High Recombination Rate of Hepatitis C Virus Revealed by a Green Fluorescent Protein Reconstitution Cell System

Genetic recombination is an important evolutionary mechanism for RNA viruses and can facilitate escape from immune and drug pressure. Recombinant hepatitis C virus (HCV) variants have rarely been detected in patients, suggesting that HCV has intrinsic low recombination rate. Recombination of HCV has been demonstrated in vitro between non-functional genomes, but its frequency and relevance for viral evolution and life cycle has not been clarified. We developed a cell-based assay to detect and quantify recombination between fully viable HCV genomes, using the reconstitution of green fluorescent protein (GFP) as a surrogate marker for recombination. Here, two GFP-expressing HCV genomes carrying different inactivating GFP mutations can produce a virus carrying a functional GFP by recombining within the GFP region. Generated constructs allowed quantification of recombination rates between markers spaced 603 and 553 nucleotides apart by flow cytometry and next-generation sequencing (NGS). Viral constructs showed comparable spread kinetics and reached similar infectivity titers in Huh7.5 cells, allowing their use in co-transfections and co-infections. Single cycle co-transfection experiments, performed in CD81-deficient S29 cells, showed GFP expression in double-infected cells, demonstrating genome mixing and occurrence of recombination. Quantification of recombinant genomes by NGS revealed an average rate of 6.1%, corresponding to 49% of maximum detectable recombination (MDR). Experiments examining recombination during the full replication cycle of HCV, performed in Huh7.5 cells, demonstrated average recombination rates of 5.0 % (40.0% MDR) and 3.6% (28.8% MDR) for markers spaced by 603 and 553 nucleotides, respectively, supporting a linear relationship between marker distance and recombination rates. First passage infections using recombinant virus supernatant resulted in comparable recombination rates of 5.9% (47.2% MDR) and 3.5% (28.0% MDR), respectively, for markers spaced by 603 and 553 nucleotides. We developed a functional cell-based assay that, to our knowledge, allows for the first-time detailed quantification of recombination rates using fully viable HCV constructs. Our data indicate that HCV recombines at high frequency between highly similar genomes, and that the frequency of recombination increases with the distance between marker sites. These results have implication for our understanding of HCV evolution and emphasize the importance of recombination in the reassortment of mutations in the HCV genome.

Analyses of HIV Proteases Variants at the Threshold of Viability Reveals Relationships Between Processing Efficiency and Fitness

Investigating the relationships between protein function and fitness provides keys for understanding biochemical mechanisms that underly evolution. Mutations with partial fitness defects can delineate the threshold of biochemical function required for viability. We utilized a previous deep mutational scan of HIV-1 protease (PR) to identify variants with 15-45% defects in replication and analyzed the biochemical function of eight variants (L10M, L10S, V32C, V32I, A71V, A71S, Q92I, Q92N). We purified each variant and assessed the efficiency of peptide cleavage for three cut sites (MA-CA, TF-PR, PR-RT) as well as a gel-based analyses of processing of purified Gag. The cutting activity of at least one site was perturbed relative to WT protease for all variants, consistent with cutting activity being a primary determinant of fitness effects. We examined the correlation of fitness defects with cutting activity of different sites. MA-CA showed the weakest correlation (R2=0.02) with fitness, suggesting relatively weak coupling with viral replication. In contrast, cutting of the TF-PR site showed the strongest correlation with fitness (R2=0.53). Cutting at the TF-PR site creates a new PR protein with a free N-terminus that is critical for activity. Our findings indicate that increasing the pool of active PR is rate limiting for viral replication making this an ideal step to target with inhibitors.

Polerovirus Genomic Variation

The polerovirus (family Solemoviridae, genus Polerovirus) genome consists of single, positive strand RNA organized in overlapping open reading frames (ORFs) that, in addition to others, code for protein 0 (P0, a gene silencing suppressor), a coat protein (CP, ORF3) and a read-through domain (ORF5) that is fused to the CP to form a CP-RT protein. The genus Polerovirus contains 26 virus species that infect a wide variety of plants from cereals to cucurbits, to peppers. Poleroviruses are transmitted by a wide range of aphid species in the genera Rhopalosiphum, Stiobion, Aphis, and Myzus. Aphid transmission is mediated both by the CP and the CP-RT. In viruses, mutational robustness and structural flexibility are necessary for maintaining functionality in genetically diverse sets of host plants and vectors. Under this scenario, within a virus genome, mutations preferentially accumulate in areas that are determinants of host adaptation or vector transmission. In this study, we profiled genomic variation in poleroviruses. Consistent with their multifunctional nature, single nucleotide variation and selection analyses showed that ORFs coding for P0 and the read-through domain within the CP-RT are the most variable and contain the highest frequency of sites under positive selection. An order/disorder analysis showed that protein P0 is not disordered. In contrast, proteins CP-RT and VPg contain areas of disorder. Disorder is a property of multifunctional proteins with multiple interaction partners. Results described here suggest that using contrasting mechanisms, P0, VPg and CP-RT mediate adaptation to host plants, to vectors, and are contributors to the broad host and vector range of poleroviruses. Profiling genetic variation across the polerovirus genome has practical applications in diagnostics, breeding for resistance, identification of susceptibility genes, and contributes to our understanding of virus interactions with their host, vectors, and environment.

Independent Epidemic Patterns of HIV-1 CRF01_AE Lineages Driven by Mobile Population in Shenzhen, an Immigrant city of China

Shenzhen, a city with > 12 million migrant population, may play a key role in the spread of HIV-1 in China. The transmission dynamics of CRF01_AE, a predominant subtype in Shenzhen, is a good model to characterize the impact of human mobility on HIV-1 epidemic locally and nationally. We used phylodynamic and phylogeographic methods to estimate the viral transmission dynamics and migration trajectory of variable lineages based on 1,423 CRF01_AE sequences in Shenzhen sampled between 2006 and 2015. Eleven lineages of CRF01_AE were detected in Shenzhen. Of those, four main lineages originated during the 1990s. The basic viral reproduction number (R0) of them ranged 1.96-3.92. The effective viral reproduction number (Re) of two lineages prevalent among heterosexuals/people who inject drugs had reduced < 1 at the end of sampling, and the main sources were the intra-provincial immigrants (72%) for one and local residents of Shenzhen (91%) for another. Within two lineages among MSM, Re had been above or close to 1 at the end of sampling, and the immigrants from Jiangxi/Shaanxi and Hubei as sources accounted for 93% and 68% of all viral migration events, respectively. Moreover, no obvious recipients were found throughout the viral migration history for any lineage. Our findings demonstrate that HIV declining epidemic in Shenzhen coincided with the initiation of the interventions during the 2000s. However, the obvious differences of the epidemic patterns between lineages emphasize the importance of further targeting interventions and continued molecular tracing, focusing on high-risk transmission sources among MSM.

Splitting of RNA-dependent RNA Polymerase is Common in Narnaviridae: Identification of a type II Divided-RdRp from Deep-Sea Fungal Isolates

Until recently, it was accepted that RNA-dependent RNA polymerase (RdRp) is the only essential gene for non-retro RNA viruses and is encoded by a single open reading frame (ORF) in their genomes. However, divided-RdRps that are coded by two ORFs were discovered in fungal RNA viruses in a few independent reports. This discovery showed higher plasticity of viral RdRp than was expected. Among these divided-RdRps, the division site was common; specifically, the first part of the RdRp contains motifs F, A, and B, whereas the latter part possesses motifs C and D. These RdRps are designated as type I divided-RdRp and have been limited to viruses in a specific clade of Narnaviridae. In this study, to further understand the plasticity of RdRp, we explored viruses from deep sea-derived fungal strains as an untapped resource with a focus on Aspergillus section Versicolores. Seven strains were found to be infected by total of 13 viruses, and the viral RNA genomes were determined by FLDS technology. Among them, six strains belong to Narnaviridae. One of the strains, Aspergillus tennesseensis narnavirus 1, which infects an Aspergillus tennesseensis, has a divided RdRp with a new division site (referred to as type II divided-RdRp). A couple of sequences for possible type II divided-RdRps were also detected in public metagenomic datasets. Our findings reveal that different types of division in RdRp are present in the virosphere, and two types of RdRp splitting occurred independently within Narnaviridae.

Epidemiological Dynamics of SARS-CoV-2 VOC Gamma in Rio de Janeiro, Brazil

The emergence and widespread circulation of SARS-CoV-2 variants of concern (VOC) or interest (VOI) imposes an enhanced threat to global public health. In Brazil, one of the countries most severely impacted throughout the pandemic, a complex dynamics involving variants co-circulation and turnover events has been recorded with the emergence and spread of VOC Gamma in Manaus in late 2020. In this context, we present a genomic epidemiology investigation based on samples collected between December 2020 and May 2021 in the second major Brazilian metropolis, Rio de Janeiro. By sequencing 244 novel genomes through all epidemiological weeks in this period, we were able to document the introduction and rapid dissemination of VOC Gamma in the city, driving the rise of the third local epidemic wave. Molecular clock analysis indicates this variant has circulated locally since the first weeks of 2021 and only seven weeks were necessary for it to achieve a frequency above 70%, consistent with rates of growth observed in Manaus and other states. Moreover, a Bayesian phylogeographic reconstruction indicates VOC Gamma spread throughout Brazil between December 2020 and January 2021, and that it was introduced in Rio de Janeiro through at least 13 events coming from nearly all regions of the country. Comparative analysis of RT-qPCR cycle threshold (Ct) values provides further evidence that VOC Gamma induces higher viral loads (N1 target; mean reduction of Ct: 2.7, 95% CI = ±0.7). This analysis corroborates the previously proposed mechanistic basis for this variant enhanced transmissibility and distinguished epidemiological behavior. Our results document the evolution of VOC Gamma and provide independent assessment of scenarios previously studied in Manaus, therefore contributing to the better understanding of the epidemiological dynamics currently being surveyed in other Brazilian regions.