Molecular epidemiology of novel swine origin influenza virus (S-OIV) from Gwalior, India, 2009

<p>Abstract</p> <p>Background</p> <p>The H1N1pandemic virus is a newly emergent human influenza A virus that is closely related to a number of currently circulating pig viruses in the 'classic North American' and 'Eurasian' swine influenza virus lineages and thus referred as S-OIV. Since the first reports of the virus in humans in April 2009, H1N1 virus has spread to 168 countries and overseas territories. India also witnessed severe H1N1 pandemic virus epidemic with considerable morbidity and mortality in different parts starting from May 2009.</p> <p>Findings</p> <p>The suspected swine flu outbreak from Gwalior India during October- December 2009 was confirmed through S-OIV HA gene specific RT-LAMP and real time RT-PCR. Positive samples through CDC real time and Lamp assay were further processed for isolation of the virus. Full HA gene sequencing of the H1N1 isolates of Gwalior, India revealed 99% homology with California and other circulating novel swine flu viruses. Three major changes were observed at nucleotide level, while two major amino acid shifts were observed at the position C9W and I30M corresponding to the ORF with prototype strain. The HA gene sequence phylogeny revealed the circulation of two genetically distinct lineages belonging to <it>Clade VII </it>and <it>Clade I </it>of S-OIV.</p> <p>Conclusions</p> <p>Our findings also supported the earlier report about circulation of mixed genogroups of S-OIV in India. Therefore continuous monitoring of the genetic makeup of this newly emergent virus is essential to understand its evolution within the country.</p

Refractoriness of Indian Aedes aegypti to Oral Infection with Yellow Fever Virus 17D Strain

Yellow fever virus (YFV) is the causative agent of yellow fever. It is one of the most important hemorrhagic arboviral infection of global public health significance. It is categorised under category ‘C’ of potential bioterrorism agent. Effect of geographical variation on vector competence in Ae. aegypti has been well documented for several viruses including YFV. In the present study, the vector competence of Ae. aegypti mosquitoes collected from Gwalior, India for YFV 17D vaccine strain was evaluated to understand the risk of its transmission. Further the risk associated with transmission of YFV 17D vaccine strain from viremic vaccinees to mosquitoes and subsequently to naive individuals was assessed. Ae. aegypti were orally infected with high titer of YFV 17D strain and the infection status was investigated at 7 and 14 day post infection (dpi) using a highly sensitive quantitative RT-PCR assay. None of the Ae. aegypti mosquito orally infected with YFV 17D strain was found to be positive for YFV. The infection rate was found to be zero per cent at both 7 dpi and 14 dpi. These results demonstrated the inability of the YFV 17D strain to cause infection or replication in the midgut of Ae. aegypti. Due to the highly attenuated replication of this strain in Ae. aegypti midgut, there is a minimal risk of its transmission. Further, it is unlikely for a mosquito that feeds on a viremic vaccine to get infected with this vaccine strain. The risk of transmission of YFV 17D strain by Indian Ae. aegypti mosquitoes is negligible. Further vector competence study using epidemic strain of YFV will aid in risk assessment analysis of YFV in India

Appearance of E1: A226V mutant Chikungunya virus in Coastal Karnataka, India during 2008 outbreak

Chikungunya has resurged in the form of unprecedented explosive epidemic in 2006 after a long gap in India affecting 1.39 million of persons. The disease continued for the next two consecutive years affecting 59,535 and 64,548 persons during 2007 and 2008 respectively. The 2008 outbreak being the second largest among these three years the information regarding the etiology and the mutations involved are useful for further control measures. Among the 2008 outbreaks the Coastal Karnataka accounts for the 46,510 persons. An in-depth investigation of Chikungunya epidemic of Coastal Karnataka, India, 2008 by serology, virus isolation, RT-PCR and genome sequencing revealed the presence and continued circulation of A226V mutant Chikungunya virus. The appearance of this mutant virus was found to be associated with higher prevalence of vector Aedes albopictus and the geographical proximity of coastal Karnataka with the adjoining Kerala state. This is the first report regarding the appearance of this mutation in Karnataka state of India. The present study identified the presence and association of A226V mutant virus with Chikungunya outbreak in India during 2008

Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses

<p>Abstract</p> <p>Background</p> <p>Dengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR) for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported.</p> <p>Results</p> <p>An optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Japanese encephalitis, West Nile, Yellow fever) and alphavirus (Chikungunya). The feasibility of M-RT-PCR assay for clinical diagnosis was validated with 620 acute phase dengue patient sera samples of recent epidemics in India. The comparative evaluation <it>vis a vis </it>conventional virus isolation revealed higher sensitivity. None of the forty healthy serum samples screened in the present study revealed any amplification, thereby establishing specificity of the reported assay for dengue virus only.</p> <p>Conclusion</p> <p>These findings clearly suggested that M-RT-PCR assay reported in the present study is the rapid and cost-effective method for simultaneous detection as well as typing of the dengue virus in acute phase patient serum samples. Thus, the M-RT-PCR assay developed in this study will serve as a very useful tool for rapid diagnosis and typing of dengue infections in endemic areas.</p

Vertical Transmission of Chikungunya virus in Aedes aegypti Mosquitoes from Northern India

Chikungunya virus is now recognised as a resurging arbovirus of global public health significance, with their circulation in both new and old world. It is horizontally transmitted among vertebrates by Aedes mosquitoes. So far, the existence of vertical transmission of Chikungunya virus in Aedes vector is riddled with conflicting reports. In this study, presence of Chikungunya virus was detected in adult Aedes aegypti mosquitoes that emerged from field-collected larvae from Gwalior, northern India during 2010. This was further confirmed through nucleotide sequencing that revealed the presence of novel east central south African (ECSA) genotype of Chikungunya virus. This provides molecular evidence for vertical transmission of Chikungunya virus in mosquitoes in nature, which may have important consequences for viral survival during inter-epidemic period and adverse climatic conditions

Phylogenetic studies reveal existence of multiple lineages of a single genotype of DENV-1 (genotype III) in India during 1956–2007

<p>Abstract</p> <p>Background</p> <p>Dengue virus type 1 (DENV-1) have been mostly circulating silently with dominant serotypes DENV-2 and DENV-3 in India. However recent times have marked an increase in DENV-1 circulation in yearly outbreaks. Many studies have not been carried out on this virus type, leaving a lacunae pertaining to the circulating genotypes, since its earliest report in India. In the present study, we sequenced CprM gene junction of 13 DENV-1 isolated from Delhi and Gwalior (North India) between 2001–2007 and one 1956 Vellore isolate as reference. For comparison, we retrieved 11 other Indian and 70 global reference sequences from NCBI database, making sure that Indian and global isolates from all decades are available for comparative analysis.</p> <p>Results</p> <p>The region was found to be AT rich with no insertion or deletion. Majority of the nucleotide substitutions were silent, except 3 non-conservative amino acid changes (I → T, A → T and L → S at amino acid positions 59,114 and 155 respectively) in the Indian DENV-1 sequences, sequenced in this study. Except two 1997–98 Delhi isolates, which group in genotype I; all other Indian isolates group in genotype III. All Indian genotype III DENV-1 exhibited diversity among them, giving rise to at least 4 distinct lineages (India 1–4) showing proximity to isolates from diverse geographic locations.</p> <p>Conclusion</p> <p>The extensive phylogenetic analysis revealed consistent existence of multiple lineages of DENV-1 genotype III during the last 5 decades in India.</p

Japanese Encephalitis Outbreak, India, 2005

An outbreak of viral encephalitis occurred in Gorakhpur, India, from July through November 2005. The etiologic agent was confirmed to be Japanese encephalitis virus by analyzing 326 acute-phase clinical specimens for virus-specific antibodies and viral RNA and by virus isolation. Phylogenetic analysis showed that these isolates belonged to genogroup 3

Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralising antibodies in rabbits and mice

Currently available vaccines for the pandemic Influenza A (H1N1) 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA) based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI) activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat

Discovery of the First Insect Nidovirus, a Missing Evolutionary Link in the Emergence of the Largest RNA Virus Genomes

Nidoviruses with large genomes (26.3–31.7 kb; ‘large nidoviruses’), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7–15.7 kb; ‘small nidoviruses’). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3′-5′exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60–80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3′-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3′-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2′-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that – in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses