Neutralization escape variant of West Nile virus associated with altered peripheral pathogenicity and differential cytokine profile.

In order to understand the factors influencing pathogenicity of a virus, two neutralization escape (NE) variants were selected from wild type lineage 1 West Nile virus (WNV) 68856 strain pathogenic by intra-peritoneal (i.p.) route using monoclonal antibodies (MAbs) against envelope (E) protein. Both NE IF1A7 1.1 and NE IVC3F10 1.2 were resistant to neutralization and were neurovirulent by intra-cranial (i.c.) inoculation. Growth kinetics in porcine stable (PS) kidney and baby hamster kidney (BHK) cells was unchanged. In contrast to parent WNV only NE IF1A7 1.1 failed to cause lethal encephalitis on i.p. inoculation and was non pathogenic. NE IF1A7 1.1 variant showed delayed replication kinetics in murine peritoneal exudate cells (PEC) and Neuro 346 cells in vitro. In comparison with parent WNV and NE IVC3F10 1.2 variant, non pathogenic variant exhibited significantly reduced tumour necrosis factor α (TNF-α) induction in infected animals and PEC. Other cytokines like Interleukin (IL)-10, IL-6 and Interferon (IFN)-ß remained unchanged. However, IL-1ß did not follow the pattern and was higher only in parent WNV-infected PEC. The E gene sequences of these NE variants showed three common amino acid substitutions at residues E50, E89 and E242. A unique E156 (ser→pro) substitution in NE IF1A7 1.1, was absent in NE IVC3F10 1.2 variant suggested probable virulence marker. Our data indicates possible role of WNV E protein in induction of TNF-α and IL-1ß and its association with WNV pathogenesis.

Spread, establishment & prevalence of dengue vector Aedes aegypti (L.) in Konkan region, Maharashtra, India.

BACKGROUND & OBJECTIVE: Aedes aegypti (L.) (Diptera: Culicidae) was surveyed in the residential biotopes of Sindhudurg, Ratnagiri and Raigadh districts, Maharashtra State during dry (January-May & November- December) and wet (June-October) months in 2002 to update information on its distribution, to analyse post invasion establishment, and to study its prevalence. METHODS: The survey was designed to unfold Ae. aegypti distributions at landscape, habitat and micro-habitat levels. Risks of distribution and establishment due to differences amongst settlements, households and habitat attributes were analysed by univariate and multivariate methods. Demographic/transport changes were surveyed for its breeding refugia during dry months and prevalence during the wet seasons. Chi square tests for difference and relative risks of container types were applied to assess container habitats preferences for Ae. aegypti breeding, thus contributing to the risk of establishment and prevalence through seasons. RESULTS: Ae. aegypti was present in 16 out of total 28 settlements in dry season and 22 of 25 in wet season; the Breteau index (BI) varied from 1.25 to 57.33 and the container index (CI) was 0.6 to 25.81 in the dry season and BI from 1.25 to 110-00 and CI - 0.2 to 11.37 in the wet season, respectively. At macro-level, rural settlements and ports showed higher odds ratios (OR>1) for presence of Ae. aegypti. At meso-level, OR were 65.8, 24.8 and 4.9 for Ae. aegypti breeding in compact houses, clustered housing and in houses with tap water source respectively. At micro-level the plastic drums and small plastic containers were the important key habitats of its breeding. In the non-residential areas Ae. aegypti breeding was noted in one port during dry season; 10-road transport tyre dumps and scrap, 5 of 7 seaports and none of the two railway station areas during wet season. INTERPRETATION & CONCLUSION: At macro-geographic level Ae. aegypti distribution increased in 3 settlements, new establishment was seen in 7, new records in ten settlements and two were negative in past and present surveys. Logistic regression analyses indicated that the distribution was found to be more associated with ports and rural areas. At meso-geographic level the house aggregations and household drinking water supplies were of risk even at lower urbanization and rural levels. At micro-level, the site and potability were confounders; outdoor non potable water storage containers posed significant breeding risk, the potable water storage was significant but it contributed little to Ae. aegypti breeding. Further, Ae. aegypti breeding showed high preference to the plastic drums and other plastic miscellany. The results signified an expansion in the risk area of diseases borne by it in the context of urbanization, transport development and changing habitats.

Outbreak of acute hemorrhagic conjunctivitis in Maharashtra and Gujarat states of India, caused by Coxsackie virus A-24 variant.

Acute hemorrhagic conjunctivitis is associated with enteroviruses. Among these, Coxsackie A-24 variant (CA-24) and Enterovirus-70 (EV-70) are known to cause epidemics and pandemics. An outbreak of acute hemorrhagic conjunctivitis occurred in August-September 2003 in Maharashtra and Gujarat states of India. The present investigation was carried out to determine the viral etiological agent associated with the epidemic. Virus isolates were obtained from 11 eye swabs of conjunctivitis patients using HeLa/ Hep-2 cell lines. The isolates were characterized by serological and mouse pathogenecity tests, RT-PCR using enterovirus common primers (VP4-VP2), CA-24 specific primers (3C-proteinase region), EV-70 primers (VP-3) followed by sequencing, and phylogenetic analysis. The virus was characterized as a Coxsackie A-24 variant (CA-24v) and none of the isolates were found to be positive for EV-70. Sequencing of the PCR products derived from all the 11 isolates revealed 98.4% (SE 0.20) nucleotide identity within the Indian strains and 98.6% (0.50) and 94.4% (0.30) nucleotide identity respectively with the West Indies and Asian strains reported worldwide. The findings suggest that the outbreak of acute hemorrhagic conjunctivitis that occurred in Maharashtra and Gujarat states of India during August-September 2003 was caused by the Coxsackie A-24 variant (CA-24v).

Venereal transmission of Chandipura virus by Phlebotomus papatasi (Scopoli).

Experiments were conducted in the laboratory on Phlebotomus papatasi to determine the possible role of males in maintaining or sustaining the Chandipura virus (CHPV) activity in nature. This study indicated that infected males are capable of passing on the virus to female sand flies while mating. The infection rate was found to be 12.5% in uninfected females when mated with infected males. The occurrence of venereal transmission of this virus may have epidemiologic importance in the natural cycle of CHPV.

Vertical and venereal transmission of Chandipura virus (Rhabdoviridae) by Aedes aegypti (Diptera: Culicidae).

Experiments in the laboratory documented vertical and venereal transmission of Chandipura virus (CHPV) in Aedes aegypti (L.). The minimum filial infection rate among the progeny of infected females was 1.2%; the rate among male and female progeny was 0.9 and 1.4%, respectively. The venereal infection rate of CHPV among inseminated females was 32.7%. Our study indicates the possible occurrence of vertical and venereal transmission of CHPV in insect vectors.

A preliminary study of multilevel geographic distribution & prevalence of Aedes aegypti (Diptera: Culicidae) in the state of Goa, India.

BACKGROUND & OBJECTIVES: Dengue virus activity has never been reported in the state of Goa. The present study was carried out to document a multilevel geographic distribution, prevalence and preliminary analysis of risk factors for the invasions of Aedes aegypti in Goa. METHODS: A geographic information system (GIS) based Ae. aegypti surveys were conducted in dry (April 2002) and wet (July 2002) seasons in the rural and urban settlements. The random walk method was used for household coverage. The non-residential area visits included ancillaries of roadways, railways, air-and seaports. Simultaneous adult mosquito collections and one-larva per container technique were adopted. RESULTS: The Ae. aegypti larval and adult prevalence was noted in all the four urban areas in both dry (Density index (DI)= 3 to 6) and wet (DI= 5 to 7) seasons and only one out of 3 villages showed Ae aegypti presence in wet season (DI= 5 to 7). In the residential areas, hutments showed higher relative prevalence indices (Breteau index, BI=100; container index, CI=11.95; adult house index, AHI=13.33) followed by close set cement houses (BI=44.1; CI=12.0; AHI=11.24). Ae aegypti relative prevalence indices were also more for households with pets (BI=85.11; CI=12.5; AHI= 42.85); those with tap had higher risk (larval house index, LHI =32.03; relative risk, RR>2, n=256). Plastic drum was the most preferred breeding place (chi(2) = 19.81; P<0.01; RR=3.41) among domestic containers and rubber tyres (chi(2) = 11.86; P<0.01; RR=3.61)among sundry/rainfilled containers. INTERPRETATION & CONCLUSION: Established Ae aegypti prevalence in the urban settlements during dry and wet seasons and its scattered distribution in a rural settlement spell risk of dengue infection at macro-level. In the residential areas nature and types of the households, tap water supply and storage and communities' attitude and practices contribute to sustained meso-level risk of Ae aegypti prevalence dependant DEN. The non-residential areas offer transient meso-level risk as Ae aegypti prevalence was seasonally unstable and monsoon dependent. Risk at micro-level was due to the preferred larval habitats of Ae aegypti breeding viz., residential plastic-ware and tyres, and transport tyres in non-residential areas.