Bing du xue bao = Chinese journal of virology最新文献

The human metapneumovirus (HMPV) is an important pathogen in respiratory-tract infections in children. We undertook genomic sequence analyses and described the genetic characteristics of an uncommon sub-genotype, the HMPV A1 strain, and provide useful data for further studies. The HMPV A1(BJ-1610)strain was identified from a nasopharyngeal aspirate collected from a 3-month-old female with bronchopneumonia. Gene fragments of BJ-1610 were amplified by reverse transcription-polymerase chain reaction(RT-PCR)and assembled by DNAStar software. Sequence alignment for BJ-1610 and other HMPV reference strains with four known genotypes available in the GenBank database was conducted by DNAStar. Phylogenetic trees were created using MEGA 6.06 software. The whole genome of BJ-1610 was 13406nt in length (GenBank accession number:KU821121).Compared with HMPV reference strains,BJ-1610 shared the highest similarities with HMPV/AUS/150229278/2003/A(KC562226)from Australia, which was classified into sub-genotype A1.The nucleotide identity of the full genome between BJ-1610 and KC562226was 98.4%.N,P,F,M2-2and L genes had great similarity with KC562226 compared with other reference strains, whereas SH and G genes shared higher similarities with other strains of sub-genotype A1.Phylogenetic analyses of the whole genome showed that BJ-1610 was clustered into sub-genotype A1 and was close to KC562226.The N,P,M,F,M2-1,M2-2and L genes of BJ-1610 showed the same genetic features as the whole genome, whereas the variable genes SH and G were closest to KC403980.The F protein of BJ-1610 showed high genetic conservation. The length of the SH protein of BJ-1610 changed from 552 bp to 567 bp due to mutations in the stop codon. The amino-acid mutations on protein G led to a decrease in the number of N-glycosylation sites. As an infrequently circulating genotype, sequence analyses of the whole genome of a HMPV A1strain(BJ-1610)will promote further studies on its epidemiology and pathogenicity, and aid the development of vaccines and antiviral drugs.

Broadly neutralizing antibodies (bNAbs) have demonstrated a protective role from experimental challenge in non-human primates and humanized mouse models. Recently, bNAbs 3BNC117 and VRC01were assessed in a phase-I clinical trial, and were shown to lower plasma viremia in human immunodeficiency virus(HIV)-1-infected individuals not receiving antiretroviral therapy. However, induction of these types of antibodies by vaccination iS extremely difficult. Moreover, the 31% protection observed in the RV144 vaccine trial in the absence of detectable bNAbs in blood samples suggested the important role of additional inhibitory functions of the antibodies that control infection and replication of HIV. Increasing evidence suggests that immunoglobulin-G Fcγ receptor-mediated inhibition of antibodies present at the mucosal site may have a protective role against mucosal transmission of HIV. Dendritic cells and macrophages express such Fc receptors on their surface, and may have a decisive role in early mucosal transmission because they have been proposed to be the first HIV target at the mucosal site. Therefore, new vaccination strategies involving induction of such non-neutralizing inhibitory antibodies and other antiviral functions, in addition to bNAbs, should be developed.

To investigate the molecular epidemiological characterization of coxsackieivirus B1(CV-B1),we performed VP1 sequencing on all isolates from acute flaccid paralysis (AFP) surveillance, environmental surveillance and viral meningitis specimens from 1994 to 2015.A total of 53CV-B1 strains were isolated, among which 41 strains,4strains,and 8strains were obtained from AFP surveillance, environmental surveillance and meningitis specimens, respectively. Phylogenetic analysis based on VP1 entire coding region revealed that Shandong CV-B1 strains were segregated into a major cluster alongside with other domestic strains, with no foreign strains existing in this cluster. Foreign strains composed two other exclusive branches.Shandong strains had VP1 nucleotide similarities of 84.4%to 100.0% with each other, and 77.9%to 85.0% with foreign strains. The results presented here demonstrate Chinese CV-B1 strains have great genetic divergence with foreign strains. EV associated surveillance should be reinforced to monitor possible importation of different EV transmission lineages.

To explore the porcine epidemic diarrhea virus(PEDV)epidemiology in domestic pigs, virus isolation and identification were carried out in pig herds in Shanghai and Jiangsu province. Based on sequences of PEDV strains in GenBank, a pair of primers targeting the M gene were designed for PEDV detection. Two clinical pig diarrhea samples were amplified and sequencing results confirmed that the fragments amplified were M gene. PEDV-positive samples were inoculated in Vero cells with different concentrations of trypsin. Our results showed that these two isolates could proliferate effectively in Vero cells with a specific concentration of trypsin. Twenty-second passages viruses were added to the feeding of newborn piglets, and animal regression test showed obvious clinical symptoms of diarrhea, confirming the the PEDV isolates, named as JSLS/PEDV/1/2014 and JS/PEDV/2/2014,were successfully obtained. Phylogenetic analysis of the M genes showed that JSLS/PEDV/1/2014 and JS/PEDV/2/2014 displayed the greatest similarity with the Chinese strains HLJ-2012 and BJ-2012-1,respectively.The S genes of the two isolates were classified in group I. There were 51 bases deletion in the ORF3 genes of these two isolates which were located in the same big branch with the DR13 strain rather than the CV777 strain.

The ultimate goal of chronic hepatitis B virus(HBV)therapy is full eradication of the virus from the liver. However, this is rarely achieved with the clinically available first-line agents (entecavir and tenofovir disoproxil fumarate) due to the inability to eliminate covalently closed circular DNA(cccDNA), which persists in the nucleus of infected hepatocyte cells,and failure of the host to induce an adequate specific immune response to control the infection. Currently, the clinical treatment for chronic HBV infection mainly includes nucleos(t)ide analogues (NAs), non-NAs and immune modulatory agents; however, each agent has individual advantages and drawbacks. It is, therefore, extremely urgent to identify novel targets involved in viral replication and develop novel anti-HBV drugs. In light of the breakthroughs in cccDNA research and host immune treatments, this review aims to summarize the state of the recent HBV drug research and development to highlight future therapeutic strategies to target the virus and host immune response.

The human immunodeficiency virus type 1(HIV-1)has spread globally and often exhibits antiviral resistance. Therefore, there is an ongoing need for the development of novel, highly efficient antiretroviral drugs with low toxicity. The capsid protein(CA),which is composed of an N-terminal domain(NTD)and C-terminal domain(CTD),plays an important role in the process of HIV-1assembly and maturation. In recent years, the structure of capsid protein has been solved. In this article, we summarizes the spatial structure of the HIV-1capsid protein determined by X-ray crystallography, and describe the structural characteristics of the NTD-NTD,NTD-CTD and CTD-CTD interfaces. This article summarizes the antiviral approaches targeting CA and expounds a new strategy in combination with CRISPR/Cas9 gene editing technology.

The purpose of this study was to develop an effective control to be applied in hepatitis E virus(HEV)nucleic acid detection.Construction of an MS2/HEV gene was performed based on an "Armored RNA technology" protocol. The gene included a partial MS2 phage genome including the 5’UTR,the maturation protein, capsid protein and initiation site of the replicase and a partially conserved sequence derived from the HEV ORF2.The target genes were synthesized and amplified by PCR, and the purified target gene products subcloned into the pET-28 b prokaryotic expression vector to obtain the pET-28b-MS2/HEV recombinant plasmid. SDS-PAGE was used for expression analysis in E. coli BL21(DE3)cells harboring the pET-28b-MS2/HEV plasmid. Centrifugal ultrafiltration was adopted for the purification and concentration of recombinant HEV RNA-loaded MS2 Bacteriophage Capsid (rHEPC). The morphological identification of the particles was subsequently performed by scanning electron microscopy. Stability of the rHEPC particles were evaluated by challenging with different concentrations of DNase I and RNase A, and also evaluated for long-term storage based on RT-PCR verification. SDS-PAGE results showed that the target MS2/HEV gene could express efficiently in recombinant E. coli BL21(DE3)and RT-PCR results revealed that the designed HEV conserved gene sequence was successfully packaged into MS2phage-like or rHEPC particles. Stability evaluation showed that the prepared rHEPC particles exhibited strong resistance to degradation by DNase I and RNase A and long-lasting protection of coated HEV RNA for at least seven months when stored at-20℃.The prepared rHEPC particles in the present study meet the basic requirements to be used as a quality control material for routine HEV nucleic acid detection.

In order to identify immunodominant linear B cell epitopes in the nucleocapsid protein N of severe fever with thrombocytopenia syndrome virus(SFTSV),bioinformatics programs were used to analyze antigenicity, hydrophilicity and surface probability of the amino acid sequence and predict possible linear B cell epitopes. PyMOL software was used to analyze the distribution of linear B cell epitopes in nucleocapsid protein N based on its crystal structure. Corresponding peptides were synthesized and examined in peptide enzyme-linked immunosorbent assay(Peptide-ELISA)individually to check whether they reacted with sera from SFTSV-infected patients. As a result, a total of six potential linear B cell epitopes were predicted as the following: A(40-KKLKETGGDDWVKDTK-55), B(71-ASGKMSNSGSKRL-83), C(94-ERAETRL-100),D(135-LKVENYPP-142),E(157-GVSEATT-163)and F(184-KMRGASKTEVYNSFRDP-200).All epitopes were located on the surface of the nucleocapsid protein N and contained flexible loops. Each of the six synthetic peptides reacted positively with sera from SFTSV-infected patients and were identified as immunodominant linear B cell epitopes. Linear regression analysis showed a positive correlation between each peptide-ELISA and commercialized N protein-based EIA. In this study, immunodominant linear B cell epitopes from the nucleocapsid protein N of SFTSV were successfully predicted and confirmed. These findings may help to establish the molecule basis of specific antigenicity and disease diagnosis.

In 2016,routine influenza virus surveillance was conducted in the live poultry markets of Wuhan, Hubei Province. An H5N2 subtype avian influenza virus(AIV)was isolated from ducks in Wuhan. The entire genome of this virus isolate was sequenced,and molecular phylogenetic analysis performed. The results indicated that the HA gene belonged to clade 2.3.4.4and contained multiple basic amino acids at the cleavage site, which is characteristic of highly pathogenic AIV. Sequence alignment revealed that the isolate shared a high degree of homology with different H5 subtype AIVs isolated from waterfowl in southern China in recent years. This isolate was likely a natural reassortant from different subtype AIVs. This study provides epidemiological evidence of influenza evolution. Continuation of molecular epidemiology studies of H5 subtype influenza viruses in live poultry markets is important for understanding their role in the variation and evolution of highly pathogenic AIVs and their potential hazardous effects on human health. Furthermore, this information is important for strengthening comprehensive AIV surveillance and control measures.

The goals of this study were to establish a scalable production method to prepare human papillomavirus(HPV)16pseudovirus (PsV) using suspension-adapted HEK-293 FT cells and to improve the purification efficiency of HPV PsV. Furthermore, we aimed to solve the cryo-electron microscopy (cryo-EM) structure of HPV16 PsV. The suspension f HEK-293 FT cells were generated from adherent cells by a stepwise decrease in serum content and the addition of an anti-clumping agent during culturing. The resultant HEK-293 FT suspension cells were transfected with an L1/L2 expression vector and pN31-EGFP plasmid to generate HPV16 PsV in the Wave Bioreactor. Following cell lysis,HPV16 PsV was purified by sucrose density gradient and subsequent CsCl iso-density gradient ultra-centrifugation The final titer of HPV16 PsV was 8.2 × 10(5) TCID(50)/μL. Purified HPV16 PsV was comfirmed to as contain L1 and L2protein by western blotting, and the L1 concentration was determined to be 156.0 μg/mL by quantitative ELISA. Finally, a FEI Tecnai G2F30 electron microscope and AUTO3 DEM were used to solve the cryoEM structure of HPV16 PsV at a resolution of 14 Å.The structure shows that HPV16 PsV exists as a T=7dicosahedral lattice, with a diameter of 600 Å. These results will be beneficial for neutralization assays and for anti-sera for HPV vaccines, the high-resolution structure determination of HPV16 PsV, and the investigation of interactions between HPV L1 and L2.