Guangqing Liu

Publications (10)31.35 Total impact

• Article: High yield expression of duck hepatitis A virus VP1 protein in Escherichia coli, and production and characterization of polyclonal antibody.

  Chuanfeng Li, Zongyan Chen, Chunchun Meng, Lu Li, Guangqing Liu
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  ABSTRACT: VP1 protein, the capsid protein of duck hepatitis A virus (DHAV), contains critical epitopes for inducing a protective immune response. Due to its low-level expression in Escherichia coli (E. coli), the function of this protein is poorly characterized. In this study, a codon-optimized VP1 gene was chemically synthesized in terms of the codon usage bias in E. coli and subcloned into pET32a (+) to increase its expression. The recombinant VP1 fusion protein was purified from inclusion body by Ni(2+) affinity chromatography His-Bind Resin and used to raise the rabbit anti-DHAV-VP1 polyclonal antibody. The expression of the codon-optimized VP1 gene in E. coli was significantly increased when compared to the wild-type VP1 gene, having an at least 17-fold increase. Western blot analysis showed that the recombinant protein was recognized by the rabbit anti-DHAV polyclonal antibody. Western blot also demonstrated that the rabbit anti-DHAV-VP1 polyclonal antibody could recognize the purified VP1 fusion protein specifically, and in the indirect immunofluorescent assays (IFA), the antibody was able to probe the VP1 protein in DHAV-1 infected cells. In conclusion, codon optimization increased dramatically DHAV VP1 expression in E. coli and the His-tagged VP1 fusion protein showed good antigenicity and immunogenicity.
  Journal of virological methods 04/2013; · 2.13 Impact Factor
• Article: Rapid Diagnosis of Goose Viral Infections by Multiplex PCR.

  Zongyan Chen, Chuanfeng Li, Guoxin Li, Hai Yu, Yifeng Jiang, Liping Yan, Chunchun Meng, Yanjun Zhou, Guangzhi Tong, Guangqing Liu
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  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
• Article: Protective immune responses in rabbits induced by a suicidal DNA vaccine of the VP60 gene of rabbit hemorrhagic disease virus.

  Yingjie Cheng, Zongyan Chen, Chuanfeng Li, Chun Meng, Run Wu, Guangqing Liu
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  ABSTRACT: A suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon was evaluated for the development of a vaccine against rabbit hemorrhagic disease virus (RHDV). The VP60 gene of RHDV was cloned and inserted into pSCA1, an SFV DNA-based replicon vector. The resultant plasmid, pSCA/VP60, was transfected into BHK-21 cells, and the antigenicity of the expressed protein was confirmed using indirect immunofluorescence and a western blot assay. In addition, immunogenicity was studied in rabbits. Fifteen rabbits were injected intramuscularly twice with pSCA/VP60 at 2-week intervals. They were challenged with an RHDV isolate 2 weeks after the second immunization. In all cases, anti-RHDV antibodies were detected by ELISA. Additionally, the lymphocyte proliferation response was tested by the 3-(4, 5-dimethyl-2- thiazolyl) -2, 5-diphenyl-2H-tetrazolium bromide method, and neutralizing antibodies were measured by microneutralization tests. Our results showed that RHDV-specific antibodies and an RHDV-specific cell-mediated immune response were strongly induced in rabbits. Furthermore, all of the rabbits were protected against challenge with wild type RHDV. In conclusion, we demonstrated that the suicidal DNA vaccine is a promising vaccine candidate that facilitates the prevention of rabbit hemorrhagic disease caused by RHDV.
  Antiviral research 01/2013; · 3.61 Impact Factor
• Article: Construction and applications of rabbit hemorrhagic disease virus replicon.

  Binbin Wang, Mingjia Zhe, Zongyan Chen, Chuanfeng Li, Chunchun Meng, Miaotao Zhang, Guangqing Liu
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  ABSTRACT: The study of rabbit hemorrhagic disease virus (RHDV) has long been hindered by the absence of an in vitro culture system. In this study, using RHDV as a model, a series of DNA-based reporter replicons were constructed in which the firefly luciferase (Fluc) gene was fused in-frame with the open reading frame of the replicon. In this construct, the Fluc gene was inserted where the coding region of viral structural protein was deleted and was under the control of a minimal cytomegalovirus (CMV) immediate-early promoter. Fluc activity analysis showed that these reporter replicons replicate efficiently in mammalian cells. On the basis of the replicon, 5'non-coding regions (5'NCR) and genome-linked protein (VPg) were deleted, and the effect on the expression of replicon was analyzed. The results showed that the expression level of Fluc was reduced in the absence of 5'NCR and VPg, suggesting that the 5'NCR and VPg may play an important role in replication and/or translation of RHDV. To further verify the speculation, we also constructed a replication deficient mutant (pRHDV-luc/Δ3D), and the impact of 5'NCR and VPg deletion on viral translation efficiency was analyzed, our results indicated that both VPg and 5'NCR were involved in RHDV translation.
  PLoS ONE 01/2013; 8(5):e60316. · 4.09 Impact Factor
• Article: Immunogenicity of virus-like particles containing modified goose parvovirus VP2 protein.

  Zongyan Chen, Chuanfeng Li, Yingqi Zhu, Binbin Wang, Chunchun Meng, Guangqing Liu
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  ABSTRACT: The major capsid protein VP2 of goose parvovirus (GPV) expressed using a baculovirus expression system (BES) assembles into virus-like particles (VLPs). To optimize VP2 gene expression in Sf9 cells, we converted wild-type VP2 (VP2) codons into codons that are more common in insect genes. This change greatly increased VP2 protein production in Sf9 cells. The protein generated from the codon-optimized VP2 (optVP2) was detected by immunoblotting and an indirect immunofluorescence assay (IFA). Transmission electron microscopy analysis revealed the formation of VLPs. These findings indicate that optVP2 yielded stable and high-quality VLPs. Immunogenicity assays revealed that the VLPs are highly immunogenic, elicit a high level of neutralizing antibodies and provide protection against lethal challenge. The antibody levels appeared to be directly related to the number of GP-Ag-positive hepatocytes. The variation trends for GP-Ag-positive hepatocytes were similar in the vaccine groups. In comparison with the control group, the optVP2 VLPs groups exhibited obviously better responses. These data indicate that the VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Thus, GPV optVP2 appears to be a good candidate for the vaccination of goslings.
  Virus Research 08/2012; 169(1):306-9. · 2.94 Impact Factor
• Article: Protective immune responses in ducklings induced by a suicidal DNA vaccine of the VP1 gene of duck hepatitis virus type 1.

  Yuzhi Fu, Zongyan Chen, Chuanfeng Li, Guangqing Liu
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  ABSTRACT: A suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon was evaluated for the development of a vaccine against duck hepatitis virus type 1 (DHV-1). The VP1 gene of DHV-1 was cloned and inserted into pSCA1, an SFV DNA-based replicon vector. The resultant plasmid, pSCA/VP1, was transfected into BHK-21 cells and the antigenicity of the expressed protein was confirmed using an indirect immunofluorescence and western blot assay. Immunogenicity was studied in ducklings. Ducklings were injected intramuscularly two times with pSCA/VP1 at 14 days intervals. Anti-DHV-1 antibodies were detected by ELISA, the lymphocyte proliferation response was also tested by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide method and neutralizing antibodies were measured by microneutralization tests. Our results showed that DHV-1-specific antibodies, neutralizing antibodies and lymphocyte proliferation were well induced in ducklings. Furthermore, all the ducklings were protected against challenge with wild DHV-1. In conclusion, we demonstrate that the suicidal DNA vaccine is a promising vaccine candidate facilitating the prevention of duck hepatitis caused by DHV-1.
  Veterinary Microbiology 07/2012; · 3.33 Impact Factor
• Source

  Available from: PubMed Central

  Article: Outbreak-associated novel duck Reovirus, China, 2011.

  Zongyan Chen, Yinqi Zhu, Chuanfeng Li, Guangqing Liu
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  ABSTRACT: TO THE EDITOR: In 2011, an unidentified disease in Pekin ducks (Anas platyrhynchos) was reported in People's Republic of China. The infection caused death in 40% of ducks of various age and 35%-40% mortality in different flocks. Clinical signs included unstable gait, weakness in legs, and diarrhea. At necropsy, large necrotic foci were observed in the spleens. All classical endemic and emerging viruses, such as duck enteritis virus, duck hepatitis virus, duck flavivirus, duck parvovirus, and avian influenza virus, could be excluded as the causative agent by PCR and serologic methods. To identify the cause of the disease, we tested tissue from affected ducks and subsequently isolated a novel duck-pathogenic orthoreovirus from the livers of affected ducks.
  Emerging Infectious Diseases 07/2012; 18(7):1209-11. · 6.79 Impact Factor
• Article: Complete genome comparison of duck hepatitis virus type 1 parental and attenuated strains.

  Yong Wang, Chuanfeng Li, Zongyan Chen, Binrui Xu, Gang Li, Guangqing Liu
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  ABSTRACT: Two complete duck hepatitis virus type 1 (DHV-1) genomes, strain SY5 and its chicken embryos passage descendent vaccine strain ZJ-A, were compared and analyzed in order to identify possible sites of attenuation. Of the 205 nucleotide changes, 22 resulted in sense mutations, 174 produced nonsense mutations. Besides, there are 7 consistent nucleotides substitutions in 5'UTR and 2 in 3'UTR. Three of these 22 sense mutations resided in VP0, 6 exists in VP1, one exists in VP3, 3 exists in 2A2, 3 exists in 2C, one was detected in 3B and 5 was in 3D. These results suggested that VP0, VP1, 3D, and 5'/3'UTR may contribute to the attenuation of DHV-1 in chicken/duck/embryos. The results provide a genetic basis for future manipulation of a DHV-1 infectious clone.
  Virus Genes 06/2012; 45(2):398-401. · 1.85 Impact Factor
• Article: Establishment of a duck cell line susceptible to duck hepatitis virus type 1.

  Yuzhi Fu, Zongyan Chen, Chuanfeng Li, Guangqing Liu
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  ABSTRACT: Until recently, there was no cell line that could produce continuously high-titer duck hepatitis virus type 1 (DHV-1). In this study, a duck embryo fibroblast (DEF) cell line was established, and the susceptibility of this cell line to DHV-1 was determined. The primary culture of DEF cells was from a duck embryo that was partially digested with trypsin. Digested tissue pieces were cultured at 37°C in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. The cultured DEF cells, which had the morphology of fibroblast, proliferated to 100% confluence four days later. An immortalized DEF cell line, named DEF-TA, was established and subcultured to passage 33, and the susceptibility of that cell line to DHV-1 was determined. In the DHV-1 susceptibility tests, cytopathic effects and the propagation of virus were observed in DEF-TA cells after DHV-1 infection. This continuous DHV-1-susceptible DEF cell line may serve as a valuable cell line for studies of cell-virus interactions and the pathogenesis of DHV-1 and may be useful for the development of an inactivated vaccine.
  Journal of virological methods 05/2012; 184(1-2):41-5. · 2.13 Impact Factor
• Source

  Available from: PubMed Central

  Article: The duck hepatitis virus 5'-UTR possesses HCV-like IRES activity that is independent of eIF4F complex and modulated by downstream coding sequences.

  Guangqing Liu, Emilio Yángüez, Zongyan Chen, Chuanfeng Li
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  ABSTRACT: Duck hepatitis virus (DHV-1) is a worldwide distributed picornavirus that causes acute and fatal disease in young ducklings. Recently, the complete genome of DHV-1 has been determined and comparative sequence analysis has shown that possesses the typical picornavirus organization but exhibits several unique features. For the first time, we provide evidence that the 626-nucleotide-long 5'-UTR of the DHV-1 genome contains an internal ribosome entry site (IRES) element that functions efficiently both in vitro and in mammalian cells. The prediction of the secondary structure of the DHV-1 IRES shows significant similarity to the hepatitis C virus (HCV) IRES. Moreover, similarly to HCV IRES, DHV-1 IRES can direct translation initiation in the absence of a functional eIF4F complex. We also demonstrate that the activity of the DHV-1 IRES is modulated by a viral coding sequence located downstream of the DHV-1 5'-UTR, which enhances DHV-1 IRES activity both in vitro and in vivo. Furthermore, mutational analysis of the predicted pseudo-knot structures at the 3'-end of the putative DHV-1 IRES supported the presence of conserved domains II and III and, as it has been previously described for other picornaviruses, these structures are essential for keeping the normal internal initiation of translation of DHV-1.
  Virology Journal 03/2011; 8:147. · 2.34 Impact Factor