Objective:To analyze the non-enterovirus A71 (non-EVA71) and non-coxsackievirus A16 (non-CVA16) enteroviruses causing hand, foot and mouth disease (HFMD) in Kunming and Qujing of Yunnan Province in 2021 by sequencing the VP4/VP2 and VP1 genes and to analyze the phylogenetic characteristics of the VP1 gene of CVA2, aiming to provide reference for the prevention and control of CVA2.Methods:The samples were made and extracted strictly according to the Laboratory Manual for Hand, Foot and Mouth Disease (China Center for Disease Control and Prevention, 2018 Edition). VP4/VP2 junction regions were firstly amplified and sequenced by MD91/OL68-1 primers. These sequences were firstly edited and then "blasted" on the GenBank to determine the virus serotype. To analyze the phylogenetic characteristics of CVA2, the entire VP1 gene sequences were amplified in two segments using enterovirus species A primers. Virus serotype was again confirmed online by "Enterovirus Genotyping Tool Version 0.1". The sequences of the reference virus genotypes/sub-genotypes were downloaded according to the reference. The phylogenetic trees were constructed by Mega5.2 software and the genetic characteristics were analyzed.Results:A total of 749 non-EVA71 and non-CVA16 enteroviruses were detected in the two areas in 2021. Group A enteroviruses were the main pathogens, with CVA16 as the predominant virus, and a small number of group B enteroviruses were reported. Only five strains of CVA2 were detected with a detection rate of 0.67% (5/749), indicating that CVA2 was a rare pathogen for HFMD in the two areas. The sequencing and serotyping results were consistent using the two genomic regions of VP4/VP2 junction region and VP1 region. Phylogenetic analysis showed that three Kunming strains belonged to genotype A, while two Qujing strains belonged to genotype D.Conclusions:The detection rate of CVA2 in Kunming and Qujing was 0.67% in 2021. CVA2 was a rare pathogen for HFMD in the two regions. Phylogenetic analysis showed genotypes A and D spread in Kunming and Qujing, respectively, but had not caused epidemics. To our knowledge, this was the first report of genotype A of CVA2 in China. Strengthening the laboratory surveillance especially molecular epidemiological surveillance is valuable for the monitor and analysis of transmission source for CVA2.

Objective:To study the genetic characteristics and genetic evolution of echovirus 30 (ECHO30) isolates in Yunnan Province, China.Methods:Virus isolation was performed on nucleic acid-positive samples for hand, foot, and mouth disease pathogen surveillance in Yunnan Province, and VP1 gene sequencing was performed. The sequences of eight ECHO30 isolates from Yunnan Province and the gene sequences of the VP1 region of the ECHO30 reference strain downloaded from GenBank were compared and analyzed using MEGA 5.0 software, and then a phylogenetic tree was constructed to measure the homology of nucleotides and amino acids between the isolates.Results:The ECHO30 virus was distributed in Wenshan, Qujing, Chuxiong, and Kunming in Yunnan Province. The ECHO30 virus was relatively common in Wenshan. ECHO30 isolates belonged to the H2 subtype of the H genotype, which was close to the local reference strain LC120939 in Yunnan Province. On the VP1 gene at site 5, the amino acid change ratio was more active, the amino acids were diverse, and mutations also occurred at sites 54, 156, 258, and so on. Nucleotide and amino acid homology were 84.0% - 100.0% and 98.4% - 100.0%, respectively.Conclusions:ECHO30 isolates from Yunnan Province have certain geographical characteristics and belong to H2 of the H genotype. The nucleotide differences in virus sequences among subtypes are small and have a close genetic relationship.

Abstract: Objective　To analyze the results of the surveillance of hand, foot and mouth disease (HFMD) pathogens in Baoshan City, Yunnan Province in 2022, and to analyze the genetic characteristics of the main epidemic strain coxsackievirus A16 (CVA16), in order to provide scientific basis for the prevention and control of HFMD. Methods　Samples of hand, foot and mouth disease cases in Baoshan City submitted to Yunnan Province in 2022 were selected, the samples were processed, and the viral nucleic acid was extracted, and the Enterovirus (EV) VP4/VP2 binding region gene was amplified with primers (MD91/OL68-1) and sequenced. The sequence was BLAST searched in GenBank to determine the virus type, and then the full sequence of VP1 region was amplified with relevant primers of each type of virus and sequenced. Viruses were identified by using Enterovirus Genotyping Tool Version 1.0. according to the reference documents, the reference sequences were downloaded for making the phylogenetic tree, and the test results were statistically analyzed. Results　A total of 307 clinical samples of hand-foot-mouth disease in Baoshan City in 2022 were detected by real-time RT-PCR. There were 280 laboratory-confirmed cases (91.21%), among which the detection rate of CVA16 was 55.05% (169/307), EV-A71 was 3.58% (11/307), and other enteroviruses were 32.57% (100/307). Of the 206 HFMD test specimens, the VP4/VP2 junction and VP1 gene sequences of enterovirus were amplified and identified, and 29 enterovirus strains were obtained, with a positive virus rate of 14.08% (29/206). All viruses belonged to group A and were divided into 3 serotypes, which included 27 strains of CVA16 (93.10%, 27/29), 1 strain of EV-A71 (3.45%, 1/29), and 1 strain of CVA10 (3.45%, 1/29). Group B, C, and D viruses were not detected. The positive rate of VP4/VP2 binding region and VP1 region of coxsackievirus A16 gene in Baoshan City in 2022 was 13.11% (27/206). The genetic evolution analysis showed that the 27 strains of CVA16 belong to B1a subtype and can be divided into two evolutionary branches. Conclusions　In 2022, the main epidemic strain of HFMD in Baoshan City, Yunnan Province is CVA16 gene, belonging to B1a subtype, which can be divided into two branches, indicating 2 transmission chains prevalent in Baoshan City. Future efforts should focus on strengthening surveillance, improving the quality of monitoring, and understanding the characteristics of viral prevalence..

Objective:To analyze the etiological characteristics of hand, foot and mouth disease (HFMD) cases and to investigate the genetic characteristics of VP1 region of coxsackievirus A6 (CVA6) and CVA16 strains in Wenshan prefecture of Yunnan Province in 2020.Methods:Virus RNA was extracted directly from stool samples of children with HFMD in Wenshan prefecture of Yunnan Province in 2020. Enterovirus (EV) VP4/VP2 junction region was amplified using MD91/OL68-1 primers and sequenced, and then the serotypes of EV isolates were preliminarily identified. Amplification and sequencing of the complete VP1 gene were performed. A phylogenetic tree was constructed by MEGA 5.2 software with the reference strains from GenBank. Genetic and molecular epidemiological characteristics of CVA6 and CVA16 were analyzed.Results:Thirty-two strains of EV and one strain of astrovirus MLB1 were detected in 317 specimens with an overall virus detection rate of 10.41% (33/317). Among the 32 EV strains, 31 (96.88%) were enterovirus species A (EVA) and one (3.12%) was EVB. EVC and EVD were not detected. CVA6 was the predominant EV, accounting for 62.50% (20/32), followed by CVA16 (18.75%, 6/32), CVA4 (9.37%, 3/32) and CVA10 (3.12%, 1/32). EVA71 was not detected. The phylogenetic analysis showed 20 CVA6 strains belonged to D3a sub-genotype and could be further divided into three clusters. Six CVA16 strains belonged to B1a sub-genotype, which was one of the predominant genotypes circulating in China, and could be divided into two clusters.Conclusions:The detection rate of pathogens causing HFMD in Wenshan prefecture in 2020 was 10.41% and the most common etiologic agents were CVA6 and CVA16. Based on the genetic analysis of the VP1 gene, the predominant genotype circulating in Wenshan prefecture in 2020 was CVA6 D3a sub-genotype, followed by CVA16 B1a sub-genotype. EVA71 was not detected.

Objective:To analyze the etiological distribution and phylogenetic characteristics of hand, foot and mouth disease (HFMD) in Qujing city of Yunnan Province in 2020.Methods:Stool samples were collected from HFMD cases in Qujing city in 2020 and virus RNA was extracted directly from treated stool suspensions. Virus VP4 gene sequences were firstly amplified using MD91/OL68-1 primer pairs and sequenced, then the virus serotypes were determined by BLAST search on the GenBank. Virus entire VP1 gene sequences were amplified and sequenced. Virus serotypes were identified online using Enterovirus Genotyping Tool Version 0.1. Sequences of reference virus genotypes/sub-genotypes were downloaded according to references. Phylogenetic trees were constructed by MEGA5.2 software and the genetic characteristics were analyzed.Results:A total of 47 strains of enteroviruses (EVs) were detected with a detection rate of 10.22% (47/460). The detected viruses were coxsackievirus A4 (CVA4, 0.65%, 3/460), CVA6 (7.83%, 36/460), CVA10 (0.87%, 4/460) and CVA16 (0.87%, 4/460). All were enterovirus species A (EVA), while other group viruses were not detected. The predominant virus was CVA6, accounting for 7.83% (36/460). EVA71 was not detected. CVA4 strains of C2 and C4 subgenotypes were co-circulating strains in Qujing city. CVA6 subgenotype D3a and CVA16 subgenotype B1a were also circulated in Qujing city. All CVA10 strains were in a separate lineage.Conclusions:Similar to the previous situation in China, the detection rates of EVA71 and CVA16 were very low, even zero. This study showed that CVA6 was the predominant virus, indicating a HFMD outbreak caused by CVA6 in Qujing city in 2020. The phylogenetic analysis showed CVA10 isolates belonged to a separate lineage, which might be unique to Qujing city. Laboratory and molecular epidemiological surveillance of non-EVA71 and non-CVA16 viruses, especially CVA6 and CVA10 should be strengthened in the future.

Objective:To investigate the predominant types of enteroviruses and the characteristics of the VP1 gene of coxsackievirus A4 (CVA4) causing hand, foot and mouth disease (HFMD) in Yunnan Province from 2018 to 2020.Methods:Throat swab and stool samples were collected from HFMD cases and tested by real-time quantitative PCR for nucleic acid detection. The samples positive for enterovirus nucleic acids were used for viral isolation and sent to the National Center for Disease Control and Prevention. The VP1 gene of the isolated strains was sequenced and analyzed.Results:From 2018 to 2020, a total of 21 757 HFMD samples were collected, 16 457 (75.64%) of which were positive for enteroviruses. Altogether 533 strains were isolated from 4 114 positive samples that were selected for viral isolation, including 89 strains of enterovirus 71 (EVA71, 16.70%), 180 strains of coxsackievirus A16 (CVA16, 33.77%), 76 strains of CVA10 (14.26%), 118 strains of CVA6 (22.14%), 26 strains of CVA4 (4.88%) and 44 strains of other types (8.26%). HFMD occurred mainly in children under five years old with higher incidence in males than in females (1.35∶1). The incidence of HFMD reached the peak in the second and third quarters. In Yunnan Province, CVA4 mainly circulated in Qujing and Kunming, and was sporadically detected in Wenshan and Honghe. The VP1 gene was 915 bp in length. Twenty-six CVA4 strains belonged to C2 subtype, which were genetically far from the prototype strain AY421762-HighPoint. Mutations in the VP1 gene were found at multiple sites including 18, 23, 34, 102, 148, 164, 200, 262, 174, 275, 285 and 303. These strains showed 80.4%-99.0% homology in nucleotide sequence and 95.6%-99.0% in amino acid sequence. Nucleotide mutations were mostly synonymous mutations.Conclusions:CVA16, CVA6, EVA71 and CVA10 were the predominant enteroviruses causing HFMD in Yunnan Province from 2018 to 2020. The prevalence of CVA4 was also worthy of attention. CVA4 isolates in Yunnan Province belonged to C2 subtype, mainly circulating in the east and southeast of Yunnan Province and gradually becoming a cocirculating predominant strain. Long-term dynamic monitoring would be of great public health significance for improving the sensitivity of HFMD early warning.

Objective To understand the dynamic changes of the pathogen spectrum of viral diarrhea in Southwest China, and to provide a scientific basis for formulating prevention and control measures. Methods A total of 2 262 fecal samples of diarrhea cases were collected from the diarrhea monitoring sites in Yunnan, Sichuan, and Chongqing from January 2017 to December 2019. The detection of rotavirus, norovirus, adenovirus, astrovirus, and sapovirus was performed by real-time PCR, and their epidemiological data were collected. Results The ratio of male to female patients was 1.30:1. The majority of patients were 3 years old and younger, accounting for 52.21%% of all cases. Children living at home accounted for 49.38% of all cases. The detection rate of rotavirus was 10.70%, followed by norovirus at 10.43% and adenovirus at 4.60%. The detection rate of rotavirus was the highest in Yunnan and Chongqing, which were 20.18% and 16.96%, respectively, while the detection rate of norovirus was the highest in Sichuan, which was 13.54%. Conclusion Rotavirus was still the main diarrheal pathogen in Southwest China. High incidence of diarrhea occurred in autumn and winter, with children living at home being the main patients. Measures should be taken to prevent outbreaks caused by norovirus and adenovirus.

Objective:To analyze the etiology of hand, foot and mouth disease (HFMD) cases collected from Wenshan prefecture from 2014 to 2018 and the molecular epidemiology of coxsackievirus A6(CV-A6).Methods:Viruses were isolated by RD cells and Hep-2 cells from stool samples collected from HFMD patients in Wenshan prefecture from 2014 to 2018. Virus RNA was extracted and virus VP4/VP2 junction region sequence was firstly amplified and sequenced by MD91 and OL68-1 primer pairs, then the virus serotype was determined. Virus entire VP1 gene sequences were determined by relative primer pairs according to the references. The reference sequences of CV-A6 virus entire VP1 gene were downloaded from the GenBank and the phylogenetic tree was constructed and the genetic characteristics and molecular epidemiology were analyzed.Results:During five years of study period, a total of 581 strains of enteroviruses (EVs) was isolated with an isolation rate of 20.40% (581/2 848). Among 581 strains, 74 strains were CV-A6, accounting for 12.74% (74/581); 124 were CV-A16, accounting for 21.34% (124/581); 374 were EV-A71, accounting for 64.37% (374/581); nine were other EVs, accounting for 1.55% (9/581). The entire VP1 sequences of 74 CV-A6 strains were filtered by constructing a phylogenetic tree and the completely same strains were excluded from analysis. We finally analyzed the phylogenetic characteristics of 22 strains isolated in this study with 52 reference strains. The results showed that all 22 Wenshan strains belonged to D3a sub-genotype, of which 21 strains belonged to cluster 1, and only one strain belonged to cluster 2.Conclusions:From 2014 to 2018, the outbreaks of HFMD in Wenshan prefecture were mainly caused by EV-A71, CV-A16 and CV-A6, accounting for 64.37%, 21.34% and 12.74% respectively. Phylogenetic analysis showed, similar to the situation in China, the sub-genotype D3a of CV-A6 was the predominant virus and the cluster 1 was the main sub-genotype in this outbreak.

Objective:To do genetic analysis on the VP1 gene of 2 Coxsackievirus A12 (CV-A12) isolated from hand, foot and mouth disease (HFMD) surveillance in Yunnan province.Methods:Coxsackievirus isolation was carried out in RD cells from the clinical samples. CV-A12 strains were identified by realtime RT-PCR and sequencing technology from the positive cultures. The VP1 region of the CV-A12 strain was further sequenced. The VP1 gene sequences were initially aligned and then used to construct the phylogenetic tree with GenBank reference strains.Results:The two CV-A12 strains had the highest homology with the Yunnan reference strains in 2018 and the amino acids in VP1 region had specific mutations with other cluster reference strains at multiple sites.Conclusions:The CV-A12 in the HFMD cases in Yunnan province has occured regional specific mutation in VP1 gene.

Objective To analyze the genetic characteristics of VP1 3'region of human coxsack-ievirus B2 (CV-B2) strains isolated from Yunnan province. Methods RT-PCR and gene sequencing were performed to analyze the VP1 3'region of 15 CV-B2 strains isolated from acute flaccid paralysis ( AFP) cases during 2005 to 2006, healthy children in 2013 and hand, foot and mouth disease (HFMD) cases in 2014 in Yunnan province. CV-B2 VP1 gene reference sequences were downloaded from the Genbank. Nucleotide (nt) and amino acid (aa) diversities were calculated by MEGA5. 2 software and a phylogenetic tree was constructed. Genetic and molecular epidemiological characteristics of CV-B2 strains circulating in Yunnan province were analyzed. Results A total of 15 CV-B2 strains were isolated, which were one from 232 AFP cases in 2005, one from 240 AFP cases in 2006, 12 from 400 healthy children in 2013 and one from 500 HFMD cases in 2014. Phylogenetic analysis of the 15 CV-B2 strains in Yunnan province and those down-loaded from the GenBank showed that CV-B2 could be genetically divided into five genotypes. The prototype strain Ohio-1 and one strain (01-1) isolated in Taiwan in 1988 belonged to genotype 1. Strains isolated in France in 2006, 2007 and 2010 belonged to genotype 2. Strains isolated in Yunnan, Shandong, Henan, Fu-jian and Taiwan belonged to genotype 3. Strains isolated in Russia, Yunnan AFP cases in 2005 and 2006 and India belonged to genotype 4. Strains isolated in Taiwan, Shandong and New South Wales, Australia be-longed to genotype 5. Different genotypes distributed in different countries/areas with some confined within specific countries/areas. Conclusions The 12 strains isolated from healthy children and one from HFMD cases in Yunnan province belonged to genotype 3, while the two strains isolated from AFP cases belonged to genotype 4. Diversities in nt and aa sequences between the strains isolated from the healthy children and HFMD case were only 0. 76％ and 0. 03％, respectively, indicating that they might come from the same transmission source. However, the nt and aa diversities between the isolates of genotype 3 ( from healthy children and HFMD case) and genotype 4 (from AFP cases in 2005 and 2006) were 15. 11％-15. 22％ and 2. 76％-2. 72％, respectively. Correlation of CV-B2 with AFP and HFMD was worthy of further study.