Novel insights into host responses to Japanese Encephalitis Virus infection: Reanalysis of public transcriptome and microRNAome datasets.

PURPOSE: Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV), is the principal cause of viral encephalitis in South-East Asian and Western Pacific countries; accounting for 68,000 cases, and up to 20,400 fatalities, annually across the world. Despite being a high-risk condition, there is no specific treatment for JE. Given rapid additions in genomics databases and the power of data reanalysis in addressing critical medical questions, the present study was designed to identify novel host factors that might have potential roles in JEV infection. METHODS: We extracted microarray and RNA-Seq data sets from NCBI-GEO and compared mock and JEV-infected samples. Raw data from all the studies were re-analyzed to identify host factors associated with JEV replication. RESULTS: We identified several coding and non-coding host factors that had no prior known role in viral infections. Of these, the coding transcripts: Myosin Heavy Chain 10 (MYH10), Progestin and AdipoQ Receptor Family Member 8 (PAQR8), and the microRNAs: hsa-miR-193b-5p, hsa-miR-3714 and hsa-miR-513a-5p were found to be novel host factors deregulated during JEV infection. MYH10 encodes a conventional non-muscle myosin, and mutations in MYH10 have been shown to cause neurological defects. PAQR8 has been associated with epilepsy, which exhibits symptoms similar to JEV infection. JE is a neuro-degenerative disease, and the known involvement of MYH10 and PAQR8 in neurological disorders strongly indicates potential roles of these host factors in JEV infection. Additionally, we observed that MYH10 and PAQR8 had a significant negative correlation with Activating transcription factor 3 (ATF3), which is a previously validated modulator of JEV infection. ATF3 is a transcription factor that binds to the promotors of genes encoding other transcription factors or interferon-stimulated genes and negatively regulates host antiviral responses during JE. CONCLUSION: Our findings demonstrate the significance of data reanalysis in the identification of novel host factors that may become targets for diagnosis/ therapy against viral diseases of major concern, such as, JE. The deregulated coding and non-coding transcripts identified in this study need further experimental analysis for validation.

Clinical Predictors of COVID-19 Severity and Mortality: A Perspective.

The COVID-19 pandemic has caused huge socio-economic losses and continues to threat humans worldwide. With more than 4.5 million deaths and more than 221 million confirmed COVID-19 cases, the impact on physical, mental, social and economic resources is immeasurable. During any novel disease outbreak, one of the primary requirements for effective mitigation is the knowledge of clinical manifestations of the disease. However, in absence of any unique identifying characteristics, diagnosis/prognosis becomes difficult. It intensifies misperception and leads to delay in containment of disease spread. Numerous clinical research studies, systematic reviews and meta-analyses have generated considerable data on the same. However, identification of some of the distinct clinical signs and symptoms, disease progression biomarkers and the risk factors leading to adverse COVID-19 outcomes warrant in-depth understanding. In view of this, we assessed 20 systematic reviews and meta-analyses with an intent to understand some of the potential independent predictors/biomarkers/risk factors of COVID-19 severity and mortality.

SARS-CoV-2 in Pregnancy: Fitting Into the Existing Viral Repertoire.

The risk of viral infection during pregnancy is well-documented; however, the intervention modalities that in practice enable maternal-fetal protection are restricted by limited understanding. This becomes all the more challenging during pandemics. During many different epidemic and pandemic viral outbreaks, worse outcomes (fetal abnormalities, mortality, preterm labor, etc.) seem to affect pregnant women than what has been evident when compared to non-pregnant women. The condition of pregnancy, which is widely understood as "immunosuppressed," needs to be re-understood in terms of the way the immune system works during such a state. The immune system gets transformed to accommodate and facilitate fetal growth. The interference of such supportive conversion by viral infection and the risk of co-infection lead to adverse fetal outcomes. Hence, it is crucial to understand the risk and impact of potent viral infections likely to be encountered during pregnancy. In the present article, we review the effects imposed by previously established and recently emerging/re-emerging viral infections on maternal and fetal health. Such understanding is important in devising strategies for better preparedness and knowing the treatment options available to mitigate the relevant adverse outcomes.

Regulation of Host Innate Immunity by Non-Coding RNAs During Dengue Virus Infection.

An estimated 3.9 billion individuals in 128 nations (about 40% of global population) are at risk of acquiring dengue virus infection. About 390 million cases of dengue are reported each year with higher prevalence in the developing world. A recent modeling-based report suggested that half of the population across the globe is at risk of dengue virus infection. In any given dengue outbreak, a percentage of infected population develops severe clinical manifestations, and this remains one of the "unsolved conundrums in dengue pathogenesis". Although, host immunity and virus serotypes are known to modulate the infection, there are still certain underlying factors that play important roles in modulating dengue pathogenesis. Advanced genomics-based technologies have led to identification of regulatory roles of non-coding RNAs. Accumulating evidence strongly suggests that viruses and their hosts employ non-coding RNAs to modulate the outcome of infection in their own favor. The foremost ones seem to be the cellular microRNAs (miRNAs). Being the post-transcriptional regulators, miRNAs can be regarded as direct switches capable of turning "on" or "off" the viral replication process. Recently, role of long non-coding RNAs (lncRNAs) in modulating viral infections via interferon dependent or independent signaling has been recognized. Hence, we attempt to identify the "under-dog", the non-coding RNA regulators of dengue virus infection. Such essential knowledge will enhance the understanding of dengue virus infection in holistic manner, by exposing the specific molecular targets for development of novel prophylactic, therapeutic or diagnostic strategies.

Natural Products as a Paradigm for the Treatment of Coxsackievirus - induced Myocarditis.

Coxsackievirus B3 (CVB3), a member of the Picornaviridae family, is considered to be one of the most important infectious agents to cause virus-induced myocarditis. Despite improvements in studying viral pathology, structure and molecular biology, as well as diagnosis of this disease, there is still no virus-specific drug in clinical use. Structural and nonstructural proteins produced during the coxsackievirus life cycle have been identified as potential targets for blocking viral replication at the step of attachment, entry, uncoating, RNA and protein synthesis by synthetic or natural compounds. Moreover, WIN (for Winthrop) compounds and application of nucleic-acid based strategies were shown to target viral capsid, entry and viral proteases, but have not reached to the clinical trials as a successful antiviral agent. There is an urgent need for diverse molecular libraries for phenotype-selective and high-throughput screening.

Cytokine gene polymorphisms among North Indians: Implications for genetic predisposition?

Variations in the production and activity of cytokines influence the susceptibility and/or resistance to various infectious agents, autoimmune diseases, as well as the post-transplant engraftment/ rejection. Differences in the production of cytokines between individuals have been correlated to single nucleotide polymorphisms (SNPs) in the promoter, coding or non-coding regions of cytokine genes. The present study aimed at understanding distribution of cytokine gene variants among HIV seropositive subjects including HIV + TB+ subjects of Indian origin. Our findings indicate significant association of pro-inflammatory (IL2, IFN-γ, TNF-α) and anti-inflammatory cytokine gene variants (IL4, IL10) with the risk to acquire the HIV infection and development of AIDS related illness in Indian population. Since distribution of genetic polymorphisms varies significantly across different populations, different genotypes might exhibit different disease-modifying effects. An understanding of the immunogenetic factors or AIDS restriction genes is important not only for elucidating the mechanisms of disease pathogenesis but also for vaccine design and its application.

Diagnostic accuracy of loop-mediated isothermal amplification assay for extra-pulmonary tuberculosis in Indian population.

BACKGROUND: Confirmatory diagnosis of extra-pulmonary tuberculosis remains a true challenge owing to difficulty in procuring appropriate specimen, inefficient laboratory methods and paucibacillary nature of infection. These obstructions become all the very difficult in pediatric EPTB cases, due to non-specific clinical signs and symptoms, low sensitivity of smear microscopy and culture, lack of awareness among clinicians, etc. AIM OF THE STUDY: The present study aimed to evaluate the diagnostic accuracy of rapid and cost-effective loop-mediated isothermal amplification (LAMP) assay for EPTB diagnosis in children. METHODS: A total of 154 cases were analyzed by EPTB-site smear microscopy, culture, PCRs for IS6110, MPB64 & Pab genes, nested PCR and LAMP assay. Single-gene PCRs were performed by custom-synthesized primers. Nested PCR was performed using the 3B BIOTUB Kit and the LAMP assay was done using the Nu-LAMP TB kit. RESULTS: We observed that the molecular tests displayed 4-fold higher positivity rate (minimum 46%) in comparison to the microbiological tests (maximum 11.03%). In contrast to the composite reference standard, LAMP assay was found to be 79.6% sensitive and 78% specific for EPTB diagnosis in childhood cases. CONCLUSIONS: Our results indicate that LAMP assay is a promising technique for efficient diagnosis of EPTB in children belonging to resource-limited regions.

Diagnostic Potential of Recombinant scFv Antibodies Generated Against Hemagglutinin Protein of Influenza A Virus.

Human influenza A viruses have been the cause of enormous socio-economic losses worldwide. In order to combat such a notorious pathogen, hemagglutinin protein (HA) has been a preferred target for generation of neutralizing-antibodies as potent therapeutic/diagnostic agents. In the present study, recombinant anti-HA single chain variable fragment antibodies were constructed using the phage-display technology to aid in diagnosis and treatment of human influenza A virus infections. Spleen cells of mice hyper-immunized with A/New Caledonia/20/99 (H1N1) virus were used as the source for recombinant antibody (rAb) production. The antigen-binding phages were quantified after six rounds of bio-panning against A/New Caledonia/20/99 (H1N1), A/California/07/2009 (H1N1)-like, or A/Udorn/307/72(H3N2) viruses. The maximum phage yield was for the A/New Caledonia/20/99 (H1N1), however, considerable cross-reactivity was observed for the other virus strains as well. The HA-specific polyclonal rAb preparation was subjected to selection of single clones for identification of high reactive relatively conserved epitopes. The high-affinity rAbs were tested against certain known conserved HA epitopes by peptide ELISA. Three recombinant mAbs showed reactivity with both the H1N1 strains and one (C5) showed binding with all the three viral strains. The C5 antibody was thus used for development of an ELISA test for diagnosis of influenza virus infection. Based on the sample size in the current analysis, the ELISA test demonstrated 83.9% sensitivity and 100% specificity. Thus, the ELISA, developed in our study, may prove as a cheaper alternative to the presently used real time RT-PCR test for detection of human influenza A viruses in clinical specimens, which will be beneficial, especially in the developing countries.

Potent Intracellular Knock-Down of Influenza A Virus M2 Gene Transcript by DNAzymes Considerably Reduces Viral Replication in Host Cells.

Influenza A virus has been known to be an important respiratory pathogen and cause of several epidemics and devastating pandemics leading to loss of life and resources across the globe. The M2 ion channel protein is highly conserved and essentially required during the trafficking, assembly, and budding processes of virus, thus an attractive target for designing antiviral drugs. We designed several 10-23 DNAzymes (Dz) targeting different regions of the M2 gene and analyzed their ability to specifically cleave the target RNA in both cell-free system as well as in cell culture using transient transfections. Dz114, among several others, directed against the predicted single-stranded bulge regions showed 70% inhibition of M2 gene expression validated by PCR and Western blot analysis. The activity was dependent on Mg(2+) (10-50 mM) in a dose-dependent manner. The mutant-Dz against M2 gene showed no down-regulation thereby illustrating high level of specificity of designed Dz114 towards the target RNA. Our findings suggest that Dz may be used as potential inhibitor of viral RNA replication and can be explored further for development of an effective therapeutic agent against influenza infection. These catalytic nucleic acid molecules may further be investigated as an alternative to the traditional influenza vaccines that require annual formulation.

Gene silencing: a therapeutic approach to combat influenza virus infections.

Selective gene silencing technologies such as RNA interference (RNAi) and nucleic acid enzymes have shown therapeutic potential for treating viral infections. Influenza virus is one of the major public health concerns around the world and its management is challenging due to a rapid increase in antiviral resistance. Influenza vaccine also has its limitations due to the emergence of new strains that may escape the immunity developed by the previous year's vaccine. Antiviral drugs are the primary mode of prevention and control against a pandemic and there is an urgency to develop novel antiviral strategies against influenza virus. In this review, we discuss the potential utility of several gene silencing mechanisms and their prophylactic and therapeutic potential against the influenza virus.

Protective immunity based on the conserved hemagglutinin stalk domain and its prospects for universal influenza vaccine development.

Influenza virus surface glycoprotein hemagglutinin (HA) is an excellent and chief target that elicits neutralizing antibodies during vaccination or natural infection. Its HA2 subunit (stem domain) is most conserved as compared to HA1 subunit (globular head domain). Current influenza vaccine relies on globular head domain that provides protection only against the homologous vaccine strains, rarely provides cross-protection against divergent strains, and needs to be updated annually. There is an urge for a truly universal vaccine that provides broad cross-protection against different subtype influenza A viruses along with influenza B viruses and need not be updated annually. Antibodies against the stem domain of hemagglutinin (HA) are able to neutralize a wide spectrum of influenza virus strains and subtypes. These stem-specific antibodies have great potential for the development of universal vaccine against influenza viruses. In this review, we have discussed the stem-specific cross-reactive antibodies and heterosubtypic protection provided by them. We have also discussed their epitope-based DNA vaccine and their future prospects in this scenario.

Diversity, molecular phylogeny, and bioactive potential of fungal endophytes associated with the Himalayan blue pine (Pinus wallichiana).

In this study, we investigated the diversity of fungal endophytes associated with Pinus wallichiana from the Western Himalayas, with emphasis on comparison of endophytic communities harbored by the stem and needle tissues of the host and their antimicrobial potential. A total number of 130 isolates, comprising of 38 different genera, were recovered from 210 fragments of the plant. Among the isolated fungi, only a single isolate, Tritirachium oryzae, belonged to the Phylum Basidiomycota whereas the rest belonged to Ascomycota. Dothideomycetes was the dominant class with the highest isolation frequency of 49.2 %. The most frequent colonizers of the host were Alternaria spp., Pestalotiopsis spp., Preussia spp., and Sclerostagonospora spp. The diversity and species richness were higher in needle tissues than in the stems. Antimicrobial activities were displayed by extracts from a total number of 22 endophytes against one or more pathogens. Endophytes designated as P1N13 (Coniothyrium carteri), P2N8 (Thielavia subthermophila), P4S6b (Truncatella betulae), P7N10 (Cochliobolus australiensis), and P8S4 (Tritirachium oryzae) were highly active against Candida albicans. Broad spectrum antimicrobial activities were obtained with the extracts of P8-S4 (Tritirachium oryzae) and P5-N26 (Coniochaeta gigantospora) that were potentially active against the Gram-positive and Gram-negative bacteria as well as the fungal pathogen, Candida albicans. The most prominent antagonistic activity against fungal pathogens was shown by P8-S4 (Tritirachium oryzae), P5-N31a (Truncatella spadicea), and P5-N20 (Fusarium larvarum). Our findings indicate that Pinus wallichiana harbors a rich endophytic fungal community with potential antimicrobial activities. Further studies are needed to understand the ecology and evolutionary context of the associations between the Himalayan pine and its endophytes.

Sequence-specific cleavage of BM2 gene transcript of influenza B virus by 10-23 catalytic motif containing DNA enzymes significantly inhibits viral RNA translation and replication.

One of the hallmarks of progression of influenza virus replication is the step involving the virus uncoating that occurs in the host cytoplasm. The BM2 ion channel protein of influenza B virus is highly conserved and is essentially required during the uncoating processes of virus, thus an attractive target for designing antiviral drugs. We screened several DNA enzymes (Dzs) containing the 10-23 catalytic motif against the influenza B virus BM2 RNA. Dzs directed against the predicted single-stranded bulge regions showed sequence-specific cleavage activities. The Dz209 not only showed significant intracellular reduction of BM2 gene expression in transient-expression system but also provided considerable protection against influenza B virus challenge in MDCK cells. Our findings suggest that the Dz molecule can be used as selective and effective inhibitor of viral RNA replication, and can be explored further for development of a potent therapeutic agent against influenza B virus infection.

Clinical presentation of patients with seasonal influenza and pandemic influenza A (H1N1-2009) requiring hospitalisation.

BACKGROUND: A sudden increase in the number of novel influenza A virus (pH1N1-2009) infection prompted us to compare the clinical presentation and outcomes of patients infected with pH1N1-2009 and seasonal influenza A virus during the post-pandemic phase. METHODS: During the period August 13 to September 27, 2010, case records of 106 patients with severe influenza like illness (ILI) and respiratory complications who underwent diagnostic testing by real-time polymerase chain reaction (RT-PCR) for confirmation of pH1N1-2009 were retrospectively studied. RESULTS: Nineteen (17.9%) patients were tested positive for pH1N1-2009 and 78 (73.6%) were tested positive for seasonal influenza A virus. The mean age of patients infected with pH1N1-2009 was 45.2 +/- 15.3 years (range of 22 to 80 years). Common presenting symptoms included fever in 17 (89.4%), cough in 16 (84.2%), myalgia in 15 (78.9%) and breathlessness in 10 (52.6%) patients. The most common comorbidities included bronchial asthma/bronchitis/chronic obstructive pulmonary disease (COPD) in 4 (21%); followed by hypertension in 3 (15.8%) and diabetes in 3 (15.8%) patients. Overall, of the 97 influenza infected patients, 9 (9.3%) needed hospitalisation to the intensive care unit (ICU); one patient with COPD died due to multi-organ failure. CONCLUSIONS: Both the pandemic and seasonal strains were found to be co-circulating in the community. Patients with severe hypoxia, hypertension, acute respiratory distress syndrome and shock required ICU care.

Cross-protective effect of antisense oligonucleotide developed against the common 3' NCR of influenza A virus genome.

The influenza A virus (IAV) has eight segmented single-stranded RNA genome containing a common and evolutionarily conserved non-coding region (NCRs) at 5' and 3' ends that are important for the virus replication. In this study, we designed an antisense oligonucleotide against the 3' NCR of vital segments of the IAV genome to inhibit its replication. The results demonstrated that the co-transfection of Madine Darby Canine Kidney (MDCK) cells with the antisense oligonucleotide and the plasmids encoding the viral genes led to the down-regulation of the viral gene expression. The designed antisense molecules reduced the cytopathic effect caused by A/PR/8/34 (H1N1), A/Udorn/307/72 (H3N2), and A/New Caledonia/20/99 (H1N1) strains of IAV for almost 48 h. Furthermore, the intra-venous delivery of this oligonucleotide significantly reduced the viral titers in the lungs of infected mice and protected the mice from lethal effects of all the strains of influenza virus. The study demonstrated that the antisense oligonucleotide designed against the NCR region inhibits the expression of the viral genome. The decrease of the cytopathic effect in the MDCK cells and increase in survival of mice confirmed the reduction of virus multiplication and pathogenesis in the presence of antisense oligonucleotide. Thus, we demonstrate that a single antisense oligonucleotide is capable of providing protection against more than one strains of the IAV.

Small interfering RNA targeting the nonstructural gene 1 transcript inhibits influenza A virus replication in experimental mice.

Nonstructural protein 1 (NS1) of influenza A viruses counteracts the host immune response against the influenza viruses by not only inhibiting the nuclear export and maturation of host cell messenger RNA (mRNA), but by also blocking the double-stranded RNA-activated protein kinase-mediated inhibition of viral RNA translation. Reduction of NS1 gene product in the host cell may be a potent antiviral strategy to provide protection against the influenza virus infection. We used small interfering RNAs (siRNAs) synthesized against the viral mRNA to down regulate the NS1 gene and observed its effect on inhibition of virus replication. When NS1 gene-specific siRNA were transfected in Madin Darby canine kidney (MDCK) cells followed by influenza A virus infection, approximately 60% inhibition in intracellular levels of NS1 RNA was observed. When siRNA was administered in BALB/c mice, 92% reduction in the levels of NS1 gene expression in mice lungs was observed. A significant reduction in the lung virus titers and cytokine levels was also detected in the presence of siRNAs as compared with the untreated control. The study was validated by the use of selectively disabled mutants of each set of siRNA. Our findings suggest that siRNA targeted against NS1 gene of influenza A virus can provide considerable protection to the virus-infected host cells and may be used as potential candidates for nucleic acid-based antiviral therapy for prevention of influenza A virus infection.

A conserved matrix epitope based DNA vaccine protects mice against influenza A virus challenge.

DNA vaccination represents a unique strategy to overcome the limitations of immunization with conventional vaccines which is restricted by the high variability of influenza viruses. We evaluated the protective efficacy of a plasmid DNA (pDNA), encoding an evolutionarily conserved epitope of viral matrix protein, against the influenza A virus infection. It was found that the mice immunized via the intra-muscular route purely elicited cell mediated immune response to the pDNA, with enhanced level of Th1 cytokines viz. IL-12 and IFNγ production in the stimulated splenocyte supernatant. The cytotoxic T lymphocytes in the spleen of immunized mice significantly lysed the virus-infected MDCK cells. A significant decrease in virus replication was also observed in the lungs of immunized mice and 83% of the mice were protected against the lethal challenge of influenza A viruses. These findings suggest that the plasmid DNA expressing a single matrix epitope may serve as a promising vaccine candidate to provide effective immunity in the susceptible (mouse) population.