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

<p>Abstract</p> <p>Background</p> <p>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 <it>Aedes aegypti </it>mosquitoes across the country. Since densonucleosis virus persistently infects mosquito populations, and is demonstrated to negatively affect multiplication of dengue virus in <it>Aedes albopictus</it>, it would be interesting to study if this virus has a role in determining the susceptibility of the vector mosquito <it>Ae. aegypti </it>to chikugunya virus.</p> <p>Methods</p> <p>Mosquito cell lines and adult <it>Ae. aegypti </it>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>Chikungunya virus neither stimulates the replication of densovirus nor is its own replication suppressed due to co-infection. <it>Ae. aegypti </it>mosquitoes with densovirus infection were as susceptible to infection by chikungunya virus as the uninfected mosquitoes.</p

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 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

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

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

<i>Serratia odorifera</i> and <i>Ae. aegypti</i> interaction.

<p>a. Significance of <i>S. odorifera</i>’s presence in the blood meal on the DENV-2 susceptibility of <i>Ae. aegypti</i>: Adult females were fed with Blood + DENV-2, Blood + DENV-2+ <i>M. oxydans</i> (Blood + DENV-2+ <i>S. odorifera</i> via blood meal. DENV-2 dissemination was detected in the head squashes on 14 post infection days by IFA. The presence of <i>S. odorifera</i> in the blood meal significantly enhanced the dengue virus susceptibility (Mann-Whitney U test; P<0.05) compared to <i>M. oxydans</i> (Mann-Whitney U test; P>0.05). Post feeding virus titers in the blood meal were determined by plaque assay (1.8× 10⁵ PFU/mL of blood). b. Overlay assay with <i>S. odorifera</i> and <i>M. oxydans</i> cell lysates: The bacterial cell lysates of <i>S. odorifera</i> (So1 and So2) and <i>M. oxydans</i> (Mo1 and Mo2) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was overlaid with BBMF of <i>Ae. aegypti.</i> The putative binding proteins were detected by mouse anti-BBMF antibody and HRP labeled-secondary antibody. c. Expression of P40 in cell lysates and cell supernatants under different temperature conditions: <i>S. odorifera</i> cell lysate (S1 and S3 30°C, S2 41°C), <i>M. oxydans</i> cell lysate (M1 and M3 30°C, M2 41°C), culture filtrate of <i>S. odorifeara</i> (S4 and S6 30°C, S5 41°C) and culture filtrate of <i>M. oxydans</i> (M4 and M6 30°C, M5 41°C) were separated by SDS-PAGE and transferred to Hybond-C membranes. The membranes were incubated with the anti P40 mouse IgG (lanes S1, S2, S4, S5, M1, M2, M4 and M5) and with PBS pH 7.4 (lanes S3, S6, M3 and M6). Presence of P40 was detected by incubating the membranes with the secondary antibody (peroxidase-conjugated goat anti mouse IgG). Reaction was developed using H₂O₂ and DABT.d. Protein-protein interaction between BBMF and <i>S. odorifera</i> cell lysate: The membrane proteins of <i>Ae. aegypti</i> midgut (Lanes L1, L2 ) were separated by SDS–PAGE and transferred to Hybond-C membranes. The membranes were incubated with <i>S. odorifera</i> cell lysate (L1) and PBS pH 7.4 (L2) at 37°C. The putative P40 binding proteins were visible after incubation with anti P40 mouse antibody and the secondary antibody (peroxidase-conjugated goat anti mouse IgG). The reaction was developed using H₂O₂ and DABT. The molecular weights of DENV-2 binding proteins are shown on the left side of the blot.</p

P40 localization in the <i>Ae. aegypti</i> gut.

<p>The midgut sections (10 µm) of <i>Ae. aegypt</i>i fourth instar larvae (a) adult female (b) and slit opened gut of adult females (c) were incubated with <i>S. odorifera</i> cell lysate and control midgut sections were incubated with PBS (pH 7.4). P40 interaction with the midgut epithelium was detected using mouse anti-P40 antibody and with a Cy3 conjugated rabbit anti mouse IgG secondary antibody. The signal was detected using a Zeiss microscope equipped with the Axiovesion detection system.</p