Antiviral drug profile of human influenza A & B viruses circulating in India: 2004-2011.

background & objectives: Recent influenza antiviral resistance studies in South East Asia, Europe and the United States reveal adamantane and neuraminidase inhibitor (NAIs) resistance. This study was undertaken to evaluate antiviral resistance in influenza viruses isolated from various parts of India, during 2004 to 2011. methods: Influenza viruses were analyzed genetically for known resistance markers by M2 and NA gene sequencing. Influenza A/H1N1 (n=206), A/H3N2 (n=371) viruses for amantadine resistance and A/H1N1 (n=206), A/H3N2 (n=272) and type B (n=326) for oseltamivir resistance were sequenced. Pandemic (H1N1) (n= 493) isolates were tested for H274Y mutation by real time reverse transcription (rRT)-PCR. Randomly selected resistant and sensitive influenza A/H1N1 and A/H3N2 viruses were confirmed by phenotypic assay. results: Serine to asparagine (S3IN) mutation was detected in six isolates of 2007-2008.One dual-resistant A/H1N1 was detected for the first time in India with leucine to phenylalanine (L26F) mutation in Mm2 gene and H274Y mutation in NA gene. A/H3N2 viruses showed an increase in resistance to amantadine from 22.5 per cent in 2005 to 100 per cent in 2008 onwards with S3IN mutation. Fifty of the 61 (82%) A/H1N1 viruses tested in 2008-2009 were oseltamivir resistant with H274Y mutation, while all A/H3N2, pandemic A/H1N1 and type B isolates remained sensitive. Genetic results were also confirmed by phenotypic analysis of randomly selected 50 resistant A/H1N1 and 40 sensitive A/H3N2 isolates. Interpretation & conclusions: Emergence of influenza viruses resistant to amantadine and oseltamivir in spite of negligible usage of antivirals emphasizes the need for continuous monitoring of antiviral resistance.

Isolation of Chandipura virus (Vesiculovirus: Rhabdoviridae) from Sergentomyia species of sandflies from Nagpur, Maharashtra, India.

Background & objectives: An outbreak of acute encephalitis syndrome was reported from Vidarbha region of Maharashtra state, India, during July 2012. Anti-IgM antibodies against Chandipura virus (CHPV) were detected in clinical samples. Sandfly collections were done to determine their role in CHPV transmission. Methods: Twenty nine pools of Sergentomyia spp. comprising 625 specimens were processed for virus isolation in Vero E6 cell line. Diagnostic RT-PCR targeting N-gene was carried out with the sample that showed cytopathic effects (CPE). The PCR product was sequenced, analysed and the sequences were deposited in Genbank database. Results: CPE in Vero E6 cell line infected with three pools was detected at 48 h post infection. However, virus could be isolated only from one pool. RT-PCR studies demonstrated 527 nucleotide product that confirmed the agent as CHPV. Sequence analysis of the new isolate showed difference in 10-12 nucleotides in comparison to earlier isolates. Interpretation & conclusions: This is perhaps the first isolation of CHPV from Sergentomyia spp. in India and virus isolation during transmission season suggests their probable role in CHPV transmission. Further studies need to be done to confirm the precise role of Sargentomyia spp. in CHPV transmission.

Influenza virus genotypes circulating in and around Lucknow, Uttar Pradesh, India, during post pandemic period, August 2010 - September 2012.

Background & objectives: During the post influenza pandemic period, continuous surveillance of influenza virus and its subtypes is mandatory to help the policy makers to take effective and appropriate decisions. Therefore, this study was planned to determine the pattern of influenza virus activity in context to various meteorological and clinical parameters in and around Lucknow, Uttar Pradesh, India, during post pandemic period August 2010 - September 2012. Methods: Nasal swabs/throat swabs/nasopharyngeal aspirates of 2669 patients were collected. One-step real time PCR for detection of influenza virus was done according to the Centers for Disease Control and Prevention (CDC) protocol. Results: Influenza positivity was 15.8 per cent (423/2669) in symptomatic patients. Of the 423 total positives, 192 (7.2%) were influenza A and 231 (8.7%) were influenza B. Positivity for influenza virus was significantly (P=0.001, OR=2.9, CI=1.9-4.3) higher in patients with Influenza like illness (ILI) (17.4%, 396/2271) than those with severe acute respiratory illness (SARI) (6.8%, 27/398). Influenza A positive samples were subtyped as; pdmH1N1 (67.2%, 129/192) and seasonal H3N2 (32.8%, 63/192). It significantly correlated with monthly mean rainfall, humidity and dew point while atmospheric pressure was inversely related. No significant association was found with temperature and wind speed. Clinical variations were observed between different strains of Influenza virus. Interpretation & conclusions: The findings provide a clear picture of different clinical presentations of various strains of influenza A and B viruses and epidemiology of influenza infection from Lucknow (UP), India. The seasonality of influenza virus infection showed variation in relation to different environmental factors. Pandemic H1N1 caused more systemic infection than seasonal influenza A/H3N2 virus.

Viral aetiology of acute lower respiratory tract illness in hospitalised paediatric patients of a tertiary hospital: One year prospective study.

Context: Acute lower respiratory tract infections (ALRI), ranked as the second leading cause of death are the primary cause of hospitalisation in children. Viruses are the most important causative agents of ALRI. Aim: To study the viral aetiology of ALRI in children at a tertiary care hospital. Setting and Design: One year prospective observational study in a tertiary care hospital of King George’s Medical University, Lucknow. Material and Methods: Nasopharyngeal aspirate (NPA) was collected from children admitted with signs and symptoms of ALRI who were aged 0-14 years. Samples were transported to the laboratory at 4°C in viral transport media and processed for detection of respiratory syncytial virus (RSV) A and B, infl uenza virus A and B, adenovirus (ADV), human Boca virus (HBoV), human metapneumo virus (hMPV) and parainfl uenzavirus 1, 2, 3 and 4 using mono/multiplex real-time polymerase chain reaction (RT-PCR). STATA was used for statistical analysis. Results: In one year, 188 NPAs were screened for respiratory viruses, of which 45.7% tested positive. RSV was most commonly detected with 21.3% positivity followed by measles virus (8.5%), infl uenza A virus (7.4%), ADV (5.3%), infl uenza B virus (1.6%), hMPV (1.1%) and HBoV (0.5%). Month wise maximum positivity was seen in December and January. Positivity rate of RSV was highest in children aged < 1 year, which decreased with increase in age, while positive rate of infl uenza virus increased with increasing age. Conclusion: The occurrence of viral predominance in ALRI is highlighted.

Molecular characterization of Chittoor (Batai) virus isolates from India.

Background & objectives: Chittoor virus (CHITV) belongs to genus Orthobunyavirus, family Bunyaviridae. It has been isolated from various species of mosquitoes and pig from different parts of India. Five isolates of CHITV were characterized at the molecular level and compared with other Batai viruses (BATV) to find out any kind of reassortment in their genome. Methods: Complete nucelocapsid (S), glycoprotein (M) and partial RNA polymerase (L) segments of CHITV were amplified and sequenced. These sequences were compared with those of Batai viruses, isolated from different geographical locations in Asia, Africa and Europe. Results: Phylogenetic analysis revealed CHITV as a variant of BATV. High level of conservation was seen among the CHITV isolates studied. The CHITV sequences showed clustering in one lineage with the sequences from Japan and Malaysia, however, BATV sequences from Europe and Africa formed a separate phylogenetic lineage. Interpretation & conclusions: The study indicates the presence of a single genotype of CHITV circulating in India, despite the involvement of different hosts in the natural cycle by this virus. Analysis of the sequences of the S, M and L segments of genome indicated that the virus has not undergone any reassortment. This virus has not caused any epidemic involving humans, however, replication of the virus in different mosquito and vertebrate hosts species suggests that it is a cause of concern.

Chandipura virus growth kinetics in vertebrate cell lines, insect cell lines & embryonated eggs.

Background & objectives: Since not much information on Chandipura virus is available, an attempt was made to study the growth kinetics of the virus in certain vertebrate, invertebrate cell lines and embryonated chicken eggs. Methods: Comparative study of Chandipura virus (CHPV) growth kinetics in three vertebrate cell lines [Vero E6, Rhabdo myosarcoma (RD), Porcine stable kidney (PS) cell lines], two insect cell lines [Aedes aegypti (AA) and Phlebotomus papatasi (PP-9) cell lines] and embryonated pathogen free chicken eggs was conducted, by tissue culture infective dose 50 per cent (TCID50) and indirect immunofluorescence assay (IFA). Results: All the cell lines and embryonated egg supported the growth of CHPV and yielded high virus titre. The vertebrate cell lines showed distinct cytopathic effect (CPE) within 4-6 h post infection (PI), while no CPE was observed in insect cell lines. PP-9 cell line was the most sensitive system to CHPV as viral antigen could be detected at 1 h PI by IFA. Interpretation & conclusions: Our results demonstrated that all the systems were susceptible to CHPV and achieved high yield of virus. However, the PP-9 cell line had an edge over the others due to its high sensitivity to the virus which might be useful for detection and isolation of the virus during epidemics.

Ganjam virus.

Ganjam virus (GANV), a member of genus Nairovirus of family Bunyavirdae is of considerable veterinary importance in India. Though, predominantly tick borne, GANV was also isolated from mosquitoes, man and sheep. Neutralizing and complement fixing antibodies to GANV have been detected in animal and human sera collected from different parts of the country. Thirty three strains of GANV have been isolated from India, mainly from Haemaphysalis ticks. The virus replicated in certain vertebrate and mosquito cell lines and found pathogenic to laboratory animals. One natural infection and five laboratoryacquired infections in men were also reported. GANV is antigenically related to Nairobi sheep disease virus (NSDV) of Africa, which is highly pathogenic for sheep and goats causing 70-90 per cent mortality among the susceptible population. Recent molecular studies have demonstrated that GANV is an Asian variant of NSDV and both these viruses are related to the dreaded Crimean Congo haemorrhagic fever (CCHF) group viruses. The versatility of the virus to replicate in different arthropod species, its ability to infect sheep, goat and man makes it an important zoonotic agent.

Effect of temperature and insecticide stresses on Aedes aegypti larvae and their influence on the susceptibility of mosquitoes to dengue-2 virus.

Two major factors, higher temperatures and the application of insecticides, can drastically alter the genetic structure of a vector mosquito population. Due to these two stresses, the majority of the population gets wiped out, but the ones that withstand the stress and survive are likely to pass on survivability, and have an altered physiology. Our study shows that exposures to higher temperatures and DDT during the larval stage affects their susceptibility as adult mosquitoes to the DEN-2 virus. The overall transcription and translation status of heat shock protein (Hsp70) in virus high- and low-susceptible was the same as that in other batches. In the case of a DDT-resistant (R-7) strain two bands were obtained during RT-PCRs after heat shock. These two alleles were obtained only with HY-1 in which R-7 males were used for the crosses, suggesting that the second allele is probably male sex linked. The higher expression of Hsp70 may provide DDT-resistant strains a better chance of survival high temperature environments, particularly in homozygotes and hybrids. It was also interesting to note that these strains have a significantly lower susceptibility to the virus. Wide-spread DDT-resistance and a rise in temperature above the average temperature during summer may result in a population with a low susceptibility to the virus. Several families of heat shock proteins are known to be expressed in mosquitoes, and may have a cumulative role in determining susceptibility to the virus, which itself is governed by several genes.

Insect cell culture in research: Indian scenario.

Insect cell cultures are widely used in viral diagnosis and biotechnology, for the production of recombinant proteins, viral pesticides and vaccines as well as in basic research in genetics, molecular biology, biochemistry, endocrinology and virology. Following KRP Singh's pioneering research in 1967, a large number of cell lines from diptera, hemiptera, and lepidopteran insects were established and characterized in India. With the availability of the modern tools in molecular biology and the advancements made in biotechnology, the indigenous cell lines may prove useful in creating a future without biohazardous chemical pesticides as well as producing life saving pharmaceuticals and vaccines for many diseases. This review summarizes information gathered regarding the insect cell lines established so far in India. It also covers the familiarization of the well characterized continuous cell lines and their potential applications. Special attention is given to virus susceptibility of the cell lines, the yield of virus with a comparative analysis with other conventional systems. The potential applications of dipteran and lepidopteran cell lines in agriculture and biotechnology are also briefly discussed for prospective studies.

Characterization of a newly established potato tuber moth (Phthorimaea operculella Zeller) cell line.

BACKGROUND & OBJECTIVE: Potato tuber moth (PTM), Phthorimaea operculella Zeller is a widely distributed, devastating pest of potatoes attacking the foliage and infest the tubers in both field and store causing serious economic damage. As application of PTM granulovirus (PTM-GV) has shown significant reduction in damage, attempts were made to develop a new cell line from this insect to grow PTM-GV for use as a biopesticide. METHODS: Approximately 100 mg of insect eggs were collected, surface sterilized and crushed gently in a boiling tube aseptically. The tissues were washed with physiological saline, suspended in growth medium and incubated stationary at 28 degrees C. Morphology of cells was studied after staining with Giemsa. Besides karyological and growth curve studies, PCR amplification was also done for rapid amplified polymorphic DNA pattern. RESULTS: A new cell line from the embryonic tissue of PTM was maintained in Mitsuhashi Maramorosch medium supplemented with 10 per cent foetal bovine serum. It is in the 78th passage level and designated as NIV-PTM-1095. Random amplified polymorphic DNA profile analysis indicated this as a new cell line from potato tuber moth and differed from the profiles of two other lepidopteran cell lines maintained in the laboratory. Three different cell types were observed at the 40th passage level and comprised of epithelial-like cells (77%), fibroblast-like cells (20%) and giant cells (3%). The chromosome number varied from 54-176. The cell line had a cell doubling time of approximately 42 h during the logarithmic phase of growth. The cell line did not support the multiplication of any of the baculoviruses used in the study. INTERPRETATION & CONCLUSION: Since the new cell line is found to replicate PTM-GV, it may be useful for the propagation of PTM-GV in large scale. Studies to scale up the production of the GV in the cell line and field trials may lead to its widespread use as an eco-friendly biopesticide.

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.

West Nile virus: the Indian scenario.

West Nile virus (WNV) is an important arthropod borne flavivirus; usually causes a mild infection called West Nile fever (WNF) in human and horses. Mosquitoes are the principal vectors of WNV. Various Culex species are found to act as vectors in different geographical regions. The virus is maintained in a bird-mosquito cycle in nature. In India, Culex mosquitoes are tentatively incriminated as vectors of WNV. Experimental studies have shown that Culex tritaeniorhynchus, Cx. vishnui, Cx. bitaeniorhynchus and Cx. univittatus, Culex pipiens fatigans and Aedes albopictus could act as potential vectors of WNV. Transovarial transmission of WNV has been experimentally demonstrated in Culex mosquitoes. Apart from mosquitoes, the role of other arthropods is also considered in the maintenance of WNV during inter-enzootic periods. The possible role of ardeid birds in the maintenance of WNV has been described in India. Though very few clinically overt cases of human encephalitis due to WNV are observed, Japanese encephalitis virus (JEV) is found to dominate in southern India. WNF in horses has not been documented in India. JEV immunized monkeys were protected from WNV challenge and the WNV immunization was found to reduce the disease severity due to JEV. Based on the limited genome sequence analysis, the Indian isolates are grouped together under the genetic lineage-I. WNV infection is diagnosed by IgM antibody capture enzyme linked immunosorbant assay, haemagglutination inhibition test, neutralization test and reverse transcriptase-polymerase chain reaction (RT-PCR). For the effective control of Culex mosquitoes, integrated vector control strategies are recommended. Specific methods are not available for the treatment of WNV infection. However, in patients with encephalitis supportive therapy is recommended. Though a few candidate vaccines are under laboratory trial, no vaccine has been available commercially for the control of WNV infection in human and animals. In view of the global interest on WNV, this paper describes the present status of WNV in India.

Establishment of two new cell lines from Bombyx mori (L.) (Lepidoptera: Bombycidae) & their susceptibility to baculoviruses.

BACKGROUND & OBJECTIVES: Lepidopteran cell cultures and baculovirus expression vector systems are becoming popular due to their potential applications in biotechnology especially for the expression of foreign proteins. Efforts were made to develop new, indigenous, cell lines from Bombyx mori larvae and pupae. METHODS: Eight to ten B. mori larvae and 10-12 pupae were surface sterilized, dissected and ovaries were removed aseptically. Ovaries were chopped finely, washed and suspended in growth medium. When the cells formed monolayers, they were subcultured and experiments were carried out. RESULTS: Two new cell lines from larval and pupal ovaries of B. mori were established in Grace's insect tissue culture medium supplemented with 20 per cent FBS (foetal bovine serum). The larval cell line consisted predominantly of epithelial-like cells (98.31%), whereas the pupal cell line had a mixed cell population of epithelial-like (71.8%) and fibroblast-like cells (27.8%). Karyology indicated a typical lepidopteran pattern in both the cell lines and had chromosome numbers ranging from 35 to 150 and 60 to 180 for larval and pupal ovaries respectively. Four-fold increase in cell number was observed in these cell lines in 7 days. Both the cell lines were found susceptible to B. mori multiple nucleopolyhedrovirus and Autographa californica multiple nucleopolyhedrovirus, but not to Helicoverpa armigera single nucleopolyhedrovirus and Spodoptera litura multiple nucleopolyhedrovirus. INTERPRETATION & CONCLUSION: These well characterized cell lines may be of immense application in biotechnology and medicine for the production of biologically active recombinant proteins to use in vaccine studies as well as in therapeutic applications.

Transovarial transmission of West Nile virus in Culex vishnui mosquito.

The phenomenon of transovarial transmission (TOT) of West Nile (WN) virus in Culex vishnui was studied. The virus was detected in the progeny of both first and second gonotropic cycles (G1 and G2). About 5.56 per cent pools of F1 progeny from the parent females infected by the oral route were found positive for WN virus. This is the first report of TOT of WN virus in this species of mosquito. The occurrence of this phenomenon is of considerable importance in view of complex natural cycle of the virus and the high density of this vector species in nature. The results suggest that this mosquito may be involved in the maintenance of this virus in nature.

Antigen distribution pattern of West Nile virus in Culex tritaeniorhynchus, Culex vishnui and Culex pseudovishnui mosquitoes.

Distribution of West Nile (WN) virus antigen in different tissues of mosquitoes was studied in three species viz., Culex tritaeniorhynchus, C. vishnui and C. pseudovishnui. Overall per cent positivity was higher in the intra thoracically inoculated as compared to the orally infected mosquitoes, suggesting the existence of a midgut barrier. In a small number of mosquitoes salivary glands were found negative even though fluorescence was seen in the respective head squashes, suggesting salivary gland barrier in these mosquitoes. There was no difference in the per cent salivary gland and salivary gland area positivity between these three species. Presence of virus antigen in the ovaries of these three species on the 3rd post infection day suggests the possibility of transovarial transmission of virus even in the first gonotrophic cycle, which is of epidemiological importance.

Antigen distribution pattern of Japanese encephalitis virus in Culex tritaeniorhynchus, C. vishnui & C. pseudovishnui.

Distribution of Japanese encephalitis (JE) virus antigen in different tissues of mosquitoes was studied in three species viz., Culex tritaeniorhynchus, C. Vishnui and C. pseudovishnui. Overall per cent positivity was higher in the intrathoracically infected as compared to the orally infected mosquitoes, suggesting the existence of a midgut barrier. The cells at the junction of the foregut-midgut, and midgut-hindgut showed intense fluorescence from the second day post feeding onwards. This suggests that the dissemination of virus takes place from these regions of the gut. A small number of salivary glands were found negative even though fluorescence was seen in the respective head squashes, suggesting involvement of the salivary gland barrier in these mosquitoes. Though there was no difference in the salivary gland positivity between these three species, the salivary gland area positivity was high in C. tritaeniorhynchus mosquitoes. Presence of virus antigen in the ovaries and developing eggs of these three species on the third day suggests the possibility of transovarial transmission of virus even in the first gonotrophic cycle which may have epidemiological importance.

Studies on the mosquito vectors of Japanese encephalitis virus in Mandya District, Karnataka, India.

Entomological investigations were carried out in areas affected by Japanese encephalitis (JE) in Mandya District, Karnataka, India, from 1983 to 1988, to determine species composition and the density of mosquito vectors, in relation to the incidence of JE cases. JE cases occurred in two spells in a year, one during April-June (summer epidemic) and another during October-December (winter epidemic). There was very high incidence of JE cases in extensively irrigated areas and a low incidence in some of the taluks with less or no irrigation systems. Among culicines, Culex tritaeniorhynchus was the most predominant species (20.54%), followed by Cx. fuscocephala (16.94), Cx. vishnui (16.48%), Cx. gelidus (10.70%) and other species. The overall mosquito population showed two peaks in a year, one during the March-April, and another during September, usually preceding the human epidemics. Relative abundance of certain species varied in different years.