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.

Incrimination of Aedes aegypti and Aedes albopictus as vectors of dengue virus serotypes 1, 2 and 3 from four states of Northeast India.

Dengue is an important vector borne disease with a great public health concern worldwide. Northeast India has experienced dengue almost every year for a decade. As studies on dengue vectors from this region are limited, we undertook an investigation to detect natural infection of the dengue virus (DENV) in potential dengue vectors of this region. Adult Aedes mosquitoes which were collected were subjected to RT-PCR for detection of infecting dengue serotype. Minimum infection rate was also determined for each positive pool. Out of the total 6229 adult Aedes mosquitoes collected, Aedes aegypti (63.3 %) was abundant in comparison to Aedes albopictus (36.7 %). These specimens (515 mosquito pools) were subjected to RT-PCR for detection of DENV-1, 2, 3 and 4. RT-PCR revealed the existence of DENV in both male as well as female mosquito pools suggesting natural transovarial transmission of DENV in this region. A total of 54 pools tested were positive for DENV-1, 2, 3 serotypes. This study revealed the occurence of DENV in both the potential dengue vectors from this region along with evidence of transovarial transmission which helps in persistence of the virus in nature.

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.

Immune response during influenza virus infection among the population of Assam, Northeast India.

Introduction: The pathogenicity of influenza virus infection is modulated by the cytokine expressions in patients. The present study was aimed to measure some important pro- and anti-inflammatory cytokines in influenza-infected population of Assam, Northeast India. Materials and Methods: Influenza viruses consisting of subtypes influenza A(H1N1)pdm09, H3N2 and influenza-B were detected in patients with symptoms of influenza-like-illness by Real-time reverse transcriptase polymerase chain reaction (RT-PCR) method. Relative messenger ribonucleic acid (mRNA) quantification of four pro-inflammatory cytokines (interleukin [IL]-6, IL-8, interferon-gamma [IFN-γ] and tumour necrosis factor-alpha [TNF-α]) and one anti-inflammatory cytokine (IL-10) were measured in influenza-positive cases and non-influenza controls, by real-time RT-PCR. The plasma concentration of the cytokines was determined using cytometric-bead-array with flow cytometry. Results: Influenza viruses were detected in 14.28% (50/350) of 350 patients screened. The expression of IL-6 was significantly raised in cases compared to controls (P = 0.018). IL-8 and IL-10 were also raised in cases, compared to controls (P = 0.284 and P = 0.018). An increased plasma TNF-α was observed in cases (1.36-fold and P = 0.289). The mRNA expression of IFN-γ was also increased in cases compared to controls (0.87-fold). However, the plasma level of IFN-γ was higher in the non-influenza controls compared to cases. Conclusions: The study revealed a differential cytokine profile during influenza virus infection in the population, which may influence disease severity. An extended study on host immune response may provide better insights for the use of cytokine antagonists in therapeutic treatments among severe cases of influenza virus infection.

Bioassay and molecular study for detection of insecticide resistance dengue causing mosquito vectors.

Nowadays, dengue infection creates a major problem across the country. The vector species carrying dengue infection has progressively started to developed resistance against most of the currently used insecticides. Hence, a study was carried out in dengue-endemic areas of Assam and Arunachal Pradesh to find the current situation of insecticide susceptibility status of dengue vectors. Based on the previous history of dengue incidence, Aedes mosquitoes were collected from Dibrugarh, Kamrup, Sivasagar, Tezpur and Tinsukia districts in Assam and Pasighat district in Arunachal Pradesh to test the insecticide resistance status through bioassay and molecular methods. The WHO standard bioassay test kits were used to detect insecticide susceptibility status among dengue vectors. In molecular study, allele-specific polymerase chain reaction (PCR) method was done for the detection of mutations in paratype voltage-gated sodium channel gene of Aedes aegypti and Aedes albopictus mosquitoes. In bioassay method, 100% A. aegypti mosquitoes were found to be resistant towards dichlorodiphenyltrichloroethane (DDT), 8% towards pyrethroid and 4% towards malathion. Similarly, 92% A. albopictus mosquitoes have shown resistance competency towards DDT, 12% towards pyrethroid and 8% towards malathion. In allele-specific PCR methods, V1016G heterozygous mutations were detected from the field collected A. aegypti and A. albopictus mosquitoes of Tinsukia, Dibrugarh and Sivasagar district. Similarly, F1534C heterozygous mutations were observed from A. aegypti mosquitoes of Tezpur, Tinsukia and Sivasagar district and A. albopictus mosquitoes of Tinsukia, Dibrugarh and Sivasagar district. From the study, it was concluded that the Aedes mosquitoes have progressively started to developed resistance towards commonly used insecticides.

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.

Molecular and phylogenetic evidence of chikungunya virus circulating in Assam, India.

PURPOSE: Northeast Region of India possesses an abundant number of Aedes mosquitoes, the common vector for Dengue and Chikungunya (CHIK). Dengue is reported every year from Assam, but active surveillance for CHIK virus (CHIKV) infection is lacking in this part of India. Therefore, this present study has been undertaken to detect any CHIKV infection during a dengue outbreak in Assam. MATERIALS AND METHODS: A total of 42 dengue negative samples collected from Guwahati were screened for the presence of CHIK IgM antibodies. Further, all the samples were processed for CHIKV RNA detection by reverse transcriptase-polymerase chain reaction (RT-PCR). Phylogenetic analysis was done by Maximum Likelihood method using Kimura-2 parameter model. RESULTS: No IgM positivity was found in the processed samples; however, 7 samples were positive for CHIKV by RT-PCR. Phylogenetic analysis revealed that the circulating CHIKV belonged to Eastern, Central and Southern African genotype. Sequence analysis showed two uniform nucleotide substitutions and very less amino acid substitution. CONCLUSION: Silent existence of CHIKV beside dengue is reported from this study. Therefore, CHIKV diagnosis should be included as a regular practice for active surveillance of the disease and its accomplishment before commencing an outbreak.

Molecular typing and phylogeny of Wolbachia: A study from Assam, North-Eastern part of India.

BACKGROUND: Wolbachia are maternally inherited endosymbiotic alphaproteobacteria, infecting 40-75% of arthropod species. Knowledge on distribution of native strains infecting mosquito vectors from endemic regions is essential for successful implementation of vector control interventions utilizing potential strains of Wolbachia. Study identified various native strains of Wolbachia inhabiting different mosquito species from field and colonised conditions of Assam. The fly Drosophila melanogaster was also included in our study. METHODS: Different mosquito species collected from field viz; Aedes albopictus, Aedes aegypti, Anopheles hyrcanus, Anopheles annularis, Culex vishnui, Toxorhynchites splendens, Armegeries obturbans and fly Drosophila melanogaster were included in the study. Anopheles stephensi and Culex quinquefasciatus were obtained from RMRC, Dibrugarh mosquito colony y for Wolbachia screening. DNA was extracted from these species, amplified using group specific wsp primers followed by sequencing and phylogenetic analysis. RESULTS: Aedes albopictus from Dibrugarh, Tinsukia and Sivasagar district showed superinfection with A and B group of Wolbachia but, Aedes albopictus from Tezpur district presented infection with A group only. Our study reports for the first time natural infection of Wolbachia A and B group from colonised Anopheles stephensi mosquito but reported no infection from field collected Anopheles hyrcanus or Anopheles annularis. Similarly Armigeres obturbans and Culex vishnui presented infection with only B group of Wolbachia. Drosophila melanogaster showed superinfection with A and B group. Toxorhynchites splendens, Aedes aegypti and Culex quinquefasciatus reported no infection with Wolbachia. CONCLUSION: To the best of our knowledge this is the first study on Wolbachia screening from Northeast part of India and also first report of natural Wolbachia infection from colonised Anopheles stephensi species. The current understanding on distribution of Wolbachia strains naturally present within insect species from this geographical region should aid future Wolbachia mediated vector control strategies.

Prevalence of multiple drug-resistant Plasmodium falciparum malaria cases in Northeast India.

Two numbers of Plasmodium falciparum field isolates from Gossingpara, Runikhata area in Chirang district of Assam had shown multiple mutations in Pfcrt-dhfr-dhps gene (up to seven mutations: One mutation in Pfcrt gene, three mutations in Pfdhfr gene and three mutations in Pfdhps gene). Similarly, two cases in Bat camp, Miao area under Changlang district of Arunachal Pradesh had shown a total of eight mutations, of which one mutation in Pfcrt gene, three mutations in Pfdhfr gene, three mutations in Pfdhps gene and one mutation in PfATPase6 gene. One case in 3 Miles, Miao area of Changlang district has shown mutations in Pfcrt(one mutation), Pfdhfr(four mutations) and Pfdhps(three mutations) gene. These results indicated that there is an existence of multiple mutant P. falciparum malaria cases in northeastern region of India.

Detection of West Nile virus in six mosquito species in synchrony with seroconversion among sentinel chickens in India.

BACKGROUND: West Nile virus (WNV) is a zoonotic flavivirus maintained in mosquito-bird transmission cycle. Although humans are accidental hosts, fatal outcomes following WNV infection have been reported from India. Studies have identified WNV as an important etiological agent causing acute encephalitis syndrome in Assam, Northeast India. While circulation of WNV is evident, the role of vectors and avian hosts involved in the transmission remains unclear. In this study we identified local mosquito species for evidence of WNV infection along with seroconversion among sentinel chickens. METHODS: Mosquitoes were collected and pooled species wise from June 2014 through December 2015. Virus was screened using reverse transcriptase PCR followed by sequencing and phylogenetic analysis. Sentinel chicken blood was screened for WNV antibody to assess their role in WNV transmission. RESULTS: A total of 52,882 mosquitoes belonging to 16 species were collected. WNV was detected in 18 pools of Culex vishnui, Culex tritaeniorhynchus, Culex quinquefasciatus, Culex whitmorei, Culex pseudovishnui and Mansonia uniformis. Phylogenetic analysis revealed that all mosquito derived sequences belonged to Lineage 5 and were 99-100% similar to the Assam strain of WNV isolated from human CSF sample in 2007. All sentinel chickens had seroconverted by the month of July that happens to be the peak WNV transmission month among humans as well. CONCLUSION: To the best of our knowledge, this is the first report of WNV identification from field-collected Cx. pseudovishnui and Mansonia uniformis in India. Our study demonstrates potential vectors which may play a crucial role in WNV transmission and should be considered in the vector control strategies. Additionally, our study highlights the role of sentinel chickens for WNV surveillance.

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.

Evidence of triple mutant Pfdhps ISGNGA haplotype in Plasmodium falciparum isolates from North-east India: An analysis of sulfadoxine resistant haplotype selection.

BACKGROUND: North-east region of India has consistent role in the spread of multi drug resistant Plasmodium (P.) falciparum to other parts of Southeast Asia. After rapid clinical treatment failure of Artemisinin based combination therapy-Sulphadoxine/Pyrimethamine (ACT-SP) chemoprophylaxis, Artemether-Lumefantrine (ACT-AL) combination therapy was introduced in the year 2012 in this region for the treatment of uncomplicated P. falciparum malaria. In a DNA sequencing based polymorphism analysis, seven codons of P. falciparum dihydropteroate synthetase (Pfdhps) gene were screened in a total of 127 P. falciparum isolates collected from Assam, Arunachal Pradesh and Tripura of North-east India during the year 2014 and 2015 to document current sulfadoxine resistant haplotypes. MATERIALS AND METHODS: Sequences were analyzed to rearrange both nucleotide and protein haplotypes. Molecular diversity indices were analyzed in DNA Sequence Polymorphism software (DnaSP) on the basis of Pfdhps gene sequences. Disappearance from selective neutrality was assessed based on the ratio of non-synonomous to synonomous nucleotide substitutions [dN/dS ratio]. Moreover, two-tailed Z test was performed in search of the significance for probability of rejecting null hypothesis of strict neutrality [dN = dS]. Presence of mutant P. falciparum multidrug resistance protein1 (Pfmdr1) was also checked in those isolates that were present with new Pfdhps haplotypes. Phylogenetic relationship based on Pfdhps gene was reconstructed in Molecular Evolutionary Genetics Analysis (MEGA). RESULTS: Among eight different sulfadoxine resistant haplotypes found, IS GNG A haplotype was documented in a total of five isolates from Tripura with association of a new mutant M538 R allele. Sequence analysis of Pfmdr1 gene in these five isolates came to notice that not all but only one isolate was mutant at codon 86 (N86 Y ; Y YSND) in the multidrug resistance protein. Molecular diversity based on Pfdhps haplotypes revealed that P. falciparum populations in Assam and Tripura were under balancing selection for sulfadoxine resistant haplotypes but population from Arunachal Pradesh was under positive selection with comparatively high haplotype diversity (h = 0.870). In reconstructed phylogenetic analysis, isolates having IS GNG A haplotype were grouped into two separate sub-clusters from the other isolates based on their genetic distances and diversities. CONCLUSION: This study suggests that sulfadoxine resistant isolates are still migrating from its epicenter to the other parts of Southeast Asia and hence control and elimination of the drug resistant isolates have become impedimental. Moreover, P. falciparum populations in different areas may undergo selection of particular sulfadoxine resistant haplotypes either in the presence of drug or after its removal to maintain their plasticity.

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.

Seroepidemiology of rickettsial infections in Northeast India.

BACKGROUND: Resurgence of scrub typhus was reported in Northeast India in 2010 after a gap of 67 years since World War II. However, the presence of other rickettsial infections remained unknown from this region. A seroepidemiological investigation was undertaken in the scrub typhus affected areas from 2013-2015 in Assam, Arunachal Pradesh and Nagaland to assess the exposure to other rickettsial diseases besides scrub typhus. METHODS: Samples were collected from people residing in scrub typhus reporting areas. Serology was performed by an indirect ELISA for the three rickettsial agents' viz., scrub typhus group orientiae (STGO), spotted fever group rickettsiae (SFGR) and typhus group rickettsiae (TGR). A sample with total net absorbance ≥1.000 was considered as positive. An entomological survey was also carried out in the affected areas. RESULTS: Overall, 1265 human blood samples were collected, of which 30.8% (n=390), 13.8% (175) and 4.2% (53) had antibodies against STGO, SFGR and TGR respectively. Presence of antibodies against more than one of the rickettsial groups was also detected. Among the arthropods collected, chiggers of Leptotrombidium deleinse, fleas belonging to Ctenocephalides felis and Pulex irritans, ticks belonging to Rhipicephalus microplus, Haemaphysalis spp. were predominant. Candidatus Rickettsia senegalensis was detected in C. felis. CONCLUSIONS: Our findings confirm wide circulation of rickettsial infections and their probable vectors in the northeast region of India.Accession numbers: KU163367, KU163368, KU499847, KU499848.