Perinatal transmission of SARS-CoV-2 and transfer of maternal IgG/neutralizing anti-SARS-CoV-2 antibodies from mothers with asymptomatic infection during pregnancy.

PURPOSE: COVID-19 pandemic remains a serious public health threat worldwide. In view of the limited data on the risk of perinatal transmission of SARS-CoV-2 and transfer of maternal anti-SARS-CoV-2 antibodies, the present study was undertaken. METHODS: A prospective study including 57 pregnant women with a positive SARS-CoV-2 RNA test (SARS-CoV-2-RNA+) and 59 neonates born to them was conducted at Pune, India. 39 viral RNA negative (SARS-CoV-2-RNA-negative) pregnant women and their 39 neonates were included as controls. Neonatal nasal swab/cord blood samples were subjected to SARS-CoV-2 RNA detection by RT-PCR for investigation of perinatal transmission. Transfer of maternal antibodies was studied using ELISA and PRNT. RESULTS: 10/57 SARS-CoV-2-RNA+ mothers were symptomatic. The duration between COVID-19 diagnosis and delivery was ≤ 7 days for 82.4%. Perinatal transmission as evidenced by viral RNA in the neonatal nasal swab/cord blood (CB) was 3.6%. IgG-anti-SARS-CoV-2 positivity was 21.6%. Of the 39 neonates born to SARS-CoV-2-RNA-negative mothers, 20 (51%) and none, respectively, were positive for IgG-anti-SARS-CoV-2 and viral RNA. Preterm deliveries were higher in SARS-CoV-2-RNA+ (18.6%) than SARS-CoV-2 RNA-negative (0/39) mothers (p < 0.005). Respiratory distress at birth (< 4 h) was higher among neonates of SARS-CoV-2-RNA+ (20/59, 33.9%) than SARS-CoV-2-RNA-negative mothers (3/39, 7.7%; p < 0.001). ~ 75% IgG-positives exhibited neutralization potential with mean PRNT titers of 42.4 ± 24 (SARS-CoV-2-RNA+) and 72.3 ± 46.7 (SARS-CoV-2 RNA-negative); higher in the latter (p < 0.05). CONCLUSION: The rate of perinatal transmission was low. Transfer of maternal antibodies was lower among SARS-CoV-2-RNA+ mothers than SARS-CoV-2-RNA-negative mothers with subclinical infection during pregnancy. Presence of neutralizing antibodies in majority of IgG-positives suggests protection from SARS-CoV-2 in early life.

Emergence of two distinct variants of SARS-CoV-2 and an explosive second wave of COVID-19: the experience of a tertiary care hospital in Pune, India.

The emergence of novel variants of SARS-CoV-2 in several countries has been associated with increased transmissibility or reduced neutralization potential of antibodies against the Wuhan virus (wild type). From August 2021 onwards, India experienced a progressive decline in the number of active SARS-CoV-2 infections, indicative of a downward trend in the explosive second wave. This prospective study was conducted quarterly for one year (May 2020 to June 2021) at a tertiary care hospital in the city of Pune in western India. Receptor-binding domain (RBD, n = 319) and full genome (n = 20) sequences from viral-RNA-positive nasopharyngeal swabs of COVID-19 patients representing the first and second waves were used for analysis. No Brazilian, South African, or California variants were detected in this study. Until December 2020, only the wild-type strain was prevalent. Concurrent with the upsurge of the second wave in March 2021, 73% (33/45) of RBD sequences harboured L452R/E484Q mutations characteristic of the Kappa variant. In April 2021, co-circulation of Kappa (37%) and Delta (L452R/T478K, 59%) variants was recorded. During May and June 2021, the Delta variant became the predominant circulating variant, and this coincided with a significant decline in the number of COVID-19 cases. Of the 20 full genome sequences, six isolates each exhibited signature mutations of the Kappa and Delta variant. With several states witnessing a reduction in the number of COVID-19 cases, continuous monitoring of newer mutations and assessment of their effect on virus transmissibility and their impact on vaccinated or previously exposed individuals is necessary.

Isolation and genetic characterization of SARS-CoV-2 from Indian patients in a single family without H/O travel abroad.

In view of the rapidly progressing COVID-19 pandemic, our aim was to isolate and characterize SARS-CoV-2 from Indian patients. SARS-CoV-2 was isolated from nasopharyngeal swabs collected from the two members of a family without any history of (H/O) travel abroad. Both the virus isolates (8003 and 8004) showed CPE on day 3 post-inoculation, viral antigens by immunofluorescence assay and produced distinct, clear and uniform plaques. Infectious virus titers were 5 × 106 and 4 × 106 Pfu/ml by plaque assay and 107.5 and 107 by CPE-based TCID50/ml, respectively. Phylogenetic analysis grouped our isolates with the Italian strains. On comparison with Wuhan strain, 3 unique mutations were identified in nsp3 (A1812D), exonuclease (P1821S) of Orf1ab and spike protein (Q677H) regions, respectively. Both the viruses grouped with Italian strains of SARS-CoV-2 suggesting possible source being the virus imported from Italy. These fully characterized virus isolates will be useful in developing neutralization/virological assays for the evaluation of vaccines/antivirals.

Performance assessment of SARS-CoV-2 IgM & IgG ELISAs in comparison with plaque reduction neutralization test.

Background & objectives: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) continues to be a devastating pandemic. This study was aimed at performance assessment of SARS-CoV-2 IgM and IgG ELISAs, and investigation of their utility for patient diagnosis and sero-epidemiologic investigations. Methods: Serum/plasma samples from COVID-19 patients or asymptomatic contacts (n=180) and healthy donors (n=90) were tested in parallel using two commercial IgM ELISAs (Erbalisa and Inbios), and four IgG ELISAs (Kavach, Euroimmun, Erbalisa and Inbios) along with an indigenous ß-propiolactone inactivated virus-based ELISA (IRSHA-IgG-ELISA). Plaque reduction neutralization test (PRNT) was used as reference test. Results: Among 180 COVID-19 patients, 125 tested positive by PRNT. Inbios-IgM-ELISA showed sensitivity (Se)/specificity (Sp)/positive predictive value (PPV)/negative predictive value (NPV) of 93.6/97.8/98.4/94.4 per cent in relation to PRNT, and performed better than Erbalisa-IgM-ELISA (Se: 48%, Sp: 95.6%, PPV: 95.2%, NPV: 65.2%). During the first week of disease, only 47.4 per cent of the COVID-19 patients tested IgM positive by Inbios-IgM-ELISA, detection improving at two weeks and beyond (~86-100%). Among IgG tests, Inbios-IgG-ELISA ranked first in terms of sensitivity (83.2%), followed by IRSHA (64.8%), Euroimmun (64%), Erbalisa (57.6%) and Kavach (56%) tests. For all IgG tests, sensitivity improved during the third (73.9-95.7%) and fourth week (100%) of illness. The specificity (96.7-100%) and PPV (96.2-100%) of all IgG tests were high; NPV ranged between 71.9 and 87.1 per cent with Inbios-IgG-ELISA scoring highest. Interpretation & conclusions: Our results show that IgM detection by the current, most sensitive ELISAs cannot replace molecular diagnosis, but may aid as a supplement test. The available IgG tests are suitable for serosurveys for the assessment of previous virus exposure.

Standardization of ELISA for anti-chikungunya-IgG antibodies and age-stratified prevalence of anti-chikungunya-IgG antibodies in Pune, India.

Chikungunya (CHIKV) reemerged in India after a gap of 32 years, in 2005-2006 and has established endemicity in Pune. To assess the degree of CHIKV exposure, we estimated age-stratified prevalence of IgG antibodies to CHIKV in Pune population. This retrospective study utilized age-stratified serum samples collected from 15 wards of Pune in 2017 for dengue (DENV) virus study. Indirect anti-CHIKV-IgG ELISA was developed and used to test 1904 samples. Exposure to CHIKV and DENV was compared in the same population. CHIKV-specific plaque reduction neutralization test (PRNT) was employed to evaluate ELISA positivity and neutralizing potential of anti-CHIKV-IgG antibodies. Indirect ELISA showed 98.5% concordance with commercial ELISA. Seropositivity to CHIKV was 46.4%, one-third children < 15 years being antibody positive. A significant increase (45%, p = 0.026-0.038) was noted at 16-25 years and varied between 48 and 56% until the age 65. In elderly (65 + years), antibody positivity was reduced (41%, p = 0.01). In children, CHIKV-PRNT50 titers increased with age and remained comparable from the age group 11-15 until > 65. Exposure to DENV was higher than CHIKV. Lower exposure of children and elderly could be due to lesser exposure to the vectors. High prevalence of IgG antibodies needs to be addressed while planning vaccine studies for CHIKV.

Comparative assessment of commercial enzyme-linked immunosorbent assay & rapid diagnostic tests used for dengue diagnosis in India.

Background & objectives: Dengue diagnosis is routinely carried out by detection of dengue virus (DENV) antigen NS1 and/or anti-DENV IgM antibodies using enzyme-linked immunosorbent assays (ELISAs) and rapid diagnostic tests (RDTs). This study was aimed at evaluation of quality of diagnostic assays currently in use in India for the identification of DENV infection. Methods: During 2016 dengue season (July-November) in Pune, India, comparative assessment of a few immunoassays was undertaken using (i) WHO-approved Panbio-Dengue-Early-(NS1)-ELISA and Panbio-Dengue-IgM-Capture-ELISA as reference tests, and (ii) Bayesian latent class analysis (BLCA) which assumes that no test is perfect. The assays included J.Mitra-Dengue-NS1-Ag-MICROLISA (JME-NS1), J.Mitra-Dengue-IgM-MICROLISA (JME-IgM), and two RDTs, namely, J.Mitra-Dengue-Day-1-Test (JM-RDT) and SD-BIOLINE-Dengue-Duo (SDB-RDT). Serum samples from patients seeking dengue diagnosis (n=809) were tested using the diagnostic kits. The presence of NS1 and/or IgM was taken as evidence for dengue-positive diagnosis. Results: Panbio-NS1/IgM-ELISAs identified 38.6 per cent patients as dengue positive. With Panbio-ELISA as reference, all the tests were less sensitive for IgM detection, while for NS1, JM-RDT was less sensitive. For combined diagnosis (both markers), sensitivity of all the tests was low (55.7-76.6%). According to BLCA, Panbio-ELISA was 84 per cent sensitive for NS1, 86 per cent specific for IgM and 87 per cent specific for combined diagnosis. Accordingly, performance of the other tests was substantially improved with BLCA; however, sensitivity of both the RDTs for IgM detection remained unacceptable. The NS1 ELISAs and RDTs detected all four DENV serotypes, JME being most efficient. All IgM tests exhibited higher sensitivity in secondary infections. Interpretation & conclusions: These results confirmed superiority of ELISAs, and testing for both NS1 and IgM markers for dengue diagnosis, and emphasized on improvement in sensitivity of RDTs.

Positive Regulation of Hepatitis E Virus Replication by MicroRNA-122.

The molecular mechanisms of liver pathology and clinical disease in hepatitis E virus (HEV) infection remain unclear. MicroRNAs (miRNAs) are known to modulate viral pathogenesis either by directly altering viral gene expression or by enhancing cellular antiviral responses. Given the importance of microRNA-122 (miR-122) in liver pathobiology, we investigated possible role of miR-122 in HEV infection. In silico predictions using HEV genotype 1 (HEV-1), HEV-2, HEV-3, and HEV-4 sequences showed that the majority of genomes (203/222) harbor at least one miR-122/microRNA-122-3p (miR-122*) target site. Interestingly, HEV-1 genomes showed a highly (97%) conserved miR-122 target site in the RNA-dependent RNA polymerase (RdRp) region (RdRpc). We analyzed the significance of miR-122 target sites in HEV-1/HEV-3 (HEV-1/3) genomes by using a replicon-based cell culture system. HEV infection did not change the basal levels of miR-122 in hepatoma cells. However, transfection of these cells with miR-122 mimics enhanced HEV-1/3 replication and depletion of miR-122 with inhibitors led to suppression of HEV-1/3 replication. Mutant HEV-1 replicons with an altered target RdRpc sequence (CACTCC) showed a drastic decrease in virus replication, whereas introduction of alternative miR-122 target sites in mutant replicons rescued viral replication. There was enrichment of HEV-1 RNA and miR-122 molecules in RNA-induced silencing complexes in HEV-infected cells. Furthermore, pulldown of miR-122 molecules from HEV-infected cells resulted in pulldown of HEV genomic RNA along with miR-122 molecules. These observations indicate that miR-122 facilitates HEV-1 replication, probably via direct interaction with a target site in the viral genome. The positive role of miR-122 in viral replication presents novel opportunities for antiviral therapy and management of hepatitis E.IMPORTANCE Hepatitis E is a problem in both developing and developed countries. HEV infection in most patients follows a self-limited course; however, 20% to 30% mortality is seen in infected pregnant women. HEV superinfections in patients with chronic hepatitis B or hepatitis C virus infections are associated with adverse clinical outcomes, and both conditions warrant therapy. Chronic HEV infections in immunocompromised transplant recipients are known to rapidly progress into cirrhosis. Currently, off-label use of ribavirin (RBV) and polyethylene glycol-interferon (PEG-IFN) as antiviral therapy has shown promising results in both acute and chronic hepatitis E patients; however, the teratogenicity of RBV limits its use during pregnancy, while alpha IFN (IFN-α) increases the risk of transplant rejections. Experimental data determined with genotype 1 virus in the current study show that miR-122 facilitates HEV replication. These observations present novel opportunities for antiviral therapy and management of hepatitis E.

Age-stratified anti-HAV positivity in Pune, India after two decades: Has voluntary vaccination impacted overall exposure to HAV?

The degree of transmission of hepatitis A virus (HAV) is inversely proportional to the socioeconomic status of a community. Serosurveys conducted at Pune, India during 1982-98 documented significant reduction in HAV exposure of paediatric, higher socioeconomic status (HSS) population. Anti-HAV positivity (ELISA) in age-stratified Pune population representing HSS and lower middle socioeconomic status (LMSS) (n = 1065) and infants till the age of 15 months (n = 690) was determined in 2017. Anti-HAV positivity in the LMSS population decreased significantly in 2017 while an increase was seen in the HSS category. The surprising rise in anti-HAV positivity in the HSS population reflected vaccine- and infection-induced antibodies while only infection-induced antibodies were present in the LMSS category. Lowest antibody prevalence in infants was at 12 months, the recommended age for hepatitis A vaccination. Improved hygiene and selective immunization practices impacted HAV exposure of the LMSS population. The data emphasize the need for hepatitis A vaccination irrespective of socioeconomic status.

Seroepidemiology of respiratory syncytial virus in western India with special reference to appropriate age for infant vaccination.

Respiratory syncytial virus (RSV) causes significant infant mortality worldwide and a vaccine may be available soon. This study determined age-stratified anti-RSV antibody positivity (enzyme-linked immunosorbent assay [ELISA]) at Pune, India (cord blood-85 years). Antibody positivity declined from 100% at birth to 71.3% (3 months), and 0.7% (6 months). A significant rise was noted at 15 months (16%), 16 to 24 months (64.5%) and 4 years (95.2%) with concomitant IgM-anti-RSV positivity indicative of recent infection. Antibody decline was higher in infants born preterm than full-term. Across subsequent age groups including the elderly, antibody positivity was similar and comparable, suggestive of repeated exposure to the virus. Early protection/vaccination is essential for the infant population.

Risk of transfusion-associated dengue: screening of blood donors from Pune, western India.

BACKGROUND: Dengue, a mosquito-borne viral disease, is endemic in >125 countries worldwide. The threat of blood-borne transmission of dengue virus (DENV) has been documented. STUDY DESIGN AND METHODS: This study was conducted to assess the potential magnitude of transfusion-associated dengue, by determination of DENV seromarkers in blood donations from Pune, India, during two dengue seasons (2016 and 2017). These included DENV nonstructural protein 1 (NS1), anti-DENV immunoglobulin (Ig) M, anti-DENV IgG (enzyme-linked immunosorbent assay), and DENV RNA (reverse transcription-polymerase chain reaction). RESULTS: NS1 (IgM) reactivity was 1 of 209, 0.48% (11/209, 5.3%) in 2016 and 2 of 311, 0.64% (20/311, 6.4%) in 2017. Of the 34 NS1/IgM reactives, 1 NS1-reactive donor and 10 IgM-reactive donors exhibited evidence of secondary infection. DENV RNA was not detected in any of the 34 NS1/IgM reactives. Among the NS1/IgM negatives, anti-DENV IgG reactivity was high in 2016 (75%) and further increased in 2017 (87%, p = 0.002). CONCLUSION: Although RNA negative, detection of 34 NS1/IgM-reactive donations, of which 11 had evidence of secondary infection, suggests the need for further evaluation on the basis of potential risk to recipients of either dengue transmission or increased risk of secondary infection. These would include multicenter studies followed by cost-benefit analyses.

Association of Toll-like receptor 4 polymorphism with hepatitis E virus-infected Indian patients.

Hepatitis E infection caused by hepatitis E virus (HEV), a major public health concern in developing countries, is responsible for sporadic and epidemic acute viral hepatitis in adults. Pathogenesis of hepatitis E infection is poorly understood. Toll-like receptors (TLRs) are the key players of innate immunity recognize pathogen-associated molecular patterns (PAMPs). Previously, we found higher TLR4 expression (at protein and gene level) with impaired cytokine response upon stimulus of PBMCs with LPS in HEV-infected patients. In view of the earlier observations of the association of polymorphisms in TLR4 genes (A299G, C399T) with liver diseases, we investigated TLR4 polymorphisms in HEV-infected patients. We observed the significant association of TLR4-399CC and CT alleles with hepatitis E (both subclinical and acute patients). Carrier frequency of TLR4-399 CT was lower in patients' categories in comparison with the controls. Higher frequency of allele TLR4-399C significantly correlated with disease progression. Acute hepatitis E patients showed the higher frequency of CG and TA haplotypes, while the rare haplotype (TG) was more frequent in controls. The other single nucleotide polymorphism (SNP) at TLR4-299 (A>G) did not show any difference. We report here for the first time the association of TLR4 polymorphism with hepatitis E and suggest that TLR 4 hyporesponsiveness during HEV infection might be related to its polymorphism.

Hepatitis E virus (HEV)-1 harbouring HEV-4 non-structural protein (ORF1) replicates in transfected porcine kidney cells.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis and a major public health problem in India. There are four mammalian HEV genotypes worldwide. In India, genotype 1 (HEV-1) is restricted to humans whereas genotype 4 (HEV-4) circulates in pigs. Studies from our laboratory have shown that HEV-4 (swine) virus can establish experimental infection in rhesus monkeys; however, HEV-1 (human) virus cannot infect pigs. Viral and/or cellular factors responsible for this host specificity are not yet known. We developed 12 different genotype 1-4 chimeric full genome clones with pSK-HEV2 as the backbone and by replacing structural (ORF2 and ORF3), non-structural (ORF1) and non-coding regions (NCR) with corresponding segments from the HEV-4 clone. S10-3 (human hepatoma) and PK-15 (pig kidney) cells were transfected with transcripts generated from the above clones to test their replication competence. Transfected cells were monitored for successful virus replication by detecting replicative intermediate RNA and capsid protein (immunofluorescence assay). All the chimeric constructs were able to replicate in S10-3 cells. However, only two chimeric clones, HEV-1 (HEV-4 5'NCR-ORF1) and HEV-1 (HEV-4 ORF1), containing 5'NCR-ORF1 and ORF1 regions from the HEV-4 clone, respectively, were able to replicate in PK-15 cells. We demonstrate for the first time the crucial role of ORF1 polyprotein in crossing the species barrier at the cellular level. These results indicate the importance of interactions between ORF1 protein domains and host cell specific factors during HEV replication and the critical role of cellular factors as post-entry barrier/s in virus establishment.

Genomic characterization of Salmonella bacteriophages isolated from India.

Salmonella are a medically important Gram-negative foodborne pathogen. Genomic diversity of Salmonella is increasingly studied but at the same time, we have limited knowledge of Salmonella phage diversity. In this study, we have isolated Salmonella phages from sewage and river water. Genomic characterization of 12 Salmonella phages was carried out using next-generation sequencing platform. Newly sequenced phages were classified based on amino acid sequence phylogenetic analysis. In newly sequenced phages, several virulence genes, DNA metabolism genes, tRNA genes, antibiotic resistance genes and genes not having known role in the life cycle of phages were identified. Annotations of newly sequenced phage genome showed the presence of polymyxin-b resistance gene and penicillin binding protein. Annotation identified number of genes which are involved in DNA metabolism. Results suggest that most of the phages having G + C content different than their host possess DNA metabolism genes. The presence of tRNAs in the genome of Salmonella_phage38-India was identified; however, we did not observe any correlation between tRNA genes and overall codon usage in the phage genome. It is suggested that the phage-encoded tRNAs may increase fitness of phages. In summary, we isolated novel Salmonella phages, determined full genome sequences and provided phylogenetic analysis-based classification.

Enhanced humoral response in pregnant mice immunized with liposome encapsulated recombinant neutralizing epitope protein of Hepatitis- E virus.

BACKGROUND: Pregnant women from developing countries are at high-risk of hepatitis E-associated high mortality and constitute priority population for vaccination. So far, candidate vaccines have not been evaluated during pregnancy. We evaluated our vaccine candidate, recombinant Neutralizing Epitope protein (rNEp) encapsulated in liposomes, in pregnant mice. METHODS: A single dose (10 µg) of the formulation was administered intramuscularly on day 7 of pregnancy. Titres of serum IgG antibodies to hepatitis E virus (IgG-anti-HEV), levels of cytokines and biochemical parameters were determined. Spleens were harvested from pregnant and non-pregnant mice for immunophenotyping (flow cytometry), cytokines (cytometric bead array, CBA) and gene expression of immune response genes (Taqman low density array, TLDA). Histopathology studies of spleen, liver, kidneys, brain and muscle was carried out. RESULTS: The vaccine was well-tolerated during pregnancy as evidenced by histopathology and serum biochemical parameters. Anti-HEV titres were significantly higher in the pregnant balb/c and C57BL/6 mice (3592 ± 802 and 1016 ± 138 respectively, than in non-pregnant groups (634 ± 191 and 320 ± 55 respectively, p < 0.001 for both) suggesting that the higher antibody response in pregnant mice was independent of the genetic makeup of the host but immunogen-driven. Pups receiving vertically transferred antibodies developed lower anti-HEV antibodies (p < 0.05) when immunized with the formulation after seronegativity than in the age-matched mice without such antibodies. In non-pregnant mice, a Th1 response and discordance between splenic and serum cytokines was evident while in pregnancy, a Th2 bias was observed irrespective of immunization. Increased CD19 levels correlated with higher anti-HEV titres in pregnant mice. CONCLUSION: The single dose of the vaccine was safe and highly immunogenic in pregnant mice. Degree and type of immune response to vaccination during pregnancy is immunogen-driven. In-depth studies are needed to understand the underlying immunologic mechanism(s). These encouraging results for a vaccine intended for use in pregnant women should be confirmed in higher animals.

Envelope specific T cell responses & cytokine profiles in chikungunya patients hospitalized with different clinical presentations.

BACKGROUND & OBJECTIVES: Since the 2006 massive outbreaks, chikungunya (CHIK) is a major public health concern in India. The aim of this study was to assess envelope specific immune responses in patients with chikungunya infection. METHODS: This study included 46 hospitalized patients with chikungunya virus infection (encephalitis, n=22, other systemic involvement, OSI, n=12, classical, n=12) and six controls from Ahmedabad city, Gujarat, India. T cell responses and the levels of Th1, pro/ anti-inflammatory cytokines against the CHIK virus envelope antigens were assessed by lymphocyte proliferation assay and by cytometric bead array in flow cytometry, respectively. RESULTS: Lymphoproliferative response was uniform among the patients. Comparisons of cytokines revealed significantly higher levels of interleukin (IL)-4 and IL-5 in encephalitis, OSI and classical patients versus controls. The levels of tumour necrosis factor (TNF)-α were higher in classical patients categories compared to the controls. Interferon (IFN)-γ levels were lower in encephalitis patients versus control. INTERPRETATION & CONCLUSIONS: Our findings showed recognition of T cell epitopes on the envelope region of chikungunya virus by all patient categories. Lower level of IFN-γ may be associated with the severity of disease in these patients.

Evaluation and Immunogenicity of Combined Liposome-Based Vaccine Candidates against Hepatitis E and B Viruses in Rhesus Monkeys.

The administration of vaccines using a combination approach ensures better coverage and reduces the number of injections and cost. The present study assessed liposome-complexed DNA-corresponding proteins of hepatitis E and B viruses (HEV and HBV) as combined vaccine candidates in rhesus monkeys. The HEV and HBV components consisted of 450 bps, neutralizing the epitope/s (NE) region, and 685 bps small (S) envelope gene-corresponding proteins, respectively. Three groups (n = 2 monkeys/group) were intramuscularly immunized with a total of three doses of NE Protein (Lipo-NE-P), NE DNA + Protein (Lipo-NE-DP), and each of NE and S DNA + Protein (Lipo-NES-DP), respectively, given one month apart. All immunized monkeys were challenged with 10,000 fifty percent monkey infectious dose of homologous HEV strain. Post-immunization anti-HEV antibody levels in monkeys were 59.4 and 148.4 IU/mL (Lipo-NE-P), 177.0 and 240.8 IU/mL (Lipo-NE-DP), and 240.7 and 164.9 IU/mL (Lipo-NES-DP). Anti-HBV antibody levels in Lipo-NES-DP immunized monkeys were 58,786 and 6213 mIU/mL. None of the challenged monkeys showed viremia and elevation in serum alanine amino transferase levels. Monkeys immunized with Lipo-NE-DP and Lipo-NES-DP exhibited a sterilizing immunity, indicating complete protection, whereas monkeys immunized with Lipo-NE-P showed limited viral replication. In conclusion, the liposome-complexed DNA-corresponding proteins of HEV and HBV induced protective humoral immune responses to both components in monkeys and are worth exploring further.

Serum immunoglobulin G subclass responses in different phases of hepatitis E virus infection.

To investigate the specific immunoglobulin (Ig) G subclass responses in patients with hepatitis E virus (HEV) infection, an open reading frame 2 (ORF2) protein based enzyme-linked immunosorbant assay was used to measure antibody levels in sera obtained at different phases of infection. Sera were collected at 2-31 days and at 6 months after the onset of symptoms corresponding to the acute (n = 48, 100% IgM-positive) and convalescent (n = 17/48, 53% IgM-positive) phases of infection, respectively. IgM-negative sera from 61 individuals infected at least ≥6 months ago (prior exposure) were also tested. IgG1, IgG2, IgG3, and IgG4 antibodies were detected in 100%, 6%, 56%, and 4% of acute phase sera, respectively, and in 100%, 0%, 0%, and 65% of convalescent phase sera, respectively. IgG1 antibody levels were significantly higher than those of the other detectable subclasses of IgG in the acute and convalescent sera (P < 0.05). The IgG3 antibodies in six acute phase patients were replaced by IgG4 antibodies in the convalescent phase of infection. Patients with prior exposure to HEV had low total IgG antibody titers and decreased IgG1 seropositivity compared with those in the acute and convalescent phases. IgG1 was the only major subclass of antibody to be detected in all the three phases of infection. Other than IgG1 antibodies, the subclass antibody response was restricted to IgG3 and IgG4 antibodies in the acute and convalescent phases of infection, respectively.

Hepatitis E virus from India exhibits significant amino acid mutations in fulminant hepatic failure patients.

In India, hepatitis E virus (HEV) is the predominant cause of acute viral hepatitis (AVH) and fulminant hepatic failure (FHF) among pregnant women and adults. The present study evaluates association, if any, of the mutations in the viral genome with disease outcome. Ten genotype-1 complete genomes (five each from AVH and FHF patients) were sequenced. Phylogenetic analysis showed a distinct cluster including all five FHF-HEV sequences from western India (present study), one FHF isolate from northern India, and one AVH isolate detected in 2010 (present study). HEV genotype-1 sequences from fulminant cases exhibited 150 significantly different (p ≤ 0.05) nucleotide substitutions when compared to all genotype-1-AVH sequences as well as isolates from the Indian subcontinent. At six positions, all FHF sequences showed identical substitutions (1 non-synonymous). Six amino acid changes in ORF1; F179S, A317T, T735I, L1110F, V1120I, and F1439Y were significantly associated with HEV-type-1 FHF. The data suggests that the nucleotide substitutions recorded and/or L1110F and V1120I amino acid substitutions in helicase domain may play important role in determining outcome of HEV infection.

Variable neutralizing antibody responses to 10 SARS-CoV-2 variants in natural infection with wild- type (B.1) virus, Kappa (B.1.617.1), and Delta (B.1.617.2) variants and COVISHIELD vaccine immunization in India: utility of the MSD platform.

For the efficacy of COVID-19 vaccines, emergence of variants accumulating immune-escape mutations remains a major concern. We analyzed the anti-variant (n = 10) neutralization activity of sera from COVID-19 patients infected with Wuhan (B.1), Kappa, and Delta variants and COVISHIELD vaccine recipients with (prepositives) or without (prenegatives) prior antibody positivity using V- PLEX ACE2 Neutralization Kit from MSD. MSD and PRNT50 correlated well (r = 0.76-0.83, p < 0.0001). Despite the least antibody positivity in Kappa patients, anti-variant neutralizing antibody (Nab) levels in the responders were comparable with Delta patients. Vaccinees sampled at 1 month (PD2-1) and 6 months (PD2-6) post-second dose showed the highest seropositivity and Nab levels against the Wuhan strain. At PD2-1, the responder rate was variant-dependent and 100% respectively in prenegatives and prepositives. Nab levels against B.1.135.1, B.1.620, B.1.1.7+E484K (both groups), AY.2 (prenegatives), and B.1.618 (prepositives) were lower than that of Wuhan. At PD2-6, positivity decreased to 15.6%-68.8% in the prenegatives; 3.5%-10.7% of prepositives turned negative for the same four variants. As against the decline in Nab levels in 9/10 variants (prenegatives), a further reduction was seen against the same four variants in the prepositives. These variants possess immune-evasion-associated mutations in the RBD/S region. In conclusion, our data show that the Nab response of patients to multiple variants depends on the infecting variant. We confirm superiority of hybrid immunity in neutralizing multiple variants. Depending on the infecting variant pre- or postvaccination, immune response to different vaccines in different populations will vary and impact protection against emerging variants. The MSD platform provides an excellent alternative to live virus/pseudovirus neutralization tests.

Platelet derived exosomes disrupt endothelial cell monolayer integrity and enhance vascular inflammation in dengue patients.

Background: Thrombocytopenia is the most notable phenomenon in dengue. Activation status of platelets and interaction of platelets with endothelium contribute towards dengue disease pathogenesis. Platelets are the major cell types known to release extracellular vesicles, especially exosomes in circulation. However, the role of platelet derived exosomes (PLT-EXOs) in endothelial dysfunction during dengue infection remains unknown. Methods: In this study, we recruited 28 healthy subjects and 69 dengue patients categorized as WS- (n=31), WS+ (n=29) and SD (n=9). Platelets were isolated from platelet rich plasma of dengue patients and their activation was assessed by flow cytometry. PLT-EXOs were isolated by ultracentrifugation method. Western blot analyses were performed to characterize the exosomes. Exosome uptake experiment was carried out to see the internalization of exosomes inside endothelial cells (HUVECs). To observe the effect of exosomes on endothelial cells, exosomes were added on HUVECs and expression of adherens and tight junctional proteins were examined by immunofluorescence assay and western blot. Expression levels of vascular injury markers were measured in the culture supernatants of Exosome-HUVEC coculture and sera of dengue patients by MSD-multiplex assay. Results: As compared to healthy subjects, CD41/CD61 expression was significantly reduced (p<0.0001) and CD62p expression was significantly increased (p<0.0001) on platelets in dengue patients. PLT-EXOs isolated from the dengue patients showed higher expression of CD63 and CD9 proteins than the healthy subjects. With in-vitro immunofluorescence assays, we illustrated the internalization of PLT-EXOs by the HUVECs and observed disruption of endothelial cell monolayer integrity in the presence of PLT-EXOs from WS+ and SD patients. Furthermore, the significant reduction in the expressions of ZO-2, VE-Cadherin and CD31 in endothelial cells following exposure to PLT-EXOs from the dengue patients provide direct evidence of PLT-EXOs mediated vascular permeability. PLT-EXOs stimulated the release of inflammatory markers CRP, SAA, sVCAM-1 and sICAM-1 in the supernatants of HUVEC cells. Importantly, significantly higher levels of CRP, sVCAM-1 and sICAM-1 in the sera of severe than mild dengue patients (p<0.0001) suggest their role in disease severity. Conclusions: In summary, our data suggest that PLT-EXOs promote vascular leakage via release of proinflammatory mediators and compromise vascular barrier integrity in dengue patients.