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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.

SummaryBackgroundThe current global pandemic of Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 led to the investigation with clinical, biochemical, immunological and genomic characterization from the patients to understand the pathophysiology of viral infection.MethodsSamples were collected from six asymptomatic and six symptomatic SARS-CoV-2 confirmed hospitalized patients in Bhubaneswar, Odisha, India. Clinical details, biochemical parameters, treatment regime were collected from hospital, viral load was determined by RT-PCR, levels of cytokines and circulating antibodies in plasma were assessed by Bioplex and isotyping respectively. In addition, the whole genome sequencing of viral strains and mutational analysis were carried out.FindingsAnalysis of the biochemical parameters highlighted the increased levels of C-Reactive protein (CRP), lactate dehydrogenase (LDH), serum SGPT, serum SGOT and ferritin in symptomatic patients indicating that patients with higher levels of few biochemical parameters might experience severe pathophysiological complications after SARS-CoV-2 infection. This was also observed that symptomatic patients were mostly with one or more comorbidities, especially diabetes (66.6%). Surprisingly the virological estimation revealed that there was no significant difference in viral load of oropharyngeal (OP) samples between the two groups. This suggests that the viral load in OP sample does not correlate with the disease severity and both asymptomatic and symptomatic patients are equally capable of transmitting the virus. Whereas, viral load was higher in plasma and serum samples of symptomatic patients suggesting that the development of clinical complications is mostly associated to high viral load in plasma and serum. This also demonstrated that the patients with high viral load in plasma and serum samples were found to develop sufficient amounts of antibodies (IgG, IgM and IgA). Interestingly, the levels of 7 cytokines (IL-6, IL-.1α, IP-10, IL-8, IL-10, IFN-α2, IL-15) were found to be highly elevated in symptomatic patients, while three cytokines (soluble CD40L, GRO and MDC) were remarkably higher in asymptomatic patients. Therefore, this data suggest that cytokines and chemokines may serve as “predictive indicator” of SARS-CoV-2 infection and contribute to understand the pathogenesis of COVID-19. The whole genome sequence analysis revealed that the current isolates were clustered with 19B, 20A and 20B clades, however acquired 11 additional changes in Orf1ab, spike, Orf3a, Orf8 and nucleocapsid proteins. The data also confirmed that the D614G mutation in spike protein is mostly linked with higher virus replication efficiency and severe SARS-CoV-2 infection as three patients had higher viral load and among them two patients with this mutation passed away.InterpretationThis is the first comprehensive study of SARS CoV-2 patients from India. This will contribute to a better understanding of the pathophysiology of SARS-CoV-2 infection and advance in the implementation of effective disease control strategies.FundingThis study was supported by the core funding of Institute of Life Sciences, Bhubaneswar, Dept of Biotechnology, India.Research in contextEvidence before this studyAsymptomatic patients are a source of concern as measures taken to control the spread of the virus are severely impacted by their undetectability. Presently, there is an inadequate information about the characteristics of the asymptomatic and symptomatic patients. The association between SARS-CoV-2 viral load, cytokines and risk of disease progression remains unclear in COVID-19 in Indian scenario. PubMed was searched for articles published up to May, 2021, using the keywords “SARS CoV-2 patients in India”, or “2019 novel coronavirus patients in India”. No published work about the patient’s data on SARS CoV-2 in Indian scenario could be identified.Added value of this studyThis investigation highlights the ability of both asymptomatic and symptomatic patients to transmit the virus equally. This study also demonstrates that the D614G mutation in the spike protein is associated with severe SARS-CoV-2 infection and enhance levels of inflammatory markers such as CRP and ferritin which can be predictive biomarkers for critical condition of patients. This is the first comprehensive study of SARS CoV-2 patients from India and will contribute to a better understanding of the pathophysiology of SARS-CoV-2 infection by advancing the implementation of effective disease control strategies.Implications of all the available evidenceThe current global pandemic of Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 led to the investigation with clinical, biochemical, immunological and viral genome sequencing to understand the pathophysiology of this virus infection. Samples were collected from six asymptomatic and six symptomatic SARS-CoV-2 confirmed hospitalized patients in Bhubaneswar, Odisha, India. This investigation highlights the ability of both asymptomatic and symptomatic patients to transmit the virus equally. This also demonstrated that the D614G mutation is mostly associated with higher virus replication capacity and severe SARS-CoV-2 infection and enhanced levels of inflammatory markers such as CRP and ferritin which are associated with critical conditions of patients. This is the first comprehensive study of SARS CoV-2 patients from India and will contribute to a better understanding of the pathophysiology of SARS-CoV-2 infection by advancing the implementation of competent disease control strategies.

Syrian golden hamsters (Mesocricetus auratus) infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) manifests lung pathology that resembles human COVID-19 patients. In this study, efforts were made to check the infectivity of a local SARS-CoV-2 isolate in 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 shows a similar type of pathophysiological manifestation of SARS-CoV-2 infection as reported earlier in COVID-19 patients and hamsters infected with other isolates. The lung-associated pathological changes were very prominent on the 4th day post-infection (dpi), mostly resolved by 14dpi. Here, we carried out quantitative proteomic analysis of the lung tissues from SARS-CoV-2-infected hamsters at day 4 and day 14 post infection. This resulted in the identification of 1,585 differentially expressed proteins of which 68 proteins were significantly altered among 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 the identification of candidate biomarkers and understanding the mechanism(s) involved in SARS-CoV-2 pathogenesis.Graphical abstract

Background: The current global pandemic of Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 led to the investigation with clinical, biochemical, immunological and genomic characterization from the patients to understand the pathophysiology of viral infection. Methods: Samples were collected from six asymptomatic and six symptomatic SARS-CoV-2 confirmed hospitalized patients in Bhubaneswar, Odisha, India. Clinical details, biochemical parameters, treatment regime were collected from hospital, viral load was determined by RT-PCR, levels of cytokines and circulating antibodies in plasma were assessed by Bioplex and isotyping respectively. In addition, the whole genome sequencing of viral strains and mutational analysis were carried out. Findings Analysis of the biochemical parameters highlighted the increased levels of C-Reactive protein (CRP), lactate dehydrogenase (LDH), serum SGPT, serum SGOT and ferritin in symptomatic patients. Symptomatic patients were mostly with one or more comorbidities, especially diabetes (66.6%). The virological estimation revealed that there was no significant difference in viral load of oropharyngeal (OP) samples between the two groups. Whereas, viral load was higher in plasma and serum samples of symptomatic patients and they develop sufficient amounts of antibodies (IgG, IgM and IgA). The levels of 7 cytokines (IL-6, IL-.1α, IP-10, IL-8, IL-10, IFN-α2, IL-15) were found to be highly elevated in symptomatic patients, while three cytokines (soluble CD40L, GRO and MDC) were remarkably higher in asymptomatic patients. The whole genome sequence analysis revealed that the current isolates were clustered with 19B, 20A and 20B clades, however acquired 11 additional changes in Orf1ab, spike, Orf3a, Orf8 and nucleocapsid proteins. The D614G mutation in spike protein is linked with higher virus replication efficiency and severe SARS-CoV-2 infection as three patients had higher viral load and among them two patients with this mutation passed away. Interpretation This is the first comprehensive study of SARS CoV-2 patients from India. This will contribute to a better understanding of the pathophysiology of SARS-CoV-2 infection and advance in the implementation of effective disease control strategies. Funding Information: This study was supported by the core funding of Institute of Life Sciences, Bhubaneswar, Dept of Biotechnology, India. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The studies involving human participants were reviewed and approved by the Institutional Human Ethics Committee, Institute of Life Sciences. Written consent informed to participate in this study was provided by the participants’ legal guardian/next of kin.

Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48 h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly.

Hepatitis B virus (HBV) suppresses innate immune signaling to establish persistent infection. Although HBV is a DNA virus, its pre-genomic RNA (pgRNA) can be sensed by RIG-I and activates MAVS to mediate interferon (IFN) λ synthesis. Despite of the activation of RIG-I-MAVS axis by pgRNA, the underlying mechanism explaining how HBV infection fails to induce interferon-αβ (IFN) synthesis remained uncharacterized. We demonstrate that HBV induced parkin is able to recruit the linear ubiquitin assembly complex (LUBAC) to mitochondria and abrogates IFN β synthesis. Parkin interacts with MAVS, accumulates unanchored linear polyubiquitin chains on MAVS via LUBAC, to disrupt MAVS signalosome and attenuate IRF3 activation. This study highlights the novel role of parkin in antiviral signaling which involves LUBAC being recruited to the mitochondria. These results provide avenues of investigations on the role of mitochondrial dynamics in innate immunity., Author Summary Hepatitis B virus (HBV) chronic infection is one of the major causes of hepatocellular carcinoma. HBV infection is associated with mitochondrial dysfunction. We previously showed that persistent infection of HBV requires rapid clearance of impaired mitochondria by mitophagy, a cellular quality control process that insures survival of HBV infected cells. During the process, Parkin, an RBR E3 ligase, is recruited to mitochondria to induce mitophagy. In this study, we show that the Parkin, plays a critical role in the modulation of innate immune signaling. Using HBV expressing cells, we show that the Parkin recruits linear ubiquitin assembly complex (LUBAC) to the mitochondria and subsequently inhibits downstream signaling of mitochondrial antiviral signaling protein (MAVS). Mitochondrial LUBAC then catalyzes linear ubiquitin chains on MAVS, which abrogates its downstream events such as MAVS-TRAFs interaction and abolishes IRF3 phosphorylation. The results of this study highlight the molecular details explaining how HBV can suppress interferon synthesis implicating a mitophagy-independent role of Parkin. HBV-induced mitochondrial damage serves as the platform for recruitment of Parkin and LUBAC, which together modify MAVS by ubiquitination and cripples its downstream signaling.

Hepatitis C virus (HCV) modulates cellular lipid metabolism to enhance its replication. HCV circulates in the blood in association with lipoproteins. HCV infection is associated with enhanced lipogenesis, reduced secretion and β-oxidation of lipids. HCV-induced imbalance in lipid homeostasis leads to steatosis. Many lipids are crucial for viral life cycle, and inhibitors of cholesterol/fatty acid biosynthetic pathways inhibit viral replication, maturation and secretion. HCV negatively modulates the synthesis and secretion of very low-density lipoproteins (VLDL). The components involved in VLDL assembly are also required for HCV morphogenesis/secretion, suggesting that HCV coopts the VLDL secretory pathway for its own secretion. This review highlights HCV-altered lipid metabolic events that aid in the viral life cycle and ultimately promote liver disease pathogenesis.

SummaryThe role of inflammation in obesity-related pathologies is well established. We investigated the therapeutic potential of LipoxinA4 (LXA4:5(S),6(R),15(S)-trihydroxy-7E,9E,11Z,13E,-eicosatetraenoic acid) and a synthetic 15(R)-Benzo-LXA4-analog as interventions in a 3-month high-fat diet (HFD; 60% fat)-induced obesity model. Obesity caused distinct pathologies, including impaired glucose tolerance, adipose inflammation, fatty liver, and chronic kidney disease (CKD). Lipoxins (LXs) attenuated obesity-induced CKD, reducing glomerular expansion, mesangial matrix, and urinary H2O2. Furthermore, LXA4 reduced liver weight, serum alanine-aminotransferase, and hepatic triglycerides. LXA4 decreased obesity-induced adipose inflammation, attenuating TNF-α and CD11c+ M1-macrophages (MΦs), while restoring CD206+ M2-MΦs and increasing Annexin-A1. LXs did not affect renal or hepatic MΦs, suggesting protection occurred via attenuation of adipose inflammation. LXs restored adipose expression of autophagy markers LC3-II and p62. LX-mediated protection was demonstrable in adiponectin−/− mice, suggesting that the mechanism was adiponectin independent. In conclusion, LXs protect against obesity-induced systemic disease, and these data support a novel therapeutic paradigm for treating obesity and associated pathologies.

Human hepatitis B and C virus (HBV and HCV) infections are the leading cause of chronic liver disease. Mitochondrial liver injury is associated with HBV and HCV infections. The objective of our stu...

Hepatitis C virus (HCV) relies on host lipid metabolic pathways for its replication, assembly, secretion, and entry. HCV induces de novo lipogenesis, inhibits β-oxidation, and lipoprotein export resulting in a lipid-enriched cellular environment critical for its proliferation. We investigated the effects of a hypolipidemic agent, nordihydroguaiaretic acid (NDGA), on host lipid/fatty acid synthesis and HCV life cycle. NDGA negated the HCV-induced alteration of host lipid homeostasis. NDGA decreased sterol regulatory element binding protein (SREBP) activation and enhanced expression of genes involved in β-oxidation. NDGA inhibited very low-density lipoprotein (VLDL) secretion by affecting mediators of VLDL biosynthesis. Lipid droplets (LDs), the neutral lipid storage organelles, play a key role in HCV morphogenesis. HCV induces accumulation and perinuclear distribution of LDs, whereas NDGA most notably reduced the overall number and increased the average size of LDs. The antiviral effects of NDGA resulted in reduced HCV replication and secretion. Conclusion: NDGA-mediated alterations of host lipid metabolism, LD morphology, and VLDL transport appear to negatively influence HCV proliferation. (HEPATOLOGY 2011;)

Hepatitis C Virus (HCV) induces intracellular events that trigger mitochondrial dysfunction and promote host metabolic alterations. Here, we investigated selective autophagic degradation of mitochondria (mitophagy) in HCV-infected cells. HCV infection stimulated Parkin and PINK1 gene expression, induced perinuclear clustering of mitochondria, and promoted mitochondrial translocation of Parkin, an initial event in mitophagy. Liver tissues from chronic HCV patients also exhibited notable levels of Parkin induction. Using multiple strategies involving confocal and electron microscopy, we demonstrated that HCV-infected cells display greater number of mitophagosomes and mitophagolysosomes compared to uninfected cells. HCV-induced mitophagy was evidenced by the colocalization of LC3 puncta with Parkin-associated mitochondria and lysosomes. Ultrastructural analysis by electron microscopy and immunoelectron microscopy also displayed engulfment of damaged mitochondria in double membrane vesicles in HCV-infected cells. The HCV-induced mitophagy occurred irrespective of genotypic differences. Silencing Parkin and PINK1 hindered HCV replication suggesting the functional relevance of mitophagy in HCV propagation. HCV-mediated decline of mitochondrial complex I enzyme activity was rescued by chemical inhibition of mitophagy or by Parkin silencing. Overall our results suggest that HCV induces Parkin-dependent mitophagy, which may have significant contribution in mitochondrial liver injury associated with chronic hepatitis C., Author Summary Hepatitis C virus (HCV) infection alters host lipid metabolism. HCV-induced mitochondrial dysfunction may promote the metabolic alterations by affecting mitochondrial β-oxidation and oxidative phosphorylation. Dysfunctional mitochondria are detrimental to cell survival and require rapid clearance to sustain cell viability. Here, we investigated the effect of HCV gene expression in promoting selective autophagy of dysfunctional mitochondria, also termed mitophagy. HCV infection stimulated the gene expression of Parkin and PINK1, the two key mediators of mitophagy. Parkin stimulation was also observed in liver biopsies of chronic hepatitis C patients. HCV infection induced the perinuclear clustering of mitochondria and triggered Parkin translocation to mitochondria, a hallmark of mitophagy. Concomitant with the mitochondrial translocation of Parkin, we observed ubiquitination of Parkin and its substrates in HCV-infected cells. We also demonstrate the formation of mitophagosomes and their subsequent delivery to lysosomes in HCV-infected cells. Silencing both Parkin and PINK1 hindered HCV replication, suggesting the functional significance of mitophagy in HCV life cycle. Furthermore, we demonstrate that Parkin-dependent mitophagy is directly associated with HCV-mediated decline in oxidative phosphorylation. Our results implicate the functional significance of Parkin and mitophagy in the persistence of HCV infection and mitochondrial injury commonly seen in patients with chronic hepatitis C.

Aims/hypothesis: In this study, we aimed to evaluate the therapeutic potential of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-activated protein kinase, for ameliorating high-fat diet (HFD)-induced pathophysiology in mice. We also aimed to determine whether the beneficial effects of AICAR were dependent on adiponectin. Furthermore, human adipose tissue was used to examine the effect of AICAR ex vivo. Methods: Six-week-old male C57BL/6J wild-type and Adipoq −/− mice were fed a standard-fat diet (10% fat) or an HFD (60% fat) for 12 weeks and given vehicle or AICAR (500 μg/g) three times/week from weeks 4–12. Diet-induced pathophysiology was examined in mice after 11 weeks by IPGTT and after 12 weeks by flow cytometry and western blotting. Human adipose tissue biopsies from obese (BMI 35–50 kg/m2) individuals were incubated with vehicle or AICAR (1 mmol/l) for 6 h at 37°C, after which inflammation was characterised by ELISA (TNF-α) and flow cytometry. Results: AICAR attenuated adipose inflammation in mice fed an HFD, promoting an M1-to-M2 macrophage phenotype switch, while reducing infiltration of CD8+ T cells. AICAR treatment of mice fed an HFD partially restored glucose tolerance and attenuated hepatic steatosis and kidney disease, as evidenced by reduced albuminuria (p