Study Of Enterovirus 71 Infection In Guangzhou

Background: Enterovirus 71 (EV71) ,the most recently described serotypeof the genus Enterovirus (family Picornaviridae), can distinguish with other Picornaviridae based on the complete VP1 gene sequence. EV71 is a common causes epidemics of hand, foot, and mouth disease , aseptic meningitis, Guillain-Barre' syndrome, acute transverse myelitis, acute cerebellar ataxia, cardiac collapse opso-myoclonus syndrome, benign intracranial hypertension, and a febrile convulsion. Since the initial description of enterovirus 71 (EV71) in 1974, outbreaks of infection with this virus have occurred periodically throughout the world. Southern of Children presented with an acute neurological illness associated with EV71 isolation and hundreds were died. In 90's later periods, there has been an increase in EV71 activity in the East Asian and the Southeast Asia area with several epidemics of HFMD being reported, including multiple cases associated with brain stem encephalitis and pulmonary edema in Malaysia, Japan, Singapore. The studies of EV71 evolution based on the complete VP1 gene sequence have demonstrated the evolution of three genogroups- A, B and C. In China, EV71 was first isolated in Wuhan in 1987, and have continued to occur throughout our country including Shanghai, Shenzhen, Chongqing, Xiamen, Taiwan, HongKong. The outbreak that occurred in 1998 in Taiwan is the largest recorded, with greater than 100,000 cases of HFMD. Of these,400 children were admitted to hospital with central nervous system (CNS) involvement and 78 died of brainstem encephalitis with neurogenic pulmonary edema. In 2000,EV71 was responsible for outbreaks of hand-foot-and-mouth disease in Taiwan that resulted in more than 80,000cases and the deaths of 41 people. In a word, EV71 is one of global virulent virus , especially in Southeast Asia. The virus's variation in the different time, the different area and the different individual caused the varied clinical manifestation. The forms of the EV71 multitudinous genotype , the structure and the function relations were still not clear.Anxiously,EV71 has more widespread nerve toxicity than polio virus, and the central system complication may cause the death. The nerve toxicity and the viral genotype accurate relations still not clear. At present does not have the security effective vaccine to prevent the EV71 infection. The epidemiological data and clinical information in our country were short of representative and were also limited . Guangzhou is the south gate, is the proliferation first station of various kinds of communicable disease disseminates from HongKong, Macao and Taiwan and the overseas. In order to prevent the explode like Southeast Asia including Taiwan in 1997 ~ 1998 , monitoring EV71 infection in Guangzhou, discussing the relations of nerve toxicity and viral genotype and clinical was extremely significant.Objectives: l.We want to reveal the incidence, distribution, clinical characteristic, molecule biology characteristic of EV71 infection and situation of EV71 nucleotide genotype epidemic in Guangzhou in 2004. It will be favor of the development of effective vaccine.2. Clarify the tissue tropism of EV71 ,discuss the relations of the clinical type (especially the nerve toxicity) with the viral genotype and the tropism in different cells.Method: All 421 specimens for HFMD , brainstem encephalitis, asepicmeningitis, herpangina, viral myocarditis and flaccid limb weakness were taken inoculated into the Vero ceils and incubated at 37C, 5%CO2. RNA was extracted directly from all the positive specimens and part of the negative specimens in virus isolation and original specimens. And then were identified for EV71 by RT-PCR using EV71 specific primers 159 and 162 ,The segment of the VP1 gene were sequenced. A phylogenetic was constructed by comparison of the sequences with other EV71 strains isolated from Guangzhou, Shenzhen, Shanghai, Wuhan, Taiwan as well as Japan, Hungary and South Korea, Malaysia and the United States. Analysis EV71 viral genotype and subgenotype in recent years our country as well as Guangzhou.2. Stochastic choices 6 EV71 strains known sequence of the segment of the VP1 gene , including 4 strains isolated from HFMD patient in Guangzhou in 2004 and 2 strains isolated from polio-paralysis in Guangzhou in 2001.The strains were used to infect threecell lines representing human neuronal cells(SK-N-SH) , human laryngeal cells (Hep-2) and monkey nephric cells(Vero) by 100 TCID50. The three cell lines were compared for presence of virus-mediated cytopathic effects under light microscope , virus replication rate by real-time PCR and the one-step growth curve.Results: l.Four hundred and twenty-one specimens were collected and detected in 2004 in Guangzhou. In the end,38 suspicious enterovirus positive specimens(9.03%), 13 EV71 positive specimens(34.21%) were found. The two months of our studied had positive specimens. It was 13 specimens(Apr), lspecimens(Mar).All strains were isolated from HFMD. There was no statistical significance between the number of male and female children. The ages of EV71-associated cases were mainly between lyear to 4 years . The sequence analysis showed that the new isolate shared 89.3 % -100 % nucleotide identity with otherstrains isolated from Chinese mainland, and shared 89.3%-95.1% with type C, the difference is smaller than 12%,but shared only 79.6%-84.7 % homology with type A and B, the difference is bigger than 15.3%.This prompts the strains isolated from Chinese mainland possibly belong to the identical genotype C. 97-56-CHN-97 may be C3, 0667-CHN-87 may be Cl,the other 15 strains isolated from mainland (including this experimental 4 strains) has composed a independent small branch, has formed C4.2.(1) 4 strains isolated from HFMD patient in Guangzhou in 2004 and 2 strains isolated from polio-paralysis in Guangzhou in 2001 ,belong to the same genotype, may cause SK-N-SH, Vero and Hep-2 cell lines virus-mediated cytopathic effects; Also in SK-N-SH and Vero cell pathological change degree is obviously heavier than Hep-2. (2) After strains isolated from HFMD infection 12h,24h,36h,48h in Vero cell ,its average viral copy number respectively is 10387, 105'89, 10791, 10972; After strains isolated from NS infection 12h,24h,36h,48h in Vero cell ,its average viral copy number respectively is 10384,10584,10788,10994, the viral duplication quantity of two kind of clinical types does not have the obvious statistical significance in the Vero cell (P>0.05). (3) After strains isolated from HFMD infection 12h,24h,36h,48h in SK-N-SH cell ,its average viral copy number respectively is 10387,105'89,107<9\ 10972; After strains isolated from NS infection 12h,24h,36h,48h in SK-N-SH cell ,its average viral copy number respectively is 10384, 105'84, 10788, 10994, the viral duplication quantity of two kind of clinical types does not have the obvious statistical significance in the SK-N-SH cell (P>0.05).(4) After strains isolated from HFMD infection 12h,24h,36h,48h in Hep-2 cell ,its average viral copy number respectively is 103-87, 105-89, lO7'91, 109-72; After strains isolated from NS infection 12h,24h,36h,48h in Hep-2 cell ,its average viral copy number respectively is 10384, 10584, 107'88,10994, the viral duplication quantity of two kind of clinical types does not have the obviousstatistical significance in the Hep-2 cell (P>0.05).(5) The viral duplication quantity in SK-N-SH and Vero infected with two kind of clinical types strains were obviously compares Hep-2 to be remarkable (P<0.05), but between SK-N-SH and Vero there does not have markable difference (P>0.05).Conclusion: 1. EV71 is one of the important pathogens of Guangzhou in2004. In April, it has a rush peak. The strains isolated from Chinese mainland have close nucleotide identity, all of them are C. Before 1997,there were several Cl and C3 in our coutry, the strains isolated from 1998 to 2004 were C4,showed that C4 was the main pathogens in the southern. Otherwise, the strains isolated from Taiwan were C2 and B. The strains isolated from mainland were closely related with most isolates from Taiwan , but from most EV71 strains reported abroadl The symptoms of EV71 infection in mainland were not as intensive those described in Taiwan's outbreak. Our study show that the strains isolated from Chinese mainland were similar and it will be favor of the development of effective vaccine.2.Consistently,viruses particles were also detected in the three cell lines. This confirms the wildly tissue tropism of EV71 and the clinical syndromes of EV71 vary from mild HFMD to severe CNS complications. We found that the strains isolated from the different clinical syndromes ,belong to the homologic genotype, have the same CPE and viral replication rate, it is reasonable to conclude from this finding that EV71 is most destructive to neurons. It would be intriguing to find out the variable relations of the viral genotype ,the tissue tropism and the clinical syndromes . Thoro- ughly discusses their relations will aid in the understanding of the neurovirulence of EV71 and in the development of new strategies to combat EV71 infection and the development of effective vaccine.