Genotypes and molecular characterization of group A rotavirus in domestic sewage in Yantai, 2014-2016 / 中华实验和临床病毒学杂志

Objective@#To understand the genotype distribution and molecular epidemiological characteristics of the group A rotavirus (RVA) in domestic sewage, and further explore the importance of environmental surveillance in investigating RVA regional circulation.@*Methods@#Sewage samples were collected monthly in the city of Yantai from January 2014 to December 2016. After concentration, total RNA was extracted, and RVA VP7 and VP4 coding regions were amplified via RT-PCR. PCR products were purified, cloned and Sanger sequenced. Genotyping and phylogenetic analysis was conducted based on the sequences.@*Results@#Thirty-six sewage samples were collected and 86.1% was positive with VP7 and VP4 sequences. A total of 205 VP7 and 239 VP4 nucleotide sequences were obtained, belonging to 4 G genotypes and 6 P genotypes. Among these, G9 (95.6%, 196/205), P[8] (58.6%, 140/239) and P[4] (28.0%, 67/239) were the most common genotypes. Phylogenetic analysis for G9, P[8] and P[4] sequences revealed co-circulation of multiple transmission chains in local population.@*Conclusions@#This study describes the genotype distribution and sequence characteristics of local RVA in Shandong province, and the result demonstrate that surveillance on environmental sewage is an effective way in investigating RVA molecular epidemiology.

Etiologic surveillance and analysis of acute meningitis and encephalitis syndrome in Jinan city in 2013-2016 / 中华预防医学杂志

Objective@#To characterize the etiology and epidemiological characteristics of the acute meningitis and encephalitis syndrome (AMES) in Jinan city in 2013-2016.@*Methods@#The epidemiological data, clinical diagnosis, serum and cerebrospinal fluid (CSF) specimens were collected from 3 577 AMES cases in 6 sentinel hospitals in Jinan city in 2013-2016. Samples of all cases were made sero-diagnosis for Immunoglobulin (Ig) M antibody to Japanese encephalitis virus (JEV) and negative cases of JEV for enterovirus (EV), mumps virus (MuV) and herpes simplex virus (HSV) by enzyme-linked immunosorbent assay (ELISA). Virus isolation and molecular identification were performed. Positive rates were analyzed by Chi-square test.@*Results@#In 2013-2016, the positive rates of JEV, EV, MuV and HSV were 9.0% (322/3 577 cases), 22.1% (643/2 916 cases), 9.9% (289/2 916 cases), 26.9% (783/2 916), respectively. Of these, the positive rates of JEV were 32.9% (261/794), 1.2% (14/1 175), 1.0% (8/807) and 4.9% (39/801 cases); EV: 19.5% (91/466), 35.1% (342/974 cases), 15.5% (115/743) and 13.0% (95/733); MuV: 9.2% (43/466), 14.4% (140/974), 9.0% (67/743) and 5.3% (39/733). HSV: 35.4% (165/466), 38.5% (375/974), 25.7% (191/743) and 7.1% (52/733). There were significant differences in positive rates of 4 kinds of viruses in 2013-2016 (P<0.001). A total of 81 EV strains belonging to 8 serotypes were isolated from 1 020 CSF specimens. The positive rates were 4.8% (6 cases), 13.1% (55 cases), 4.1% (7 cases) and 4.2% (13 cases) from 2013 to 2016. Coxsackievirus (CV) B5, echovirus (E) 6 and E30 accounted for 46% (37 isolates), 22% (18 isolates) and 21% (17 isolates) of all strains.@*Conclusion@#The AMES cases in Jinan city in 2013-2016 were mainly caused by HSV, EV, MuV, JEV. CVB5, E6 and E30 were the dominant serotypes of EV associated with AMES cases in Jinan city.

Surveillance and genetic characteristics of echovirus 6 strains isolated from environmental sewage and aseptic encephalitis cases / 中华微生物学和免疫学杂志

Objective To analyze the genetic characteristics of echovirus 6 ( E-6) strains isolated from patients with acute meningitis/encephalitis syndrome ( AMES) in 2014 and sewage samples in 2013—2014 in Shandong province and to investigate their correlations.Methods Enterovirus strains were isolated from cerebrospinal fluid, stool and throat swab samples collected from 940 cases of AMES and 96 sewage samples used for environmental surveillance.The positive isolates were identified by molecular typing meth-od.Homologous and phylogenetic analyses based on the VP1 sequences of E-6 isolates were performed.Re-sults Altogether 47 E-6 strains were isolated from patients with AMES in 2014, accounting for 29.56%of all isolated enteroviruses ( EVs) strains.No E-6 strains were isolated from AMES cases in 2013.Data of the environmental surveillance showed that E-6 virus strains had been frequently detected in sewage samples since the summer of 2013 to the end of 2014.In total, 40 E-6 virus strains were isolated (7.87% of total isolated EVs strains) in 2013 and 139 E-6 virus strains (26.18%) in 2014.Phylogenetic analysis indicated that the E-6 isolates recruited in this study belonged to clusters A and C with high intracluster sequence iden-tities between AMES and environmental isolates.The nucleotide identities were 98.3%-100% among cluster A E-6 virus strains isolated from AMES cases in 2014 and 96.6%-100% among cluster A E-6 virus environ-mental isolates during the surveillance year 2013—2014.The cluster A E-6 virus strains shared 97.1%-100% nucleotide identities between the AMES and environmental isolates.For cluster C E-6 virus strains, the nucleotide identities were 100%, 98.7%-100% and 99.1%-100%, respectively.Conclusion The epidemic of viral encephalitis in Shandong province in 2014 was associated the transmission of two lineages of E-6 virus.Environmental surveillance revealed the potential epidemic of E-6 virus even before the epidemic of viral encephalitis in Shandong province, indicating the possibility of using environmental surveillance for early warning of related diseases.

Investigation of a Patient with Pre-vaccine-derived Poliovirus in Shandong Province, China / 病毒学报

To analyze the genetic characteristics of a polio-I highly variant vaccine recombinant virus in Shandong Province (China) in 2011 and to identify isolates from healthy contacts, two stool specimens from one patient with acute flaccid paralysis (AFP) and 40 stool specimens from his contacts were collected for virus isolation. The complete genome of poliovirus and VP1 coding region of the non-polio enterovirus were sequenced. Homologous comparison and phylogenetic analyses based on VP1 sequences were undertaken among coxsackievirus (CV) B1, CV-B3 isolates, and those in GenBank. One poliovirus (P1/11186), CV-A4 and CV-A8 were isolated from the AFP patient; one CV-A2, Echovirus 3 (E-3), E-12 and E-14, ten CV-B1, and five CV-B3 strains were isolated from his contacts. These results led us to believe that there may be a human enterovirus epidemic in this area, and that surveillance must be enhanced. P1/11186 was a type-1 vaccine-related poliovirus; it combined with type-2 and type-3 polioviruses in 2A and 3A regions, respectively. There were 25 nucleotide mutations with 9 amino-acid alterations in the entire genome. There were 8 nucleotide mutations with 5 amino-acid alterations in the VP1 region compared with the corresponding Sabin strains. Homology analyses suggested that the ten CV-B1 isolates had 97.0%-100% nucleotide and 98.9%-100% amino-acid identities with each other, as well as 92.6%-100% nucleotide and 99.2%-100% amino-acid identities among the five CV-B3 isolates. Phylogenetic analyses on the complete sequences of VP1 among CV-B1 and CV-B3 isolates showed that Shandong strains, together with strains from other provinces in China, had a close relationship and belonged to the same group.

Primary researches on human genetically engineered antibodies to HIV-1 / 中华微生物学和免疫学杂志

Objective To pan and characterize anti-HIV-1 Fab by the phage antibody library technology. Methods Total RNA were extracted from lymphocytes which were isolated from peripheral blood collected from asymptomatic HIV-1 infected donors with high titer antibody against HIV-1. The genes of heavy chains Fd fragment and light chains of antibody were amplified by RT-PCR. The phagmids pComb3X cloned Fd and light chain genes were transformed into E. coli XL1-Blue by electroporation to construct phage Fab library. By three runs of "absorption-elution-neutralization-enrichment", the clones were induced by IPTG and characterized by ELISA. The positive clones were sequenced and analyzed the sequences. Subsequently, Fab antibodies of these positive clones were induced to expressed and purified, then the recombinant virus neutralization assay was performed. Results A phage Fab library was constructed with 8×106 members, and 11 positive clones were obtained by detecting IPTG-induced-expressing Fabs with ELISA. By analysis of the sequences, 10 light chain genes and 8 Fd genes were ensured to be obtained. Compared with the genes of anti-HIV-1 antibodies in HIV sequence database, the gene sequences we obtained were highly homologous to some patent genes of anti-HIV-1 gp120 antibodies in HIV sequence database( light chains with 60%-90% identity, Fd with 71%-85% identity); The CDRs of these positive clones were determined by comparing the positive clone genes with antibodies' genes in V base database, furthermore, CDRH3 of these positive clones has the length of 12-22 aa. Strand shift had little effect to improving affinity of our Fab clones. Fab antibodies were induced to express at the concentration of ＞ 10 mg/L. Three Fab antibodies neutralize HIV-1 virus to some extent. Conclusion The studies will provide the basis on further study on the anti-HIV-1 Fabs obtained successfully.

The impact of the modification of 2G12 and 2F5 neutralizing epitopes of the HIY-1 envelope (Env) on its assembly of functional pseudovirus and neutralizing activity / 中华微生物学和免疫学杂志

Objective To study the influence of the modification of the special neutralizing epitopes of the HIV-1 envelope (Env) on its assembly of functional pseudovirus and neutralizing activity. Methods Site-directed mutations were performed using cycling mutagenesis and selection of mutants with Dpn I . With this method, the 2G12 and 2F5 neutralizing epitopes were integrated into Env of subtype BC which was without the two epitopes, then the capability of forming pseudovirus and the neutralizing activity against 2G12 and 2F5 were compared with pre-modified Env. Results The special Env neutralizing epitopes of five HIV pseudovirus (BC02, BC03, BC04, BC05 and BC12) were modified. Among the five pseudovirus, BC04 and BC12 pseudovirus can't be formed after the 2G12 epitope was modified, whilst the BC02, BC03 and BC05 pseudovirus can be formed after the 2G12 and 2F5 epitopes were added, and there was no variation of the pseudovirus titer; On the aspect of neutralizing activity, BG03 pseudovirus against 2G12 and 2F5 was enhanced, BC02 and BC05 pseudovirus against 2F5 was enhanced while which against 2G12 was not changed. Conclusion The modification of 2G12 epitope influences the forming of pseudovirus and the addition of neutralizing epitopes can enhance the neutralizing activity of pseudovirus, which offers new approach for the optimization of HIV immunogen.