Prevalence of mixed genotype hepatitis C virus infections in the UK as determined by genotype‐specific PCR and deep sequencing

The incidence of mixed genotype hepatitis C virus infections in the UK is largely unknown. As the efficacy of direct acting antivirals is variable across different genotypes, treatment regimens are tailored to the infecting genotype, which may pose issues for the treatment of underlying genotypes within undiagnosed mixed genotype HCV infections. There is therefore a need to accurately diagnose mixed genotype infections prior to treatment. PCR-based diagnostic tools were developed to screen for the occurrence of mixed genotype infections caused by the most common UK genotypes, 1a and 3, in a cohort of 506 individuals diagnosed with either of these genotypes. The overall prevalence rate of mixed infection was 3.8% however this rate was unevenly distributed, with 6.7% of individuals diagnosed with genotype 3 harbouring genotype 1a strains and only 0.8% of samples from genotype 1a patients harbouring genotype 3 (p<0.05). Mixed infection samples consisted of a major and a minor genotype, with the latter constituting less than 21% of the total viral load and, in 67% of cases, less than 1% of the viral load. Analysis of a subset of the cohort by Illumina PCR-next generation sequencing resulted in a much greater incidence rate than obtained by PCR. This may have occurred due to the non-quantitative nature of the technique and despite the designation of false positive thresholds based on negative controls

Retrocyclin Rc-101 Overcomes Cationic Mutations On The Heptad Repeat 2 Of Hiv-1 Gp41

Retrocyclin RC-101, a θ-defensin with lectin-like properties, potently inhibits infection by many HIV-1 subtypes by binding to the heptad repeat (HR)-2 region of gp41 and preventing six-helix bundle formation. In the present study, we used in silico computational exploration to identify residues of HR2 that interacted with RC-101 and then analyzed the HIV-1 Sequence Database at LANL for residue variations in the HR1 and HR2 segments that could plausibly impart in vivo resistance. Docking RC-101 to gp41 peptides in silico confirmed its strong preference for HR2 over HR1, and implicated residues crucial for its ability to bind HR2. We mutagenized these residues in pseudotyped HIV-1 JR.FL reporter viruses, and subjected them to single round replication assays in the presence of 1.25-10ug/ml RC-101. Except for one mutant that was partially resistant to RC-101, the other pseudotyped viruses with single-site cationic mutations in HR2 manifested absent or impaired infectivity or retained wild-type susceptibility to RC-101. Overall, these data suggest that most mutations capable of rendering HIV-1 resistant to RC-101 will also exert deleterious effects on the ability of HIV-1 to initiate infections - an interesting and novel property for a potential topical microbicide

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10

Tempus et Locus: a tool for extracting precisely dated viral sequences from GenBank, and its application to the phylogenetics of primate erythroparvovirus 1 (B19V)

The presence of data in the collection_date field of a GenBank sequence record is of great assistance in the use of that sequence for Bayesian phylogenetics using tip-dating. We present Tempus et Locus (TeL), a tool for extracting such sequences from a GenBank-formatted sequence database. TeL shows that 60% of viral sequences in GenBank have collection date fields, but that this varies considerably between species. Primate erythroparvovirus 1 (human parvovirus B19 or B19V) has only 40% of its sequences dated, of which only 112 are of more than 4 kb. 100 of these are from B19V sub-genotype 1a and were collected from a mere 6 studies conducted in 5 countries between 2002 and 2013. Nevertheless, Bayesian phylogenetic analysis of this limited set gives a date for the common ancestor of sub-genotype 1a in 1990 (95% HPD 1981-1996) which is in reasonable agreement with estimates of previous studies where collection dates have been assembled by more laborious methods of literature search and direct enquiries to sequence submitters. We conclude that although collection dates should become standard for all future GenBank submissions of virus sequences, accurate dating of ancestors is possible with even a small number of sequences if sampling information is high quality

The p12 Domain Is Unstructured in a Murine Leukemia Virus p12-CAN Gag Construct

The Gag polyproteins of gammaretroviruses contain a conserved p12 domain between MA and CA that plays critical roles in virus assembly, reverse transcription and nuclear integration. Here we show using nuclear magnetic resonance, that p12 is unstructured in a Moloney murine leukemia virus (MMLV) Gag fragment that includes the N-terminal domain of CA (p12-CAN). Furthermore, no long range interactions were observed between the domains, as has been previously predicted. Flexibility appears to be a common feature of Gag “late” domains required for virus release during budding. Residues near the N-terminus of CAN that form a β-hairpin in the mature CA protein are unfolded in p12-CAN, consistent with proposals that hairpin formation helps trigger capsid assembly

Proactive Detection of Unknown Binary Executable Malware

To detect unknown malware, heuristic methods or more generally statistical approaches are the most promising research trends nowadays, but their computing and detection performances are generally not compatible with what users do accept. Hence, most commercial AV products still heavily rely on signature-based detection (opcodes, control flow graph, and so on). This implies that frequent and prior updates must be performed. May their analysis techniques be fully static of dynamic (using sandboxing or virtual machines), commercial AVs do not capture what defines malware compared to benign files: their intrinsic actions. In this chapter, we focus on binary executables and we describe how to effectively synthetize these actions and what are the differences between malware and nonmalicious files. We extract and analyze two tables that are present in executable files: the import address table (IAT) and export address table (EAT). These tables summarize the different interactions of the executable with the operating system. We show how this information can be used in supervised learning to provide effective detection algorithms, which have proven to be very accurate and proactive with respect to unknown malware detection

Descoberta de novos vírus vegetais e estudo da diversidade viral intrahospedeiro a partir de dados gerados por sequenciamento em larga escala

Dissertação (mestrado)—Universidade de Brasília, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2018.As tecnologias de sequenciamento em larga escala permitem a caracterização genômica das comunidades virais presentes em tecidos vegetais e animais e em amostras ambientais com alta sensibilidade e acurácia. Devido ao sequenciamento simultâneo de várias sequências genômicas, essa técnica também permite o estudo da alta diversidade genética intra-hospedeiro apresentada pelos vírus de RNA. Nesse trabalho, estudamos e estabelecemos um pipeline para a análise de viroma em planta utilizando o modelo de pepino, reportamos a descoberta de dois novos vírus em videiras, Grapevine enamovirus1 (GEV-1) e Grapevine virga-like virus (GVLV). Após ensaios de amplificação rápida das extremidades do cDNA (rapid amplification of cDNA ends – RACE) da extremidade 5' do genoma do GEV-1, foi descrito a sequência genômica quase completa desse vírus (6227 bp), possibilitando a sua classificação como um membro do gênero Enamovirus (família Luteoviridae) com base na sua organização genômica, estudos filogenéticos e critérios estabelecidos pelo Comitê Internacional de Taxonomia de Vírus (International Committee on Taxonomy of Viruses – ICTV). Entretanto, o genoma do GVLV permanece parciamente sequenciado em duas partes: um contig de 3348 bp que contém os domínios metiltransferase (Met) e helicase (Hel); e um contig de 1272 bp que corresponde à RNA polimerase dependente de RNA (RdRp) parcial. Com base em estudos filogenéticos não foi possível classificar esse vírus, que mostra baixa identidade com ambas as famílias Virgaviridae e Bromoviridae. Adicionalmente, esse trabalho apresenta um estudo da diversidade genética intra-hospedeiro dos vírus associados ao enrolamento da folha da videira (Grapevine leafroll-associated virus – GLRaV), com foco na poliproteína dos GLRaV-2 e -3 (gêneros Closterovirus e Ampelovirus, respectivamente), assim como a detecção in silico de uma molécula defectiva de RNA do GLRaV-4 (Ampelovirus), a partir de dados gerados por HTS. As populações intra-hospedeiro encontradas em dois isolados de GLRaV-2 mostraram apenas 11 polimorfismos de único nucleotídeo (single nucleotide polymorphisms – SNPs) em comum (~14% dos SNPs em cada isolado). A diversidade intra-hospedeiro encontrada em dois isolados de GLRaV-3 foi baixa se comparada com os isolados de GLRaV-2.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) e Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).High-throughput sequencing technologies allow for the genomic characterization of viral communities present in plant and animal tissues and environmental samples with high accuracy and sensibility. The simultaneous sequencing of various genomic sequences by this technique also makes it useful for the study of the high intrahost genetic diversity presented by RNA viruses. In this work, we studied and established the conditions of analysis of plant virome using the cucumber model, the discovery of two novel grapevine viruses, Grapevine enamovirus-1 (GEV-1) and Grapevine virga-like virus (GVLV). After rapid amplification of cDNA ends (RACE) assays of the 5' end of GEV-1 genome, we obtained the near full genomic sequence of this virus (6227 bp), enabling its classification as a member of the genus Enamovirus (family Luteoviridae) based on its genomic properties, phylogenetic studies and criteria stablished by the International Committee on Taxonomy of Viruses (ICTV). However, the genome of GVLV remains only partially sequenced, separated in two parts: a 3348 bp contig containing the methyltranferase (Met) and helicase (Hel) domains; and a 1272 bp contig which corresponds to the partial RNA dependent RNA polimerase (RdRp). Based on phylogenetic studies, were not able to classify this novel virus, which shows low identity with viruses in the families Virgaviridae and Bromoviridae. Additionally, this works presents a study on the intrahost genetic diversity of Grapevine leafroll-associated viruses (GLRaVs), focusing on the polyprotein of GLRaV-2 and -3 (genera Closterovirus and Ampelovirus, respectively), as well as an in silico detection of a defective RNA molecule of GLRaV-4 (Ampelovirus). The intrahost population of two isolates of GLRaV-2 showed only 11 single nucleotide polymorphisms (SNPs) in common (~14 of the SNPs found on each isolate). The intrahost genetic diversity found on two isolates of GLRaV3 was low compared to GLRaV-2