Complete Genome Analysis of an Enterovirus EV-B83 Isolated in China.

Enterovirus B83 (EV-B83) is a recently identified member of enterovirus species B. It is a rarely reported serotype and up to date, only the complete genome sequence of the prototype strain from the United States is available. In this study, we describe the complete genomic characterization of an EV-B83 strain 246/YN/CHN/08HC isolated from a healthy child living in border region of Yunnan Province, China in 2008. Compared with the prototype strain, it had 79.6% similarity in the complete genome and 78.9% similarity in the VP1 coding region, reflecting the great genetic divergence among them. VP1-coding region alignment revealed it had 77.2-91.3% with other EV-B83 sequences available in GenBank. Similarity plot analysis revealed it had higher identity with several other EV-B serotypes than the EV-B83 prototype strain in the P2 and P3 coding region, suggesting multiple recombination events might have occurred. The great genetic divergence with previously isolated strains and the extremely rare isolation suggest this serotype has circulated at a low epidemic strength for many years. This is the first report of complete genome of EV-B83 in China.

[Complete Genome Characterization of Vaccine-derived Polioviruses Isolated in Yunnan,China].

To explore the genomic characterization of 4vaccine-derived poliovirus(VDPV)strains isolated from 2acute flaccid paralysis(AFP)cases in Yunnan Province in 2010 and 2012,respectively,the complete genome sequences of the 4strains were determined. Sequence analysis revealed that the complete genome length of the type Ⅱ and type Ⅰ VDPV was 7439nt and 7441 nt, respectively. Nucleotide and amino acid sequence similarities of type II VDPV were 95.4% and 97.7%,respectively,and type I VDPV were93.9% and 97.9%,respectively as compared with those of Sabin strains. Nucleotide substitutions were found at two important attenuation sites (nt 481 and nt in type Ⅱ VDPV, and three important attenuation sites(nt480,nt2795 and nt6203)in type I VDPV. Type 2 and type 1VDPV strains had 1.0% and2.3% divergence with Sabin strains, respectively. Similarity plot analysis showed multiple recombination events in the genome of the 4strains,which showed that the recombination was common and complex. Analysis of the characteristics of VDPVs on molecular level could provide valuable information on evolutionary dynamics and lay foundation for developing scientific and feasible strategy to control VDPV.

Molecular epidemiology and recombination of human enteroviruses from AFP surveillance in Yunnan, China from 2006 to 2010.

The study represents the genetic overview of non-polio enteroviruses (NPEV) isolated from acute flaccid paralysis (AFP) cases in Yunnan Province from 2006 to 2010. Molecular typing based on VP1 nucleotide sequence was carried out on 98 NPEV isolates, and 33 serotypes were identified. EV-B was detected most frequently with an overall prevalence of 71.4%, followed by EV-A (18.4%) and EV-C (10.2%). No EV-D was identified. NPEV positive rate was higher in children <3 years of age and in summer and autumn months. Clinically, 68.4% patients presented with fever, and 16 cases (16.3%) were classified as Guillain-Barré syndrome, followed by myositis (13.3%). The phylogenetic analysis on the VP1 and 3D regions of prevalent serotypes provided evidence for recombination events among them. EV-A71, an important pathogen previously demonstrated to be associated with paralysis, had also been detected (n = 8) in this study and they all belonged to genotype C4. Great genetic divergence between Yunnan isolates and strains from other regions of the world was revealed. The findings of the study are of great importance for further research on molecular evolution of EV under the circumstance of no specialized EV surveillance system in China.

[Molecular epidemiological analysis of ECHO7 virus isolated from sewage water in Yunnan Province, China].

To investigate the epidemic and evolutionary trends of enterovirus (EV) in the external environment of Yunnan Province, China, molecular typing was performed on 4 EV strains that were isolated from environmental sewage in Yunnan. The VP1 region of isolates was amplified by RT-PCR using universal enterovirus primers, and the amplified VP1 region was sequenced for GenBank BLAST search and genotype analysis. The 4 EV strains were identified as ECHO7. Their nucleotide and amino acid homologies with the VP1 sequences of 68 ECHO7 strains retrieved from GenBank were measured by Mega software analysis. Our findings showed that ECHO7 strains from environmental sewage and population samples were in different evolutionary branches. These strains showed typical geographical and temporal differences; In addition, there were different transmission chains at the same time and in the same area. ECHO7 strains isolated from sewage water and patients with acute flaccid paralysis during the same period in Yunnan belonged to different clusters and evolved at different speeds. Special concerns are needed for this problem. Continuous molecular biological surveillance of human EV in the external environment of Yunnan will provide strong support for early warning of EV diseases.

Complete genome characterization of a novel enterovirus type EV-B106 isolated in China, 2012.

Human enterovirus B106 (EV-B106) is a recently identified member of enterovirus species B. In this study, we report the complete genomic characterization of an EV-B106 strain (148/YN/CHN/12) isolated from an acute flaccid paralysis patient in Yunnan Province, China. The new strain had 79.2-81.3% nucleotide and 89.1-94.8% amino acid similarity in the VP1 region with the other two EV-B106 strains from Bolivia and Pakistan. When compared with other EV serotypes, it had the highest (73.3%) VP1 nucleotide similarity with the EV-B77 prototype strain CF496-99. However, when aligned with all EV-B106 and EV-B77 sequences available from the GenBank database, two major frame shifts were observed in the VP1 coding region, which resulted in substantial (20.5%) VP1 amino acid divergence between the two serotypes. Phylogenetic analysis and similarity plot analysis revealed multiple recombination events in the genome of this strain. This is the first report of the complete genome of EV-B106.

[Study on the molecular typing and epidemiology of non-polio enterovirus in Yunnan Province, China during 2006-2010].

In order to explore the genotype distribution and molecular evolution of non-polio enterovirus (NPEVs)in Yunnan Province,the People's Republic of China, we sequenced and analyzed the partial VP1 coding region of 105 NPEVs isolated from acute flaccid paralysis (AFP) surveillance in Yunnan province during a 5- year study period from 2006 to 2010. The viral genomes of 105 NPEVs were translated to corresponding amino acid sequences and compared with those of the prototype strains, and the phylogenetic tree was constructed among these VP1 nucleotide sequences and other prototype strains from GenBank. Analysis showed that 18 isolates were classified into 7 serotypes of human enterovirus A species, while 77 isolates into 22 serotypes of B and 10 isolates into 4 serotypes of C species. However, we did not isolate any viruses which belonged to human enterovirus D species. Thus, under AFP surveillance, human enterovirus B species accounted for 73. 3% of the 105 isolates and was considered as the predominant one,followed by human enterovirus A(17. 1%) and human enterovirus C(9. 5%). Phylogenetic analysis showed that various serotypes of the virus and the corresponding prototype strains or other representative strains clustered into the same grooup, however, Yunnan strains and prototype strains were located in the different branches (except CA2,EV90 and EV76). The degree of variation was different even among the same genotype strains. This report showed that different genotype strains spread widely in Yunnan Province.

[Genetic characteristics of EV71 in Yunnan Province from 2009 to 2010].

In order to learn about the genetic characteristic of human enterovirus type71 (HEV71) isolated from cases of Hand, Foot and Mouth Disease (HFMD) in Yunnan Province from 2009 to 2010. 50 isolates form HFMD cases were performed entire VP1 coding region amplification by reverse transcription-polymerase chain reaction and sequencing the nucleotide sequences; then the phylogenetic tree was constructed. The complete nucleotide sequences of region VP1 of the 50 strains were all 891nt length coding 297 amino acids. The result of molecular identification of the 50 strains is HEV71. Phylogenetic analysis indicated that 48 EV71 isolates belonged to subgenotype C4a and 2 EV71 isolates belonged to genotype A. From 2009 to 2010, the pathogen of HFMD cases were EV71 strains in Yunnan province, which were co-evolved with isolates from other provinces in mainland of China. There was no significant difference found in the whole sequence of VP1 gene of the strains isolated from different regions or under different diseases occurred, but the spread of genetype A appared in Yunnan Province in 2009.

[Molecular typing of enteroviruses from healthy children in the border areas of Yunnan Province and Myanmar and the genetic characteristics of ECHO7 and ECHO13 in 2009].

To explore the enteroviruses surveillance among healthy children under 15 years old in the border areas of Yunnan Province and Myanmar in 2009. The stool samples were collected from the healthy children under 15 years old who came from the border areas of Myanmar and Yunnan Province, virus isolation and sequencing were conducted for all the 271 samples. 6 strains of polioviruses (PVs) were detected from 271 stools with an isolation rate of 2.8%, which belonged to vaccine strains and 24 non-polioviruses (NPVs) were detected with an isolation rate of 8.9%. 24 NPVs belonged to human enterovirus group B (HEV-B) with 6 serotypes, HEV-A, HEV-C and HEV-D viruses were not isolated. Among them, 13 NPVs were E7 (54.17%) and 5 NPVs were E13 (20.83%). Our results showed that the enterovirus carrying rate in the border areas of Yunnan province was higher than the rate of routine acute flaccid paralysis (AFP) detection system. The HEV-B viruses were the only enteroviruses isolated. The phylogenetical analysis showed that Echovirus 7(E7) and 13 (E13) exhibited genetic polymorphism.

[Distribution of hepatitis B virus genotypes and serotypes in people who had a physical examination in Yunnan Province].

OBJECTIVE: To know genotypes and serotypes of hepatitis B virus (HBV) detected from hepatitis B infected people in Yunnan Province. METHODS: Serum samples were collected from HBsAg carriers detected from people who had a physical examination at Yunnan Provincial Center for Disease Control and Prevention. The S genes of HBV were amplified by nested PCR and the PCR products were sequenced. The viral genotype was identified by phylogenetic analysis. 27 reference sequences corresponding to HBV genotype A to I were obtained from GenBank. According to the amino acid sequences deduced from the nucleotide sequences of S gene, the dominant serotype of HBV detected from these people were confirmed. RESULTS: 39 HBsAg positive serum samples were detected from 2216 people who had a physical examination. The results shows that 76.9% were C genotype; 15.4% were B genotype; 5.1% were D genotype; 2.5% were I genotype. Three serotypes were found. The rates of adw2, adrq+ and ayr serotypes are 71.8%, 17.9% and 10.3% respectively. All of adw2 subtype specimens are C genotype. Among the serum specimens in which both HBsAg and HBeAg are positive, 75% were C genotype and adw2 subtype. CONCLUSION: It is determined that the main genotype and subtype of HBV prevailed in Yunnan province is C genotype and adw2 subtype.

[Molecular identification of hepatitis B virus genotype I in Yunnan Province, the People's Republic of China].

Molecular typing was conducted according to the reported method for one HBsAg positive carrier who had a physical examination in Yunnan Province. The S gene of this HBV sample was amplified by nested PCR and the PCR products were directly sequenced. Blast searching was done on the Genbank database and the sequence were compared with the HBV reference sequences in database. The phylogenetic tree was constructed. Homology analysis of nucleotide and smino acid were performed between the sequences from the sample and the reference sequences corresponding to HBV genotype A to I. Analysis of nucleotide and amino acid identities suggested that the sample belonged to HBV genotype I. The HBV genotype I is the first reported in China.

[Investigation on one vaccine-derived poliovirus (VDPV) case in Yunnan Province].

To find out epidemiological feature of paralytic cases caused by type 2 vaccine-derived poliovirus (VDPV) and the excretion status of the case and to explore the enterovirus infection status among healthy children under five years old around the case in Zhaotong city, Yunnan Province in 2010. Field epidemiological studies at the epidemic area were conducted and a total of 108 stool samples were collected, three from the case, seven from the close contacts and 98 from the healthy children. VDPV was not isolated again from the case; Sabin-like PV strains or VDPV were not isolated from the close contacts and the healthy children; An active search for acute flaccid paralysis cases was conducted in the area, which indicated that the VDPV did not cause virus circulation in local area. Twenty one (20.0%) NPEVs were isolated from 105 stool samples. Among the 21 NPEV isolates, 11 isolates (52.4%) were HEV-A (3 serotypes), 10 isolates (47.6%) were HEV-B (4 serotypes).

[Status of enterovirus infection and molecular identification among healthy children at the areas bordering Myanmar, in Yunnan province, China].

OBJECTIVE: To explore the enterovirus infection status among healthy children under 15 years old in the border areas of Yunnan province that connecting Myanmar. METHODS: A total of 319 stool samples were collected from healthy children in the 10 entrance ports. Enterovirus was isolated from these stool samples and then poliovirus and adenovirus were serotyped by neutralization test using specific anti-sera. All the non-polio enteroviruses (NPEVs) were identified by partial sequencing of VP1 gene. RESULTS: All 53 enterovirus were isolated from 319 stool samples and 16.6% of them carried the virus. 23 polio virus (PVs) and 30 NPEVs were isolated with rates of carrying the virus were 7.2% and 9.4% respectively. 4 adenovirus were also isolated with a rate as 1.25%. 1 isolate could not be amplified by any Pan-enterovirus primers or by RT-PCR so was not able to be sequenced. The results of NPEVs sequencing showed that:1 isolate (3.3%) was classified into 1 serotype of HEV-A while 20 isolates (66.7%) were classified into 11 serotypes of HEV-B and 8 isolates (26.7%) were classified into 3 serotypes of HEV-C. However, we could not isolate any viruses that belong to HEV-D. nt. Result from the aa identify calculation showed that the nt and aa identification between isolates and corresponding standard strains were more than 75% and 85% respectively. The findings were similar to the international standards. CONCLUSION: Our results showed that the rate of carrying the enterovirus especially poliovirus in some areas of Yunnan province that bordering Myanmar was higher than that of rate through the routine acute flaccid paralysis detection system. Of the enterovirus isolated, HEV-B group appeared the predominant with the wide spread of enterovirus serotype. Some newer enterovirus were also detected such as EV73 (2 strains), EV75 (1 strain), EV80 (1 strain) and EV96 (4 strains).

[Molecular identification of human enterovirus 73 in Yunnan Province, the People's Republic of China].

Molecular typing was conducted according to the reported methods for 2 enteroviruses that were isolated from healthy children in the border areas of Yunnan Province with Myanmar. RT-PCR and sequencing were performed with 292/222 primers according to the Oberste's methods. The resulting sequences were blasted against the Genbank database and compared with all available enterovirus database. Analysis of homology at nucleotide and amino acid level identically suggested that the two enteroviruses are human enterovirus 73.

[Study on the molecular typing and epidemiology of non-polio enteroviruses isolated from Yunnan province, China].

OBJECTIVE: This report presented an overview on the epidemiology of enterovirus in Yunnan province, the People's Republic of China. METHODS: A total of 210 strains of non-polioviruses isolated under acute flaccid paralysis surveillance during a 5-year study period from 1997 to 2000 and 2004 were examined. Of the 210 non-polioviruses strains, a total of 12 strains of adenoviruses were serologically identified. The remaining 198 isolates were used for molecular typing, and the viral genomes of 195 nonpolio enteroviruses (NPEVs) were translated to corresponding amino acid sequences and compared with those of the prototype strains. RESULTS: Based on molecular typing, 5 isolates were classified into 5 serotypes of human enterovirus A species while 158 isolates into 34 serotypes of B and 32 isolates into 6 serotypes of C species. However, we did not isolate any viruses which belonged to human enterovirus D species. Thus, under acute flaccid paralysis surveillance, human enterovirus B species accounted for 75.2% of the 210 isolates and was considered as the predominant one, followed by human enterovirus C (12.2%), adenovirus (5.7%), and human enterovirus A (2.4%). CONCLUSION: Although the epidemiological characteristics of NPEVs from Yunnan province remained "unknown", the molecular typing method had provided us a breakthrough to understand the epidemiology of these viruses.