Whole-genome analysis of coxsackievirus B3 reflects its genetic diversity in China and worldwide.

BACKGROUND: Coxsackievirus B3 (CVB3) has emerged as an active pathogen in myocarditis, aseptic meningitis, hand, foot, and mouth disease (HFMD), and pancreatitis, and is a heavy burden on public health. However, CVB3 has not been systematically analyzed with regard to whole-genome diversity and recombination. Therefore, this study was undertaken to systematically examine the genetic characteristics of CVB3 based on its whole genome. METHODS: We combined CVB3 isolates from our national HFMD surveillance and global sequences retrieved from GenBank. Phylogenetic analysis was performed to examine the whole genome variety and recombination forms of CVB3 in China and worldwide. RESULTS: Phylogenetic analysis showed that CVB3 strains isolated worldwide could be classified into clusters A-E based on the sequence of the entire VP1 region. The predominant CVB3 strains in China belonged to cluster D, whereas cluster E CVB3 might be circulated globally compared to other clusters. The average nucleotide substitution rate in the P1 region of CVB3 was 4.82 × 10-3 substitutions/site/year. Myocarditis was more common with cluster A. Clusters C and D presented more cases of acute flaccid paralysis, and cluster D may be more likely to cause HFMD. Multiple recombination events were detected among CVB3 variants, and there were twenty-three recombinant lineages of CVB3 circulating worldwide. CONCLUSIONS: Overall, this study provides full-length genomic sequences of CVB3 isolates with a wide geographic distribution over a long-term time scale in China, which will be helpful for understanding the evolution of this pathogen. Simultaneously, continuous surveillance of CVB3 is indispensable to determine its genetic diversity in China as well as worldwide.

Isolation of an imported subgenotype B5 strain of human enterovirus A71 in Chongqing City, China, 2014.

BACKGROUND: Enterovirus A71 (EV-A71) is the main pathogen responsible for large outbreaks of hand, foot, and mouth disease (HFMD) in mainland China, and the dominant EV-A71 strains belong to subgenotype C4. To date, only one imported subgenotype B5 of EV-A71 has been reported in Xiamen City Fujian Province, 2009. RESULTS: Here, we report on another imported subgenotype B5 of EV-A71 isolated from a HFMD patient in Chongqing City in 2014 (strain CQ2014-86/CQ/CHN/2014, hereafter refer as CQ2014-86). The VP1 coding sequence and the whole genome sequence revealed that strain CQ2014-86 shares the high nucleotide identity with Vietnamese strains isolated in 2011-2013, suggesting that strain CQ2014-86 may have been imported from Vietnam. In the 5'UTR, P2 and P3 regions, recombination events were found between strain CQ2014-86 and other EV-A, such as coxsackievirus A4 (CV-A4), CV-A5, CV-A14 and CV-A16. CONCLUSIONS: This is the second report on importation of subgenotype B5 of EV-A71 in China, implying that we need to pay more attention to the importation of different subgenotypes of EV-A71.

Acupuncture for Meige syndrome: A case report.

INTRODUCTION: Meige syndrome (MS) is a cranial dystonia disorder characterized by blepharospasm and oromandibular dystonia. Current treatments include botulinum toxin therapy, oral medications, and surgical interventions. Here, we present a case that the patient's symptoms improved significantly after treatment with acupuncture. CASE DESCRIPTION: A 72-year-old woman came to our acupuncture clinic with eyelid twitching and jaw muscle tension. She described being diagnosed with MS two years ago due to the same symptoms, and her symptoms gradually worsened over the past six months. After 4 weeks of acupuncture treatment, the patient's MS symptoms improved significantly. CONCLUSION: Acupuncture may be a potentially salvage treatment option for MS.

Molecular Epidemiology and Evolution of Coxsackievirus A14.

As the proportion of non-enterovirus 71 and non-coxsackievirus A16 which proportion of composition in the hand, foot, and mouth pathogenic spectrum gradually increases worldwide, the attention paid to other enteroviruses has increased. As a member of the species enterovirus A, coxsackievirus A14 (CVA14) has been epidemic around the world until now since it has been isolated. However, studies on CVA14 are poor and the effective population size, evolutionary dynamics, and recombination patterns of CVA14 are not well understood. In this study, 15 CVA14 strains were isolated from HFMD patients in mainland China from 2009 to 2019, and the complete sequences of CVA14 in GenBank as research objects were analyzed. CVA14 was divided into seven genotypes A-G based on an average nucleotide difference of the full-length VP1 coding region of more than 15%. Compared with the CVA14 prototype strain, the 15 CVA14 strains showed 84.0-84.7% nucleotide identity in the complete genome and 96.9-97.6% amino acid identity in the encoding region. Phylodynamic analysis based on 15 CVA14 strains and 22 full-length VP1 sequences in GenBank showed a mean substitution rate of 5.35 × 10-3 substitutions/site/year (95% HPD: 4.03-6.89 × 10-3) and the most recent common ancestor (tMRCA) of CVA14 dates back to 1942 (95% HPD: 1930-1950). The Bayesian skyline showed that the effective population size had experienced a decrease-increase-decrease fluctuation since 2004. The phylogeographic analysis indicated two and three possible migration paths in the world and mainland China, respectively. Four recombination patterns with others of species enterovirus A were observed in 15 CVA14 strains, among which coxsackievirus A2 (CVA2), coxsackievirus A4 (CVA4), coxsackievirus A6 (CVA6), coxsackievirus A8 (CVA8), and coxsackievirus A12 (CVA12) may act as recombinant donors in multiple regions. This study has filled the gap in the molecular epidemiological characteristics of CVA14, enriched the global CVA14 sequence database, and laid the epidemiological foundation for the future study of CVA14 worldwide.

Molecular evidence of persistent epidemic and evolution of subgenotype B1 coxsackievirus A16-associated hand, foot, and mouth disease in China.

The molecular epidemiology of CVA16 in China between 1999 and 2008 reflects a pattern of endemic cocirculation of clusters B1a and B1b within subgenotype B1 viruses. The annual evolution rate of CVA16 was estimated as approximately 0.91 x 10(-2) substitutions per synonymous nucleotide/year and is slightly lower than that of HEV71.

Retrospective seroepidemiology indicated that human enterovirus 71 and coxsackievirus A16 circulated wildly in central and southern China before large-scale outbreaks from 2008.

BACKGROUND: Large nationwide outbreaks of hand, foot, and mouth disease (HFMD) occurred in China from 2008; most of the cases were in children under 5 years. This study aims to identify the situation of natural human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) infections in children before 2008 in China. RESULTS: Retrospective seroepidemiologic studies of HEV71 and CVA16 were performed with 900 serum samples collected from children ≤5 years of age in 2005. The samples were collected from 6 different geographical areas (Anhui, Guangdong, Hunan, Xinjiang, Yunnan, and Heilongjiang provinces) in mainland China. Of the 900 samples, 288 were positive for HEV71; the total positive rate was 32.0% and the geometric mean titer (GMT) was 1:8.5. Guangdong (43.7% and 1:10.8), Xinjiang (45.4% and 1:11.1), and Yunnan (43.4% and 1:12.0) provinces had relatively high rates of infection, while Heilongjiang province (8.1% and 1:4.9) had the lowest rate of infection. On the other hand, 390 samples were positive for CVA16; the total positive rate was 43.4% and the GMT was 1:9.5. Anhui (62.2% and 1:16.0) and Hunan (61.1% and 1:23.1) had relatively high rates, while Heilongjiang (8.0% and 1:4.6) had the lowest rate. Although there is a geographical difference in HEV71 and CVA16 infections, low neutralizing antibody positive rate and titer of both viruses were found in all 6 provinces. CONCLUSIONS: This report confirmed that HEV71 and CVA16 had wildly circulated in a couple provinces in China before the large-scale outbreaks from 2008. This finding also suggests that public health measures to control the spread of HEV71 and CVA16 should be devised according to the different regional characteristics.

Genetic Diversity Analysis of Coxsackievirus A8 Circulating in China and Worldwide Reveals a Highly Divergent Genotype.

Coxsackievirus A8 (CV-A8) is one of the pathogens associated with hand, foot and mouth disease (HFMD) and herpangina (HA), occasionally leading to severe neurological disorders such as acute flaccid paralysis (AFP). Only one study aimed at CV-A8 has been published to date, and only 12 whole-genome sequences are publicly available. In this study, complete genome sequences from 11 CV-A8 strains isolated from HFMD patients in extensive regions from China between 2013 and 2018 were determined, and all sequences from GenBank were retrieved. A phylogenetic analysis based on a total of 34 complete VP1 sequences of CV-A8 revealed five genotypes: A, B, C, D and E. The newly emerging genotype E presented a highly phylogenetic divergence compared with the other genotypes and was composed of the majority of the strains sequenced in this study. Markov chain Monte Carlo (MCMC) analysis revealed that genotype E has been evolving for nearly a century and somehow arose in approximately 2010. The Bayesian skyline plot showed that the population size of CV-A8 has experienced three dynamic fluctuations since 2001. Amino acid residues of VP1100N, 103Y, 240T and 241V, which were embedded in the potential capsid loops of genotype E, might enhance genotype E adaption to the human hosts. The CV-A8 whole genomes displayed significant intra-genotypic genetic diversity in the non-capsid region, and a total of six recombinant lineages were detected. The Chinese viruses from genotype E might have emerged recently from recombining with European CV-A6 strains. CV-A8 is a less important HFMD pathogen, and the capsid gene diversity and non-capsid recombination variety observed in CV-A8 strains indicated that the constant generation of deleterious genomes and a constant selection pressure against these deleterious mutations is still ongoing within CV-A8 quasispecies. It is possible that CV-A8 could become an important pathogen in the HFMD spectrum in the future. Further surveillance of CV-A8 is greatly needed.

Thermal stability and degradation kinetics of polystyrene/organically-modified montmorillonite nanocomposites.

Organically-modified montmorillonite (MMT) clays have been prepared using ammonium salts containing quinoline, pyridine, benzene, and styrenic groups. The nanocomposites were prepared by melt blending and the formation of nanocomposites was characterized using X-ray diffraction (XRD) and transmission electron microscopy (TEM). Thermal stability and flammability were evaluated by thermogravimetric analysis (TGA) and cone calorimetry measurements, respectively. The presence of modified MMT at 5% loading resulted in significant improvement in thermal stability compared to the virgin polymer. Effective activation energies for mass loss were determined via a model-free isoconversional approach from TGA data obtained under N2 and under air. The additives served to raise the activation energy, with a more significant impact observed under pyrolysis conditions. The onset temperature of degradation and temperature of maximum decomposition rate are increased, while the peak heat release rate and mass loss rates are significantly reduced in the presence of three of the modified clays. No reduction in the total heat released is observed.

Two Genotypes of Coxsackievirus A2 Associated with Hand, Foot, and Mouth Disease Circulating in China since 2008.

Coxsackievirus A2 (CV-A2) has been frequently detected and commonly associated with hand, foot, and mouth disease (HFMD) in China since 2008. However, limited sequences of CV-A2 are currently available. As a result, we have been focusing on the genetic characteristics of CV-A2 in the mainland of China during 2008-2015 based on national HFMD surveillance. In this study, 20 CV-A2 strains were isolated and phylogenetic analyses of the VP1 sequences were performed. Full-length genome sequences of two representative CV-A2 isolates were acquired and similarity plot and bootscanning analyses were performed. The phylogenetic dendrogram indicated that all CV-A2 strains could be divided into four genotypes (Genotypes A-D). The CV-A2 prototype strain (Fleetwood) was the sole member of genotype A. From 2008 to 2015, the CV-A2 strains isolated in China dispersed into two different genotypes (B and D). And the genotype D became the dominant circulating strains in China. Strains isolated in Russia and India from 2005 to 2011 converged into genotype C. Intertypic recombination occurred between the Chinese CV-A2 strains and other enterovirus-A donor sequences. This result reconfirmed that recombination is a common phenomenon among enteroviruses. This study helps expand the numbers of whole virus genome sequence and entire VP1 sequence of CV-A2 in the GenBank database for further researcher.

[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.

The persistent circulation of enterovirus 71 in People's Republic of China: causing emerging nationwide epidemics since 2008.

Emerging epidemics of hand-foot-and-mouth disease (HFMD) associated with enterovirus 71 (EV71) has become a serious concern in mainland China. It caused 126 and 353 fatalities in 2008 and 2009, respectively. The epidemiologic and pathogenic data of the outbreak collected from national laboratory network and notifiable disease surveillance system. To understand the virological evolution of this emerging outbreak, 326 VP1 gene sequences of EV71 detected in China from 1987 to 2009 were collected for genetic analyses. Evidence from both traditional and molecular epidemiology confirmed that the recent HFMD outbreak was an emerging one caused by EV71 of subgenotype C4. This emerging HFMD outbreak is associated with EV71 of subgenotype C4, circulating persistently in mainland China since 1998, but not attributed to the importation of new genotype. Originating from 1992, subgenotype C4 has been the predominant genotype since 1998 in mainland China, with an evolutionary rate of 4.6∼4.8×10⁻³ nucleotide substitutions/site/year. The phylogenetic analysis revealed that the majority of the virus during this epidemic was the most recent descendant of subgenotype C4 (clade C4a). It suggests that the evolution might be one of the potential reasons for this native virus to cause the emerging outbreak in China. However, strong negative selective pressure on VP1 protein of EV71 suggested that immune escape might not be the evolving strategy of EV71, predicting a light future for vaccine development. Nonetheless, long-term antigenic and genetic surveillance is still necessary for further understanding.

Emergence and transmission pathways of rapidly evolving evolutionary branch C4a strains of human enterovirus 71 in the Central Plain of China.

BACKGROUND: Large-scale outbreaks of hand, foot, and mouth disease (HFMD) occurred repeatedly in the Central Plain of China (Shandong, Anhui, and Henan provinces) from 2007 until now. These epidemics have increased in size and severity each year and are a major public health concern in mainland China. PRINCIPAL FINDINGS: Phylogenetic analysis was performed and a Bayesian Markov chain Monte Carlo tree was constructed based on the complete VP1 sequences of HEV71 isolates. These analyses showed that the HFMD epidemic in the Central Plain of China was caused by at least 5 chains of HEV71 transmission and that the virus continued to circulate and evolve over the winter seasons between outbreaks. Between 1998 and 2010, there were 2 stages of HEV71 circulation in mainland China, with a shift from evolutionary branch C4b to C4a in 2003-2004. The evolution rate of C4a HEV71 was 4.99×10(-3) substitutions per site per year, faster than the mean of all HEV71 genotypes. The most recent common ancestor estimates for the Chinese clusters dated to October 1994 and November 1993 for the C4a and C4b evolutionary branches, respectively. Compared with all C4a HEV71 strains, a nucleotide substitution in all C4b HEV71 genome (A to C reversion at nt2503 in the VP1 coding region, which caused amino acid substitution of VP1-10: Gln to His) had reverted. CONCLUSIONS: The data suggest that C4a HEV71 strains introduced into the Central Plain of China are responsible for the recent outbreaks. The relationships among HEV71 isolates determined from the combined sequence and epidemiological data reveal the underlying seasonal dynamics of HEV71 circulation. At least 5 HEV71 lineages circulated in the Central Plain of China from 2007 to 2009, and the Shandong and Anhui lineages were found to have passed through a genetic bottleneck during the low-transmission winter season.

[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.