We compared genetic variations in the VP1 gene of foot-and-mouth disease viruses (FMDVs) isolated since 2000 from various region of the world. We analyzed relative synonymous codon usage (RSCU) and phylogenetic relationship between geographical regions, and calculated the genetic substitution patterns between Korean isolate and those from other countries. We calculated the ratios of synonymously substituted codons (SSC) to all observed substitutions and developed a new analytical parameter, EMC (the ratio of exact matching codons within each synonymous substitution group) to investigate more detailed substitution patterns within each synonymous codon group. We observed that FMDVs showed distinct RSCU patterns according to phylogenetic relationships in the same serotype (serotype O). Moreover, while the SSC and EMC values of FMDVs decreased according to phylogenetic distance, G + C composition at the third codon position was strictly conserved. Although there was little variation among the SSC values of 18 amino acids, more dynamic differences were observed in EMC values. The EMC values of 4- and 6-fold degenerate amino acids showed significantly lower values while most 2-fold degenerate amino acids showed no significant difference. Our findings suggest that different EMC patterns among the 18 amino acids might be an important factor in determining the direction of evolution in FMDV.
Animals ; Capsid Proteins/*genetics ; Cattle ; Codon/*genetics ; Evolution, Molecular ; Foot-and-Mouth Disease/diagnosis/epidemiology/*virology ; Foot-and-Mouth Disease Virus/*genetics/isolation & purification ; Gene Frequency ; Geography ; Korea ; Phylogeography ; Polymorphism, Genetic ; RNA, Viral/*analysis ; Species Specificity

We compared genetic variations in the VP1 gene of foot-and-mouth disease viruses (FMDVs) isolated since 2000 from various region of the world. We analyzed relative synonymous codon usage (RSCU) and phylogenetic relationship between geographical regions, and calculated the genetic substitution patterns between Korean isolate and those from other countries. We calculated the ratios of synonymously substituted codons (SSC) to all observed substitutions and developed a new analytical parameter, EMC (the ratio of exact matching codons within each synonymous substitution group) to investigate more detailed substitution patterns within each synonymous codon group. We observed that FMDVs showed distinct RSCU patterns according to phylogenetic relationships in the same serotype (serotype O). Moreover, while the SSC and EMC values of FMDVs decreased according to phylogenetic distance, G + C composition at the third codon position was strictly conserved. Although there was little variation among the SSC values of 18 amino acids, more dynamic differences were observed in EMC values. The EMC values of 4- and 6-fold degenerate amino acids showed significantly lower values while most 2-fold degenerate amino acids showed no significant difference. Our findings suggest that different EMC patterns among the 18 amino acids might be an important factor in determining the direction of evolution in FMDV.
Animals ; Capsid Proteins/*genetics ; Cattle ; Codon/*genetics ; Evolution, Molecular ; Foot-and-Mouth Disease/diagnosis/epidemiology/*virology ; Foot-and-Mouth Disease Virus/*genetics/isolation & purification ; Gene Frequency ; Geography ; Korea ; Phylogeography ; Polymorphism, Genetic ; RNA, Viral/*analysis ; Species Specificity

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.
Base Sequence ; Cell Line, Tumor ; Child, Preschool ; China ; epidemiology ; DNA Primers ; genetics ; DNA, Viral ; chemistry ; isolation & purification ; Diagnosis, Differential ; Disease Outbreaks ; Enterovirus ; genetics ; isolation & purification ; Exanthema ; Female ; Fever ; Genotype ; Hand, Foot and Mouth Disease ; diagnosis ; virology ; Herpes Simplex ; diagnosis ; transmission ; virology ; Herpesvirus 1, Human ; genetics ; isolation & purification ; Humans ; Infant ; Male ; Molecular Sequence Data ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo observe the duration of enterovirus-71 (EV71) and coxsackievirus A 16 (CoxA16) viral shedding in stool samples of children with hand, foot and mouth disease (HFMD) infected with EV71 and CoxA16 and to explore the relationship between the duration of intestinal virus shedding and the severity of illness of children with HFMD.

METHODTotally 113 laboratory-confirmed cases of children with HFMD infected with EV71 and CoxA16 were followed up. The stool samples were collected with the interval of 4 to7 days and the viral nucleic acids were detected by fluorescent PCR until the stool viral nucleic acids of infected children turned to be negative. The cases in EV71 group were further divided into "ordinary EV71 group" and "severe EV71 group" according to the severity of the illness. The positive rates of viral nucleic acid and the differences of distribution among different groups were analyzed by Kaplan-Meier survival analysis during the follow-up period.

RESULTThe 113 cases of infected children were grouped as follows: 65 cases of EV71 positive children, 44 cases of CoxA16 positive children, 4 cases of EV71/CoxA16 mixed infection. The median duration of the stool viral nucleic acids turning to negative was 26 (18.25-32.50) days in EV71 group and 27 (14.50-33.75) days in CoxA16 group (Z = 1.51, P > 0.05). At 1, 4, 6 and 10 weeks, the positive rates of stool viral nucleic acid of children with HFMD in EV71 group were 100%, 48.1%, 17.2% and 0 respectively. At 1, 4 and 6 weeks, the positive rates of stool viral nucleic acid of children with HFMD in CoxA16 group were 95.5%, 53.8% and 0 respectively (χ(2) = 0.18, P > 0.05). At 1, 4 and 6 weeks, the positive rates of stool viral nucleic acid of children with HFMD in ordinary EV71 group were 100%, 23.5% and 0 respectively, while at 1, 4, 6 and 10 weeks, the positive rates of stool viral nucleic acid of children with HFMD in severe EV71 group were 100%, 62.4%, 26.0% and 0 respectively (χ(2) = 5.689, P < 0.05).

CONCLUSIONThe duration of enterovirus shedding in stool samples of children with HFMD lasted for a long period. The maximum duration of EV71 and CoxA16 in stool of children with HFMD was 10 weeks and 6 weeks, respectively. The duration of intestinal virus shedding of children with HFMD infected with EV71 was related with the severity of the illness.

Child ; Child, Preschool ; China ; epidemiology ; Coxsackievirus Infections ; diagnosis ; epidemiology ; Enterovirus ; genetics ; isolation & purification ; Enterovirus A, Human ; genetics ; isolation & purification ; Feces ; virology ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; prevention & control ; virology ; Humans ; Infant ; Male ; Nucleic Acids ; isolation & purification ; Polymerase Chain Reaction ; RNA, Viral ; genetics ; Virus Shedding

OBJECTIVEIt was noticed that coxsackievirus A16 (CA16) and enterovirus 71 (EV71) were two major etiological agents of hand, foot and mouth disease (HFMD) in children. Recently there were several large outbreaks of HFMD in the Asia-Pacific region, and there was a propensity to cause severe complications or death in children under 5 years of age. The severe forms were associated with EV71 infection. Although epidemics of HFMD have been reported in the mainland of China, few reports about EV71 as the pathogen of HFMD epidemics are available. The present study was conducted to investigate the causal agent of an HFMD epidemic in children in Shanghai from April to June of 2002.

METHODSTotally 102 specimens (including vesicle fluid, stool and throat swabs) were collected from 72 patients with HFMD. The specimens were inoculated into Vero and/or RD cells. At first all the isolates were respectively neutralized by the RIVM pools of enterovirus antiserum, the type-specific antisera to EV71 or to CA16. Secondly all untyped isolates were tested by RT-PCR assay with two specific primer pairs for VP1 genes of EV71 and CA16 respectively. The EV71 and CA16 were identified depending on the size of PCR products. Sequence analyses of VP1 genes of 9 virus strains were performed by the laboratory of China CDC.

RESULTSViruses were isolated from 91 specimens from 67 patients. Serotyping by neutralization failed for all the isolates. But the RT-PCR results indicated that the viruses isolated from 78 specimens from 58 patients were identified as positive for CA16 and the isolates from 13 specimens from 9 patients were identified as positive for EV71, the ratio between CA16 and EV71 was 6.4:1. The results of sequence analyses were consistent with those of PCR assay. Two EV71 strains isolated in this study belonged to a new lineage (C4) within genogroup C. One patient with EV71-associated HFMD had a complication of encephalitis with convulsion, shock, coma and dyspnea.

CONCLUSIONCA16 and EV71 were the primary causes of HFMD during the epidemic. It was the first report of EV71-associated severe encephalitis occurred in patients with HFMD in Shanghai.

Animals ; Cercopithecus aethiops ; Child ; Child, Preschool ; China ; epidemiology ; Coxsackievirus Infections ; diagnosis ; epidemiology ; Disease Outbreaks ; Enterovirus ; isolation & purification ; Enterovirus A, Human ; isolation & purification ; Female ; Genes, Viral ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Infant ; Male ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Vero Cells