A serological survey of arboviral diseases among the human population of the Andaman and Nicobar Islands,India

In an attempt to determine the prevalence of certain arthropod-borne viruses of public health importance amongst the human population of the Andaman and Nicobar Islands of India, 2,401 sera were collected from six major localities. The sera were analysed by the hemagglutination inhibition (HI) and neutralization (N) tests, using Chikungunya (CHIK), Japanese encephalitis (JE), West Nile (WN), dengue (DEN-2), Langat (TP-21) and Kyasanur Forest disease (KFD) viral antigens. The highest prevalence of HI antibodies was detected against KFD virus (22.4%), followed by Langat (20.2%), JE (5.9%), DEN-2 (3.1%), CHIK (2.9%) and WN (0.8%) viruses. Cross-reactions to the viral antigens were also noted. The results of N tests indicated a high prevalence of DEN-2 (25.4%) virus, followed by Langat (17.5%), CHIK (15.3%), KFD (12%), JE (2.19%) and WN (1.8%). These results are discussed in relation to important epidemiological parameters like age, sex and geographical location. To our knowledge, this is the first report of an extensive serosurvey of arthropod-borne viruses on these islands.     

Recurrent outbreaks of Japanese encephalitis in Nagaland (1985-1989)--a seroepidemiological study.

Recurrent epidemics of encephalitis in Nagaland, a North-Eastern State of India, following its first appearance in 1985, were investigated both epidemiologically and virologically. Although, no viral agent could be isolated from any of the clinical samples and mosquitoes, detection of JE specific IgM antibodies in many of the CSF and acute blood samples, together with presence of HI and CF antibodies to JE antigen in a number of acute and convalescent sera established the etiologic role of JE virus in this region. A total number of 83 clinically diagnosed cases could only be investigated virologically between 1985-89, where evidence of JE could be established in 34 (40.9 per cent) and flavivirus (including JE in majority) in 17 (21.5 per cent) cases. A limited serological survey among the close contacts of the victims in 1985 revealed JE antibody in 26.6 per cent of them. Analysis of the epidemiological and serological findings of different years revealed that while the outbreaks of 1985, 1987 and 1988 were due to JE infection, the episodes of 1986 and 1989, on the other hand, had chiefly features of high and prolonged fever with limited number of CNS involvement of undetermined origin, where the possibility of malarial infection has been a suspect apart from JE etiology.     

Absence of dengue 2 infection enhancement in human sera containing Japanese encephalitis antibodies

Sera from 52 young adults resident in a rural area in North Thailand were studied for plaque-reducing neutralizing antibodies against dengue (DEN) viruses types 1-4 and Japanese encephalitis (JE), and for DEN-2 infection-enhancing antibodies using a newly described microtest in the human monocyte cell line, U-937. Infection-enhancing antibody titers in U-937 cells using a simplified micromethod were similar to results obtained by published methods using human peripheral blood leukocytes and a macrotest using U-937 cells. In the sample, there were 23 with antibodies to one or more DEN viruses with or without accompanying JE antibodies; 16 sera demonstrated antibodies only to JE and 13 had no detectable antibodies to any flavivirus. All but two DEN antibody-containing sera enhanced DEN-2 infections in U-937 cells, often to titers of 1:10,000 or greater. By contrast, only one of 16 JE-immune sera enhanced DEN-2 infection in monocytes, and that at a dilution of 1:100. None of the flavivirus-negative sera had DEN-2 enhancing activity. The failure of human anti-JE contrasts with the ability of rabbit anti-JE to enhance DEN-2 infections, but correlates with the absence of recorded instances of dengue shock syndrome in human beings sequentially infected with JE and then a DEN virus. This report seemingly reconciles in vitro and in vivo phenomena, and may provide an opportunity to study mechanisms involved.     

Seroprevalence of West Nile virus in wild birds in far eastern Russia using a focus reduction neutralization test

West Nile (WN) virus has been spreading geographically to non-endemic areas in various parts of the world. However, little is known about the extent of WN virus infection in Russia. Japanese encephalitis (JE) virus, which is closely related to WN virus, is prevalent throughout East Asia. We evaluated the effectiveness of a focus reduction neutralization test in young chicks inoculated with JE and WN viruses, and conducted a survey of WN infection among wild birds in Far Eastern Russia. Following single virus infection, only neutralizing antibodies specific to the homologous virus were detected in chicks. The neutralization test was then applied to serum samples from 145 wild birds for WN and JE virus. Twenty-one samples were positive for neutralizing antibodies to WN. These results suggest that WN virus is prevalent among wild birds in the Far Eastern region of Russia.     

Prevalence of Japanese encephalitis and West Nile viral infections in pig population in and around Chandigarh

A study on sero prevalence of Japanese Encephalitis (JE) and West Nile (WN) viruses in pigs was carried out in and around Chandigarh. A total of 158 blood samples collected (June, 1995-March, 1996) from batches of pigs were screened for antibodies against JE and WN viral antigens by haemagglutination inhibition and complement fixation tests. In Chandigarh out of 13 pigs, 5 had JE and one WN HAI antibodies. Similarly, the sera of 14 and 1 pigs in Punjab and 29 and 3 pigs in Haryana were positive for JE and WN HAI antibodies respectively. In total JE specific HAI antibodies were found in 30.3% and CF antibodies in 12.5% of pigs whereas WN specific HAI antibodies were obtained in 3.2% and only one pig had CF antibodies. The results show that JE and WN viral infections are prevalent in pig population in and around Chandigarh.     

Short report: absence of protective neutralizng antibodies to West Nile virus in subjects following vaccination with Japanese encephalitis or dengue vaccines

Protection of individuals against West Nile (WN) encephalitis is an emerging concern in the United States and Europe. We investigated whether immunization with licensed inactivated Japanese encephalitis (JE) vaccine or experimental live attenuated dengue vaccines resulted in induction of cross-neutralizing antibodies against WN virus. Protective neutralizing antibody titers to WN virus were not detected in any volunteer despite successful immunization to related flaviviruses. Vaccination against JE or dengue is unlikely to prevent WN virus infection but may still protect against disease.     

Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay

Type-specific monoclonal antibodies prepared against the four dengue (DEN) virus serotypes were evaluated for their ability to identify low-passage human and mosquito isolates from Jamaica and West Africa by an indirect immunofluorescence assay. Serotyped human isolates from Jamaican dengue fever patients included 12 DEN-1, two DEN-2, and five DEN-4 viruses. Viruses from West Africa included 84 DEN-2 mosquito strains as well as two DEN-1 and one DEN-2 from humans. Results obtained using the immunofluorescence assay were consistent with virus identifications obtained using the more classical but costly and time-consuming plaque-reduction neutralization test. More viral isolates and higher virus yields were obtained using the C6/36 clone of Aedes albopictus cells rather than LLC-MK2 (monkey kidney) cells. Dengue type-specific monoclonal antibodies detected prototype viral antigens 24-48 hours postinfection in C6/36 cells. This is the first time that monoclonal antibodies have been used to serotype low-passage flavivirus isolates.     

Seroprevalence and distribution of Flaviviridae, Togaviridae, and Bunyaviridae arboviral infections in rural Cameroonian adults

Arboviruses from the families Flaviviridae, Togaviridae, and Bunyaviridae are suspected to cause widespread morbidity in sub-Saharan African populations, but little research been done to document the burden and distribution of these pathogens. We tested serum samples from 256 Cameroonian adults from nine rural villages for the presence of Dengue-2 (DEN-2), West Nile (WN), Yellow fever (YF), Chikungunya (CHIK), O'nyong-nyong (ONN), Sindbis (SIN), and Tahyna (TAH) infection using standard plaque-reduction neutralization tests (PRNT). Of these samples, 12.5% were DEN-2 positive, 6.6% were WN positive, 26.9% were YF positive, 46.5% were CHIK seropositive, 47.7% were ONN positive, 7.8% were SIN positive, and 36.3% were TAH positive. DEN-2, YF, and CHIK seroprevalence rates were lower among individuals living in dwellings with grass or thatched roofs versus corrugated tin and in villages isolated from urban centers. Seroprevalence rates of YF and CHIK increased with age. These results suggest that inter-epidemic arboviral infection is common in central African populations.     

Longitudinal studies in South Indian villages on Japanese encephalitis virus infection in mosquitoes and seroconversion in goats

Japanese encephalitis (JE) is endemic in Cuddalore district, Tamil Nadu, where Culex tritaeniorhynchus Giles was the major vector. We screened 45 100 adult female Cx. tritaeniorhynchus (902 pools) by enzyme-linked immunosorbent assay and isolated and confirmed JE virus (JEV) by using an insect bioassay system. We had 69 isolates of which 62 (90%) were identified as JEV. The average vector abundance per man hour for Cx. tritaeniorhynchus was 324.5 per month for the period June 1998-May 2000. The average minimum infection rate (MIR) per month in Cx. tritaeniorhynchus was 1.4 (range 0.0-5.6). Every year, a new batch of goats, 20 in the first year and 31 in the second year, born during the non-JE transmission period (January-June), aged <6 months and negative for haemagglutination inhibition (HI) antibodies were procured and placed in the villages as sentinels. Fortnightly, blood specimens were collected from these goats and tested for JE antibodies by HI test. Seroconversions (SCs) were recorded in 14 goats (70%) in the first year and 23 goats (74%) in the second year. JE HI antibody titres in goats were low (1:10-1:80) and these levels declined to undetectable levels in about 4 weeks following SCs. The time sequence of events indicated that four of five peaks of MIR in mosquitoes were followed 1-3 months later by peaks in the proportion of seroconverted goats. We suggest the screening of goats and cattle as a more feasible tool to stratify areas according to JE infection risk to the human population through the regular health system rather than screening mosquitoes using monoclonal antibodies, which is possible only in specialized laboratories.     

猪感染乙型脑炎与人乙型脑炎关系的研究

我们用HI试验和温敏试验,检测了320份猪血清中的乙脑抗体,HI和IgM抗体阳性率分别为79.1％(GMT29.8)和40％。HI和IgM抗体阳转率的高峰期分别在7月中旬(100％)和6月上旬(54.3％)。猪乙脑抗体阳转率的高低与人乙脑疫情(96例)具有相应的关系(K=53,P<0.05),可用它来预测和预报人乙脑疫情。     

Flavivirus type-specific antigens produced from fusions of a portion of the E protein gene with the Escherichia coli trpE gene.

Based on previous data showing that JEV and DEN-1 fusion proteins can be used as antigens, we engineered analogous fusion proteins for the same regions of the E proteins of DEN-2, DEN-3, and DEN-4, using the polymerase chain reaction. The resulting fusion proteins were tested in a modified ELISA for their reactivity with mouse immune ascitic fluids (MIAF) generated against 10 different flaviviruses. The results showed that these recombinant antigens could be used as type-specific immunological reagents, and suggest that these antigens could serve as the basis for rapid, safe, and inexpensive flavivirus serosurveys.     

Comparison of haemagglutination inhibition (HI) and indirect fluorescent antibody (IFA) techniques for the serological diagnosis of certain flavivirus infections

Paired serum samples from 25 patients having clinical evidence of arboviral infections and 15 controls were examined for antibodies against dengue, Japanese encephalitis and West nile viruses by haemagglutination inhibition (HI) and indirect fluorescent antibody (IFA) methods. The two tests were comparable in specificity but the IFA test was found to be more sensitive than the HI test. Like the HI test, the antibodies detected by IFA test cross-reacted within the flavivirus group under test. Use of IFA test is recommended to detect flavivirus infections as the test is simple, sensitive and can be carried out rapidly.     

酶联免疫吸附试验检测猪血清的乙型脑炎抗体的评价

本文报告用酶联免疫吸附试验捕获法和间接法分别检测猪血清乙型脑炎IgM和IgG抗体。酶标法测得的IgM和IgG滴度与血凝抑制试验滴度显著相关。但前者可测出70％的IgG型抗体和63.3％的IgM型抗体,而后者仅56.7％阳性。在乙脑流行季节,两种类型的乙脑抗体出现几乎同样快。IgG可持续终身。IgM仅在短期内检测到,当表示猪乙脑新感染。     

Serological evidence of persistence of Japanese encephalitis virus activity in Bihar, India

A total of 64 sera samples from ducks, 212 from 245 fowls, 1 from 16 sparrows and 5 from human were examined for HI and CF antibodies to Japanese encephalitis (JE), West Nile (NW), Dengue-2 (DN-2), and Chikungunya (CHIK) viruses. The results were compared with those obtained earlier while examining pigs' sera from the same area. The results revealed that ducks, fowls and peridomestic sparrows possessed antibodies to JEV. JEV activity in ducks and fowls was significantly higher compared to its activity in pigs in the same area reported earlier indicating persistence of its activity.     

Surveillance of Chikungunya virus activity in some North-eastern states of India

Objective: To detect the prevalence pattern of Chikungunya virus in three states of Northeast India. Methods: A total of 1 510 samples were collected from different private and government hospitals of Assam, Arunachal Pradesh and Meghalaya. Serum was tested for the presence of IgM antibodies against Chikungunya virus followed by RT-PCR for amplification of Chikungunya E1 gene region using specific primers. Results: Overall, 11.83%(172/1 454) clinical samples were positive by MAC-ELISA and/or RT-PCR assay. Asymptomatic infection was seen in 17.86%. Males were more affected than females and age group 16-30 years was mostly affected. Fever(100.00%) was the primary symptom followed by headache(72.03%) and arthralgia(41.53%). Only 118 Chikungunya positive cases could be traced, of which 25.42% complained about sequelae of infection. In entomological investigation, Aedes aegypti was more predominant(92.10%) than Aedes albopictus(7.90%). No mosquito pools could be incriminated for Chikungunya virus. Conclusions: In this study, Chikungunya was observed to be prevalent in Assam, Arunachal Pradesh and Meghalaya. Though Chikungunya is a selflimiting infection, increasing morbidity by CHIKV infection is affecting social and economic status of individual. Thus, a community empowerment to effectively control mosquito population by employing different mosquito control measures along with personal protection is mandatory to tackle future outbreak of the disease.     

Detection of West Nile Virus infection in birds in the United States by blocking ELISA and immunohistochemistry

A blocking ELISA targeting an immunodominant West Nile epitope on the West Nile Virus NS1 protein was assessed for the detection of West Nile-specific antibodies in blood samples collected from 584 sentinel chickens and 238 wild birds collected in New Jersey from May-December 2000. Ten mallard ducks (Anas platyrhynchos) experimentally infected with West Nile virus and six uninfected controls were also tested. The ELISA proved specific in detecting WNV antibodies in 9/10 chickens and 4/4 wild birds previously confirmed as positive by Plaque Reduction Neutralization test (PRNT) at the Center for Disease Control, Division of Vector Borne Diseases, Fort Collins, CO, USA (CDC). Nine out of the ten experimentally infected mallard ducks also tested positive for WN antibodies in the blocking ELISA, while 6/6 uninfected controls did not. Additionally, 1705 wild birds, collected in New Jersey from December 2000-November 2001 and Long Island, New York between November 1999 and August 2001 were also tested for WN antibodies by the blocking ELISA. These tests identified 30 positive specimens, 12 of which had formalin-fixed tissues available to allow detection of WN specific viral antigen in various tissues by WNV-specific immunohistochemistry. Our results indicate that rapid and specific detection of antibodies to WN virus in sera from a range of avian species by blocking ELISA is an effective strategy for WN Virus surveillance in avian hosts. In combination with detection of WN-specific antigens in tissues by immunohistochemistry (IHC) the blocking ELISA will also be useful for confirming WN infection in diseased birds.     

Investigation of an outbreak of chikungunya in Malegaon Municipal areas of Nasik district, Maharashtra (India) and its control

BACKGROUND & OBJECTIVES: An outbreak of chikungunya fever occurred in Malegaon town of Nasik district of Maharashtra state, India during February and March 2006. A total of 4530 fever cases were reported during this period including 1781 cases which were admitted in different hospitals of the town. An entomological and epidemiological investigation was carried out in the affected villages during the outbreak to study the possible causes of the outbreak and to isolate the virus responsible. METHODS: Entomological evaluation was done as per WHO guidelines. Sera samples were collected by venipuncture from clinically suspected chikungunya patients in hospitals and also during house-to-house survey in affected villages. IgM antibodies to dengue virus were detected using IgM capture ELISA (PANBIO) and by "Haemagglutination inhibition test" for detection of antibodies against Chikungunya virus. Acute sera samples were inoculated in cell lines for virus isolation. The isolates were confirmed by RT-PCR. RESULTS: On investigation, it was found that water storage containers like cement tanks, plastic containers or earthen pots placed in front of the individual houses were the potential breeding sites for Aedes aegypti. Entomological survey carried out in the most affected areas revealed high Aedes indices. House, container and breteau indices were found to be 27.2, 16.19 and 35.1, respectively. Out of the 13 acute sera samples collected, virus was isolated in 10 samples. The isolates were confirmed by RT-PCR and sequencing using primers from nsP1 gene of Chikungunya virus (CHIKV, Accession No. EF077609, EF077610). Of the 17 convalescent sera tested, significant level of HI antibodies to CHIKV was detected in five samples. One sample was positive for IgM antibodies against dengue virus. Based on clinico-epidemiological features and laboratory findings, the illness was confirmed to be of chikungunya viral disease. CONCLUSION: Control measures targeting the vector population and personal protective measures against the mosquito bites were instituted. Extensive IEC campaign with the involvement of community and religious leaders helped in containment of the disease.     

Epidemiological profile of Japanese encephalitis in Gorakhpur district, Uttar Pradesh, 1982-1988.

An in-depth study of Japanese Encephalitis (JE) situation in Gorakhpur district of Uttar Pradesh from 1982-1988 showed increasing trend in the incidence of JE. Total number of annual cases and case fatality rate (CFR) rose from 118 and 23.7 per cent in 1982 to 772 and 32.2 per cent in 1988 respectively. A definite increase was noticed in the number of cases per block following lull years in 1984 and 1987. Among the total affected 1201 villages, 1083 were affected only once. All age groups were affected and the disease showed marked seasonality during August to November. JE, which came in epidemic form in earlier years has established in the area in endemic form.     

The neurovirulence of flaviviruses in crab-eating monkeys (Macaca fascicularis)

The neurovirulent properties of attenuated dengue-2 and yellow fever (YF) vaccines, dengue-2 (DEN-2) and Japanese encephalitis (JE) viruses were studied in crab-eating monkeys (Macaca fascicularis). Number of central nervous system sites (as proportion affected) with neurovirulence (NV) lesions were compared. The results indicate that these monkeys reliably developed NV-lesion when inoculated with either JE or YF vaccine viruses (87%). NV-lesions occurred in a minority when inoculated with DEN-2 vaccine virus, were of minimal severity (9%), were probably biologically insignificant, and were of equal or less severity than lesions produced by its parental virus (10%).