Distinguishing features of long COVID identified through immune profiling.

Post-acute infection syndromes may develop after acute viral disease1. Infection with SARS-CoV-2 can result in the development of a post-acute infection syndrome known as long COVID. Individuals with long COVID frequently report unremitting fatigue, post-exertional malaise, and a variety of cognitive and autonomic dysfunctions2-4. However, the biological processes that are associated with the development and persistence of these symptoms are unclear. Here 275 individuals with or without long COVID were enrolled in a cross-sectional study that included multidimensional immune phenotyping and unbiased machine learning methods to identify biological features associated with long COVID. Marked differences were noted in circulating myeloid and lymphocyte populations relative to the matched controls, as well as evidence of exaggerated humoral responses directed against SARS-CoV-2 among participants with long COVID. Furthermore, higher antibody responses directed against non-SARS-CoV-2 viral pathogens were observed among individuals with long COVID, particularly Epstein-Barr virus. Levels of soluble immune mediators and hormones varied among groups, with cortisol levels being lower among participants with long COVID. Integration of immune phenotyping data into unbiased machine learning models identified the key features that are most strongly associated with long COVID status. Collectively, these findings may help to guide future studies into the pathobiology of long COVID and help with developing relevant biomarkers.

Predominance of genomically defined A lineage of HPV16 over D lineage in Indian patients from eastern India with squamous cell carcinoma of the cervix in association with distinct oncogenic phenotypes.

Human papillomavirus type-16 (HPV16) is classified into lineages, A, B, C and D and 10 sub-lineages portraying variable infectivity, persistence, and cytological outcomes, however, with geographical variations. Our objective was to delineate the distinctive features of lineages among cervical squamous cell carcinoma (SCC) in the eastern region of India. A total of 145 SCC cases and 24 non-malignant specimens, harboring episomal HPV16, were included. The presence of higher proportion of lineage A over D was observed among SCC cases (86.89% A1, 8.97% D1 and 4.14% D2), while only A1 sub-lineage viruses were found among control specimens. Among the A1 viruses, an association of variants in the E5 (Y44L, I65V), E6 (L83V) genes and LCR: C7577T with SCC, with combined Odd's ratio (95% CI) of 20.5(4.61-91.25) was observed. Network analyses revealed the presence of 10 clades of lineage A viruses comprising of 64 HPV16 genomes harboring the risk alleles. High episomal HPV16 DNA copy numbers (adjusted p-value= 0.0271) and E7 mRNA expression (p-value=0.000017) predominated in SCC with lineage A, over D. Our study highlights the distinctive modalities of oncogenicity among different HPV16 lineages.

Complete Genome Sequences of Eight Human Papillomavirus Type 16 Asian American and European Variant Isolates from Cervical Biopsies and Lesions in Indian Women.

Human papillomavirus type 16 (HPV16), a member of the Papillomaviridae family, is the primary etiological agent of cervical cancer. Here, we report the complete genome sequences of four HPV16 Asian American variants and four European variants, isolated from cervical biopsies and scrapings in India.

RNA and ß-hemolysin of group B Streptococcus induce interleukin-1ß (IL-1ß) by activating NLRP3 inflammasomes in mouse macrophages.

The inflammatory cytokine IL-1ß is critical for host responses against many human pathogens. Here, we define Group B Streptococcus (GBS)-mediated activation of the Nod-like receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, ß-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1ß production.

Differential expression of HPV16 L2 gene in cervical cancers harboring episomal HPV16 genomes: influence of synonymous and non-coding region variations.

We tested the hypothesis that (i) synonymous variations within the coding regions, and (ii) variations within the non-coding regions of HPV, influence cervical cancer (CaCx) pathogenesis under the impact of intact HPV16 genomes. Whole genome sequence analysis of HPV16 isolates within 70 CaCx cases and 25 non-malignant samples revealed that synonymous variations were significantly higher within the E6 (p = 0.014), E5 (p = 0.001) and L2 (p = 0.0002) genes of HPV16 isolates within cases, compared to isolates within non-malignant samples. All of the 25 (100%) humanized codons identified within L2 ORF of the samples analyzed, were harbored by CaCx cases, while 8 out of 25 (32%) were harbored by HPV16 positive non-malignant samples (p = 3.87105E-07). L2 (mRNA and protein) expression was evident only among cases with episomal viral genomes and L2 mRNA expression correlated significantly with E2 gene copy numbers suggesting expression from all episomal genomes. Among such cases, Asian American (AA) isolates portrayed all of the humanized codons (100%; 4-6/sample) recorded within L2, which was significantly higher (p = 2.02E-7) compared to the European (E) isolates (22.8%; none or 1-2/sample). Additionally, majority of E variant isolates within cases (54/57; 94.7%) portrayed a variation (T4228C) within the short non-coding region (NCR2) between E5 and L2 genes, which portrays a weak promoter activity specific for L2 mRNA expression. This resulted in loss of 9 out of 14 miRNA binding sites (hsa-miR-548 family), despite the significant overexpression of miR548a-5p and miR548d-5p among such cases (28.64 and 36.25 folds, respectively), in comparison to HPV negative control samples. The findings exemplify the biological relevance of sequence variations in HPV16 genomes and highlight that episomal HPV16 in CaCx cases employ multiple mechanisms to sustain L2 expression, thereby justifying the potential role of L2 in such cancers, as opposed to those harboring viral integration.

Some novel insights on HPV16 related cervical cancer pathogenesis based on analyses of LCR methylation, viral load, E7 and E2/E4 expressions.

This study was undertaken to decipher the interdependent roles of (i) methylation within E2 binding site I and II (E2BS-I/II) and replication origin (nt 7862) in the long control region (LCR), (ii) expression of viral oncogene E7, (iii) expression of the transcript (E7-E1/E4) that encodes E2 repressor protein and (iv) viral load, in human papillomavirus 16 (HPV16) related cervical cancer (CaCx) pathogenesis. The results revealed over-representation (p<0.001) of methylation at nucleotide 58 of E2BS-I among E2-intact CaCx cases compared to E2-disrupted cases. Bisulphite sequencing of LCR revealed overrepresentation of methylation at nucleotide 58 or other CpGs in E2BS-I/II, among E2-intact cases than E2-disrupted cases and lack of methylation at replication origin in case of both. The viral transcript (E7-E1/E4) that produces the repressor E2 was analyzed by APOT (amplification of papillomavirus oncogenic transcript)-coupled-quantitative-RT-PCR (of E7 and E4 genes) to distinguish episomal (pure or concomitant with integrated) from purely integrated viral genomes based on the ratio, E7 C(T)/E4 C(T). Relative quantification based on comparative C(T) (threshold cycle) method revealed 75.087 folds higher E7 mRNA expression in episomal cases over purely integrated cases. Viral load and E2 gene copy numbers were negatively correlated with E7 C(T) (p = 0.007) and E2 C(T) (p<0.0001), respectively, each normalized with ACTB C(T), among episomal cases only. The k-means clustering analysis considering E7 C(T) from APOT-coupled-quantitative-RT-PCR assay, in conjunction with viral load, revealed immense heterogeneity among the HPV16 positive CaCx cases portraying integrated viral genomes. The findings provide novel insights into HPV16 related CaCx pathogenesis and highlight that CaCx cases that harbour episomal HPV16 genomes with intact E2 are likely to be distinct biologically, from the purely integrated viral genomes in terms of host genes and/or pathways involved in cervical carcinogenesis.

Genetic analysis of cytomegalovirus in malignant gliomas.

Human cytomegalovirus (HCMV) has been found in malignant gliomas at variable frequencies with efforts to date focused on characterizing the role(s) of single gene products in disease. Here, we reexamined the HCMV prevalence in malignant gliomas using different methods and began to dissect the genetics of HCMV in tumors. HCMV DNA was found in 16/17 (94%) tumor specimens. Viral DNA copy numbers were found to be low and variable, ranging from 10(2) to 10(6) copies/500 ng of total DNA. The tumor tissues had incongruences between viral DNA copy numbers and protein levels. However, nonlatent protein expression was detected in many tumors. The viral UL83 gene, encoding pp65, was found to segregate into five cancer-associated genotypes with a bias for amino acid changes in glioblastoma multiforme (GBM) in comparison to the low-grade tumors. Deep sequencing of a GBM-associated viral population resulted in 81,224 bp of genome coverage. Sequence analysis revealed the presence of intact open reading frames and higher numbers of high-frequency variations within the repeat long region compared to the unique long region, which harbors many core genes, and the unique short region (P = 0.001). This observation was in congruence with phylogenetic analyses across replication-competent viral strains in databases. The tumor-associated viral population was less variable (π = 0.1% and π(AA) = 0.08%) than that observed in other clinical infections. Moreover, 42/46 (91.3%) viral genes analyzed had dN/dS scores of <1, which is indicative of high amino acid sequence conservation. Taken together, these findings raise the possibility that replication-competent HCMV may exist in malignant gliomas.

Association of viral load with HPV16 positive cervical cancer pathogenesis: causal relevance in isolates harboring intact viral E2 gene.

We tested the hypothesis that cervical cancers (CaCx) harbor high HPV16 viral load compared to controls and this is influenced by E2 status and age of subjects. Viral load (natural log transformed values) per 100ng genomic DNA was estimated (152 cases and 87 controls) by Taqman assay. Median viral load was significantly higher (Mann-Whitney U test) among cases (17.21) compared to controls (9.86), irrespective of E2 status or upon considering E2 status as a covariate in logistic regression model (p<0.001). Viral load of E2 intact cases (17.80) was significantly higher (p<0.001) compared to those with disrupted E2 (9.78). At equivalent probability of being a case, viral load was higher among individuals (i) of lower age, irrespective of E2 status, and (ii) with intact E2 but of similar age as those with disrupted E2. Thus viral load in association with E2 status and/or age might be of causal relevance in CaCx pathogenesis.

Characterization of sequence variations within HPV16 isolates among Indian women: prediction of causal role of rare non-synonymous variations within intact isolates in cervical cancer pathogenesis.

We re-sequenced HPV16 genome (~6 kb) implicated in cervical carcinogenesis (LCR, E2, E5, E6, E7, L1, L2) to prioritize sequence variants for functional validation as biomarkers, using CaCx cases (n=74) and asymptomatic controls (n=24). Of the nucleotide variations recorded (n=271), non-synonymous changes in L2 region were significantly higher (p=0.005) among cases (2.67%) compared to controls (1.27%). Using SIFT database, 29 non-synonymous changes (frequency=0.01-0.03) predicted as deleterious to protein functions were identified. Haplotype analysis considering 110 polymorphic variations (frequency> or =0.05) within intact viral isolates (53 CaCx cases and 21 controls) using NETWORK software, confirmed Asian-American (AA, 14.86%) and European (E, 85.14%) variants, differing at 78 positions. The E-variants portrayed thirty-six haplotypes, of which, E-12 was most prevalent within cases (38.1%; 16/42) and controls (28.57%; 6/21) harboring polymorphic variations at 10 positions, in contrast to HPV16R. Cases of the E-12 haplotype harbored 7 deleterious mutations distributed within L1 (n=1), E2 (n=1), E5 (n=1), and L2 (n=4), while none within similar controls. Thus rare deleterious variations within genes implicated in productive infection over the E-12 haplotype background of intact HPV16 isolates might be of causal relevance for CaCx development.

CpG methylation of HPV 16 LCR at E2 binding site proximal to P97 is associated with cervical cancer in presence of intact E2.

Human papillomavirus type 16 (HPV-16) E2 protein negatively regulates transcription of the E6 and E7 genes. This study was done to test the hypothesis that methylation of the HPV 16 long control region (LCR) is overrepresented among cervical cancer (CaCx) cases compared to cytologically normal controls harboring intact E2 gene. Methylation of the E2 binding site (E2BS-I), proximal to the P97 promoter, was assessed by HpaII/ MspI restriction digestion while McrBC digestion was used to assess LCR-E6 (7289-540) for 57 CaCx samples and 15 normal controls. E2BS-I methylation was found to be significantly higher (56.14%) in cases compared to (20%) controls [OR(age-adjusted) (95% CI): 4.53 (1.05-19.43) p=0.042]. The difference between cases (54.39%) and controls (40%) with respect to LCR-E6 methylation status [OR(age-adjusted) (95% CI): 1.77(0.5-6.3); p=0.38] was not significant. Sequencing of a randomly selected set of 13 methylated malignant samples revealed absence or rare presence, of methylation at CpGs 7579, 7535, 7683 and 7862 respectively. Methylation was found to be more at CpGs within E2 binding sites proximal to the P97 promoter. These results indicate the involvement of E2 binding site methylation in presence of intact E2, leading to loss of E2 repressor activity in CaCx.

HPV16 E2 gene disruption and polymorphisms of E2 and LCR: some significant associations with cervical cancer in Indian women.

OBJECTIVES: We evaluated the status of the HPV16 E2 gene (disrupted or intact), nucleotide sequence alterations within intact E2 genes and LCR of HPV16 isolates in a group of CaCx cases (invasive squamous cell carcinomas, n = 81) and population controls (normal cervical scrapes, n = 27) from Indian women. METHODS: E2 disruption was detected by amplifying the entire E2 gene with single set of primers, while overlapping primers were used to determine if any particular region got selectively disrupted. Nucleotide variations in E2 and LCR were analyzed by PCR amplification followed by bi-directional sequencing. The associations between the viral factors and CaCx were analyzed using Fisher's Exact or Chi-squared test and interpreted as OR (95% CI) and P values. RESULTS: E2 disruption was significantly higher among the cases [3.38 (1.07-10.72); P = 0.02], which was maximum in the region between nucleotides 3650 and 3872 (DNA-binding region). The European (E) variant was found to be the prevalent subgroup (87.76% among cases and 96.30% among the controls), and the remaining samples were Asian-American variants. Among the E subgroup, variation at position 7450 (T > C) within the E2-binding site-IV was found to be significantly higher among the E2 undisrupted cases (21/37; 56.76%), compared to controls (5/18; 27.78%) [3.41 (1.01-11.55); P = 0.03]. CONCLUSIONS: Besides HPV16 E2 disruption, LCR 7450T > C variation within undisrupted E2 of E subgroup appears to be a major factor contributing to the risk of CaCx development in Indian women. Furthermore, polymorphisms in the E2 gene of HPV16 may not be significant for disease risk.