Objective: To study the genome molecular characteristics of Getah virus (SC1210) which isolated in Sichuan province in 2012. Methods: Reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify the isolate and the genome was sequenced by the second Ion Torrent PGM. Computer softwares, including Mega Align and Mega 6, were used to analyze the nucleotide and deduced amino acid sequence, and draw phylogenetic trees. Results: SC1210 was identified as Getah virus. The full genome sequence was 11 690nt, the nucleotide and amino acid homology of the full sequence with other strains were 99.2%-99.7% and 96.5%-99.4%.The capsid protein of SC1210 consisting of 804 nucleotides, encoding 268 amino acids and the full-length of E2 protein, had 1 266 nucleotides, encoding 422 amino acids. The nucleotide homology of the capsid protein and the E2 protein with other strains were 94.9%-99.2% and 94.6%-99.6%, and the amino acid were 97%-99.6% and 97.1%-99.5%. The 3′ UTR of the virus included 402 nucleotides and there were three repeat sequence elements and 19 nucleotides conservation sequence. Conclusions The first GETV isolate SC1210 in Sichuan province has a closer relationship with Yunnan strain YN040 and a far genetic relationship with MM2021.

Objective: To establish a method of correlative light and electron microscopy (CLEM) to study the intracellular location of adenovirus protein IX. Methods: MiniSOG (mini singlet oxygen generator) is a recently-invented genetically-encoded tag for CLEM. MiniSOG-fused adenovirus IX gene (IXSOG) was cloned by PCR, and inserted into pcDNA3 plasmid to form pTPL-IXSOG, which was used to transfect 293 cells. IXSOG expressing cells could be distinguished under fluorescence microscope due to the emission of green fluorescence of miniSOG. The transfected cells were fixed in 2.5% glutaraldehyde in situ, stained with diaminobenzidine(DAB) through the photooxidation activity of miniSOG, and used to prepare ultrathin sections. Intracellular location of IXSOG was studied by observing the sections under transmission electron microscope. Results: Eukaryotic expression plasmid carrying IXSOG fusion gene was constructed. IXSOG expressing cells were selected for DAB photooxidation and preparation of ultrathin sections. IXSOG fusion mainly formed punctate aggregations or inclusions in the nucleus. Conclusions The correlative light and electron microscopy method based on miniSOG was successfully established, and it could be used to study the intracellular localization of viral proteins.

Objective: To analysis the alterations of CaM and its downstream factors in the brains of scrapie infected mice. Methods: Using the methods of Western blot and immunohistochemistry assay to detect the levels and distributions of CaM, as well as the expressing alterations of the downstream substrates of CaM in the brains of mice infected with scrapie. Results: Compared with the normal controls, the levels of CaM are significantly increased in the brains of scrapie-infected mice and particularly distributing in the regions of cortex, thamalus and cerebellum. Remarkable high levels of CaMKII, p-CaMKII and p-CaMKIV are observed in the brain homogenates of scrapie-infected mice. The regulatory protein of cAMP response element binding protein (CREB) and p-CERB are also increased, while the levels of BDNF which is regulated by p-CREB are obeviously downregulated. Conclusions The synthesis of BDNF may be influenced by the prion replication in neuron and further attenuates its neuronal protective features.

Objective: To compare the hepatobiliary injury difference of newborn BALB/c mice infected by different titers of rhesus rotavirus(RRV). Methods: Neonatal mice(n=80) were randomly separated into 4 groups and were intraperitoneally inoculated with different titers of rotavirus: High titer group(1×10⁷ PFU/ml); Medium titer group(1×10⁶ PFU/ml); Low titer group(2.5×10⁵ PFU/ml); Control group (only culture medium) within the first 24 hours after birth. All mice were sacrificed at day 12 after RRV inoculation then the liver and blood samples were collected. Meanwhile, mice were observed daily for at least 12 days, including their weight, skin color and survival situation. Liver functions were examined by serum biochemical test and morphologic changes in the biliary tract were observed. Tissue sections underwent H&E staining and immunohistochemically analysis for the presence of CK19. Results: Compared with the normal mice, the mice in the experimental group had different degrees of skin jaundice, weight lost, survival rate decreased, liver function damage. In the experimental group, the symptom of low titer group was light, and could be restored to normal, however, when compared with the low titer group, the mice in the high titer group were serious, their skin jaundice was more obvious, weight was significantly reduced and irreversible, survival rate was lower(50%), liver function of TBIL, DBIL, TBA, ALT, ALP were significantly increased.Further analysis showed that the high titer group had high bile duct obstruction rate (80%), with no case of obstruction in the low titer group. Histologic analysis also showed intrahepatic bile duct atresia in the high titer group, a large number of inflammatory cell infiltrated around the portal area, while the morphology of intrahepatic bile duct was almost normal and just a small amount of inflammatory cell infiltrated around the portal area in the low titer group. Conclusions Different titers of rotavirus had different effects on the newborn mice hepatobiliary system: high titer was easy to cause biliary atresia, and low titer caused hepatitis.

Objective: To understand the serotypes, genotypes and transmission source of dengue viruses(DENV) isolated in Yunnan from 2013 to 2015. Methods: Viral RNA was extracted from serum samples of dengue fever(DF) cases at the acute stage in Yunnan, then the gene fragments of envelope protein(E) region were amplified by RT-PCR. The homology and phylogenetic analysis was made on the nucleotide and deduced amino acid sequences by bioinformatics softwares including Clustal X, DNAStar and MEGA5. Results: Viral nucleic acid detection and sequencing indicated that 40 E genes of DENV were obtained. The serotypes and genotypes of DENV were revealed by homology and phylogenetic analysis based on E genes of DENV. Fifteen virus strains belonged to DENV serotype 1(DENV-1), of these, 14(11 from Ruili, 1 from Lincang and 2 from Kunming) were genotype I(G-I), 1 from Kunming was G-V. Twenty-two virus strains belonged to DENV serotype 2(DENV-2), of these, 10 from Ruili were G-I and 12 from Xishuangbanna were G-IV. Two virus strains belonged to DENV serotype 3(DENV-3) and G-II. One virus strain belonged to DENV serotype 4(DENV-4) and G-I. All detected DENV genotypes were mainly predominant in Southeast Asia. All the 40 Yunnan DENV strains shared high homology with the DENV strains in Southeast Asia countries. Conclusions Four serotypes and multiple genotypes of DENV had been co-circulating in Yunnan from 2013 to 2015. The DENV transmitted from Southeast Asia countries was the main cause of DF in Yunnan. It is necessary to strengthen the surveillance and management on the imported cases of DF in Yunnan.

Objective: To investigate the genetic characteristics of Lamivudine-resistant mutation patterns and HBV S gene mutants in patients with chronic hepatitis disease of different disease progression. Methods: Blood samples of LAM-resistant patients with chronic hepatitis disease were collected. HBV RT gene nucleotide sequences were obtained, and then differences in drug-resistant mutation patterns, drug susceptibility and HBV S gene mutants characteristics between the two groups were analyzed. Results: Forty-seven chronic hepatitis B (CHB) patients and 16 HBV-related liver cirrhosis (LC)/HBV-related hepatocellular carcinoma (HCC) patients were included in this study. M204I single point mutation and L180M+ M204I/V were the most common pattern during patients with chronic hepatitis disease (35/63, 55.56%). The numbers of resistant to three nucleos(t)ide analogues in LC/HCC group was higher than CHB group’s (62.50% vs 34.04%, P=0.046). In HBV S gene, more immune associated HBsAg-escape mutations were detected in LC/HCC group than that in CHB group (62.50% vs 31.91%, P=0.031). I126T/V and G145A (for LCC/HCC group, 60%), I126S/T and S117T (for CHB group, 46.67%) were showed as the most common form for HBsAg escape mutations in the two groups. The two groups both detected RT mutations concomitantly with stop codon mutations in S gene (rtA181T/sW172* and rtM204I/sW196*). Conclusions Different characteristics in Lamivudine-resistant mutations and associated HBV S gene mutants were found in patients with chronic hepatitis disease of different disease progression, and LC/HCC patients exhibit more multi-drug resistant variants and immune associated HBsAg-escape mutants than CHB patients.

Objective: To learn the prevalence of HPV infection and its genotypes distribution in perianal warts, discuss the association between HPV infection and age and gender, and assess the methodology performance of PCR-reverse dot blot(PCR-RDB) to detect HPV. Methods: HPV genotypes were detected in 316 paraffin specimens of confirmed or suspected perianal condyloma acuminata(CA) patients, including 58 cases for HPV DNA sequencing. Results: The HPV infection rate in 316 patients was 81.6%. The first fifth genotypes of single HPV infection were HPV11, 6, 18, 16 and 33. HPV6+ 11, HPV6+ 11+ 16 and HPV6+ 18 were more common in mixed infection. The highest HPV infection rate was from the males aged below 35 years. Regarding the sequencing result as a gold standard, the sensitivity, specificity and accuracy of PCR-RDB in HPV were 92.31%, 89.47% and 91.38% respectively. Conclusions The proportion of HPV crissum infection in young male was quite high, the major pathogenic types were HPV 11, 6, 18, 16 and 33. The PCR-RDB method can meet the needs of clinical diagnosis of HPV.

Objective: To analyze the infection characteristics of patients in acute gastroenteritis outbreaks caused by noroviruses. Methods: Between April 2014 and March 2016, the clinical data and samples were collected from the patients in acute gastroenteritis outbreaks caused by noroviruses in Beijing. Noroviruses were detected and genotyped using real time RT-PCR, and the infection characteristics of norovirus gastroenteritis were analyzed using the descriptive epidemiological method. Results: A total of 1743 clinical diagnosed cases of norovirus gastroenteritis were collected, and children under 12 years old accounted for 77.68% (1354/1743). The detection rate of noroviruses was 73.98% (509/688). The detection rates of noroviruses in fecal, swab and vomitus samples were gradually decreased (χ²=67.798, P<0.001). Among these clinical diagnosed cases, vomiting was the most common symptom (93.98%), followed by abdominal pain (40.34%), diarrhea (30.35%) and fever (27.94%). The most common symptom of patients under 6 years old was vomiting(98.14%), whereas diarrhea was most common among over 18 years old patients (68.12%). With the increase of age of the patients, the incidence of vomiting was gradually decreased (χ²=100.913, P<0.001), whereas the incidence of diarrhea was gradually increased (χ²=261.164, P<0.001). There was no statistical difference in vomiting, diarrhea and abdominal pain symptoms between patients infected with genogroup Ⅰ and Ⅱ noroviruses, and patients infected with genogroup Ⅱ (34.49%, 149/432) had the higher incidence rate of fever than that of genogroup Ⅰ (18.18%, 14/77) (χ²=7.985, P<0.01). Conclusions Noroviruses mainly infected children under 12 years old in acute gastroentertis outbreaks. The most common symptom of patients with acute gastroenteritis caused by noroviruses was vomiting, and the incidences of vomiting and diarrhea were significantly correlated with age of the patients. Patients infected with genogroup Ⅱ had the higher incidence rate of fever than genogroup Ⅰ infection.

Objective: To understand the viral etiology of a clustered case of human infection outbreak in the middle school of Huai’an city. Methods: Nasopharyngeal swab samples from patients were collected and rapidly detected by Real-time RT-PCR and the target virus isolated in cells. Furthermore, HA1 segments of target virus were amplified by RT-PCR and sequenced. The genetic and phylogenetic analysis based on HA1 genes was computed. Results: Influenza A(H1N1)pdm09 viral nucleic acid in 11 nasopharyngeal swab samples from patients in the outbreak were positive. Compared to the vaccine strains A/California/07/2009, the Huai’an isolates, nucleotide identity was 97.7%-98.1%, and amino acid identity was 96.6%-97.4%. Phylogenetic analysis of HA1 segment sequences indicated that the Huai’an strains from the outbreak were related closely to the viruses isolated in the year of 2014. Sequence analysis indicated that the Huai’an isolates had no amino acid substitution in the receptor binding sites and glycosylation sites, while in the Ca1 of antigenic determinant of HA1 the Huai’an isolates had an amino acid substitution of S for T at 220. Conclusions The pathogen of the clustered case of human infection was Influenza A(H1N1)pdm09 virus. Though the Huai’an isolates had one animo acid substitution in the Ca1 of antigenic determinant, the antigenicity characteristic remained unchanged.

Objective: To understand the epidemiological and virological features of influenza B viruses and the difference between the vaccine strains and epidemic strains, the antigenic and genetic characteristics on hemagglutinin (HA) gene of influenza B viruses circulating in Fujian during 2010-2015. Methods: The representative strains were selected randomly according to the lineage of influenza B viruses isolated from network laboratory in Fujian, 2010-2015. Viral RNA was extracted and gene fragments were amplified by reverse transcription polymerase chain reaction (RT-PCR ) and the PCR products were sequenced. The complete HA gene sequence was obtained and analyzed via bioinformatics. Results: Compared to the vaccine strains recommended by WHO, there were significant changes in genetic and antigenic characteristics on HA gene of B Yamagata lineage viruses from 2010 to 2015, especially in 2010, 2014 and 2015. There were major five amino acid residues substitutions (116, 150, 165, 196 and 202) involved in antigenic determinants, and the variable sites gradually increased as time on over. However, the variability of B Victoria lineage viruses on HA gene was less and there was no obvious trend over time. The results showed that the B Yamagata vaccine strains of 2010 and 2015 recommended by WHO had poor protective effect on influenza virus infection, while the B Victoria vaccine strain still play a satisfactory protective effect on humans in Fujian. Conclusions With time on, influenza B Yamagata lineage viruses had gradually mutated, causing a poorly match with vaccine strains in part of year, and emerging antigenic drift phenomenon. Strengthening further surveillance of mutations of B influenza virus remains essential to allow for early warning of influenza epidemic.