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Viral populations are large and highly heterogeneous. Despite the evolutionary relevance of such heterogeneity, statistical approaches to quantifying the extent to which viruses maintain a high genotypic and/or phenotypic diversity have been rarely pursued. Here, we address this issue by analyzing a nucleotide-to-protein sequence map through deep sequencing of populations of the Qβ phage adapted to high temperatures. Tens of thousands of different sequences corresponding to two fragments of the gene coding for the viral replicase were recovered. A diversity analysis of two independent populations consistently revealed that about 40% of the mutations identified caused changes in protein amino acids, leading to an almost complete exploration of the protein neighborhood of (non-silent) mutants at a distance of one. The functional form of the empirical distribution of phenotype abundance agreed with analytical calculations that assumed random mutations in the nucleotide sequence. Our results concur with the idea that viral populations maintain a high diversity as an efficient adaptive mechanism and support the hypothesis of universality for a lognormal distribution of phenotype abundances in biologically meaningful genotype–phenotype maps, highlighting the relevance of entropic effects in molecular evolution.

Parvovirus B19 (B19V) is a ssDNA human virus, responsible for an ample range of clinical manifestations. Sequencing of B19V DNA from clinical samples is frequently reported in the literature to assign genotype (genotypes 1–3) and for finer molecular epidemiological tracing. The increasing availability of Next Generation Sequencing (NGS) with its depth of coverage potentially yields information on intrinsic sequence heterogeneity; however, integration of this information in analysis of sequence variation is not routinely obtained. The present work investigated genomic sequence heterogeneity within and between B19V isolates by application of NGS techniques, and by the development of a novel dedicated bioinformatic tool and analysis pipeline, yielding information on two newly defined parameters. The first, α-diversity, is a measure of the amount and distribution of position-specific, normalised Shannon Entropy, as a measure of intra-sample sequence heterogeneity. The second, σ-diversity, is a measure of the amount of inter-sample sequence heterogeneity, also incorporating information on α-diversity. Based on these indexes, further cluster analysis can be performed. A set of 24 high-titre viraemic samples was investigated. Of these, 23 samples were genotype 1 and one sample was genotype 2. Genotype 1 isolates showed low α-diversity values, with only a few samples showing distinct position-specific polymorphisms; a few genetically related clusters emerged when analysing inter-sample distances, correlated to the year of isolation; the single genotype 2 isolate showed the highest α-diversity, even if not presenting polymorphisms, and was an evident outlier when analysing inter-sample distance. In conclusion, NGS analysis and the bioinformatic tool and pipeline developed and used in the present work can be considered effective tools for investigating sequence diversity, an observable parameter that can be incorporated into the quasispecies theory framework to yield a better insight into viral evolution dynamics.

The Seneca Valley virus (SVV) is an oncolytic virus from the picornavirus family, characterized by a 7.3-kilobase RNA genome encoding for all the structural and functional viral proteins. Directed evolution by serial passaging has been employed for oncolytic virus adaptation to increase the killing efficacy towards certain types of tumors. We propagated the SVV in a small-cell lung cancer model under two culture conditions: conventional cell monolayer and tumorspheres, with the latter resembling more closely the cellular structure of the tumor of origin. We observed an increase of the virus-killing efficacy after ten passages in the tumorspheres. Deep sequencing analyses showed genomic changes in two SVV populations comprising 150 single nucleotides variants and 72 amino acid substitutions. Major differences observed in the tumorsphere-passaged virus population, compared to the cell monolayer, were identified in the conserved structural protein VP2 and in the highly variable P2 region, suggesting that the increase in the ability of the SVV to kill cells over time in the tumorspheres is acquired by capsid conservation and positively selecting mutations to counter the host innate immune responses.

BACKGROUND Different forms of pregenomic and other hepatitis B virus (HBV) RNA have been detected in patients’ sera. These circulating HBV-RNAs may be useful for monitoring covalently closed circular DNA activity, and predicting hepatitis B e-antigen seroconversion or viral rebound after nucleos(t)ide analog cessation. Data on serum HBV-RNA quasispecies, however, is scarce. It is therefore important to develop methodologies to thoroughly analyze this quasispecies, ensuring the elimination of any residual HBV-DNA. Studying circulating HBV-RNA quasispecies may facilitate achieving functional cure of HBV infection. AIM To establish a next-generation sequencing (NGS) methodology for analyzing serum HBV-RNA and comparing it with DNA quasispecies. METHODS Thirteen untreated chronic hepatitis B patients, showing different HBV-genotypes and degrees of severity of liver disease were enrolled in the study and a serum sample with HBV-DNA > 5 Log10 IU/mL and HBV-RNA > 4 Log10 copies/mL was taken from each patient. HBV-RNA was treated with DNAse I to remove any residual DNA, and the region between nucleotides (nt) 1255-1611 was amplified using a 3-nested polymerase chain reaction protocol, and analyzed with NGS. Variability/conservation and complexity was compared between HBV-DNA and RNA quasispecies. RESULTS No HBV-DNA contamination was detected in cDNA samples from HBV-RNA quasispecies. HBV quasispecies complexity showed heterogeneous behavior among patients. The Rare Haplotype Load at 1% was greater in DNA than in RNA quasispecies, with no statistically significant differences (P = 0.1641). Regarding conservation, information content was equal in RNA and DNA quasispecies in most nt positions [218/357 (61.06%)]. In 102 of the remaining 139 (73.38%), HBV-RNA showed slightly higher variability. Sliding window analysis identified 4 hyper-conserved sequence fragments in each quasispecies, 3 of them coincided between the 2 quasispecies: nts 1258-1286, 1545-1573 and 1575-1604. The 2 hyper-variable sequence fragments also coincided: nts 1311-1344 and 1461-1485. Sequences between nts 1519-1543 and 1559-1587 were only hyper-conserved in HBV-DNA and RNA, respectively. CONCLUSION Our methodology allowed analyzing HBV-RNA quasispecies complexity and conservation without interference from HBV-DNA. Thanks to this, we have been able to compare both quasispecies in the present study.

Background : RNA viruses evolve very fast, with a mutation rate of 103 to 105 base substitution per nucleotides per copy. The mutation is a survival strategy for the viruses, which leads them to survive in the new host. Fitness is defined as the replication capacity of the virus in an experimental setup. Generally, the large population passage of the virus leads to fitness gain, but the world data of the coronavirus infection and death shows the flattened curve with time. It is contradictory to the principle of fitness gain due to large population passage. The coronavirus is losing its potency but remains infectious as it is passaging into millions that leads to a decline in the death of COVID patients and high recovery rates. Fitness loss of coronaviruses attributed to a high level of mutation in the RNA genome as well as host immune response. The current outbreak of SARS CoV-2 is surfaced in December 2019 in Hubei province of China and considered as bats/pangolin origin, spreading 235 countries of the world, infecting nearly 31,664,104 people, and claimed nearly 972,221 lives as of September 24, 2020 (Death rate approximately 3%). This coronavirus has passaged into 31,664,104 people from the beginning of this pandemic until September 24, 2020. Now the virus is losing potency rather than being monotonous and continuous in producing virusrelated complications. The population is still getting infected at the same rate, but the severity of the disease is reduced due to the potency of the virus diminished due to the passage effect as well as fitness loss of the virus due to high mutation rates. The death rate is reduced to 3% as compared to 6% in June 2020, when this paper was first submitted. Objective: The purpose of the study is to prove the fact that the coronavirus loses its potency with time but, they remain infective. It becomes more infectious due to mutation of the gene but loses the capacity to kill the host. Methods: Since the WHO announces the COVID-19 outbreak is an emergency of international concern, every country in the world is taking many measures to mitigate the viral load to their population. Simultaneously, the WHO, CDC USA, CDC Europe, and much other organization is updating the COVID cases and death online daily as reported by the respective country. With the help of the COVID-19 outbreak data published by the European CDC and ourworldindata.org, we correlate the total cases of coronavirus and total death in the top ten affected countries in the world. We also link the trends of total cases vs. total death and total new cases vs. total new death related to COVID-19 in Germany, Spain, the United Kingdom, Italy, and New Zealand from January 30, 2020, until September 24, 2020. The reason to select these countries for the study is that these countries updating the COVID cases and deaths regularly and said to achieve the peak of COVID related infections and recovering from the pandemic. Results: We have tried to correlate the high mutation rate of the virus that leads to losing its potency to severe infection and death in the human. Viral extinction through high mutation could be considered as the new anti-viral strategies. Conclusion: Coronavirus is losing its potency to causing death to the human. The new infection is still being reported from every corner of the world, but the death rate is significantly decreasing.

Since the identification of the SARS-CoV-2, genus Beta-Coronavirus, in January 2020, the virus quickly spread in less than 3 months to all continents with a susceptible human population of about a 7.9billion, and still in active circulation. In the process, it has accumulated mutations leading to genetic diversity. Regular emergence of variants of concern/significance in different ecology shows genetic heterogeneity in the base population of SARS-CoV-2 that is continuously expanding with the passage of the virus in the vast susceptible human population. Natural selection of mutant occurs frequently in a positive sense (+) single-stranded (ss) RNA virus upon replication in the host. The Pressure of sub-optimal levels of virus-neutralizing antibodies and also innate immunity influence the process of genetic/ antigenic selection. The fittest of the mutants, that could be more than one, propagate and emerge as variants. The existence of different lineages, clades, and strains, as well as genetic heterogeneity of plaque purified virus population, justifies SARS-CoV-2 as 'Quasispecies‘ that refers to swarms of mutant sequences generated during replication of the viral genome, and all mutant sequences may not lead to virion. Viruses having a quasispecies nature may end up with progressive antigenic changes leading to antigenic plurality that is driven by ecology, and this phenomenon challenges vaccination-based control programs. © 2021, Editorial board of Journal of Experimental Biology and Agricultural Sciences. All rights reserved.

SARS-CoV-2; Mutacions; Quasiespècie SARS-CoV-2; Mutaciones; Cuasiespecie SARS-CoV-2; Mutations; Quasispecies A common trait among RNA viruses is their high capability to acquire genetic variability due to viral and host mechanisms. Next-generation sequencing (NGS) analysis enables the deep study of the viral quasispecies in samples from infected individuals. In this study, the viral quasispecies complexity and single nucleotide polymorphisms of the SARS-CoV-2 spike gene of coronavirus disease 2019 (COVID-19) patients with mild or severe disease were investigated using next-generation sequencing (Illumina platform). SARS-CoV-2 spike variability was higher in patients with long-lasting infection. Most substitutions found were present at frequencies lower than 1%, and had an A → G or T → C pattern, consistent with variants caused by adenosine deaminase acting on RNA-1 (ADAR1). ADAR1 affected a small fraction of replicating genomes, but produced multiple, mainly non-synonymous mutations. ADAR1 editing during replication rather than the RNA-dependent RNA polymerase (nsp12) was the predominant mechanism generating SARS-CoV-2 genetic variability. However, the mutations produced are not fixed in the infected human population, suggesting that ADAR1 may have an antiviral role, whereas nsp12-induced mutations occurring in patients with high viremia and persistent infection are the main source of new SARS-CoV-2 variants. This study was supported by the Direcció General de Recerca i Innovació en Salut (DGRIS) of the Catalan Health Ministry, Generalitat de Catalunya, through the Vall d’Hebron Institut de Recerca (VHIR); the European Regional Development Fund (ERDF) “A way to achieve Europe” by the Spanish Network for Research in Infectious Diseases [grant number REIPI RD16/0016/0003]; Instituto de Salud Carlos III [grant number FIS PI19/00301]; and Centro para el Desarrollo Tecnologico Industrial (CDTI) from the Ministry of Economic Affairs and Digital Transformation [grant number IDI-20200297].

espanolObjetivos. Argentina ha informado de altos niveles de farmacorresistencia transmitida (TDR), en mujeres embarazadas infectadas por el VIH por secuenciacion poblacional. Nuestro objetivo fue describir, en pacientes con TDR, el porcentaje de cuasiespecies que albergan mutaciones de resistencia (RAM) y la carga mutacional (ML). Pacientes y metodos. Estudio retrospectivo en una cohorte de 40 mujeres embarazadas VIH positivas naive, cuyas muestras pretratamiento habian sido genotipificadas por TRUGENE (periodo 2008-2014). Las muestras se genotipificaron por secuenciacion ultraprofunda y se calculo la ML considerando la carga viral multiplicada por la frecuencia de las cuasiespecies que albergan RAMs. Resultados. La TDR para ITINN, ITIAN e IP fue del 17,5% (n=7 pacientes), 10% (n=4), 12,5% (n=5) respectivamente. Las RAMs ITINN predominantes fueron K103N (n=4; 10%) y G190A/E/ S (n=3; 7,5%). Para los ITINN, el 78% de los RAM estaban presentes en >93,5% de la poblacion viral y la ML fue >1000 copias/ml (c/mL) para el 89%, con una mediana (IQR) de 8330 c/ml (7738-29796). Se describieron los siguientes RAM de ITIAN (% de cuasiespecies, ML): T215I (99,7%, 11014 c/ml); D67G (1,28%, 502 c/ml); M41L (79,8%, 88578 c/mL) y M184I (1,02%, 173 c/mL). Las IP-RAMs mas frecuentes fueron I85V, M46I, I50V y L90M (n=2, 5% cada una). Para los IP, las cuasiespecies con RAMs fueron Conclusion. Los ITINN-RAMs son predominantes dentro de la poblacion viral, por lo general superan el umbral de 1000 c/mL, que indicaria un riesgo potencial mas alto de transmision perinatal. Por el contrario, las mutaciones de IP aparecen principalmente como variantes minoritarias, con un riesgo menor de transmision. Para ITIAN, las cuasiespecies que albergan RAM y los valores de ML fueron variables. EnglishObjective. Argentina has reported high levels of transmitted drug resistance (TDR), in HIV-infected pregnant women by population sequencing. We aimed to describe, in patients with TDR, the percentage of quasispecies harboring resistance mutations (RAMs) and mutational load (ML). Patients and Methods. Retrospective study in a cohort of 40 naive HIV-infected pregnant women, whose pretreatment samples had been genotyped by TRUGENE (period 2008-2014). Samples were re-sequenced with Ultra-deep Sequencing and ML was calculated considering baseline HIV-1 RNA load multiplied by the frequency of quasispecies harboring RAMs. Results. TDR for NNRTIs, NRTIs and PIs was 17.5% (n=7 patients), 10% (n=4), 12.5% (n=5) respectively. Predominant NNRTI RAMs were K103N (n=4; 10%) and G190A/E/S (n=3; 7.5%). For NNRTIs, 78% of RAMs were present in >93.5% of viral population and ML was >1000 copies/mL (c/mL) for 89%, with a median (IQR) of 8330 c/ml (7738-29796). The following NRTI RAMs were described (per patient: % of quasispecies, ML): T215I (99.7%, 11014 c/ml); D67G (1.28%, 502 c/mL); M41L (79.8%, 88578 c/mL) and M184I (1.02%, 173 c/mL). Most frequent PI-RAMs were I85V, M46I, I50V and L90M (n=2, 5% each). For PIs, quasispecies with RAMs were Conclusion. NNRTI-RAMs are predominant within the viral population, usually exceeding the threshold of 1000 c/mL, indicating potential higher risk of perinatal transmission. Conversely, PI mutations appear mostly as minority variants, with potential lower risk of transmission. Among NRTI, quasispecies harboring RAMs and ML values were variable.

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed., This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call COV20/00181), and co-financed by European Development Regional Fund ‘A way to achieve Europe’. The work was also supported by grants CSIC-COV19-014 from Consejo Superior de Investigaciones Científicas (CSIC), project 525/C/2021 from Fundació La Marató de TV3, PID2020-113888RB-I00 from Ministerio de Ciencia e Innovación, BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 and PI21/00139 from Instituto de Salud Carlos III, and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P., M.C., and P.M. are supported by the Miguel Servet programme of the Instituto de Salud Carlos III (CPII19/00001, CPII17/00006, and CP16/00116, respectively) cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) cofinanced by Fondo Social Europeo (FSE). R.L.-V. is supported by predoctoral contract PEJD-2019-PRE/BMD-16414 from Comunidad de Madrid. C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. P.S. is supported by postdoctoral contract “Margarita Salas” CA1/RSUE/2021 from MCIU. B.S. was supported by a predoctoral research fellowship (Doctorados Industriales, DI-17-09134) from Spanish MINECO.

Purpose of Review Major waterborne viruses comprise numerous variants rather than only a master sequence and form a genetically diverse population. High genetic diversity is advantageous for adaptation to environmental changes because the highly diverse population likely includes variants resistant to an adverse effect. Disinfection is a broadly employed tool to inactivate pathogens, but due to virus evolvability, waterborne viruses may not be inactivated sufficiently in currently applied disinfection conditions. Here, by focusing on virus population genetics, we explore possibility and factor of emergence of disinfection sensitivity change. Recent Findings To test whether virus population obtains disinfection resistance, the evolutionary experiment developed in the field of population genetics has been applied, indicating the change in disinfection sensitivity. It has been also confirmed that the sensitivity of environmental strains is lower than that of laboratory strains. In some of these studies, genetic diversity within a population less sensitive to disinfection is higher. Researches in virus population genetics have shown the contribution of intra-population genetic diversity to virus population phenotype, so disinfection sensitivity change may attribute to the genetic diversity. Summary The research elucidating a relationship between virus evolution and disinfection has only recently begun, but significant information about the relationship has been accumulated. To develop an effective disinfection strategy for the control of waterborne virus spread, we need to clarify whether disinfection practice truly affects virus outbreaks by refining both laboratory and field experiments related to virus evolution in the disinfection-exerted environment.

Background & Aims HBV genotype G (HBV/G) is mainly found in co-infections with other HBV genotypes and was identified as an independent risk factor for liver fibrosis. This study aimed to analyse the prevalence of HBV/G co-infections in healthy European HBV carriers and to characterize the crosstalk of HBV/G with other genotypes. Methods A total of 560 European HBV carriers were tested via HBV/G-specific PCR for HBV/G co-infections. Quasispecies distribution was analysed via deep sequencing, and the clinical phenotype was characterized regarding qHBsAg-/HBV-DNA levels and frequent mutations. Replicative capacity and expression of HBsAg/core was studied in hepatoma cells co-expressing HBV/G with either HBV/A, HBV/D or HBV/E using bicistronic vectors. Results Although no HBV/G co-infection was found by routine genotyping PCR, HBV/G was detected by specific PCR in 4%-8% of patients infected with either HBV/A or HBV/E but only infrequently in other genotypes. In contrast to HBV/E, HBV/G was found as the quasispecies major variant in co-infections with HBV/A. No differences in the clinical phenotype were observed for HBV/G co-infections. In vitro RNA and DNA levels were comparable among all genotypes, but expression and release of HBsAg was reduced in co-expression of HBV/G with HBV/E. In co-expression with HBV/A and HBV/E expression of HBV/G-specific core was enhanced while core expression from the corresponding genotype was markedly diminished. Conclusions HBV/G co-infections are common in European inactive carriers with HBV/A and HBV/E infection, but sufficient detection depends strongly on the assay. HBV/G regulated core expression might play a critical role for survival of HBV/G in co-infections.

New SARS-CoV-2 mutants have been continuously indentified with enhanced transmission ever since its outbreak in early 2020. As an RNA virus, SARS-CoV-2 has a high mutation rate due to the low fidelity of RNA polymerase. To study the single nucleotide polymorphisms (SNPs) dynamics of SARS-CoV-2, 158 SNPs with high confidence were identified by deep meta-transcriptomic sequencing, and the most common SNP type was C > T. Analyses of intra-host population diversity revealed that intra-host quasispecies’ composition varies with time during the early onset of symptoms, which implicates viral evolution during infection. Network analysis of co-occurring SNPs revealed the most abundant non-synonymous SNP 22,638 in the S glycoprotein RBD region and 28,144 in the ORF8 region. Furthermore, SARS-CoV-2 variations differ in an individual’s respiratory tissue (nose, throat, BALF, or sputum), suggesting independent compartmentalization of SARS-CoV-2 populations in patients. The positive selection analysis of the SARS-CoV-2 genome uncovered the positive selected amino acid G251V on ORF3a. Alternative allele frequency spectrum (AAFS) of all variants revealed that ORF8 could bear alternate alleles with high frequency. Overall, the results show the quasispecies’ profile of SARS-CoV-2 in the respiratory tract in the first two months after the outbreak.

Although chemotherapy is generally indicated for treatment of enterovirus infections, antivirals are currently not used in clinical practice. The use of monotherapy is the main reason for this unfavourable state. This is related to the fact that enterovirus progeny consist of innumerable quasispecies, allowing the virus to develop drug resistance quickly. Here, we present a consecutive alternating administration (CAA) treatment scheme for combining enterovirus inhibitors. Applying the CAA approach with a combination of pleconaril (capsid binder), guanidine HCl (viral 2C inhibitor), and oxoglaucine (PI4KB inhibitor) (PGO) was found to be effective in the treatment of newborn mice infected with a massive inoculum (20 MLD50) of the coxsackievirus B3 cardiotropic Woodruff or neurotropic Nancy strain. In addition to preventing drug resistance, the CAA approach resulted in the parallel development of increased susceptibility to the compounds in the PGO combination. These observations demonstrate the therapeutic potential of the CAA approach for treatment of enterovirus infections.

Hepatitis C virus (HCV) persistently infects more than 175-185 million people globally. Drug resistance to anti-HCV DAAs therapeutics particularly NS3/4A has been a major concern for efficacy of HCV treatment due to generation of HCV quasispecies. Risk determination of the resistance of HCV to anti-NS3/4A inhibitors was accomplished using ViPR algorithm that computes and analyzes resistance-associated amino acid substitutions in HCV NS3 protein target site and reveal altered response to NS3/4A PIs. Spot comparison and data visualization was carried out using ncbi.nlm.nih.gov protein sequence graphics and R-package for Ubuntu version 12.04. Resistance to multiple NS3/4A PIs (telaprevir, simeprevir, faldaprevir and asunaprevir) was exhibited by HCV genotype 1 variant D1194A/E/T/G-NS3 (a substitution hot spot) while the risk of resistance to telaprevir (V1062 L NS3 variant) and simeprevir (S1148R variant) was demonstrated by HCV genotype 2. Genotype 3 & 4 revealed resistance against only telaprevir. Another almost pan-genotype (genotype 2,3,4,5 and unclassified) substitution hot spot was identified as anti-telaprevir variant V1062 L. Single substitution conferring multiple anti-NS3/4A PIs resistance was observed in D1194A NS3 variant (anti-simeprevir, anti-faldaprevir, anti-asunaprevir in genotype 1), Q1106K NS3 variant (anti-simeprevir, anti-faldaprevir in genotype 5 and 6) and T1080S NS3 variant (anti-faldaprevir, anti-telaprevir in genotype 5 and unclassified NS3 sequence). In addition to that, variant S1148G exhibited increased sensitivity to simeprevir in case of genotype 1. Because of a higher degree of HCV genomic variability, NS3 variants with decreased susceptibility to NS3/4A PIs exists, and therefore the resistance profile along with potency, adverse effect of PIs is noteworthy and should be well-considered while developing different protease inhibitors for effective treatment of HCV patients.

Objective:To stop transmission of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in association with myocardial perfusion imaging (MPI) at a cardiology clinic.Design:Outbreak investigation and quasispecies analysis of HCV hypervariable region 1 genome.Setting:Outpatient cardiology clinic.Patients:Patients undergoing MPI.Methods:Case patients met definitions for HBV or HCV infection. Cases were identified through surveillance registry cross-matching against clinic records and serological screening. Observations of clinic practices were performed.Results:During 2012–2014, 7 cases of HCV and 4 cases of HBV occurred in 4 distinct clusters among patients at a cardiology clinic. Among 3 case patients with HCV infection who had MPI on June 25, 2014, 2 had 98.48% genetic identity of HCV RNA. Among 4 case patients with HCV infection who had MPI on March 13, 2014, 3 had 96.96%–99.24% molecular identity of HCV RNA. Also, 2 clusters of 2 patients each with HBV infection had MPI on March 7, 2012, and December 4, 2014. Clinic staff reused saline vials for >1 patient. No infection control breaches were identified at the compounding pharmacy that supplied the clinic. Patients seen in clinic through March 27, 2015, were encouraged to seek testing for HBV, HCV, and human immunodeficiency virus. The clinic switched to all single-dose medications and single-use intravenous flushes on March 27, 2015, and no further cases were identified.Conclusions:This prolonged healthcare-associated outbreak of HBV and HCV was most likely related to breaches in injection safety. Providers should follow injection safety guidelines in all practice settings.

Haplotipos; Enfermedades infecciosas; Mutación Haplotips; Enfermetats infeccioses; Mutació Haplotypes; Infectious diseases; Mutation 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

Background HIV-1C has been shown to have a greater risk of virological failure and reduced susceptibility towards boosted protease inhibitors (bPIs), a component of second-line combination antiretroviral therapy (cART) in South Africa. This study entailed an evaluation of HIV-1 drug resistance-associated mutations (RAMs) among minor viral populations through high-throughput sequencing genotypic resistance testing (HTS-GRT) in patients on the South African national second-line cART regimen receiving bPIs. Methods During 2017 and 2018, 67 patient samples were sequenced using high-throughput sequencing (HTS), of which 56 samples were included in the final analysis because the patient’s treatment regimen was available at the time of sampling. All patients were receiving bPIs as part of their cART. Viral RNA was extracted, and complete pol genes were amplified and sequenced using Illumina HiSeq2500, followed by bioinformatics analysis to quantify the RAMs according to the Stanford HIV Drug Resistance Database. Results Statistically significantly higher PI RAMs were observed in minor viral quasispecies (25%; 14/56) compared to non-nucleoside reverse transcriptase inhibitors (9%; 5/56; p = 0.042) and integrase inhibitor RAM (4%; 2/56; p = 0.002). The majority of the drug resistance mutations in the minor viral quasispecies were observed in the V82A mutation (n = 13) in protease and K65R (n = 5), K103N (n = 7) and M184V (n = 5) in reverse transcriptase. Conclusions HTS-GRT improved the identification of PI and reverse transcriptase inhibitor (RTI) RAMs in second-line cART patients from South Africa compared to the conventional GRT with ≥20% used in Sanger-based sequencing. Several RTI RAMs, such as K65R, M184V or K103N and PI RAM V82A, were identified in

Time space between waves of Covid-19 is linked to selection of most appropriate virus mutant(s)/variant(s), in the Spike (S) gene, generated during massive and error prone replication of the virus in the human populations that are either naive or partially immune due to infection immunity or vaccinal immunity, having higher transmissibility with immune-escape feature and increased pathogenicity. The mutations in the S gene have been found to be progressive to resist prevalent virus neutralizing antibodies in the hosts. The first mutation of significance was D (Asp) to G (Gly) at the position 614 of the Spike protein, over the original Wuhan strain/virus. Subsequently, with in a short time span of less than 18 months since December 2019, many Variants/mutants of SARS-CoV-2, viz., B.1.1.7, B.1.427, B.1.1.28.2, B.1.429, B.1.617, B.1.351, and P.1 have been found associated with the current waves of the Covid-19 pandemic in different countries. The latest significant S variants identified in the second wave in India and elsewhere are B.1.617 and its Kappa (k), Delta (8) and Delta plus (8+; Lys to Asn at position 417) strains, in the order of appearance. The lambda (X) variant is prevalent in South America. The P.2 variant B.1.1.28.2 was antigenically dominant over B.1 D614G virus. The k and 8 variants of the S gene were partially resistant to neutralization by existing vaccinal antibodies, and the 8 variant has been feared to ignite third wave in many countries of Americas, Asia and Europe. The SARS-CoV-2 possibly has ‘Quasispecies’ structure, like the transboundary animal pathogen Foot and Mouth Disease (FMD) virus, and continuous forward mutations in the S gene has been the cause of the fact that "previous immunity acquired during the course of the natural infection (or vaccination) is not fully competent to protect against subsequent mutants selected (in the nature) that leads to new waves of the disease". The waves of Covid-19 have been guided by emergence of sequential antigenic variants, e.g. B.1 D614G virus ^B.1 8 mutant ^B.1 8+ mutant. Intermittent/inter-wave drop in incidence of the infection between the Covid-19 waves could be attributed to very short term (less than 3 months?) infection/vaccination immunity that is breached by the successive antigenic mutant (s) selected. Vaccination and virus circulation happening simultaneously, can facilitate faster selection of neutralization-escape mutants, which is now evident and may lead to new waves of the disease/infection at short intervals of less than 6 months. All variants described here are antigenically dissimilar, whereas the current Covid-19 vaccines are monovalent. Therefore, it may be essential and beneficial to have multivalent vaccines or with higher antigenic mass in monovalent vaccines. Further, it is required that nasal swabs of a certain percentage of vaccinates be collected at the time of vaccination and preserved for retrospect analysis of their infection status, that in turn will reveal the infection status of the population in villages/cities/districts/State.

Currently, a lot of attention is given to SARS-CoV-2 subpopulations and their coexistence with different genomic variants within the same patient. In this study, we performed next-generation whole-genome sequencing and assembly of viruses from samples representing swabs or autopsy specimens obtained from patients diagnosed with СOVID-19, which were initially confirmed by the real-time polymerase chain reaction (Ct = 10.4–19.8). Samples were prepared for sequencing by using the SCV-2000bp protocol. The obtained data were checked for presence of more than one SARS-CoV-2 genetic variants in a sample. Variants of nucleotide substitutions, coverage for each variant, and location of the variable position in the reference genome were detected with tools incorporated in the CLC Genomics Workbench program. In our search for variable nucleotide positions, we assumed that the sample had two genetic variants (not more); the threshold value ≥ 90% was set for probability of the identified variant. Variants represented by less than 20% of the reads in the total coverage were not taken into consideration. The obtained results showed that 5 samples had variability, i.e. they had several genetic variants of SARS-CoV-2. In 4 samples, both of the detected genomic variants differed only in one nucleotide position. The fifth sample demonstrated more substantial differences: a total of 3 variable positions and one three-nucleotide deletion. Our study shows that different genetic variants of SARS-CoV-2 can coexist within the same patient.

Few non-invasive models were established for precisely identifying the immune tolerant (IT) phase from chronic hepatitis B (CHB). This study aimed to develop a novel approach that combined next-generation sequencing (NGS) and machine learning algorithms using our recently published viral quasispecies (QS) analysis package. 290 HBeAg positive patients from whom liver biopsies were taken were enrolled and divided into a training group (n = 148) and a validation group (n = 142). HBV DNA was extracted and QS sequences were obtained by NGS. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) based on viral operational taxonomic units (OTUs) were performed to explore the correlations among QS and clinical phenotypes. Three machine learning algorithms, including K-nearest neighbour, support vector machine, and random forest algorithm, were used to construct diagnostic models for IT phase classification. Based on histopathology, 90 IT patients and 200 CHB patients were diagnosed. HBsAg titres for IT patients were higher than those of CHB patients (p < 0.001). HCA and PCA analysis grouped IT and CHB patients into two distinct clusters. The relative abundance of viral OTUs differed mainly within the BCP/precore/core region and was significantly correlated with liver inflammation and fibrosis. For the IT phase classification, all machine-learning models showed higher AUC values compared to models based on HBsAg, APRI, and FIB-4. The relative abundance of viral OTUs reflects the severity of liver inflammation and fibrosis. The novel QS quantitative analysis approach could be used to diagnose IT patients more precisely and reduce the need for liver biopsy.

Hand, Foot and Mouth Disease (HFMD) is usually a self-limiting, mild childhood disease that is caused mainly by Coxsackie virus A16 (CVA16) and Enterovirus A71 (EV-A71), both members of the Picornaviridae family. However, recurring HFMD outbreaks and epidemics due to EV-A71 infection in the Western Pacific region, and the propensity of EV-A71 strains to cause severe neurological complications have made this neurotropic virus a serious public health concern in afflicted countries. High mutation rate leading to viral quasispecies combined with frequent intra- and inter-typic recombination events amongst co-circulating EV-A71 strains have contributed to the great diversity and fast evolution of EV-A71 genomes, making impossible any accurate prediction of the next epidemic strain. Comparative genome sequence analyses and mutagenesis approaches have led to the identification of a number of viral determinants involved in EV-A71 fitness and virulence. These viral determinants include amino acid residues located in the structural proteins of the virus, affecting attachment to the host cell surface, receptor binding, and uncoating events. Critical residues in non-structural proteins have also been identified, including 2C, 3A, 3C proteases and the RNA-dependent RNA polymerase. Finally, mutations altering key secondary structures in the 5’ untranslated region were also found to influence EV-A71 fitness and virulence. While our current understanding of EV-A71 pathogenesis remains fragmented, these studies may help in the rational design of effective treatments and broadly protective vaccine candidates.

Background Human immunodeficiency virus (HIV)/hepatitis B virus (HBV) co-infection can accelerate HBV-induced liver disease. A previous study showed that variation in the HBV pre-S region and quasispecies heterogeneity (Sn, mean genetic distance, dS, dN, and dS/dN) are both related to HBV-induced terminal liver disease in HBV mono-infection. Currently, data are lacking on quasispecies variation of the HBV pre-S region in HIV/HBV co-infection. Investigating the quasispecies variation of the HBV pre-S region and its related factors in HIV/HBV co-infection will help to better explore the pathogenic mechanism of HIV/HBV co-infection. Methods According to the HIV antibody results obtained before treatment, chronic HBV-infected patients were divided into HIV/HBV co-infected and HBV mono-infected groups. The clinical characteristics of all patients were collected, and DNA was extracted from the serum. The HBV pre-S region was amplified by nested PCR and was further TA cloned. BioEdit software 7.0 was used for sequence alignment with reference to the standard sequence of the matched HBV genotype. We used 1:1 propensity score matching (PSM) to control for baseline confounding factors between the two groups. Results After 1:1 PSM, we identified 100 patients with similar propensities: 50 HIV/HBV co-infected patients and 50 HBV mono-infected patients. HBV quasispecies indices were lower in the HIV/HBV co-infected group than those in the HBV mono-infected group. A significant correlation was observed between all quasispecies indices and soluble cluster of differentiation 163 (sCD163) and interleukin-18 (IL-18) in the HIV/HBV co-infected group; however, this phenomenon was not found in the HBV mono-infected group. Conclusion Combined HIV infection reduces quasispecies heterogeneity in the HBV pre-S region, and the quasispecies heterogeneity is related to the sCD163 and IL-18 levels.

Objective Short-read next-generation sequencing (NGS) has been implemented to study the resistance profile of HIV as it provides a higher sensitivity than Sanger sequencing. However, short-reads only generates a consensus view of the viral population rather than a reconstruction of the viral haplotypes. In this study, we evaluated the resistance profile of HIV quasispecies in patients undergoing treatment failure using SMRT sequencing. Design Whole-pol RT-PCR was performed on viral RNA extracted from plasma samples of 38 HIV-positive individuals undergoing treatment failure, and sequenced in the RSII instrument. Error correction and viral haplotype phasing was performed with the Multilayer Directed Phasing and Sequencing (MDPSeq) algorithm. Presence of resistance mutations reported by the IAS-USA in 2017 was assessed using an in-house script. Results The SMRT sequencing-based test detected 131/134 resistance mutations previously detected using a Sanger sequencing-based test. However, the SMRT test also identified seven additional mutations present at an estimated frequency lower than 30%. The intra-host phylogenetic analysis showed that seven samples harbored at least one resistance variant at 20--80% frequency. The haplotype-resolved sequencing revealed viral diversification and selection of new resistance during suboptimal treatment, an overall trend toward selection and accumulation of new resistance mutations, as well as the co-existence of resistant and susceptible variants. Conclusion Our results validate the SMRT sequencing-based test for detection of HIV drug resistance. In addition, this method unraveled the complex dynamic of HIV quasispecies during treatment failure, which might have several implications on clinical management.

Motivation RNA viruses exhibit a high mutation rate and thus they exist in infected cells as a population of closely related strains called viral quasispecies. The viral quasispecies assembly problem asks to characterize the quasispecies present in a sample from high-throughput sequencing data. We study the de novo version of the problem, where reference sequences of the quasispecies are not available. Current methods for assembling viral quasispecies are either based on overlap graphs or on de Bruijn graphs. Overlap graph-based methods tend to be accurate but slow, whereas de Bruijn graph-based methods are fast but less accurate. Results We present viaDBG, which is a fast and accurate de Bruijn graph-based tool for de novo assembly of viral quasispecies. We first iteratively correct sequencing errors in the reads, which allows us to use large k-mers in the de Bruijn graph. To incorporate the paired-end information in the graph, we also adapt the paired de Bruijn graph for viral quasispecies assembly. These features enable the use of long-range information in contig construction without compromising the speed of de Bruijn graph-based approaches. Our experimental results show that viaDBG is both accurate and fast, whereas previous methods are either fast or accurate but not both. In particular, viaDBG has comparable or better accuracy than SAVAGE, while being at least nine times faster. Furthermore, the speed of viaDBG is comparable to PEHaplo but viaDBG is able to retrieve also low abundance quasispecies, which are often missed by PEHaplo. Availability and implementation viaDBG is implemented in C++ and it is publicly available at https://bitbucket.org/bfreirec1/viadbg. All datasets used in this article are publicly available at https://bitbucket.org/bfreirec1/data-viadbg/. Supplementary information Supplementary data are available at Bioinformatics online.

International audience; Objective: HIV-1 transmission leads to a genetic bottleneck, with one or a few variants of the donor quasispecies establishing an infection in the new host. We aimed to characterize this bottleneck in more detail, by comparing the properties of HIV envelope glycoproteins from acute and chronic infections within the particular context of a male-to-male transmission cluster. Design: We compared the genotypic and phenotypic properties of envelope glycoproteins from viral variants derived from five study participants from the same transmission cluster. Methods: We used single-genome amplification to generate a collection of full-length env sequences. We then constructed pseudotyped viruses expressing selected Env variants from the quasispecies infecting each study participant and compared their infectivities and sensitivities to various entry inhibitors. Results: The genotypic analyses confirmed the genetic bottleneck expected after HIV transmission, with a limited number of variants identified in four study participants during acute infection. However, the transmitted sequences harbored no evident common signature and belonged to various genetic lineages. The phenotypic analyses revealed no difference in infectivity, susceptibility to the CCR5 antagonist maraviroc, the fusion inhibitor enfurvitide or type-I interferon between viruses from participants with acute and chronic infections. The key property distinguishing transmitted viruses was a higher resistance to soluble CD4 þ , correlated with greater sensitivity to occupation of the CD4 þ receptor by the anti-CD4 þ antibodies LM52 and SK3. Conclusion: These results suggest that envelope glycoproteins from transmitted/ founder viruses bind CD4 þ less efficiently than those of viruses from chronic infections.

Lotus (Nelumbo nucifera Gaertn.) is an economically important perennial aquatic herb plant in China. Presently, viral diseases caused by Cucumber mosaic virus (CMV) and Dasheen mosaic virus (DsMV) are detected in lotus. In this study, the putatively novel badnaviruses from lotus plants (LBVs) were identified using next-generation sequencing of siRNAs and conventional Sanger sequencing based on the RT/RNase H coding region sequences. Lotus plants infected by LBVs harbored virus quasispecies. A unique recombination event of LBVs was found in a single lotus plant. Sap inoculation showed that LBVs could actively replicate in the lotus plants, but without a wide host range. The field survey of 43 lotus plants in Jiangsu Province showed a prevalence of 62.8% for LBVs, confirming that it is widely distributed in Jiangsu Province of China.

The aim of this study was to investigate the influence of variant coat proteins (CPs) from different quasispecies of betanodavirus on diverse aspects of nodavirus-induced pathogenesis. It is known that variant CPs can acquire either nuclear or cytoplasmic localization, depending on the nodavirus CP genotype, and this variation may arise during viral replication and influence the regulation of host and viral gene transcription. To investigate the role of these variant CPs in pathogenesis, six variant CP expression plasmids were constructed, each containing different quasispecies CP variants from nodavirus genotype red spotted grouper nervous necrosis virus (RGNNV). The CP expression plasmids were transiently transfected into grouper GF-1 cells. At different times, the cell cycle and cell proliferation were assayed using flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. The proportion of G2/M-phase GF-1 cells transfected with CP expression plasmids was higher than that of cells transfected with the blank plasmid, especially in regards to quasispecies 2 (QS2). The proliferation ratio of cells transfected with the CP expression plasmids was significantly higher than that of cells transfected with the blank plasmid, with the exception of QS6. We also found that the different quasispecies CPs downregulated the promoter activity of the tumor necrosis factor (TNF) gene to different degrees. In addition, this is the first report showing the betanodavirus CP derived from different quasispecies of RGNNV provide evidence of a chronically nodavirus-infected grouper. Overall, this study represents the first comprehensive analysis of variant CPs from grouper with persistent nodavirus infections and their effects on different aspects of pathogenesis.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains the furin cleavage Proline-Arginine-Arginine-Alanine (PRRA) motif in the S1/S2 region, which enhances viral pathogenicity but is absent in closely related bat and pangolin coronaviruses. Whether bat-like coronaviral variants without PRRA (∆PRRA) can establish natural infections in humans is unknown. Methods Here, we developed a duplex digital polymerase chain reaction assay to examine ∆PRRA variants in Vero-E6-propagated isolates, human organoids, experimentally infected hamsters, and coronavirus disease 2019 (COVID-19) patients. Results We found that SARS-CoV-2, as currently transmitting in humans, contained a quasispecies of wild-type, ∆PRRA variants and variants that have mutations upstream of the PRRA motif. Moreover, the ∆PRRA variants were readily detected despite being at a low intra-host frequency in transmitted founder viruses in hamsters and in COVID-19 patients, including in acute cases and a family cluster, with a prevalence rate of 52.9%. Conclusions Our findings demonstrate that bat-like SARS-CoV-2ΔPRRA not only naturally exists but remains transmissible in COVID-19 patients, which has significant implications regarding the zoonotic origin and natural evolution of SARS-CoV-2.

Background Combined HIV infection can accelerate HBV-induced liver disease. It is known that HBV Pre-S deletion is closely related to HBV-associated terminal liver disease in HBV mono-infection. Currently, data on HBV Pre-S quasispecies feature deletion in HIV/HBV co-infected patients are lacking. Methods The characteristics and blood samples of patients with chronic HBV infection were collected and classified into an HIV/HBV co-infection group and an HBV mono-infection group according to HIV antibody results before treatment. HBV DNA in serum was extracted. The HBV Pre-S region was amplified by nested-PCR and was further T-A cloned. Using the standard sequence of the matched genotype HBV as a reference, BioEdit 7.0 software was employed for sequence alignment. Results HBV Pre-S regions were successfully amplified from 147 patients, including 71 cases in the HIV/HBV co-infected group and 76 cases in the HBV mono-infected group. The proportion of the HIV/HBV co-infected group with Pre-S quasispecies deletion was lower than that of the HBV mono-infected group. By analyzing the frequency of Pre-S quasispecies in the two groups, the frequency of Pre-S quasispecies in HIV/HBV co-infected patients with Pre-S quasispecies was higher than HBV mono-infected patients. The frequency of Pre-S quasispecies deletion of the S protein promoter region in the HIV/HBV co-infected group was significantly higher than that in the HBV mono-infected group. Conclusion High-frequency Pre-S quasispecies deletions are predominant in HIV/HBV co-infected patients; however, low-frequency Pre-S deletions are predominant in HBV mono-infected patients, providing a reference for the pathogenesis of the accelerated progression of liver disease in HIV/HBV co-infection.

Background Viruses have high mutation rates and generally exist as a mixture of variants in biological samples. Next-generation sequencing (NGS) approaches have surpassed Sanger for generating long viral sequences, yet how variants affect NGS de novo assembly remains largely unexplored. Results Our results from > 15,000 simulated experiments showed that presence of variants can turn an assembly of one genome into tens to thousands of contigs. This “variant interference” (VI) is highly consistent and reproducible by ten commonly-used de novo assemblers, and occurs over a range of genome length, read length, and GC content. The main driver of VI is pairwise identities between viral variants. These findings were further supported by in silico simulations, where selective removal of minor variant reads from clinical datasets allow the “rescue” of full viral genomes from fragmented contigs. Conclusions These results call for careful interpretation of contigs and contig numbers from de novo assembly in viral deep sequencing.

Background COVID-19 (coronavirus disease 2019) has caused a major epidemic worldwide; however, much is yet to be known about the epidemiology and evolution of the virus partly due to the scarcity of full-length SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genomes reported. One reason is that the challenges underneath sequencing SARS-CoV-2 directly from clinical samples have not been completely tackled, i.e., sequencing samples with low viral load often results in insufficient viral reads for analyses. Methods We applied a novel multiplex PCR amplicon (amplicon)-based and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of SARS-CoV-2 from serials dilutions of a cultured isolate, and eight clinical samples covering a range of sample types and viral loads. We also examined and compared the sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner. Results We demonstrated that both amplicon and capture methods efficiently enriched SARS-CoV-2 content from clinical samples, while the enrichment efficiency of amplicon outran that of capture in more challenging samples. We found that capture was not as accurate as meta and amplicon in identifying between-sample variations, whereas amplicon method was not as accurate as the other two in investigating within-sample variations, suggesting amplicon sequencing was not suitable for studying virus-host interactions and viral transmission that heavily rely on intra-host dynamics. We illustrated that meta uncovered rich genetic information in the clinical samples besides SARS-CoV-2, providing references for clinical diagnostics and therapeutics. Taken all factors above and cost-effectiveness into consideration, we proposed guidance for how to choose sequencing strategy for SARS-CoV-2 under different situations. Conclusions This is, to the best of our knowledge, the first work systematically investigating inter- and intra-individual variations of SARS-CoV-2 using amplicon- and capture-based whole-genome sequencing, as well as the first comparative study among multiple approaches. Our work offers practical solutions for genome sequencing and analyses of SARS-CoV-2 and other emerging viruses.

HIV drug resistance is a major threat to achieving long-term viral suppression in HIV-positive individuals. Drug resistant HIV variants, including minority variants, can compromise response to antiretroviral therapy. Many studies have investigated the clinical relevance of drug resistant minority variants, but the level at which minority variants become clinically relevant remains unclear. A combination of Primer-ID and deep sequencing is a promising approach that may quantify minority variants more accurately compared to standard deep sequencing. However, most studies that used the Primer-ID method have analyzed clinical samples directly. Thus, its sensitivity and quantitative accuracy have not been adequately validated using known controls. Here, we constructed defined proportions of artificial RNA and virus quasispecies and measured their relative proportions using the Primer-ID based, quantitative single-variant sequencing (qSVS) assay. Our results showed that minority variants present at 1% of quasispecies were detected reproducibly with minimal variations between technical replicates. In addition, the measured frequencies were comparable to the expected frequencies. These data validate the accuracy and reproducibility of the qSVS assay in quantifying authentic HIV minority variants, and support the use of this approach to examine the impacts of minority HIV variants on virologic response and clinical outcome.

HIV has remarkable genetic diversity among populations. The diversity has critical impacts on transmission, immune escape, pathogenesis, and clinical management. HIV-1 diversity originates from frequent mutation and recombination during reverse transcription. This work focuses on the quasispecies genetic dynamics within individuals with primary infections. Eleven men who have sex with men from the Beijing PRIMO Clinical Cohort were identified as primary infection and had three or four series of their anticoagulant blood samples collected. Viral RNA was extracted and amplified using single-genome amplification. Products of the gp120 gene that met single-genome amplification requirements were sequenced. Subtype assortment of all collected sequences was performed using both the jumping profile hidden Markov model (jpHMM) and REGA. Quasispecies diversity at each time was estimated using Mega 6. Intrapatient recombination was analyzed using RDP4. According to the Fiebig classification system, YA-81 belongs to stage III and YA-113 belongs to stage IV. The other samples are all associated with the infection stage of V/VI. YA113 had a dual infection with subtype B and a new unique recombinant form involving CRF01_AE and C. The other eight were infected with CRF01_AE, one was infected with B/C recombinant, and the last one with B. Of the 10 single infections, 8 were caused by 1 founder virus. They all displayed a sharp increase of quasispecies diversity during the sampling times. Two were caused by at least two founder viruses. The diversity of these strains starts at a significantly high level and is followed by a relatively steady trend. Critically, the separate subtypes YA113-B and YA113-CRF01_AE/C both showed a similar trend to those infected by a single founder virus. Recombination analysis revealed that 5 of 11 cases underwent detectable intrapatient recombination. These findings indicate that tracing the dynamics of HIV-1 quasispecies during early infection may be relevant and valuable for understanding pathways of viral diversification and immune escape.

Mother-to-child transmission of hepatitis B virus (HBV) is the main route of transmission in Asia, and characterization of HBV quasispecies is needed to further understand virus evolution and adaptation. To understand changes in HBV during mother-to-child transmission, we enrolled nine pairs of mothers and children in the study, including a set of twins. Three groups were infected with HBV genotype C, and six groups were infected with HBV genotype B. The full-length HBV genome was amplified by PCR from serum samples before antiviral treatment, the whole viral genomes from each pair were sequenced, and the complexity and diversity of the quasispecies were analyzed. The entropy of transmitted HBV in children was found to be lower than their mothers, suggesting that there was a bottleneck effect during HBV transmission from the mother to the child. Selective evolution was shown by calculating πN and πS in the whole genomes, and the highest values were obtained for the X gene, which plays a role in viral replication and immune escape. All genotype C patients and only one genotype B pair had a πN/πS greater than 1 ratio, indicating that positive selection had occurred. In addition, quasispecies were found to be different between the twin children despite having the same mother, indicating that virus evolution is host-specific.

The fidelity of flaviviruses is thought to be tightly regulated for optimal fitness within and between hosts. West Nile virus (WNV) high-fidelity (HiFi) mutations V793I and G806R within the RNA-dependent RNA polymerase, and low-fidelity (LoFi) mutation T248I within the methyltransferase, were previously shown to attenuate infectivity and replicative fitness in Culex mosquitoes and Culex tarsalis (CXT) cells but not in mammalian cells. We hypothesized that fidelity alterations would modify adaptation and maintenance in a host-specific manner. To test this hypothesis, wild-type (WT), HiFi (V793I/G806R) and LoFi (T248I) variants were sequentially passaged eight times in avian (PDE) or mosquito cells, or alternately between the two. Initial characterization confirmed that fidelity mutants are attenuated in mosquito, but not avian, cells. Deep sequencing revealed mutations unique to both cell lines and fidelity mutants, including ENV G1378A, a mutation associated with avian cell adaptation. To characterize maintenance and adaptation, viral outputs were monitored throughout passaging and viral fitness was assessed. The results indicate that fidelity mutants can at times recover fitness during mosquito cell passage, but remain attenuated relative to WT. Despite similar initial fitness, LoFi mutants were impaired during sequential passage in avian cells. Conversely, HiFi mutants passaged in avian cells showed increased adaptation, suggesting that increased fidelity may be advantageous in avian hosts. Although some adaptation occurred with individual mutants, the output titres of fidelity mutants were on average lower and were often lost during host switching. These data confirm that arbovirus fidelity is likely fine-tuned to maximize survival in disparate hosts.

International audience; Objectives: To evaluate the impact of neutralizing monoclonal antibody (mAb) treatment and to determine whether the selective pressure of mAbs could facilitate the proliferation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with spike protein mutations that might attenuate mAb effectiveness.Patients and methods: We evaluated the impact of mAbs on the nasopharyngeal (NP) viral load and virus quasispecies of mAb-treated patients using single-molecule real-time sequencing. The mAbs used were: Bamlanivimab alone (four patients), Bamlanivimab/Etesevimab (23 patients) and Casirivimab/Imdevimab (five patients).Results: The NP SARS-CoV-2 viral load of mAb-treated patients decreased from 8.2 log10 copies/mL before administration to 4.3 log10 copies/mL 7 days after administration. Five immunocompromised patients given Bamlanivimab/Etesevimab were found to have mAb activity-reducing spike mutations. Two patients harboured SARS-CoV-2 variants with a Q493R spike mutation 7 days after administration, as did a third patient 14 days after administration. The fourth patient harboured a variant with a Q493K spike mutation 7 days post-treatment, and the fifth patient had a variant with a E484K spike mutation on day 21. The emergence of the spike mutation was accompanied by stabilization or rebound of the NP viral load in three of five patients.Conclusion: Two-mAb therapy can drive the selection of resistant SARS-CoV-2 variants in immunocompromised patients. Patients given mAbs should be closely monitored and measures to limit virus spread should be reinforced.

Chronic infection with Hepatitis B virus (HBV) is a major risk factor for the development of advanced liver disease including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The relative contribution of virological factors to disease progression has not been fully defined and tools aiding the deconvolution of complex patient virus profiles is an unmet clinical need. Variable viral mutant signatures develop within individual patients due to the low-fidelity replication of the viral polymerase creating ‘quasispecies’ populations. Here we present the first comprehensive survey of the diversity of HBV quasispecies through ultra-deep sequencing of the complete HBV genome across two distinct European and Asian patient populations. Seroconversion to the HBV e antigen (HBeAg) represents a critical clinical waymark in infected individuals. Using a machine learning approach, a model was developed to determine the viral variants that accurately classify HBeAg status. Serial surveys of patient quasispecies populations and advanced analytics will facilitate clinical decision support for chronic HBV infection and direct therapeutic strategies through improved patient stratification.

RNA viruses replicate as complex mutant spectra termed viral quasispecies. The frequency of each individual genome in a mutant spectrum depends on its rate of generation and its relative fitness in the replicating population ensemble. The advent of deep sequencing methodologies allows for the first-time quantification of haplotype abundances within mutant spectra. There is no information on the haplotype profile of the resident genomes and how the landscape evolves when a virus replicates in a controlled cell culture environment. Here, we report the construction of intramutant spectrum haplotype landscapes of three amplicons of the NS5A-NS5B coding region of hepatitis C virus (HCV). Two-dimensional (2D) neural networks were constructed for 44 related HCV populations derived from a common clonal ancestor that was passaged up to 210 times in human hepatoma Huh-7.5 cells in the absence of external selective pressures. The haplotype profiles consisted of an extended dense basal platform, from which a lower number of protruding higher peaks emerged. As HCV increased its adaptation to the cells, the number of haplotype peaks within each mutant spectrum expanded, and their distribution shifted in the 2D network. The results show that extensive HCV replication in a monotonous cell culture environment does not limit HCV exploration of sequence space through haplotype peak movements. The landscapes reflect dynamic variation in the intramutant spectrum haplotype profile and may serve as a reference to interpret the modifications produced by external selective pressures or to compare with the landscapes of mutant spectra in complex in vivo environments. IMPORTANCE The study provides for the first time the haplotype profile and its variation in the course of virus adaptation to a cell culture environment in the absence of external selective constraints. The deep sequencing-based self-organized maps document a two-layer haplotype distribution with an ample basal platform and a lower number of protruding peaks. The results suggest an inferred intramutant spectrum fitness landscape structure that offers potential benefits for virus resilience to mutational inputs.

BackgroundThis study aimed to explore the molecular mechanism of the coexistence of hepatitis B surface antigen (HBsAg) and hepatitis B surface antibody (HBsAb) serological pattern via intensive characterization of HBV s gene in both chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC) patients.MethodA total of 73 HBsAg+/HBsAb+ patients (CHB = 36, HCC = 37) and 96 HBsAg+/HBsAb− patients (CHB = 47, HCC = 49) were enrolled from 13 medical centers in China. The sequence features were elaborated based on the combination of next-generation sequencing (NGS) and multidimensional bioinformatics analysis.ResultsThe 16 high-frequency missense mutations, changes of stop codon mutation, clustering, and random forest models based on quasispecies features demonstrated the significant discrepancy power between HBsAg+/HBsAb+ and HBsAg+/HBsAb− in CHB and HCC, respectively. The immunogenicity for cytotoxic T lymphocyte (CTL) epitope Se and antigenicity for the major hydrophilic region (MHR) were both reduced in HBsAg+/HBsAb+ patients (CTL Se: p < 0.0001; MHR: p = 0.0216). Different mutation patterns were observed between HBsAg+/HBsAb+ patients with CHB and with HCC. Especially, mutations in antigenic epitopes, such as I126S in CHB and I126T in HCC, could impact the conformational structure and alter the antigenicity/immunogenicity of HBsAg.ConclusionBased on NGS and bioinformatics analysis, this study indicates for the first time that point mutations and quasispecies diversities of HBV s gene could alter the MHR antigenicity and CTL Se immunogenicity and could contribute to the concurrent HBsAg+/HBsAb+ with different features in HCC and CHB. Our findings might renew the understanding of this special serological profile and benefit the clinical management in HBV-related diseases.

Background Genome assembly of viruses with high mutation rates, such as Norovirus and other RNA viruses, or from metagenome samples, poses a challenge for the scientific community due to the coexistence of several viral quasispecies and strains. Furthermore, there is no standard method for obtaining whole-genome sequences in non-related patients. After polyA RNA isolation and sequencing in eight patients with acute gastroenteritis, we evaluated two de Bruijn graph assemblers (SPAdes and MEGAHIT), combined with four different and common pre-assembly strategies, and compared those yielding whole genome Norovirus contigs. Results Reference-genome guided strategies with both host and target virus did not present any advantages compared to the assembly of non-filtered data in the case of SPAdes, and in the case of MEGAHIT, only host genome filtering presented improvements. MEGAHIT performed better than SPAdes in most samples, reaching complete genome sequences in most of them for all the strategies employed. Read binning with CD-HIT improved assembly when paired with different analysis strategies, and more notably in the case of SPAdes. Conclusions Not all metagenome assemblies are equal and the choice in the workflow depends on the species studied and the prior steps to analysis. We may need different approaches even for samples treated equally due to the presence of high intra host variability. We tested and compared different workflows for the accurate assembly of Norovirus genomes and established their assembly capacities for this purpose.

Fibrosis; Hepatitis C virus; Viral load Fibrosis; Virus de la hepatitis C; Carga viral Fibrosi; Virus de l'hepatitis C; Càrrega viral Cirrhosis derived from chronic hepatitis C virus (HCV) infection is still a common indication for liver transplantation (LT). Reinfection of the engrafted liver is universal in patients with detectable viral RNA at the time of transplant and causes fast progression of cirrhosis (within 5 years) in around one-third of these patients. To prevent damage to the liver graft, effective direct-acting antiviral (DAA) therapy is required as soon as possible. However, because of post-LT clinical instability, it is difficult to determine the optimal time to start DAAs with a low risk of complications. Evaluate changes in quasispecies complexity following LT and seek a predictive index of fast liver damage progression to determine the timing of DAA initiation. HCV genomes isolated from pre-LT and 15-day post-LT serum samples of ten patients, who underwent orthotopic LT, were quantified and sequenced using a next-generation sequencing platform. Sequence alignments, phylogenetic trees, quasispecies complexity measures, biostatistics analyses, adjusted R2 values, and analysis of variance (ANOVA) were carried out. Three different patterns of reinfection were observed (viral bottlenecking, conserved pre-LT population, and mixed populations), suggesting that bottlenecking or homogenization of the viral population is not a generalized effect after liver graft reinfection. None of the quasispecies complexity measures predicted the future degree of liver damage. Higher and more uniform viral load (VL) values were observed in all pre-LT samples, but values were more dispersed in post-LT samples. However, VL increased significantly from the pre-LT to 15-day post-LT samples in patients with advanced fibrosis at 1-year post-LT, suggesting that a VL increase on day 15 may be a predictor of fast liver fibrosis progression. HCV kinetics after LT differ between patients and are not fibrosis-dependent. Higher VL at day 15 post-LT versus pre-LT samples may predict fast liver fibrosis progression. This study was supported by grants from Instituto de Salud Carlos III cofinanced by the European Regional Development Fund (ERDF) with grant numbers PI19/00533, PI19/00301, Clinical Trial Gov. Identifier: NCT01707849, and from Centro para el Desarrollo Tecnológico Industrial-CDTI of the Spanish Ministry of Economics and Competitiveness (MINECO) grant number, IDI-20200297. C.P. is supported by the Miguel Servet program of Instituto de Salud Carlos III, grant CP14/00121, cofinanced by the ERDF. Astellas Pharma Inc and Novartis Pharma also provided funding for the study, but these companies had had no role in the study design, data collection or analysis, decision to publish, or preparation of the manuscript.

Enterovirus A71 (EV-A71) and many members of the Picornaviridae family are neurotropic pathogens of global concern. These viruses are primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. An animal model of oral infection was developed using transgenic mice expressing human SCARB2 (hSCARB2 Tg), murine-adapted EV-A71/MP4 virus, and EV-A71/MP4 virus with an engineered nanoluciferase gene that allows imaging of viral replication and spread in infected mice. Next-generation sequencing of EV-A71 genomes in the tissues and organs of infected mice was also performed. Oral inoculation of EV-A71/MP4 or nanoluciferase-carrying MP4 virus stably induced neurological symptoms and death in infected 21-day-old weaned mice. In vivo bioluminescence imaging of infected mice and tissue immunostaining of viral antigens indicated that orally inoculated virus can spread to the central nervous system (CNS) and other tissues. Next-generating sequencing further identified diverse mutations in viral genomes that can potentially contribute to viral pathogenesis. This study presents an EV-A71 oral infection murine model that efficiently infects weaned mice and allows tracking of viral spread, features that can facilitate research into viral pathogenesis and neuroinvasion via the natural route of infection. IMPORTANCE Enterovirus A71 (EV-A71), a positive-strand RNA virus of the Picornaviridae, poses a persistent global public health problem. EV-A71 is primarily transmitted through the fecal-oral route, and thus suitable animal models of oral infection are needed to investigate viral pathogenesis. We present an animal model of EV-A71 infection that enables the natural route of oral infection in weaned and nonimmunocompromised 21-day-old hSCARB2 transgenic mice. Our results demonstrate that severe disease and death could be stably induced, and viral invasion of the CNS could be replicated in this model, similar to severe real-world EV-A71 infections. We also developed a nanoluciferase-containing EV-A71 virus that can be used with this animal model to track viral spread after oral infection in real time. Such a model offers several advantages over existing animal models and can facilitate future research into viral spread, tissue tropism, and viral pathogenesis, all pressing issues that remain unaddressed for EV-A71 infections.

Background Previous works have observed that younger infants with chronic hepatitis B virus (HBV) infection are more responsive to antiviral treatment. However, the underlying mechanism remains unclear. In this study, the dynamic changes of HBV quasispecies in infants with immunoprophylaxis failure were investigated to provide virological explanations for clinical management on infantile antiviral therapy. Methods Thirteen 7-month-old infants with immunoprophylaxis failure and their mothers were enrolled from a prospective cohort, and 8 of them were followed up to 3 years old. The sequences of HBV quasispecies were analyzed by the full-length genome clone-based sequencing, and compared among mothers and their infants at different ages. Results The results revealed that the complexity, mutation frequency and genetic distance of HBV quasispecies decreased significantly at full-length, partial open reading frames and regulatory regions of HBV genome at nucleotide level in 7-month-old infants comparing with their mothers, whereas increased significantly to near the maternal level when infants grew up to 3 years old. Furthermore, similar changes were also found in Core, PreS2, RT and P regions of HBV genome at amino acid level, especially for potential NAs-resistant mutants in RT region and immune-escape mutants in Core and PreS2 regions. Conclusions This study uncovered the evolution of HBV quasispecies in infancy after mother-to-child transmission, which may provide the virological evidence for explaning that younger children are more responsive to antiviral therapy.

Enterovirus, like the majority of RNA viruses, evolves to survive the changeable environments by a variety of strategies. Here, we showed that HY12 virus evolved to alter its characteristics and pathogenicity by employing a non-synonymous mutation. Analyses of 5′UTR, VP1 and VP2 gene sequences revealed the existence of HY12 virus in an array of mutants defined as quasispecies. The determination of diversity and complexity showed that the mutation rate and complexity of HY12 virus quasispecies increased, while the proportion of HY12 VP1 and VP2 consensus (master) sequences decreased with increasing passages. Synonymous mutation and non-synonymous mutation analysis displayed a positive selection for HY12 quasispecies evolution. A comparison of HY12 virus in different passages demonstrated that HY12 virus altered its characteristic, phenotype, and pathogenicity via non-synonymous mutation. These findings revealed the evolution pattern for HY12 virus, and the alteration of HY12 virus characteristics and pathogenicity by mutation.

Within the family Geminiviridae, the emergence of new species results from their high mutation and recombination rates. In this study, we report the variability and evolution of digitaria streak virus (DSV), a mastrevirus isolated in 1986 from the grass Digitaria setigera in an island of the Vanuatu archipelago. Viral DNA of DSV samples was amplified from D. setigera specimens, derived from the naturally infected original plant, which were propagated in different laboratories in France and Italy for more than 20 years. From the consensus sequences, the nucleotide substitution rate was estimated for the period between a sample and the original sequence published in 1987, as well as for the period between samples. In addition, the intra-host genetic complexity and diversity of 8 DSV populations with a total of 165 sequenced haplotypes was characterized. The evolutionary rate of DSV was estimated to be between 1.13 × 10−4 and 9.87 × 10−4 substitutions/site/year, within the ranges observed in other single-stranded DNA viruses and RNA viruses. Bioinformatic analyses revealed high variability and heterogeneity in DSV populations, which confirmed that mutant spectra are continuously generated and are organized as quasispecies. The analysis of polymorphisms revealed nucleotide substitution biases in viral genomes towards deamination and oxidation of single-stranded DNA. The differences in variability in each of the genomic regions reflected a dynamic and modular evolution in the mutant spectra that was not reflected in the consensus sequences. Strikingly, the most variable region of the DSV genome, encoding the movement protein, showed rapid fixation of the mutations in the consensus sequence and a concomitant dN/dS ratio of 6.130, which suggests strong positive selection in this region. Phylogenetic analyses revealed a possible divergence in three genetic lineages from the original Vanuatu DSV isolate.

Viral quasispecies are dynamic distributions of nonidentical but closely related mutant and recombinant viral genomes subjected to a continuous process of genetic variation, competition, and selection that may act as a unit of selection. The quasispecies concept owes its theoretical origins to a model for the origin of life as a collection of mutant RNA replicators. Independently, experimental evidence for the quasispecies concept was obtained from sampling of bacteriophage clones, which revealed that the viral populations consisted of many mutant genomes whose frequency varied with time of replication. Similar findings were made in animal and plant RNA viruses. Quasispecies became a theoretical framework to understand viral population dynamics and adaptability. The evidence came at a time when mutations were considered rare events in genetics, a perception that was to change dramatically in subsequent decades. Indeed, viral quasispecies was the conceptual forefront of a remarkable degree of biological diversity, now evident for cell populations and organisms, not only for viruses. Quasispecies dynamics unveiled complexities in the behavior of viral populations,with consequences for disease mechanisms and control strategies. This review addresses the origin of the quasispecies concept, its major implications on both viral evolution and antiviral strategies, and current and future prospects., SAF2014-52400-R from Ministerio de Economía y Competitividad (MINECO), SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER), and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121 and CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander

Mother-to-child transmission (MTCT) through breastfeeding remains a major source of pediatric HIV-1 infection worldwide. To characterize plasma HIV-1 subtype C populations from infected mothers during pregnancy that related to subsequent breast milk transmission, an exploratory study was designed to apply next generation sequencing and a custom bioinformatics pipeline for HIV-1 gp41 extending from heptad repeat region 2 (HR2) through the membrane proximal external region (MPER) and the membrane spanning domain (MSD). MPER harbors linear and highly conserved epitopes that repeatedly elicits HIV-1 neutralizing antibodies with exceptional breadth. Viral populations during pregnancy from women who transmitted by breastfeeding, compared to those who did not, displayed greater biodiversity, more frequent amino acid polymorphisms, lower hydropathy index and greater positive charge. Viral characteristics were restricted to MPER, failed to extend into flanking HR2 or MSD regions, and were unrelated to predicted neutralization resistance. Findings provide novel parameters to evaluate an association between maternal MPER variants present during gestation and lactogenesis with subsequent transmission outcomes by breastfeeding. Importance HIV-1 transmission through breastfeeding accounts for 39% of MTCT and continues as a major route of pediatric infection in developing countries where access to interventions for interrupting transmission is limited. Identifying women who are likely to transmit HIV-1 during breastfeeding would focus therapies, such as broad neutralizing HIV monoclonal antibodies (bn-HIV-Abs), during the breastfeeding period to reduce MTCT. Findings from our pilot study identify novel characteristics of gestational viral MPER quasispecies related to transmission outcomes and raise the possibility for predicting MTCT by breastfeeding based on identifying mothers with high-risk viral populations.

The error rate displayed during template copying to produce viral RNA progeny is a biologically relevant parameter of the replication complexes of viruses. It has consequences for virus–host interactions, and it represents the first step in the diversification of viruses in nature. Measurements during infections and with purified viral polymerases indicate that mutation rates for RNA viruses are in the range of 10 to 10 copying errors per nucleotide incorporated into the nascent RNA product. Although viruses are thought to exploit high error rates for adaptation to changing environments, some of them possess misincorporation correcting activities. One of them is a proofreading-repair 3 to 5 exonuclease present in coronaviruses that may decrease the error rate during replication. Here we review experimental evidence and models of information maintenance that explain why elevated mutation rates have been preserved during the evolution of RNA (and some DNA) viruses. The models also offer an interpretation of why error correction mechanisms have evolved to maintain the stability of genetic information carried out by large viral RNA genomes such as the coronaviruses., Ministerio de Economía y Competitividad (MINECO), grant number SAF2014-52400-R, SAF2017-87846-R and BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 from Instituto de Salud Carlos III, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER), and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CPII19/00001), cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged

International audience; Hepatitis E virus (HEV) infection can lead to a variety of neurological disorders. While HEV RNA is known to be present in the central nervous system, HEV quasispecies in serum and cerebrospinal fluid (CSF) have rarely been explored. We studied the virus’ quasispecies in the blood and the CSF of five patients at the onset of their neurological symptoms. The samples of three patients suffering from meningitis, neuralgic amyotrophy and acute inflammatory polyradiculoneuropathy were taken at the acute phase of the HEV infection. The samples from the other two patients were taken during the chronic phase (5 years after HEV diagnosis) when they presented with clinical signs of encephalitis. We sequenced at least 20 randomly polyproline regions of the selected virus clones. Phylogenetic analysis of the virus variants in the blood and the CSF revealed no virus compartmentalization for the three acute-phase patients but there was clear evidence of HEV quasispecies compartmentalization in the CSF of the two patients during chronic infection. In conclusion, prolonged infection in the immunocompromised condition can lead to independent virus replication in the liver and the tissues, producing viruses in CSF.