indicate that ub is a superior adjuvant for a PCV2 DNA vaccination than the hsp70c and IL-2 molecules.
[Show abstract][Hide abstract] ABSTRACT: SYBR Green coupled to melting curve analysis has been suggested to detect RNA viruses showing high genomic variability. Here, a SYBR Green-based real-time RT-PCR assay was developed for simultaneous detection and differentiation of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and classical type 2 PRRSV (C-PRRSV). The different strains were identified by their distinctive melting temperatures: 82.98 ± 0.25 °C and 85.95 ± 0.24 °C for HP-PRRSVs or 82.74 ± 0.26 °C for C-PRRSVs. Specificity was tested using nine other viral and bacterial pathogens of swine. The detection limit was 1 TCID(50) for HP- or C-PRRSV. Furthermore, the detection results for samples from an animal trial with HP- or C-PRRSV infections showed that the SYBR Green-based real-time RT-PCR was more sensitive than the conventional RT-PCR. Additionally, an analysis of 319 field samples from North China, Central China and Northeast China showed that HP- and C-PRRSVs co-circulated in pig herds. Thus, the SYBR Green-based real-time RT-PCR, which can be performed within one hour, is a rapid, sensitive and low-cost diagnostic tool for rapid differential detection and routine surveillance of HP- and classical type 2 PRRSVs in China.
Archives of Virology 10/2012; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is the most abundant viral structural protein with high immunogenicity. Previously, the nonessential sequences for virus infectivity were identified at both N and C terminal ends of N protein. Here, by means of reverse genetics, a marker virus (v7APMa) was generated with a mutant N protein that differs from the wild-type strains (vAPRRS, type 2 PRRSV). v7APMa shows stable inheritance in cell culture and the virologic characteristics of the marker virus in vitro showed that v7APMa replicates well as its parental strain. In the pig model, the v7APMa marker virus induced a similar level of anti-N protein antibodies and robust antibodies against the marker peptide, from 14 days post infection. In addition, a peptide-based ELISA was developed to detect the specific antibodies for the introduced 7APMa peptide. This approach, using a rationally designed marker virus, provides a new potential mutant basis for further development of PRRSV novel vaccines.