[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].

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.

[Identification and genetic characteristics of five non-polio enteroviruses (NPEVs) multiplicated on L20B cell line].

OBJECTIVE: To identify and describe the genetic characteristics of non-polio enteroviruses (NPEVs) which could multiplicated on L20B cell line, and discuss the impact to isolation rate of polioviruses. METHODS: 5 L20B positive isolates were collected from stool samples of patient with acute flaccid paralysis (AFP) and health children in Guangxi Zhuang autonomous region and Ningxia Hui autonomous region. These isolates were analysed using VP4 nucleotide sequencing method. The completed VP, coding regions were amplified and sequenced. Finally, phylogenetic tree were constructed base on completed VP1 coding regions sequences of these L20B positive isolates and those downloaded form GenBank database. RESULTS: The 5 NPEVs were all identified as coxsackieviruses group A (CVA). Among them, 1 was identified as CVA4, 1 was identified as CVA8 and 3 were identified as CVA10, and all of them belonged to human enterovirus species A (HEV-A). Nucleotide homology analysis and phylogenetic analysis base on completed VP1 coding regions sequences showed that all these 3 serotypes of NPEVs clustered independently, and they were all native circulated viruses in mainland China. CONCLUSIONS: 5 NPEVs in this study belonged to HEV-A, covered 3 serotypes, CVA4, CVA8 and CVA10. Due to the high sensitivity of primarily inoculation of poliovirus onto L20B cell, the isolation rate of polioviruses could not be affected so much. Hand, foot, and mouth disease (HFMD) outbreaks in mainland China in recent years, and it was found that HEV-A is also pathogen of HFMD. Identification of these viruses have current significance to differential diagnosis of poliovirus infection and HFMD control and prevention.

[Genetic characterization of coxsackievirus A16 isolated in Ningxia Hui Municipality in 2008].

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 transcription-polymerase 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 B1b 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 B1a and B1b 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 confirmed that these strains co-evolved and co-circulated with those from neighboring countries and regions.

[Multiple viral transmission chains of human enterovirus 71 co-circulated in Gansu Province in 2008].

OBJECTIVE: To study the molecular evolution of Human enterovirus 71 (HEV71) isolated from clinical specimens of Hand, Foot and Mouth Disease (HFMD)patients in Gansu in 2008. METHODS: Clinical samples including stools, throat swabs and vesicle fluids were collected from HFMD patients in outpatient service in Gansu province and then viral isolation was performed. Reverse Transcription-Polymerase Chain Reactions (RT-PCR) specific for HEV71 were performed with the viral isolates. Then 20 HEV71 isolates were selected randomly for entire VP1 coding gene amplification and sequencing, finally phylogenetic tree was constructed among these 20 Gansu HEV71 strains and 38 HEV71 representative strains of known genotypes and subgenotypes. RESULTS: The nucleotide acid and amino acid of 20 Gansu HEV71 strains were closed to HEV71 strains isolated in Mainland China since 1998, especially has the highest homology to HEV71 stains circulated in mainland China in 2008 and it showed that all strains clustered within the C4b evolution branch of C4 Subgenotype. But there was some difference in the nucleotide and the amino acid in VP1 region among the 20 Gansu HEV71 strains, the homology were 92.5%-100.0% and 96.9%-100% respectively. 20 HEV71 strains lie in several lineages in the phylogenetic tree, and it revealed that these strains belonged to at least 7 viral transmission chains. CONCLUSIONS: The HEV71 strains circulated in Gansu province in 2008 were all belong to C4b evolution branch of C4 subgenotype. Phylogenetic analysis revealed these HEV71 strains located in different lineages,and a close phylogenetic and chronological relationship with HEV71 isolated in other provinces in mainland China had been observed in 2008. This confirmed Gansu HEV71 strains had not evolved independently, but coevolved with the HEV71 strains in other provinces in mainland China.

[Seroepidemiology investigation of HEV71 in healthy children of 1-6 years old in three counties of China in 2005].

OBJECTIVES: To investigate human enterovirus 71(HEV71) antibody levels among children in China in recent years, to find the epidemic status of hand, foot and mouth diseases (HFMD) in children, and to provide a scientific base for the HFMD prevention and control. METHODS: 371 sera specimens were colleted from 371 children of 1-6 years old in 3 counties of 3 provinces in 2005. All sera proposed to be detected for measles antibodies investigation. HEV71 antibody was detected by using neutralization test, and then the results were analyzed by statistic methods. RESULTS: 164 sera were positive for HEV71 antibody, the positive rate was 44.2%. The distribution of HEV71-positive rate was associated with age of the children, the positive rate was increasing with the age increasing. Children under 3 years old had lower antibody positive rate. The GMT decreased with the increasing of ages. CONCLUSION: HEV71 infections occurred among children in 3 counties of 3 provinces before 2005, and children under 3 years old were main susceptible population due to their low HEV71 antibody level, It is important to take the preventive measures to protect them from HFMD.

[Genetic characterization of enterovirus 71 stains isolated in Ningxia province in 2008].

OBJECTIVE: To study the genetic characterization of enterovirus 71 (EV71) strains isolated from specimens of patients with hand-foot-mouth disease (HFMD) in Ningxia province in 2008. METHODS: All the stool, throat swab and vesicle samples that collected from patients with HFMD were cultured. The positive isolates were identified by reverse transcriptase PCR (RT-PCR) with specific primers of EV71. Complete VP1 gene sequences (891 nucleotides) of 29 strains (part of 93 EV71 strains) were determined and compared with A, B and C genotype reference EV71 strains while EV71 China isolates by homogeneity and phylogenetic tree analyses. RESULTS: 215 strains of EV were isolated from 439 specimens. Results from RT-PCR indicated that 93 strains belong to EV71. Phylogenetic tree analysis revealed that the selected 29 stains were clustered with reference strains of C4 subgenotype. The nucleotide identity with C4 reference strains was 91.7%-99.4%. The amino acid homogeneity was 96.6%-100.0%. CONCLUSION: The recently identified EV71 strains in Ningxia province belonged to subgenotype C4 which resembled to most of the isolates in China.

An outbreak of hand, foot, and mouth disease associated with subgenotype C4 of human enterovirus 71 in Shandong, China.

BACKGROUND: An outbreak of hand, foot, and mouth disease (HFMD) included 1149 people in Linyi City, Shandong Province, China, in 2007: three children died. OBJECTIVES: To characterize the pathogens responsible for this outbreak and to analyze their genetic features. STUDY DESIGN: A total of 233 clinical specimens were collected from 105 hospitalized patients, including 11 patients with severe HFMD. Virological investigations (direct RT-PCR, viral isolation and molecular identification) and phylogenetic analysis were performed. RESULTS: Human enterovirus 71 (HEV71) was the main pathogen that caused this outbreak, based on clinical manifestations, epidemiological data, and laboratory results. Phylogenetic analysis indicated that the Shandong HEV71 isolates belonged to 3 lineages in subgenotype C4. Subgenotype C4 could be further divided into two clusters (C4a and C4b), which corresponded to two time periods. Cluster C4a HEV71 has been the predominant virus circulating in mainland China in the past 5 years. CONCLUSIONS: The 2007 HFMD outbreak was mainly caused by HEV71 subgenotype C4 with 3 transmission chains. This virus has been continuously circulating in China since 1998. The Shandong strains co-evolved with isolates from other provinces in mainland China and neighboring countries.

[The effect of liposome-carried metallothionein on secondary venous ischemia-reperfusion injury in a rat flap].

OBJECTIVE: To investigate the effect of liposome-carried metallothionein (lipo-MT) on secondary ischemia-reperfusion injury in the rat island flap. METHODS: An abdominal island flap was created in the Wistar rat. The animals were divided into four groups: the sham group, the secondary ischemia-reperfusion group, the group treated with blank liposome and the group treated with lipo-MT. The malondialdehyde (MDA) content, the myeloperoxidase (MPO) activity was assayed immediately, at 30 minutes and 7 days after the secondary venous ischemia-reperfusion. The level of endothelin (ET) and lactic dehydrogenase (LDH) of the rat plasma was measured at 30 minutes after secondary venous ischemia-reperfusion. The content of MT of the flap was assayed by Cd-hemoglobin saturation method at 7 days after the operation. RESULTS: The treatment of lipo-MT significantly decreased the content of MDA, MPO of the flap, decreased the activity of ET, LDH of the rat palsma, increased the content of MT of the flap and improved the flap viability. CONCLUSION: Lipo-MT can improve flap survival by reducing ischemia-reperfusion injury.