Mechanism of Japanese encephalitis virus genotypes replacement based on human, porcine and mosquito-originated cell lines model

OBJECTIVE@#To examine the multiplication efficiency Japanese encephalitis virus (JEV) genotype I (GI) and genotype III (GIII) of different cell lines which originated from human, porcine, mosquitoes in order to prove mechanism of JEV GI replacement JEV GIII since it emerging in nature recent decades.@*METHODS@#The mixture of GI and GIII JEV isolates was inoculated on human rhabdomyosarcoma (RD), pig kidney epithelial (PS) and Aedes albopictus C6/36 clone (C6/36) which originated from human, porcine and mosquitoes, respectively. Plaque assays were performed to calculate virus titer and real-time RT-PCR with GI and GIII specific primer sets to quantify the number of GI and GIII RNA copies.@*RESULTS@#The highest virus titer reached at the 3rd day of post infection when GI and GIII mixture was inoculated on RD and PS and that of C6/36 was at the 4th day. JEVs were amplified and maintained by C6/36 cells after 10 passages whereas that by RD and PS only limited within 8 and 6 passages, respectively. GI strain amplified and maintained more efficiently on C6/36 and PS but not RD, whereas GIII strain amplified and maintained more efficiently on RD.@*CONCLUSIONS@#There is a correlation between the multiplication efficiency of GI and GIII JEV strains when these two genotype strains co-infected on different cell lines with the predominance of GI strains in C6/36 and PS and the limited detection of GI strains in RD cells proving a possible mechanism of shift JEV genotypes in nature recent decades since GI emerging.

Mechanism of Japanese encephalitis virus genotypes replacement based on human, porcine and mosquito-originated cell lines model

Objective: To examine the multiplication efficiency Japanese encephalitis virus (JEV) genotype I (GI) and genotype III (GIII) of different cell lines which originated from human, porcine, mosquitoes in order to prove mechanism of JEV GI replacement JEV GIII since it emerging in nature recent decades. Methods: The mixture of GI and GIII JEV isolates was inoculated on human rhabdomyosarcoma (RD), pig kidney epithelial (PS) and Aedes albopictus C6/36 clone (C6/36) which originated from human, porcine and mosquitoes, respectively. Plaque assays were performed to calculate virus titer and real-time RT-PCR with GI and GIII specific primer sets to quantify the number of GI and GIII RNA copies. Results: The highest virus titer reached at the 3rd day of post infection when GI and GIII mixture was inoculated on RD and PS and that of C6/36 was at the 4th day. JEVs were amplified and maintained by C6/36 cells after 10 passages whereas that by RD and PS only limited within 8 and 6 passages, respectively. GI strain amplified and maintained more efficiently on C6/36 and PS but not RD, whereas GIII strain amplified and maintained more efficiently on RD. Conclusions: There is a correlation between the multiplication efficiency of GI and GIII JEV strains when these two genotype strains co-infected on different cell lines with the predominance of GI strains in C6/36 and PS and the limited detection of GI strains in RD cells proving a possible mechanism of shift JEV genotypes in nature recent decades since GI emerging.

Study on epidemiological characteristics of the Japanese Encephalitis in Ha Nam during 2001 - 2006

Background: Viet Nam is located in the epidemiological region of Japanese Encephalitis (JE). JE vaccine manufactured in Viet Nam has been used in the national vaccination program since 2003, but there has been no scientific study on the effectiveness of this program in Ha Nam province. Objectives: To investigate the epidemiology of JE and effectiveness of vaccinations in Ha Nam province from 2001-2006. Subjects and method: All residents in Ha Nam province, with the key focus group being JE cases. The retrospective and prospective epidemiological descriptive method was used. Results: The records of 148 cases of JE were collected. Among them, 42.5% (63 cases) were confirmed as JE by MAC-ELISA. The incidence and mortality rate were 1.29/100,000 and 0.12/100,000, respectively. Confirmed JE cases were found annually in all districts of Ha Nam province. However, it has been in decline, especially since the vaccination program was implemented for 1-5 year old children. The disease occurred in the summer with the peak in May and June (80.9% of total cases). Most of the cases were children under 15 years old (98.4%). The highest incidence rate was 6.4/100,000; 5.5/100,000 and 5.3/100,000 in the children aged 5-9, 1-4 and under 1 year old, respectively. Conclusion: The vaccination program appeared effective in reducing the mortality and morbidity of JE among children in Ha Nam province.

Detection of Japanese encephalitis genotype 1 in central, southern and highland of Viet Nam

Background: In recent year, Japanese Encephalitis Virus (JEV) genotype 1 has been detected among isolates from mosquitoes and pig\u2019s blood samples in northern Viet Nam, but there has been no information on the presence of this genotype in the Central, Southern and Highland regions. Objectives: This study aims to detect the Japanese encephalitis genotype 1 in various different geographic regions of Viet Nam. Material and method: Sequence analysis\u2019s of whole E gene of 18 strains isolated from human, mosquitoes and pig\u2019s blood during 2001-2007. Results: 7 strains isolated from pig\u2019s blood and mosquito samples in the Northern, Central, Southern and Highland fell into genotype 1, but 11 others isolated from humans in the Northern and Central regions belonged to genotype 3. Conclusion: This is the first time that JEV genotype 1 was detected in the central, northern, highland Viet Nam and further studies on genotype 1 causing human diseases needs to be carried out.\r\n', u'\r\n', u'

Evaluation of the quality of the IgM antibody capture ELISA diagnostic kit for the Japanese encephalitis virus produced by National Institute of Hygiene and Epidemiology

Background: IgM antibody capture ELISA (MAC-ELISA) technique has been widely applied for Japanese Encephalitis Virus (JEV) diagnosis. So far rare internationally commercial kits are available. Thus, the international evaluation of the kit is required as per the recommendation of the WHO. Objectives: To evaluate the quality of the IgM antibody capture ELISA diagnostic kit for JEV produced by the Vietnam National Institute of Hygiene and Epidemiology (NIHE). Subjects and method: In this study, NIID kit was used as control to check the kit from NIHE. Both NIHE and NIID kits were used to detect JEV IgM among 38 serum and 6 CFS samples, which belongs to 5 sample groups (JE patients group, dengue patients group, other viral encephalitis patients group, Tick Born Encephalitis (TBE) patient group and healthy JE vaccinated donors group). Results: The detection of JEV IgM by NIHE kit was concurrent with the NIID kit. There is no positive with the JE in the groups of Dengue patients, TBE, other virus encephalitis patients and JE vaccinated donors. Conclusion: MAC-ELISA kit of NIHE can be used for different diagnosis of JEV and Dengue virus (both viruses are in Flavivirus genus), as well as other viruses caused by encephalitis.

Japanese encephalitis disease and the efficacy of vaccination in Thai Binh province, 2003 - 2007

Background: Japanese Encephalitis (JE) is common in the plains and mountainous areas in Asia \u2013 Pacific. Japanese encephalitis vaccine shows effectiveness in protecting children from JE in some countries such as Japan and Korea. Objective: To evaluate the efficacy of Japanese Encephalitis (JE) vaccination in Thai Binh province during 2003-2007. Subject and Method: Prospective, retrospective and sero-epidemiological methods were carried out on 329 samples collected from viral encephalitis patients and tested by JE MAC-ELISA, the positive average was 41.6% (137/329). Result: It had dramatically dropped from 85.2% in 2003 to 8.5 % in 2007 related to the rate of JE vaccination for children from 1 to 5 years old increasing from 49 % in 2003 to 77 % in 2007. Most of JE confirmed cases were un-vaccinated. Conclusion: JE etiology cause viral encephalitis in children in Thai Binh province was reduced thanks to JE vaccination in EPI program for 1 to 5 year old children. But more than 96% (131/136) of viral encephalitis in 15 years old upward was unknown etiology, the need for further study of the etiology cause viral encephalitis in adults.

Study fluctuation and confirmation of Japanese encephalitis vector in Ha Nam province, 2006-2007 \r\n', u'\r\n', u'

Background: Japanese Encephalitis (JE) virus is a leading cause of encephalitis in children with high mortality and complication. JE is a dangerous infectious disease via Culex mosquitoes. Objective: To identify the density of some mosquitoes and vectors that transmit JE virus in Ha Nam province. Subject and Method:There were 30.333 mosquitoes including 05 genera and 15 species which were collected at night from April 2006 to March 2007 in the following communes: Tanson (midland area) Hungcong (plain area) and Liemchinh (suburban area). Results: A total of 06 Culex species, the highest rate is Culex tritaeniohychus (41.20%); Culex annulus (15.56%); other Culex species occupied around 0.15% -2.49%. In these communes, the density of Culex tritaeniohychus was higher than Culex annulus. They were found to be active all year round, but the highest density of Culex tritaeniohychus and Culex annulus is 5.86 and 2.15, respectively in April and fall to 4.35% and 1.71% in July. The collected mosquitoes during April, May, June and July includes 133 and 28 Culex tritaeniohychus and Culex annulus pools, respectively; all were to be processed for the isolation of Japanese Encephalitis (JE) virus. There were 12 Culex tritaeniohychus and 2 Culex annulus pools which were collected in Tanson commune yielding positive. Conclusion: The results were confirmed that Culex tritaenioohychus and Culex annulus are the major vectors that transmit JE virus in Ha Nam, 2006 - 2007. All 14 JE virus strains were isolated from collected mosquito pools during April, May and June; whereas JE virus strain was not yet isolated from collected mosquito pool in July. Thus, in order to control JE disease, it is necessary to control JE vectors in April, May and June.

Detection of Japanese encephalitis frequency in the pig population in Ha Nam province by GAC-ELISA

Background: Mosquitoes and pigs play important roles in maintaining and increasing the Japanese Encephalitis (JE) virus in nature and which is then transmitted to humans. Thus, surveillance of the JE infection frequency in the pig population may predict the human JE cases. \r\n', u'Objectives: The study aimed to determine IgG antibody against the JE virus in the pig population in Hanam province \r\n', u'Subjects and methods: The study included 1791 pig serum samples collected from 3 districts of Hanam province from Apr 2006 to Mar 2007. GAC-ELISA technique was used to determine the JE virus infection in the swine population.\r\n', u'Results: The average positive rate in pig population was 34.9 % (626/1791); with the highest frequency occurring in the summer (37.7%- 84.0 %), co-incident with the JE season in Northern Vietnam. On the contrary, in winter JE case are rare, frequency of IgG antibody against JE virus in the swine population was low, ranging from 9.2% to 22.0.%. \r\n', u'Conclusions: These results have shown the ecologically close relationship between the amplification of the JE virus in the swine population, vector and JE cases in northern Vietnam. \r\n', u'

Detection of IgM anti Japanese Encephalitis (JE) virus by antigen genotype 1 & genotype 3

Background: Recently Japanese Encephalitis (JE) virus type 1 has surfaced and is co-circulated with JE virus type 3 in the northern areas of Viet Nam, so a sensitivity of JE viral antigen genotype 3 to detect IgM is required. Objectives: To compare the sensitivity of JE viral antigen genotype 1 and 3 to detect IgM against the JE virus. Materials and method: 783 cerebrospinal fluid and serum samples from viral encephalitis cases from 1999-2005 were collected and examined by MAC-ELISA for JE viral antigen genotype 1 and 3. Results: The agreement on the diagnosis of these kinds of antigen was 99.7% and the sensitivity of JE viral antigen genotype 3 was higher than that of genotype 1. Thus, JE viral antigen genotype 3 could be considered as the selected antigen for JE diagnosis in Viet Nam. IgM titer determined by JE viral antigen genotype 1 was higher than that of genotype 3 in 2003 and 2005 and lower in 1999, 2000, 2001, 2002 and 2004. Conclusion: The dominant phenomenon of JE viral genotypes differing over the years might be due to the interaction of the virus and its vectors. Further study is required to clarify this observation.

Observation of Nam Dinh virus multiplication on Aedes albopictus cell line clone C6/36 by electron microscopy

Background: Virus is one of main causes of children acute encephalitis syndrome in countries of Asia south-east. In Vietnam, apart from Japanese Encephalitis Virus which is considered as main cause of children acute encephalitis syndrome, there are other viral pathologies, of which is Nam Dinh virus. Nam Dinh virus \ufffd?a novel Arbovirus was isolated from acute encephalitis syndrome patient in northern Vietnam, 2002. The circulation of this virus has been recognized in the north, central, and highland regions. Objective: To observe the multiplication of Nam Dinh virus on the Aedes albopictus cell line clone C6/36. Materials and method: In this study the ultra-thin section method was used to observe the multiplication of the Nam Dinh virus on the Aedes albopictus cell line clone C6/36, 48 hours post-infection. Results and Conclusion: Nam Dinh encephalitis virus got used to Aedes albopictus cell line clone C6/36 and damaged cells, 24-48 hours post-infection. Its multiplication is taking place in the cytoplasm, a typical characteristic of RNA virus. Nucleocapsids of the virus were found in vacuoles of the cell. Proteincapsid of the virus was synthesized in a rough endoplasmic reticulum (rER). After assembling in the cytoplasm, the virus is released from the cell by budding and used the cell membrane as its envelope.

Emerging a member of reoviridae family associated with acute encephalitis syndrome in Gia Lai province, 2005

Background: In recent years, some arbo viruses which causes acute encephalitis syndrome (AES) have been identified in serveral countries in the world such as Chandipura virus belonging to Rhabdoviridae family in India, Banna virus belonging to Reoviridae family in China. In Vietnam, apart from Japanese Encephalitis Virus which is considered as main cause of AES, there are a few intestinal viruses like Herpes symplex virus type 1 and 4 and Nam Dinh virus considering other causes of AES. Objective: To identify the hyppothesis that one virus strain parasitizing in mosquito in Gia Lai province causes AES in order to provide more information about virus strains which cause AES in Vietnam. Subjects and method: Aedes albopictus cell line clone C6/36 was used for the isolation of virus in 43 cerebrospinal fluid samples of patients who were treated in Gia Lai hospital, from January/2005 to July/2005. Result and Conclusion: One virus strain from a 3-year old girl in Gia Lai province was isolated in 2005. The virus coded 05VN225 has the morphology similar to other viruses belonging to Reoviridea family.The nucleic acid sequence of the virus was checked with specific primers of alphavirus and flavirus groups, Nam Dinh virus and Conti virus group B (reovirus) of the Reoviridae. The positive result was confirmed with reovirus primers. This member of the Reoviridae family was isolated from acute encephalitis syndrome in Vietnam in 2005. Further study on pathology of the virus is very necessary.

Sensitivity of particle agglutination assay with MAC-ELISA for Japanese encephalitis rapid diagnosis: a comparative study

Particle agglutination assay (PA) was developed and applied for detecting specific IgM antibodies to Japanese encephalitis virus in recent years. In this study, the sensitivity of PA and MAC-ELISA were 91.11 and 97.77%, respectively

Changes in results of Japanese encephalitis (JE) serodiagnosis in Northern Vietnam, 1998

During the 1998 Japanese Encephalitis epidemic in Northern Vietnam the authors have examined 402 patients which were clinically diagnosed as "acute encephalitis syndrome". The IgM anti JE was evidenced in only 132 cases. The seropositivity is patients under 5 years of age was 33.0 - 42.1% in 1998, compared with 64.2 - 75.7% during the period of 1989 - 1995.