Transient expression is the major method to express foot-and-mouth disease virus (FMDV) capsid proteins in mammalian cells. To achieve stable expression of FMDV capsid proteins and efficient assembly of virus like particles (VLPs) in cells, the plasmids of piggyBac (PB) transposon-constitutive expression and PB transposon-tetracycline (Tet) inducible expression vectors were constructed. The function of the plasmids was tested by fluorescent proteins. By adding antibiotics, the constitutive cell pools (C-WT, C-L127P) expressing P12A3C (WT/L127P) genes and the inducible cell pools (I-WT, I-L127P) expressing P12A3C (WT/L127P) genes were generated. The genes of green fluorescent protein, 3C protease and reverse tetracycline transactivator (rtTA) were integrated into chromosome, which was confirmed by fluorescence observation and PCR testing. The cell pool I-L127P has a stronger production capacity of capsid proteins and VLPs, which was confirmed by Western blotting and enzyme linked immunosorbent assay (ELISA), respectively. In conclusion, inducing the chromosomal expression of FMDV capsid proteins was firstly reported, which may facilitate the technical process of mammalian production of FMDV VLPs vaccine and the construction of mammalian inducible expression systems for other proteins.
Animals ; Foot-and-Mouth Disease Virus/genetics* ; Capsid Proteins ; Viral Proteins/metabolism* ; Foot-and-Mouth Disease/prevention & control* ; Tetracyclines/metabolism* ; Viral Vaccines ; Antibodies, Viral ; Mammals/metabolism*

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMax^TM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×10^9.1 TCID₅₀/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.
Adenoviridae/genetics* ; Adenoviruses, Human/genetics* ; Animals ; Antibodies, Viral ; Capsid/metabolism* ; Capsid Proteins ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus/genetics* ; HEK293 Cells ; Humans ; Mice ; Recombinant Proteins/genetics* ; Serogroup ; Swine ; Viral Proteins ; Viral Vaccines/genetics*

OBJECTIVE: To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody. METHODS: The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay. RESULTS: The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10. CONCLUSION We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.
Animals ; Antibodies ; Capsid ; Capsid Proteins/genetics* ; Dependovirus/genetics* ; Prokaryotic Cells ; Rabbits ; Recombinant Proteins/genetics*

The stability of virus-like particles (VLPs) is currently the main factor affecting the quality of foot-and-mouth disease VLPs vaccines. In order to further improve the quality of the VLPs vaccine of foot-and-mouth disease (FMD), three amino acid modification sites were designed and screened through kinetic analysis software, based on the three-dimensional structure of FMDV. The three mutant recombinant plasmids were successfully prepared by the point mutation kit, transformed into Escherichia coli strain BL21 and expressed in vitro. After purification by Ni ion chromatography column, SDS-PAGE proved that the three amino acid mutations did not affect the expression of the target protein. The results of the stability study of three FMD mutant VLPs obtained by in vitro assembly show that the introduction of internal hydrophobic side chain amino acids made the morphology of VLPs more uniform (N4017W), and their stability was significantly improved compared to the other two VLPs. The internal hydrophobic force of the capsid contributes to the formation of VLPs and helps to maintain the stability of the capsid, providing new experimental ideas for improving the quality of VLPs vaccines, and helping to promote the development of VLPs vaccines.
Amino Acids ; Animals ; Capsid Proteins/genetics* ; Foot-and-Mouth Disease/prevention & control* ; Foot-and-Mouth Disease Virus/genetics* ; Kinetics ; Vaccines, Virus-Like Particle/genetics* ; Viral Vaccines/genetics*

Objective: To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases. Methods: A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID Results: The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID Conclusion This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.
Capsid Proteins/genetics* ; Enterovirus A, Human/genetics* ; Enterovirus B, Human/genetics* ; Enterovirus C, Human/genetics* ; Enterovirus D, Human/genetics* ; Humans ; Molecular Epidemiology/methods* ; Molecular Typing/methods* ; Reverse Transcriptase Polymerase Chain Reaction/methods*

Progressive multifocal leukoencephalopathy (PML) is a rare and lethal central nervous demyelinating disease caused by JC polyomavirus (JCV), particularly in patients with impaired immune system. The variation of JCV plays an important role in the pathogenesis of PML, including the recombination of non-coding regulatory region (NCCR), which is closely related to binding sites of transcription factors and affect the level of gene transcription. Nucleotide mutations in VP1 region determine the antigenicity and receptor specificity of JCV, play an important role in cell adsorption, immune-mediation and pathogenicity. In addition, immune cells are also involved in the pathogenesis of PML. T lymphocytes can recognize virus antigens, clear JCV, which are directly related to the prognosis of PML. B lymphocytes can serve as latent sites of JCV, and participate in viral transmission, replication, and coordination of the expression of transcription factors. This paper summarizes the roles of JCV variation and immune cells in pathogenesis of PML.
B-Lymphocytes ; immunology ; virology ; Capsid Proteins ; genetics ; immunology ; Humans ; JC Virus ; immunology ; Leukoencephalopathy, Progressive Multifocal ; pathology ; virology ; Mutation ; T-Lymphocytes ; immunology ; virology

Objective: To analyze the genetic characterization of norovirus isolated in an outbreak of gastroenteritis in Jiangsu province. Methods: Extracted viral RNA from the swab samples of cases of acute gastroenteritis outbreak in Jiangsu province on December 16-27, 2016 was reversely transcribed to cDNA, and partial RNA-dependent RNA polymerase sequence and complete capsid sequence (VP1) were amplified by RT-PCR. Amplification products were sequenced for the analysis of genetic characteristics. Results: Based on sequence alignment, the variant shared a high level of identity with the strain GⅡ.g isolated in Spain and Finland (98.7%) in the RNA-dependent RNA polymerase region, and with the strain GⅡ.1 isolated in American (99.4%) in the VP1. The recombination was determined by using software Simplot, and the breakpoint of recombination was located in the ORF1/2 overlap region at position 5 106 of VP1. The result of amino acids alignment in capsid region showed that there were no mutations in the amino acids of the predicted epitopes and receptor binding site Ⅰ-Ⅲ, but a unique amino acid change was detected at position 132 (N-S). Conclusion: The norovirus isolated in the outbreak of gastroenteritis in Jiangsu province was a rare recombinant norovirus variant GⅡ.g-GⅡ.1.
Caliciviridae Infections/epidemiology* ; Capsid Proteins ; Disease Outbreaks ; Gastroenteritis/epidemiology* ; Genotype ; Humans ; Norovirus/isolation & purification* ; Phylogeny ; RNA, Viral/genetics* ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo understand the molecular characteristics of a dengue virus outbreak in China-Myanmar border region, Yunnan province, 2015 and provide etiological evidence for the disease control and prevention.

METHODSSemi-nested RTPCR was conducted to detect the capsid premembrane (CprM) gene of RNA of dengue virus by using dengue virus NS1 positive serum samples collected in Mengdin township, Gengma county, Yunnan province in July, 2015. Some positive samples were then detected by using PCR with specific primers to amplify the full E gene. The positive PCR products were directly sequenced. Then sequences generated in this study were BLAST in NCBI website and aligned in Megalign in DNAstar program. Multiple sequence alignments were carried out by using Mega 5.05 software based on the sequences generated in this study and sequences downloaded from GenBank, including the representative strains from different countries and regions. Phylogenetic trees were constructed by using Neighbor-Joining tree methods with Mega 5.05 software.

RESULTSTwenty one of 25 local cases and 10 of 14 imported cases from Myanmar were positive for DENV-1. Eight serum samples were negative for dengue virus. A total of 13 strains with E gene (1485 bp), including 8 local strains and 5 imported strains, were sequenced, which shared 100% nucleotide sequence identities. Twelve strains with CprM gene (406 bp) from 9 local cases and 3 imported cases shared 100% nucleotide sequence identities. Phylogenetic analyses based on E gene showed that the new 13 strains clustered in genotype I of dengue virus and formed a distinct lineage.

CONCLUSIONSThis outbreak was caused by genotype I of DENV-1, which had the closest phylogenetic relationships with dengue virus from neighboring Burma area. Comprehensive measures of prevention and control of dengue fever should be strengthened to prevent the spread of dengue virus.

Capsid Proteins ; China ; epidemiology ; DNA Primers ; Databases, Nucleic Acid ; Dengue ; epidemiology ; virology ; Dengue Virus ; genetics ; Disease Outbreaks ; Genotype ; Humans ; Myanmar ; epidemiology ; Phylogeny ; Polymerase Chain Reaction ; Sequence Alignment ; Software

This study bioinformatically analyzed the non-VP1 capsid proteins (VP2-VP4) of Coxasckievirus A6 (CVA6), with an attempt to predict their basic physicochemical properties, structural/functional features and linear B cell eiptopes. The online tools SubLoc, TargetP and the others from ExPASy Bioinformatics Resource Portal, and SWISS-MODEL (an online protein structure modeling server), were utilized to analyze the amino acid (AA) sequences of VP2-VP4 proteins of CVA6. Our results showed that the VP proteins of CVA6 were all of hydrophilic nature, contained phosphorylation and glycosylation sites and harbored no signal peptide sequences and acetylation sites. Except VP3, the other proteins did not have transmembrane helix structure and nuclear localization signal sequences. Random coils were the major conformation of the secondary structure of the capsid proteins. Analysis of the linear B cell epitopes by employing Bepipred showed that the average antigenic indices (AI) of individual VP proteins were all greater than 0 and the average AI of VP4 was substantially higher than that of VP2 and VP3. The VP proteins all contained a number of potential B cell epitopes and some eiptopes were located at the internal side of the viral capsid or were buried. We successfully predicted the fundamental physicochemical properties, structural/functional features and the linear B cell eiptopes and found that different VP proteins share some common features and each has its unique attributes. These findings will help us understand the pathogenicity of CVA6 and develop related vaccines and immunodiagnostic reagents.
Amino Acid Sequence ; Capsid Proteins ; genetics ; immunology ; Computational Biology ; Enterovirus ; genetics ; pathogenicity ; Epitopes, B-Lymphocyte ; genetics ; immunology ; Humans

In order to develop a combined live vaccine that will be used to prevent against porcine parvovirus (PPV) and Pseudorabies virus (PRV) infection, the VP2 gene of PPV was inserted into the transfer vector plasmid pG to produce the recombinant plasmid pGVP2. The plasmid pGVP2 and the genome of PRV HB98 attenuated vaccine were transfected by using lipofectamine into swine testis cells for the homologous recombination. The recombinant virus rPRV-VP2 was purified by selection of green fluorescence plaques for five cycles. 6-week-old female Kunming mice were immunized intramuscularly with attenuated PRV parent HB98 strain, commercial inactivated vaccine against PPV, recombinant virus, DMEM culture solution. The injections were repeated with an equivalent dose after 2 weeks in all of the groups, and then challenged with the virulent PRV NY strain at 7 weeks after the first immunization. The recombinant virus rPRV-VP2 was successfully generated, and the recombinant virus could effectively elicite anti-PPV and PRV antibody and significant cellular immune response as indicated by anti-PPV ELISA and HI, PRV-neutralizing assay and flow cytometry. The challenge assay indicated that recombinant virus could protect the mice against the virulent PRV challenge. These results demonstrated that the recombinant virus can be a candidate recombinant vaccine strain for the prevention of PRV and PPV.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; administration & dosage ; genetics ; immunology ; Capsid Proteins ; administration & dosage ; genetics ; immunology ; Female ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Herpesvirus 1, Suid ; genetics ; metabolism ; Mice ; Parvovirus, Porcine ; genetics ; immunology ; Swine ; Swine Diseases ; immunology ; prevention & control ; virology ; Viral Vaccines ; administration & dosage ; genetics ; immunology