Influence of COVID-19 over seasonal influenza activity in southern India.

Basic epidemiological data is urgently needed in order to ascertain the changes brought about by COVID-19 pandemic, and help researchers, clinicians, and policy makers in addressing these issues. Data on influenza positivity from 2009 to 2019 was collected from Regional Influenza laboratory, JIPMER. Being COVID testing centre we tested samples (2020-2023) from Tamilnadu and Pondicherry. All SARI samples which were negative for COVID-19 were subjected to Influenza testing as regular surveillance activity was suspended. On comparison we found significant reduction in detection rates of influenza A H1N1, H3N2 and influenza B (p value = <0.01).

Defining the key intrahepatic gene networks in HCV infection driven by sex.

OBJECTIVE: The transcriptional response in the liver during HCV infection is critical for determining clinical outcomes. This issue remains relatively unexplored as tissue access to address this at scale is usually limited. We aimed to profile the transcriptomics of HCV-infected livers to describe the expression networks involved and assess the effect on them of major predictors of clinical outcome such as IFNL4 (interferon lambda 4) host genotype and sex. DESIGN: We took advantage of a large clinical study of HCV therapy accompanied by baseline liver biopsy to examine the drivers of transcription in tissue samples in 195 patients also genotyped genome-wide for host and viral single nucleotide polymorphisms. We addressed the role of host factors (disease status, sex, genotype, age) and viral factors (load, mutation) on transcriptional responses. RESULTS: We observe key modules of transcription which can be impacted differentially by host and viral factors. Underlying cirrhotic state had the most substantial impact, even in a stable, compensated population. Notably, sex had a major impact on antiviral responses in concert with IL28B (interleukin 28B)/IFNL4 genotype, with stronger interferon and humoral responses in females. Males tended towards a dominant cellular immune response. In both sexes, there was a strong influence of the underlying host disease status and of specific viral mutations, and sex-specific expression quantitative trait loci were also observed. CONCLUSION: These features help define the major influences on tissue responses in HCV infection, impacting on the response to treatment and with broader implications for responses in other sex-biased infections.

Molecular characterization of porcine circovirus 2 circulating in Assam and Arunachal Pradesh of India.

PCV2 is the primary etiological agent of porcine circovirus-associated diseases (PCVADs) which affect pigs worldwide. Currently, there is a worldwide genotype prevalence switch from PCV2b to PCV2d, which has led to increased virulence of the circulating virus strains leading to vaccine failures and selection pressure. In the present study, the PCV2 genotypes circulating in north eastern region (NER) of India particularly the states of Assam and Arunachal Pradesh was characterized by isolation, sequencing and phylogenetic analysis of cap gene. The phylogenetic analysis revealed that the PCV2 isolates circulating in pigs of Assam and Arunachal Pradesh were mostly of PCV2d genotype. Hence, it can be concluded that PCV2d genotype is the most dominating genotype in NER and priority should be given to this genotype for development of future vaccine candidate against PCV2 in India.

Newcastle Disease Virus Vectored Chicken Infectious Anaemia Vaccine Induces Robust Immune Response in Chickens.

Newcastle disease virus (NDV) strain R2B, with an altered fusion protein cleavage site, was used as a viral vector to deliver the immunogenic genes VP2 and VP1 of chicken infectious anaemia virus (CIAV) to generate a bivalent vaccine candidate against these diseases in chickens. The immunogenic genes of CIAV were expressed as a single transcriptional unit from the NDV backbone and the two CIA viral proteins were obtained as separate entities using a self-cleaving foot-and-mouth disease virus 2A protease sequence between them. The recombinant virus (rR2B-FPCS-CAV) had similar growth kinetics as that of the parent recombinant virus (rR2B-FPCS) in vitro with similar pathogenicity characteristics. The bivalent vaccine candidate when given in specific pathogen-free chickens as primary and booster doses was able to elicit robust humoral and cell-mediated immune (CMI) responses obtained in a vaccination study that was conducted over a period of 15 weeks. In an NDV and CIAV ELISA trial, there was a significant difference in the titres of antibody between vaccinated and control groups which showed slight reduction in antibody titre by 56 days of age. Hence, a second booster was administered and the antibody titres were maintained until 84 days of age. Similar trends were noticed in CMI response carried out by lymphocyte transformation test, CD4+ and CD8+ response by flow cytometry analysis and response of real time PCR analysis of cytokine genes. Birds were challenged with virulent NDV and CIAV at 84 days and there was significant reduction in the NDV shed on the 2nd and 4th days post challenge in vaccinated birds as compared to unvaccinated controls. Haematological parameters comprising PCV, TLC, PLC and PHC were estimated in birds that were challenged with CIAV that indicated a significant reduction in the blood parameters of controls. Our findings support the development and assessment of a bivalent vaccine candidate against NDV and CIAV in chickens.

Evaluation of the oncolytic property of recombinant Newcastle disease virus strain R2B in 4T1 and B16-F10 cells in-vitro.

Recombinant Newcastle disease virus vectors have gained a lot of interest for its oncolytic virus therapy and cancer immune therapeutic properties due to its selective replication to high titers in cancer cells. The aim of this study was to find out the oncolytic effects of mesogenic recombinant NDV strain R2B-GFP on murine mammary tumor cell line 4T1 and murine melanoma cell line B16-F10. The anti-tumor effects of R2B-GFP virus were studied via expression of virus transgene GFP in cancer cells, evaluating its cytotoxicity and cell migration efficacies by MTT and wound healing assays respectively. In addition, the underlying apoptotic mechanism of R2B-GFP virus was estimated by TUNEL assay, colorimetric estimation of Caspase-3, 8 and 9 and the estimation of Bax to Bcl-2 ratio. The results showed a significant decrease in viability of both 4T1 and B16-F10 cells infected with R2B-GFP virus at 0.1 and 1 MOI. R2B-GFP virus could significantly induce apoptosis in the 4T1 and B16-F10 cells as compared to the uninfected control. Further, a flow cytometry analysis on apoptotic cells percentage and mitochondria membrane permeability test was also studied in R2B-GFP virus treated 4T1 and B16-F10 cell lines. The R2B-GFP virus caused an increase in loss of mitochondrial membrane permeability in both 4T1 and B16-F10 cells indicating the involvement of mitochondrial regulated cell death. Thus, the recombinant virus R2B-GFP virus proved to be a valid candidate for oncolytic viral therapy in 4T1 and B16-F10 cells.

T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses.

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.

Newcastle disease virus vectored rabies vaccine induces strong humoral and cell mediated immune responses in mice.

Rabies is a devastating disease affecting almost all mammalian animal species including humans. Vaccines are available to combat the disease. Protection against the disease is rendered by assessing the humoral immune response. Recent reports suggest the role of cell mediated immune response (CMI) in assessing vaccine efficacy. In the present study, two live vectored vaccine candidates containing glycoprotein G of rabies virus were generated using the mesogenic Newcastle disease virus (NDV) strain R2B and another with NDV with an altered fusion protein cleavage site as backbones. The efficacy of these vaccine candidates on testing in experimental mouse model indicated generation of robust humoral and CMI responses. The recombinant NDV containing the altered fusion protein cleavage site with glycoprotein G showed the highest CMI response in mice indicating its usage as a potential live vectored vaccine candidate against the disease.

Detection of neutralising antibodies to SARS-CoV-2 to determine population exposure in Scottish blood donors between March and May 2020.

BackgroundThe progression and geographical distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the United Kingdom (UK) and elsewhere is unknown because typically only symptomatic individuals are diagnosed. We performed a serological study of blood donors in Scotland in the spring of 2020 to detect neutralising antibodies to SARS-CoV-2 as a marker of past infection and epidemic progression.AimOur objective was to determine if sera from blood bank donors can be used to track the emergence and progression of the SARS-CoV-2 epidemic.MethodsA pseudotyped SARS-CoV-2 virus microneutralisation assay was used to detect neutralising antibodies to SARS-CoV-2. The study comprised samples from 3,500 blood donors collected in Scotland between 17 March and 18 May 2020. Controls were collected from 100 donors in Scotland during 2019.ResultsAll samples collected on 17 March 2020 (n = 500) were negative in the pseudotyped SARS-CoV-2 virus microneutralisation assay. Neutralising antibodies were detected in six of 500 donors from 23 to 26 March. The number of samples containing neutralising antibodies did not significantly rise after 5-6 April until the end of the study on 18 May. We found that infections were concentrated in certain postcodes, indicating that outbreaks of infection were extremely localised. In contrast, other areas remained comparatively untouched by the epidemic.ConclusionAlthough blood donors are not representative of the overall population, we demonstrated that serosurveys of blood banks can serve as a useful tool for tracking the emergence and progression of an epidemic such as the SARS-CoV-2 outbreak.

Systemic iron reduction by venesection alters the gut microbiome in patients with haemochromatosis.

BACKGROUND & AIMS: Iron reduction by venesection has been the cornerstone of treatment for haemochromatosis for decades, and its reported health benefits are many. Repeated phlebotomy can lead to a compensatory increase in intestinal iron absorption, reducing intestinal iron availability. Given that most gut bacteria are highly dependent on iron for survival, we postulated that, by reducing gut iron levels, venesection could alter the gut microbiota. METHODS: Clinical parameters, faecal bacterial composition and metabolomes were assessed before and during treatment in a group of patients with haemochromatosis undergoing iron reduction therapy. RESULTS: Systemic iron reduction was associated with an alteration of the gut microbiome, with changes evident in those who experienced reduced faecal iron availability with venesection. For example, levels of Faecalibacterium prausnitzii, a bacterium associated with improved colonic health, were increased in response to faecal iron reduction. Similarly, metabolomic changes were seen in association with reduced faecal iron levels. CONCLUSION: These findings highlight a significant shift in the gut microbiome of patients who experience reduced colonic iron during venesection. Targeted depletion of faecal iron could represent a novel therapy for metabolic and inflammatory diseases, meriting further investigation. LAY SUMMARY: Iron depletion by repeated venesection is the mainstay of treatment for haemochromatosis, an iron-overload disorder. Venesection has been associated with several health benefits, including improvements in liver function tests, reversal of liver scarring, and reduced risk of liver cancer. During iron depletion, iron absorption from the gastrointestinal (GI) tract increases to compensate for iron lost with treatment. Iron availability is limited in the GI tract and is crucial to the growth and function of many gut bacteria. In this study we show that reduced iron availability in the colon following venesection treatment leads to a change in the composition of the gut bacteria, a finding that, to date, has not been studied in patients with haemochromatosis.

Infectious bursal disease virus in chickens: prevalence, impact, and management strategies.

Infectious bursal disease (IBD), also known as Gumboro disease, is a highly contagious, immunosuppressive disease of young chickens. Although first observed about 60 years ago, to date, the disease is responsible for major economic losses in the poultry industry worldwide. IBD virus (IBDV), a double-stranded RNA virus, exists as two serotypes with only serotype 1 causing the disease in young chickens. The virus infects the bursa of Fabricius of particularly the actively dividing and differentiating lymphocytes of the B-cells lineage of immature chickens, resulting in morbidity, mortality, and immunosuppression. Immunosuppression enhances the susceptibility of chickens to other infections and interferes with vaccination against other diseases. Immunization is the most important measure to control IBD; however, rampant usage of live vaccines has resulted in the evolution of new strains. Although the immunosuppression caused by IBDV is more directed toward the B lymphocytes, the protective immunity in birds depends on inducement of both humoral and cell-mediated immune responses. The interference with the inactivated vaccine induced maternally derived antibodies in young chicks has become a hurdle in controlling the disease, thus necessitating the development of newer vaccines with improved efficacy. The present review illustrates the overall dynamics of the virus and the disease, and the recent developments in the field of virus diagnosis and vaccine research.

Recombinant Newcastle Disease Virus (NDV) Expressing Sigma C Protein of Avian Reovirus (ARV) Protects against Both ARV and NDV in Chickens.

Newcastle disease (ND) and avian reovirus (ARV) infections are a serious threat to the poultry industry, which causes heavy economic losses. The mesogenic NDV strain R2B is commonly used as a booster vaccine in many Asian countries to control the disease. In this seminal work, a recombinant NDV strain R2B expressing the sigma C (σC) gene of ARV (rNDV-R2B-σC) was generated by reverse genetics, characterized in vitro and tested as a bivalent vaccine candidate in chickens. The recombinant rNDV-R2B-σC virus was attenuated as compared to the parent rNDV-R2B virus as revealed by standard pathogenicity assays. The generated vaccine candidate, rNDV-R2B-σC, could induce both humoral and cell mediated immune responses in birds and gave complete protection against virulent NDV and ARV challenges. Post-challenge virus shedding analysis revealed a drastic reduction in NDV shed, as compared to unvaccinated birds.

Evaluation of a fusion gene-based DNA prime-protein boost vaccination strategy against Newcastle disease virus.

The low potency of genetic immunization has to date impeded development of commercial vaccines against major infectious diseases. The aim of this study was to develop and evaluate a fusion gene-based DNA prime-protein boost vaccination strategy to improve the efficacy of both DNA and subunit vaccines against Newcastle disease virus (NDV). The fusion (F) protein, a viral surface glycoprotein, is responsible for the cell membrane fusion and spread, also is one of the major targets for immune response. In this study, groups of chickens were vaccinated twice intramuscularly at 14-day interval either with plasmid DNA encoding F protein gene of NDV or with recombinant F protein alone or with plasmid DNA and boosted with the recombinant F protein and compared with birds that were vaccinated with live NDV vaccine. The immune response was evaluated by indirect ELISA, lymphocyte transformation test, virus neutralization test, cytokine analysis, immunophenotyping of peripheral blood mononuclear cells, and protective efficacy study against virulent NDV challenge virus infection. Chickens in prime-boost group developed a higher level of humoral and cellular immune responses as compared with those immunized with plasmid or protein alone. The DNA prime-protein boost using F protein of NDV yielded 91.6% protection against virulent NDV challenge infection better than immunization with DNA vaccine (66.6%) or rF protein (83.3%) alone. These findings suggest that the "DNA prime-protein boost" approach using full-length F gene could enhance the immune response against NDV in the chickens.

Serological profiling of rabies antibodies by enzyme-linked immunosorbent assay and its comparative analysis with rapid fluorescent focus inhibition test in mouse model.

AIM: In this study, we have used enzyme-linked immunosorbent assay (ELISA) as an alternative test to replace the cumbersome rapid fluorescent focus inhibition test (RFFIT) to ascertain the immune status of immunized mice against rabies virus. MATERIALS AND METHODS: Rabies is a devastating disease worldwide caused by rabies virus. Proper usage of pre- or post-exposure rabies vaccine can prevent the disease transmission. In this study, mice were immunized with Vero cell-adapted inactivated rabies vaccine. RFFIT was used as a test to determine the serum neutralizing titers in infected/vaccinated mice. Seroprofiling of mice sera was done in vitro by ELISA. RESULTS: Twenty-one days post-immunization, both ELISA and RFFIT assays indicated similar antibody levels in mice sera that were immunized with Vero cell-adapted inactivated rabies vaccine. Both the tests were correlated, and the linearity was verified by the regression line (R²=0.979). CONCLUSION: In this study, we profiled the serological status of Vero cell-adapted inactivated rabies vaccine through ELISA in mice model that correlated well with the OIE gold standard test RFFIT.

Antiviral activity of bone morphogenetic proteins and activins.

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.

Impact of Interferon Lambda 4 Genotype on Interferon-Stimulated Gene Expression During Direct-Acting Antiviral Therapy for Hepatitis C.

New directly acting antivirals (DAAs) provide very high cure rates in most patients infected by hepatitis C virus (HCV). However, some patient groups have been relatively harder to treat, including those with cirrhosis or infected with HCV genotype 3. In the recent BOSON trial, genotype 3, patients with cirrhosis receiving a 16-week course of sofosbuvir and ribavirin had a sustained virological response (SVR) rate of around 50%. In patients with cirrhosis, interferon lambda 4 (IFNL4) CC genotype was significantly associated with SVR. This genotype was also associated with a lower interferon-stimulated gene (ISG) signature in peripheral blood and in liver at baseline. Unexpectedly, patients with the CC genotype showed a dynamic increase in ISG expression between weeks 4 and 16 of DAA therapy, whereas the reverse was true for non-CC patients. Conclusion: These data provide an important dynamic link between host genotype and phenotype in HCV therapy also potentially relevant to naturally acquired infection. (Hepatology 2018; 00:000-000).

Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication.

Peptide nucleic acids (PNAs) are potentially attractive antisense agents against hepatitis B virus (HBV), although poor cellular uptake limits their therapeutic application. In the duck HBV (DHBV) model, we evaluated different cell-penetrating peptides (CPPs) for delivery to hepatocytes of a PNA-targeting hepadnaviral encapsidation signal (ε). This anti-ε PNA exhibited sequence-specific inhibition of DHBV RT in a cell-free system. Investigation of the best in vivo route of delivery of PNA conjugated to (D-Arg)8 (P1) showed that intraperitoneal injection to ducklings was ineffective, whereas intravenously (i.v.) injected fluorescein-P1-PNA reached the hepatocytes. Treatment of virus carriers with i.v.-administered P1-PNA resulted in a decrease in viral DNA compared to untreated controls. Surprisingly, a similar inhibition of viral replication was observed in vivo as well as in vitro in primary hepatocyte cultures for a control 2 nt mismatched PNA conjugated to P1. By contrast, the same PNA coupled to (D-Lys)4 (P2) inhibited DHBV replication in a sequence-specific manner. Interestingly, only P1, but not P2, displayed anti-DHBV activity in the absence of PNA cargo. Hence, we provide new evidence that CPP-PNA conjugates inhibit DHBV replication following low-dose administration. Importantly, our results demonstrate the key role of CPPs used as vehicles in antiviral specificity of CPP-PNA conjugates.

MAIT cells are activated during human viral infections.

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/ß. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.

Impact of IL-27 on hepatocyte antiviral gene expression and function.

Background: Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines. It is a potent cytokine, with potential antiviral impact, and has been shown to play a role in modulating functions of diverse cell types, including Th1, Th2, and NK and B cells, demonstrating both pro- and anti-inflammatory roles.  In hepatocytes, it is capable of inducing signal transducer and activator of transcription (STAT)1, STAT3 and interferon-stimulated genes. Methods: To address its role in viral hepatitis, the antiviral activity of IL-27 against hepatitis C virus (HCV) and hepatitis B virus (HBV) was tested in vitro using cell-culture-derived infectious HCV (HCVcc) cell culture system and the HepaRG HBV cell culture model. To further investigate the impact of IL-27 on hepatocytes, Huh7.5 cells were treated with IL-27 to analyse the differentially expressed genes by microarray analysis. Furthermore, by quantitative PCR, we analyzed the up-regulation of chemokine (CXCL)-10 in response to IL-27. Results: In both HCV and HBV infection models, we observed only a modest direct antiviral effect. Microarray analysis showed that the up-regulated genes mostly belonged to antigen presentation and DNA replication pathways, and involved strong up-regulation of CXCL-10, a gene associated with liver inflammation. Overall, gene set enrichment analysis showed a striking correlation of these genes with those up-regulated in response to related cytokines in diverse cell populations. Conclusion: Our data indicate that IL-27 can have a significant pro-inflammatory impact in vitro, although the direct antiviral effect is modest. It may have a potential impact on hepatocyte function, especially chemokine expression and antigen presentation.