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 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 analyze the genetic characteristics of the complete genome of a strain of human respiratory syncytial virus (HRSV) in Qinghai province in 2024.Methods:A total of 300 samples were collected during 2024 influenza surveillance in Qinghai province sentinel hospitals from patients with fever accompanied by severe respiratory infection symptoms. We used real-time fluorescent quantitative reverse transcription polymerase chain reaction RT-PCR) method to screen out HRSV subtype B (HRSVB) positive specimens, whole genome sequencing was performed on positivespecimens meeting the requirements for the sequencing. After downloading the global representative HRSVB genotypes at GenBank database, sequence alignment was performed, related evolutionary tree was built and the calculation and analyses of genetic distance were done, analyses of HRSVB sequencing of sequence homology of nucleotides, amino acids and amino acid mutation were performed.Results:The first strain in Qinghai, China/qinghai/2024-03 had a complete sequence of 15 140 bp nucleotides, with HRSV′s all structural characteristics, and subtype HRSVA prototype strain Long strains of nucleotide the lowest homology was 80.0%, and subtype HRSVB prototype strain nucleotide homology was above 94.7%. The result indicated that the first strain in Qinghai belonged to HRSVB subtype. Genetic evolution shows China/qinghai/2024-03 and USA/WA-S23450/2021 (OR326803.1) and Germany/2021 (OR795235.1) all belong to a branch, they have the closest relationship. Phylogenetic analysis of G gene showed that the strain belonged to BA9 genotype of HRSVB subtype, and the hypervariable regions of the genome were SH and G genes.Conclusions:In this study, the complete genome sequence of HRSV China/qinghai/2024-03 was obtained for the first time, and the basic molecular structural characteristics were elucidated, which filled the gaps in the gene and amino acid data of HRSV in our province, and also provided a basis for HRSV epidemiology.

Objective:To study the epidemic status about genotype of the astrovirus, and to provide the epidemic data and control epidemic of infectious disease.Methods:Screening for astrovirus positive nucleic acids was performed using an astrovirus real-time fluorescent PCR kit. RT-PCR amplification was performed with astrovirus-specific primers Mn289/270. The positive products were recovered and purified and directly sequenced. Sequence analysis and phylogenetic analysis of astrovirus sequences were performed using Clustal and MEGA3.0 biological software.Results:From 2016 to 2018, we collected 496 fecal specimens from children with diarrhea from different regions, of them 136 cases had viral diarrhea, and in 17 of them astrovirus (3.4%) was detected, there were more males than females. All the 17 cases were under 5 years of age, and 12 cases (70.58%) were younger than 1 year old. In regional distribution, samples from Xining accounted for 64.70% (11/17), Huangzhong county for 29.41% (5/17), Huangnan Prefecture for 5.88%(1/17). The positive cases were mainly found in April to June, and there was another peak from October to December, which was consistent with the time distribution of rotavirus in past years. Seven strains were HAstV-1, one strain was HAstV-2, and the homology among four strains of HAstV-1 was 99.0%-100%. The homology with the other two strains of the same type was 88.4-95.7%.Conclusions:The dominant genotype of Astrovirus was HAstV-1 in infants with diarrhea in Qinghai, meanwhile there was also HAstV-2.

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 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: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 evaluate the running status of Qinghai provincial hand foot and mouth disease (HFMD) laboratory network in 2017. Methods: The surveillance database developed in Qinghai provincial HFMD laboratory network in 2017 were analyzed, and the indicators for the running status of HFMD laboratory network in Qinghai province were evaluated. Results: It was shown that 574 samples of suspected HFMD cases were detected by real time RT-PCR in 2017, and 368 were positive, the positive rate was 64.11%. Then 121 virus strains were isolated, the results of sequencing and analysis showed that 100 strains were EV71 with C4a genotype, 16 strains were CA16 with B1b genotype, and 5 strains belonged to the other enterovirus. In addition, Qinghai provincial HFMD network labs passed all the confirmatory test and proficiency test, and on-site review held by national HFMD laboratory in 2017, respectively. Conclusions Qinghai provincial HFMD laboratory network has been established and running well. It provided important scientific basis for HFMD surveillance in Qinghai province.

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.

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.