Analysis on the epidemiology and etiology characteristics of first imported Chikungunya fever case in Henan Province in 2017 / 中华预防医学杂志

To study the epidemiology and etiology characteristics of first imported Chikungunya fever case in Henan province, China, 2017. The patient was confirmed by Chikungunya virus (CHIKV) infected as CHIKV ribonucleotide was continuously detected in his serum specimens. BHK-21 cell line was used for virus isolation, the strain was named CHIKV/Henan001/2017. CHIKV/Henan001/2017 belonged to genotype ECSA. The highest ribonucleotide homology sequence of highly conserved region E1 with CHIKV/Henan001/2017 was hk02 strain (99.8%), who was an imported strain to Hong Kong, China, 2016. Epidemiological information and laboratory testing confirmed it was an imported Chikungunya fever case in Henan province, 2017. No secondary case has been reported.

Etiology analysis on a laboratory confirmed human rabies case with atypical symptoms / 中华实验和临床病毒学杂志

Objective@#To investigate and analyze the clinical features, epidemiologic information and pathogenic characteristics of a rabies patient.@*Methods@#Clinical data of the patient(boy) was collected and epidemiological survey was conducted, fluorescence quantitative reverse transcription-polymerase chain reaction (FQRT-PCR) and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the samples of saliva, cerebrospinal fluid (CSF), skin tissue with hair follicle at the back of the neck for rabies laboratory diagnosis.@*Results@#Early symptoms of the boy were vomiting, diarrhea, fever and irritability, followed by coma and death. The boy had nasal trauma one month ago and the domestic dog died of illness during the same period. He did not accept the rabies post-exposure prophylaxis (PEP). The result of the saliva sample was positive by FQRT-PCR. The predicted segments of the glycoprotein(G), nucleoprotein (N) genes of rabies virus were amplified from the positive saliva sample of the patient by RT-PCR. Compared with rabies virus strains in Henan province, the nucleotide homology and amino acid homology in G gene segment were 96.5%-98.8% and 96.5%-99.2% respectively.@*Conclusions@#The case was diagnosed in laboratory as rabies case. The pathogenic rabies virus strain was endemic in Henan province. The nasal trauma, the dead domestic dog were probably related to the infection of the boy.

Molecular epidemiological analysis of coxsackievirus A16 strains causing neurologic complications / 中华微生物学和免疫学杂志

Objective To analyze the VP1 sequences of coxsackievirus A16(CA16) causing neu-rologic complications. Methods Clinical samples and epidemiological information were collected from pa-tients with viral encephalitis. Coxsackievirus A16 in these samples were first detected with real time RT-PCR and then isolated. RT-PCR was performed to amplify VP1 sequences and the amplified products were se-quenced. DNAStar 5.0 and Mega 5 were used for sequence analysis. All data was analyzed with SPSS statis-tical software. Results Fifteen samples were collected from 12 patients with hand, foot and mouth disease (HFMD) complicated by neurologic complications. Eight patients had the symptoms of fever, skin rash, signs of meningeal irritation and neck rigidity. No typical cluster was associated with clinical features or the time of onset. Both pharyngeal/anal swab and serum samples were collected from three patients (patient′s number:01111,01169 and 01130). The two samples collected from both 01111 and 01130 patients shared 100% similarity in nucleotide and amino acid based on VP1 sequences,while those from 01169 patient dif-fered in only one base. The 15 CA16 isolates were highly similar in VP1 gene, sharing 94.5%-100% ho-mology in nucleotide sequences and 98.0%-100% homology in amino acid sequences. These 15 isolates showed 68.5%-70.5% identities in nucleotide sequences and 90.5%-91.9% identities in amino acid se-quences with the CA16 prototype strain G10. Phylogenetic analysis revealed that based upon VP1 sequences, all of the 15 CA16 isolates grouped into genotype B subtype 1b (B1b), which was further classified into three clusters. Conclusion All of the 15 CA16 isolates causing neurologic complications belonged to B1b sub-genotype. Understanding the molecular epidemiology of CA16 would be essential for controlling morbidi-ty rates of HFMD and vaccine research.

Etiology study on severe cases caused by hand-foot-mouth disease in children from Henan province, 2014 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the etiology of severe hand-foot-mouth disease (HFMD) in children in Henan province.</p><p><b>METHODS</b>A total of 244 HFMD cases admitted to a hospital in Zhengzhou from April to June of 2014 were recruited for research sampling, Real-time RT-PCR, virus isolation, VP1 sequencing and alignment methods were used to test the enterovirus-related etiology. SPSS 17.0 was used in performing statistical analysis.</p><p><b>RESULTS</b>There were 109 severe and 135 mild cases among all the 244 HFMD cases. The number of enterovirus positive stool samples was 229, with positive rate as 93.85%. EV71, Cox A16 and Cox A10 made up 83.84%, 5.68% and 8.30% of the enterovirus etiologicy, strains, respectively. EV71 infection caused 8 HFMD cases with heart-lung failure and 2 death, Cox A10 infection led to 1 HFMD case with heart-lung failure and death. There were statistically differences seen regarding the enterovirus infection rates between severe and the mild HFMD cases (χ(2)=5.312,P=0.021). Statistically significant difference was seen in the constituent ratio of EV71, Cox A16 and the others by Fisher' s exact test (P=0.048). There was statistically significant difference seen between the cardiorespiratory failure rate and the fatality rate by EV71 and Cox A10 infection (χ(2)=0.051,P=0.821; χ(2)=2.198,P=0.138). Cox A10 strains idenfied in Henan in 2014 belonged to genotype 6. The rates on homology of nucleotide and amino acid among the Cox A10 strains in Henan in 2014 were 94.3%-99.7% and 96.3%-100.0% respectively.</p><p><b>CONCLUSIONS</b>EV71 still remained the most common pathogen that causing severe HFMD in children, with the increasing Cox A10 percentage in the pathogens spectrum of HFMD infection. Cox A10 strains in Henan in 2014 belonged to genotype 6. Genotype 6 Cox A10 had appeared and widely distributed in Henan for long time, but not yet variated or reconstructed. Cox A10 infection could lead to cardio-respiratory failure thus called for the monitoring program on non-EV71 and non-Cox A16 enterovirus, especially Cox A10 to be strenthened.</p>

Expression and activity determination of recombinant capsid protein VP2 gene of enterovirus type 71 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen.</p><p><b>METHODS</b>VP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected.</p><p><b>RESULTS</b>VP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay.</p><p><b>CONCLUSIONS</b>VP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.</p>

Identification and phylogenetic analysis of Coxsackie-virus B5 that caused an outbreak of viral encephalitis in Henan area / 中华微生物学和免疫学杂志

Objective To identify the pathogen that caused an outbreak of viral encephalitis in Henan area in 2011.Phylogenic analysis was carried out on Coxsackie-virus B5 (CVB5) which was isolated during this outbreak.Methods Five throat swab,21 stool and 14 cerebrospinal fluid (CSF) specimens were collected from 29 inpatients during this outbreak.Viral isolation and real time RT-PCR were then performed for all specimens.Viral nucleic acid of enterovirus 71 (EV71),coxsackievirus A 16 (CA16) and pan-enterovirus (PE)were detected by real time RT-PCR.Phylogenetic tree based on entire VP1 sequences was constructed among CVB5 isolates from 2 stool and 3 CSF specimens of 5 inpatients and others published data retrieved from GenBank.Results The real time RT-PCR results showed that the PE nucleic acid positive rates of throat swab,stool and CSF specimens were 60.0％ (3/5),61.9％ (13/21) and 85.7％ (12/14) respectively.All of these specimens were negative for EV71 and CA16.The isolation rates of throat swab,stool and CSF specimens were 20.0％ (1/5),25.0％ (5/21) and 29.0％ (4/14),respectively.BLAST with both VP1 and 5′-UTR sequences and molecular typing indicated that CVB5 was the main pathogen.Analysis among the 5 positve isolates based on the complete VP1 sequences showed 97.9％-99.5％ homology.Data from homologous comparisons indicated that these isolates had the highest nucleotide acid identity with the Changchun CVB5 CC10/10/Changchun strain (97.1％-98.1％) which caused hand,foot,and mouth disease (HFMD) outbreak in Changchun in 2010,and lower identity (89.0％-89.6％ and 91.8％-92.5％) with the COXB5/Henan/2010 and 03001N strain isolated from Pingdingshan,Henan in 2010 and 2012,respectively.Phylogenetic tree in VP1 region showed that isolates of this outbreak belonged to genotype D,the same clade with Changchun strain.Conclusion CVB5 was the major etiological agent correlated with this outbreak.The shift of predominant genotype might serve as one of the causes that associated with this outbreaks.