Human Adenovirus Type 55 strains

Arboviruses continue to be a significant global health concern. The unbiased metagenomic analyses of mosquito-borne and mosquito-specific viruses are useful to understand viral diversity and for the surveillance of pathogens of medical and veterinary importance. Metagenomic analysis was conducted on 6368 mosquitoes (736 pools), covering 16 species from 18 locations throughout the Republic of Korea (ROK) in 2016. In this report, we describe three viruses detected in a single pool of Aedes vexans nipponii collected at Yongsan U.S. Army Garrison, located in a densely populated district of Seoul, the ROK. The three novel viruses, designated as Yongsan bunyavirus 1 (YBV1), Yongsan picorna-like virus 3 (YPLV3) and Yongsan sobemo-like virus 1 (YSLV1), share sequence and structural characteristics with members belonging to the family Bunyaviridae, order Picornavirales, and family Solemoviridae, with shared RNA-dependent RNA polymerase (RdRp) amino acid identities of 40%, 42% and 86%, respectively. The real-time reverse transcription and polymerase chain reaction (RT-PCR) of 3493 Aedes vexans nipponii (257 pools) showed a high prevalence of YBV1 and YSLV1 viruses, which were present in 65% and 62% of tested pools, respectively. This study highlighted the utility of a metagenomic sequencing approach for arbovirus discovery and for a better understanding of the virome of potential medically relevant vectors.

Dengue is one of the most widespread vector-borne viral diseases in the world. However, the size, heterogeneity, and temporal dynamics of the cell-associated viral reservoir during acute dengue virus (DENV) infection remains unclear. In this study, we analyzed cells infectedin vitrowith DENV and PBMC from an individual experiencing a natural DENV infection utilizing 5’ capture single cell RNA sequencing (scRNAseq). Both positive- and negative-sense DENV RNA was detected in reactions containing either an oligo(dT) primer alone, or in reactions supplemented with a DENV-specific primer. The addition of a DENV-specific primer did not increase the total amount of DENV RNA captured or the fraction of cells identified as containing DENV RNA. However, inclusion of a DENV-specific cDNA primer did increase the viral genome coverage immediately 5’ to the primer binding site. Furthermore, while the majority of intracellular DENV sequence captured in this analysis mapped to the 5’ end of the viral genome, distinct patterns of enhanced coverage within the DENV polyprotein coding region were observed. The 5’ capture scRNAseq analysis of PBMC not only recapitulated previously published reports by detecting virally infected memory and naïve B cells, but also identified cell-associated genomic variants not observed in contemporaneous serum samples. These results demonstrate that oligo(dT) primed 5’ capture scRNAseq can detect DENV RNA and quantify virus-infected cells in physiologically relevant conditions, and provides insight into viral sequence variability within infected cells.

Influenza within-host viral populations are the source of all global influenza diversity and play an important role in driving the evolution and escape of the influenza virus from human immune responses, antiviral treatment, and vaccines, and have been used in precision tracking of influenza transmission chains. Next Generation Sequencing (NGS) has greatly improved our ability to study these populations, however, major challenges remain, such as accurate identification of intra-host single nucleotide variants (iSNVs) that represent within-host viral diversity of influenza virus. In order to investigate the sources and the frequency of called iSNVs in influenza samples, we used a set of longitudinal influenza patient samples collected within a University of Maryland (UMD) cohort of college students in a living learning community. Our results indicate that technical replicates aid in removal of random RT-PCR, PCR, and platform sequencing errors, while the use of clonal plasmids for removal of systematic errors is more important in samples of low RNA abundance. We show that the choice of reference for read mapping affects the frequency of called iSNVs, with the sample self-reference resulting in the lowest amount of iSNV noise. The importance of variant caller choice is also highlighted in our study, as we observe differential sensitivity of variant callers to the mapping reference choice, as well as the poor overlap of their called iSNVs. Based on this, we develop an approach for identification of highly probable iSNVs by removal of sequencing and bioinformatics algorithm-associated errors, which we implement in phylogenetic analyses of the UMD samples for a greater resolution of transmission links. In addition to identifying closely related transmission connections supported by the presence of highly confident shared iSNVs between patients, our results also indicate that the rate of minor variant turnover within a host may be a limiting factor for utilization of iSNVs to determine patient epidemiological links.

Intra-host single nucleotide variants (iSNVs) have been increasingly used in genomic epidemiology to increase phylogenetic resolution and reconstruct fine-scale outbreak dynamics. These analyses are preferably done on sequence data from direct clinical samples, but in many cases due to low viral loads, there might not be enough genetic material for deep sequencing and iSNV determination. Isolation of the virus from clinical samples with low-passage number increases viral load, but few studies have investigated how dengue virus (DENV) culture isolation from a clinical sample impacts the consensus sequence and the intra-host virus population frequencies. In this study, we investigate consensus and iSNV frequency differences between DENV sequenced directly from clinical samples and their corresponding low-passage isolates. Twenty five DENV1 and DENV2 positive sera and their corresponding viral isolates (T. splendens inoculation and C6/36 passage) were obtained from a prospective cohort study in the Philippines. These were sequenced on MiSeq with minimum nucleotide depth of coverage of 500×, and iSNVs were detected using LoFreq. For both DENV1 and DENV2, we found a maximum of one consensus nucleotide difference between clinical sample and isolate. Interestingly, we found that iSNVs with frequencies ≥5 % were often preserved between the samples, and that the number of iSNV positions, and sample diversity, at this frequency cutoff did not differ significantly between the sample pairs (clinical sample and isolate) in either DENV1 or DENV2 data. Our results show that low-passage DENV isolate consensus genomes are largely representative of their direct sample parental viruses, and that low-passage isolates often mirror high frequency within-host variants from direct samples.

With the emergence and spread of SARS-CoV-2 variants, genomic epidemiology and surveillance have proven invaluable tools for variant tracking. Here, we analyzed SARS-CoV-2 samples collected from personnel located at the US/NATO bases across Afghanistan. Sequencing and phylogenetic analyses revealed at least 16 independent introductions of SARS-CoV-2 into four of these relatively isolated compounds during April and May 2021, including multiple introductions of Alpha and Delta variants. Four of the introductions resulted in sustained spread of the virus within, and in two cases between, the compounds. Three of these outbreaks, one Delta and two Alpha, occurred simultaneously. Even in rigorously controlled and segregated environments, SARS-CoV-2 introduction and spread may occur frequently.