Ankita Agarwal

Evidence of experimental vertical transmission of emerging novel ECSA genotype of Chikungunya Virus in Aedes aegypti.

Background Chikungunya virus (CHIKV) has emerged as one of the most important arboviruses of public health significance in the past decade. The virus is mainly maintained through human-mosquito-human cycle. Other routes of transmission and the mechanism of maintenance of the virus in nature are not clearly known. Vertical transmission may be a mechanism of sustaining the virus during inter-epidemic periods. Laboratory experiments were conducted to determine whether Aedes aegypti, a principal vector, is capable of vertically transmitting CHIKV or not. Methodology/Principal Findings Female Ae. aegypti were orally infected with a novel ECSA genotype of CHIKV in the 2nd gonotrophic cycle. On day 10 post infection, a non-infectious blood meal was provided to obtain another cycle of eggs. Larvae and adults developed from the eggs obtained following both infectious and non-infectious blood meal were tested for the presence of CHIKV specific RNA through real time RT-PCR. The results revealed that the larvae and adults developed from eggs derived from the infectious blood meal (2nd gonotrophic cycle) were negative for CHIKV RNA. However, the larvae and adults developed after subsequent non-infectious blood meal (3rd gonotrophic cycle) were positive with minimum filial infection rates of 28.2 (1∶35.5) and 20.2 (1∶49.5) respectively. Conclusion/Significance This study is the first to confirm experimental vertical transmission of emerging novel ECSA genotype of CHIKV in Ae. aegypti from India, indicating the possibilities of occurrence of this phenomenon in nature. This evidence may have important consequence for survival of CHIKV during adverse climatic conditions and inter-epidemic periods.

Emergence of influenza A(H1N1)pdm09 genogroup 6B and drug resistant virus, India, January to May 2015

To investigate the aetiology of the 2015 A(H1N1)pdm09 influenza outbreak in India, 1,083 nasopharyngeal swabs from suspect patients were screened for influenza A(H1N1)pdm09 in the state of Madhya Pradesh. Of 412 positive specimens, six were further characterised by phylogenetic analysis of haemagglutinin (HA) sequences revealing that they belonged to genogroup 6B. A new mutation (E164G) was observed in HA2 of two sequences. Neuraminidase genes in two of 12 isolates from fatal cases on prior oseltamivir treatment harboured the H275Y mutation.

Complete genome sequencing and evolutionary analysis of Indian isolates of Dengue virus type 2

Dengue is the most important arboviral infection of global public health significance. It is now endemic in most parts of the South East Asia including India. Though Dengue virus type 2 (DENV-2) is predominantly associated with major outbreaks in India, complete genome information of Indian DENV-2 is not available. In this study, the full-length genome of five DENV-2 isolates (four from 2001 to 2011 and one from 1960), from different parts of India was determined. The complete genome of the Indian DENV-2 was found to be 10,670 bases long with an open reading frame coding for 3391 amino acids. The recent Indian DENV-2 (2001-2011) revealed a nucleotide sequence identity of around 90% and 97% with an older Indian DENV-2 (1960) and closely related Sri Lankan and Chinese DENV-2 respectively. Presence of unique amino acid residues and non-conservative substitutions in critical amino acid residues of major structural and non-structural proteins was observed in recent Indian DENV-2. Selection pressure analysis revealed positive selection in few amino acid sites of the genes encoding for structural and non-structural proteins. The molecular phylogenetic analysis based on comparison of both complete coding region and envelope protein gene with globally diverse DENV-2 viruses classified the recent Indian isolates into a unique South Asian clade within Cosmopolitan genotype. A shift of genotype from American to Cosmopolitan in 1970s characterized the evolution of DENV-2 in India. Present study is the first report on complete genome characterization of emerging DENV-2 isolates from India and highlights the circulation of a unique clade in South Asia.

Application of real-time RT-PCR in vector surveillance and assessment of replication kinetics of an emerging novel ECSA genotype of Chikungunya virus in Aedes aegypti

Chikungunya has emerged as one of the most important arboviral infection of global significance. Expansion of Chikungunya virus endemic areas can be ascribed to naive population, increasing vector population and adaptability of virus to new vector. In this study, a SYBR Green I based quantitative RT-PCR assay was developed. The assay was found to be 10-fold more sensitive than conventional RT-PCR and no cross reactivity was observed with related alphaviruses and flaviviruses. The detection efficiency of the assay was impervious to mosquitoes of different pool sizes. Vector surveillance has resulted in detection of CHIKV RNA in Aedes aegypti, confirming its vectorial potential for CHIKV in northern India. The assessment of the assay was further carried out by studying the competence of Indian Ae. aegypti for CHIKV, which revealed 100% infection rate and dissemination rate with 60% transmission rate. The replication kinetics of CHIKV in different anatomical sites of Ae. aegypti revealed highest titre at day 6 post infection in midgut and at day 10 post infection in saliva, legs and wings. The implementation of the assay in detecting lower viral load makes it a remarkable tool for surveillance of virus activity in mosquitoes.

Two novel epistatic mutations (E1:K211E and E2:V264A) in structural proteins of Chikungunya virus enhance fitness in Aedes aegypti.

Expansion of CHIKV outbreaks with appearance of novel mutations are reported from many parts of the world. Two novel mutations viz. E1:K211E and E2:V264A in background of E1:226A are recently identified from Aedes aegypti dominated areas of India. In this study, the role of these mutations in modulation of infectivity, dissemination and transmission by two different Aedes species was studied. Mutations were sequentially constructed in CHIKV genome and female Ae. aegypti and Aedes albopictus mosquitoes were orally infected with eight different CHIKV mutants. Double mutant virus containing E1:K211E and E2:V264A mutations in background of E1:226A revealed remarkably higher fitness for Ae. aegypti, as indicated by significant increase in virus infectivity (13 fold), dissemination (15 fold) and transmission (62 fold) compared to parental E1:226A virus. These results indicate that adaptive mutations in CHIKV are leading to efficient CHIKV circulation in Ae. aegypti endemic areas, contributing and sustaining the major CHIKV outbreaks.

Mosquito saliva induced cutaneous events augment Chikungunya virus replication and disease progression.

Chikungunya virus (CHIKV) is transmitted when infected mosquito probes the host skin. While probing, mosquito saliva is expectorated into host skin along with virus which contains cocktail of molecules having anti-hemostatic and immunomodulatory properties. As mosquito saliva is a critical factor during natural arboviral infection, therefore we investigated mosquito saliva induced cutaneous events that modulate CHIKV infection. The effect of mosquito saliva on CHIKV infection was examined through inoculation of suckling mice subcutaneously with either CHIKV alone or uninfected mosquito bite followed by CHIKV. Histopathological evaluation of skin revealed infiltration of transmigrated inflammatory cells. Dermal blood vessels were hyperemic and adnexa showed degenerating lesions. Severe hemorrhage was observed in dermis and hypodermis in mosquito bite+CHIKV group compared to CHIKV group. Analysis of cytokines in skin showed significant downregulation of inflammatory genes like TLR-3, IL-2, IFN-γ, TNF-α and IFN-β in mosquito bite+CHIKV group compared to CHIKV group. In contrast, significant upregulation of anti-inflammatory genes like IL-4 and IL-10 was observed. These early events might have been responsible for increased dissemination of CHIKV to serum and peripheral organs as demonstrated through >10-fold higher viremia, antigen localization, cellular infiltration and degenerative changes. Thus mosquito saliva induced early cellular infiltration and associated cytokines augment CHIKV pathogenesis in a mouse model. This mosquito improved CHIKV mouse model simulates the realistic conditions that occur naturally during infected mosquito bite to a host. It will lead to better understanding of CHIKV pathobiology and promote the evaluation of novel medical countermeasures against emerging CHIKV.

Expression and Characterization of Yeast Derived Chikungunya Virus Like Particles (CHIK-VLPs) and Its Evaluation as a Potential Vaccine Candidate

Chikungunya virus (CHIKV) has emerged as a global health concern due to its recent spread in both old and new world. So far, no CHIKV specific drug or vaccine is licensed for human use. In this study, we report production of Chikungunya virus like particles (CHIK-VLPs) using novel yeast expression system (Pichia pastoris) and its evaluation as vaccine candidate. The gene encoding structural polyprotein of CHIKV from a recent epidemic strain was cloned into yeast expression system. The multicopy integrants were processed for expression of CHIK-VLPs. The VLPs were purified and confirmed through electron microscopic analysis for their morphological identity with CHIKV. The in vitro and in vivo evaluation of CHIK-VLPs as vaccine candidate was determined in Balb/c mice. Induction of both humoral and cellular immune response was observed with different doses of CHIK-VLPs. The humoral immune response was studied through different techniques like enzyme linked immunosorbent assay, IgG Isotyping and plaque reduction neutralization test. CHIK-VLPs were found to elicit high titer of antibodies that are able to recognize native CHIKV. Higher level of IgG2a and IgG1 subtypes was identified suggestive of balanced Th1/Th2 response. Both in vitro and in vivo neutralization activity of CHIK-VLPs antibodies was observed even with low concentration, which shows its high specificity and neutralizing activity against two different CHIKV strains. Neonatal mice receiving anti-CHIK-VLPs antibodies were protected from CHIKV challenge. Induction of cellular immune response was confirmed through higher level of TNF-α, IL-10 and substantial level of IL-2, IL-4 and IFN-γ indicating a balanced response. This is the first report, where CHIK-VLPs has been expressed by Pichia pastoris and evaluated for neutralizing activity against CHIKV. These promising results indicate the utility of CHIK-VLPs as a promising vaccine candidate against emerging CHIKV.

Molecular and Virological Investigation of a Focal Chikungunya Outbreak in Northern India

Chikungunya (CHIK) fever is one of the most important arboviral infections of medical significance. The objective of the present study is to identify and characterize the etiology of a focal febrile arthritis outbreak from Gwalior, northern India, during October-November 2010. A detailed virological (isolation) and molecular (end-point RT-PCR, quantitative RT-PCR, and nucleotide sequencing) investigation of this outbreak was carried out by collecting and studying 52 clinical samples and 15 mosquito pools from the affected region. The investigation revealed the presence of CHIK viral RNA in 29% of clinical samples and 13% mosquito pool by RT-PCR. The quantification of CHIK viral RNA in samples varied from 102.50 to 106.67 copies/mL, as demonstrated through quantitative RT-PCR. In addition, six CHIK viruses were isolated from RT-PCR positive samples. The nucleotide sequences of partial E1 gene of five representative CHIK viruses were deciphered, which revealed that all the viral strains from this outbreak belong to the recently emerging ECS African genotype. Identification of Chikungunya virus ECSA African genotype as the etiology of the present outbreak confirms the continued circulation of the novel genotype, since 2006, in India. The identification of CHIK virus in Aedes aegypti also confirmed it as the major vector in northern India.

Development of a SYBR green I-based quantitative RT-PCR for Ross River virus: Application in vector competence studies and antiviral drug evaluation

Abstract Ross River virus (RRV) is an emerging Alphavirus and is presently endemic in many parts of Oceania. Keeping in mind its emergence, we developed a molecular detection system and utilized it to study vector competence and evaluate activity of antiviral compounds against RRV. A SYBR Green I-based quantitative RT-PCR for detection of RRV was developed targeting the E2 gene, with a detection limit of 100 RNA copies/reaction. The specificity was confirmed with closely related Alphaviruses and Flaviviruses. The assay was applied to study the vector competence of Indian Aedes aegypti for RRV, which revealed 100% infection and dissemination rate with 75% transmission rate. Viral RNA was found in saliva as early as 3day post infection (dpi). Further application of the assay in antiviral drug evaluation revealed the superior in vitro activity of ribavirin compared to chloroquine in Vero cells. Successful demonstration of this assay to detect RRV in low titre mosquito samples makes it a sensitive tool in vector surveillance. This study also showed that Indian Ae. aegypti are well competent to transmit RRV highlighting the risk of its introduction to naïve territories across continents. Further validation of this assay, revealed its utility in screening of potential antivirals against RRV.

Impact of transmission cycles and vector competence on global expansion and emergence of arboviruses

Arboviruses are transmitted between arthropod vectors and vertebrate host. Arboviral infection in mosquitoes is initiated when a mosquito feeds on a viremic host. Following ingestion of a viremic blood meal by mosquitoes, virus enters midgut along with the blood, infects and replicates in midgut epithelial cells, and then escapes to the hemocoel, from where it disseminates to various secondary organs including salivary glands. Subsequently, when mosquito bites another host, a new transmission cycle is initiated. The midgut and salivary glands act as anatomical barriers to virus infection and escape. These complex interactions between the virus and vector dictate the vector competence. Thus, vector competence reflects the success in overcoming different barriers within the vector. Along with these, other intrinsic factors like midgut microbiota and immune responses, extrinsic factors like temperature and humidity, and genetic factors like vector genotype and viral genotype have been discussed in this review. Recent advancement on novel molecular tools to study vector competence is also included. Different modes of arboviral transmission like horizontal, vertical, and venereal and how these play role in sustenance and emergence of arboviruses in nature are also discussed. These factors can be exploited to reduce the susceptibility of vectors for the viruses, so as to control arboviral diseases to certain extent.