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Background: Dengue is the most rapidly spreading arboviral disease in the world. Strain variations within the four dengue serotypes classify the virus into genetically distinct groups within serotypes called genotypes. Several studies have shown that disease outcome is associated with the genotype involved. Northeast Region of India comprises of 8 states and has witnessed several Dengue outbreaks in recent years. During 2015, a massive Dengue outbreak occurred in Pasighat, Arunachal Pradesh, India reporting around 2000 cases. All four serotypes have been reported from this region but studies on the genotype of the virus is lacking. This study was undertaken with an objective to elucidate the genotype of Dengue virus circulating in Arunachal Pradesh during the outbreak. Methods&Materials: A total of 115 Dengue suspected samples were collected from Pasighat General Hospital, Arunachal Pradesh. Screening of Dengue NS1 antigen or Anti- Dengue IgM antibody was done dependinguponthe duration of sample collection and onset of symptom. IgG ELISA was done to determine whether the infection was primary or secondary. RT-PCR was performed using specific primers to amplify C-prM gene region. The obtained sequences were analyzed using BioEdit software and a phylogenetic tree was constructed in MEGA 6 software. Results: Total 50 samples were Dengue positive by either NS1 or IgM ELISA of which eight (16%) cases were due to secondary infection. Dengue virus RNA was detected in 28 samples. Serotype 1 was found to be predominant during the outbreak. Phylogenetic analysis of the obtained sequences using Maximum Likelihood method and Kimura-2 parameter model revealed that the circulating Dengue virus-1 during the outbreak belonged to Genotype III. Conclusion: This is the maiden report on genotyping of Dengue virus 1 from this region of India. Studies have shown that the emergence of newer dengue serotype/ genotype after a gap have always been associated with massive outbreaks; therefore it is necessary to carry out active surveillance to monitor Dengue virus epidemiology. Further study on pathogenecity of Dengue virus 1 as well as other serotypes circulating in this region is in scope of this study. [ABSTRACT FROM AUTHOR]

Background: In India, scrub typhus (ST) is considered to be a reemerging infectious disease. From northeast India, ST cases have recently been reported (2012) in Assam after a gap of 68 years. The causative organism, Orientia tsutsugamushi is known to be genetically and antigenically highly variable. Sequence analysis of 56-kilodalton (kda) type-specific antigen (TSA) gene has become an important tool for genetic characterization of Orientia. This variable gene sequence is useful for analysis of genetic diversity of Orientia isolates. Although re-emergence of ST has been reported, circulating strains and their origin have yet to be identified. In the present study, we determined the prevalent strains of O. tsutsugamushi circulating in Assam and their origin with respect to 56 kDa TSA gene. Methods & Materials: 370 clinical serum samples from suspected scrub typhus and other unidentified fever cases were received from tertiary hospitals in Assam. Serological screeningwas done using Scrub Typhus Detect IgM ELISA (InBios International, Inc., USA). Positive and equivocal samples were subjected to PCR. Nested PCR was performed using primers specific for O. tsutsugamushi 56 kDa gene. Phylogenetic analysis was performed using MEGA 6 software. Results: 19.4% sera (72/370) were found to be IgM positive and 6.4% (24/370) were equivocal. 13 of these samples were PCR positive for 56 kDa gene. Phylogenetic analysis of study strains showed variations in sequence homologies that formed 3 distinct clades that clustered with reference strains from: 1) India 2) Taiwan and 3) Thailand. Conclusion: In summary, we have identified strains of O. tsutsugamushi circulating in Assam and established their evolutionary relationship with reference strains by analyzing a variable portion of 56 kDa gene. Understanding the evolution of the prevalent strains is important to understand the genetic differences which may help in planning control strategies as well as prophylactic measures including development of vaccines. [ABSTRACT FROM AUTHOR]

Background: Co-circulation of Japanese encephalitis Virus (JEV) and West Nile Virus (WNV) has been accounted in India. Both viruses are antigenically related and belong to the Japanese encephalitis (JE) serocomplex. Recently, the Government of India introduced the live attenuated JE vaccine SA 14-14-2 in routine immunization program for children and mass vaccination campaign among adults in highly JE endemic areas of Assam, Northeast India. However, the protection elicited by the JE vaccine against the circulating JEV and WNV in this region have not been studied. Thus, we investigated whether a single dose of this vaccine provided protection against local JEV and WNV isolates in animal model. Methods & Materials: Eight groups (n = 6) of four-six week old Swiss albino mice were inoculated sub-cutaneously with the JE vaccine. Four weeks post immunization, three mice groups were challenged intra-peritoneally with three JEV and four WNV each comprising of both archival and circulating strains. One mice group served as a control with no virus challenge. Mice were observed for 21 days. Results: The protection rates against three JEV strains (genotype III) were 100%. However, we noticed limited protection against the fourWNVstains (Lineage V). But, interestingly, the protection rates against archival WNV strains 804994 and circulating WNIRGC07 were 50% and 33.33% respectively. Whereas, no protection was conferred by the vaccine to WNV archival G22886 and circulating WNIRTC08 strains. Conclusion: The study showed total protection against JEV strains which may be due to the same genotype of the vaccine (Genotype III) as that of the local JEV strains. However, JEV vaccine was found to elicit partial cross protection to a circulating WNV strain which was reported to be a variant. It is noteworthy that no protection was observed against the other circulating WNV strain. Thus for immunization strategies, limited cross protection against heterologous viruses of the JE serocomplex must be considered. [ABSTRACT FROM AUTHOR]

Background: West Nile virus (WNV) is a flavivirus transmitted by mosquitoes. The prevalence of WNV antibodies in Indian population has been known since 1952. Following phylogenetic analysis, Indian isolates were classified into Lineage 1 and 5. Since 2006, with the identification of WNV as an aetiology causing acute encephalitis syndrome (AES) in Northeast India, scattered cases have been recorded in Assam every year during the months of June to October. However, the mosquito species involved in transmission of WNV in this region requires detailed study. Methods & Materials: We analysed human sera from hospitalised AES cases and mosquitoes sampled in four West Nile reporting areas during June 2014 - June 2015. Mosquitoes were caught using suction tube and flash light. A total of 970 pools from 37246 mosquitoes were based on uniformity of species, collection site and date. Clinical samples were tested for presence of WNVspecific IgM antibodies and confirmed by neutralizing antibody test for the acute and convalescent phase serum specimen. Both clinical samples and mosquito pools were also tested for the presence of WNV RNA using NS1 gene specific primers. Results: Of the 222 AES acute case serum samples tested for the presence of WNV specific IgM antibodies, 48 were found positive. Six IgM positive samples demonstrated a fourfold rise in neutralizing antibody titre againstWNVin convalescent sera.WNVRNA was detected in the cerebrospinal fluid of one human sample. Vector incrimination showed 14 pools (4- Culex vishnui, 1- Culex pseudovishnui, 2- Culex tritaeniorhynchus, 2- Culex quinquefasciatus, 1- Culex whitmorei and 4 -Mansonia uniformis) to be positive for WNV RNA. Interestingly, 13 of the positive pools were collected during November- December 2014. Mosquito and human derived WNV sequence were similar to Indian Lineage 5 WNV isolates. Conclusion: Our results provide molecular evidence for the persistence and maintenance of Lineage 5WNVin Northeast India. This study confirms the role of Culex and Mansonia mosquito species in the transmission of WNV in Assam. Further studies are required to address WNV transmission and maintenance during winters for implementation of vector intervention strategies. [ABSTRACT FROM AUTHOR]

Background: In India, rickettsial infections were first reported from Assam and West Bengal lay back during 1947. Since then, sporadic cases have been reported from different parts of the country except in the north eastern (NE) states. However, after a gap of 68 years, scrub typhus was reported to resurge in three states of NE region in 2012. This long serenity may be due to lack of surveillance and low index of suspicion for scrub typhus and other rickettsial infections. Therefore, a two year study was undertaken to study the sero-epidemiology of rickettsial infections viz., Scrub typhus (ST), Spotted fever group rickettsia (SFGR) and typhus group rickettsia (TGR) in Northeast India. Methods & Materials: We investigated scrub typhus reporting areas of three states viz., Assam, Arunachal Pradesh and Nagaland during 2013-2014. About 908 human blood samples were collected from healthy volunteers with informed consent. All collected human sera were screened for antibodies against ST, SFGR and TGR using a four step indirect ELISA with respective antigens. A sample whose total net absorbance was = 1.000 was considered as sero-positive. Positive samples were further titrated using 1:100, 1:400, 1:1600 and 1:6400 sample dilutions. Results: Overall, 33.5% (305/908), 11.2% (102/908) and 3.9% (35/908) were found to be seropositive for ST, SFGR andTGRrespectively. Co-circulation of ST and SFGRwasfound in all the three states whereas TGR were detected in samples collected from Arunachal Pradesh only. People engaged in agricultural and forest sectors were predominantly affected with a higher male to female ratio. Rural setting and lack of hygiene was a notable feature in the affected areas. Conclusion: Our findings indicated a wide circulation of rickettsial infections in this region. This is also the first evidence of SFGR and TGR circulation in NE region of India. Continuous surveillance, understanding eco-epidemiology of these diseases and consideration of these agents for diagnosis of febrile illness by public health workers is warranted in future. [ABSTRACT FROM AUTHOR]

Background: The interaction of nanoparticles with microorganisms is an emerging field. Within this, an area that has been limitedly explored is the interaction of nanoparticles with viruses. Recently studies on silver nanoparticles (AgNPs) showed potent antiviral activity against HIV, HBV, influenza and dengue virus. However, synthesis of metallic nanoparticles involves physical and chemical approach utilizing toxic chemicals, which may hinder its application for human use. Therefore, development of novel ecofriendly nanoparticles is of great interest. In the present study, we synthesized green AgNPs with catechin as reducing agent. Catechin, a polyphenol being itself having antiviral propertymayenhance the efficiency of AgNPs. We evaluated biogenic catechin AgNPs against Japanese encephalitis virus (JEV), which is a major public health problem particularly in Asia. Methods & Materials: Green nanoparticles were synthesized by adding purified catechin intoAgNO3solution by following standard method. Standard circulating JEV strain JERG07 (Source: Human, 2007, Assam, India) was used. In vitro assays were done using VERO cell line. Cell viability assay for 2 fold diluted green AgNPs was done using MTT standard protocol assay. Cytopathetic effect (CPE) inhibition assay was performed with 1 Multiplicity of infection (MOI) of JEV. Cells were also observed daily under inverted microscope and were assigned a score based on appearance of CPE. Further, virus yield reduction assay was done to quantify the level of protection of nanoparticles treated both at pre & post JEV infection. Results: Transmission Electron Microscopy (TEM) analysis revealed formation of biogenic AgNPs within the size of 50nm. Maximum (100%) cell viability for green nanoparticles was observed in the range from 0.04g/ml to 5.85g/ml. Within thesamerange CPE inhibition assay also showed full protection against JEV. Further, virus yeild reduction assay showed reduction in plaques number in comparison to virus control in both pre & post JEV infection cells. Conclusion: The present study demonstrated the ability of catechin reduced biogenic AgNPs to prevent JEV infection by inhibition of virus attachment and post infection spread. Future studies should be focused on elucidating the mechanism of nanoparticle-virus interaction at cellular and microscopic level. [ABSTRACT FROM AUTHOR]