Comparative virome analysis of individual shedding routes of Miniopterus phillipsi bats inhabiting the Wavul Galge cave, Sri Lanka.

Bats are described as the natural reservoir host for a wide range of viruses. Although an increasing number of bat-associated, potentially human pathogenic viruses were discovered in the past, the full picture of the bat viromes is not explored yet. In this study, the virome composition of Miniopterus phillipsi bats (formerly known as Miniopterus fuliginosus bats in Sri Lanka) inhabiting the Wavul Galge cave, Sri Lanka, was analyzed. To assess different possible excretion routes, oral swabs, feces and urine were collected and analyzed individually by using metagenomic NGS. The data obtained was further evaluated by using phylogenetic reconstructions, whereby a special focus was set on RNA viruses that are typically associated with bats. Two different alphacoronavirus strains were detected in feces and urine samples. Furthermore, a paramyxovirus was detected in urine samples. Sequences related to Picornaviridae, Iflaviridae, unclassified Riboviria and Astroviridae were identified in feces samples and further sequences related to Astroviridae in urine samples. No viruses were detected in oral swab samples. The comparative virome analysis in this study revealed a diversity in the virome composition between the collected sample types which also represent different potential shedding routes for the detected viruses. At the same time, several novel viruses represent first reports of these pathogens from bats in Sri Lanka. The detection of two different coronaviruses in the samples indicates the potential general persistence of this virus species in M. phillipsi bats. Based on phylogenetics, the identified viruses are closely related to bat-associated viruses with comparably low estimation of human pathogenic potential. In further studies, the seasonal variation of the virome will be analyzed to identify possible shedding patterns for particular viruses.

First Complete Cytochrome B Sequences and Molecular Taxonomy of Bat Species from Sri Lanka.

This is the first report on the molecular identification and phylogeny of the Rousettus leschenaultii Desmarest, 1810, Rhinolophus rouxii Temminck, 1835, Hipposideros speoris Schneider, 1800, Hipposideros lankadiva Kelaart, 1850, and Miniopterus fuliginosus Kuhl, 1817, bat species in Sri Lanka, inferred from analyses by mitochondrially encoded cytochrome b gene sequences. Recent research has indicated that bats show enormous cryptic genetic diversity. Moreover, even within the same species, the acoustic properties of echolocation calls and morphological features such as fur color could vary in different populations. Therefore, we have used molecular taxonomy for the accurate identification of five bat species recorded in one of the largest cave populations in Sri Lanka. The bats were caught using a hand net, and saliva samples were collected non-invasively from each bat by using a sterile oral swab. Nucleic acids were extracted from the oral swab samples, and mitochondrial DNA was amplified by using primers targeting the mitochondrially encoded cytochrome b gene. This study reports the first molecular evidence for the identification of five bat species in Sri Lanka. Our findings will contribute to future conservation and systematic studies of bats in Sri Lanka. This study will also provide the basis for a genetic database of Sri Lankan bats which will contribute significantly to the investigation of potentially zoonotic bat viruses.

Transcriptomics of Acute DENV-Specific CD8+ T Cells Does Not Support Qualitative Differences as Drivers of Disease Severity.

While several lines of evidence suggest a protective role of T cells against disease associated with Dengue virus (DENV) infection, their potential contribution to immunopathology in the acute phase of DENV infection remains controversial, and it has been hypothesized that the more severe form of the disease (dengue hemorrhagic fever, DHF) is associated with altered T cell responses. To address this question, we determined the transcriptomic profiles of DENV-specific CD8+ T cells in a cohort of 40 hospitalized dengue patients with either a milder form of the disease (dengue fever, DF) or a more severe disease form (dengue hemorrhagic fever, DHF). We found multiple transcriptomic signatures, one associated with DENV-specific interferon-gamma responding cells and two other gene signatures, one specifically associated with the acute phase and the other with the early convalescent phase. Additionally, we found no differences in quantity and quality of DENV-specific CD8+ T cells based on disease severity. Taken together with previous findings that did not detect altered DENV-specific CD4 T cell responses, the current analysis argues against alteration in DENV-specific T cell responses as being a correlate of immunopathology.

Paramyxovirus Diversity within One Population of Miniopterus fuliginosus Bats in Sri Lanka.

Bats are known as typical reservoirs for a number of viruses, including viruses of the family Paramyxoviridae. Representatives of the subfamily Orthoparamyxovirinae are distributed worldwide and can cause mild to fatal diseases when infecting humans. The research on Paramyxoviruses (PMVs) from different bat hosts all over the world aims to understand the diversity, evolution and distribution of these viruses and to assess their zoonotic potential. A high number of yet unclassified PMVs from bats are recorded. In our study, we investigated bat species from the families Rhinolophidae, Hipposiderae, Pteropodidae and Miniopteridae that are roosting sympatrically in the Wavul Galge cave (Koslanda, Sri Lanka). The sampling at three time points (March and July 2018; January 2019) and screening for PMVs with a generic PCR show the presence of different novel PMVs in 10 urine samples collected from Miniopterus fuliginosus. Sequence analysis revealed a high similarity of the novel strains among each other and to other unclassified PMVs collected from Miniopterus bats. In this study, we present the first detection of PMVs in Sri Lanka and the presence of PMVs in the bat species M. fuliginosus for the first time.

Mild venom-induced consumption coagulopathy associated with thrombotic microangiopathy following a juvenile Russell's viper (Daboia russelii) envenoming: A case report.

Russell's viper envenoming causes venom-induced consumption coagulopathy (VICC) within hours of a bite, which is associated with thrombotic microangiopathy (TMA) and acute kidney injury (AKI) in a proportion of cases. We report a juvenile Russell's viper bite in which a patient developed mild VICC after the usual 24-h observation period, which was subsequently associated with severe AKI due to TMA. This shows the clinical importance of detecting and treating mild VICC, which may be delayed or not detected with bedside clotting tests.

Full Genome of batCoV/MinFul/2018/SriLanka, a Novel Alpha-Coronavirus Detected in Miniopterus fuliginosus, Sri Lanka.

Coronaviruses (CoV) are divided into the genera α-CoVs, ß-CoVs, γ-CoVs and δ-CoVs. Of these, α-CoVs and ß-CoVs are solely capable of causing infections in humans, resulting in mild to severe respiratory symptoms. Bats have been identified as natural reservoir hosts for CoVs belonging to these two genera. Consequently, research on bat populations, CoV prevalence in bats and genetic characterization of bat CoVs is of special interest to investigate the potential transmission risks. We present the genome sequence of a novel α-CoV strain detected in rectal swab samples of Miniopterus fuliginosus bats from a colony in the Wavul Galge cave (Koslanda, Sri Lanka). The novel strain is highly similar to Miniopterus bat coronavirus 1, an α-CoV located in the subgenus of Minunacoviruses. Phylogenetic reconstruction revealed a high identity of the novel strain to other α-CoVs derived from Miniopterus bats, while human-pathogenic α-CoV strains like HCoV-229E and HCoV-NL63 were more distantly related. Comparison with selected bat-related and human-pathogenic strains of the ß-CoV genus showed low identities of ~40%. Analyses of the different genes on nucleotide and amino acid level revealed that the non-structural ORF1a/1b are more conserved among α-CoVs and ß-CoVs, while there are higher variations in the structural proteins known to be important for host specificity. The novel strain was named batCoV/MinFul/2018/SriLanka and had a prevalence of 50% (66/130) in rectal swab samples and 58% (61/104) in feces samples that were collected from Miniopterus bats in Wavul Galge cave. Based on the differences between strain batCoV/MinFul/2018/SriLanka and human-pathogenic α-CoVs and ß-CoVs, we conclude that there is a rather low transmission risk to humans. Further studies in the Wavul Galge cave and at other locations in Sri Lanka will give more detailed information about the prevalence of this virus.

A Population of CD4+CD8+ Double-Positive T Cells Associated with Risk of Plasma Leakage in Dengue Viral Infection.

According to the WHO 2009 classification, dengue with warning signs is at the risk of developing severe form of dengue disease. One of the most important warning signs is plasma leakage, which can be a serious complication associated with higher morbidity and mortality. We report that the frequency of CD4+CD8+ double-positive (DP) T cells is significantly increased in patients at risk of developing plasma leakage. Transcriptomic analysis demonstrated that CD4+CD8+ DP cells were distinct from CD4+ Single Positive (SP) T cells but co-clustered with CD8+ SP cells, indicating a largely similar transcriptional profile. Twenty significant differentially expressed (DE) genes were identified between CD4+CD8+ DP and CD8+ SP cells. These genes encode OX40 and CCR4 proteins as well as other molecules associated with cell signaling on the cell surface (NT5E, MXRA8, and PTPRK). While comparing the profile of gene expression in CD4+CD8+ DP cells from patients with and without warning signs of plasma leakage, similar expression profile was observed, implying a role of CD4+CD8+ DP cells in plasma leakage through a quantitative increase rather than functional alteration. This study provided novel insight into the host immune response during the acute febrile phase of DENV infection and the role of CD4+CD8+ DP T cells in the pathogenesis of plasma leakage.

Predictive value of hepatic transaminases during febrile phase as a predictor of a severe form of Dengue: analysis of adult Dengue patients from a tertiary care setting of Sri Lanka.

OBJECTIVES: Dengue viral infection is an ongoing epidemic in Sri Lanka, causing significant mortality and morbidity. A descriptive-analytical study was carried out using serologically confirmed Dengue patients during a 6 month period. The relationship between the elevation of hepatic enzymes and severity of Dengue was assessed after stratifying recorded maximum AST/ALT (SGOT/SGPT) values 2-15 times elevated and by the phases of the illness. Sensitivity, specificity, predictive values, and ROC curves were assessed using maximum values for AST and ALT. RESULTS: Out of 255 patients, 107(42%) were females. The majority (52.9%) were in the 20-39 year age group. Only 19.6% had DHF. No statistically significant difference was noticed in the values of maximum transaminases during the febrile phase among DF and DHF patients. Higher sensitivity and low specificity with the 1-5 times elevation range was noticed, and a higher cut-off level of more than 5 times elevation showed low sensitivity and higher specificity. The combination of both transaminases cut-offs with age and sex also does not show clinically significant predictability of severe disease. The AST and ALT elevations are not showing discriminatory predictive value on dengue severity. As different serotypes cause different epidemics, it is important to carry out large-scale specific studies considering the serotypes.

Detection of Alpha- and Betacoronaviruses in Miniopterus fuliginosus and Rousettus leschenaultii, two species of Sri Lankan Bats.

Bats are known to be potential reservoirs of numerous human-pathogenic viruses. They have been identified as natural hosts for coronaviruses, causing Severe Acute Respiratory Syndrome (SARS) in humans. Since the emergence of SARS-CoV-2 in 2019 interest in the prevalence of coronaviruses in bats was newly raised. In this study we investigated different bat species living in a sympatric colony in the Wavul Galge cave (Koslanda, Sri Lanka). In three field sessions (in 2018 and 2019), 395 bats were captured (Miniopterus, Rousettus, Hipposideros and Rhinolophus spp.) and either rectal swabs or fecal samples were collected. From these overall 396 rectal swab and fecal samples, the screening for coronaviruses with nested PCR resulted in 33 positive samples, 31 of which originated from Miniopterus fuliginosus and two from Rousettus leschenaultii. Sanger sequencing and phylogenetic analysis of the obtained 384-nt fragment of the RNA-dependent RNA polymerase revealed that the examined M. fuliginosus bats excrete alphacoronaviruses and the examined R. leschenaultii bats excrete betacoronaviruses. Despite the sympatric roosting habitat, the coronaviruses showed host specificity and seemed to be limited to one species. Our results represent an important basis to better understand the prevalence of coronaviruses in Sri Lankan bats and may provide a basis for pursuing studies on particular bat species of interest.

High Levels of Serum Angiopoietin 2 and Angiopoietin 2/1 Ratio at the Critical Stage of Dengue Hemorrhagic Fever in Patients and Association with Clinical and Biochemical Parameters.

Longitudinal changes of serum angiopoietin 1 (Ang-1) and angiopoietin 2 (Ang-2) associated with endothelial stability in dengue patients with different disease stages were studied. Serum Ang-1 and Ang-2 levels were measured in confirmed dengue fever (DF) patients on admission (DFA, n = 40) and discharge (DFD, n = 20); in dengue hemorrhagic fever (DHF) patients on admission (DHFA, n = 40), at critical stage (DHFC, n = 36), and on discharge (DHFD, n = 20); and in healthy controls (HC, n = 25). DHFC had the highest serum Ang-2 and lowest Ang-1 levels compared to DFA, DHFA, and HC (P < 0.050). The ratio of serum Ang-2/Ang-1 in DHFC was the highest among all study categories tested (P < 0.001). Significant positive correlations were observed between serum Ang-1 and platelet count in DHFA (Pearson r = 0.653, P < 0.001) and between Ang-1 and pulse pressure in DHFC (r = 0.636, P = 0.001). Using a cutoff value of 1.01 for the Ang-2/Ang-1 ratio for DHFC, a sensitivity of 83.2% and a specificity of 81.2% discerning DF from DHF were obtained. Therefore, serum Ang-2/Ang-1 could be used as a biomarker for endothelial dysfunction in severe dengue at the critical stage.

Genetic risk for dengue hemorrhagic fever and dengue fever in multiple ancestries.

BACKGROUND: Genetic risk factors for dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) and dengue fever (DF) are limited, in particular there are sparse data on genetic risk across diverse populations. METHODS: We conducted a genome-wide association study (GWAS) in a derivation and validation sample of 7, 460 participants of Latin American, South Asian, and South East Asian ancestries. We then developed a weighted polygenic risk score (PRS) for each participant in each of the validation cohorts of the three ancestries to predict the risk of DHF/DSS compared to DF, DHF/DSS compared to controls, and, DF compared to controls. FINDINGS: The risk of DHF/DSS was significantly increased, odds ratio [OR] 1.84 (95%CI 1.47 to 2.31) (195 SNPs), compared to DF, fourth PRS quartile versus first quartile, in the validation cohort. The risk of DHF/DSS compared to controls was increased (OR=3.94; 95% CI 2.84 to 5.45) (278 SNPs), as was the risk of DF compared to controls (OR=1.97; 95%CI 1.63 to 2.39) (251 SNPs). Risk increased in a dose-dependent manner with increase in quartiles of PRS across comparisons. Significant associations persisted for PRS built within ancestries and applied to the same or different ancestries as well as for PRS built for one outcome (DHF/DSS or DF) and applied to the other. INTERPRETATION: There is a strong genetic effect that predisposes to risk of DHF/DSS and DF. The genetic risk for DHF/DSS is higher than that for DF when compared to controls, and this effect persists across multiple ancestries.

Molecular Signatures of Dengue Virus-Specific IL-10/IFN-γ Co-producing CD4 T Cells and Their Association with Dengue Disease.

Dengue virus (DENV) can cause diseases ranging from dengue fever (DF) to more severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Whether antiviral T cells contribute to the protection against or pathogenesis of severe disease is not well defined. Here, we identified antigen-specific IL-10+IFN-γ+ double-positive (DP) CD4 T cells during acute DENV infection. While the transcriptomic signatures of DP cells partially overlapped with those of cytotoxic and type 1 regulatory CD4 T cells, the majority of them were non-cytotoxic/Tr1 and included IL21, IL22, CD109, and CCR1. Although we observed a higher frequency of DP cells in DHF, the transcriptomic profile of DP cells was similar in DF and DHF, suggesting that DHF is not associated with the altered phenotypic or functional attributes of DP cells. Overall, this study revealed a DENV-specific DP cell subset in patients with acute dengue disease and argues against altered DP cells as a determinant of DHF.

Circulating T cell-monocyte complexes are markers of immune perturbations.

Our results highlight for the first time that a significant proportion of cell doublets in flow cytometry, previously believed to be the result of technical artifacts and thus ignored in data acquisition and analysis, are the result of biological interaction between immune cells. In particular, we show that cell:cell doublets pairing a T cell and a monocyte can be directly isolated from human blood, and high resolution microscopy shows polarized distribution of LFA1/ICAM1 in many doublets, suggesting in vivo formation. Intriguingly, T cell-monocyte complex frequency and phenotype fluctuate with the onset of immune perturbations such as infection or immunization, reflecting expected polarization of immune responses. Overall these data suggest that cell doublets reflecting T cell-monocyte in vivo immune interactions can be detected in human blood and that the common approach in flow cytometry to avoid studying cell:cell complexes should be re-visited.

Dengue type 1 viruses circulating in humans are highly infectious and poorly neutralized by human antibodies.

The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses of humans. The interactions between DENVs and the human host that lead to asymptomatic, mild, or severe disease are poorly understood, in part, because laboratory models are poor surrogates for human DENV disease. Virologists are interested in how the properties of DENVs replicating in people compare with virions propagated on laboratory cell lines, which are widely used for research and vaccine development. Using clinical samples from a DENV type 1 epidemic in Sri Lanka and new ultrasensitive assays, we compared the properties of DENVs in human plasma and after one passage on laboratory cell lines. DENVs in plasma were 50- to 700-fold more infectious than cell culture-grown viruses. DENVs produced by laboratory cell lines were structurally immature and hypersensitive to neutralization by human antibodies compared with DENVs circulating in people. Human plasma and cell culture-derived virions had identical genome sequences, indicating that these phenotypic differences were due to the mature state of plasma virions. Several dengue vaccines are under development. Recent studies indicate that vaccine-induced antibodies that neutralized DENVs in cell culture assays were not sufficient for protecting people from DENV infections. Our results about structural differences between DENVs produced in humans versus cell lines may be key to understanding vaccine failure and developing better models for vaccine evaluation.

Is Total Serum Nitrite and Nitrate (NOx) Level in Dengue Patients a Potential Prognostic Marker of Dengue Hemorrhagic Fever?

Potential use of total nitrite plus nitrate (NOx) and nitrite (NO2-) separately as surrogate markers for serum nitric oxide in severe dengue and their longitudinal changes along with the progression of infection was studied. Deproteinized sera from confirmed dengue fever (DF, n = 145) and dengue hemorrhagic fever (DHF, n = 74) patients on admission-A, critical-C, discharge-D, and convalescence-CON stages and from age-gender matched healthy individuals (HC, n = 77) were taken to assess NO2- and NOx levels using Griess and modified Griess assays. Serum NOx in DHFA was significantly lower compared to DFA (p < 0.001). HC had the lowest NOx and NO2- compared to all patient categories (p < 0.001) except NO2- in DF-CON and DHF-CON and NOx in DHF-CON. Serum NOx and NO2- in DHF patients admitted on fever day 3 (DHFA-3) was significantly lower compared to DFA-3 (p < 0.05). Cut-off values of 4.46 µM for NOx (91.3% sensitivity and 80.1% specificity) and 1.25 µM for NO2- (75.0% sensitivity and 73.3% specificity) were obtained for day 3 of fever. Serum NOx may be used as potential prognostic marker of DHF in patients presenting with DF in the early stage (on day 3 of fever) of the disease.

Sequence-based HLA-A, B, C, DP, DQ, and DR typing of 714 adults from Colombo, Sri Lanka.

DNA sequence-based typing at the HLA-A, -B, -C, -DPB1, -DQA1, -DQB1, and -DRB1 loci was performed on 714 healthy adult blood bank donors from Colombo, Sri Lanka, to characterize allele frequencies in support of studies on T cell immunity against pathogens, including Dengue virus. Deviations from Hardy Weinberg proportions were not detected at any locus. Several alleles were found in >30% of individuals, including the class II alleles DPB1 * 04:01, DPB1 * 02:01, DQB1 * 06:01 and DRB1 * 07:01, and the class I alleles A * 33:03 and A * 24:02. Genotype data will be available in the Allele Frequencies Net Database.

Positive melioidosis serology in a patient with adult onset Still's disease: a case report of a diagnostic dilemma.

BACKGROUND: Autoimmune disorders are known to produce false positives in serological tests for infections. Aetiological association between infections and autoimmunity, increased susceptibility to infectious and autoimmune disorders with immune dysregulation and non-specific polyclonal expansion of B cells with autoimmunity may cause confusion in diagnosis and patient management. We report a patient with Adult Onset Still's Disease (AOSD) presenting with rising melioidosis antibody titres that caused diagnostic confusion. CASE PRESENTATION: A forty-nine-year-old female presented with prolonged fever, sore-throat, large joint arthritis, lymphadenopathy, hepatomegaly and transient rash. She had elevated inflammatory markers and a rising melioidosis antibody titre. The patient responded poorly to prolonged course of appropriate antimicrobials but showed rapid and sustained improvement with glucocorticoids. CONCLUSION: Positive melioidosis serology could have been due to a co-infection or false positive antibody reaction due to non-specific B cell expansion or an indicator of true infection that triggered the immune dysregulation to develop AOSD.

Unique phenotypes and clonal expansions of human CD4 effector memory T cells re-expressing CD45RA.

The expression of CD45RA is generally associated with naive T cells. However, a subset of effector memory T cells re-expresses CD45RA (termed TEMRA) after antigenic stimulation with unknown molecular characteristics and functions. CD4 TEMRA cells have been implicated in protective immunity against pathogens such as dengue virus (DENV). Here we show that not only the frequency but also the phenotype of CD4 TEMRA cells are heterogeneous between individuals. These cells can be subdivided into two major subsets based on the expression of the adhesion G protein-coupled receptor GPR56, and GPR56+ TEMRA cells display a transcriptional and proteomic program with cytotoxic features that is distinct from effector memory T cells. Moreover, GPR56+ TEMRA cells have higher levels of clonal expansion and contain the majority of virus-specific TEMRA cells. Overall, this study reveals the heterogeneity of CD4 TEMRA cells and provides insights into T-cell responses against DENV and other viral pathogens.

A case report of co-infection of Melioidosis and cutaneous Leishmaniasis.

BACKGROUND: Leishmaniasis and melioidosis are frequently reported from the North Central Province of Sri Lanka. However, only one case of co-infection of the two diseases has been reported to date over the world. This is a case report of a patient who had co-infection with cutaneous leishmaniasis and melioidosis and was successfully treated and recovered from the illness. CASE PRESENTATION: A 61 year old female patient with diabetes mellitus presented with fever for one month's duration and was found to have hepatosplenomegaly and an ulcer over the left arm. She had elevated inflammatory markers and blood culture grew Burkholderia pseudomallei and serum was highly positive for melioidosis antibodies. A slit skin smear of the ulcer showed Leishmania amastigotes. CONCLUSION: Melioidosis and leishmaniasis are emerging infectious diseases in endemic countries and can be severe. The high prevalence rates in Sri Lanka should keep the treating physicians' threshold for suspicion low for these two diseases.

HLA-DRB1 Alleles Are Associated With Different Magnitudes of Dengue Virus-Specific CD4+ T-Cell Responses.

BACKGROUND: Each year dengue virus (DENV) infects 400 million human but causes symptomatic disease in only a subset of patients, suggesting that host genetic factors may play a role. HLA molecules that restrict T-cell responses are one of the most polymorphic host factors in humans. METHODS: Here we map HLA DRB1-restricted DENV-specific epitopes in individuals previously exposed to DENV, to identify the breadth and specificity of CD4(+) T-cell responses. To investigate whether HLA-specific variations in the magnitude of response might predict associations between dengue outcomes and HLA-DRB1 alleles, we assembled samples from hospitalized patients with known severity of disease. RESULTS: The capsid protein followed by nonstructural protein 3 (NS3), NS2A, and NS5 were the most targeted proteins. We further noticed a wide variation in magnitude of T-cell responses as a function of the restricting DRB1 allele and found several HLA alleles that showed trends toward a lower risk of hospitalized disease were associated with a higher magnitude of T-cell responses. CONCLUSIONS: Comprehensive identification of unique CD4(+) T-cell epitopes across the 4 DENV serotypes allows the testing of T-cell responses by use of a simple, approachable technique and points to important implications for vaccine design.