Inhibition of early RNA replication in Chikungunya and Dengue virus by lycorine: In vitro and in silico studies.

Arboviruses such as chikungunya virus (CHIKV) and dengue virus (DENV) collectively afflict millions of individuals worldwide particularly in endemic countries like India, leading to substantial morbidity and mortality. With the lack of effective vaccines for both CHIKV and DENV in India, the search for antiviral compounds becomes paramount to control these viral infections. In line with this, our investigation was focused on screening natural compounds for their potential antiviral activity against CHIKV and DENV. Using different assays, including plaque assay, immunofluorescence, and reverse transcription-quantitative real-time PCR (qRT-PCR), out of 109 natural compounds tested, we confirmed lycorine's in vitro antiviral activity against CHIKV and DENV at low micromolar concentrations in different cell types. Time of addition assays indicated that lycorine does not impede viral entry. Additionally, qRT-PCR results along with time of addition assay suggested that lycorine interferes with the synthesis of negative strand viral RNA. Molecular docking analysis was done to understand the mode of inhibition of viral replication. The results revealed that the most likely binding site with the highest binding affinity of lycorine, was at the palm and finger domains, in the vicinity of the catalytic site of CHIKV and DENV RNA-dependent RNA polymerase (RdRp). Collectively, our data underscores the potential of lycorine to be developed as a direct acting inhibitor for DENV and CHIKV, addressing the critical need of requirement of an antiviral in regions where these viruses pose significant public health threats.

In vitro comparative evaluation of apical leakage using a bioceramic sealer with three different obturating techniques: A glucose leakage model.

Context: Bioceramic sealers have improved sealing ability by forming an interfacial apatite layer that chemically bonds the sealer and radicular dentin thus decrease apical leakage. Aim: This study aims to evaluate and compare the apical leakage of Cerafill RCS bioceramic sealer and gutta percha when used with three different obturating techniques. Materials and Methods: Thirty-four extracted single-rooted premolars were decoronated and prepared up to size F3. Then, the specimens were randomly divided into 3 experimental groups (n = 10) cold lateral obturation technique, warm vertical obturation technique, single-cone obturation technique, positive and negative control groups (n = 2), according to the obturation technique used along with a bioceramic sealer. To evaluate apical leakage, all specimens were mounted in a glucose leakage model and assessed at 7 and 14 days using an ultraviolet-visible spectrophotometer. Statistical Analysis: The results were subjected to ANOVA/Kruskal-Wallis ANOVA; followed by post hoc analysis using Bonferroni correction. Results: Significant differences were found in the cumulative leakage of all the three experimental groups. Significantly higher leakage was found in groups obturated using single-cone obturation technique as compared to warm vertical compaction technique at both 7 and 14 days. Conclusions: Warm vertical compaction showed a better sealing result than single-cone obturation techniques at all observation periods.

Correction: Bispidine-Amino Acid Conjugates Act as a Novel Scaffold for the Design of Antivirals That Block Japanese Encephalitis Virus Replication.

[This corrects the article DOI: 10.1371/journal.pntd.0002005.].

Neutralizing antibodies from prior exposure to dengue virus negatively correlate with viremia on re-infection.

BACKGROUND: India is hyperendemic to dengue and over 50% of adults are seropositive. There is limited information on the association between neutralizing antibody profiles from prior exposure and viral RNA levels during subsequent infection. METHODS: Samples collected from patients with febrile illness was used to assess seropositivity by indirect ELISA. Dengue virus (DENV) RNA copy numbers were estimated by quantitative RT-PCR and serotype of the infecting DENV was determined by nested PCR. Focus reduction neutralizing antibody titer (FRNT) assay was established using Indian isolates to measure the levels of neutralizing antibodies and also to assess the cross-reactivity to related flaviviruses namely Zika virus (ZIKV), Japanese encephalitis virus (JEV) and West Nile virus (WNV). RESULTS: In this cross-sectional study, we show that dengue seropositivity increased from 52% in the 0-15 years group to 89% in >45 years group. Antibody levels negatively correlate with dengue RNAemia on the day of sample collection and higher RNAemia is observed in primary dengue as compared to secondary dengue. The geometric mean FRNT50 titers for DENV-2 is significantly higher as compared to the other three DENV serotypes. We observe cross-reactivity with ZIKV and significantly lower or no neutralizing antibodies against JEV and WNV. The FRNT50 values for international isolates of DENV-1, DENV-3 and DENV-4 is significantly lower as compared to Indian isolates. CONCLUSIONS: Majority of the adult population in India have neutralizing antibodies to all the four DENV serotypes which correlates with reduced RNAemia during subsequent infection suggesting that antibodies can be considered as a good correlate of protection.

India is one of the hotspots of dengue infection. The objective of the study was to assess whether having previous exposure to dengue virus could influence how the body will respond to repeat infections with dengue virus. Here, we analysed samples from febrile patients to measure the amount of dengue virus genetic material in the blood, the type of virus and the amount of antibodies, which are proteins produced by the host in response to dengue virus infection. The majority of patient samples demonstrated the capability to restrict all four types of dengue virus in circulation within the country, but reduced capacity to restrict when it comes to international dengue virus types. These data will help to inform future dengue vaccine design and clinical studies in India.

Humoral cross-reactivity towards SARS-CoV-2 in young children with acute respiratory infection with low-pathogenicity coronaviruses.

Background: SARS-CoV-2 infection in children frequently leads to only asymptomatic and mild infections. It has been suggested that frequent infections due to low-pathogenicity coronaviruses in children, impart immunity against SARS-CoV-2 in this age group. Methods: From a prospective birth cohort study prior to the pandemic, we identified children with proven low-pathogenicity coronavirus infections. Convalescent sera from these children were tested for antibodies against respective seasonal coronaviruses (OC43, NL63, and 229E) and SARS-CoV-2 by immunofluorescence and virus microneutralization assay respectively. Results: Forty-two children with proven seasonal coronavirus infection were included. Convalescent sera from these samples demonstrated antibodies against the respective seasonal coronaviruses. Of these, 40 serum samples showed no significant neutralization of SARS-CoV-2, while 2 samples showed inconclusive results. Conclusion: These findings suggest that the antibodies generated in low-pathogenicity coronavirus infections offer no protection from SARS-CoV-2 infection in young children.

Higher circulating natural killer cells and lower lactate levels at admission predict spontaneous survival in non-acetaminophen induced acute liver failure.

Massive cellular necrosis in acute liver failure (ALF) is dominantly immune mediated and innate immune cells are major pathophysiological determinants in liver damage. In fifty ALF and fifteen healthy, immune cells phenotyping by flow-cytometry, DAMPs using ELISA were analysed and correlated with clinical and biochemical parameters. ALF patients (aged 27 ± 9 yr, 56% males, 78% viral aetiology) showed no difference in neutrophils and classical monocytes, but significantly increased intermediate monocytes (CD14+CD16+) (p < 0.01), decreased non-classical monocytes (CD14-CD16+) and CD3-veCD16+CD56+ NK cells compared to HC. ALF patients who survived, showed higher NK cells (9.28 vs. 5.1%, p < 0.001) among lymphocytes and lower serum lactate levels (6.1 vs. 28, Odds ratio 2.23, CI 1.27-3.94) than non- survivors had higher. Logistic regression model predicted the combination of lactate levels with NK cell percentage at admission for survival. In conclusion, Combination of NK cell frequency among lymphocytes and lactate levels at admission can reliably predict survival of ALF patients.

PNPLA3 polymorphisms are associated with raised alanine aminotransferase levels in hepatitis C virus genotype 3.

BACKGROUND AND STUDY AIMS: Hepatitis C virus (HCV) infection is one of the leading causes of end-stage liver diseases. This study aimed to determine the association between polymorphisms in interleukin 28B (IL28B), PNPLA3, toll-like receptor 7 (TLR7), nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and retinoic inducible gene-I (RIG-I) and HCV genotype and clinical presentation in an Indian population. PATIENTS AND METHODS: A total of 500 patients with chronic HCV were enrolled in 19 centres across India. Genomic DNA was extracted from whole blood samples, and single nucleotide polymorphisms (SNPs) for IL28B, PNPLA3, TLR7, NOD2 and RIG-I genes were genotyped by real-time PCR using a TaqManSNP genotyping assay. RESULTS: The mean age of the patients was 45 + 13 years, and the most common genotype observed was HCV genotype 3 (54%), followed by genotype 1 (24%). Although the allelic frequencies of TLR7, NOD2 and RIG-I were in significant disequilibrium in HCV patients compared with those in controls, the PNPLA3 polymorphism correlated significantly with higher viral load and alanine aminotransferase (ALT) levels in genotype 3 patients. Patients with PNPLA3 CG/GG genotypes, along with IL28B genotype CC, had higher levels of ALT than those with other genotypes. CONCLUSION: These results indicate that PNPLA3 polymorphisms are associated with higher ALT levels in HCV genotype 3 patients in India and can help in identifying people who are at greater risk of developing HCV-associated liver diseases.

Natural Killer Cells Contribute to Pathogenesis of Severe Alcoholic Hepatitis by Inducing Lysis of Endothelial Progenitor Cells.

BACKGROUND: Endothelial progenitor cells (EPCs) help in neovascularization and endothelial repair during injury. Patients with cirrhosis show increased number and function of EPCs in circulation. METHODS: Since natural killer (NK) cells regulate EPCs, we investigated the relationship between the 2 in alcoholic cirrhosis (AC, n = 50) and severe alcoholic hepatitis (SAH, n = 18) patients and compared with nonalcoholic cirrhosis (n = 15) and healthy controls (HC, n = 30). Levels of systemic inflammatory cytokines were measured, and coculture assays were performed between EPCs and NK cells in contact-dependent and contact-independent manner. NK cell-mediated killing of EPCs was evaluated, and expression of receptors including fractalkine (FKN) on EPCs and its cognate receptor CX3CR1 on NK cells was studied by RT-PCR assays. RESULTS: Patients with SAH had higher regulated on activation, normal T cell expressed and secreted (RANTES) (p = 0.01), vascular endothelial growth factor (VEGF) (p = 0.04), IL-1ß (p = 0.04), and IL-6 (p = 0.00) growth factors and proinflammatory cytokines as compared to AC and HC. Distinct populations of CD31+ CD34+ EPCs with low and high expression of CD45 were significantly lower in SAH than HC (CD45low , p = 0.03; CD45hi , p = 0.04) and AC (CD45low , p = 0.05; CD45hi , p = 0.02). SAH patients, however, showed increased functional capacity of EPCs including colony formation and LDL uptake. NK cells were reduced in SAH compared with AC (p = 0.002), however with higher granzyme ability (p < 0.001 and p = 0.04, respectively). In SAH, EPC-NK cell interaction assays showed that NK cells lysed the EPCs in both contact-dependent and contact-independent assays. Expression of interaction receptor CX3CR1 was significantly higher on NK cells (p = 0.0005), while its cognate receptor, FKN, was increased on EPCs in SAH patients as compared to HC (p = 0.0055). CONCLUSION: We conclude that in SAH, NK cells induce killing of EPCs via CX3CR1/FKN axis that may be one of the key events contributing to disease severity and proinflammatory responses in SAH.

CD8 T cells protect adult naive mice from JEV-induced morbidity via lytic function.

Following Japanese encephalitis virus (JEV) infection neutralizing antibodies are shown to provide protection in a significant proportion of cases, but not all, suggesting additional components of immune system might also contribute to elicit protective immune response. Here we have characterized the role of T cells in offering protection in adult mice infected with JEV. Mice lacking α/ß-T cells (TCRß-null) are highly susceptible and die over 10-18 day period as compared to the wild-type (WT) mice which are resistant. This is associated with high viral load, higher mRNA levels of proinflammatory cytokines and breach in the blood-brain-barrier (BBB). Infected WT mice do not show a breach in BBB; however, in contrast to TCRß-null, they show the presence of T cells in the brain. Using adoptive transfer of cells with specific genetic deficiencies we see that neither the presence of CD4 T cells nor cytokines such as IL-4, IL-10 or interferon-gamma have any significant role in offering protection from primary infection. In contrast, we show that CD8 T cell deficiency is more critical as absence of CD8 T cells alone increases mortality in mice infected with JEV. Further, transfer of T cells from beige mice with defects in granular lytic function into TCRß-null mice shows poor protection implicating granule-mediated target cell lysis as an essential component for survival. In addition, for the first time we report that γ/δ-T cells also make significant contribution to confer protection from JEV infection. Our data show that effector CD8 T cells play a protective role during primary infection possibly by preventing the breach in BBB and neuronal damage.

N-Desmethylclozapine, Fluoxetine, and Salmeterol Inhibit Postentry Stages of the Dengue Virus Life Cycle.

Around 10,000 people die each year due to severe dengue disease, and two-thirds of the world population lives in a region where dengue disease is endemic. There has been remarkable progress in dengue virus vaccine development; however, there are no licensed antivirals for dengue disease, and none appear to be in clinical trials. We took the approach of repositioning approved drugs for anti-dengue virus activity by screening a library of pharmacologically active compounds. We identified N-desmethylclozapine, fluoxetine hydrochloride, and salmeterol xinafoate as dengue virus inhibitors based on reductions in the numbers of infected cells and viral titers. Dengue virus RNA levels were diminished in inhibitor-treated cells, and this effect was specific to dengue virus, as other flaviviruses, such as Japanese encephalitis virus and West Nile virus, or other RNA viruses, such as respiratory syncytial virus and rotavirus, were not affected by these inhibitors. All three inhibitors specifically inhibited dengue virus replication with 50% inhibitory concentrations (IC50s) in the high-nanomolar range. Estimation of negative-strand RNA intermediates and time-of-addition experiments indicated that inhibition was occurring at a postentry stage, most probably at the initiation of viral RNA replication. Finally, we show that inhibition is most likely due to the modulation of the endolysosomal pathway and induction of autophagy.

Identification and characterization of the role of c-terminal Src kinase in dengue virus replication.

We screened a siRNA library targeting human tyrosine kinases in Huh-7 cells and identified c-terminal Src kinase (Csk) as one of the kinases involved in dengue virus replication. Knock-down of Csk expression by siRNAs or inhibition of Csk by an inhibitor reduced dengue virus RNA levels but did not affect viral entry. Csk partially colocalized with viral replication compartments. Dengue infection was drastically reduced in cells lacking the three ubiquitous src family kinases, Src, Fyn and Yes. Csk knock-down in these cells failed to block dengue virus replication suggesting that the effect of Csk is via regulation of Src family kinases. Csk was found to be hyper-phosphorylated during dengue infection and inhibition of protein kinase A led to a block in Csk phosphorylation and dengue virus replication. Overexpression studies suggest an important role for the kinase and SH3 domains in this process. Our results identified a novel role for Csk as a host tyrosine kinase involved in dengue virus replication and provide further insights into the role of host factors in dengue replication.

Japanese encephalitis virus disrupts cell-cell junctions and affects the epithelial permeability barrier functions.

Japanese encephalitis virus (JEV) is a neurotropic flavivirus, which causes viral encephalitis leading to death in about 20-30% of severely-infected people. Although JEV is known to be a neurotropic virus its replication in non-neuronal cells in peripheral tissues is likely to play a key role in viral dissemination and pathogenesis. We have investigated the effect of JEV infection on cellular junctions in a number of non-neuronal cells. We show that JEV affects the permeability barrier functions in polarized epithelial cells at later stages of infection. The levels of some of the tight and adherens junction proteins were reduced in epithelial and endothelial cells and also in hepatocytes. Despite the induction of antiviral response, barrier disruption was not mediated by secreted factors from the infected cells. Localization of tight junction protein claudin-1 was severely perturbed in JEV-infected cells and claudin-1 partially colocalized with JEV in intracellular compartments and targeted for lysosomal degradation. Expression of JEV-capsid alone significantly affected the permeability barrier functions in these cells. Our results suggest that JEV infection modulates cellular junctions in non-neuronal cells and compromises the permeability barrier of epithelial and endothelial cells which may play a role in viral dissemination in peripheral tissues.

Adaptor protein complexes-1 and 3 are involved at distinct stages of flavivirus life-cycle.

Intracellular protein trafficking pathways are hijacked by viruses at various stages of viral life-cycle. Heterotetrameric adaptor protein complexes (APs) mediate vesicular trafficking at distinct intracellular sites and are essential for maintaining the organellar homeostasis. In the present study, we studied the effect of AP-1 and AP-3 deficiency on flavivirus infection in cells functionally lacking these proteins. We show that AP-1 and AP-3 participate in flavivirus life-cycle at distinct stages. AP-3-deficient cells showed delay in initiation of Japanese encephalitis virus and dengue virus RNA replication, which resulted in reduction of infectious virus production. AP-3 was found to colocalize with RNA replication compartments in infected wild-type cells. AP-1 deficiency affected later stages of dengue virus infection where increased intracellular accumulation of infectious virus was observed. Therefore, our results propose a novel role for AP-1 and AP-3 at distinct stages of infection of some of the RNA viruses.

Bispidine-amino acid conjugates act as a novel scaffold for the design of antivirals that block Japanese encephalitis virus replication.

BACKGROUND: Japanese encephalitis virus (JEV) is a major cause of viral encephalitis in South and South-East Asia. Lack of antivirals and non-availability of affordable vaccines in these endemic areas are a major setback in combating JEV and other closely related viruses such as West Nile virus and dengue virus. Protein secondary structure mimetics are excellent candidates for inhibiting the protein-protein interactions and therefore serve as an attractive tool in drug development. We synthesized derivatives containing the backbone of naturally occurring lupin alkaloid, sparteine, which act as protein secondary structure mimetics and show that these compounds exhibit antiviral properties. METHODOLOGY/PRINCIPAL FINDINGS: In this study we have identified 3,7-diazabicyclo[3.3.1]nonane, commonly called bispidine, as a privileged scaffold to synthesize effective antiviral agents. We have synthesized derivatives of bispidine conjugated with amino acids and found that hydrophobic amino acid residues showed antiviral properties against JEV. We identified a tryptophan derivative, Bisp-W, which at 5 µM concentration inhibited JEV infection in neuroblastoma cells by more than 100-fold. Viral inhibition was at a stage post-entry and prior to viral protein translation possibly at viral RNA replication. We show that similar concentration of Bisp-W was capable of inhibiting viral infection of two other encephalitic viruses namely, West Nile virus and Chandipura virus. CONCLUSIONS/SIGNIFICANCE: We have demonstrated that the amino-acid conjugates of 3,7-diazabicyclo[3.3.1]nonane can serve as a molecular scaffold for development of potent antivirals against encephalitic viruses. Our findings will provide a novel platform to develop effective inhibitors of JEV and perhaps other RNA viruses causing encephalitis.

Expression of TLR 2, TLR 4 and iNOS in cervical monocytes of Chlamydia trachomatis-infected women and their role in host immune response.

PROBLEM: To study the innate immune response -TLR2 TLR 4 and iNOS expression in female genital Chlamydia trachomatis infection. METHOD: TLR 2, TLR 4, and iNOS expression was evaluated by real-time PCR in C. trachomatis-infected asymptomatic, mucopurulent cervicitis (MPC), and fertility disorders (FD) women. Expression of TLR signaling pathway genes was checked in vivo in C. trachomatis-infected cervical monocytes. Further, inos gene expression and nitric oxide release was assessed in vitro in THP-1 cell line upon chlamydial infection. RESULTS: TLR2, TLR4, and iNOS expression was significantly (P < 0.05) higher in C. trachomatis-positive women with FD, MPC, and asymptomatic women, respectively, than in control. Chlamydial infection significantly upregulates CD86, TLR4, MyD88, IRAK2, nF-κB, IL-1,ß and IL-12 genes. Expression of iNOS gene was found to be significantly (P < 0.05) high 12 hrs post-infection. CONCLUSIONS: Chlamydia trachomatis stimulates innate immune cells by activation of TLR2/TLR 4. Overall data indicate that recognition by TLR4 helps in initiation of immune response while recognition by TLR2 leads to secretion of inflammatory cytokines while iNOS-induced nitric oxide production helps in clearing Chlamydia. These results are first to provide initial insights into how innate immune response operates in human cervical monocytes upon chlamydial infection.

Characterization of apoptotic activities during chlamydia trachomatis infection in primary cervical epithelial cells.

Chlamydiae are obligate intracellular bacteria that infect human epithelial cells. It has been reported that Chlamydia trachomatis, induces apoptosis in epithelial cells, however, the molecular mechanisms responsible for host cell death especially in primary epithelial cells remained largely unknown as most of the studies are in cell line like HeLa. In this study we demonstrated that C. trachomatis induces apoptosis signaling pathway and apoptosis in primary cervical epithelial cells in a time and dose dependent manner. Live cervical epithelial cells were isolated from endocervical cells and induction was done with chlamydial EBs. Our results demonstrated that apoptosis in infected epithelial cells was associated with an increased activity of caspase 8; however, caspase 9 was activated to a lesser extent. Analysis of apoptosis pathway revealed that expression level of McL-1, Bcl-2, CASP8, and TRADD genes were found to be significantly upregulated (P < 0.01), where as levels of Caspase 1, Caspase 10 and BRIC2 were found to be significantly downregulated (p < 0.01). Our results showed that Chlamydia induces apoptosis and caspase activation in epithelial cells through caspase 8, with an increased expression of the McL-1, which confers a block at the mitochondrial level.

The mucosal immune response to Chlamydia trachomatis infection of the reproductive tract in women.

Sexually transmitted Chlamydia trachomatis infection is an important public health concern with major adverse effects on female reproductive tract health and function. The magnitude of reproductive morbidity associated with sexually transmitted C. trachomatis infection is enormous, however to date no prophylactic vaccine is available. In part this is due to the lack of information on the mucosal immunobiology of the host-pathogen interaction and correlates of protective immunity during genital C. trachomatis infection. In this review, we focus on current knowledge of mucosal innate and adaptive immune responses in the female genital tract during C. trachomatis infection, which will eventually help in the development of a vaccine for prevention of chlamydial infection.

Determination of chlamydial load and immune parameters in asymptomatic, symptomatic and infertile women.

The regulation of immune response and chlamydial infectious load in the cervix of human females is largely unknown. Infectious load in terms of inclusion-forming units (IFUs) was determined by quantitative cultures in Chlamydia-positive women, in asymptomatic women, women with mucopurulent cervicitis (MPC) and women with fertility disorders (FD). CD4(+), CD8(+), CD14(+) cells, myeloid and plasmacytoid dendritic cells (mDCs and pDCs) in the cervix were quantified by flow cytometry. Cervical cytokines, levels of beta-estradiol and C-reactive protein (CRP) in serum and cervical immunoglobulin A antibody to chlamydial major outer membrane protein antigen, chlamydial heat shock protein 60 and 10 antigens were measured by an enzyme-linked immunosorbent assay. In asymptomatic women, chlamydial load showed significant positive correlations with CD4, mDCs, interleukin-12 (IL-12) and IL-2; however, negative correlations were found with CD8 and IL-8 levels. In women with MPC, chlamydial IFUs correlated positively with CD8, pDC number, IL-8, CRP and interferon-gamma (IFN-gamma). In women with FD, chlamydial load showed a significant positive correlation with the pDC number, IL-10 and estradiol level and a negative correlation with CD4 and IFN-gamma. Overall, these results suggest that the interplay between chlamydial infectious load and host immune responses may be the deciding factor for the clinical condition presented during Chlamydia trachomatis infection.