Objective　 To conduct surveillance on sapovirus among hospitalized children under five years old with diarrhea in Qinghai Province in 2023, to preliminarily study the positive rate and prevalent genotypes of sapovirus, and to accumulate molecular epidemiological data on sapovirus infections in the province. Methods　A surveillance sentinel was established at the Qinghai Provincial Women and Children's Hospital. According to the "National Viral Diarrhea Surveillance Program" (2021 edition), surveillance of viral diarrhea in hospitalized children under five with diarrhea was conducted. Fluorescent quantitative PCR was used to test patient samples, and whole-genome sequencing was performed on positive samples with a CT value ≤32. Sequence analysis of the VP1 gene of sapovirus was conducted, including phylogenetic tree analysis, homology analysis, average genetic distance analysis, and amino acid variation site analysis. Results　The positive rate of sapovirus surveillance in Qinghai Province in 2023 was 3.89% (7/180). The VP1 gene sequence analysis showed that the virus had 6 amino acid substitutions and was the closest to the isolates from China in 2013 (MK111629.1), 2014 (MK111630.1), 2015 (MH477433.1), and 2016 (KX980412.1). This virus belongs to the GⅠ.1 genotype, which is currently the predominant genotype currently infecting China. The nucleotide homology between the five reference strains was 93.71%-98.81%, and the amino acid homology was 99.29%-99.82%. The average nucleotide genetic distance was 0.012-0.067. Conclusions　The study found that the epidemic level of sapovirus diarrhea in Qinghai Province in 2023 was similar to that reported in several provinces from 2020 to 2023. This is the first time that whole-genome sequencing has been used to identify the genotype of sapovirus in the province, and sequence analysis indicates that the virus strain (23-14) is the dominant strain prevalent in China. This research provides sequence references for molecular evolution studies of sapovirus infections and serves as a reference for future pathogen detection of viral diarrhea and molecular epidemiological analysis of sapovirus.

Objective:Based on targeted amplicon technology combined with high-throughput sequencing technology and bioinformatic analysis technology, to understand the characteristics of the whole genome of the monkeypox virus and its variation, and to construct a method for the analysis of monkeypox virus variation and molecular traceability of the case in Qinghai province, and to provide technical support for the prevention and control of monkeypox epidemic in the future.Methods:The extracted viral DNA was used as a template, and the genome of monkeypox virus was specifically amplified by Ion AmpliSeq Monkeypox Panel with the number of amplicons 1 609 and the length of 125 bp-275 bp, and the sequencing library was constructed by Ion AmpliSeq Library Kit Plus, and sequenced by Ion Torrent GeneStudio S5. The sequencing library was constructed by Ion AmpliSeq Library Kit Plus, and the monkeypox virus genome was sequenced using Ion Torrent GeneStudio S5 sequencer. Monkeypox virus was analyzed for genomic profiling and mutation site analysis using the online analysis tool Nextclade. The genomic sequence of the case virus in this study was compared with some sequences in the GIASID monkeypox virus database and a phylogenetic tree was constructed to analyze the potential origin of the case virus.Results:The Ct values of monkeypox virus genes in the rash swab and oropharyngeal swab samples were 32.13 and 36.91, respectively. The rash swab sample had a reads number match of 99.99% and a genome coverage of 99.45% after whole-genome sequencing of monkeypox virus, and the sequences belonged to the IIb (West African branch) B. 1.3 type. The analysis of nucleotide mutation sites and phylogenetic tree showed that the sequences were in the same branch with four monkeypox virus genome sequences recently submitted by China and Japan in the GISAID monkeypox virus database, and had the closest evolutionary relationship with the sequence EPI_ISL_18059184 (sampled on 2023-07-03) submitted by Yunnan, China, which shared 82 single-nucleotide mutation sites, among which the sequence from Yunnan was only present in all of the shared 82 single-nucleotide mutation sites. The sequence in this study has 2 additional nucleotide mutation sites on top of the shared 82 single nucleotide mutation sites. The sequence submitted by Japan, EPI_ISL_17692269 (sampled on 2023-04-28), is more closely related in evolution, sharing 78 single nucleotide mutation sites, with 7 single nucleotide mutation site differences, and the Japanese sequence shares 78 single nucleotide mutation sites. The Japanese sequence shared 78 mutation sites with one additional nucleotide mutation site (G57786A), while the present sequence had six additional nucleotide mutation sites (G13563A, C21062T, G101241A, C142797T, G152866A, T169721A).Conclusions:The whole genome sequence of monkeypox virus of 197 084 bp was successfully obtained from a sample with low viral load, and the average. We constructed a method for sequencing and analyzing the whole genome of monkeypox virus.

Objective:To understand the prevalence and genotype characteristics of enterovirus and Coxsackie virus in hand, foot and mouth disease (HFMD) patients in Qinghai province from 2015 to 2018.Methods:The throat swabs of HFMD patients were collected for virus isolation and RT-PCR in Qinghai province. The nucleotide sequence of the amplified products was determined and analyzed, and the gene evolution tree was constructed with reference to the sequence of some strains of NCBI.Results:From 2015 to 2018, 1 738 samples of clinical diagnosis positive cases were collected, including 326 EV-A71 cases, accounting for 18.76%, 237 CV-A16 cases, accounting for 13.64%, 628 CV-A6 cases, accounting for 36.13%. There were statistically significant differences among different genotypes in 4 years (EV-A71, χ2=245.315, P<0.001; CV-A16, χ2=27.680, P<0.001; CV-A6, χ2=702.713, P<0.001). A total of 317 cell culture isolates were obtained after isolation with RD cells. After sequence, DNA sequences of typical genotypes were selected for sequence analysis, from 2017 to 2018. The homology of all genotypes of VP1 was between 59% and 100%, the homology was 93% to 100% in 19 strains of EV-A71, the homology was 92% to 100% in 20 strains of CV-A16, the homology was 97% to 99% in 6 strains of CV-A6. According to the evolutionary tree, the sequences of EV-A71 strains are all on the C4a branch of the evolutionary tree in Qinghai province; 18 strains of CV-A16 were B1b and 2 stains were 1D; 5 strains of CV-A6 were D3 and 1 was B1. Conclusions:From 2015 to 2018, the prevalent genotypes of HFMD shifted from EV-A71 to CV-A16 and CV-A6 in Qinghai province, The EV-A71 genotype in Qinghai province has always been C4a genotype. There are different genotypes of CV-A16 and CV-A6. The nucleotide sequence differences between different genotypes are large, and the sequence variation among the same gene is small.

Objective:To analyze the epidemiological characteristics of rubella and the genetic characteristics of rubella virus (RV) circulated in Qinghai province in 2020, so as to provide scientific basis for optimization and improvement of local rubella prevention and control strategy.Methods:The rubella epidemiological characteristics were analyzed by summarizing the data on the rubella incidence in Qinghai province in 2020 from the National Notifiable Disease Reporting System. Through Qinghai provincial measles and rubella laboratory network, throat swab samples from susceptible rubella outbreak and sporadic cases in 2020 were collected and identified. RV strains were obtained after three passages of virus isolation from positive samples. After extracting the viral RNA, the 739 nucleotide fragments within the E1 gene were amplified and determined to identify the genotype and sub-genotype of the Qinghai strains in 2020 and further analyzed the molecular differences between Qinghai strains and the RV strains circulated in China.Results:In 2020, the rubella incidence in Qinghai province had shown an obvious upward trend, and the age of onset had shifted to adolescents in the 10-19 years of age group (accounting for 94.9%). Totally 29 RV strains were isolated from four high incidence areas of rubella in Qinghai province. All RV strains were identified as sub-genotype 2B-L2c, which is also the dominant subtype of RV circulated in China. In addition, virological surveillance data showed that there were different transmission chains of sub-genotype 2B-L2c in Qinghai province in 2020, and an outbreak might be caused by different transmission chain viruses.Conclusions:The accumulation of rubella susceptible population aged 10-19 years and the transmission of new imported 2B-L2c virus had led to the rubella reemergence and outbreaks in several cities in Qinghai province in 2020.

Objective:To investigate the epidemiological and pathogenic characteristics of a mumps outbreak in a primary school in Xining, Qinghai province.Methods:The epidemiological investigation was carried out to analyze the epidemiological distribution of mumps cases in the outbreak. Serum and throat swab samples were collected from 9 suspected mumps cases for laboratory testing. The throat swab samples detected positive for nucleic acid of mumps virus were subjected to virus isolation. Then the SH gene was amplified by RT-PCR for positive virus isolates, and the genotypes of mumps virus were identified and gene characteristics were analyzed.Results:A total of 13 cases were reported in this outbreak. The age of cases was mainly 7-11 years old, and the cases were mainly concentrated at 8 years old (69.23%. 9/13). The male to female ratio is 1.6: 1. None of the 13 cases had a history of mumps vaccination. And there was an obvious in-class clustering in this mumps outbreak. Of the 9 suspected mumps cases, 8 were double positive for mumps specific IgM antibody and viral nucleic acid. Two positive mumps virus isolates were obtained and identified by genotyping as F genotype, and the SH gene sequence of the two mumps virus isolates had 100% homology.Conclusions:This outbreak is caused by genotype F mumps virus. MuV immunization activities were recommended to conduct among unvaccinated students in primary and secondary schools.

Objective:To understand the genotype and genetic characteristics of rubella virus (RV) circulated in Qinghai province.Methods:The throat swabs were collected from suspected rubella cases in two cities of Qinghai province in 2010 and 2019, respectively. After nucleic acid detection and virus isolation, the target genotyping sequences (739 nucleotide fragments of E1 gene) of positive virus isolates were amplified and determined. The sequences of the viruses were compared with 32 reference strains of 13 genotypes recommended by World Health Organization(WHO) to determine the genotype, and the genetic relationship between Qinghai isolates and RV strains from other provinces of China was also analyzed.Results:In this study, four RV virus strains were isolated, and the genetic relationship analysis showed that these virus strains were classified into 1E-Cluster A sub-genotype and 2B-Cluster C sub-genotype, which was basically consistent with the epidemic trend of RV in other provinces of China. The four virus strains in Qinghai province were highly conserved at the amino acid level, but region-specific mutation sites were also found.Conclusions:This study provides some laboratory data for the formulation of rubella prevention and control strategy in Qinghai province..

Objective: To evaluate 2017 poliovirus surveillance in Qinghai Province. Methods: According to the World Health Organization (WHO) 4 th edition of the polio laboratory manual procedure for virus isolation, the isolated L20B positive strain was identified as intratypic differentiation (ITD) by the China Center for Disease Control and Prevention, CDC). The National Polio Laboratory performed the nucleotide sequence determination of the capsid protein VP1 coding region of poliovirus (PV) and analyzed the poliovirus surveillance and the result of analysis of the cases with acute flaccid paralysis (AFP) reported in Qinghai Province in 2017 and stool samples of healthy children. Results: In 2017, Qinghai CDC Polio Laboratory received specimens of 211 AFP cases and healthy stool samples. PV2 strains were isolated with a separation rate of 0.95%. Non-polio enterovirus (NPEV) strains were isolated from 25 strains with the isolation rate of 11.85%. Two PVs were used for ITD. All of them were vaccine-associated strains. Conclusions In 2017, the Qinghai CDC Polio Laboratory did not find any poliovirus and vaccine-derived poliovirus in the AFP cases and stool samples from healthy persons, maintaining the polio-free status.

Objective: To investigate the genetic characteristics of VP1 coding region of enterovirus Coxsackievirus A16(CV-A16) and etiological features of hand, foot and mouth disease (HFMD) in 2017 in Xining city. Methods: The pharyngeal swab specimens were collected from HFMD patients, and detected by real-time reverse transcription-polymerase chain reaction (RT-PCR). For CV-A16 positive samples, virus isolation was performed. Then RNA was extracted, and then VP1 coding region was amplified by RT-PCR. The phylogenetic tree was constructed by comparing with other genotypes and sub-genotypes strains of EV-A71. Results: It was shown that 70 strains of CV-A16 were isolated from 2017 to 2018 in Xining city. In 2017, 10 strains were isolated and divided into two different lineages by phylogenetic analysis, 3 strains of B1a and 7 stains of B1b. In 2018, 60 stains were isolated, which were all belong to B1b. Conclusions B1a and B1b of CV-A16 are prevalent in Xining city from 2017 to 2018, in which B1b is the prominent isolates.

Objective: To investigate the genetic characteristics of enterovirus A 71 (EV-A71) and etiological features of hand, foot and mouth disease (HFMD) in Qinghai province from 2016 to 2017. Methods: Specimens were collected from HFMD patients in Qinghai province, and detected by real-time reverse transcription-polymerase chain reaction (RT-PCR). For EV-A71 positive samples, virus was isolated and RNA was extracted, and then VP1 coding region was amplified by RT-PCR. The phylogenetic tree was constructed by comparing with other genotypes and sub-genotypes strains of EV-A71. Results: It was shown that 114 strains of EV-A71 were isolated in Qinghai province from 2016 to 2017, which all belonged to genotype C4a, and could be divided to two different lineages by phylogenetic analysis. From 2016 to 2017, the epidemic strains of EV-A71 in the different transmission chains of Qinghai province was closely related to other provinces of China. Conclusions C4a was the dominant genotype of EV-A71 in Qinghai province from 2016 to 2017, and no other genotype was detected. In addition, EV-A71 isolated from Qinghai province co-evolved with EV-A71 in other provinces of China.

Objective: To evaluate the running status of Qinghai provincial measles laboratory network during 2012—2017. Methods: To analyze serological and virological surveillance database developed in Qinghai provincial measles laboratory network in 2012—2017, and evaluate the indicators for the running status of measles laboratory network in Qinghai Province. Results: It was shown that 5 763 suspected measles cases were reported in Qinghai Province during 2012—2017, and 4 167 serum samples were collected from suspected measles cases, the collection rate is 72.31%; among them, 3 697 were IgM positive for measles and the positive rate was 88.72%; 68 were IgM positive for rubella and the positive rate was 1.63%. And 515 throat swab specimens were collected from suspected measles cases, 82 measles virus isolates were obtained and the positive rate was 15.92%. The result of sequencing and analysis showed that all the measles viruses belonged to genotype H1 and subgenotype H1a, which were predominant genotype circulated in China in recent years. In addition, Qinghai provincial measles network labs passed all the serological confirmatory test and proficiency test, and on-site review held by national or provincial measles laboratory and WHO during 2012—2017, respectively. Conclusions Qinghai provincial measles laboratory network has been established and running well. It provided important scientific basis for measles elimination in Qinghai province.