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Abstract Patients with HBeAg-negative chronic infection (CI) have not been extensively studied because of low viremia. The HBx protein, encoded by HBX, has a key role in viral replication. Here, we analyzed the viral quasispecies at the 5′ end of HBX in CI patients and compared it with that of patients in other clinical stages. Fifty-eight HBeAg-negative patients were included: 16 CI, 19 chronic hepatitis B, 16 hepatocellular carcinoma and 6 liver cirrhosis. Quasispecies complexity and conservation were determined in the region between nucleotides 1255 and 1611. Amino acid changes detected were tested in vitro. CI patients showed higher complexity in terms of mutation frequency and nucleotide diversity and higher quasispecies conservation (p

Deletions in the 3′ end region of the hepatitis B virus (HBV) X open reading frame (HBX) may affect the core promoter (Cp) and have been frequently associated with hepatocellular carcinoma (HCC). The aim of this study was to investigate the presence of variants with deletions and/or insertions (Indels) in this region in the quasispecies of 50 chronic hepatitis B (CHB) patients without HCC. We identified 103 different Indels in 47 (94%) patients, in a median of 3.4% of their reads (IQR, 1.3–8.4%), and 25% (IQR, 13.1–40.7%) of unique sequences identified in each quasispecies (haplotypes). Of those Indels, 101 (98.1%) caused 44 different altered stop codons, the most commonly observed were at positions 128, 129, 135, and 362 (putative position). Moreover, 39 (37.9%) Indels altered the TATA-like box (TA) sequences of Cp; the most commonly observed caused TA2 + TA3 fusion, creating a new putative canonical TATA box. Four (8%) patients developed negative clinical outcomes after a median follow-up of 9.4 (8.7–12) years. In conclusion, we observed variants with Indels in the HBX 3′ end in the vast majority of our CHB patients, some of them encoding alternative versions of HBx with potential functional roles, and/or alterations in the regulation of transcription.

The hepatitis delta virus (HDV) genome has an autocatalytic region called the ribozyme, which is essential for viral replication. The aim of this study was to use next-generation sequencing (NGS) to analyze the ribozyme quasispecies (QS) in order to study its evolution and identify highly conserved regions potentially suitable for a gene-silencing strategy. HDV RNA was extracted from 2 longitudinal samples of chronic HDV patients and the ribozyme (nucleotide, nt 688–771) was analyzed using NGS. QS conservation, variability and genetic distance were analyzed. Mutations were identified by aligning sequences with their specific genotype consensus. The main relevant mutations were tested in vitro. The ribozyme was conserved overall, with a hyper-conserved region between nt 715–745. No difference in QS was observed over time. The most variable region was between nt 739–769. Thirteen mutations were observed, with three showing a higher frequency: T23C, T69C and C64 deletion. This last strongly reduced HDV replication by more than 1 log in vitro. HDV Ribozyme QS was generally highly conserved and was maintained during follow-up. The most conserved portion may be a valuable target for a gene-silencing strategy. The presence of the C64 deletion may strongly impair viral replication, as it is a potential mechanism of viral persistence.

Objectives: The study aim was to describe the etiological profile and clinical characteristics of pneumonia among children hospitalized in Thimphu, Bhutan. Methods: This prospective study enrolled children aged 2–59 months admitted to the Jigme Dorji Wangchuck National Referral Hospital with World Health Organization (WHO)-defined clinical pneumonia. Demographic and clinico-radiological data were collected through questionnaires, physical examination, and chest radiography. Blood samples and nasopharyngeal washing were collected for microbiological analysis including culture and molecular methods. Results: From July 2017 to June 2018, 189 children were enrolled, of which 53.4% were infants. Pneumonia-related admissions were less frequent over the winter. Chest radiographies were obtained in 149 children; endpoints included pneumonia in 39 cases (26.2%), other infiltrates in 31 (20.8%), and were normal in 79 children (53.0%). Non-contaminated bacterial growth was detected in 8/152 (5.3%) blood cultures, with only two cases of Streptococcus pneumoniae. Viral detection in upper respiratory secretions was common, with at least one virus detected in 103/115 (89.6%). The three most-commonly isolated viruses were respiratory syncytial virus (52/115; 45.2%), rhinovirus (42/115; 36.5%), and human parainfluenza virus (19/115; 16.5%). A third of patients with viral infections showed mixed infections. Case fatality rate was 3.2% (6/189). Conclusion: Respiratory viral infections predominated among this cohort of WHO-defined clinical pneumonia cases, whereas bacterial aetiologies were uncommon, highlighting the epidemiologic transition that Bhutan seems to have reached.

ABSTRACT The measurement and interpretation of HBV DNA and RNA levels in HBV infected patients treated with antiviral therapy supports the objective of HBV disease management. Here, we quantified circulating HBV RNA through a standardized and sensitive assay in follow-up samples from both naive and treated patients as a marker of infection evolution. HBV DNA (HBV DNA for use in Cobas 6800/8800 Automated Roche Molecular Systems), RNA (Roche HBV RNA Investigational Assay for use in the Cobas 6800/8800; Roche), HBeAg and HBsAg (Elycsys HBsAg chemiluminescence immunoassay by Cobas 8000; Roche), and core-related antigen (Lumipulse G chemiluminescence assay; Fujirebio) levels were measured in cohorts of untreated or nucleos(t)ide treated, HBV-infected subjects in an outpatient hospital setting. HBV DNA levels in untreated people were 3.6 log10 higher than corresponding RNA levels and were stable over 5 years of observation. While only five of 52 treated patients had DNA levels below the lower limit of quantification (10 IU/mL) at the end of follow-up, 13 had HBV RNA levels persistently above this limit, including eight with undetectable DNA. In samples with undetectable core-related antigen we observed a median HBsAg titer 2.7-fold higher than in samples with undetectable RNA (adjusted P = 0.012). Detectable HBV RNA with undetectable HBV DNA was a negative predictor of HBsAg decrease to a level ≤100 IU/mL (P = 0.03). In naive patients the difference between HBV DNA and RNA was higher than previously reported. HBV RNA rapidly decreased during treatment. However, in some cases, it was detectable even after years of effective therapy, being a negative predictor of HBsAg decrease. The investigational RNA assay for use on the Cobas 6800/8800 instruments is a sensitive and standardized method that could be applied in general management of HBV infection. IMPORTANCE This study focused on the quantification of circulating HBV RNA by using a standardized and sensitive assay. Thanks to this system we observed a higher difference between circulating HBV DNA and RNA than previously reported. In treated patients, HBV RNA decreased together with DNA, although some patients presented detectable levels even after years of successful antiviral treatment, suggesting a persistent viral transcription. Of note, the detection of viral RNA when HBV DNA is undetectable was a negative predictor of HBsAg decrease to a level ≤100 IU/mL. This assay could be extremely helpful in HBV patients management to study viral transcription and to identify those treated patients that may achieve sustained viral suppression.

Universal history is characterized by continuous evolution, in which civilizations are born and die. This evolution is associated with multiple factors, among which the role of microorganisms is often overlooked. Viruses and bacteria have written or decisively contributed to terrible episodes of history, such as the Black Death in 14th century Europe, the annihilation of pre-Columbian American civilizations, and pandemics such as the 1918 Spanish flu or the current COVID-19 pandemic caused by the coronavirus SARS-CoV-2. Nevertheless, it is clear that we could not live in a world without these tiny beings. Endogenous retroviruses have been key to our evolution and for the regulation of gene expression, and the gut microbiota helps us digest compounds that we could not otherwise process. In addition, we have used microorganisms to preserve or prepare food for millennia and more recently to obtain drugs such as antibiotics or to develop recombinant DNA technologies. Due to the enormous importance of microorganisms for our survival, they have significantly influenced the population genetics of different human groups. This paper will review the role of microorganisms as “villains” who have been responsible for tremendous mortality throughout history but also as “friends” who help us survive and evolve.

BACKGROUND:The objective of this study is to describe incidence and shifts of serotype and clonal distribution of invasive Streptococcus pneumoniae strains in four different age groups (65 years) during a period of intermediate PCV13 vaccination coverage (2011-2016) in Catalonia, Spain. METHODS:We included all pneumococcal strains systematically sent to the Catalan support laboratory for molecular surveillance of invasive pneumococcal disease (IPD) located at Hospital Sant Joan de Deu, Barcelona. Two study periods were considered: 2011-13, early PCV13 vaccination period (EVP) and 2014-2016, late vaccination period (LVP). RESULTS:A total of 2142 strains were included in the study. Five years after intermediate introduction of PCV13 in our population, a significant decrease of overall incidence of IPD in children 65 years. Results found when comparing both periods were consistent with IRRs observed year by year. In children 65 years the most frequently isolated serotypes were 3, 19A and 7F vs 3, 14 and 12F, respectively. Regarding clonal complexes (CCs) expressing mainly PCV13 serotypes, significant decreases of the proportions of CC306, CC191 and CC320 were observed, while CC156 showed a significant increase. As for CCs expressing mostly non-PCV13 serotypes, significant increases in ST989, CC53 and CC404 were showed. CONCLUSIONS:Despite low vaccine coverage in our setting a significant decrease of incidence of IPD was observed in children younger than 5 years. The modest indirect protection against vaccine serotypes causing IPD in elderly indicate the need for the inclusion of more serotypes in future high-valent PCV and vaccinating old adults should be considered.