Temporal and coevolutionary analyses reveal the events driving the emergence and circulation of human mamastroviruses

ABSTRACTCharacterized by high genetic diversity, broad host range, and resistance to adverse conditions, coupled with recent reports of neurotropic astroviruses circulating in humans, mamastroviruses pose a threat to public health. The current astrovirus classification system based on host source prevents determining whether strains with distinct tropism or virulence are emerging. By using integrated phylogeny, we propose a standardized demarcation of species and genotypes, with reproducible cut-off values that reconcile the pairwise sequence distribution, genetic distances between lineages, and the topological reconstruction of the Mamastrovirus genus. We further define the various links established by co-evolution and resolve the dynamics of transmission chains to identify host-jump events and the sources from which different mamastrovirus species circulating in humans have emerged. We observed that recombination is relatively infrequent and restricted to within genotypes. The well-known “human” astrovirus, defined here as mamastrovirus species 7, has co-speciated with humans, while there have been two additional host-jumps into humans from distinct hosts. Newly defined species 6 genotype 2, linked to severe gastroenteritis in children, resulted from a marmot to human jump taking place ∼200 years ago while species 6 genotype 7 (MastV-Sp6Gt7), linked to neurological disease in immunocompromised patients, jumped from bovines only ∼50 years ago. Through demographic reconstruction, we determined that the latter reached coalescent viral population growth only 20 years ago and is evolving at a much higher evolutionary rate than other genotypes infecting humans. This study constitutes mounting evidence of MastV-Sp6Gt7 active circulation and highlights the need for diagnostics capable of detecting it

Emergence of a Distinct Picobirnavirus Genotype Circulating in Patients Hospitalized with Acute Respiratory Illness

Picobirnaviruses (PBV) are found in a wide range of hosts and typically associated with gastrointestinal infections in immunocompromised individuals. Here, a divergent PBV genome was assembled from a patient hospitalized for acute respiratory illness (ARI) in Colombia. The RdRp protein branched with sequences previously reported in patients with ARI from Cambodia and China. Sputa from hospitalized individuals (n = 130) were screened by RT-qPCR which enabled detection and subsequent metagenomic characterization of 25 additional PBV infections circulating in Colombia and the US. Phylogenetic analysis of RdRp highlighted the emergence of two dominant lineages linked to the index case and Asian strains, which together clustered as a distinct genotype. Bayesian inference further established capsid and RdRp sequences as both significantly associated with ARI. Various respiratory-tropic pathogens were detected in PBV+ patients, yet no specific bacteria was common among them and four individuals lacked co-infections, suggesting PBV may not be a prokaryotic virus nor exclusively opportunistic, respectively. Competing models for the origin and transmission of this PBV genotype are presented that attempt to reconcile vectoring by a bacterial host with human pathogenicity. A high prevalence in patients with ARI, an ability to reassort, and demonstrated global spread indicate PBV warrant greater public health concern

Advanced molecular surveillance approaches for characterization of blood borne hepatitis viruses.

Defining genetic diversity of viral infections directly from patient specimens is the ultimate goal of surveillance. Simple tools that can provide full-length sequence information on blood borne viral hepatitis viruses: hepatitis C, hepatitis B and hepatitis D viruses (HCV, HBV and HDV) remain elusive. Here, an unbiased metagenomic next generation sequencing approach (mNGS) was used for molecular characterization of HCV infections (n = 99) from Israel which yielded full-length HCV sequences in 89% of samples, with 7 partial sequences sufficient for classification. HCV genotypes were primarily 1b (68%) and 1a (19%), with minor representation of genotypes 2c (1%) and 3a (8%). HBV/HDV coinfections were characterized by suppressed HBV viral loads, resulting in sparse mNGS coverage. A probe-based enrichment approach (xGen) aiming to increase HBV and HDV coverage was validated on a panel of diverse genotypes, geography and titers. The method extended HBV genome coverage a median 61% (range 8-84%) and provided orders of magnitude boosts in reads and sequence depth for both viruses. When HBV-xGen was applied to Israeli samples, coverage was improved by 28-73% in 4 samples and identified HBV genotype A1, A2, D1 specimens and a dual B/D infection. Abundant HDV reads in mNGS libraries yielded 18/26 (69%) full genomes and 8 partial sequences, with HDV-xGen only providing minimal extension (3-11%) of what were all genotype 1 genomes. Advanced molecular approaches coupled to virus-specific capture probes promise to enhance surveillance of viral infections and aid in monitoring the spread of local subtypes

Purifying selection decreases the potential for Bangui orthobunyavirus outbreaks in humans

Pathogens carried by insects, such as bunyaviruses, are frequently transmitted into human populations and cause diseases. Knowing which spillover events represent a public health threat remains a challenge. Metagenomic next-generation sequencing (mNGS) can support infectious disease diagnostics by enabling the detection of any pathogen from clinical specimens. mNGS was performed on blood samples to identify potential viral coinfections in human immunodeficiency virus (HIV)-positive individuals from Kinshasa, the Democratic Republic of the Congo (DRC), participating in an HIV diversity cohort study. Time-resolved phylogenetics and molecular assay development assisted in viral characterization. The nearly complete genome of a novel orthobunyavirus related to Nyangole virus, a virus previously identified in neighboring Uganda, was assembled from a hepatitis B virus-positive patient. A quantitative polymerase chain reaction assay was designed and used to screen >2,500 plasma samples from Cameroon, the DRC, and Uganda, failing to identify any additional cases. The recent sequencing of a US Center for Disease Control Arbovirus Reference Collection revealed that this same virus, now named Bangui virus, was first isolated in 1970 from an individual in the Central African Republic. Time-scaled phylogenetic analyses of Bangui with the related Anopheles and Tanga serogroup complexes indicate that this virus emerged nearly 10,000 years ago. Pervasive and episodic models further suggest that this virus is under purifying selection and that only distant common ancestors were subject to positive selection events. This study represents only the second identification of a Bangui virus infection in over 50 years. The presumed rarity of Bangui virus infections in humans can be explained by its constraint to an avian host and insect vector, precluding efficient transmission into the human population. Our results demonstrate that molecular phylogenetic analyses can provide insights into the threat posed by novel or re-emergent viruses identified by mNGS

Discovery of a Novel Human Pegivirus in Blood Associated with Hepatitis C Virus Co-Infection

<div><p>Hepatitis C virus (HCV) and human pegivirus (HPgV), formerly GBV-C, are the only known human viruses in the <i>Hepacivirus</i> and <i>Pegivirus</i> genera, respectively, of the family <i>Flaviviridae</i>. We present the discovery of a second pegivirus, provisionally designated human pegivirus 2 (HPgV-2), by next-generation sequencing of plasma from an HCV-infected patient with multiple bloodborne exposures who died from sepsis of unknown etiology. HPgV-2 is highly divergent, situated on a deep phylogenetic branch in a clade that includes rodent and bat pegiviruses, with which it shares <32% amino acid identity. Molecular and serological tools were developed and validated for high-throughput screening of plasma samples, and a panel of 3 independent serological markers strongly correlated antibody responses with viral RNA positivity (99.9% negative predictive value). Discovery of 11 additional RNA-positive samples from a total of 2440 screened (0.45%) revealed 93–94% nucleotide identity between HPgV-2 strains. All 12 HPgV-2 RNA-positive cases were identified in individuals also testing positive for HCV RNA (12 of 983; 1.22%), including 2 samples co-infected with HIV, but HPgV-2 RNA was not detected in non-HCV-infected individuals (p<0.0001), including those singly infected by HIV (p = 0.0075) or HBV (p = 0.0077), nor in volunteer blood donors (p = 0.0082). Nine of the 12 (75%) HPgV-2 RNA positive samples were reactive for antibodies to viral serologic markers, whereas only 28 of 2,429 (1.15%) HPgV-2 RNA negative samples were seropositive. Longitudinal sampling in two individuals revealed that active HPgV-2 infection can persist in blood for at least 7 weeks, despite the presence of virus-specific antibodies. One individual harboring both HPgV-2 and HCV RNA was found to be seronegative for both viruses, suggesting a high likelihood of simultaneous acquisition of HCV and HPgV-2 infection from an acute co-transmission event. Taken together, our results indicate that HPgV-2 is a novel bloodborne infectious virus of humans and likely transmitted via the parenteral route.</p></div

Phylogenetic analysis of HPgV-2 relative to other pegiviruses and hepaciviruses.

<p><b>(A)</b> Pairwise amino acid identity plots comparing HPgV-2 (UC0125.US) with other representative pegiviruses (red) and hepaciviruses (blue). The sliding window size is 50 nt. <b>(B)</b> Phylogenetic trees of the NS3 (left) and NS5B (right) proteins were constructed for 10 newly sequenced HPgV-2 strains (boldface red), representative hepaciviruses, and all fully sequenced pegiviruses in the NCBI nt database except for members of the simian pegivirus clade, for which 5 representative strains are shown (triangle). Each tree is rooted with yellow fever virus (YFV) as an outgroup.</p

Molecular and Serologic Characterization of HPgV-2 Strains

<p>Abbreviations: NA, not applicable; NT, not tested.</p><p>*These two samples are from the same individual with bleed dates 26 days apart (sample ABT0030P.US is the earlier sample).</p><p>**These two samples are from the same individual with bleed dates 60 days apart (sample ABT0035P.US is the earlier sample).</p><p>^†Italicized numbers show elevated signals, but below the cutoff value.</p><p>Molecular and Serologic Characterization of HPgV-2 Strains</p

Discovery and whole-genome characterization of HPgV-2.

<p><b>(A)</b> Three NGS reads with remote amino acid homology (<60%) to SPgV-A / GBV-A, of which two were overlapping, were detected in a plasma sample from a patient with HCV infection who died of abdominal sepsis (the index case). <b>(B)</b> The initial set of contigs generated from <i>de novo</i> assembly of HPgV-2 reads that were identified using BLASTx alignment to other pegivirus genomes. <b>(C)</b> Gap closure using PCR followed by Sanger sequencing. <b>(D)</b> Coverage plot showing mapping of the initial NGS data to the nearly complete (>98%) assembled draft genome. (<b>E)</b> Coverage plot showing mapping of the NGS data from a subsequent sequencing run to the complete HPgV-2 genome, after the 5' and 3' ends were recovered using RACE [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005325#ppat.1005325.ref020" target="_blank">20</a>]. (<b>F)</b> Genomic arrangement of HPgV-2. Putative cleavage sites within the polyprotein are indicated with black triangles (structural proteins) or hollow triangles (non-structural proteins). Arrows denote predicted N-linked (red) or O-linked (blue) glycosylation sites.</p

Prevalence of HPgV-2 RNA in HCV and non-HCV infected groups.

<p>*P-value of differences in HPgV-2 RNA (top) and antibody (bottom) prevalence between reference and comparison groups. Index case (HCV Ab+ /NAT+) has been excluded from totals screened.</p><p>Prevalence of HPgV-2 RNA in HCV and non-HCV infected groups.</p