Contemporary and historical human migration patterns shape hepatitis B virus diversity.

Infection by hepatitis B virus (HBV) is responsible for approximately 296 million chronic cases of hepatitis B, and roughly 880,000 deaths annually. The global burden of HBV is distributed unevenly, largely owing to the heterogeneous geographic distribution of its subtypes, each of which demonstrates different severity and responsiveness to antiviral therapy. It is therefore crucial to the global public health response to HBV that the spatiotemporal spread of each genotype is well characterized. In this study, we describe a collection of 133 newly sequenced HBV strains from recent African immigrants upon their arrival in Belgium. We incorporate these sequences-all of which we determine to come from genotypes A, D, and E-into a large-scale phylogeographic study with genomes sampled across the globe. We focus on investigating the spatio-temporal processes shaping the evolutionary history of the three genotypes we observe. We incorporate several recently published ancient HBV genomes for genotypes A and D to aid our analysis. We show that different spatio-temporal processes underlie the A, D, and E genotypes with the former two having originated in southeastern Asia, after which they spread across the world. The HBV E genotype is estimated to have originated in Africa, after which it spread to Europe and the Americas. Our results highlight the use of phylogeographic reconstruction as a tool to understand the recent spatiotemporal dynamics of HBV, and highlight the importance of supporting vulnerable populations in accordance with the needs presented by specific HBV genotypes.

Respiratory Viruses in Wastewater Compared with Clinical Samples, Leuven, Belgium.

In a 2-year study in Leuven, Belgium, we investigated the use of wastewater sampling to assess community spread of respiratory viruses. Comparison with the number of positive clinical samples demonstrated that wastewater data reflected circulation levels of typical seasonal respiratory viruses, such as influenza, respiratory syncytial virus, and enterovirus D68.

Exploring the relationship between anellovirus load and clinical variables in hospitalized COVID-19 patients: Implications for immune activation and inflammation.

Objectives: Anelloviruses have been linked with host-immunocompetence and inflammation. Here, we studied the anellovirus load in hospitalized COVID-19 patients. Methods: We collected samples of patients recruited in the DAWN-Plasma trial that received convalescent plasma (CP) therapy (four plasma units) combined with standard of care (SOC) or SOC of alone. Plasma samples were collected on day 0 and 6 of hospitalization and we quantified anellovirus load. With multivariate models, clinical variables were associated with changes in anellovirus load. Results: Samples were collected on day 0 and 6 of 150 patients (103 CP + SOC and 47 SOC). Anellovirus load was higher on day 0 compared to day 6 and we found a significant drop in SOC patients. Patients receiving immunosuppressive drug had a lower anellovirus load (coefficient: 1.021, 95% confidence interval [CI] 0.270-1.772, P = 0.008), while patients admitted to the emergency room displayed a higher abundance on day 0 (1.308, 95% CI 0.443-2.173, P = 0.003). Unspecific markers of inflammation and organ damage, D-dimer (0.001, 95% CI <0.001-0.001, P = 0.001) and lactate dehydrogenase (0.002, 95% CI 0.001-0.004, P = 0.044), were positively associated with anellovirus load. Finally, anellovirus load on day 0 (-39.9, 95% CI -75.72 to -4.27, P = 0.029) was negatively associated with SARS-CoV-2 antibody response on day. Conclusion: The results showed associations between clinical variables and anellovirus load in COVID-19 patients. Many variables share properties related to host immunocompetence or inflammation. Therefore, we expect that anellovirus abundance displays the net state of immune activation.

Expansion of Betatorquevirus and/or Gammatorquevirus in Patients with Severe Clinical Outcomes of the Liver Diseases.

Anellovirus (AV) is a ubiquitous virus in the human population. Individuals can be infected with multiple AV genera and species to form a heterogeneous repertoire, termed the anellome. Using advanced methods, we examined the anellomes from 12 paired serum and liver samples, as well as 2701 subjects with different clinical diagnoses. Overall, anellomes are remarkably individualized, with significant among-group differences (Kruskal-Wallis test p = 6.6 × 10-162 for richness and p = 7.48 × 10-162 for Shannon entropy). High dissimilarity scores (beta diversity) were observed between patient groups, except for paired serum and liver samples. At the population level, the relative abundance of combinational AV genus Betatorquevirus (torque teno mini viruses, TTMV), and Gammatorquevirus (torque teno midi viruses, TTMDV) exhibited an exponential distribution with a low bound point at 32%. Defined by this value, the AV TTMV/TTMDV-expanded anellome was significantly enriched among patients with acute liver failure (31.7%) and liver transplantation (40.7%), compared with other patient groups (χ2 test: p = 4.1 × 10-8-3.2 × 10-3). Therefore, anellome heterogeneity may be predictive of clinical outcomes in certain diseases, such as liver disease. The consistency of anellome between paired serum and liver samples indicates that a liquid biopsy approach would be suitable for longitudinal studies to clarify the causality of the AV TTMV/TTMDV-expanded anellome in the outcomes of liver disease.

Characterization of the Human Blood Virome in Iranian Multiple Transfused Patients.

Blood transfusion safety is an essential element of public health. Current blood screening strategies rely on targeted techniques that could miss unknown or unexpected pathogens. Recent studies have demonstrated the presence of a viral community (virobiota/virome) in the blood of healthy individuals. Here, we characterized the blood virome in patients frequently exposed to blood transfusion by using Illumina metagenomic sequencing. The virome of these patients was compared to viruses present in healthy blood donors. A total number of 155 beta-thalassemia, 149 hemodialysis, and 100 healthy blood donors were pooled with five samples per pool. Members of the Anelloviridae and Flaviviridae family were most frequently observed. Interestingly, samples of healthy blood donors harbored traces of potentially pathogenic viruses, including adeno-, rota-, and Merkel cell polyomavirus. Viruses of the Anelloviridae family were most abundant in the blood of hemodialysis patients and displayed a higher anellovirus richness. Pegiviruses (Flaviviridae) were only observed in patient populations. An overall trend of higher eukaryotic read abundance in both patient groups was observed. This might be associated with increased exposure through blood transfusion. Overall, the findings in this study demonstrated the presence of various viruses in the blood of Iranian multiple-transfused patients and healthy blood donors.

Plasma virome dynamics in chronic hepatitis B virus infected patients.

The virome remains an understudied domain of the human microbiome. The role of commensal viruses on the outcome of infections with known pathogens is not well characterized. In this study we aimed to characterize the longitudinal plasma virome dynamics in chronic hepatitis B virus (HBV) infected patients. Eighty-five longitudinal plasma samples were collected from 12 chronic HBV infected individuals that were classified in the four stages of HBV infection. The virome was characterized with an optimized viral extraction protocol and deep-sequenced on a NextSeq 2500 platform. The plasma virome was primarily composed of members of the Anello- Flavi-, and Hepadnaviridae (HBV) families. The virome structure and dynamics did not correlate with the different stages of chronic HBV infection nor with the administration of antiviral therapy. We observed a higher intrapersonal similarity of viral contigs. Genomic analysis of viruses observed in multiple timepoint demonstrated the presence of a dynamic community. This study comprehensively assessed the blood virome structure in chronic HBV infected individuals and provided insights in the longitudinal development of this viral community.

Indoor air surveillance and factors associated with respiratory pathogen detection in community settings in Belgium.

Currently, the real-life impact of indoor climate, human behaviour, ventilation and air filtration on respiratory pathogen detection and concentration are poorly understood. This hinders the interpretability of bioaerosol quantification in indoor air to surveil respiratory pathogens and transmission risk. We tested 341 indoor air samples from 21 community settings in Belgium for 29 respiratory pathogens using qPCR. On average, 3.9 pathogens were positive per sample and 85.3% of samples tested positive for at least one. Pathogen detection and concentration varied significantly by pathogen, month, and age group in generalised linear (mixed) models and generalised estimating equations. High CO2 and low natural ventilation were independent risk factors for detection. The odds ratio for detection was 1.09 (95% CI 1.03-1.15) per 100 parts per million (ppm) increase in CO2, and 0.88 (95% CI 0.80-0.97) per stepwise increase in natural ventilation (on a Likert scale). CO2 concentration and portable air filtration were independently associated with pathogen concentration. Each 100ppm increase in CO2 was associated with a qPCR Ct value decrease of 0.08 (95% CI -0.12 to -0.04), and portable air filtration with a 0.58 (95% CI 0.25-0.91) increase. The effects of occupancy, sampling duration, mask wearing, vocalisation, temperature, humidity and mechanical ventilation were not significant. Our results support the importance of ventilation and air filtration to reduce transmission.

Monitoring of SARS-CoV-2 concentration and circulation of variants of concern in wastewater of Leuven, Belgium.

Wastewater surveillance plays an important role in the management of the coronavirus disease 2019 (COVID-19) pandemic all over the world. Using different wastewater collection points in Leuven, we wanted to investigate the use of wastewater surveillance as an early warning system for an uprise of infections and as a tool to follow the circulation of specific variants of concern (VOCs) in particular geographic areas. Wastewater samples were collected from local neighborhood sewers and from a large regional wastewater treatment plant (WWTP) in the area of Leuven, Belgium. After virus concentration, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was quantified by real-time quantitative polymerase chain reaction (RT-qPCR) and normalized with the human fecal indicator pepper mild mottle virus (PMMoV). A combination of multiplex RT-qPCR assays was used to detect signature mutations of circulating VOCs. Fecal virus shedding of SARS-CoV-2 variants was measured in feces samples of hospitalized patients. In two residential sampling sites, a rise in wastewater SARS-CoV-2 concentration preceded peaks in positive cases. In the WWTP, viral load peaks were seen concomitant with the consecutive waves of positive cases caused by the original Wuhan SARS-CoV-2 strain and subsequent VOCs. During the Omicron BA.1 wave, the wastewater viral load increased to a lesser degree, even after normalization of SARS-CoV-2 concentration using PMMoV. This might be attributable to a lower level of fecal excretion of this variant. Circulation of SARS-CoV-2 VOCs Alpha, Delta, Omicron BA1/BA.2, and BA.4/BA.5 could be detected based on the presence of specific key mutations. The shift in variants was noticeable in the wastewater, with key mutations of two different variants being present simultaneously during the transition period. Wastewater-based surveillance is a sensitive tool to monitor SARS-CoV-2 circulation levels and VOCs in larger regions. In times of reduced test capacity, this can prove to be highly valuable. Differences in excretion levels of various SARS-CoV-2 variants should however be taken into account when using wastewater surveillance to monitor SARS-CoV-2 circulation levels in the population.

Reconstruction of the origin and dispersal of the worldwide dominant Hepatitis B Virus subgenotype D1.

Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV). HBV-D1 is the dominant subgenotype in the Mediterranean basin, Eastern Europe, and Asia. However, little is currently known about its evolutionary history and spatio-temporal dynamics. We use Bayesian phylodynamic inference to investigate the temporal history of HBV-D1, for which we calibrate the molecular clock using ancient sequences, and reconstruct the viral global spatial dynamics based, for the first time, on full-length publicly available HBV-D1 genomes from a wide range of sampling dates. We pinpoint the origin of HBV subgenotype D1 before the current era (BCE) in Turkey/Anatolia. The spatial reconstructions reveal global viral transmission with a high degree of mixing. By combining modern-day and ancient sequences, we ensure sufficient temporal signal in HBV-D1 data to enable Bayesian phylodynamic inference using a molecular clock for time calibration. Our results shed light on the worldwide HBV-D1 epidemics and suggest that this originally Middle Eastern virus significantly affects more distant countries, such as those in mainland Europe.

Tissue presentation of human pegivirus infection in liver transplanted recipients.

Human pegivirus-1 (HPgV-1) is known for its protective role in HIV co-infected individuals. This immunomodulatory effect raised questions concerning the possible role of HPgV-1 infection and the risk of rejection in liver transplanted patients. We aimed to evaluate the possible protective effect of HPgV-1 on graft outcome of liver transplanted patients. A total of 283 patients were recruited. Formalin-fixed paraffin-embedded tissue samples were collected from the explanted liver. HBV-DNA, HCV-RNA, and HPgV-1-RNA were determined using PCR and multiplex RT-PCR assays. The clinical course of patients including the occurrence of acute cellular rejection was compared between HPgV-1-infected vs. uninfected patients. HBV-DNA, HCV-RNA and HPgV-1-RNA were detected in 42.6%, 4.9%, and 7.8% of samples, respectively. None of the HPgV-1-infected patients experienced graft rejection. Group LASSO logistic regression revealed that HPgV-1 infection was the only factor which significantly reduced the odds of graft rejection (OR = 0.5, 95% CI = 0.29-0.89). No significant association was found between the presence of HPgV-1 with HBV and HCV infections. The lack of graft rejection in HPgV-1-infected liver transplanted patients might indicate a possible role of this virus for graft surveillance. Since these are still preliminary findings, prospective studies should further elucidate the role of HPgV-1 in liver transplantation outcomes.

Genomic and serological assessment of asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in child labor.

Since working children have limited access to testing and monitoring for COVID-19, we decided to measure SARS-CoV-2 prevalence among them and compare it to non-working children. Our objective is to compare the frequency of SARS-CoV-2 genome and anti-SARS-CoV-2 antibody among working and non-working children. Volunteer child labor studying at Defense of Child Labor and Street Children and randomly selected 5-18-year-old (same range as child labor group) unemployed children participated in this study. The groups, respectively, had 65 and 137 members. This is an analytical cross-sectional study that surveys molecular prevalence of SARS-CoV-2 infection by RT-PCR, and seroprevalence of SARS-CoV-2 antibody by ELISA in working and non-working children. The IBM SPSS statistics software version 25 was used for data analysis. The χ2 or Fisher's exact test was used to analyze categorical dependent variables, for calculating odds ratios and 95% confidence intervals. Among the children enrolled in this study, molecular prevalence of SARS-CoV-2 turned out to be 18.5% in working children while it was 5.8% in unemployed children [aOR: 3.00 (CI95%: 1.00-7.00); P value: 0.003] and seroprevalence turned out to be 20% in working children vs 13.9% in non-working children [aOR: 1.000 (CI95%: 0.00-2.00); > P 0.001]. Equal SARS-CoV-2 viral load as adults and no symptoms or mild ones in children, coupled with working children's strong presence in crowded areas and their higher rate of COVID-19 prevalence, make them a probable source for spread of the virus.

A perspective on the applications of furin inhibitors for the treatment of SARS-CoV-2.

Currently, the world is facing a pandemic of the new coronavirus SARS-CoV-2 that causes COVID-19. Identifying key targets in the viral infection lifecycle is urgently needed for designing therapeutic strategies to combat the virus. Furin is a subtilisin-like proprotein convertase with diverse cellular functions. Emerging evidence suggests that furin plays a critical role in the activation and/or infectivity of SARS-CoV-2. In this perspective, we discuss the potential role of furin in the entry SARS-CoV-2 into host cells. Furthermore, we evaluate available peptide and non-peptide furin inhibitors and potential outcomes, including immune responses.

Exploration of the Ixodes ricinus virosphere unveils an extensive virus diversity including novel coltiviruses and other reoviruses.

Recent metagenomics studies have revealed several tick species to host a variety of previously undiscovered RNA viruses. Ixodes ricinus, which is known to be a vector for many viral, bacterial, and protozoan pathogens, is the most prevalent tick species in Europe. For this study, we decided to investigate the virosphere of Belgian I. ricinus ticks. High-throughput sequencing of tick pools collected from six different sampling sites revealed the presence of viruses belonging to many different viral orders and families, including Mononegavirales, Bunyavirales, Partitiviridae, and Reoviridae. Of particular interest was the detection of several new reoviruses, two of which cluster together with members of the genus Coltivirus. This includes a new strain of Eyach virus, a known causative agent of tick-borne encephalitis. All genome segments of this new strain are highly similar to those of previously published Eyach virus genomes, except for the fourth segment, encoding VP4, which is markedly more dissimilar, potentially indicating the occurrence of a genetic reassortment. Further polymerase chain reaction-based screening of over 230 tick pools for 14 selected viruses showed that most viruses could be found in all six sampling sites, indicating the wide spread of these viruses throughout the Belgian tick population. Taken together, these results illustrate the role of ticks as important virus reservoirs, highlighting the need for adequate tick control measures.

Human gene polymorphisms and their possible impact on the clinical outcome of SARS-CoV-2 infection.

The SARS-CoV-2 pandemic has become one of the most serious health concerns globally. Although multiple vaccines have recently been approved for the prevention of coronavirus disease 2019 (COVID-19), an effective treatment is still lacking. Our knowledge of the pathogenicity of this virus is still incomplete. Studies have revealed that viral factors such as the viral load, duration of exposure to the virus, and viral mutations are important variables in COVID-19 outcome. Furthermore, host factors, including age, health condition, co-morbidities, and genetic background, might also be involved in clinical manifestations and infection outcome. This review focuses on the importance of variations in the host genetic background and pathogenesis of SARS-CoV-2. We will discuss the significance of polymorphisms in the ACE-2, TMPRSS2, vitamin D receptor, vitamin D binding protein, CD147, glucose-regulated protein 78 kDa, dipeptidyl peptidase-4 (DPP4), neuropilin-1, heme oxygenase, apolipoprotein L1, vitamin K epoxide reductase complex 1 (VKORC1), and immune system genes for the clinical outcome of COVID-19.

Convalescent Plasma against COVID-19: A Broad-Spectrum Therapeutic Approach for Emerging Infectious Diseases.

In the lack of an effective vaccine and antiviral treatment, convalescent plasma (CP) has been a promising therapeutic approach in past pandemics. Accumulating evidence in the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic corroborates the safety of CP therapy and preliminary data underline the potential efficacy. Recently, the Food and Drug Administration (FDA) permitted CP therapy for coronavirus disease 2019 (COVID-19) patients under the emergency use authorization, albeit additional clinical studies are still needed. The imminent threat of a second or even multiple waves of COVID-19 has compelled health authorities to delineate and calibrate a feasible preparedness algorithm for deploying CP as an immediate therapeutic intervention. The success of preparedness programs depends on the interdisciplinary actions of multiple actors in politics, science, and healthcare. In this review, we evaluate the current status of CP therapy for COVID-19 patients and address the challenges that confront the implementation of CP. Finally, we propose a pandemic preparedness framework for future waves of the COVID-19 pandemic and unknown pathogen outbreaks.

Clinical relevance of plasma virome dynamics in liver transplant recipients.

BACKGROUND: The role of the microbiome in liver transplantation (LT) outcome has received a growing interest in the past decades. In contrast to bacteria, the role of endogenous viral communities, known as the virome, is poorly described. Here, we applied a viral metagenomic approach to study the dynamic evolution of circulating viruses in the plasma of LT recipients and its effect on the clinical course of patients. METHODS: Patients chronically infected with hepatitis B virus (HBV) that received a LT due to endstage liver disease were included in this study. Longitudinal plasma samples were collected pre- and post-LT. Intact viral particles were isolated and sequenced on an Illumina HiSeq 2500 platform. Short read libraries were analysed with an in-house bioinformatics pipeline. Key endpoints were the dynamics of viral families and post-LT complications. FINDINGS: The initiation of immunosuppression induced a bloom of the Anelloviridae that dominated the post-LT plasma virome. A variety of post-LT complication were observed. Nephrotoxicity was reported in 38% of the patients and was associated with a high abundance of anelloviruses. Besides nephrotoxicity, 16 (67%) patients experienced flares of viral or bacterial infections in post-transplant follow-up. These flares were recognized by an increased burden of anelloviruses (p < 0.05). Interestingly, no mortality was observed in patients infected with human pegivirus. INTERPRETATION: These findings suggest a diagnostic potential for the Anelloviridae family in post-LT complications. Furthermore, the impact of human pegivirus infection on post-transplant survival should be further investigated. FUNDING: This trial was supported by Gilead Sciences grant number BE-2017-000133.

Novel hepatitis B virus subgenotype A8 and quasi-subgenotype D12 in African-Belgian chronic carriers.

BACKGROUND: Hepatitis B virus (HBV) is a public health threatening virus and is classified into more than eight genotypes and more than forty subgenotypes. OBJECTIVES: To characterize and propose novel strains assigned as A8 and D12. METHODS: Four out of 133 HBV complete genome sequences, isolated from Belgian chronic carriers with African origin were phylogenetically analyzed. RESULTS: Phylogenetic analyses of HBV genotypes A and D strains exhibited separate clusters supported by significant bootstrap values. The two genotype A strains isolated from Congolese patients, and two genotype D strains isolated from Ghanaian carriers clustered separately from the other known subgenotypes A (A1-A6 and quasi-subgenotypes) and subgenotypes D (D1-D11). The mean inter-subgenotypic nucleotide divergence over the full-length genome sequence between the novel strains (A8 and D12) and A1-A7 and D1-D11 subgenotypes was higher than 4%. CONCLUSIONS: Phylogenetic analysis of the full-length HBV genome sequences revealed a novel subgenotype and quasi-subgenotype based on the nucleotide divergence and identification of novel amino acids motifs in different ORFs. We identified two strains of the novel subgenotype A8 and two strains of the novel quasi-subgenotype D12. Notably, the analysis demonstrated that the subgenotype A8 strains are a basal lineage that diverged before the other African subgenotypes A.

Molecular epidemiology, phylogenetic analysis and genotype distribution of hepatitis B virus in Saudi Arabia: Predominance of genotype D1.

Despite the implementation of various vaccination programs, hepatitis B virus (HBV) poses a considerable health problem in Saudi Arabia. Insight on HBV evolutionary history in the region is limited. We performed a comprehensive epidemiological and phylogenetic reconstruction based on a large cohort of HBV infected patients. Three hundred and nineteen HBV-infected patients with different clinical manifestations, including inactive and active chronic carriers and patients with cirrhosis and hepatocellular carcinoma (HCC), were enrolled in this study. The full-length large S gene was amplified and sequenced. Phylogenetic analysis was performed to determine the genotype and subgenotypes of the isolates. Phylogenetic tree analysis revealed that genotype D is the most dominant genotype among patients. Moreover, this analysis identified two strains with genotype E isolated from active carriers. Detailed phylogenetic analyses confirmed the presence of four HBV D subgenotypes, D1 (93%, n = 296), D2 (0.02%, n = 5), D3 (0.003%, n = 1), and D4 (0.003%, n = 1). In addition, six genotype D strains were not assigned to any existing HBV D subgenotype. The large S gene of eight strains showed signatures of genotype recombination between the genotypes D and A and between D and E. Several strains harbored medically important point mutations at the protein level. Along with the dominance of the HBV genotype D, isolation of the E genotype and several recombinant strains from patients with Saudi Arabian origin is an essential result for decisions involving therapeutic measures for patients. Development of vaccines and detection of diagnostic escape mutations at antigenic epitopes on the HBsAg will be valuable to public health authorities. Furthermore, the diversity at the nucleotide and amino acid levels and different proportions of dN/dS at the PreS1, PreS2, and HBsAg reveal the selective pressure trend from inactive status towards advanced liver diseases.

Signature of natural resistance in NS3 protease revealed by deep sequencing of HCV strains circulating in Iran.

A tremendous upscale of screening and treatment strategies is required to achieve elimination of the hepatitis C virus (HCV) in Iran by 2030. Among treated patients, at least 5-10% is expected to experience treatment failure. To efficiently retreat cases with prior exposure to NS5A and NS5B drugs, knowledge on the natural prevalence of NS3 resistance is key. The NS3 region of 32 samples from sixteen Iranian HCV patients, among which 6 injecting drug users, was amplified and subjected to deep sequencing. Amplification and sequencing were successful in 29 samples. The reads were assembled to consensus sequences and showed that 6 patients were infected with HCV1a (37.5%), 7 with HCV1b (43.8%) and 3 with HCV3a (18.7%). Nucleotide identities were shared for >97% between intra-host sequences. Two patients were infected with natural resistant viruses, of which one solely comprising low frequency variants. Inferred phylogenies showed that Iranian sequences clustered together for HCV1a and HCV1b, while for HCV3a a potential recombination event was detected. We firstly report the use of deep sequencing for HCV in Iran, demonstrate the use of NS3 inhibitors as salvage therapy in case of retreatment and stress the importance for Iran to prioritize drug users for screening and treatment.