We wished to study the dynamic change in variation of rubella viruses circulating during 1999-2015 in mainland China at the molecular level. Molecular evolution of Chinese rubella viruses collected during 1999 ~ 2015 was analyzed according to a surveillance database of measles/rubella laboratory networks in China. A total of 1737 rubella viruses were obtained from 20 of 31 provinces (except Xinjiang and Tibet) during 1999 ~ 2015. Four genotypes (1E, IF, 2A, 2B) were detected. The genotype-1E rubella virus was detected first in 2001. Subsequently, genotype 1E became the predominant genotype circulating during 2001~2013, and could be divided into two closely related clusters (A (2004-2015) and B (2001-2009)). However, the detection rate of the genotype-1E rubella virus decreased year-by-year from 2011, and reached the lowest level (1. 3%) in 2015. The genotype-1F rubella virus was restricted geographically to China, and no longer found after 2002; presumably its circulation in China was interrupted. All genotype-2A rubella viruses were derived from vaccine-related cases. At least four genotypes of 2B rubella viruses (lineage 1 ~ 4) circulated in mainland China during 2000 ~ 2015. The genotype-2B rubella virus was detected sporadically and was in a weak position until 2010. However, the detection rate of imported genotype-2B rubella viruses (lineage 3) was increased and became the predominant genotype during 2014~2015. Through the study of 16 consecutive years in mainland China, the evolution and epidemic situation of the rubella virus was obtained to aid virology surveillance for rubella control in China.

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.

Loop mediated isothermal amplification(LAMP)technology is a newly developed isothermal amplification technology for in vitro detection of nucleic acids. Although the LAMP assay is rapid, specific, sensitive, simple and has been widely applied for rapid detection of nucleic acids, it continues to improve and develop. In this paper, we summarize approaches to addressing amplification product contamination, primer design to avoid false positives, and the development of related techniques based on LAMP technology. This paper could serve as a reference for the application of the assay at the grassroots level.

Enterovirus 71（EV71）is one of the major pathogens of hand, foot and mouth disease (HFMD). The EV71 genome encodes an RNA-dependent RNA polymerase（RdRp）,3D(pol),which is critical for genome transcription and translation. However, how the 3D(pol) interacts with the host remains unclear. Yeast two-hybrid systems provide an effective approach for detecting protein-protein interactions. In this report, we inserted the DNA sequence of 3D(pol) into the pGBKT7 vector as the bait plasmid for the yeast two-hybrid experiment and transformed the plasmid into the yeast AH109 strain. We detected the expression,cytotoxicity and self-activity of 3D(pol).The 3D(pol) expressed well without affecting cell growth but exhibited strong transcriptional activation in yeast cells. We further constructed a series of pGBKT7-3D(pol) deletion mutants and identified the shortest transcriptional activation domain（1-94aa）using a self-activation assay. The results provide a molecular basis for screening the host proteins that interact with 3D(pol) using the yeast two hybrid system.

To explore the mechanisms of influenza H7N9 virus pathogenesis, influenza H7N9 virus and H1N1 influenza A virus(H1N1pdm09)-infected A549 cellular models were established, and differential protein expression in A549 cells infected with the two strains were investigated.A549 cells were infected with H7N9 and H1N1pdm09influenza virus at a multiplicity of infection(MOI)of 0.001.The temporal response of A549 cells infected with the two strains was evaluated using the proteomics approaches(2DDIGE combined with MALDI-TOF-MS/MS)at 24,48 and 72hours post infection(hpi).There were 11,12 and 33proteins with significantly different expression at 24,48 and 72hpi,respectively.Compared with H1N1pdm09 infection, functional analysis revealed that the down-regulation of proteins in H7N9 infection including F-actin-capping protein subunit alpha-1(CapZ-α1), ornithine aminotransferase(OAT),poly(rC)-binding protein 1(PCBP1)and eukaryotic translation initiation factor 5A-1(eIF5A)produced cytopathic effects. The down-regulation of platelet-activating factor acetylhydrolaseIb subunit beta(PAFAH1B2)in H7N9-infection may be related to the clinical symptoms of patients infected by the influenza H7N9 virus.

To understand the prevalence and molecular typing of enterovirus D68 among children with severe acute respiratory infection(SARI)in Beijing and Shanghai,259 respiratory samples were collected from in Beijing during 2008-2010,and 441 respiratory samples were collected in Shanghai city between 2013and2014.All the samples were used for the screening of EV-D68 by nest RT-PCR and sequencing, and then EV-D68-positive samples were used for the complete genome sequencing through overlapping PCR. All available EV-D68full-length genomes collected from GenBank were used for phylogenetic analysis and comparison of EV-D68 types prevalent in China and America. One(0.4%)from 259 respiratory samples in Beijing was positive for EV-D68,and 4(0.9%)among the 441 samples from Shanghai were positive for EV-D68.Phylogenetic analysis of full length genome indicated that the EV-D68 prevalent in Beijing belong to Clade A2 and Clade B2,different from the American popular strains(Clade A1,Clade B1,Clade B4 and Clade B5).Partial sequence analysis declared phylogenetic conflict among different gene sequences. We concluded that the prevalence rate of EV-D68 among SARI Children in Beijing and Shanghai currently was lower(5/700;<1%),and the EV-D68 genotype prevalent in China and America belong to different clusters. Partial sequence analysis indicated that intratypic recombinant events may occur in EV-D68 prevalent in China.

To explore the genomic characterization of 4vaccine-derived poliovirus(VDPV)strains isolated from 2acute flaccid paralysis(AFP)cases in Yunnan Province in 2010 and 2012,respectively,the complete genome sequences of the 4strains were determined. Sequence analysis revealed that the complete genome length of the type Ⅱ and type Ⅰ VDPV was 7439nt and 7441 nt, respectively. Nucleotide and amino acid sequence similarities of type II VDPV were 95.4% and 97.7%,respectively,and type I VDPV were93.9% and 97.9%,respectively as compared with those of Sabin strains. Nucleotide substitutions were found at two important attenuation sites (nt 481 and nt in type Ⅱ VDPV, and three important attenuation sites(nt480,nt2795 and nt6203)in type I VDPV. Type 2 and type 1VDPV strains had 1.0% and2.3% divergence with Sabin strains, respectively. Similarity plot analysis showed multiple recombination events in the genome of the 4strains,which showed that the recombination was common and complex. Analysis of the characteristics of VDPVs on molecular level could provide valuable information on evolutionary dynamics and lay foundation for developing scientific and feasible strategy to control VDPV.

Our objective was to establish a robust method for the expression and purification of hepatitis E virus(HEV)p495protein using a baculovirus-based insect cell expression system; to determine the properties and cryo-EM structure of the resulting virus-like particles(VLPs);and to compare their immunogenicity with p239 particles in the commercial hepatitis E vaccine (Hecolin). The sequence spanning HEV ORF2 amino acids 112-606 in the genotype I HEV isolate was cloned into baculovirus to express recombinant p495 protein. ELISA, analytical ultracentrifugation, size-exclusion chromatography and negative-staining transmission electron microscopy(TEM)were carried out to characterize the physicochemical properties of p495.Recombinant p495 VLPs were obtained successfully from the insect cell expression system with purity of>95%and yield of 15mg/L.The recombinant HEV p495 protein was homogeneous in solutions. The 3Dstructure of p495 VLPs was determined by cryo-EM;it was icosahedral with T=1arrangement,and showed good congruency with the crystal structure in the literature(PDB ID:2ZZQ).In mouse vaccination experiments,p495 conferred comparable immunogenicity with that of p239 antigen in Hecolin. Thus, a robust and scalable approach to obtain homogeneous, immunogenic HEV p495 VLPs has been established. This study may assist investigations of HEV receptors, epitope mapping, vaccine improvement and so on.

To determine the lysis spectrum of Yersinia enterocolitica bacteriophage phiYe-F10 and to analyze the relationship between the lysis ability of phiYe-F10 and the virulence gene of Yersinia enterocolitica. To observe the lysis ability of the phage phiYe-F10 to the different Yersinia strains with the double-layer technique. The strains used in this study including 213 of Yersinia enterocolitica and 36 of Yersinia pseudotuberculosis and 1 of Yersinia pestis. The virulence genes of these Yersinia enterocolitica (attachment invasion locus (ail) and enterotoxin (ystA, ystB) and yersinia adhesin A (yadA), virulence factor (virF), specific gene for lipopolysaccharide O-side chain of serotype O : 3 (rfbc) were all detected. Among the 213 Yersinia enterocolitica, 84 strains were O : 3 serotype (78 strains with rfbc gene), 10 were serotype O : 5, 13 were serotype O : 8, 34 were serotype O : 9 and 72 were other serotypes. Of these, 77 were typical pathogenic Yersinia enterocolitica harboring with virulence plasmid (ail+, ystA+, ystB-, yadA+, virF+), and 15 were pathogenic bacterial strains deficiency virulence plasmid (ail+, ystA+, ystB-, yadA-, virF-) and the rest 121 were non pathogenic genotype strains. PhiYe-F10 lysed the 71 serotype O : 3 Yersinia enterocolitica strains which were all carried with rfbc+, including 52 pathogenic Yersinia enterocolitica, 19 nonpathogenic Y. enterocolitica. The phiYe-F10 can not lysed serotype O : 5, O : 9 and other serotype Y. enterocolitica, the lysis rate of serotype O : 3 was as high as 84.5%. The phiYe-F10 can not lysed Yersinia pseudotuberculosis and Yersinia pestis. Yersinia phage phiYe-F10 is highly specific for serotype O : 3 Yersinia enterocolitic at 25 degrees C, which showed a typical narrow lysis spectrum. Phage phiYe-F10 can lysed much more pathogenic Y. enterocolitica than nonpathogenic Y. enterocolitica.

We wished to undertake molecular genetic typing and evaluate recombinants of the hepatitis-B virus (HBV) in Tibet (China). Multistage random sampling was used to collect HBsAg-positive samples. Nested polymerase chain reactions were used to amplify the whole sequence of the HBV. DNAstar, MEGA6 and SimPlot were used to assemble sequences, create phylogenetic trees, and undertake recombination analyses. Twelve whole sequences of the HBV of a Tibetan population were collected using these methods. Results showed that all 12 strains were C/D recombinants. Nine of the recombinations were at nt750, and the other three at nt1526. Therefore, the 12 strains could be divided into two types of recombinants: C/Da and C/Db. Analyses of the sequence of the whole genome revealed that the 12 strains belonged to genotype C, and that the nucleotide distance was > 4% between the 12 strains and sub-genotypes C1 to C15 in Genbank. The most likely sub-genotype was C1. Individuals with C/Da were from central and northern Tibet (e.g., Lasa, Linzhi, Ali) and those with C/Db recombinants were from Shannan in southern Tibet. These data suggest that the two types of recombinants had a good distribution in Tibet. Also, they can provide important information for studies on HBV recombination, gene features, virus evolution, as well as the control and prevention of HBV infection in Tibet.