Experimental study of dengue virus infection in Aedes aegypti and Aedes albopictus: A comparative analysis on susceptibility, virus transmission and reproductive success.

Epidemiology of dengue fever has substantially changed over the years with respect to prevalent strains, affected geographical locations and severity of disease. Mosquito vectors show variable response in terms of susceptibility to four different serotypes of dengue virus. Although studies have postulated that, the vectors Ae. aegypti and Ae. albopictus are crucial for transmission of dengue virus, comparative efficacy of these species for viral transmission and tolerance is still enigmatic. In this study, these two vectors were infected orally with four serotypes of the dengue virus viz. DENV-1 to DENV-4 and their co-infection. It was observed that Ae. aegypti harbors multiple serotype infections more efficiently than Ae. albopictus. We suggest that transovarial transmission is of low importance in the epidemiology of the virus due to low infection rates in the filial generation, and also that reduced fecundity and fertility in both vectors after dengue virus infection affect the ecology of the pathogen.

Dengue virus serotypes and genotypic characterization from northeast India.

Dengue is a rapidly spreading acute arboviral infection transmitted through a human and Aedes mosquito cycle. Though northeast region of India has been experiencing dengue outbreaks regularly for over a decade, reports on genetic characterization of the virus from this region are limited. The present study was undertaken to detect the genotype and genetic composition of circulating dengue virus (DENV) in this region. Blood samples were collected from 918 suspected dengue patients of five northeast Indian states. Serological investigations, viz, nonstructural 1 (NS1) enzyme-linked immunosorbent assay (ELISA), immunoglobulin M (IgM) ELISA, and immunoglobulin G (IgG) ELISA were performed followed by molecular detection. Sequence analysis and phylogenetic tree construction based on capsid-premembrane (C-prM) gene junction was done by BioEdit and MEGA6 software, respectively. Serological detection showed 35.34% NS1 and 18.12% IgM positivity. Secondary infection was observed in 24.53%. All four serotypes were detected. Phylogenetic analysis demonstrated circulation of genotype III of DENV-1, genotype IV of DENV-2, and genotype III of DENV-3. Sequences from this region form distinct clades in the phylogenetic tree. Characterization of the C-prM gene junction reveals divergence among the DENV strains. As genetic variation within the DENV is known to be associated with diverse clinical outcomes, information regarding the genetic composition of circulating virus could be beneficial in designing an effective intervention strategy.

Polymorphism of HLA class I and class II alleles in influenza A(H1N1)pdm09 virus infected population of Assam, Northeast India.

Human leucocyte antigen (HLA) represents one of the most highly polymorphic systems which plays a central role in the immune response. Genetic polymorphism of HLA in influenza A(H1N1)pdm09 infected population may be an important factor in disease progression and severity that needs further probing. In this study, a total of 110 Influenza like illness patients were recruited from the population of Assam, Northeast India, from which 35 cases infected by A(H1N1)pdm09 viruses and 35 controls were typed for HLA-A, B and DRB1 locus by PCR-SSP method. A total of seven alleles of HLA-A, 16 alleles of HLA-B, and 11 alleles of HLA-DRB1 locus were identified. The most common alleles within each locus in cases were HLA-A*11 (85.71%, P = 0.046), HLA-B*35 (25%, P = 0.0001), and HLA-DRB1*15 (49.35%, P = 0.133) as compared to the controls, HLA-A*11 (40.82%), HLA-B*35 (0.00%), and HLA-DRB1*15 (67.53%). The frequency of HLA-A*11 and HLA-B*35 were significantly higher in cases as compared to the controls. In DRB1 locus, HLA-DRB1*10 was significantly higher in cases (20.78%, P = 0.005) than that of controls (0.00%). Whereas, HLA-DRB1*15 showed a higher frequency in controls than in cases. In addition, HLA-DRB3*01 (P = 0.053), DRB4*01 (P = 1.000), and DRB5*01(P = 0.591) were also identified along with HLA-DRB1 haplotype. From this preliminary study, it is suspected that there may be a role of HLA-A*11, HLA-B*35 and HLA-DRB1*10 in conferring susceptibility to influenza A(H1N1)pdm09 infection in the study population. A larger extended study on HLA polymorphism may explain the association between HLA and influenza A(H1N1)pdm09 infection and provide insights for HLA restricted peptide based vaccines.

Scrub Typhus Leading to Acute Encephalitis Syndrome, Assam, India.

To determine the contribution of Orientia tsutsugamushi, the agent of scrub typhus, as a cause of acute encephalitis syndrome (AES) in Assam, India, we conducted a retrospective study of hospital patients with symptoms of AES during 2013-2015. Our findings suggest that O. tsutsugamushi infection leads to AES and the resulting illness and death.

Immunogenicity & safety of a single dose of live-attenuated Japanese encephalitis vaccine SA 14-14-2 in adults.

BACKGROUND & OBJECTIVES: Japanese encephalitis (JE) caused by mosquito-borne Flavivirus is one of the leading causes of viral encephalitis in Asia. Control strategies include vector control and human vaccination. Due to lack of immunization programmes in endemic regions, there are still high mortality and morbidity. A live-attenuated SA 14-14-2 JE vaccine (LAJEV) has been licensed and used in Asian countries, including India. We report the assessment of immunogenicity and safety of the vaccine in adults during the first mass adult vaccination campaign carried out in Assam, India. METHODS: One thousand and seventy five adults (aged ≥15 yr) who received LAJEV were monitored for adverse events following immunization for one year. The safety assessment of vaccinated population was evaluated till 28 days and at 6 and 12 months. Blood samples collected from the enrolled participants were tested by plaque reduction neutralization test (PRNT 50 ) to assess the neutralizing antibody titres (NATs) before vaccination and 28 days, six and 12 months post-vaccination (PV). RESULTS: Among the 1075 vaccinated individuals, four reported minor adverse effects from 30 min to 28 days PV. Based on the pre-vaccination NAT, the study participants were categorized as seronegative, moderately seropositive and strongly seropositive. Nearly 85.5 per cent of JE seronegative participants seroconverted by 28 days PV. The geometric mean titre (GMT) in all the three groups increased by 28 days and decreased by six and 12 months PV. Nearly 60 per cent of the moderately positive individuals exhibited four-fold rise in GMT, 28 days PV. Almost 95.5 per cent of the participants in the study population remained seroprotected at the end of 12 months PV. INTERPRETATION & CONCLUSIONS: This study on immunogenicity and safety of LAJEV in adults showed that a single dose of the live-attenuated vaccine was safe and induced protective immunity to both JE seronegative and naturally seropositive adults. Further study is required to find out long term protective efficacy of this vaccine.

Genetic characterization and molecular phylogeny of Aedes albopictus (Skuse) species from Sonitpur district of Assam, India based on COI and ITS1 genes.

BACKGROUND & OBJECTIVES: Aedes albopictus (Skuse) is one of the major vectors of dengue which is an emerging threat in Northeast part of India. The morphological characterisation of mosquitoes is time consuming and lacks accuracy for distinguishing closely related species. Hence, molecular methods of mosquito identification, genetic diversity and molecular phylogeny have gained increased importance. This study was aimed to identify and characterize the most abundant species of Aedes vectors collected from different breeding spots in Assam, Northeast India employing molecular as well as bioinformatics tools. METHODS: Ae. albopictus species was genetically characterized with internal transcribed spacer1 (ITS1) and cytochrome c oxidase subunit I (COI) genes and sequence analysis was carried out following molecular methods like PCR amplification, DNA sequencing and multiple sequence analysis. Maximum likelihood molecular phylogeny was reconstructed to define the evolutionary relationship among studied isolates and isolates from other parts of Southeast Asia. RESULTS: Molecular study revealed that all five subject specimens belonged to Ae. albopictus species as per both ITS1 and COI genes. Maximum likelihood tree based on ITS1 and COI genes showed that isolates were distinctly grouped into separate clusters. Almost similar pattern of amino acid frequencies in COI gene was found amongst the five studied isolates. However, amino acid frequency in ITS1 gene was found to be dissimilar, indicating polymorphisms in this gene, among the isolates. INTERPRETATION & CONCLUSION: This is the first report among the Northeastern states of India describing the genetic make-up of Ae. albopictus species by virtue of highly conserved mitochondrial (mt) DNA and ribosomal (r) DNA gene sequences. This study also illustrates that the sequence diversity of these two genes in this mosquito species differs geographically which differentiate a population and brings unique identity.

Chikungunya outbreak in Garo Hills, Meghalaya: An epidemiological perspective.

BACKGROUND & OBJECTIVES: Chikungunya (CHIK) fever is a mosquito-borne disease caused by chikungunya virus (CHIKV). Chikungunya infection was first reported from India in 1963 from Kolkata. We report the serological and molecular evidence of an outbreak of chikungunya in northeast India that occurred in Tura, a hilly and forested terrain in Garo Hills district of Meghalaya. METHODS: Blood samples (3 ml) collected from hospitalized patients during the outbreak were tested for IgM antibodies against CHIKV and followed up four months later. A repeat survey was carried out in the same area after four months from where cases had been reported. Blood samples were also collected from people with history of fever and body ache in the last four months. Persons showing IgM positivity against CHIKV in the repeat survey were followed up one and a half years later. All samples were also processed by RT-PCR assay for CHIK Envelope (E) 1 gene. Immature mosquitoes were collected, link reared and identified with standard keys. Virus incrimination studies were done on Aedes aegypti and Ae. albopictus mosquitoes collected during the survey. RESULTS: Fever, headache and joint pain were the primary clinical presentations. Twenty three (35.93 %) of 64 samples reported during the outbreak were IgM positive for CHIK. Three samples showed PCR amplification. All these were IgM positive. The sequenced E1 gene revealed that the strains belonged to East Central South African (ECSA) genotype. INTERPRETATION & CONCLUSIONS: Field survey done after four months revealed that some individuals still had joint pain associated with episodes of headache and fever. It could be inferred that these persons might have contracted infection during the CHIK outbreak four months ago or during the intervening period which caused persistence of sequelae. ECSA genotype was found to be involved in the outbreak. Aedes albopictus was the predominant mosquito species collected during the outbreak.

Molecular determination of antifolate resistance associated point mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes among the field samples in Arunachal Pradesh.

BACKGROUND & OBJECTIVES: Antimalarial resistance in P. falciparum malaria parasite creates a serious obstacle in malaria control programme. Keeping this in mind, in the present study antifolate resistance associated point mutations in P. falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) genes among the field samples in Arunachal Pradesh were determined. METHODS: Blood samples were collected from 145 suspected malaria patients/healthy control subjects in malarious areas of Lohit and Changlang districts of Arunachal Pradesh, India during January 2012 to December 2013. RESULTS: In microscopic slide examination, 51.03% (74/145) were found malaria positive. Plasmodium falciparum mono-infection was observed in 62.16% (46/74) of total malaria positive cases. Polymerase chain reaction (PCR) was employed in all the P. falciparum positive samples for detection of 648 bp of Pfdhfr and 710 bp of Pfdhps genes. All the amplified products were analysed for detection of single nucleotide polymorphisms in dhfr and dhps genes. A total of four different genotypes of Pfdhfr gene were observed, of which double mutant allele ANRNI was mostly prevalent and it was found in 65.22% (30/46) cases. Likewise, four different haplotypes of Pfdhps gene were detected, of which triple mutant allele AGEAA shares 69.57% (32/46) followed by other haplotypes. In Pfdhfr-Pfdhps two locus mutations analysis, two isolates in Changlang district had shown quintuple mutant haplotype AIRNL-AGEAA, likely to be associated with treatment failure. The P. falciparum two locus dhfr-dhps haplotype (ANRNI-AGEAA) was observed in 56.52% (26/46) cases. INTERPRETATION & CONCLUSION: Overall, high grade of sulphadoxine-pyrimethamine resistance associated genetic polymorphisms were observed among the P. falciparum parasite population in Arunachal Pradesh during the study period.

First evidence of dengue virus infection in wild caught mosquitoes during an outbreak in Assam, Northeast India.

BACKGROUND & OBJECTIVES: Dengue is one of the major public health problems worldwide, transmitted mainly by Aedes aegypti and Ae. albopictus mosquitoes. Rapid urbanisation and industrialisation have led to an increase in vector population in Northeastern states of India. In 2013, Guwahati, the capital city of Assam, India experienced an outbreak of dengue. This study was undertaken with an objective to determine infection rates of dengue viruses (DENV) in both the established vectors present in this region. METHODS: During the outbreak (2013), adults and larvae of both the vector species were collected from different container habitats found in case reporting areas and container index was also recorded. The mosquitoes were first pooled, homogenised and processed for NS1-ELISA. This was followed by RT-PCR of the mosquito pools. RESULTS: Both Ae. aegypti and Ae. albopictus were found breeding in containers with container index in the range of 29.41 to 80%. Six pools of Ae. aegypti were found to be positive for NS1 antigen. RT-PCR assay revealed positivity in only the NS1-ELISA positive pools, exhibiting circulation of serotype DENV-2. Minimum infection rate of female and male Ae. aegypti was recorded as 10.87 and 11.03 respectively. INTERPRETATION & CONCLUSION: This is the maiden report of detection of DENV in wild caught Ae. aegypti mosquitoes from Northeastern Region of India. The study also demonstrates the presence of transovarial transmission of dengue virus in this part of country. This information is useful in respect of both entomological as well as epidemiological point of view for taking appropriate vector control measures.

Spatiotemporal distribution of dengue vectors & identification of high risk zones in district Sonitpur, Assam, India.

BACKGROUND & OBJECTIVES: Dengue is an arboviral disease of public health importance in many parts of India and recently many cases have been reported from northeastern India. Aedes mosquitoes, which are the vectors of dengue, are widely prevalent in the region. A study was initiated in Sonitpur district of Assam to understand the spatiotemporal distribution and seasonal prevalence of dengue vectors and to identify the high risk zones. METHODS: Ovitrap surveys were conducted in three randomly selected villages under each of the eight public health centres (PHC) in district Sonitpur of Assam, northeastern India during March 2011-February 2012. Three risk zones (high, medium and low) were identified on the basis of per trap density of Aedes mosquitoes. Meteorological data were collected to study the temporal distribution of dengue vectors. RESULTS: Aedes albopictus (99.3%) was the predominant dengue vector followed by Ae. aegypti (0.7%) recorded in the ovitraps. The highest vector density was observed during the post-monsoon (60.1±18 per trap) while the lowest during the winter (7.6±4.9 per trap) and the season-wise differences in the vector density were significant (P=0.005). Maximum temperature (correlation coefficient, r=0.45) and minimum temperature (r=0.408) showed the highest positive correlation with the vector density, whereas the number of rainy days showed high positive correlation (r=0.185) than the total rainfall (r=0.117). The high risk zone (Dekhiajuli, Behali, Bihaguri and Gohpur PHC) as indicated by the high larval densities of dengue vectors, 45.3±18, 42.1±22.3, 36.9±29.1, 35.3±22.6 per trap, respectively, was validated by dengue epidemiological data collected during 2012. INTERPRETATION & CONCLUSIONS: Yearlong monitoring of dengue vectors was done for the first time in this region. Monthly maximum temperature and the number of rainy days could be used for the prediction of larval density of Aedes mosquitoes. The identification high dengue risk zones would help in adopting targeted interventions for disease management in future.

Dengue outbreak in a hilly state of Arunachal Pradesh in Northeast India.

Dengue has been reported from plains as well as hilly regions of India including some parts of Northeast India. In July-August 2012, outbreak of fever with unknown origin (FUO) indicative of Dengue was reported in Pasighat, East Siang district of Arunachal Pradesh (AP) state. Serum samples (n = 164) collected from patients from Health Training and Research Centre General Hospital, Pasighat, were tested for NS1 antigen and IgM antibodies. NS1-positive samples were analyzed by RT-PCR assay and entomological surveys were carried out. The majority of suspected cases reported NS1 antigen positivity. Females and young adults were mostly affected. The majority of the amplified NS1-positive samples showed Dengue serotype 3 infection. Aedes (Stegomyia) albopictus, known as semiurban breeding mosquitoes, was the only potential vector species identified from the affected areas of Pasighat which single handedly contributed to the outbreak. Thus, the present work identifies Dengue as an emerging arboviral infection in hilly state of AP along with a looming risk of its spread to neighbouring areas.

Detection of point mutation in Plasmodium falciparum ATPase6 gene associated with artemisinin resistance from Assam and Arunachal Pradesh.

BACKGROUND & OBJECTIVES: Resistance against partner drugs of artemisinin has been reported from different parts of India. The study aims to find out the single nucleotide polymorphisms in Plasmodium falciparum ATPase6 gene associated with artemisinin resistance. METHODS: Blood samples were collected from 141 patients with P. falciparum monoinfection in malaria endemic zones of Assam and Arunachal Pradesh. A 645 bp portion of PfATPase6 gene was amplified and sequenced to determine the frequency of mutations associated with resistance to artemisinin. RESULTS: Mutations at codon S769N, which have been proposed to confer artemisinin resistance, were not detected in our study samples. Instead of that a novel non-synonymous mutation (C-T) at 1847 bp position resulting in serine to phenylalanine alteration at codon S616F was detected from the P. falciparum field isolates in Changlang district of Arunachal Pradesh, whereas no mutation was detected in any of the analyzed samples in Assam indicating that wild type PfATPase6 genotype was found circulating in this region. Overall, based on the mutational pattern, two haplotypes of PfATPase6 gene were observed during the study, the wild type and mutant S616F allele. The overall haplotype diversity (Hd) was found to be: 0.069 and nucleotide diversity (per site Pi): 0.00012. Highest haplotype diversity was recorded in Changlang district of Arunachal Pradesh having Hd value of 0.33333 along with single polymorphic site and nucleotide diversity (Pi): 0.00060. A pair-wise fixation index (FST) value of 0.16667 indicates great genetic differentiation within the parasite population of Changlang district with the population of Karbi Anglong, Chirang, Tinsukia, Sivasagar, Jorhat, NC Hills, Lakhimpur, Golaghat and Dibrugarh districts of Assam and Lohit district of Arunachal Pradesh. INTERPRETATION & CONCLUSION: A better understanding of the distribution of antimalarial drug resistance with malaria parasite may provide insight into some of the epidemiological determinants of the increasing case burden.

Physicochemical characteristics of habitats in relation to the density of container-breeding mosquitoes in Asom, India.

BACKGROUND & OBJECTIVES: Container-breeding mosquitoes, especially Aedes spp are vectors of diseases such as dengue and chikungunya. The abundance of these disease vectors in an area depends on the availability of container habitats and their physicochemical characteristics. The species composition of container-breeding mosquitoes in Asom, India was studied and the larval density was correlated with the habitat characteristics. METHODS: Natural and man-made water-holding containers in Sonitpur district of Asom were surveyed for the presence of mosquito larvae. The percent composition of container-breeding mosquitoes and container index were calculated. The physicochemical characteristics of breeding water such as pH, conductivity, salinity, total dissolved solids, turbidity and dissolved oxygen were measured. RESULTS: Aedes albopictus (93.7%) was the predominant species in the container-breeding habitats whereas Culex quinquefasciatus (2.77%), Armigeres subalbatus (2.26%), Ae. aegypti (0.76%), Toxorhynchites sp (0.4%) and Lutzia sp (0.11%) were recorded in relatively low numbers. The larval density (mean ± SEmean) of the container breeding mosquitoes ranged from 4.4 ± 1.8 to 15.4 ± 8.2, while the container index ranged from 1.58 to 5.68%. The mean (± SEmean) pH, conductivity, salinity, total dissolved solids, turbidity, and dissolved oxygen of water in the container habitats were 7.15 ± 0.11; 396.1 ± 58.5 µS/cm; 0.24 ± 0.04 ppt; 207.1 ± 30.4 mg/l; 32.3 ± 5.1 NTU; and 1.42 ± 0.12% respectively. The mosquito larval density in the container habitats was having significant negative correlation with the conductivity of breeding water (r = - 0.89; p = 0.003). Salinity, total dissolved solids and turbidity of water in the habitats were negatively correlated, whereas pH and dissolved oxygen were positively correlated with the larval density. INTERPRETATION & CONCLUSION: The studies indicated the predominance of Ae. albopictus in the container-breeding habitats and reiterated its importance as a potential vector of dengue and chikungunya in the region. The spread of Ae. aegypti, the principal vector of dengue, in the semi-urban areas probably through road transport is a matter of public health concern. The use of conductivity of breeding water as an index for the proliferation of container breeding mosquitoes in the region could be explored further.

Nation-wide vector surveillance on Zika and Dengue did not indicate transmission of the American lineage-pandemic Zika virus in India.

OBJECTIVES: Following the Public Health Emergency of International Concern declared on Zika by the World Health Organization during 2016, the Indian Council of Medical Research carried out nationwide vector surveillance for Zika and Dengue viruses (ZIKV and DENV) in India as a preparedness measure in 2016-19. METHODS: High-risk zones distributed to 49 Districts in 14 states/union territories were included in the study. Seven ICMR institutions participated, following a standard operating protocol. Aedes specimens sampled weekly were processed by multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) for ZIKV/DENV and random samples crosschecked with real-time RT-PCR for ZIKV. RESULTS: Altogether, 79 492 Aedes specimens in 6492 pools were processed; 3 (0.05%) and 63 (0.97%) pools, respectively, were found positive for ZIKV and DENV. ZIKV infections were recorded in Aedes aegypti sampled during the 2018 sporadic Zika outbreak in Jaipur, Rajasthan. However, these belonged to the Asian lineage of the virus, already circulating in the country. Both Ae. aegypti and Aedes albopictus distributed to 8 states/union territories were found to be infected with DENV. Both sexes of Ae. albopictus were infected, indicating transovarial transmission. CONCLUSION: This investigation evinced no active transmission of the American lineage-pandemic Zika virus in India during the pandemic period.