One step TaqMan real-time reverse transcription polymerase chain reaction (R/T RT-PCR)using a set of primers/probes was developed for the detection of foot-and-mouth disease (FMD)virus. The gene-specific probes labeled fluorogen for the internal ribosomal entry site, Leader sequence and 2B regions were used to detect FMD virus (FMDV). This assay specifically detected FMDV both in cell culture preparations and clinical samples, and was capable of distinguishing FMD from other viral diseases similar to clinical signs (swine vesicular disease, vesicular stomatitis and bovine viral diarrhea). This assay was shown to be 1000-fold more sensitive than the conventional RT-PCR method. The detection limits of this assay was 1 TCID 50 /ml of the FMDV RNA concentration. Quantification was obtained by a standard curves plotting threshold cycle values versus known infectivity titer. The assay was sensitive, specific and rapid enough to detect FMDV RNA genome in probang samples. As such, the described method is reliable and provides faster disease diagnostics than the conventional RT-PCR procedure to detect FMDV.
Animals ; Foot-and-Mouth Disease/*diagnosis/virology ; Foot-and-Mouth Disease Virus/*isolation&purification ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; Sensitivity and Specificity ; Taq Polymerase

Animals ; Critical Illness ; Enterovirus A, Human ; pathogenicity ; Enterovirus Infections ; diagnosis ; therapy ; Hand, Foot and Mouth Disease ; diagnosis ; therapy ; virology ; Humans

OBJECTIVETo explore the development and prognosis of the acute flaccid paralysis (AFP) associated with enterovirus 71 (EV71) infection through clinical follow-up study for clinical and magnetic resonance imaging (MRI) features based on the research progress of virology and pathology.

METHODSixteen children with HFMD associated with AFP in hospital from May 1, 2011 to August 31, 2011 were investigated and the patients received intensive rehabilitation training. The 16 cases were divided into two groups (the recovery or the sequela) by if the muscle strength recovered to level 4 after intensive rehabilitation. The MRI findings of 15 children were analyzed and among them, 6 patients were reexamined after one month. The clinical markers were compared between groups including course of disease, WBC, WBC in cerebrospinal fluid (CSF), ventilator support, therapy, the worst muscle strength, the initial tendon reflex, the muscle atrophy, and multi-limb paralysis. The data were analyzed by t test and χ2 test with SPSS10.0.

RESULTAll the 16 children were infected with enterovirus 71 (EV71). The myodynamia of 7 children were level 0, 4 children had serious upper limbs paralysis. The neck muscle in 3 cases and the brain stem motor ruckus in 4 cases were involved. The ankle clonus of non-completely paralyzed limbs in 14 cases occurred during rehabilitation. Eight children had the better prognosis, the other 8 children had sequela. 0 level muscle strength (0 case vs. 7 cases, χ2=12.4), the initial tendon reflex (2 cases vs. 8 cases, χ2=9.6), obvious muscle atrophy (0 case vs. 8 cases, χ2=16), were significantly different in the children with the recovery when compared to the sequela (P<0.01). The severe upper limbs paralysis had the worse prognosis than the severe lower limbs paralysis. MR imaging showed signs of spinal nerve root inflammation and the bilateral hyperintense lesions, symmetrical in the posterior portions of the medulla, pons, and asymmetrical in the ventral horns of cervical spinal cord. Signal enhancement occurred only in the early MRI examination.

CONCLUSIONIn the evolution of AFP due to EV71 infection, the upper motor neuron damage is common, the prognosis is related with the severity of early paralysis and neuron damage. MR imaging is helpful to understand the pathological mechanism of AFP.

Child, Preschool ; Enterovirus A, Human ; pathogenicity ; Female ; Follow-Up Studies ; Hand, Foot and Mouth Disease ; diagnosis ; pathology ; virology ; Humans ; Infant ; Magnetic Resonance Imaging ; Male ; Paralysis ; diagnosis ; pathology ; virology ; Prognosis

OBJECTIVETo facilitate early recognition of ominous clinical manifestations, to understand pathophysiology and assess treatment effects in patients with severe enterovirus 71 (EV71) associated hand, foot and mouth disease (HFMD).

METHODA retrospective analysis was performed based on the clinical records, laboratory data and treatment effects which were collected from twelve severe EV71 infected cases from nine hospitals in 2008, in Hubei province, China.

RESULTOf the 12 severe cases, ten (83.3%) were male and two female. The median age was 1.96 yrs (8 m to 7 yrs). The mean hyperthermic duration was 6 days with the peak temperature over 38.5 degrees C, and mean rash duration was 7 days. Fever and rash emerged simultaneously in 4 of 5 cases with cardiopulmonary failure. The severe complications included encephalitis (10 cases), pulmonary edema or hemorrhage (5 cases). Eleven cases were checked with magnetic resonance imaging (MRI) and four cases showed characteristics of encephalitis or meningitis, two with images of naso sinusitis and ethmoid-mastoid inflammation. Chest X-ray examination showed with pulmonary edema on single or both sides (5 cases), bronchitis (4 cases), and normal image (3 cases). There was no specific finding in the cardiac ultrasound and electrocardiogram in any of the patients, as well as the white blood cell count, blood glucose, prothrombin time, partial thromboplastin time and D-dimer. Cerebrospinal fluid showed aseptic meningitis with the increase of cell count in 7 cases. All patients were treated with antibiotics and/or antivirals, such as cephalosporins, ribavirin etc. Eleven patients were treated with intravenous immunoglobulin (total dose 2 - 4.5 g/kg) for 2 - 5 days, and the highest blood concentration of immunoglobulin was detected increasing at 7 g/L. Seven cases were also treated with methylprednisolone 10 - 30 mg/(kg x d), four with dopamine, dobutamine, or digitalis. In addition, by using continuous positive airway pressure by nasal catheter and maintenance of circulation in the cases with cardiopulmonary failure could not relieve the symptoms of dyspnoea, and mechanical ventilation was required to maintain for a mean of 72 hrs (24 - 96 hrs). Except one case died of pulmonary edema in the early stage, others were cured without sequelae.

CONCLUSIONSevere EV71 infection is more common in children younger than 3 years old, in which the profound complications include encephalitis and pulmonary edema. The mechanical ventilation should be critically urged for child with complicating cardiopulmonary failure as soon.

Child ; Child, Preschool ; Enterovirus A, Human ; classification ; Enterovirus Infections ; diagnosis ; therapy ; virology ; Female ; Hand, Foot and Mouth Disease ; diagnosis ; therapy ; virology ; Humans ; Infant ; Male ; Retrospective Studies ; Treatment Outcome

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

A multiplex RT-PCR assay based on GeXP system was developed in order to detect simultaneously human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) and other coxsackieviruses (CVA4, 5, 9 and 10, CVB1, 3 and 5). Enterovirus detection was performed with a mixture of 12 pairs of oligonucleotide primers including one pair of published primers for amplifying all known pan-enterovirus genomes and eleven primer pairs specific for detection of the VP1 genes of EV71, C A16, CVA4, CVA5, CVA9, CVA10, CVB1, CVB3 and CVB5, respectively. The specificity of multiplex RT-PCR system was examined using enterovirus cell cultures and positive strains identified previously from hand-foot-and-mouth disease (HFMD) patients. Serial dilution of titrated EV71 and C A16 cell cultures and in vitro transcripted RNA of enterovirus VP1 regions were used to detect the sensitivity of the multiplex RT-PCR system. The limit of detection for this multiplex RT-PCR system was 10(0.5) TCID50/microL for EV71 and C A16 cell cultures and 1000 copies for in vitro transcripted RNA of nine viruses per assay. This multiplex RT-PCR assay is a rapid, sensitive and specific assay for the diagnosis of common enterovirus infection in cases of HFMD outbreak and is also potentially useful for molecular epidemiological investigation.
DNA Primers ; genetics ; Enterovirus ; classification ; genetics ; isolation & purification ; Hand, Foot and Mouth Disease ; diagnosis ; virology ; Humans ; Reverse Transcriptase Polymerase Chain Reaction ; methods

OBJECTIVETo discuss the treatment strategy of severe hand-foot-and-mouth disease (HFMD) cases, prevent the severe cases from progressing to fatal condition and enhance the survival rate of critically ill patients.

METHODSEighty HFMD cases were divided into four groups, A, B, C and D, according to the severity of patients' nervous system manifestation and other system involved. Different intensive care and treatments were used and the effect and outcome were analyzed for each group. All statistical analyses were performed by using SPSS software 13.0. One-way ANOVA and Chi-square test were used for data analysis.

RESULTSThe most common symptoms were continuous fever (69/70) and myoclonic jerk (67/70). The fewer the rashes were, the more severe the patient's condition was, heart rate >200/min, hypertension, increase of white blood cells in peripheral blood and hyperglycemia were common in patients with lesions in brain stem and pulmonary edema. There were no relations between patient's conditions and CSF white blood cells and CRP. CNS involvement was highly associated with EV71 infection. There were 69 cases in group A, B and C in total and all recovered. Of 11 patients in group D, 6 got complicated neurogenic pulmonary edema and circulatory failure, 2 cases died and 9 cases survived, 8 cases recovered without sequelae while one case had sequelae of mental retardation and dyscinesia.

CONCLUSIONAdministration of mannitol, methylprednisolone, IVIG and other supportive treatments in time and reasonably might have advantages in avoiding aggravation of the condition and enhancing the rate of successful rescue in patients with nervous system involvement.

Adolescent ; Child ; Child, Preschool ; Critical Care ; Female ; Hand, Foot and Mouth Disease ; diagnosis ; drug therapy ; mortality ; Humans ; Infant ; Intensive Care Units, Pediatric ; Male ; Nervous System ; virology ; Survival Rate

OBJECTIVETo investigate the etiology of hand, foot and mouth disease (HFMD) in Beijing during 2013, and study the clinical characteristics of HFMD caused by the main serotypes of enterovirus in the study.

METHODClinical data and 128 stool samples were collected from 128 hospitalized children with HFMD in Beijing Ditan Hospital during 2013. One step RT-PCR method was used for enterovirus genotyping to investigate the etiology of HFMD. Clinical characteristics of HFMD caused by the main serotypes of enterovirus were analyzed. And VP1 segments of the main virus were amplified to construct phylogenetic tree for the phylogenetic analysis.

RESULTA total of 128 hospitalized children with HFMD were included. HFMD was more likely developed in children under 2 years of age (81.6%, 102/125); 11 different enteroviruses were genotyped, with a total enterovirus positive rate of 76.6% (98/128); the positive rate of coxsackievirus A6 (CA6), 43.0% ( 55/128), was the highest, followed by enterovirus 71 (EV71), accounting for 14.8% (19/128). HFMD caused by CA6 was atypical, the rashes of which involved the perioral, trunk, limbs, face and neck (47%, 26/55), besides the common parts. Of the 55 cases caused by CA6, 6 children had clinical manifestations of nervous system involvement, one of whom even displayed type 2 respiratory failure. Mental status change more likely to occur in EV71-infected children than in CA6-infected ones (42% (8/19) vs. 11% (6/55) (χ(2)=7.041, P=0.008)); 13 children displayed onychomadesis, including 12 CA6 cases (23%, 12/53) and 1 CA10 cases (17%, 1/6), in the convalescence of hand, foot and mouth disease, and the correlation between onychomadesis and CA6 infection was significant (χ(2)=9.297, P=0.002). Phylogenetic analysis of 33 CA6 VP1 showed that the CA6 isolates of this study were highly similar to that of Taiwan and the nucleotide similarity was 95.91%-98.89%.

CONCLUSIONCA6 was the major pathogen of hospitalized children with hand, foot and mouth disease in Beijing during 2013, followed by EV71. The rashes caused by CA6 involved a wide range of skin sites and patients with CA6 infection displayed manifestations of neurological involvement or pulmonary edema similar to EV71 infection. Mental status change more likely occurred in EV71-infected children when neurological system was involved..

Child, Hospitalized ; Child, Preschool ; Enterovirus ; classification ; Enterovirus Infections ; diagnosis ; virology ; Exanthema ; pathology ; Genotype ; Hand, Foot and Mouth Disease ; diagnosis ; virology ; Hospitals ; Humans ; Infant ; Phylogeny ; Pulmonary Edema ; pathology ; Skin ; pathology ; Taiwan

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