Nipah Virus Outbreak in Kerala State, India Amidst of COVID-19 Pandemic.

We report here a Nipah virus (NiV) outbreak in Kozhikode district of Kerala state, India, which had caused fatal encephalitis in a 12-year-old boy and the outbreak response, which led to the successful containment of the disease and the related investigations. Quantitative real-time reverse transcription (RT)-PCR, ELISA-based antibody detection, and whole genome sequencing (WGS) were performed to confirm the NiV infection. Contacts of the index case were traced and isolated based on risk categorization. Bats from the areas near the epicenter of the outbreak were sampled for throat swabs, rectal swabs, and blood samples for NiV screening by real-time RT-PCR and anti-NiV bat immunoglobulin G (IgG) ELISA. A plaque reduction neutralization test was performed for the detection of neutralizing antibodies. Nipah viral RNA could be detected from blood, bronchial wash, endotracheal (ET) secretion, and cerebrospinal fluid (CSF) and anti-NiV immunoglobulin M (IgM) antibodies from the serum sample of the index case. Rapid establishment of an onsite NiV diagnostic facility and contact tracing helped in quick containment of the outbreak. NiV sequences retrieved from the clinical specimen of the index case formed a sub-cluster with the earlier reported Nipah I genotype sequences from India with more than 95% similarity. Anti-NiV IgG positivity could be detected in 21% of Pteropus medius (P. medius) and 37.73% of Rousettus leschenaultia (R. leschenaultia). Neutralizing antibodies against NiV could be detected in P. medius. Stringent surveillance and awareness campaigns need to be implemented in the area to reduce human-bat interactions and minimize spillover events, which can lead to sporadic outbreaks of NiV.

Detection of Nipah virus in Pteropus medius in 2019 outbreak from Ernakulam district, Kerala, India.

BACKGROUND: In June 2019, Nipah virus (NiV) infection was detected in a 21-year-old male (index case) of Ernakulum, Kerala, India. This study was undertaken to determine if NiV was in circulation in Pteropus species (spp) in those areas where the index case had visit history in 1 month. METHODS: Specialized techniques were used to trap the Pteropus medius bats (random sampling) in the vicinity of the index case area. Throat and rectal swabs samples of 141 bats along with visceral organs of 92 bats were collected to detect the presence of NiV by real-time reverse transcriptase-polymerase chain reaction (qRTPCR). Serum samples of 52 bats were tested for anti-NiV Immunoglobulin (Ig) G antibodies by Enzyme-Linked Immunosorbent Assay (ELISA). The complete genome of NiV was sequenced by next-generation sequencing (NGS) from the tissues and swab samples of bats. RESULTS: One rectal swab sample and three bats visceral organs were found positive for the NiV. Interestingly, 20.68% (12/58) of Pteropus were positive for anti-NiV IgG antibodies. NiV sequences of 18,172; 17,200 and 15,100 nucleotide bps could be retrieved from three Pteropus bats. CONCLUSION: A distinct cluster of NiV sequences, with significant net-evolutionary nucleotide divergence, was obtained, suggesting the circulation of new genotype (I-India) in South India. NiV Positivity in Pteropus spp. of bats revealed that NiV is circulating in many districts of Kerala state, and active surveillance of NiV should be immediately set up to know the hotspot area for NiV infection.

Experimental Zika virus infection in Aedes aegypti: Susceptibility, transmission & co-infection with dengue & chikungunya viruses.

BACKGROUND & OBJECTIVES: There are reports about the susceptibility of Aedes mosquitoes to ZIKV from various countries, however, no such information is available from Indian sub-continent, although, high level of group cross-reactivity of ZIKV with other flaviviruses has been reported. During outbreak situations, many cases of Dengue (DEN) and Chikungunya (CHIK) are reported. In such scenario, vector mosquitoes are likely to get co-infection/secondary-infection with one or other virus. The present study was carried out to determine the susceptibility of Indian strain of Aedes aegypti to Zika virus (ZIKV) strain (MR-766) and the effect of co-infection/super-infection with either dengue virus (serotype-2) (DENV) or chikungunya virus (CHIKV) on ZIKV replication. METHODS: Ae. aegypti mosquitoes used in this study were reared for many generations since 1980 at laboratory colony maintained at the ICMR-National Institute of Virology, Pune, India. Transmissibility of ZIKV from infected mosquitoes to suckling mice was also studied. Mosquitoes were experimentally infected with ZIKV and super-infected with either DENV or CHIKV via membrane-feeding route and incubated for 14 days at 28±2°C and humidity of 85±5 per cent. Replication of these viruses in mosquitoes was confirmed using real-time reverse transcription-polymerase chain reaction and immunofluorescence assay. Twenty infected mosquitoes were allowed to feed upon four suckling CD1 mice for about 30 min. Transmission of the ZIKV by infected mosquitoes to suckling mice was confirmed by the appearance of clinical signs and the presence of viral RNA in different organs. RESULTS: Concomitant infection of mosquitoes with all the three viruses showed simultaneous propagation of all three viruses, confirmed by real time RT-PCR and IFA. Infection of mosquitoes with CHIKV followed by ZIKV showed positivity in individual head squashes (7%) for both viruses using IFA; only 8.3 per cent showed dual positivity with primary infection of ZIKV followed by DENV; 8.3 per cent dual infection positivity was observed when infected with DENV followed by ZIKV; 5 per cent showed dual infection was observed when infected with ZIKV followed by CHIKV. Ae. aegypti was found to be susceptible to ZIKV strain as ZIKV could be detected from the second post-infection day (PID) in infected mosquitoes. Transmission of ZIKV to mice by the bite of infected Ae. aegypti establishes this species as a potential vector. INTERPRETATION & CONCLUSIONS: From super-infection experiments, it was concluded that ZIKV might have a relative advantage in replication dynamics over DENV. Vertical transmission was not observed for ZIKV in experimentally infected mosquitoes (n=920 larvae). Further studies are required to understand the possibility of silently circulating ZIKV in India, which remain non-detected because of lack of surveillance.

Kyasanur Forest Disease Prevalence in Western Ghats Proven and Confirmed by Recent Outbreak in Maharashtra, India, 2016.

INTRODUCTION: Kyasanur forest disease (KFD) outbreak was confirmed in Dodamarg Taluka, Sindhudurga district (Maharashtra) in India during the year 2016. The rise in suspected KFD cases was reported in January 2016, peaked during March, and then declined gradually from April 2016. The outbreak was thoroughly investigated considering different socio-clinical parameters. METHODS: Total, 488 suspected KFD cases were investigated using KFD specific real-time RT-PCR and anti-KFDV IgM enzyme-linked immunosorbent assay (ELISA). Sero-epidemiological survey was carried out in the affected area using anti-KFDV IgG ELISA. RESULTS: Among suspected KFD cases, high age-specific attack rate (105.1 per 1000 persons) was observed in adults (aged 40-59 years). Out of 488 suspected KFD cases, 130 were laboratory confirmed. Of these, 54 cases were KFDV real-time RT-PCR positive, 66 cases were anti-KFDV IgM ELISA positive and 10 cases were positive by both the assays. Case fatality ratio among laboratory-confirmed KFD cases were 2.3% (3/130). Majority of laboratory-confirmed KFD cases (93.1%) had visited Western Ghats forest in Dodamarg for activities like working in cashew nut farms (79.8%), cashew nut fruit collection (76.6%), collection of firewood (68.5%) and dry leaves/grass (40.3%), etc., before the start of symptoms. Common clinical features included fever (100%), headache (93.1%), weakness (84.6%), and myalgia (83.1%). Hemorrhagic manifestations were observed in nearly one-third of the laboratory-confirmed KFD cases (28.5%). A seroprevalence of (9.7%, 72/745) was recorded in KFD-affected area and two neighboring villages (9.1%, 15/165). Serosurvey conducted in Ker village showed clinical to subclinical ratio of 6:1 in KFD-affected areas. CONCLUSION: This study confirms the outbreak of KFD Sindhudurg district with 130 cases. Detection of anti-KFDV IgG antibodies among the healthy population in KFD-affected area during the KFD outbreak suggested the past exposure of KFD infection. This outbreak investigation has helped health authorities in adopting KFD vaccination strategy for the population at risk.

Zika virus: Current concerns in India.

With confirmation of Zika virus (ZIKV) presence in India, screening of a large number of febrile illness samples yielded only four positive cases. In this review, we address the current concern with context to India. The possible reasons for low level of Zika prevalence in India have been discussed, by extracting some probable explanations from previous experience of chikungunya virus-vector model/studies. In the current context, it is hypothesized that Indian mosquito strains have lower susceptibility gradient/threshold for ZIKV. The very low positivity in the humans also indicates low levels of mosquito-human-mosquito transmission cycle. There is also a need to look for the existence of any such animal cycle/sylvatic involvement in India. The recently detected four cases in India show local transmission of ZIKV suggesting that ZIKV might have been present in India since long time. The earlier vector-virus relationship studies with chikungunya suggested that in due course of time, ZIKV might become a major public health concern in the future.

Chikungunya virus susceptibility & variation in populations of Aedes aegypti (Diptera: Culicidae) mosquito from India.

BACKGROUND & OBJECTIVES: Although having immense clinical relevance, yet only a few studies have been targeted to understand the chikungunya virus (CHIKV) susceptibility and growth in Aedes aegypti populations from India. This study was undertaken to investigate CHIKV susceptibility and growth kinetics in Ae. aegypti along with genetic heterogeneity of Ae. aegypti populations. METHODS: Dose dependent CHIKV susceptibility and growth kinetic studies for three CHIKV strains reported from India were carried out in Ae. aegypti mosquito populations. The phenotypic variation and genetic heterogeneity in five Ae. aegypti populations were investigated using multivariate morphometrics and allozyme variation studies. RESULTS: The dissemination and growth kinetics studies of the three CHIKV strains showed no selective advantage for a particular strain of CHIKV in Ae. aegypti. At 100 per cent infection rate, five geographic Ae. aegypti populations showed differences in dissemination to three CHIKV strains. Morphometric studies revealed phenotypic variation in all the studied populations. The allelic frequencies, F statistics, and Nei's genetic identity values showed that genetic differences between the populations were small, but significant. INTERPRETATION & CONCLUSIONS: The results obtained in this study suggest that genetic background of the vector strongly influences the CHIKV susceptibility in Ae. aegypti.

Serratia odorifera mediated enhancement in susceptibility of Aedes aegypti for chikungunya virus.

BACKGROUND & OBJECTIVES: The susceptibility of the mosquito to the invading pathogen is predominantly dictated by the complex interactions between the mosquito midgut and the surface proteins of the invading pathogen. It is well documented that the midgut microbiota plays an important role in determining the susceptibility of the mosquito to the pathogen. In the present study, we investigated the influence of Serratia odorifera, an endogenous cultivable midgut inhabitant of Aedes aegypti on the chikungunya virus (CHIKV) susceptibility to this mosquito. METHODS: Ae. aegypti females free of gutflora were co-fed with CHIKV and either of the two midgut inhabitants namely, S. odorifeara and Microbacterium oxydans. CHIKV dissemination was checked on 10 th day post feeding (DPF) using indirect immunoflurescence assay and plaque assay. CHIKV interacting proteins of the mosquito midgut were identified using virus overlay protein binding assay and MALDI TOF/TOF analysis. RESULTS: The observations revealed that co-feeding of S. odorifera with CHIKV significantly enhanced the CHIKV susceptibility in adult Ae. aegypti, as compared to the mosquitoes fed with CHIKV alone and CHIKV co-fed with another midgut inhabitant, M. oxydans. Virus overlay protein binding assay (VOPBA) results revealed that porin and heat shock protein (HSP60) of Ae. aegypti midgut brush border membrane fraction interacted with CHIKV. INTERPRETATION & CONCLUSIONS: The results of this study indicated that the enhancement in the CHIKV susceptibility of Ae. aegypti females was due to the suppression of immune response of Ae. aegypti as a result of the interaction between S. odorifera P40 protein and porin on the gut membrane.

Administration of E2 and NS1 siRNAs inhibit chikungunya virus replication in vitro and protects mice infected with the virus.

BACKGROUND: Chikungunya virus (CHIKV) has reemerged as a life threatening pathogen and caused large epidemics in several countries. So far, no licensed vaccine or effective antivirals are available and the treatment remains symptomatic. In this context, development of effective and safe prophylactics and therapeutics assumes priority. METHODS: We evaluated the efficacy of the siRNAs against ns1 and E2 genes of CHIKV both in vitro and in vivo. Four siRNAs each, targeting the E2 (Chik-1 to Chik-4) and ns1 (Chik-5 to Chik-8) genes were designed and evaluated for efficiency in inhibiting CHIKV growth in vitro and in vivo. Chik-1 and Chik-5 siRNAs were effective in controlling CHIKV replication in vitro as assessed by real time PCR, IFA and plaque assay. CONCLUSIONS: CHIKV replication was completely inhibited in the virus-infected mice when administered 72 hours post infection. The combination of Chik-1 and Chik-5 siRNAs exhibited additive effect leading to early and complete inhibition of virus replication. These findings suggest that RNAi capable of inhibiting CHIKV growth might constitute a new therapeutic strategy for controlling CHIKV infection and transmission.

Serratia odorifera a midgut inhabitant of Aedes aegypti mosquito enhances its susceptibility to dengue-2 virus.

Mosquito midgut plays a crucial role in its vector susceptibility and pathogen interaction. Identification of the sustainable microflora of the midgut environment can therefore help in evaluating its contribution in mosquito-pathogen interaction and in turn vector competence. To understand the bacterial diversity in the midgut of Aedes aegypti mosquitoes, we conducted a screening study of the gut microbes of these mosquitoes which were either collected from fields or reared in the laboratory "culture-dependent" approach. This work demonstrated that the microbial flora of larvae and adult Ae. aegypti midgut is complex and is dominated by gram negative proteobacteria. Serratia odorifera was found to be stably associated in the midguts of field collected and laboratory reared larvae and adult females. The potential influence of this sustainable gut microbe on DENV-2 susceptibility of this vector was evaluated by co-feeding S. odorifera with DENV-2 to adult Ae. aegypti females (free of gut flora). The observations revealed that the viral susceptibility of these Aedes females enhanced significantly as compared to solely dengue-2 fed and another gut inhabitant, Microbacterium oxydans co-fed females. Based on the results of this study we proposed that the enhancement in the DENV-2 susceptibility of Ae. aegypti females was due to blocking of prohibitin molecule present on the midgut surface of these females by the polypeptide of gut inhabitant S. odorifera.

Evidence of co-infection of chikungunya and densonucleosis viruses in C6/36 cell lines and laboratory infected Aedes aegypti (L.) mosquitoes.

BACKGROUND: Densonucleosis viruses are the etiological agents of insect's disease. We have reported the isolation of densovirus from India and its distribution among the natural populations of Aedes aegypti mosquitoes across the country. Since densonucleosis virus persistently infects mosquito populations, and is demonstrated to negatively affect multiplication of dengue virus in Aedes albopictus, it would be interesting to study if this virus has a role in determining the susceptibility of the vector mosquito Ae. aegypti to chikugunya virus. METHODS: Mosquito cell lines and adult Ae. aegypti mosquitoes infected with densovirus were superinfected with Chikungunya virus and both the viruses were quantitated by determining their genomic copy number by real time amplification. Comparison was made between the log of genomic copy numbers of the viruses in the presence and absence of each other. RESULTS: The log of copy number of the viruses did not vary due to co-infection. Even though the RNA copy number of chikungunya virus increased over the period of time, no change was observed in the RNA copy number between the control and the co-infected group on any given day. Similarly, DNA copy number of densovirus also remained unchanged between the control and the co-infected groups. CONCLUSION: Chikungunya virus neither stimulates the replication of densovirus nor is its own replication suppressed due to co-infection. Ae. aegypti mosquitoes with densovirus infection were as susceptible to infection by chikungunya virus as the uninfected mosquitoes.

Morphometric and allozyme variation in Culex tritaeniorhynchus mosquito populations from India.

Four populations of Culex tritaeniorhynchus (Giles) (Diptera: Culicidae), collected from Bellary, Cuddalore, Pune, and the Microbial Containment Complex laboratory culture in India were analyzed for morphological and allozyme variation. Multivariate analysis based on eight morphological characteristics and three morphometric indices was used to investigate the morphological variations among the four populations. Principal component analysis of the data suggested that siphon, saddle, and anal gills related variables were most important. Discriminant factor analysis of morphological data revealed that the four populations form significantly different clusters which can be differentiated from each other based on siphon, saddle, and pectin teeth related variables. Allozyme electrophoresis of the four populations revealed that the mean heterozygosity per locus value had high variation, ranging from 0.0879 to 1.794. Fst values between 0 and 0.519 suggested genetic differentiation within these populations. Fis values ranged from 0 to 1 with most of the values closer to 1. The allelic frequencies and Nei's genetic identity values showed that genetic differences between populations were small, but significant. Some of the morphological and allozyme variations in the Cx. tritaeniorhynchus populations could be partly attributed to the environmental conditions. The findings suggested that transition of morphological characters and allozyme variations in Cx. tritaeniorhynchus populations seem to be consequences of influence and selection by the environmental conditions. These results indicated that populations of Cx. tritaeniorhynchus in non-endemic areas of Japanese encephalitis (JE) virus infection have higher adaptability as compared to endemic areas of JE infection.

Dengue-2-virus-interacting polypeptides involved in mosquito cell infection.

For the design of effective antiviral strategies, understanding the fundamental steps of the virus life cycle, including virus-host interactions, is essential. We performed a virus overlay protein binding assay followed by proteomics for identification of proteins from membrane fractions of A7 (Aedes aegypti) cells, C6/36 (Aedes albopictus) cells and the midgut brush border membrane fraction of Ae. aegypti mosquito that bind to dengue-2 virus. Actin, ATP synthase ß subunit, HSc 70, orisis, prohibitin, tubulin ß chain, and vav-1 were identified as dengue-2-virus-binding proteins. Our results suggest that dengue-2 virus exploits an array of housekeeping proteins for its entry in mosquito cells.