Transgenic mouse models support a protective role of type I IFN response in SARS-CoV-2 infection-related lung immunopathology and neuroinvasion.

Type I interferon (IFN-I) response is the first line of host defense against invading viruses. In the absence of definite mouse models, the role of IFN-I in SARS-CoV-2 infection remains perplexing. Here, we develop two mouse models, one with constitutively high IFN-I response (hACE2; Irgm1-/-) and the other with dampened IFN-I response (hACE2; Ifnar1-/-), to comprehend the role of IFN-I response. We report that hACE2; Irgm1-/- mice are resistant to lethal SARS-CoV-2 infection. In contrast, a severe SARS-CoV-2 infection along with immune cell infiltration, cytokine storm, and enhanced pathology is observed in the lungs and brain of hACE2; Ifnar1-/- mice. The hACE2; Irgm1-/-Ifnar1-/- double-knockout mice display loss of the protective phenotype observed in hACE2; Irgm1-/- mice, suggesting that heightened IFN-I response accounts for the observed immunity. Taking the results together, we demonstrate that IFN-I protects from lethal SARS-CoV-2 infection, and Irgm1 (IRGM) could be an excellent therapeutic target against SARS-CoV-2.

The Early Secretory Pathway Is Crucial for Multiple Aspects of the Hepatitis C Virus Life Cycle.

Although most of the early events of the hepatitis C virus (HCV) life cycle are well characterized, our understanding of HCV egress is still unclear. Some reports implicate the conventional endoplasmic reticulum (ER)-Golgi route, while some propose noncanonical secretory routes. Initially, the envelopment of HCV nucleocapsid occurs by budding into the ER lumen. Subsequently, the HCV particle exit from the ER is assumed to be mediated by coat protein complex II (COPII) vesicles. COPII vesicle biogenesis also involves the recruitment of cargo to the site of vesicle biogenesis via interaction with COPII inner coat proteins. We investigated the modulation and the specific role of the individual components of the early secretory pathway in HCV egress. We observed that HCV inhibits cellular protein secretion and triggers the reorganization of the ER exit sites and ER-Golgi intermediate compartments (ERGIC). Gene-specific knockdown of the components of this pathway such as SEC16A, TFG, ERGIC-53, and COPII coat proteins demonstrated the functional significance of these components and the distinct role played by these proteins in various aspects of the HCV life cycle. SEC16A is essential for multiple steps in the HCV life cycle, whereas TFG is specifically involved in HCV egress and ERGIC-53 is crucial for HCV entry. Overall, our study establishes that the components of the early secretory pathway are essential for HCV propagation and emphasize the importance of the ER-Golgi secretory route in this process. Surprisingly, these components are also required for the early stages of the HCV life cycle due to their role in overall intracellular trafficking and homeostasis of the cellular endomembrane system. IMPORTANCE The virus life cycle involves entry into the host, replication of the genome, assembly of infectious progeny, and their subsequent release. Different aspects of the HCV life cycle, including entry, genome replication, and assembly, are well characterized; however, our understanding of the HCV release is still not clear and subject to debate due to varied findings. Here, we attempted to address this controversy and enhance our understanding of HCV egress by evaluating the role of the different components of the early secretory pathway in the HCV life cycle. To our surprise, we found that the components of the early secretory pathway are not only essential for HCV release but also contribute to many other earlier events of the HCV life cycle. This study emphasizes the importance of the early secretory pathway for the establishment of productive HCV infection in hepatocytes.

Defective Mitochondrial Quality Control during Dengue Infection Contributes to Disease Pathogenesis.

Mitochondrial fitness is governed by mitochondrial quality control pathways comprising mitochondrial dynamics and mitochondrial-selective autophagy (mitophagy). Disruption of these processes has been implicated in many human diseases, including viral infections. Here, we report a comprehensive analysis of the effect of dengue infection on host mitochondrial homeostasis and its significance in dengue disease pathogenesis. Despite severe mitochondrial stress and injury, we observed that the pathways of mitochondrial quality control and mitochondrial biogenesis are paradoxically downregulated in dengue-infected human liver cells. This leads to the disruption of mitochondrial homeostasis and the onset of cellular injury and necrotic death in the infected cells. Interestingly, dengue promotes global autophagy but selectively disrupts mitochondrial-selective autophagy (mitophagy). Dengue downregulates the expression of PINK1 and Parkin, the two major proteins involved in tagging the damaged mitochondria for elimination through mitophagy. Mitophagy flux assays also suggest that Parkin-independent pathways of mitophagy are also inactive during dengue infection. Dengue infection also disrupts mitochondrial biogenesis by downregulating the master regulators PPARγ and PGC1α. Dengue-infected cells release mitochondrial damage-associated molecular patterns (mtDAMPs) such as mitochondrial DNA into the cytosol and extracellular milieu. Furthermore, the challenge of naive immune cells with culture supernatants from dengue-infected liver cells was sufficient to trigger proinflammatory signaling. In correlation with our in vitro observations, dengue patients have high levels of cell-free mitochondrial DNA in their blood in proportion to the degree of thrombocytopenia. Overall, our study shows how defective mitochondrial homeostasis in dengue-infected liver cells can drive dengue disease pathogenesis. IMPORTANCE Many viruses target host cell mitochondria to create a microenvironment conducive to viral dissemination. Dengue virus also exploits host cell mitochondria to facilitate its viral life cycle. Dengue infection of liver cells leads to severe mitochondrial injury and inhibition of proteins that regulate mitochondrial quality control and biogenesis, thereby disrupting mitochondrial homeostasis. A defect in mitochondrial quality control leads to the accumulation of damaged mitochondria and promotes cellular injury. This leads to the release of mitochondrial damage-associated molecular patterns (mt-DAMPs) into the cell cytoplasm and extracellular milieu. These mt-DAMPs activate the naive immune cells and trigger proinflammatory signaling, leading to the release of cytokines and chemokines, which may trigger systemic inflammation and contribute to dengue disease pathogenesis. In correlation with this, we observed high levels of cell-free mitochondrial DNA in dengue patient blood. This study provides insight into how the disruption of mitochondrial quality control in dengue-infected cells can trigger inflammation and drive dengue disease pathogenesis.

Isolation and Characterization of Five Severe Acute Respiratory Syndrome Coronavirus 2 Strains of Different Clades and Lineages Circulating in Eastern India.

The emergence of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as a serious pandemic has altered the global socioeconomic dynamics. The wide prevalence, high death counts, and rapid emergence of new variants urge for the establishment of research infrastructure to facilitate the rapid development of efficient therapeutic modalities and preventive measures. In agreement with this, SARS-CoV-2 strains were isolated from patient swab samples collected during the first COVID-19 wave in Odisha, India. The viral isolates were adapted to in vitro cultures and further characterized to identify strain-specific variations in viral growth characteristics. The neutralization susceptibility of viral isolates to vaccine-induced antibodies was determined using sera from individuals vaccinated in the Government-run vaccine drive in India. The major goal was to isolate and adapt SARS-CoV-2 viruses in cell culture with minimum modifications to facilitate research activities involved in the understanding of the molecular virology, host-virus interactions, drug discovery, and animal challenge models that eventually contribute toward the development of reliable therapeutics.

Comodulation of Dengue and Chikungunya Virus Infection During a Coinfection Scenario in Human Cell Lines.

The Dengue virus (DENV) and Chikungunya virus (CHIKV) are the arboviruses that pose a threat to global public health. Coinfection and antibody-dependent enhancement are major areas of concern during DENV and CHIKV infections, which can alter the clinical severity. Acute hepatic illness is a common manifestation and major sign of disease severity upon infection with either dengue or chikungunya. Hence, in this study, we characterized the coexistence and interaction between both the viruses in human hepatic (Huh7) cells during the coinfection/superinfection scenario. We observed that prior presence of or subsequent superinfection with DENV enhanced CHIKV replication. However, prior CHIKV infection negatively affected DENV. In comparison to monoinfection, coinfection with both DENV and CHIKV resulted in lower infectivity as compared to monoinfections with modest suppression of CHIKV but dramatic suppression of DENV replication. Subsequent investigations revealed that subneutralizing levels of DENV or CHIKV anti-sera can respectively promote the ADE of CHIKV or DENV infection in FcγRII bearing human myelogenous leukemia cell line K562. Our observations suggest that CHIKV has a fitness advantage over DENV in hepatic cells and prior DENV infection may enhance CHIKV disease severity if the patient subsequently contracts CHIKV. This study highlights the natural possibility of dengue-chikungunya coinfection and their subsequent modulation in human hepatic cells. These observations have important implications in regions where both viruses are prevalent and calls for proper management of DENV-CHIKV coinfected patients.

Role of Lipid Transfer Proteins (LTPs) in the Viral Life Cycle.

Viruses are obligate parasites that depend on the host cell machinery for their replication and dissemination. Cellular lipids play a central role in multiple stages of the viral life cycle such as entry, replication, morphogenesis, and egress. Most viruses reorganize the host cell membranes for the establishment of viral replication complex. These specialized structures allow the segregation of replicating viral RNA from ribosomes and protect it from host nucleases. They also facilitate localized enrichment of cellular components required for viral replication and assembly. The specific composition of the lipid membrane governs its ability to form negative or positive curvature and possess a rigid or flexible form, which is crucial for membrane rearrangement and establishment of viral replication complexes. In this review, we highlight how different viruses manipulate host lipid transfer proteins and harness their functions to enrich different membrane compartments with specific lipids in order to facilitate multiple aspects of the viral life cycle.

Quantitative proteomics of hamster lung tissues infected with SARS-CoV-2 reveal host factors having implication in the disease pathogenesis and severity.

Syrian golden hamsters (Mesocricetus auratus) infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests lung pathology. In this study, efforts were made to check the infectivity of a local SARS-CoV-2 isolate in a self-limiting and non-lethal hamster model and evaluate the differential expression of lung proteins during acute infection and convalescence. The findings of this study confirm the infectivity of this isolate in vivo. Analysis of clinical parameters and tissue samples show the pathophysiological manifestation of SARS-CoV-2 infection similar to that reported earlier in COVID-19 patients and hamsters infected with other isolates. However, diffuse alveolar damage (DAD), a common histopathological feature of human COVID-19 was only occasionally noticed. The lung-associated pathological changes were very prominent on the 4th day post-infection (dpi), mostly resolved by 14 dpi. Here, we carried out the quantitative proteomic analysis of the lung tissues from SARS-CoV-2-infected hamsters on day 4 and day 14 post-infection. This resulted in the identification of 1585 proteins of which 68 proteins were significantly altered between both the infected groups. Pathway analysis revealed complement and coagulation cascade, platelet activation, ferroptosis, and focal adhesion as the top enriched pathways. In addition, we also identified altered expression of two pulmonary surfactant-associated proteins (Sftpd and Sftpb), known for their protective role in lung function. Together, these findings will aid in understanding the mechanism(s) involved in SARS-CoV-2 pathogenesis and progression of the disease.

Identification of multipotent drugs for COVID-19 therapeutics with the evaluation of their SARS-CoV2 inhibitory activity.

The SARS-CoV2 is a highly contagious pathogen that causes COVID-19 disease. It has affected millions of people globally with an average lethality of ~3%. There is an urgent need of drugs for the treatment of COVID-19. In the current studies, we have used bioinformatics techniques to screen the FDA approved drugs against nine SARS-CoV2 proteins to identify drugs for repurposing. Additionally, we analyzed if the identified molecules can also affect the human proteins whose expression in lung changed during SARS-CoV2 infection. Targeting such genes may also be a beneficial strategy to curb disease manifestation. We have identified 74 molecules that can bind to various SARS-CoV2 and human host proteins. We experimentally validated our in-silico predictions using vero E6 cells infected with SARS-CoV2 virus. Interestingly, many of our predicted molecules viz. capreomycin, celecoxib, mefloquine, montelukast, and nebivolol showed good activity (IC50) against SARS-CoV2. We hope that these studies may help in the development of new therapeutic options for the treatment of COVID-19.

Analysis of Indian SARS-CoV-2 Genomes Reveals Prevalence of D614G Mutation in Spike Protein Predicting an Increase in Interaction With TMPRSS2 and Virus Infectivity.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has emerged as a global pandemic worldwide. In this study, we used ARTIC primers-based amplicon sequencing to profile 225 SARS-CoV-2 genomes from India. Phylogenetic analysis of 202 high-quality assemblies identified the presence of all the five reported clades 19A, 19B, 20A, 20B, and 20C in the population. The analyses revealed Europe and Southeast Asia as two major routes for introduction of the disease in India followed by local transmission. Interestingly, the19B clade was found to be more prevalent in our sequenced genomes (17%) compared to other genomes reported so far from India. Haplotype network analysis showed evolution of 19A and 19B clades in parallel from predominantly Gujarat state in India, suggesting it to be one of the major routes of disease transmission in India during the months of March and April, whereas 20B and 20C appeared to evolve from 20A. At the same time, 20A and 20B clades depicted prevalence of four common mutations 241 C > T in 5' UTR, P4715L, F942F along with D614G in the Spike protein. D614G mutation has been reported to increase virus shedding and infectivity. Our molecular modeling and docking analysis identified that D614G mutation resulted in enhanced affinity of Spike S1-S2 hinge region with TMPRSS2 protease, possibly the reason for increased shedding of S1 domain in G614 as compared to D614. Moreover, we also observed an increased concordance of G614 mutation with the viral load, as evident from decreased Ct value of Spike and the ORF1ab gene.

Emergence of novel reassortant H6N2 avian influenza viruses in ducks in India.

The recent reports of human infection due to H6 subtype avian influenza viruses (AIV), which are prevalent in terrestrial poultry, indicate evolution of the virus to a possible pandemic strain. Here, we report antigenic and genetic characterization of two H6N2 viruses isolated from apparently healthy domestic ducks in Kerala and Assam, India during 2014 and 2015, respectively. Hemagglutination inhibition assay revealed antigenic divergence between the two isolates, which was corroborated by amino acid differences at 55 positions (15.98%) between their hemagglutinin (HA) 1.The sequence analyses indicated that both the viruses are avian origin with avian receptor specificity, low pathogenic to poultry and sensitive to oseltamivir. However, Kerala14 had V27I mutation marker for amantadine resistance in M2. The Assam15 virus had an additional N-linked glycosylation on HA2 (position 557) compared to Kerala14 virus. Phylogenetic analysis of the HA gene revealed that both the viruses belonged to distinct lineages (Eurasian and Asia II). Phylogeny of neuraminidase and internal gene segments revealed that both the viruses are novel reassortants and are genetically distinct with different gene constellations. The results suggest independent introductions of the two H6N2 viruses into India and migratory wild birds in the Central Asian flyway might be the source of H6N2 viruses in ducks in India. Therefore, continued AIV surveillance in poultry and wild birds is essential for early detection of emergence of novel strains with pandemic potential and control of their spread.

Multiple introductions of a reassortant H5N1 avian influenza virus of clade 2.3.2.1c with PB2 gene of H9N2 subtype into Indian poultry.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are a threat to poultry in Asia, Europe, Africa and North America. Here, we report isolation and characterization of H5N1 viruses isolated from ducks and turkeys in Kerala, Chandigarh and Uttar Pradesh, India between November 2014 and March 2015. Genetic and phylogenetic analyses of haemagglutinin gene identified that the virus belonged to a new clade 2.3.2.1c which has not been detected earlier in Indian poultry. The virus possessed molecular signature for high pathogenicity to chickens, which was corroborated by intravenous pathogenicity index of 2.96. The virus was a reassortant which derives its PB2 gene from H9N2 virus isolated in China during 2007-2013. However, the neuraminidase and internal genes are of H5N1 subtype. Phylogenetic and network analysis revealed that after detection in China in 2013/2014, the virus moved to Europe, West Africa and other Asian countries including India. The analyses further indicated multiple introductions of H5N1 virus in Indian poultry and internal spread in Kerala. One of the outbreaks in ducks in Kerala is linked to the H5N1 virus isolated from wild birds in Dubai suggesting movement of virus probably through migration of wild birds. However, the outbreaks in ducks in Chandigarh and Uttar Pradesh were from an unknown source in Asia which also contributed gene pools to the outbreaks in Europe and West Africa. The widespread incidence of the novel H5N1 HPAI is similar to the spread of clade 2.2 ("Qinghai-like") virus in 2005, and should be monitored to avoid threat to animal and public health.

Efficacy of chitooligosaccharides for the management of diabetes in alloxan induced mice: a correlative study with antihyperlipidemic and antioxidative activity.

The present study evaluates the effects of chitooligosaccharides (COS) for the management of alloxan induced diabetes in mice. For the management of the carbohydrate metabolism in diabetes by the COS, the amount of glucose in blood along with quantification of glycogen in liver were measured and noted a significant recovery in respect to diabetic control group. As hyperlipidemia and oxidative stress are the disorders of diabetes so, we have also assessed the serum levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDLc), very low density lipoprotein cholesterol (VLDLc) and high density lipoprotein cholesterol (HDLc). For the recovery of oxidative stress the SOD MDA catalase in liver and GOT and GPT activities in serum were measured. The COS results a significant recovery in the levels of above mentioned biosensors of lipid profile when treated to experimentally induce diabetic mice. The effect of COS at the dose of 10mg/kg body weight was found to be a potent agent for diabetes and complication associated with this disease. The COS has no toxic effect in general which has been focused here by the monitoring of COS dose in normal healthy mice. The results of this study enlighted that the COS has antidiabetic, antihyperlipidemic and antioxidative activities.