Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence complete genomes comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimised library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved starting with clinical samples in 1-2 days following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas.

Transmission of Zika virus (ZIKV) in the Americas was first confirmed in May 2015 in northeast Brazil1. Brazil has had the highest number of reported ZIKV cases worldwide (more than 200,000 by 24 December 20162) and the most cases associated with microcephaly and other birth defects (2,366 confirmed by 31 December 20162). Since the initial detection of ZIKV in Brazil, more than 45 countries in the Americas have reported local ZIKV transmission, with 24 of these reporting severe ZIKV-associated disease3. However, the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of this information for interpreting observed trends in reported microcephaly. Here we address this issue by generating 54 complete or partial ZIKV genomes, mostly from Brazil, and reporting data generated by a mobile genomics laboratory that travelled across northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Analyses of viral genomes with ecological and epidemiological data yield an estimate that ZIKV was present in northeast Brazil by February 2014 and is likely to have disseminated from there, nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates for the international spread of ZIKV from Brazil indicate the duration of pre-detection cryptic transmission in recipient regions. The role of northeast Brazil in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the basic reproduction number of the virus.

A vigorous hepatitis C virus (HCV)-specific Th cell response is regarded as essential to the immunological control of HCV viremia. The aim of this study was to comprehensively define the breadth and specificity of dominant HCV-specific CD4(+) T cell epitopes in large cohorts of subjects with chronic and spontaneously resolved HCV viremia. Following in vitro stimulation of PBMC, HCV-specific cell cultures from each subject were screened with an overlapping panel of synthetic 20-mer peptides spanning the entire HCV polyprotein. Of 22 subjects who spontaneously controlled HCV viremia, all recognized at least one of a group of six epitopes situated within the nonstructural (NS) proteins NS3, NS4, and NS5, each of which was detected by >30% of subjects, but most subjects recognized additional, more heterogeneous specificities. In contrast, none of the most frequently targeted epitopes was detected by >5% of persons with chronic infection. The most frequently recognized peptides showed promiscuous binding to multiple HLA-DR molecules in in vitro binding assays and were restricted by different HLA-DR molecules in functional assays in different persons. These data demonstrate that predominant CD4(+) T cell epitopes in persons with resolved HCV infection are preferentially located in the nonstructural proteins and are immunogenic in the context of multiple class II molecules. This comprehensive characterization of CD4(+) T cell epitopes in resolved HCV infection provides important information to facilitate studies of immunopathogenesis and HCV vaccine design and evaluation.

Early after symptom onset, HCV-specific CD4+ T cell responses are primed and detectable in patients regardless of clinical outcome, but without early antiviral therapy these T cells become exhausted or deleted in chronically infected patients.

CD8(+)-T-cell responses play an important role in the containment and clearance of hepatitis C virus (HCV) infection, and an association between viral persistence and development of viral escape mutations has been postulated. While escape from CD8+ -T-cell responses has been identified as a major driving force for the evolution of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), a broader characterization of this relationship is needed in HCV infection. To determine the extent, kinetics, and driving forces of HCV sequence evolution, we sequenced the entire HCV genome longitudinally in four subjects monitored for up to 30 months after acute infection. For two subjects the transmission sources were also available. Of 53 total non-envelope amino acid substitutions detected, a majority represented forward mutations away from the consensus sequence. In contrast to studies in HIV and SIV, however, only 11% of these were associated with detectable CD8+ T-cell responses. Interestingly, 19% of non-envelope mutations represented changes toward the consensus sequence, suggesting reversion in the absence of immune pressure upon transmission. Notably, the rate of evolution of forward and reverse mutations correlated with the conservation of each residue, which is indicative of structural constraints influencing the kinetics of viral evolution. Finally, the rate of sequence evolution was observed to decline over the course of infection, possibly reflective of diminishing selection pressure by dysfunctional CD8+ T cells. Taken together, these data provide insight into the extent to which HCV is capable of evading early CD8+ T-cell responses and support the hypothesis that dysfunction of CD8+ T cells may be associated with failure to resolve HCV infections.

ObjectiveChronic HBV infection affects more than 250 million people worldwide and remains a global healthcare problem in part because we lack curative treatment. Sustained viral control requires HBV-specific T cells, but these become functionally impaired in chronic infection. Clinical evidence indicates that functional cure of HBV infection by the host immune response is feasible. Developing T cell-based therapies able to achieve functional cure will require identification of the requirements for a successful T cell response against HBV and the relative contribution of individual T cell specificities to HBV control.DesignThe phenotype and function of HBV-specific T cells were studied directly ex vivo using fluorochrome-labelled multimers. We studied multiple HBV-specific T cell specificities targeting different HBV proteins in individuals with either an acute self-limiting or chronic HBV infection.ResultsWe detected strong T cell responses targeting multiple HBV viral proteins in acute self-limiting and low-frequency core and polymerase-specific T cells in chronic infection. Expression of the T cell inhibitory receptor PD-1, as well as T cell differentiation, T cell function and T cell regulation differed by stages and outcomes of infection. In addition, these features differed significantly between T cells targeting different HBV specificities.ConclusionHBV-specific T cells with different target specificities are characterised by distinct phenotypical and functional profiles. These results have direct implications for the design of immunological studies in HBV infection, and are potentially relevant for informing immunotherapeutic approaches to induce functional cure.

We monitored expression of PD-1 (a mediator of T-cell exhaustion and viral persistence) on hepatitis C virus (HCV)-specific CD8؉ and CD4 ؉ T cells from blood and liver during acute and chronic infections and after the resolved infection stage. PD-1 expression on HCV-specific T cells was high early in acute infection irrespective of clinical outcome, and most cells continued to express PD-1 in resolved and chronic stages of infection; intrahepatic expression levels were especially high. Our results suggest that an analysis of PD-1 expression alone is not sufficient to predict infection outcome or to determine T-cell functionality in HCV infection.

Serological evidence of hepatitis E virus infection (HEV) has been observed in both humans