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.

Substantial progress in the use of chemo-photodynamic nano-drug delivery systems (nano-DDS) for the treatment of the malignant breast cancer has been achieved. The inability to customize precise nanostructures, however, has limited the therapeutic efficacy of the prepared nano-DDS to date. Here, we report a structurally defined tandem-responsive chemo-photosensitive co-nanoassembly to eliminate primary breast tumor and prevent lung metastasis. This both-in-one co-nanoassembly is prepared by assembling a biocompatible photosensitive derivative (pheophorbide-diphenylalanine peptide, PPA-DA) with a hypoxia-activated camptothecin (CPT) prodrug [(4-nitrophenyl) formate camptothecin, N-CPT]. According to computational simulations, the co-assembly nanostructure is not the classical core-shell type, but consists of many small microphase regions. Upon exposure to a 660 nm laser, PPA-DA induce high levels of ROS production to effectively achieve the apoptosis of normoxic cancer cells. Subsequently, the hypoxia-activated N-CPT and CPT spatially penetrate deep into the hypoxic region of the tumor and suppress hypoxia-induced tumor metastasis. Benefiting from the rational design of the chemo-photodynamic both-in-one nano-DDS, these nanomedicines exhibit a promising potential in the inhibition of difficult-to-treat breast tumor metastasis in patients with breast cancer.

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 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.

Objective: In this study we analyzed the genetic characteristics of echovirus 30 (E-30) VP1 gene sequences from Yunnan province isolated from viral meningitis (VM) cases in 2010-2013. Methods: RT-PCR and VP1 gene sequencing were done for 9 E-30 strains isolated from VM cases in 2010-2013. VP1 gene sequences of E-30 reference strains were downloaded from the GenBank and their nucleotide (nt) and amino acid (aa) diversities were calculated by MEGA 5.1 software, the phylogenetic tree was constructed and the genetic characteristics and molecular epidemiology were analyzed. Results: In 2010-2013, 9 strains of E-30 viruses were detected from 79 VM cases caused by echoviruses, accounting for 11.39%(9/79), the overall positive rate was 1.63%(9/553). Phylogenetic analysis revealed that E-30 strains can be divided into four genotypes (genotype A, B, C and D), and genotype D can be further divided into seven sub-genotypes. Nine Yunnan VM isolates were distributed in D7 sub-genotype, and can be further clustered into 3 branches: 5 strains isolated in 2010 were clustered in branch 1, it is evident that these viruses were responsible for an aseptic meningitis outbreak in Kunming in that year; one 2011 isolate, together with 2013 isolate and one isolate from healthy children in 2010 were clustered in branch 2, these two branches were Yunnan special branches, and two 2011 isolates had the highest homology with 2003 VM outbreaks′ strains isolated from Shandong, Jiangsu, and Zhejiang, showing that these strains may have the same evolutionary sources. Conclusions Nine Yunnan VM isolates were distributed in D7 sub-genotype, and these strains have different evolutionary sources, showing that at different times E-30 viruses in the same sub-genotypes branch might prevail in different areas.

Objective: To detect the enterovirus VP4 and VP1 genes in 510 stool samples collected from hand, foot and mouth disease (HFMD) cases and analyze the phylogenetic characteristics of the entire VP1 genes of coxsackievirus A6 (CV-A6) strains in six prefectures/cities of Yunnan Province in 2018. Methods: Viral RNA was abstracted from the stool samples. VP4 gene sequences were amplified by RT-PCR and sequenced using the MD91/OL68-1 primer pair to identify viral genotypes. Whole VP1 gene sequences were amplified and sequenced using appropriate primer pairs. The whole VP1 gene sequences of CV-A6 reference strains were downloaded from GenBank. MEGA5.2 software was used to analyze the similarity in nucleotide and amino acid sequences between different strains and phylogenetic tree was constructed for analysis of genetic characteristics and molecular epidemiology. Results: VP4 and VP1 gene sequences were obtained from 57 out of 510 stool samples with a positive rate of 11.17% (57/510). There were 43 CV-A6 (8.43%, 43/510), six CV-A10 (1.17%, 6/510), two enterovirus A71 (EV-A71, 0.39%, 2/510) and two CV-A9 (0.39%, 2/510) strains. The other four strains were CV-A4 (0.19%, 1/510), CV-A5 (0.19%, 1/510), CV-B1 (0.19%, 1/510) and E11 (0.19%, 1/510). The phylogenetic analysis showed that all 43 CV-A6 strains belonged to sub-genotype D3. Conclusions In the 510 HFMD samples, CV-A6 strains were mostly detected with a detection rate of 8.43% and accounted for 75.44% (43/57) of all isolates, followed by CV-A10 (1.17%, 6/510) and EV-A71 (0.39%, 2/510). There was a large HFMD outbreak mainly caused by CV-A6 in Yunnan Province in 2018. The outbreak was caused by CV-A6 of sub-genotype D3, as was the case with pervious outbreaks in China.

Objective To isolate and analyze the genetic characteristics of a new strain of entero-virus EV-C99 from a healthy child in Yunnan Province in 2016. Methods Virus isolation was performed ac-cording to the World Health Organization ( WHO) recommended procedures. Viral RNA was extracted from the supernatant of cell culture. RT-PCR and sequencing analysis of VP1 gene were used for virus identifica-tion. VP1 sequence was edited and stitched by Sequencher 5. 0 software. The edited sequence was BLAST searched in GenBank and the preliminary result indicated that it was an EV-C99 virus. Nucleotide (nt) and amino acid (aa) identities were calculated by MEGA5. 2 software. Serotype of the virus was identified ac-cording to the standard for enterovirus sequence typing. Results The virus could be amplified by 494/496 and 495/497 primer pairs and the edited sequence was about 1 200 bp. Result of the " BLAST" search showed that it was a new strain of enterovirus EV-C99. Comparative analysis with the prototype strain BAN00-10461 (GenBank ID:EF015008) by MEGA5. 2 software showed that the VP1 gene of that virus was 909 bp and the identities between them were 77. 05% in nt sequence and 90. 04% in aa sequence. According to the standard for enterovirus sequence typing,the virus was a new strain of enterovirus EV-C99 belonging to hu-man enterovirus species C (EV-C). Conclusion A new strain of enterovirus EV-C99 was isolated during an investigation of enteroviruses among healthy children in Yunnan Province in 2016. To our knowledge,this is the third report of EV-C99 in China. Phylogenetic analysis reveals that the EV-C99 Yunnan strain,Xinjiang strains and Shandong strains all belong to genotype B,but group into different clusters,indicating that Chinese strains have diverse genetic characteristics.