Objective:To study the complete genome characterization of Human Astrovirus (HAstV) in Shandong Province.Methods:Stool samples from acute flaccid paralysis (AFP) surveillance in Shandong Province from 2020 to 2022 were collected, and HAstV nucleic acid was examined by real-time quantitative PCR (qPCR). Next-generation sequencing (NGS) was conducted for the positive samples to obtain complete genome sequences and identify the genotype. Homology comparison and phylogenetic analysis were performed by using BioEdit and Mega software.Results:A total of 667 samples were examined by qPCR, of which 14 were HAstV-positive (2.1%), including HAstV-1 ( n=6), MLB1 ( n=6), MLB2 ( n=1), and VA2 ( n=1). The complete genome sequences were obtained from 11 samples. The six HAstV-1 sequences of this study had 98.2% to 99.9% nt similarities with each other and 87.6% to 98.6% with those from other regions. The four MLB1 sequences of this study had 99.1% to 99.9% nt similarities with each other and 92.2% to 99.4% with those from other regions. The VA2 sequence of this study had 96.0% to 96.3% nt similarities with those from other regions. Phylogenetic analysis based on ORF2 region showed that the local HAstV-1 sequences were most closely related to Japanese strains, and had distinct topology with phylogenies based on ORF1a and ORF1b regions. Conclusion:The complete genome sequences of 11 HAstV strains are obtained, and the VA2 complete genome is found.

Objective:The investigation and research on the application status of Hepatic Venous Pressure Gradient (HVPG) is very important to understand the real situation and future development of this technology in China.Methods:This study comprehensively investigated the basic situation of HVPG technology in China, including hospital distribution, hospital level, annual number of cases, catheters used, average cost, indications and existing problems.Results:According to the survey, there were 70 hospitals in China carrying out HVPG technology in 2021, distributed in 28 provinces (autonomous regions and municipalities directly under the central Government). A total of 4 398 cases of HVPG were performed in all the surveyed hospitals in 2021, of which 2 291 cases (52.1%) were tested by HVPG alone. The average cost of HVPG detection was (5 617.2±2 079.4) yuan. 96.3% of the teams completed HVPG detection with balloon method, and most of the teams used thrombectomy balloon catheter (80.3%).Conclusion:Through this investigation, the status of domestic clinical application of HVPG has been clarified, and it has been confirmed that many domestic medical institutions have mastered this technology, but it still needs to continue to promote and popularize HVPG technology in the future.

Objective:To explore newer enteroviruses (EVs) circulating in China and research their genetic characterization.Methods:Sewage samples were collected in Jinan in January and July of 2018, respectively. PCR amplification of the P1 coding region of EV and next-generation sequencing (NGS) were conducted. Homologous and phylogenetic analyses were performed on the newly identified newer EVs.Results:The EV-A89 (n=2) and EV-C96 (n=1) nucleic acid sequences were detected in the sewage in January and July, respectively and obtained complete P1 coding sequence. The two EV-A89 sequences had 6.1% nucleotide divergence among themselves, and had 88.2%-95.3% homologies with other strains. No close genetic relationship was obtained with the sequences from Xinjiang, China and those from other countries. The EV-C96 sequence in this study had 76.2%-89.4% nucleotide similarities with other isolates throughout the world. Phylogenetic analysis showed that global EV-C96 strains were divided into 5 branches, i. e, A to E. Except for the Shandong 1991 isolate, the other Shandong and domestic strains were all located in branch D.Conclusions:This study added the molecular epidemiologic data of newer enteroviruses EV-A89 and EV-C96, and provides basic data for future research on the epidemic trend of EV-A89 and EV-C96 as well as their association with diseases.

Objective:To establish virus culture method and full genome sequencing method for severe acute respiratory syndrome coronavirus (2019-nCoV), and to illuminate the variation of 2019-nCoV.Methods:The pharyngeal swab specimens were inoculated into Vero-E6 cells for isolation, and the cytopathic effect were observed day by day, and the result of virus isolation were confirmed by Real-Time RT-PCR. Some isolated viruses were diluted 10 times to detect the virus titer. Full-genome of 2019-nCoV was sequenced with the whole genome capture technology and next generation sequencing technology. Phylogenetic analysis and molecular characterization for obtained 2019-nCoV sequences were undertaken.Results:Twenty-two 2019-nCoV strains were isolated with Vero-E6 cell line in the Biosafety Level-3 Laboratory (BSL-3). Eighteen full-genome sequences obtained (almost 29 000 nucleotide) were analyzed with 99.94%~100% identity and 28 nucleotides and 20 amino acids variations. Phylogenetic analysis showed that the 2019-nCoV sequences belonged to different clades, including clade S and clade L with Europe lineage L. I and Europe lineage L. II.1. Furthermore, 5 of 18 2019-nCoV harbor the D614G mutation.Conclusions:2019-nCoV were successfully isolated with Vero-E6 cells, and partially illuminated the phylogenetic characteristics, which provided a basis for subsequent drug screening and biological characteristics research. Continuous monitoring and analysis of the sequences of new cases would be vital to understand the genetic evolution and rates of substitution of the 2019-nCoV.

Rotaviruses infection is one of the main causes of diarrhea among infants and children in the world, resulting in rotavirus gastroenteritis, which is very harmful and has caused a huge disease burden and no specific drug treatment. This paper reviews the rotavirus etiology, epidemiological characteristics, disease burden of rotavirus gastroenteritis and rotavirus vaccines. RV in group A is the main cause of acute gastroenteritis in infants, people at all ages are generally susceptible to RV, 3 to 24 months infants have the most severe symptoms of diarrhea, RVGE epidemic is seasonal and peaks in winter, increasing RV vaccination can reduce the incidence and mortality of rotavirus diarrhea in infants to reduce the burden of corresponding disease. This article focuses on RV vaccines currently in use and their effect on preventing RV infection, and put forward thoughts and suggestions on technical issues related to the application of RV vaccine in China. Provide support for improving the RV vaccine immunization strategy and Chinese-specific immunization strategy for eventually incorporating RV vaccine into the national child immunization program.

Objective:To understand the detection of group A rotavirus (RVA) in domestic sewage and its molecular epidemiological characteristics, and further explore the feasibility and necessity of RVA environmental surveillance.Methods:From 2016 to 2018, we collected domestic sewage samples monthly in Jinan city, and concentrated them via anion membrane adsorption-elution method. Then RNA extraction and RVA VP7 and VP4 coding region RT-PCR amplification were performed. After purification, TA cloning and sequencing, homology analysis and phylogenetic analysis were conducted on the obtained sequences.Results:RVA G gene was detected in 31 of the 36 sewage samples (86.1% detection rate); RVA P genotype was detected in 33 samples (91.7% detection rate). A total of 536 RVA sequences were obtained, of which 225 G-type sequences belonged to 6 genotypes, and the G9 accounted for 92.4% (208/225); 311 P-type sequences were obtained, which belonged to 4 genotypes. The dominant P[8] accounted for 50.1% (156/311), followed by P[4] with 41.8% (130/311). Phylogenetic analysis shows that there were multiple transmission chains circulating in the dominant genotypes G9 and P[8].Conclusion:The genotype, homology, and phylogenetic characteristics of sequences obtained from domestic sewage in Jinan area were described, which further confirmeing that RVA environmental surveillance is not feasible but also necessary.

Rotaviruses infection is one of the main causes of diarrhea among infants and children in the world, resulting in rotavirus gastroenteritis, which is very harmful and has caused a huge disease burden and no specific drug treatment. This paper reviews the rotavirus etiology, epidemiological characteristics, disease burden of rotavirus gastroenteritis and rotavirus vaccines. RV in group A is the main cause of acute gastroenteritis in infants, people at all ages are generally susceptible to RV, 3 to 24 months infants have the most severe symptoms of diarrhea, RVGE epidemic is seasonal and peaks in winter, increasing RV vaccination can reduce the incidence and mortality of rotavirus diarrhea in infants to reduce the burden of corresponding disease. This article focuses on RV vaccines currently in use and their effect on preventing RV infection, and put forward thoughts and suggestions on technical issues related to the application of RV vaccine in China. Provide support for improving the RV vaccine immunization strategy and Chinese-specific immunization strategy for eventually incorporating RV vaccine into the national child immunization program.

Objective:To understand the detection of group A rotavirus (RVA) in domestic sewage and its molecular epidemiological characteristics, and further explore the feasibility and necessity of RVA environmental surveillance.Methods:From 2016 to 2018, we collected domestic sewage samples monthly in Jinan city, and concentrated them via anion membrane adsorption-elution method. Then RNA extraction and RVA VP7 and VP4 coding region RT-PCR amplification were performed. After purification, TA cloning and sequencing, homology analysis and phylogenetic analysis were conducted on the obtained sequences.Results:RVA G gene was detected in 31 of the 36 sewage samples (86.1% detection rate); RVA P genotype was detected in 33 samples (91.7% detection rate). A total of 536 RVA sequences were obtained, of which 225 G-type sequences belonged to 6 genotypes, and the G9 accounted for 92.4% (208/225); 311 P-type sequences were obtained, which belonged to 4 genotypes. The dominant P[8] accounted for 50.1% (156/311), followed by P[4] with 41.8% (130/311). Phylogenetic analysis shows that there were multiple transmission chains circulating in the dominant genotypes G9 and P[8].Conclusion:The genotype, homology, and phylogenetic characteristics of sequences obtained from domestic sewage in Jinan area were described, which further confirmeing that RVA environmental surveillance is not feasible but also necessary.

Objective: To sequence the 3′UTR of enterovirus 71 strains, investigate its foundation and impact in virulence by constructing a 3′UTR-replaced recombinant cDNA infectious clone. Methods: Viral RNA of EV-A71 isolated viruses were extracted, and the nucleotide analysis was performed after sequencing. The 3′UTR of a full-length infectious clone of SDLY107 strain was replaced by its corresponding part of SDLY1 strain, and then the recombinant virus was constructed and identified. Results: The nine isolated strains were classified into sub-genotype C4a of enterovirus (EV)-A71 by analysis, and nucleotide sequence homology for 3′UTR were 94%-100%. 3′UTR of EV-A71 strains may be associated with its pathogenicity. Identification of the rescued virus by sequencing and indirect immunofluorescence confirmed the successful construction of infectious recombinant virus. Conclusions Sequence analysis indicated that the 3′UTR may be involved in the pathogenicity of EV-A71. The recombinant virus SDLY107(1-3′UTR) was rescued successfully. Our study may provide evidence for further research on the influence of 3′UTR on the virulence of enterovirus 71.

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.