Previous SARS-CoV-2 Infection Status Among the Current RT-PCR-Positive Individuals Affected During the Second Wave of COVID-19 Infections in Chennai, India.

India witnessed a very strong second wave of coronavirus disease 2019 (COVID-19) during March and June 2021. Newly emerging variants of concern can escape immunity and cause reinfection. We tested newly diagnosed COVID-19 cases during the second wave in Chennai, India for the presence of Immunoglobulin G (IgG) antibodies to estimate the extent of re-infection. Of the 902 unvaccinated COVID-19 positive individuals, 53 (26.5%) were reactive for IgG antibodies and non-reactive for Immunogobulin M (IgM) antibodies. Among the 53 IgG-positive individuals, the interval between symptom onset (or last contact with the known case in case of asymptomatic) was <5 days in 29 individuals, ≥5 days in 11 individuals, while 13 asymptomatic individuals did not know their last contact with a positive case. The possible re-infections ranged between 3.2% (95% CI: 2.2-4.5%) and 4.3% (95% CI: 3.4-6.2%). The findings indicate that re-infection was not a major reason of the surge in cases during second wave. The IgG seropositivity among recently diagnosed unvaccinated COVID-19 patients could provide early indications about the extent of re-infections in the area.

Seroprevalence of Dengue Infection Using IgG Capture ELISA in India, 2017-2018.

We conducted a nationally representative population-based survey in 60 districts from 15 Indian states covering all five geographic regions during 2017-2018 to estimate the age specific seroprevalence of dengue. Of the 12,300 sera collected, 4,955 were positive for IgG antibodies against dengue virus using IgG Indirect ELISA indicating past dengue infection. We tested 4,948 sera (seven had inadequate volume) positive for IgG antibodies on indirect ELISA using anti-dengue IgG capture ELISA to estimate the proportion of dengue infections with high antibody titers, suggestive of acute or recent secondary infection. Of the 4,948 sera tested, 529 (10.7%; 95% CI: 9.4-12.1) were seropositive on IgG capture ELISA. The proportions of dengue infections with high titers were 1.1% in the northeastern, 1.5% in the eastern, 6.2% in the western, 12.2% in the southern, and 16.7% in the northern region. The distribution of dengue infections varied across geographic regions, with a higher proportion of infections with high antibody titer in the northern and southern regions of India. The study findings could be useful for planning facilities for clinical management of dengue infections.

Burden of dengue infection in India, 2017: a cross-sectional population based serosurvey.

BACKGROUND: The burden of dengue virus (DENV) infection across geographical regions of India is poorly quantified. We estimated the age-specific seroprevalence, force of infection, and number of infections in India. METHODS: We did a community-based survey in 240 clusters (118 rural, 122 urban), selected from 60 districts of 15 Indian states from five geographical regions. We enumerated each cluster, randomly selected (with an Andriod application developed specifically for the survey) 25 individuals from age groups of 5-8 years, 9-17 years, and 18-45 years, and sampled a minimum of 11 individuals from each age group (all the 25 randomly selected individuals in each age group were visited in their houses and individuals who consented for the survey were included in the study). Age was the only inclusion criterion; for the purpose of enumeration, individuals residing in the household for more than 6 months were included. Sera were tested centrally by a laboratory team of scientific and technical staff for IgG antibodies against the DENV with the use of indirect ELISA. We calculated age group specific seroprevalence and constructed catalytic models to estimate force of infection. FINDINGS: From June 19, 2017, to April 12, 2018, we randomly selected 17 930 individuals from three age groups. Of these, blood samples were collected and tested for 12 300 individuals (5-8 years, n=4059; 9-17 years, n=4265; 18-45 years, n=3976). The overall seroprevalence of DENV infection in India was 48·7% (95% CI 43·5-54·0), increasing from 28·3% (21·5-36·2) among children aged 5-8 years to 41·0% (32·4-50·1) among children aged 9-17 years and 56·2% (49·0-63·1) among individuals aged between 18-45 years. The seroprevalence was high in the southern (76·9% [69·1-83·2]), western (62·3% [55·3-68·8]), and northern (60·3% [49·3-70·5]) regions. The estimated number of primary DENV infections with the constant force of infection model was 12 991 357 (12 825 128-13 130 258) and for the age-dependent force of infection model was 8 655 425 (7 243 630-9 545 052) among individuals aged 5-45 years from 30 Indian states in 2017. INTERPRETATION: The burden of dengue infection in India was heterogeneous, with evidence of high transmission in northern, western, and southern regions. The survey findings will be useful in making informed decisions about introduction of upcoming dengue vaccines in India. FUNDING: Indian Council of Medical Research.

Large-scale synthesis and functional elements for the antimicrobial activity of defensins.

Human neutrophil defensins, and their analogues incorporating anionic, hydrophobic or cationic residues at the N- and C-termini, were synthesized by solid-phase procedures. The synthetic defensins were examined for their microbicidal activity against Candida albicans, two Gram-negative bacteria (Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis) and two Gram-positive bacteria (Streptococcus gordonii and Streptococcus mutans). The human neutrophil peptide 1 (HNP1) and HNP2 were found to be potent candidacidal agents. HNP3, which differs by one amino acid at the N-terminus of its sequence, was totally inactive. The Gram-negative bacteria A. actinomycetemcomitans and P. gingivalis and the Gram-positive bacteria S. gordonii and S. mutans were insensitive to human defensins. However, the insertion of two basic residues, such as arginine, at both the N-terminus and the C-terminus of HNP2 significantly enhanced antifungal and antibacterial activity. The addition of anionic residues, such as aspartic acid, at the N- and C-termini rendered the molecule totally inactive. The presence of two hydrophobic amino acids, such as valine, at the N-terminus of HNP2 and of two basic arginine residues at its C-terminus resulted in molecules that were optimally active against these oral pathogens. The results suggest that the N- and C-terminal residues in defensin peptides are the crucial functional elements that determine their microbicidal potency. The three-dimensional structure of all defensins constitutes the same amphiphilic beta-sheet structure, with the polar face formed by the N- and C-terminal residues playing an important role in defining microbicidal potency and the antimicrobial spectrum. The enhanced microbicidal activity observed for defensin peptides with two basic residues at both the N- and C-termini could be due to optimization of the amphiphilicity of the structure, which could facilitate specific interactions with the microbial membranes.

Synthesis, microbicidal activity, and solution structure of the dodecapeptide from bovine neutrophils.

The dodecapepetide sequence R-L-C-R-I-V-V-I-R-V-C-R with a disulfide bridge between the cysteine residues found in bovine neutrophils was synthesized by solid-phase procedures. Its antimicrobial activity against oral microorganisms such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus mutans, and Streptococcus gordonii was examined, and its structural features were examined by CD and determined by two-dimensional (2D) nmr. The strains P. gingivalis (W50 and 381), A. actinomycetemcomitans (Y4 and 67), S. gordonii (DL1), and S. mutans (GS5) are found to be highly sensitive to this peptide at 2-2.5 microM concentrations, suggesting that the dodecapeptide is a potent antibiotic for oral pathogens. The weak negative n-sigma* band observed at approximately 265-270 nm in the CD spectra of this peptide provides evidence for the presence of a disulfide bridge. The negative n-pi* band at approximately 200 nm and the positive pi-pi* band at 185 nm suggest a folded structure for this peptide. The negative n-pi* shifts from 200 to 206 nm with an increase in intensity in dipalmitoylphosphotidylcholine vesicles, suggesting that the peptide might associate to form higher order aggregates in lipid medium. The assignment of backbone and side-chain proton resonances has been accomplished by the combined analysis of 2D total correlated and nuclear Overhauser effect spectroscopy. The temperature dependence of amide NH chemical shifts and (1)H-(2)H exchange effect on amide NH resonances indicate the involvement of amide NH groups of Cys3, Ile5, Ile8, Val10, and Arg12 in intramolecular hydrogen bonding. The coupling constant (J(NH-C(alpha)H)) values, the set of medium-, short-, and long-range nuclear Overhauser effects, and the results of restrained structure calculation using the distance geometry algorithm for nmr applications provide evidence for a folded, loop-like structure with a type I (III) beta-turn involving Ile5, Val6, Val7, and Ile8, and two antiparallel beta-strands involving the N-terminal Arg1, Leu2, Cys3, and Val4 and the C-terminal Arg9, Val10, Cys11, and Arg12 residues. The structure of the dodecapeptide mimics the amphiphilic structure of large 30-35 residue defensins and the peptide appears to exhibit similar antimicrobial potency.

Regulation of protease expression in Porphyromonas gingivalis.

Although the strong protease activity of Porphyromonas gingivalis appears to be an important virulence property of these organisms, little information is currently available regarding the regulation of expression of the multiple protease genes. Utilizing the lacZ reporter gene strategy, the environmental factors which regulate the expression of the Arg-gingipain gene rgpA and the prtT protease gene were investigated. These two genes are reciprocally regulated since factors which retarded growth (iron depletion and nutrient limitation) appeared to upregulate rgpA expression while down-regulating prtT expression. However, inactivation of the major rgpA gene resulted in increased transcription of the prtT and tpr protease genes while decreasing expression of the Lys-gingipain kgp gene as detected by Northern blot analysis. By contrast, inactivation of the prtT gene did not significantly affect kgp expression but moderately decreased rgpA mRNA levels. These results indicate that the protease genes of P. gingivalis are not coordinately regulated and suggest that some of these enzymes play specific roles in the physiology and/or virulence of these organisms.

Bactericidal activity and poly-L-proline II conformation of the tandem repeat sequence of human salivary mucin glycoprotein (MG2).

The tandem repeat 23-residue sequence [TRS23 (145-167): T-T-A-A-P-P-T-P-S-A-T-T-P-A-P-P-S-S-S-A-P-P-E] of human salivary mucin glycoprotein MG2 was examined for its in vitro bactericidal activity against four oral microorganisms, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus gordonii, and Streptococcus mutans. The conformational features of the proline-rich peptide were determined by circular dichroism (CD) and 600 MHz two-dimensional (2D) nuclear magnetic resonance (NMR) in aqueous solution. The strains of P. gingivalis (W50 and 381), A. actinomycetemcomitans (Y4 and 67), S. gordonii (DL1), and S. mutans (GS5) are highly sensitive to this peptide at 1.5-3.0 microM concentrations, suggesting that the proline-rich repeat sequence is a potent bactericidal agent for oral pathogens. The assignment of backbone and side-chain proton resonances was accomplished by the combined analysis of 2D total correlated spectroscopy and nuclear Overhauser effect spectroscopy. The temperature dependence of amide NH chemical shifts and the 1H-2H exchange effect on amide NH resonances suggest the absence of intramolecularly hydrogen-bonded NH groups. The coupling constant (JNH-CalphaH) values, conformational restriction offered by the proline residues (phi = -60 degrees +/- 15 degrees), the set of medium- and short-range nuclear Overhauser effects observed for this sequence, and the results of restrained structure calculation using DIANA, the distance geometry algorithm for NMR applications, provide evidence for the existence of a significant population of poly-L-proline II-type helices in aqueous solution. The CD spectra of the peptide in phosphate buffer (pH 7.2) and in methanol are reminiscent of the CD spectrum of the poly-L-proline II helical conformation and are consistent with the NMR data. The bactericidal activity of the proline-rich repeat sequence suggests that bacterial colonization, facilitated by the adsorbed salivary mucins on tooth surface, could be partly controlled and cleared by proteolytically degraded proline-rich peptides of MG2 in saliva before the colonized organisms turn into pathogens. It appears that the poly-L-proline II helix is the biologically active backbone conformation for bactericidal activity of the tandem repeat sequences of salivary MG2.

Role of Arg-gingipain A in virulence of Porphyromonas gingivalis.

In order to access the role of the Porphyromonas gingivalis Arg-gingipain proteases in the virulence of this organism, a mutant defective in the rgpA gene was constructed in strain 381. This mutant, MT10, displayed only 40% of the Arg-specific cysteine protease activity of the wild-type strain. In addition, MT10, as well as the recently characterized protease mutant G-102, which is defective in the rgpB gene, displayed reduced self-aggregation, hemagglutination, and the ability to bind to immobilized type I collagen compared to levels of the wild-type parent. However, unlike mutant G-102, the rgpA mutant displayed increased binding to epithelial cells relative to that of the parental organism. Mutant MT10 also did not express detectable levels of the FimA protein as assessed by both Western and Northern blotting or fimbriae visible by electron microscopy of the cells. Furthermore, the ability of MT10 to degrade rat tail collagen fibers when it was cultured at 37 degrees C was markedly attenuated compared to that of strain 381. These results suggest that Arg-gingipain A may play a significant role in the pathogenicity of P. gingivalis by altering the colonization and toxic properties of the organism.

Properties of adenosine monophosphate deaminase of Candida albicans.

Adenosine monophosphate deaminase (AMPD; EC 3.5.4.6) catalyses the hydrolysis of adenosine monophosphate (AMP) to commensurate amounts of inosine monophosphate (IMP) and ammonia. The production of AMP deaminase in Candida albicans was measured in Lee's medium grown cultures. The highest AMPD activity was observed at 24 h of growth. The enzyme had an optimum pH and temperature at 6-7 and 28 degrees C, respectively. This enzyme was inhibited under iron-limited growth conditions as well as by protease inhibitors. The AMPD of C. albicans showed a moderate increase in activity when cultures were grown in the presence of the divalent cations Mg2+, Ca2+, and Zn2+. Moreover, ADP, ATP, adenine, adenosine, deoxyribose and hypoxanthine increased the enzyme activity. Cultures grown in trypticase soy broth exhibited maximum AMPD activity compared with those grown in Sabouraud dextrose broth or Lee's medium.

Isolation and characterization of a hemin-regulated gene, hemR, from Porphyromonas gingivalis.

An hemR (hemin-regulated) gene from Porphyromonas gingivalis ATCC 53977 has been isolated and characterized. This gene is present downstream from the prtT gene, previously cloned in this laboratory. In addition, another putative gene, ORF1, was identified between hemR and prtT. The complete nucleotide sequences of ORF1 and hemR were determined, and the deduced amino acid sequence of ORF1 and HemR proteins corresponded to 16- and 48-kDa proteins, respectively. The amino termini of the HemR protein exhibited significant homology with iron-regulated, TonB-dependent outer membrane receptor proteins from various bacteria, while the carboxyl terminus of the HemR protein displayed almost complete identity with a P. gingivalis PrtT protease domain. PCR analyses confirmed the existence of such extensive homology between the carboxyl termini of both the prtT and hemR genes on the P. gingivalis chromosome. Northern blots indicated that ORF1 was part of a 1.0-kb mRNA and was positively regulated by hemin levels. On the other hand, the hemR gene was apparently a part of a 3.0-kb polycistronic message and was negatively regulated at the transcriptional level by hemin. Primer extension analysis of the hemR gene revealed that the transcription start site was at a C residue located within ORF1. An examination of HemR::lacZ constructs in both Escherichia coli and P. gingivalis confirmed hemin repression of hemR expression in both organisms. Moreover, the HemR protein expressed in E. coli was detected by an antiserum from a periodontitis patient heavily colonized with P. gingivalis but not by serum from a periodontally healthy patient or by antisera against hemin-grown P. gingivalis cells. Therefore, it is likely that the 48-kDa HemR protein can be expressed only under hemin-restricted conditions. These results suggest that we have isolated a hemin-regulated gene, hemR, which encodes a 48-kDa protein that may be a TonB-dependent outer membrane protein.

Proteolytic activity of Aeromonas caviae.

Aeromonas strains produce a variety of virulence factors including proteases. Studies on the kinetics of growth of Aeromonas caviae NRRL B-966 and its proteases suggest that the proteolytic activities are produced throughout the growth phase, with peak level occurring at stationary phase. A. caviae synthesize both intracellular and extracellular proteases with the latter account for major portion of the total activity. Optimum pH for the A. caviae proteolytic activity is at 7.0. A. caviae produces a thermoresistant protease, whose activity is dependent on Mg++ and Ca++ ions. Inhibition of proteolytic activity by phenyl methyl sulfonyl fluoride suggest the presence of a serine protease in A. caviae. Nitrogenous compounds enhance the proteolytic activity while carbohydrates tested in this study inhibit the activity.

Cloning and expression of hemolysin genes from Treponema denticola strains ATCC 35404 (TD-4) and human clinical isolate GM-1 in Escherichia coli.

The oral spirochete, Treponema denticola is a putative etiologic agent in adult periodontitis, and acute necrotizing ulcerative gingivitis. In vitro, the oral treponeme produces several factors including proteases, hemolysins, hemin-binding proteins, which could potentially be involved in the virulence of this spirochete. Our laboratory has been investigating the pathobiology of T. denticola, and has demonstrated the production of several hemolysins by T. denticola. In this report two hemolysin genes from T. denticola strains ATCC 35404 (TD-4) and GM-1 were isolated by screening genomic DNA libraries of T. denticola on sheep blood agar plates. Physical maps of the insert fragments were not identical. Southern blot analyses suggested some degree of homology in the nucleotide sequence. Maxicell analyses of [35S]-methionine-labeled polypeptides from the recombinant plasmids have suggested the synthesis of an approximately 62.5 kDa polypeptide. Biochemical characterization of the T. denticola hemolysin genes indicated the activity to be inhibited by Mg2+, Ca2+ and Zn2+ but not by EDTA. Dithiothreitol and glutathione moderately enhanced the hemolytic activity of the recombinant plasmids. Iron partially inhibited the hemolytic activities. Addition of 2-2' bipyridyl moderately enhanced the activities, possibly by iron limitation. These results suggest the isolation of an identical hemolysin gene from T. denticola strains TD-4 and GM-1.

Characterisation of haemolytic activity from Aeromonas caviae.

Aeromonas caviae, an enteropathogen associated with gastroenteritis, displays several virulence characteristics. Studies on the kinetics of growth of A. caviae and expression of beta-haemolytic toxin revealed that A. caviae produced maximum haemolytic activity extracellularly during the stationary phase. Preliminary studies on the properties of A. caviae haemolysin suggested that divalent cations (Mg2+ and Ca2+) and thiol compounds, dithiothreitol and mercaptoethanol enhanced the haemolytic activity. Addition of L-cysteine, glutathione and EDTA reduced the haemolytic activity. The iron chelator, 2-2' bipyridyl, significantly inhibited the growth of A. caviae possibly by iron limitation, with parallel enhancement of haemolysin production compared to A. caviae grown in excess of iron. These results suggest that A. caviae produces only beta-haemolysin, which resembles the haemolysins reported for several other bacteria and the activity might be regulated by environmental factors especially iron.

Factors influencing beta-galactosidase activity of Aeromonas caviae.

Aeromonas caviae, often reported to be associated with diarrhoeal patients, elaborates several virulence factors as well as catabolic enzymes such as xylanase and beta-galactosidase. Studies on the kinetics of growth of A. caviae and synthesis of beta-galactosidase suggested that the activity was cell associated and reached a peak during the late logarithmic phase of growth. The optimum pH for beta-galactosidase activity was 7.0 and required Ca2+ and glutathione for enhancement of its activity; IPTG also slightly improved the activity. Aerobic cultivation of A. caviae in LB containing glucose, fructose, maltose and sucrose completely inhibited the activity possibly due to acetic acid production. Addition of 100 mM cAMP to the media containing glucose (0.25%, w/v) restored the relative activity by 8.8%; however, the final pH of the media remained acidic. Aerobic growth of A. caviae with other carbon sources did not affect beta-galactosidase activity, probably as there was no acid production and thereby the final pH of the media unaltered. Arabinose, xylose and galactose induced the A. caviae beta-galactosidase activity by several folds and lactose moderately enhanced its activity.

Cloning of two distinct hemolysin genes from Porphyromonas (Bacteroides) gingivalis in Escherichia coli.

Hemolysin is considered a potent virulence factor in a large number of Gram-positive and Gram-negative bacterial pathogens. The hemolysin produced by the oral pathogen Porphyromonas gingivalis functions to provide the cell with its required heme-containing molecules for growth in the periodontal pocket. Two distinct P. gingivalis genes, each of which confers a hemolytic phenotype in Escherichia coli, were isolated by screening genomic DNA libraries of P. gingivalis on sheep blood agar plates. The results obtained from physical maps and Southern blots indicated a considerable degree of divergence in the nucleotide sequences of these two genes. Maxicell analyses of the recombinant plasmids in E. coli suggested that plasmid pPGH5 encoded a polypeptide of molecular weight 48 kDa, while an 18-kDa polypeptide was obtained with pPGH1 and pPGH7. When E. coli harboring these hemolysin genes were subjected to iron starvation, the levels of hemolysin activity increased. Biochemical characterization of hemolytic activities indicated that the activity of both hemolysins was inhibited by Mg2+ and Ca2+; but not by EDTA. Elevated levels of hemolytic activity were obtained from the E. coli recombinant strains in the presence of glutathione, DTT and 2-mercaptoethanol. Cholesterol inhibited the activity.

Cloning and expression in Escherichia coli of an alkaline phosphatase (phoA) gene from Zymomonas mobilis.

An alkaline phosphatase (phoA) gene from Zymomonas mobilis was isolated in Escherichia coli CC118 by use of the plasmid Bluescript KS+. The origin of the 6.4-kb DNA fragment in pZAP1 from the chromosome of Z. mobilis was confirmed by Southern blotting and hybridization studies. The Z. mobilis phoA gene was localized at one end of the chromosomal insert on plasmid pZAP1. The Z. mobilis phoA gene was expressed from its own promoter in E. coli, and the enzyme was localized to the periplasmic space. Z. mobilis alkaline phosphatase activity in E. coli was repressed in high-phosphate media and derepressed under a phosphate-limited growth condition. These results suggest that Z. mobilis alkaline phosphatase is subjected to normal regulation in E. coli.

Characterization of Zymomonas mobilis alkaline phosphatase activity in Escherichia coli.

Zymomonas mobilis phoA gene encoding alkaline phosphatase was expressed in Escherichia coli CC118 carrying the recombinant plasmid pZAP1. The pH optimum for this enzyme was 9.0 and showed a peak activity at 42 degrees C. This enzyme required Zn2+ for its catalytic activity; however, Mg2+ or Ca2+ significantly affected the activity. This enzyme was found to be ethanolabile, and ethanol inhibition was reversed by addition of Zn2+. Kinetics of Z. mobilis alkaline phosphatase production in E. coli CC118 (pZAP1) showed that the enzyme activity was growth associated and localized in the cellular fraction, and the maximum activity was found in the stationary phase.

Cloning and expression in Escherichia coli of mercuric ion resistance coding genes from Zymomonas mobilis.

From a genomic library of Zymomonas mobilis prepared in Escherichia coli, two clones (carrying pZH4 and pZH5) resistant to the mercuric ion were isolated. On partial restriction analysis these two clones appeared to have the same 2.9 kb insert. Mercuric reductase activity was assayed from the Escherichia coli clone carrying pZH5 and it was Hg(2+)-inducible, NADH dependent and also required 2-mercaptoethanol for its activity. The plasmid pZH5 encoded three polypeptides, mercuric reductase (merA; 65 kDa), a transport protein (merT 18-17 kDa) and merC (15 kDa) as analysed by SDS-PAGE. Southern blot analysis showed the positive signal for the total DNA prepared from Hgr Z. mobilis but not with the Hgs strain which was cured for a plasmid (30 kb). These results were also confirmed by isolating this plasmid from Hgr Z. mobilis and transforming into E. coli. Moreover the plasmid pZH5 also hybridized with the mer probes derived from Tn21.

Cloning and sequencing of the sacA gene: characterization of a sucrase from Zymomonas mobilis.

The Zymomonas mobilis gene (sacA) encoding a protein with sucrase activity has been cloned in Escherichia coli and its nucleotide sequence has been determined. Potential ribosome-binding site and promoter sequences were identified in the region upstream of the gene which were homologous to E. coli and Z. mobilis consensus sequences. Extracts from E. coli cells, containing the sacA gene, displayed a sucrose-hydrolyzing activity. However, no transfructosylation activity (exchange reaction or levan formation) could be detected. This sucrase activity was different from that observed with the purified extracellular protein B46 from Z. mobilis. These two proteins showed different electrophoretic mobilities and molecular masses and shared no immunological similarity. Thus, the product of sacA (a polypeptide of 58.4-kDa molecular mass) is a new sucrase from Z. mobilis. The amino acid sequence, deduced from the nucleotide sequence of sacA, showed strong homologies with the sucrases from Bacillus subtilis, Salmonella typhimurium, and Vibrio alginolyticus.