National Institute of Virology Dengue es 1. Epidemiological investigations on dengue virus 51 infections in Karnataka and neighbouring areas 2. Studies on Ae aegypti (Linneaus) in relation to 54 dengue virus infection in rural areas of Karnataka State, India 3. Studies on distribution of Aedes aegypti in the towns 55 and villages along the western coastal region of India 4. Detection of dengue virus antigen in Aedes aegypti 57 mosquitoes following outbreaks of dengue like illness 5. Studies on molecular aspects of transovarial 59 transmission of dengue virus in Aedes aegypti mosquito 6. Phylogenetic analysis of dengue viruses 61 7. Detection/Serotyping of dengue viruses using 63 molecular techniques 8. Dengue virus-host cell interactions 65 9. Athymic mice as model to study immunology of 67 dengue virus
Epidemiological investigations on dengue virus infections in Karnataka and neighbouring areas P Yergolkar K Hanumayya karnivbng@bgl.vsnl.net.in Dengue virus infections continues to be of public health concern in Karnataka and neighbouring states. There is a need to improve early diagnosis for patient management and for undertaking suitable prevention and control measures. Objectives To determine the incidence of dengue in the referred cases of dengue-like disease in Karnataka and neighboring states. Achievements During the year 2004-05, a total of 740 specimens were received from suspected cases of dengue-like illness and tested for dengue IgM antibodies by MAC-ELISA against dengue-2 antigen. Of these 498 had symptoms of dengue fever and 83 were of suspected DHF/DSS cases. One hundred fifty nine cases were from villages. Of the total cases, 694 were from Karnataka, 39 from Andhra Pradesh and 2 from Tamil Nadu and address was not known in five cases. Of the 262 cases diagnosed with dengue infection, 242 were from Karnataka, 18 from Andhra Pradesh and one each from Tamil Nadu and one whose address was not knows. Of the 159 rural cases, 55% cases tested positive for dengue. Dengue cases were diagnosed in all months excepting March. Positive cases were seen in all age groups of both sexes. The ratio of males:female sera tested was 1:1.01. This was consistent with those of dengue positive cases, which was 1:0.96. Among the total suspected cases of dengue, 83 had clinical symptoms of DHF/DSS as per the clinical information recorded on laboratory request form at the time of specimen sending. Clinical grading of these cases were DHF I-5, DHF II-62, DSS III-6 and DSS IV-10. Dengue was confirmed in 55 of these cases. District wise, and age wise DHF/DSS cases are presented in Tables 1, & 2 respectively. 51
Table-1 : Serological diagnosis of suspected dengue cases from Karnataka and bordering districts of Andhra Pradesh & Tamil Nadu, Apr-2004 to Mar-2005.. No. District/State No. of cases +ve/no. tested No. of DF cases Pos. for Dengue No. of DHF/DSS cases Pos. for Dengue Karnataka 242/694 108 46 1 Bangalore City 31/92 23 8 2 Bangalore Urban 3/22 3-3 Bangalore Rural 5/54 3 2 4 Bagalkot 6/6 1 5 5 Belgaum 5/11 4 1 6 Bellary 20/76 12-7 Bijapur 40/67 25 15 8 Chikmagalur 0/32 - - 9 Chitradurga 1/1 1-10 Davanagere 5/7 4 1 11 Dakshina Kannada 1/11 1-12 Dharwad 5/38 2 3 13 Gadag 1/1-1 14 Gulbarga 7/11 4 3 15 Hassan 0/3 - - 16 Haveri 5/9 3 2 17 Kolar 75/156 7 1 18 Koppal 0/2 - - 19 Mandya 3/17 3-20 Raichur 13/26 11 2 21 Shimoga 1/2 1-22 Tumkur 1046-1 23 Udupi 0/1 - - 24 Uttara Kannada 1/3-1 25 Andhra Pradesh 18/39 10 8 26 Tamil Nadu 1/2 1-27 Address not known 1/5-1 Grand Total 740 119 55 Note: Case histories of all patients were not available so the totals of DF and DHF/DSS do not tally with the total Dengue sero positives 52
Table 2: Age wise distribution of dengue cases from Karnataka and bordering districts of Andhra Pradesh & Tamil Nadu, Apr-2004 to Mar-2005 Age group in years No. of DF cases No. of DHF/DSS cases No. of cases tested Pos. for Dengue Pos. for Dengue 1 5 188 48 32 6 10 155 38 13 11 15 71 17 3 16 85 320 103 6 Age not known 6 1 1 Total 740 207 55 Conclusion Continued activity of dengue and DHF cases in Karnataka and bordering areas was confirmed with diagnosis of 262 dengue cases. This included 55 DHF/DSS cases. Future plan The studies will be continued. It will help in identifying endemic areas of Karnataka and studying the nature of clinical disease pattern associated with dengue infection. 53
Studies on Ae aegypti (Linneaus) in relation to dengue virus infection in rural areas of Karnataka State, India JP George P Yergolkar nivbng@bgl.vsnl.net.in During the previous years, increasing number dengue cases have been reported from several villages of Karnataka State. Therefore, it is necessary to study the ecology of Aedes aegypti, with a view to plan control strategies against this species. Objectives Pattern of Ae. aegypti distribution and seasonal prevalence and dengue virus infection in the villages. Factors governing the distribution of Ae. aegypti mosquitoes. Identification of important breeding sources. Achievements During the year 2004-05 several villages in Karnataka State reported febrile illness suspected to be due to dengue virus infection. Entomological investigations were carried out using single larva survey and adult collections in 12 localities, 11 from Kolar and 1 from Tumkur districts. All these villages were found postive for Ae. aegypti breeding. The positive villages were from Kolar (7), Bangarpet (2), Srinivasapura and Mulbagal (1 each), taluks of Kolar district and Sira taluk of Tumkur district. In the villages the Breteau Index ranged from 25.00 to 61.54, Container Index from 7.40 to 33.33 and Adult House Index from 5.00 to 26.66. Among the 120 positive containers 77 (64.17%) were from indoor and 43 (35.83%) from outdoor habitats. Cement water storage tanks were the main breeding sources. They accounted for 80.83% of the total positive containers followed by were mud pots (5%), drums (7.5%), metal (2.5%), old tyres (1.66%) and miscellaneous containers (2.56% )respectively. In addition to Ae aegypti 30 larvae/pupae of Ae. vittatus (17), Ae. albopictus (5), Cx. quinquefasciatus (4), Cx.(L) fucanus (4) were encountered mainly in outdoor containers. A total of 66 Ae. aegypti adults consisting of 19 males and 47 females were collected from these villages. Attempts for virus isolations are under way in 64 wild-caught Ae. aegypti adults and 60 larvae reared unfed adults. Future plan Investigations will be continued with an emphasis on the habitat linked species frequencies. 54
Studies on distribution of Aedes aegypti in the towns and villages along the western coastal region of India PVM Mahadev AC Mishra, DT Mourya mahadevpvm@yahoo.co.in In view of the discontinuous distribution of Aedes aegypti and increasing incidences of dengue activity in rural and urban areas in 2002, these surveys were continued upto November 2004. Objectives To study the landscape distribution of Ae aegypti in the coastal region covering the states of Tamil Nadu, Kerala, and Karnataka, To conduct case studies in selected villages, towns / cities to study the pattern of Ae aegypti prevalence and the impact of ecological changes brought about due to urbanization and transport development Achievements In all 18576 water containers were examined in 2406 households visited in Karnataka, Kerala and Tamil Nadu States up to the end of November 2004. A total of 191 Ae aegypti and 679 Ae albopicta were encountered in the residential areas (table 3 fig 2). Table 3: Summary of the containers with breeding of Ae aegypti & Ae albopictus in the residential biotopes of towns (U) and villages (R) in the states of Karnataka, Kerala and Tamil Nadu during 2004 Season State / UT Urban/ No of No of No of Nos with Nos with Rural Houses containers containers Ae Ae aegypti per house albopictus Karnataka 11 U & 7 R 560 2725 4.87 316 124 Dry Kerala 16 U & 10 R 900 6785 7.54 TamilNadu Nil Mahe (UT) 1 U 50 412 8.24 (Pondicherry) Total 28 U & 17 R 1510 9922 6.57 Karnataka 10 U & 2 R 504 5439 10.8 363 67 Wet Kerala 3 U & 2 R 259 2033 7.85 TamilNadu 2 U & 3 R 133 1182 8.89 Total 15 U & 7 R 896 8654 9.66 55
D engue 1 8 1 1 Karnataka:Ae aegypti in 8/16 settlements Kerala: Ae aegypti in24/25 Figure 2 : Ae. aegypti distribution in Karnataka and Kerala In addition 61 non-residential areas associated with transport systems of road, rail, sea and airports were surveyed in all these states. A total 1258 containers including indoor iron drums, tyres, outdoor iron drum, plastic drums, metals containers and miscellaneous containers (i.e. plant containers, cement tanks, earthenware, grinding stones, coconut shells etc.) were examined. In Tamil Nadu, the plantations and road/rail depots in three towns/cities were examined. The Aedes spp. positivity was distributed evenly among the different kinds of containers. In Kerala, ten towns/cities were visited. The localities included waterfronts, rail/road depots and airports. Higher positivity was seen in outdoor tyres and metal containers. In Karnataka, which included 9 towns/cities. A large number of positive containers belonged to outdoor tyres, metal containers and miscellaneous containers. Future plan Data processing is in progress 56
Detection of Dengue virus antigen in Aedes aegypti mosquitoes PVM Mahadev G Geevarghese mahadevpvm@icmr.org.in The entomological surveillance of dengue viruses in the wake of disease outbreaks is being conducted since 1986. In addition to continuationof this activity we took a stock of Ae. aegypti pools processed thus far. The variations in virus activity across >5yrs.is also mapped. Objectives Detection of dengue viruses in the wild caught Ae aegypti mosquitoes from areas in Maharashtra reporting dengue like illness. Mapping dengue activity in Maharashtra state Achievements During the year 2004-05 a total of 83M and 341F Ae aegypti in 47 pools were processed for dengue antigen detection. Of the pools from Maharashtra state, 6 were collected from the urban areas and 40 were from the rural areas. Region-wise breakup of these was as follows: 21 (45.65%) were from the central region, 9 (19.56%) from Vidarbha, 14 (30.43%) from Marathwada and 2 (4.35%) from the coastal regions. Dengue virus antigen was noted in 3 urban areas viz., Nashik city (1/5F) Latur city (0/3M, 1/5F) and Amaravati (0/5M, 2/14F); and in 5 rural settlements viz industrial village of Sansar, Pune district (0/1M, 1/3F), Bobade Takli, Parbhani district (0/1M; 1/8F); Mauje-Karla, Jalna district (0/2M; 1/11F); Pangari, Beed district (0/7M, 1/6F) and Wangi, Aurangabad district 0/2M, 3/ 11F). All these districts had earlier history of dengue virus activity. Mosquitoes (4M and 54F) were also received from Ahmedabad, Gujarat of which 4F had dengue antigen. in addition Ae albopictus (2M, 5F) and Ae vittatus (10F) were also tested fand found negative for dengue antigen. A retrospective analysis was carried out on Ae aegypti pools collected during the last twenty years. A total of 428 pools consisting of Ae aegypti mosquitoes were received from Maharashtra state, and >50 showed dengue positivity. 10% of the positive mosquitoes were further processed and virus was isolated either in infant Swiss albino mice or in colonized Ae aegypti and type of dengue was identified. So far majority of the isolates were dengue-2 followed by dengue-3. These pools were mostly from the rural areas and <10% came from urban areas. Rate of pools received varied as follows: hot dry months of March to May an average of 3.85 (77pools/20years); during the wet months i.e., monsoon and post monsoon hot humid months of June to November- 15.8 (316/20); during the cool dry months of December to February-1.75 (35/20). The trend of dengue spread in the villages 57
D engue of Maharashtra state can be surmised from the maps with>=5 year lag as given in Figures 3A, B and C. A B C Figure 3: Dengue activity in various parts of Maharashtra (A) 1986-1990 (B) 1991-1995 (C) ³1996 Future plans To continue and monitor mosquito positivity rates in the wake of climate warming 58
Studies on molecular aspects of transovarial transmission of dengue virus in Aedes aegypti mosquito D T Mourya P Yadav, MD Gokhale, J Bhat, PV Barde mouryadt@vsnl.net A possible mechanism of survival of dengue viruses in the vector mosquitoes during nonepidemic seasons is suggested to be through Transovarial Transmission route (TOT). The following study was undertaken to explore this research problem further. Objectives Monitoring of TOT in Aedes aegypti populations in an urban and rural area throughout the year to understand its epidemiological significance. To understand environmental influence like higher temperatures and stress on the rate of TOT. To determine if dengue virus undergoes any genomic changes while undergoing the TOT. Achievements Transovarial transmission of Dengue virus in the field caught mosquito Field caught Aedes aegypti larvae collected from Loni Village, Pune district, were tested for dengue antigen by antigen capture ELISA. A total of 18 pools were processed with 100 larvae per pool. Among these only one pool was positive by ELISA. This positive sample was inoculated in mosquitoes and tested by IFA and ELISA. This sample was then inoculated in C6/36 cell line and after 4 passage infected cells were harvested. The cell lysate was used for RT- PCR with dengue primers to identify the serotype. 196 BP 146 BP Lane 1 & 2; Amplified 196bp (DEN-3) and 146bp (DEN-4) products, lane 2; 100bp molecular weight ladder Figure 4: Dengue virus E gene sequences amplified by PCR and resolved in agarose gel 70 59
Interestingly, the sample was positive for both dengue-3 and dengue-4 serotypes (Fig-4) confirmed by nucleotide sequencing of the amplicons. Sequences of dengue-4 showed maximum homology with AY152112S1, a 1992 isolate from Puerto-Rico while dengue-3 with AB111085, a 1973 isolate from Japan, sequences obtained from the GenBank. Sequence analysis of dengue virus obtained after transovarial passage Laboratory experiments were carried out with dengue-2 (TR1751) strain to determine TOT in Aedes aegypti mosquitoes. obtained after TOT passage was detected using RT-PCR. The pree (291bp) and E gene (346bp) specific primers for dengue-2 virus were used and TM the amplicons sequenced. Sequence alignments were done using the Clustal X and compared with the original virus used for the experiments. Results showed that there were no significant changes in the E protein of the virus after TOT passage. 1 2 3 60
Phylogenic analysis of dengue viruses Cecilia D, Shah PS, Pawar JA, Bachal RVcdayaraj@hotmail.com It is important to understand the evolution of dengue viruses in the country in view of the changing scenario of epidemiology and disease severity. Last year it was shown that the dengue-2 strains from 1960s (genotype V) and 1990s (genotype IV) were different. Objectives To determine when the switch from genotype V to IV occurred in the Dengue 2 strains circulating in the country. To carry out full genome sequence analysis on all four serotypes of dengue viruses. Achievements The E gene of three strains, one each from 1970, 1980 and 2004 were sequenced. The 1970 and 1980 strains were genotype V (American) while the strain isolated in 2004 was genotype IV (closer to Chinese). Thus the virus seems to have been reintroduced into the country after 1980. (Fig. 5) For full genome sequencing two strains for each serotype were amplified in mouse brain and C6/36. The sequences for all four serotypes were downloaded from Genbank and aligned. Full genome sequences, 13 for DEN1, 11 for Dengue 2, 8 for Dengue 3, and 5 for Dengue 4, were used for deriving the consensus sequences. Full genome sequencing of Dengue 2 has been initiated. Future Plans The full genome sequencing of all four serotypes. 61
G V PRico69 Vell57 Vell60 Cal80 Trinidad-53 Rajas71 Tai-87 D-1 Phillis83 D-3 NewGui44 Br-90 D-4-ROOT 0.0 0.1 0.2 Jama-83 Hissar96A Tha-64 HISSAR96B Indonesia-76 JAMMU93 SW-Pune-02 China99 Dhule94A Dhule94B Vietnam62 Seychelles-77 SriLanka85 G IV Figure 5: Phylodendrogram of Dengue - 2 virus isolates Maximum likelyhood tree showing the phylogenetic relationship among 23 strains of dengue 2 viruses using 1500 nucleotides of the E gene. Four Indian strains isolated before 1980 are labeled in red and six strains isolated after 1980 are labeled in green. Neighbour-joining bootstrap values (1000 replications) were used in the analysis. 62
Detection and Serotyping of dengue viruses using molecular techniques Shah PS, Pawar JA, Rupali R V, Cecilia D Although the laboratory diagnosis of dengue virus infection is routinely carried out using a MAC-ELISA method, it is imperative to have more sensitive and accurate molecular serotyping methods for the virus. A single reaction multiplex PCR assay was successfully developed and the details are reported in the following section. Objective To determine the utility of a single reaction multiplex RT-PCR in conjunction with MAC- ELISA for the laboratory diagnosis of dengue virus infection. Achievement Samples were collected from an outbreak in Saswad, Pune district from suspected dengue cases. The early samples (POD 0-7) were tested by both RT-PCR and MAC-ELISA while the later samples were tested only by MAC-ELISA. Thirteen of 48 samples tested were positive by RT-PCR (Table 7). Two serotypes were detected, eleven dengue-2 and two dengue-3. Only one of the 13 RT-PCR-positive samples was positive for IgM, indicating that the RT-PCR was able to detect Dengue virus RNA before seroconversion. Convalescent samples were obtained only for 8 RT-PCR-positive samples, all of them showed seroconversion proving thereby the specificity of the test. For RT-PCR/IgM-negative cases, convalescent samples were received only for 10 individuals. Three of these seroconverted indicating thereby that the RT-PCR test had either failed to detect virus or that the time of sample collection was not suitable. Thus, the sensitivity of the test needs to be improved. By including the RT-PCR test the percentage of cases confirmed to be dengue from the suspected cases rose from 34% (if only MAC-ELISA had been done) to 51%. More importantly the RT-PCR test was able to detect cases before seroconversion, which served as an early warning and was thus very helpful to the health managers. 63
Table 7. Detection of dengue cases by RT-PCR Post-onset day No. of samples tested RT-PCR IgM (1st sample) IgM (2nd sample) 13 positive 0-7 48 (11 DEN2, 1 postive 8/8 positive* 2DEN3) 12 negative 35 negative 9 postive 3/10 positive 26 negative 8-10 9 ND 6 postive 3 negative >11day 19 ND 10 positive 9 negative * Four second samples could not be obtained. 64
D engue Although dengue viruses have been shown to replicate in different cell types, the mechanisms by which the virus ineracts with the host cell is incompletely understood. The present study aims in studying the molecular and cellular basis of dengue virus-host cell interactions like nature of receptors; virus replication strategies and identifying cytoorganelles involved in dengue replication. Dengue virus-host cell interactions Objective Samatha S, and Cecilia D cdayaraj@hotmail.com l Investigate the involvement of cellular organelles important to cellular activation, virus translation and maturation in dengue infection using confocal microscopy Achievements The effect of dengue virus infection on mitochondria, endoplasmic reticulum and golgi apparatus was investigated in insect (C6/36) and mammalian (PS) cells. Soon after infection the mitochondria in C6/36 and PS cells underwent a change in their morphological state from punctate to reticulate. The reticulate organization is associated with activation of the cell and helps to disseminate energy throughout the cell. However no dengue antigen colocalized with mitochondria all through the period of infection even when tested till the 5th PID. (Fig. 6) Control (Punctate) Infected (2h pi.) (Reticulate) Infected (5PID) (A) (B) (C) Effect of dengue infection on mitochondria (A) control C6/36 cells showing punctate pattern of mitochondria, (B) infected C6/36 cells at 4 h p.i. showing reticulate pattern and (C) dual staining of infected cells (dengue viral proteins green and mitochondria red) no colocalization observed till day 5 p.i. Figure 6: Dengue viral antigen distribution in dengue virus infected cells 65
D engue Studies with ER and Golgi showed that both organelles were closely involved in dengue replication. Cells infected with dengue virus were stained for either ER or Golgi with direct probes followed by staining of dengue antigen using mouse immune serum followed by anti-mouse IgG-FITC (dual with Golgi probe) or anti-mouseigg-tritc (dual with ER probe). The virus antigen could be seen in the ER as early as 12 h post infection and in the Golgi apparatus by day 3-post infection. ER Dengue Colocalization (a) Golgi Dengue Colocalization (b) Fig. 7 Association of Dengue antigen (a)with ER at 12h post infection (b) Golgi Apparatus on 3rd PID. Future plans Further studies will include cells of different lineages and viruses of different serotypes. 66
Athymic mice as model to study immunology of dengue virus S.A. Mahamuni M.M. Gore, G.N. Sapkal, V.M. Ayachit, milind_gore@hotmail.com Objectives To develop an animal model for dengue virus infection in athymic nude mice Achivements In order to study multiplication of dengue virus in athymic mice by peripheral route adult BL6 Nude (congenitally athymic) (nu/nu), heterozygous euthymic (nu/+) and adult Swiss albino mice were inoculated with 2 log dose of dengue-2 virus (P23085) in 0.1 ml by sc route. Various organs were harvested on 5, 10, 20 and 30 days post infection. Dengue virus antigen was detected by antigen capture ELISA. Infectivity of virus from various organs was assayed using Vero cell culture. 3 Swiss Blood Liver Brain Spleen OD at 492 nm 2 1 0 Control 5th day 10th day 20th day 30th day PI day 3 nu/nu Blood Liver Brain Spleen nu/+ 3 Blood Liver Brain Spleen OD at 492 nm 2 1 OD at492 nm 2 1 0 Control 5th day 10th day 20th day 30th day PI day 0 Control 5th day 10th day 20th day 30th day PI day Figure 8 : Detection of Dengue 2 viral antigens in the viscera of experimentally infected nude mice and controls 67
None of the mice showed sickness except one nu/+ mouse. Dengue antigen detected in different organs was a result of virus multiplication as live virus could be amplified in Vero cells. Infectious dengue virus could be detected in nu/nu and nu/+mice brain by ic inoculation in infant Swiss mice. Passive transfer of Dengue virus immune spleenocytes in virus infected nu/nu mice Heterozygous (nu /+)BL6 nude mice were immunized with BPL inactivated dengue-1 and dengue-2 virus at weekly interval by i.p route. After two doses the mice were bled and sera were tested for neutralizing antibody-using Immunofluorescence based NT. Results indicated only homologous neutralization. To study effect of immune cell transfer pre and post challenge, cells were transferred one day before or on 5th PI day after subcutaneous challenge. Additionally homologous and heterologous protection was studied using Den1 and Den2 viruses for challenge. On 10th and 20th day post challenge different organs were harvested to check the effect of immune cells on viral load. The work on histopathology and antigen capture ELISA of these organs is in progress. Future Plans Immunohistology and antigen capture ELISA on harvested samples would be undertaken to observe the effect of homologous and heterolologus immunity on challenge. 68