Estudi de la composició de la quasiespècies del virus de la hepatitis B a les regions codificants de les proteïnes de l'envolta i la polimerasa viral per seqüenciació massiva : associació de les seves variants genètiques amb el tractament antiviral de la hepatitis B crònica amb anàlegs de nucleòsids/nucleòtids /

Aquesta tesi doctoral es basa en dos estudis en els que s'analitzen les variants genètiques de la quasiespècies del virus de la hepatitis B (VHB) en una regió de la pauta oberta de lectura (ORF) de la polimerasa viral (P) on poden acumular-se mutacions relacionades amb la resistència a certs tractaments de la família dels anàlegs de nucleòsids/nucleòtids (NUCs). Aquesta anàlisi s'ha realitzat per seqüenciació massiva basada en tècniques d'"Ultra-deep pyrosequencing" (UDPS). Les mutacions seleccionades a causa del tractament antiviral a l'ORF P també poden produir canvis d'aminoàcid (aa) a l'ORF de les proteïnes de superfície (S) [tots dos ORF estan solapats a la mateixa seqüència de nucleòtids (nt)]. Alguns d'aquests canvis poden donar lloc a la pèrdua de l'acció protectora de la vacunació i/o les immunoglobulines contra el VHB (HBIg) i també a falsos negatius en els mètodes diagnòstics del VHB. En el primer estudi s'han analitzat la quasiespècies del VHB en quatre pacients amb hepatitis B crònica abans de rebre qualsevol tractament amb NUCs. Un d'aquests pacients va rebre tractament seqüencial amb diferents NUCs i es va analitzar una mostra obtinguda en finalitzar cada un d'ells. Aquestes quasiespècies virals van ser estudiades amb la primera versió de la tecnologia d'UDPS, que permetia lectures de seqüència fins a 250 nt. La metodologia desenvolupada en aquest estudi va permetre determinar la composició de variants genètiques en proporcions fins al 0,1% de la quasiespècies. Es va analitzar la variabilitat i conservació dels ORFs P i S en la quasiespècies viral abans del primer tractament amb NUCs dels quatre pacients inclosos a l'estudi [lamivudina (LAM)]. En general, la freqüència de les mutacions relacionades amb resistència a LAM no va permetre preveure la seva selecció durant aquest tractament. En el pacient analitzat longitudinalment, al llarg dels diferents tractaments es van analitzar les divergències globals de les seqüències de nt i aa i la desviació estàndard (SD) de les freqüències de les mutacions a l'ORF P. La SD va permetre identificar les més rellevants per explicar la fallada a cada un d'aquests (per tant aquests resultats tenen un valor potencialment fenotípic). L'anàlisi del lligament d'aquestes mutacions va evidenciar l'acumulació de varies d'elles en una sola variant genètica (per exemple rtL180M-rtS202G-rtM204V-rtV207I), a excepció de rtA181T/sW172stop que sol ser la única mutació en les variants que la contenen. En totes les mostres analitzades es van observar proporcions relativament elevades de variants defectives amb codons stop prematurs a l'ORF S. En el segon estudi es va analitzar la quasiespècies viral en quatre pacients amb recurrència de la infecció per VHB després del trasplantament hepàtic ortotòpic (OLT) a pesar de la profilaxi amb LAM o HBIg+LAM. En aquest estudi es va utilitzar una nova metodologia descrita prèviament pel grup del doctorand, que va permetre estudiar les variants presents en proporcions fins al 0,25% de la quasiespècies del VHB. Aquest procediment està basat en una versió de la tecnologia d'UDPS que permet obtenir seqüències fins a 400 nt, cosa que va fer possible explorar la seqüència sencera del principal epítop de l'antigen de superfície del VHB (HBsAg), el determinant "a". Així en els quatre casos estudiats es va observar un efecte coll d'ampolla de l'OLT sobre les poblacions de variants que formen la quasiespècies del VHB, que va donar lloc a diferències rellevants entre les quasiespècies d'abans i després de l'OLT: a nivell de mutacions individuals dels ORFs P i S, que en alguns casos van donar lloc a la recurrència de la infecció sense HBsAg detectable, i a través de canvis en el genotip del VHB i en diferents índexs de diversitat de la quasiespècies viralThis doctoral thesis is based on two studies in which the genetic variant composition of the hepatitis B virus (HBV) quasispecies was analysed in a region of the viral polymerase (P) open reading (ORF) where mutations associated with resistance to certain treatments from the nucleoside/nucleotide analogues (NUCs) family can accumulate. This analysis was performed by massive sequencing techniques based on Ultra-deep pyrosequencing (UDPS). Mutations selected in the P ORF due to antiviral treatment can also cause amino acid (aa) changes in the surface proteins (S) ORF (both ORF are overlapping in the same nucleotide [nt] sequence). Some of these changes may result in the loss of the protective action of vaccination and immunoglobulins against HBV (HBIg), and also to false negative results in HBV diagnostic methods. In the first study, the HBV quasispecies was analysed in four chronic hepatitis B patients prior to receiving any treatment with NUCs. One of these patients received sequential treatment with different NUCs and a sample obtained at the end of each of them was analysed. These viral quasispecies were studied using the first version of the UDPS technology, which made it possible to obtain sequence reads up to 250 nt. The methodology developed in this study enabled us to determine the genetic variant composition in proportions up to 0.1% of the quasispecies. In the viral quasispecies from the four patients included in this study the variability and conservation of P and S ORF was analysed before they received the first NUCs treatment (lamivudine [LAM]). Overall, the frequency of mutations associated with LAM resistance did not make it possible to predict their selection during this treatment. Throughout the different treatments of the longitudinally analysed patient, the global divergences of nt and aa sequences and the standard deviation (SD) of the frequencies of mutations in the P ORF were analysed. The SD identified the most relevant of them to explain failure in each of these treatments (therefore these results have a potential phenotypic value). The linkage analysis of these mutations showed the accumulation of several of them in a single genetic variant (e.g. rtL180M-rtS202G-rtM204V-rtV207I); except for rtA181T/sW172stop which is usually the only mutation in variants that contain it. All the samples showed relatively high proportions of defective variants with premature stop codons in the S ORF. In the second study the viral quasispecies was analysed in four patients who showed recurrence of HBV infection after orthotopic liver transplantation (OLT) despite the prophylaxis with LAM or HBIg+LAM. In this study a new methodology previously described by the candidate's group was used, which allowed for the study of the variants present in proportions up to 0.25% of the HBV quasispecies. This procedure is based on a version of the UDPS technology which made it possible to obtain sequences of up to 400 nt in length, which allowed for the exploration of the whole sequence of the main epitope of HBV surface antigen (HBsAg), the "a" determinant. In the four cases studied a bottleneck effect was observed over the variant populations that form the HBV quasispecies due to the OLT, which resulted in significant differences between the quasispecies from before and after OLT: at the level of individual mutations in P and S ORFs, which in some cases resulted in recurrence of infection without detectable HBsAg, and through changes in the HBV genotype and in different indices of viral quasispecies diversity

Microorganisms as Shapers of Human Civilization, from Pandemics to Even Our Genomes: Villains or Friends? A Historical Approach

SARS-CoV-2; Influenza; MicrobiotaSARS-CoV-2; Influenza; MicrobiotaSARS-CoV-2; Influenza; MicrobiotaUniversal 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

Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection

Hepatitis B X gene; Hepatitis B virus; Hepatitis delta virusHepatitis B gen X; Virus d'hepatitis B; Virus d'hepatitis deltaHepatitis B gen X; Virus de hepatitis B; Virus de hepatitis deltaBACKGROUND: Hepatitis delta virus (HDV) seems to strongly suppress hepatitis B virus (HBV) replication, although little is known about the mechanism of this interaction. Both these viruses show a dynamic distribution of mutants, resulting in viral quasispecies. Next-generation sequencing is a viable approach for analyzing the composition of these mutant spectra. As the regulatory hepatitis B X protein (HBx) is essential for HBV replication, determination of HBV X gene (HBX) quasispecies complexity in HBV/HDV infection compared to HBV mono-infection may provide information on the interactions between these two viruses. AIM: To compare HBV quasispecies complexity in the HBX 5' region between chronic hepatitis delta (CHD) and chronic HBV mono-infected patients. METHODS: Twenty-four untreated patients were included: 7/24 (29.2%) with HBeAg-negative chronic HBV infection (CI, previously termed inactive carriers), 8/24 (33.3%) with HBeAg-negative chronic hepatitis B (CHB) and 9/24 (37.5%) with CHD. A serum sample from each patient was first tested for HBV DNA levels. The HBX 5' region [nucleotides (nt) 1255-1611] was then PCR-amplified for subsequent next-generation sequencing (MiSeq, Illumina, United States). HBV quasispecies complexity in the region analyzed was evaluated using incidence-based indices (number of haplotypes and number of mutations), abundance-based indices (Hill numbers of order 1 and 2), and functional indices (mutation frequency and nucleotide diversity). We also evaluated the pattern of nucleotide changes to investigate which of them could be the cause of the quasispecies complexity. RESULTS: CHB patients showed higher median HBV-DNA levels [5.4 logIU/mL, interquartile range (IQR) 3.5-7.9] than CHD (3.4 logIU/mL, IQR 3-7.6) (P = n.s.) or CI (3.2 logIU/mL, IQR 2.3-3.5) (P < 0.01) patients. The incidence and abundance indices indicated that HBV quasispecies complexity was significantly greater in CI than CHB. A similar trend was observed in CHD patients, although only Hill numbers of order 2 showed statistically significant differences (CHB 2.81, IQR 1.11-4.57 vs CHD 8.87, 6.56-11.18, P = 0.038). There were no significant differences in the functional indices, but CI and CHD patients also showed a trend towards greater complexity than CHB. No differences were found for any HBV quasispecies complexity indices between CHD and CI patients. G-to-A and C-to-T nucleotide changes, characteristic of APOBEC3G, were higher in CHD and CI than in CHB in genotype A haplotypes, but not in genotype D. The proportion of nt G-to-A vs A-to-G changes and C-to-T vs T-to-C changes in genotype A and D haplotypes in CHD patients showed no significant differences. In CHB and CI the results of these comparisons were dependent on HBV genotype. CONCLUSION: The lower-replication CHD and CI groups show a trend to higher quasispecies complexity than the higher-replication CHB group. The mechanisms associated with this greater complexity require elucidation.Supported by the Instituto de Salud Carlos III, grants PI15/00856 and PI17/02233; co-financed by the European Regional Development Fund (ERDF

ACE Score Identifies HBeAg-negative Inactive Carriers at a Single-point Evaluation, Regardless of HBV Genotype

HBV DNA; Hepatitis B virus; Inactive carrierADN del VHB; Virus de la hepatitis B; Portador inactivoADN del VHB; Virus de l'hepatitis B; Portador inactiuBackground and Aims Hepatitis B virus (HBV) biomarkers have been used for a better categorization of patients, even though the lack of simple algorithms and the impact of genotypes limit their application. Our aim was to assess the usefulness of noninvasive markers for the identification of HBV inactive carriers (ICs) in a single-point evaluation and to design a predictive model for their identification. Methods This retrospective-prospective study included 343 consecutive HBeAg-negative individuals. Clinical, analytical, and virological data were collected, and a liver biopsy was performed if needed. Subjects were classified at the end of follow-up as ICs, chronic hepatitis B and gray zone.A predictive model was constructed, and validated by 1000-bootstrap samples. Results After 39 months of follow-up, 298 subjects were ICs, 36 were chronic hepatitis B CHB, and nine were gray zone. Eighty-nine (25.9%) individuals required a liver biopsy. Baseline HBV DNA hazard ratio (HR) 6.0, p<0.001), HBV core-related antigen (HBcrAg) (HR 6.5, p<0.001), and elastography (HR 4.6, p<0.001) were independently associated with the IC stage. The ACE score (HBV DNA, HBcrAg, elastography), obtained by bootstrapping, yielded an area under the receiver operating characteristics (AUROC) of 0.925 (95% CI: 0.880–0.970, p<0.001) for identification of ICs. The AUROC for genotype D was 0.95, 0.96 for A, 0.90 for E, and 0.88 for H/F. An ACE score of <1 had a positive predictive value of 99.5%, and a score ≤12 points had a diagnostic accuracy of 93.8%. Conclusions Low baseline HBV DNA, HBcrAg, and liver stiffness were independently associated with the IC phase. A score including those variables identified ICs at a single-point evaluation, and might be applied to implement less intensive follow-up strategies.This study received partial financial support from Instituto de Salud Carlos III (PI17/02233 and PI20/01692)

Estudi de la composició de la quasiespècies del virus de la hepatitis B a les regions codificants de les proteïnes de l’envolta i la polimerasa viral per seqüenciació massiva: associació de les seves variants genètiques amb el tractament antiviral de la hepatitis B crònica amb anàlegs de nucleòsids/nucleòtids

Aquesta tesi doctoral es basa en dos estudis en els que s’analitzen les variants genètiques de la quasiespècies del virus de la hepatitis B (VHB) en una regió de la pauta oberta de lectura (ORF) de la polimerasa viral (P) on poden acumular-se mutacions relacionades amb la resistència a certs tractaments de la família dels anàlegs de nucleòsids/nucleòtids (NUCs). Aquesta anàlisi s’ha realitzat per seqüenciació massiva basada en tècniques d’“Ultra-deep pyrosequencing” (UDPS). Les mutacions seleccionades a causa del tractament antiviral a l’ORF P també poden produir canvis d’aminoàcid (aa) a l’ORF de les proteïnes de superfície (S) [tots dos ORF estan solapats a la mateixa seqüència de nucleòtids (nt)]. Alguns d’aquests canvis poden donar lloc a la pèrdua de l’acció protectora de la vacunació i/o les immunoglobulines contra el VHB (HBIg) i també a falsos negatius en els mètodes diagnòstics del VHB. En el primer estudi s’han analitzat la quasiespècies del VHB en quatre pacients amb hepatitis B crònica abans de rebre qualsevol tractament amb NUCs. Un d’aquests pacients va rebre tractament seqüencial amb diferents NUCs i es va analitzar una mostra obtinguda en finalitzar cada un d’ells. Aquestes quasiespècies virals van ser estudiades amb la primera versió de la tecnologia d’UDPS, que permetia lectures de seqüència fins a 250 nt. La metodologia desenvolupada en aquest estudi va permetre determinar la composició de variants genètiques en proporcions fins al 0,1% de la quasiespècies. Es va analitzar la variabilitat i conservació dels ORFs P i S en la quasiespècies viral abans del primer tractament amb NUCs dels quatre pacients inclosos a l’estudi [lamivudina (LAM)]. En general, la freqüència de les mutacions relacionades amb resistència a LAM no va permetre preveure la seva selecció durant aquest tractament. En el pacient analitzat longitudinalment, al llarg dels diferents tractaments es van analitzar les divergències globals de les seqüències de nt i aa i la desviació estàndard (SD) de les freqüències de les mutacions a l’ORF P. La SD va permetre identificar les més rellevants per explicar la fallada a cada un d’aquests (per tant aquests resultats tenen un valor potencialment fenotípic). L’anàlisi del lligament d’aquestes mutacions va evidenciar l’acumulació de varies d’elles en una sola variant genètica (per exemple rtL180M-rtS202G-rtM204V-rtV207I), a excepció de rtA181T/sW172stop que sol ser la única mutació en les variants que la contenen. En totes les mostres analitzades es van observar proporcions relativament elevades de variants defectives amb codons stop prematurs a l’ORF S. En el segon estudi es va analitzar la quasiespècies viral en quatre pacients amb recurrència de la infecció per VHB després del trasplantament hepàtic ortotòpic (OLT) a pesar de la profilaxi amb LAM o HBIg+LAM. En aquest estudi es va utilitzar una nova metodologia descrita prèviament pel grup del doctorand, que va permetre estudiar les variants presents en proporcions fins al 0,25% de la quasiespècies del VHB. Aquest procediment està basat en una versió de la tecnologia d’UDPS que permet obtenir seqüències fins a 400 nt, cosa que va fer possible explorar la seqüència sencera del principal epítop de l’antigen de superfície del VHB (HBsAg), el determinant “a”. Així en els quatre casos estudiats es va observar un efecte coll d’ampolla de l’OLT sobre les poblacions de variants que formen la quasiespècies del VHB, que va donar lloc a diferències rellevants entre les quasiespècies d’abans i després de l’OLT: a nivell de mutacions individuals dels ORFs P i S, que en alguns casos van donar lloc a la recurrència de la infecció sense HBsAg detectable, i a través de canvis en el genotip del VHB i en diferents índexs de diversitat de la quasiespècies viral.This doctoral thesis is based on two studies in which the genetic variant composition of the hepatitis B virus (HBV) quasispecies was analysed in a region of the viral polymerase (P) open reading (ORF) where mutations associated with resistance to certain treatments from the nucleoside/nucleotide analogues (NUCs) family can accumulate. This analysis was performed by massive sequencing techniques based on Ultra-deep pyrosequencing (UDPS). Mutations selected in the P ORF due to antiviral treatment can also cause amino acid (aa) changes in the surface proteins (S) ORF (both ORF are overlapping in the same nucleotide [nt] sequence). Some of these changes may result in the loss of the protective action of vaccination and immunoglobulins against HBV (HBIg), and also to false negative results in HBV diagnostic methods. In the first study, the HBV quasispecies was analysed in four chronic hepatitis B patients prior to receiving any treatment with NUCs. One of these patients received sequential treatment with different NUCs and a sample obtained at the end of each of them was analysed. These viral quasispecies were studied using the first version of the UDPS technology, which made it possible to obtain sequence reads up to 250 nt. The methodology developed in this study enabled us to determine the genetic variant composition in proportions up to 0.1% of the quasispecies. In the viral quasispecies from the four patients included in this study the variability and conservation of P and S ORF was analysed before they received the first NUCs treatment (lamivudine [LAM]). Overall, the frequency of mutations associated with LAM resistance did not make it possible to predict their selection during this treatment. Throughout the different treatments of the longitudinally analysed patient, the global divergences of nt and aa sequences and the standard deviation (SD) of the frequencies of mutations in the P ORF were analysed. The SD identified the most relevant of them to explain failure in each of these treatments (therefore these results have a potential phenotypic value). The linkage analysis of these mutations showed the accumulation of several of them in a single genetic variant (e.g. rtL180M-rtS202G-rtM204V-rtV207I); except for rtA181T/sW172stop which is usually the only mutation in variants that contain it. All the samples showed relatively high proportions of defective variants with premature stop codons in the S ORF. In the second study the viral quasispecies was analysed in four patients who showed recurrence of HBV infection after orthotopic liver transplantation (OLT) despite the prophylaxis with LAM or HBIg+LAM. In this study a new methodology previously described by the candidate’s group was used, which allowed for the study of the variants present in proportions up to 0.25% of the HBV quasispecies. This procedure is based on a version of the UDPS technology which made it possible to obtain sequences of up to 400 nt in length, which allowed for the exploration of the whole sequence of the main epitope of HBV surface antigen (HBsAg), the “a” determinant. In the four cases studied a bottleneck effect was observed over the variant populations that form the HBV quasispecies due to the OLT, which resulted in significant differences between the quasispecies from before and after OLT: at the level of individual mutations in P and S ORFs, which in some cases resulted in recurrence of infection without detectable HBsAg, and through changes in the HBV genotype and in different indices of viral quasispecies diversity