Epidemiology of hand, foot, and mouth disease and genetic characterization of enterovirus A71: a survey from 2007 to 2012 in Linyi of Shandong Province, China / 病毒学报

To investigate the epidemiology of hand, foot, and mouth disease (HFMD) and the genetic characteristics of enterovirus A71 (EV-A71) in Linyi of Shandong Province, China during 2007-2012. The number of reported HFMD cases were obtained from the National Notifiable Disease Reporting System (NNDRS) were analyzed by descriptive epidemiology method; the VP1 region of EV-A71 isolated from HFMD patients in Linyi was amplified and sequenced. Finally, the genetic variability and phylogenecity of VP1 sequences of EV-A71 were analyzed by MEGA 5.0. The results showed that HFMD incidence was reported in each year from 2007 to 2012 in Linyi, and the highest incidence and mortality were reported in 2009, when there were total 14697 cases and 9 of death. The reported incidence was 140.28/100000, and the mortality was 0.086/100000. The peak incidence usually occurred between April and July, and the summit occurred in May. Scattered children accounted for 77.37%-92.00% of all cases. The peak age was 2.5 years during 2007-2009 and 1.5 years during 2010-2012. A total of 1365 laboratory-confirmed HFMD cases were reported in the 6 consecutive years, accounting for 2.98% of the gross number. Among these reports, the ratio of EV-A71 was 44.18%, and the ratio of coxsackievirus A16 (CVA16) was 46.59%. All EV-A71 strains isolated in Linyi during 2007-2012 belonged to the C4a evolutionary branch of C4 genotype. In conclusion, HFMD outbreaks occurred every year in Linyi during 2007-2012. Incidence varied significantly among different counties. The peak incidence in each year lasted from April to July. Most of the patients were children under 3 years of age, and scattered children took the highest proportion. Co-circulation of EV-A71 and CVA16 was the major cause of HFMD in each year. Since the first report of HFMD prevalence caused by EV-A71 (C4a) in 2007, the virus has been prevalent continuously in Linyi for 6 years.

Rash and fever illness caused by herpes simplex virus type 1 needs to be distinguished from hand, foot and mouth disease / 病毒学报

An epidemic of rash and fever illnesses suspected of hand, foot and mouth disease (HFMD) occurred in Gansu Province of China in 2008, laboratory tests were performed in order to identify the pathogen that caused this epidemic. Eight clinical specimens collected from the 4 patients (each patient has throat swab and herpes fluid specimens) with rash and febrile illness, were inoculated onto RD and HEp-2 cells for virus isolation, and the viral nucleic acid was then extracted with the positive virus isolates, the dual-channel real-time reverse transcript-polymerase chain reaction (RT-PCR) was performed to detect the nucleic acid of human enterovirus (HEV) in the viral isolates at the same time. For the viral isolates with the negative results of HEV, a sequence independent single primer amplification technique (SISPA) was used for "unknown pathogen" identification. Totally, 6 viral isolates were identified as herpes simplex virus type 1 (HSV-1). Comprehensive analyses results of the clinical manifestations of the patients, epidemiological findings and laboratory test indicated that this epidemic of rash and febrile illness was caused by HSV-1. The differences among the gG region of 6 HSV-1 isolates at nucleotide level and amino acid level were all small, and the identities were up to 98. 8% and 97.9%, respectively, showing that this outbreak was caused by only one viral transmission chain of HSV-1. HSV-1 and other viruses that cause rash and febrile illnesses need differential diagnosis with HFMD. The etiology of rash and febrile illness is sometimes difficult to distinguish from the clinical symptoms and epidemiological data, the laboratory diagnosis is therefore critical.

Epidemiology and etiology of hand-foot-and-mouth disease in Hainan, 2010 / 病毒学报

To reveal the epidemiological and pathogenic characteristics of Hand-foot-and-mouth disease (HFMD) in Hainan province in 2010, epidemiology data of HFMD reporting cases were analyzed, clinical specimens from 1346 HFMD cases were collected for enterovirus (EV) detection. Viral isolation was performed for EV nucleic acid positive samples. Complete VP1 encoding region of EV71 were sequenced and analyzed with Sequencher (version 5.0) and MEGA software (version 5.0). The epidemiology data showed that all 18 prefectures in Hainan had reporting cases during 2010, with higher incidence in the northeast; and the children less than 4 years old accounted for the majority of the suffered; the epidemic reached peak during September to October, which was different from other Provinces in China. The laboratory results indicated that EV71 and CA16 were identified as the major causative pathogens in Hainan in 2010, however, EV71 infection was absolutely dominant among severe and fatal cases. In addition, some HFMD cases were identified associated with other serotypes of EV infections. Molecular epidemiological analysis showed that all the EV71 strains belonged to C4a evolutionary branch, which is the dominant evolutionary branch in China in recent years, and at least three transmission chains existed. This study has an important information in clarifying the characteristics of epidemics and transmission of HFMD in Hainan, and to provide the guidance for HFMD prevention and control in the future.

Transmission and prevalence patterns of C4a evolutionary lineage of human enterovirus 71 circulating in mainland China, 2008-2010 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To understand the evolutionary relationship between the C4a evolutionary lineage of human enterovirus 71 (HEV71) strains circulating in mainland of China during 2008-2010 and 2008 Fuyang strains and study the prevalence and transmission patterns of 2008 Fuyang strains.</p><p><b>METHODS</b>Download all the complete VP1 ( > or = 891 bp) or approximate complete VP1 (> or = 876 bp) gene nucleotide sequences from GenBank of HEV71 strains circulating in Mainland of China during 2008-2010. And analyze the phylogenetic relationship between Fuyang strains and other provinces' strains using the MEGA software, version 5.0.</p><p><b>RESULTS</b>All of the HEV71 isolates circulating in Mainland of China during 2008-2010 were clustered into evolutionary lineage C4a except for eight strains grouped in the genotype A and one isolate belongs to evolutionary lineage C4b; the homology analysis showed there were 96.5%-100% identity between C4a viruses circulating in mainland China during 2008-2010 and 2008 Fuyang strains, and they were evolved from C4b viruses of 1998. The transmission chains of Fuyang strains were mainly transmitted in Guangdong, Jiangsu, Shanghai, Hunan, Shandong provinces.</p><p><b>CONCLUSION</b>The predominant viruses circulating in Mainland of China during 2008-2010 were evolutionary lineage C4a of human Enterovirus 71; Fuyang transmission chains mainly distributed in southern of China and the Central China around Anhui provinces.</p>

Analysis of genetic characteristics of type II non-wild poliovirus in mainland China, 2010 / 病毒学报

To study the genetic characteristics of 123 type II non-wild polioviruses isolated from acute flaccid paralysis (AFP) cases in mainland China in 2010, provide the scientific basis for maintaining the "polio-free" status, and the switching use of polio vaccine for China. VP1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and the PCR products were then sequenced. The sequence results were analyzed with Sequencher 4.8, BioEdit 7.0.9 and MEGA 5.0. Of 65 strains, nt2909 was found to be a mutation hotspot, and also a neurovirulence determinant in VP1 region. During 2010, two vaccine-derived polioviruses (VDPVs) were isolated from Yunnan province, China and no wild poliovirus (WPV) was isolated. The epidemiological studies and laboratory results of the two VDPVs showed that they were newly discovered VDPVs because of the genetic difference from other VDPVs strains isolated in the world, implying the sensitive poliovirus surveillance network could timely detect the transmission of VDPVs and the importation of WPV.

Genetic characterization of poliovirus isolates from environmental sewage surveillance in Shandong, 2010 / 病毒学报

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.

Genetic characterization of coxsackievirus A16 isolated in Ningxia Hui Municipality in 2008 / 中华流行病学杂志

Objective To study the genetic characterization of coxsackievirus A16 (CVA16)strains isolated during an epidemic of hand, foot, and mouth disease (HFMD) in Ningxia Hui Municipality in 2008. Methods Clinical samples were collected from HFMD patients in Ningxia Hui Municipality and CVA16 strains were isolated by viral isolation methods. Reverse transcriptionpolymerase chain reactions (RT-PCR), specific for CVA16 were performed with these CVA16 strains.Entire VP1 coding region amplification and sequencing were then performed and finally phylogenetic tree was constructed among Ningxia CVA16 strains and CVA16 representative strains of known genotypes and subgenotypes. Results 70 Ningxia CVA16 strains were isolated from HFMD patients in Ningxia in 2008 and the homology of nucleotide and amino acid were 90.8%-100.0% and 98.9%-100.0%, respectively. Phylogenetic characteristics of the strains reconfirmed that they could be divided into two distinct genotypes-A and B. Genotype B could be further divided into the subgenotypes B1 and B2, while all the 70 Ningxia CVA16 strains belonged to the co-circulated clusters B1a and Blb within subgenotype B1, which belonged to 2 viral transmission chains.Conclusion Our results indicated that subgenotype B1 CVA16 strains continued to circulate over a wide geographic area of mainland China since the first reported episode in Shenzhen city in 1999. Like other CVA16 strains isolated elsewhere in China, both Bla and Blb evolution branches were co-circulating in Ningxia Hui Municipality. Based on the close phylgenetic and chronological relationship with CVA16 isolated in other countries and regions near China. Our data confimed that these strains co-evolved and co-circulated with those from neighboring countries and regions.

Outbreak of acute hemorrhagic conjunctivitis in Beijing City in 2007 caused by coxsackievirus A24 variant: molecular identification and phylogenetic analysis / 病毒学报

In 2007, an outbreak of acute hemorrhagic conjunctivitis (AHC) occurred in Beijing. In order to identify the etiology of this outbreak, 57 eye conjunctival swabs were collected from 57 outpatient patients, and detected for adenovirus, human enterovirus 70 (HEV70) and Coxsackievirus A24 variant (CVA24v) genes by using RT-PCR or PCR methods. The results showed that 38 were positive for CVA24v, the positive rate was 66.7%, but none was positive for HEV70 and adenovirus, showing that this outbreak was caused by CVA24v. 9 viral isolates were obtained from 57 clinical specimens by using viral isolation method, and all were identified as CVA24v by molecular typing method. All 9 CVA24v isolates were performed by VP1 sequencing, the results showed that except for strain 0744/BJ/CHN/2007, the variability at nucleotide acid level and amino acid level among other 8 CVA24v were relatively low, and the homologies were more than 99.6% and 100.0%, respectively; the homologies of nucleotide acid and amino acid between strain 0744/BJ/CHN/2007 and other 8 CVA24v were 96.8%-97.2% and 99.7%, respectively. Phylogenetic analysis of 9 CVA24v revealed that they represented the Clade 4 and Clade 5 in Group I, showed that this outbreak was caused by at least 2 viral transmission chains. Comparing to 3C region of CVA24v frequently used before, VP1 region was considered as the most rigorous target for molecular epidemiology study of CVA24v. To enhance the research of sero-epidemiology and molecular epidemiology of CVA24v and to know the genetic characterizations and molecular evolution of CVA24v are most important to prevent and control the outbreaks of AHC in China.

The epidemiologic and virological analysis of an outbreak of hand, foot, and mouth disease in Inner Mongolia in 2007 / 病毒学报

In 2007, an outbreak of hand, foot, and mouth disease (HFMD) occurred in Jungar Banner, Erdos city, Inner Mongolia Autonomous Region, China Fever, vesicular exanthema on the hands, feet, mouth, and buttocks were presented in most of the patients. Most of the patients were infants less than 5 years old, and an obvious peak period appeared in the disease outbreak. From 28 hospitalized patients, 23 stool specimens and 6 throat swab specimens were collected for enterovirus isolation, and 15 enteroviruses were isolated, 9 were identified as Human Enterovirus 71 (HEV71, the isolation rate is 31.03%) and 1 was identified as Coxsackievirus A16 (CVA16). According to the comprehensive analysis of clinical manifestation, epidemiology data and laboratory results, this outbreak was probably mainly caused by HEV71. The variability at nucleotide acid level and amino acid level among 9 HEV71 was relatively low, and the homology was more than 99.4% and 99.0% respectively, showing that this outbreak was caused by only one viral transmission chain. Phylogenetic analysis of 9 HEV71 strains isolated during this outbreak revealed that they all belonged to subgenotype C4, which has been continuously circulating in mainland China since its first reported occurrence in Shenzhen City in 1998. It was also suggested that subgenotype C4 HEV71 had a widely distribution and transmission in mainland China.

Seroepidemical study of Coxsackievirus A 16, in four provinces, China, 2005 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the situation of 1- 5-years-old children's antibody against Coxsackievirus A group 16 strain (CVA16) in Guangdong, Heilongjiang,Yunnan Province and Xinjiang Uygur Autonomous Regions, China, 2005, it can offer scientific evidences for preventing and controlling CVA16 causative hand-food and mouth disease.</p><p><b>METHODS</b>Using microneutrilization test, to study 503 serum samples randomly selected from sera collected in 2005.</p><p><b>RESULTS</b>Positive rate of anti-CVA16 antibody were 41.90%, 9.40%, 40.00% and 34.40% in Guangdong, Heilongjiang,Yunnan and Xinjiang, respectively. Antibody titer was relative low (average, 1: 6.1) and there was no statistical difference of geometry mean of antibody titer (GMT) among Guangdong, Heilongjiang, Yunnan (F = 0.97, 0.40, 1.06, respectively; P > 0.05), while there had statistical difference of GMT between Heilongjiang and other three regions( F = 10.61, P < 0.00).</p><p><b>CONCLUSIONS</b>There had probably existed local epidemic in some regions of Guangdong, Heilongjiang, Yunnan Province and Xinjiang Uygur Autonomous Regions, China, 2005 or even before, but the area and degree of transmission and epidemic had difference. Children aged from 1- 5-years-old were relatively susceptible population of CVA16 infection.</p>

In vitro study on the effect of ribavirin reducing the EV71 replication / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Observe the effect of Ribavirin on reducing the EV71 replication, inactivating EV71 and protecting the RD-A cells against the EV71 infection in vitro.</p><p><b>METHODS</b>Using the EV71 isolated from Anhui Fuyang HFMD outbreak, the effect of Ribavirin on RD-A cells during the EV71 infection was observed.</p><p><b>RESULTS</b>In the experiment of Ribavirin inhibiting the EV71 replication, comparing with the no-Ribavirin-dealed virus control group, the group of 1:640 dilution of Ribavirin delayed the CPE for 2 days, while the normal cell group was growing very well. In the experiment of protecting cell from EV71 infection, comparing with the no-Ribavirin-dealed virus control group, the group of 1:8 dilution of Ribavirin delayed the CPE for 4 days. In the experiment of Ribavirin effect on the inactivation of EV71, the group of 1:40 dilution of Ribavirin delayed the CPE for 2 days comparing with the virus control group.</p><p><b>CONCLUSION</b>Ribavirin could inhibit the replication of the EV71 and inactivate the EV71 in vitro. Additionally, Ribavirin could protect RD-A cells from EV 71 infection. The observation will contribute to EV71 infection control and quick medicine therapy.</p>

Reevaluate the effect of G-480 point mutation that determines the neurovirulence of type I vaccine polioviruses / 病毒学报

Whole genome sequencing of 9 type I circulating vaccine-derived polioviruses (cVDPVs) isolated in Guizhou Province in China revealed that reverse mutations did not occur in G-480 and U-525 which are known as the most important neurovirulence determinate sites, while other known neurovirulence determinate sites such as A-2438, A-2795, C-6203 and G-7441 did revert to Mahoney type. 5 type I cVDPVs were selected for neurovirulence test on PVR-Tg21 transgenic mice which express human poliovirus receptor gene based on their different nucleotide sequences, they all showed higher neurovirulence than P1/Sabin strain, and the neurovirulence of CHN8184 and CHN8229-1. 1 were comparable to that of wild type P1/Mahoney. The neurovirulence of CHN8229-1.1, CHN8229-2 and CHN8229-3 presented a trend of decreasing, but still laid in high level. There were 7 nucleotide mutations between CHN8229-1.1 and CHN8229-2, and only 2 between CHN8229-2 and CHN8229-3 in their whole genomes, but the neurovirulence among them were relatively different, showing that there must be some unknown neurovirulence determinate sites among these mutations. Computer predicted RNA secondary structure of stem-loop V of the poliovirus 5' NCR of Guizhou type I cVDPVs was relatively stable. In the situation that no reverse mutation occurred in G-480, some type I cVDPVs already showed high neurovirulence nearly equal to P1/Mahoney, it meant that the effect of G-480 point mutation that determined neurovirulence of P1/Sabin strain has been overestimated, G-480 was not the only important site to determine neurovirulence in P1/Sabin strain, others also may play the very important role. More details are needed to elucidate the mechanism of attenuation in type I polioviruses.