Evolutionary phylogeography reveals novel genotypes of coxsackievirus A24 variant and updates the spatiotemporal dynamics in the population with acute hemorrhagic conjunctivitis.

OBJECTIVES: The coxsackievirus A24 variant (CVA24v) has raised a remarkable concern because of its main etiological role in acute hemorrhagic conjunctivitis. METHODS: We conducted a retrospective study to summarize CVA24v isolated from acute hemorrhagic conjunctivitis outbreaks and acute flaccid paralysis surveillance in Shandong province, China during 1988-2020. Phylogenetic and phylogeographic methods based on the VP1 coding region were used to determine the CVA24v origin, spatiotemporal dynamics, and evolution. Also, the positive selection sites in the VP1 gene were identified and exhibited in the tertiary structure. RESULTS: The global CVA24vs were classified into eight genotypes (GⅠ-GⅧ). Here, 12 CVA24v isolates were detected, of which five strains were typed as two novel genotypes (GⅦ and GⅧ) and reported first in the world. The time to the most recent common ancestor of the global CVA24v was estimated around March 1965 and evolved with 5.573 × 10-3 substitutions/site/year. Four residues under positive selection were detected, and residue 146T might be adapted in the CVA24v pandemic. Phylogeographic analysis indicated that China was the main source sink for CVA24v dispersion in a long-lasting global pattern. CONCLUSION: Our study updated the epidemiological characteristics of CVA24v and enabled a better understanding of the molecular mechanism underlying different genotypes. The results provided new insights into the CVA24v origin, spatiotemporal dynamics, and possibly, the determinants of viral tropism and pathogenicity.

[Genetic characterization of echovirus 6 isolated from meningitis and encephalitis cases in Shandong Province, China].

To analyze the genetic characteristics of echovirus 6 (E6) isolated from meningitis and encephalitis cases in Shandong Province, China, we collected cerebrospinal fluid samples from meningitis and encephalitis cases in Shandong Province from 2007 to 2012 for virus isolation. Viral RNAs were extracted from positive isolates, and complete VP1 coding regions were amplified by RT-PCR and sequenced. Homology comparison and phylogenetic analysis were performed. Six isolates were identified as E6 by microneutralization assay and molecular typing. The homology analysis showed that the six isolates had 78. 6%-99. 8% nucleotide and 95. 5%-100. 0% amino acid identities with each other, as well as 76. 9%-78. 4% nucleotide and 92. 3%-95. 1% amino acid identities with the prototype strain (D' Amori). The phylogenetic analysis based on the integrated VP1 sequences indicated that all Shandong E6 isolates could be separated into four clusters, designated as A, B, C, and D. The six E6 isolates belonged to clusters A, B, and D. Our study reveals high genetic differences between Shandong E6 isolates and suggests different transmission lineages of E6 co-circulated in Shandong Province.

[Research progress in human enterovirus recombination].

Complex genetic variation has been known to occur during the transmission of human enterovirus (HEV), and the HEV virulence and pathogenicity enhanced by genetic recombination also pose a serious threat to human health. In recent years, the interest in recombination mechanism of genetic plasticity has been renewed with the emergence of pathogenic recombinant circulating vaccine-derived polioviruses, which were implicated in poliomyelitis outbreaks in several regions of the world with insufficient vaccination coverage. This paper reviews recent research progress in HEV genome, including evolutionary characteristics, recombination types, and in vitro recombinant construction.

[Temporal fluctuation and genetic characterization of echovirus 11 isolates upon surveillance of environmental sewage in Shandong Province, China].

We wished to analyze the genetic characterization of echovirus 11 (Echo11) from samples of environmental sewage in Shandong Province (China). The VP1 coding region was typed as the strains were amplified. Phylogenetic analyses on the VP1 sequences from these isolates, strains isolated from AFP cases in the period 1994-2010 and others published in GenBank were conducted. From 2011 to 2012, 94 Echo11 strains were isolated from samples of environmental sewage in Jinan and Linyi City in Shandong Province. Numbers of Echo11 were seasonal and reached peaks in the summer and autumn in both cities; A- mong these isolates, nucleotide (nt) identities were 89.5%-100.0% whereas amino acid (aa) identities were 95.4%-100.0%. The nt and aa identities were 76.6%-79.7% and 90.4%-92.5% between those strains and the prototype (Gregory) strain of Echo11, respectively. All isolates from Shandong Province were the A genotype and the strains evolved very rapidly, which suggested that several transmission chains was co-circulating. We described the temporal fluctuation and genetic characterization of Echo11 isolates from surveillance of environmental sewage in Shandong Province, thereby providing important information for exploring the dynamic change and genetic variation of circulating human enteroviruses in this Province in China.

[Seroprevalence of the newer enterovirus types A71, A90 and B87 in healthy individuals in Shandong Province, China].

This study aimed to investigate antibody levels of the newer human enteroviruses (EV) A71, A90, and B87 in the population of Shandong Province, and provide a scientific basis for the development of prevention and control measures. In this study, serum specimens were collected from 400 individuals living in Yantai city, Shandong Province in 2010. EV-A71, A90, and B87 antibodies were detected using neutralization tests, and the results were analyzed by statistical methods. It was found that the positive neutralizing antibody rates of EV-A71, A90 and B87 in the population were 46.0%, 8.8%, and 47.0%, respectively. Their geometric mean titers (GMT) were 1 : 5.20, 1 : 1.49, and 1 : 4.02, respectively. Positive antibody rates for EV-A71 and EV-B87 were lowest in the 1-yr and 7-mo age groups, respectively. Positive rates increased gradually with age, and become consistent in the population aged >5 years. Positive antibody rates of EV-A90 were consistent across all age groups. Maternal antibody levels of EV-A71 declined rapidly after birth, and the increase in seroprevalence among 3-7 years old children implied that most EV-A71 infections occurred in preschool and early elementary school children. High positive antibody rates of EV-B87 in healthy individuals, especially children, implied that there may be an immune barrier within the general population. The population monitoring of EV-A90 should be strengthened, as its positive antibody rate is low.

[Isolation and characterization of a human enterovirus 74 isolated in Shandong Province, China].

Human Enterovirus HEV 74 is a new member of species Human enterovirus B (HEV-B). To understand its evolution and restructuring characteristics, we report the complete genome sequence of a HEV74 strain 05293/SD/CHN/2005(abbreviated as 05293) isolated from an acute flaccid paralysis (AFP) case in Shangdong Province, China, 2005. Analysis of the complete genomic sequence of 05293 showed that its genome was collinear with that of previously described 2 HEV74 strains, except for insertions and deletions at the 5'NTR and the 3 NTR regions. The complete genome sequence of strain 05293 displayed 80. 8% nucleotide and 96% amino acid identity to the prototype strain USA/CA75-10213, and 80. 6% and 95. 9% to another isolated strain Rikaze-136. The P1, P2 and P3 coding regions of strain 05293 displayed 81. 5%, 80. 0%, 79. 7% nucleotide and 95. 9%, 96. 0%, 96.2% amino acid identity to the prototype strain USA/CA75-10213, and 81. 9%, 78. 8%, 79. 5% and 95. 9%, 96. 1%, 95. 7% to strain Rikaze-136, respectively. The phylogenetic tree and Simplot analysis on 05293 and HEV-B genome sequences were performed, and the result indicated frequent recombination within HEV-B.

[Progress in research of new human enterovirus types].

More and more new human enteroviruses (HEVs) types were identified with the broad application of the molecular serotyping methods for enteroviruses. Since enterovirus 71 (EV71) was first reported in 1969, numerous epidemic outbreaks associated with new enteroviruses have occurred all around the world, and pose a significant threat to public health . The epidemics of hand, foot and mouth disease (HFMD) caused by EV71 infection in China have raised great concern of global scholars. This paper reviewed research progress in recent years of the molecular typing, evolution, epidemiology, and pathogenesis attributable to new enterovirus types.

[Identification and genetic characterization of coxsackieviruses A2, 6, 8 and 12 isolated in Shandong Province].

In previous study, molecular typing method was performed to identify human enteroviruses (HEVs) isolates collected from acute flaccid paralysis (AFP) cases from 1989 to 2011 and hand, foot and mouth disease (HFMD) patients, and 8 HEV-A serotypes were identified. In order to explore the genotypes and molecular evolution characteristics of HEV-A in Shandong province, viral RNA of the remaining isolates was extracted and entire VP1 coding region was amplified, sequenced and identified with HEV-A primers. Another 7 HEV-A Shandong isolates were obtained, and identified as Coxsackievirus A (CVA) 2, 6, 8 and 12 by molecular typing method. Homologous comparison showed that the nucleotide acid identities of Shandong strains ranged from 80.8% to 85.0% with prototype strains. Phylogenetic analysis based on VP1 sequences indicated that CVA8 and CVA12 strains were genetically related with domestic strains. However, CVA2 and CVA6 strains were distinct from both domestic and foreign strains. In addition, multiple transmission chains of CVA2 and CVA6 existed within Shandong province. So far, a total of 12 HEV-A serotypes were identified in Shandong province. This study enriched the distribution of serotypes and genetic evolution characteristics of HEV-A isolates in Shandong, and revealed different transmission chains of CVA2, 6, 8, 12 serotypes co-circulated in Shandong province or in China.

[Identification and molecular characterization of human enteroviruses type 74, 80, and 87 isolated in Shandong Province, China].

An increasing number of new types of human enteroviruses (HEV) have been identified with the application of molecular typing method based on VP1 sequence analysis. In this study, the non-polio enteroviruses (NPEV) isolated from acute flaccid paralysis (AFP) cases in Shandong Province, China were typed via molecular typing method, and 1 EV74, 3 EV80 and 1 EV87 strains were identified. Homologous comparison revealed EV74, EV80 and EV87 Shandong strains had 81.4%, 76.4%-81.7%, and 80.3% VP1 identities with prototype strains. Phylogenetic analysis suggested a remote genetic distance with other strains. This is the first report of EV74 and EV87 in mainland China, and the low isolation rate in AFP surveillance suggested these three serotypes has not been the predominant viruses in China.

[Identification and genetic characterization of coxsackievirus A24 isolated from patients with acute hemorrhagic conjunctivitis in Shandong Province].

To identify the pathogen of acute hemorrhagic conjunctivitis (AHC) in Shandong Province in 2010, eye mucous swab samples were collected from 26 patients in Qingdao and Linyi City. Real time-PCR assays for EV70, CVA24 and Adenovirus were performed on these samples. The result showed 17 samples (65.39%) were CVA24 positive while all the samples for HEV70 and Adenovirus detection were negative, which implied that CVA24 was the causative pathogen of this outbreak. A total of 10 virus strains isolated on Hep-2 cells were identified as CVA24 through VP1 amplification and nucleotide sequence analysis. The nucleotide and amino acid homologies on VP1 region among these isolates were 99.3%-100.0% and 99.5%-100.0%, respectively, and the strains aggregated together to one clade in phylogenetic tree. These results showed that the CVA24 circulating in Qingdao and Linyi City belonged to one transmission chain. Shandong CVA24s segregated into 5 different clades, and great nucleotide divergence was observed be tween AHC isolates and others.

[Genetic characterization of poliovirus isolates from environmental sewage surveillance in Shandong, 2010].

To investigate the genetic characteristics of poliovirus isolates from environmental sewage surveillance in Shandong province, we collected sewage samples in Jinan and Linyi City. Serotyping and VP1/ 3D sequencing were performed on polioviruses isolated from the concentrated sewage samples, and VP1 mutation and recombination were analyzed. Thirty-two of sewage samples were collected, and polioviruses were detected in 10 of the samples with a positive rate of 31.3%. Eighteen Sabin strains were isolated including three type 1, nine type 2, and six type 3 polioviruses, and the number of nucleotide substitutions in VP1 coding region varied from 0 to 4. Recombination was found in three Sabin 2 and four Sabin 3 polioviruses. Analysis of neurovirulence sites of VP1 revealed that one Sabin 1 vaccine strain had a nucleotide change of A to G at nt 2749, one Sabin 2 strain had a nucleotide change of A to G at nt 2908, three Sabin 2 strains had a nucleotide change of U to C at nt 2909, and all six Sabin 3 strains had a nucleotide change of C to U at nt 2493. Poliovirus vaccine strains could be isolated from environmental sewage with a high rate of gene recombination and back mutation of neuvirulence-associated sites. None of wild-type poliovirus or vaccine-derived poliovirus was detected.

[Study on the genetic characterization and molecular epidemiology of echovirus 11 strains isolated in Shandong province, China].

OBJECTIVE: To analyze the evolution and genetic characterization of echovirus 11 (Echo11) from the acute flaccid paralysis (AFP) cases in Shandong province. METHODS: Isolation of Enterovirus was performed from stool samples of AFP cases from 1994 to 2009. All positive strains were sero-typed by neutralization test. Entire VP1 coding region from 27 strains typed as Echo11 was amplified by reverse transcription-polymerase chain reaction (RT-PCR), and subsequently phylogenetic analyse on VP1 sequences from these strains and others published in GenBank were conducted. RESULTS: From 1994 to 2009, altogether 915 non-polio enterovirus (NPEV) strains were isolated with 79 (8.6%) isolates serotyped as Echo11. There were 876 nucleotides (nt) in the complete VP1 genes, encoding 292 amino acids (aa). The nt identities were 76.4% - 100.0% among those Echo11 Shandong strains with the aa identities as 91.4% - 100.0%. The nt and aa identities were 77.7% - 80.7% and 90.7% - 94.8% between Shandong strains and prototype strains, respectively. CONCLUSION: All Echo11 strains could be divided into four genotypes. Shandong strains that forming three (A1, A2 and C1) new sub-genotypes, with every sub-genotype had several brands. Sub-genotype A1 appeared to be the lately circulating one.

[Genetic analysis of echovirus 30 isolated from acute meningitis/encephalitis syndrome cases in Linyi city of Shandong province, China].

OBJECTIVE: To investigate the pathogen spectrum constitution of HEV related to acute meningitis/encephalitis syndrome (AMES) and to analyze the genetic characterization of the echovirus 30 (Echo30) isolates. METHODS: Cerebrospinal fluid, stool, and/or throat swab specimens from 101 AMES cases were collected for virus isolation with RD and HEp-2 cell lines in Linyi city from April to December, 2010. After typing by neutralization test, the entire VP1 gene of the isolates were amplified by RT-PCR and sequenced. Homologous comparison was carried out and phylogenetic analysis were performed. RESULTS: HEVs were isolated from 47 specimens of 34 patients (4 from cerebrospinal fluid, 18 from throut swab and 25 from stool), with 11 Echo30 isolates identified by microneutralization assay and molecular typing method. In alignment of VP1 sequences with global Echo30 isolates, relative high nucleotide homologies (91.2% - 95.4%) with Taian, Zhangqiu and Jiangsu isolates and high divergences (17.9% -19.6%) with prototype strain were observed. Nucleotide divergences among Linyi isolates were 0 - 10.2%, and 3 lineages were revealed via phylogenetic analysis, reflecting 3 transmission chains of Echo30 co-circulated in Linyi city, 2010. CONCLUSION: Echo30 was the predominant serotype of AMES cases in Linyi city, 2010. These isolates possessed considerable divergence with Echo30 from other countries.

[Genotype distribution of enterovirus group B isolated in Shandong Province, China].

In order to explore the genotype distribution of human enterovirus group B (HEV-B) in Shandong Province and to study the correlation between HEV-B serotypes and disease outbreaks, we sequenced and analyzed the entire VP1 coding region of HEV-B isolated from acute flaccid parolysis (AFP) system and disease outbreaks in Shandong province during 1998-2008. All together twenty nine HEV-B serotypes were identified, including twenty Echovirus (ECHO) serotypes, five Coxsackievirus B (CVB1-5) serotypes, one Coxsackievirus A9(CVA9) serotype, and newer enteroviruses EV73, EV75, and EV97. E11, CVB3, E6, E14 and E25 were the five frequently isolated serotypes from AFP surveillance system. CVB3, CVB5 and ECHO30 were the major causative agent of aseptic meningitis in Shandong province. Comparison of nucleotide sequence homology showed 75.4%-99.6% inter-typic identities within Shandong strains, and 73.8%-85.2% identities with prototype strains. Amino acid sequence comparison showed the differentiation was not much. Our research showed different serotypes possessed distinct time-cycling pattern, and different sub-genotypes could be further classified according to the inter-typic genetic distance. Thereby the route and range of transmission of HEV could be determined.

[Genotype distribution of enterovirus group C in Shandong Province, China].

Human Enterovirus C group (HEV-C) includes 17 serotypes, which can not be serotype-identified by neutralization test using antiserum pool for NPEV. In order to elucidate the genotypes and molecular evolution of HEV-C in Shandong Province, We selected the strains isolated from AFP cases between 1994-2009 to perform reverse transcriptase-polymerase chain reactions (RT-PCR) by the primers specific for entire VP1 coding gene of HEV-C and sequencing. The phylogenetic tree was then constructed among these VP1 nucleotide sequences and other prototype strains. Totally 12 Shandong local strains were obtained and separated into 4 genotypes, CVA20, CVA21,CVA24 and EV 96. The homologous comparison and phylogenetic analysis showed Shandong strains were distinct from prototype strains in each genotype. This report showed that different genotype HEV-C strains spread widely in Shandong Province.

[Identification and phylogenic analysis of Coxsackie-virus B5 during an outbreak of aseptic meningitis in Shandong].

OBJECTIVE: To identify the pathogen that caused an outbreak of aseptic meningitis in Shandong province in 2005. Phylogenic analysis was carried out on Coxsackie-virus B5 (CVB5) which was isolated during this outbreak. METHODS: 78 stool and 58 cerebrospinal fluids (CSF) specimens were collected from some inpatients during this outbreak. Virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) was then performed. Phylo-genetic trees based on entire and partial VP1 sequences were constructed among CVB5 isolates and others published in GenBank. RESULTS: The isolation rates of stool and CSF specimens were 38.5% (30/78) and 48.3% (28/58) respectively. Among the results of serotype identification and molecular typing of 58 positive isolates, 54 were identified as CVB5, 2 as ECHO24, 1 as CVB3 and 1 as CVA9. Results from viral investigation showed that CVB5 was the main pathogen causing this outbreak. Data from homological comparisons indicated that Shandong strains had the highest nucleotide acid identity with the Zhejiang/12/02 strain (97.5% - 97.8%), and lower identity (78.3% - 78.6%) with the prototype strain (Faulkner strain). Phylogenic tree in VP1 region showed that CVB5 could be separated into four genotypes. Isolates of this outbreak belonged to genotype D. CONCLUSION: CVB5 was the major etiological agent correlated with this outbreak. The shift of predominant genotype might serve as one of the causes that associated with the outbreaks of aseptic meningitis.

[Identification and genetic characterization of human enterovirus type 73, 75, and 97 strains of specie B isolated in Shandong province].

Molecular typing was conducted for three human enteroviruses (HEV) isolated from acute flaccid paralysis (AFP) cases in Shandong province, China. RNAs from virus supernatants were extracted and complete VP1 genes were amplified by RT-PCR and sequenced. Genotypes of these isolates were identified as HEV type 73, 75 and 97, respectively by BLAST program. Homology and phylogenetic tree analyses were performed. Sequence analysis of VP1 gene showed significant variation compared with prototype strains. This study presents the genetic characteristics of HEV 73, 75 and 97 of specie B in Shandong Province, and the first report of HEV97 in China.

[Genotype distribution of enterovirus A species isolated in Shandong Province, China].

In order to study the genotypes and molecular evolution of human enterovirus (HEV) A species in Shandong Province, Stool samples were collected from AFP and HFMD patients in Shandong Province and virus isolation was performed. Reverse Transcription-Polymerase Chain Reactions (RT-PCR) specific for EV71 and CVA16 were performed with the virus isolates from HFMD patients. Positive isolates were selected for entire VP1 coding gene amplification and sequencing. Isolates with negative PCR results and isolates from AFP patients were selected for entire VP1 coding gene amplification and sequencing using primers specific for HEV A species. Phylogenetic tree was constructed among these VP1 nucleotide sequences and of other strains. Altogether 293 strains classified into 8 genotypes were isolated. The homologous comparison and phylogenetic analysis showed Shandong strains were distinct with prototype strains in every genotype. This report presents an overview of HEV-A in Shandong Province.

[Molecular characterization of coxsackievirus B3 isolated from an outbreak of aseptic meningitis in Shandong Province, China].

OBJECTIVE: To identify the pathogen caused an outbreak of aseptic meningitis in Tancheng county of Shandong province in 2008, and to analyze the molecular characterization of VP1 gene of the Coxsackievirus B3(CVB3) isolates. METHODS: Stool and cerebrospinal fluid(CSF) specimens were collected from this outbreak for virus isolation with RD and Hep-2 cell. After typing by neutralization test, the VP1 gene of the isolates were amplified by RT-PCR and sequenced. Homologous comparison and phylogenetic analysis were performed. RESULTS: 35 strains of enteviruse were isolated from 22 stools and 120 CSFs(7 from stools and 28 from CSFs), 34 strains identified as CVB3 and 1 as Echovirus 30(ECHO30) by neutralization test. The nucleotide homologies were 90.5%-100.0% in the partial VP1 gene (381 bp) among 34 CVB3 isolates. Homology comparisons indicated that Shandong strains have the identity of 79.5%-81.6% with the CVB3 prototype strain Nancy. 012/2008TC/SD/CHN and 177/2008TC/SD/CHN showed the highest nucleotides homologies (98.2% and 91.0% respectively) with Fuyang19 strain of Anhui province in 2008 in complete VP1 gene. The phylogenetic tree based on complete VP1 genes showed that all the CVB3 correlated with aseptic meningitis in China recently came from the same evolution linkage and formed a monophyletic cluster. CONCLUSION: The causative agent of this outbreak of aseptic meningitis was CVB3. CVB3 circulated in China was genetically different from other countries.

[Cloning and sequence analysis of envelope glycoprotein G2 gene of hantavirus in Shandong province].

OBJECTIVE: To construct the cloning vector of glycoprotein G2 gene of hantavirus (HV), to analyze the sequence of G2 gene by the phylogenetic tree, and to study the differences among glycoprotein G2 genes from the world around. METHODS: Envelope glycoprotein G2 gene was amplified from four specimens of Shandong province by RT-PCR, and the product recombined into the PMD-18T vector. The clones that carry the G2 gene were identified. After sequencing, the gene sequence was handled with the software DNASTAR, compared with 24 strains worldwide and the phylogenetic tree was drawn. RESULTS: HV G2 gene was amplified by RT-PCR from 4 specimens, named GM04-38.G2, ZB8.G2, JUN5-14.G2, RCH5.G2, respectively. The map of the phylogenetic tree showed that all the 4 strains belonged to SEO-type hantavirus. The analysis of the sequence showed that all the four HV strains had the highest rates of homology with Z37 strain. The sequence homology of SEO-type HV strains was from 82.3% to 99.8%. CONCLUSION: The four cloning vectors containing the glycoprotein G2 genes were successfully constructed. Envelope glycoprotein G2 gene of four specimens from Shandong province had high homology rates.