Deep Sequencing to Reveal Phylo-Geographic Relationships of Juquitiba Virus in Paraguay.

Several hantaviruses result in zoonotic infections of significant public health concern, causing hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in the Old and New World, respectively. Given a 35% case fatality rate, disease-causing New World hantaviruses require a greater understanding of their biology, genetic diversity, and geographical distribution. Juquitiba hantaviruses have been identified in Oligoryzomys nigripes in Brazil, Paraguay, and Uruguay. Brazil has reported the most HCPS cases associated with this virus. We used a multiplexed, amplicon-based PCR strategy to screen and deep-sequence the virus harbored within lung tissues collected from Oligoryzomys species during rodent field collections in southern (Itapúa) and western (Boquerón) Paraguay. No Juquitiba-like hantaviruses were identified in Boquerón. Herein, we report the full-length S and M segments of the Juquitiba hantaviruses identified in Paraguay from O. nigripes. We also report the phylogenetic relationships of the Juquitiba hantaviruses in rodents collected from Itapúa with those previously collected in Canindeyú. We showed, using the TN93 nucleotide substitution model, the coalescent (constant-size) population tree model, and Bayesian inference implemented in the Bayesian evolutionary analysis by sampling trees (BEAST) framework, that the Juquitiba virus lineage in Itapúa is distinct from that in Canindeyú. Our spatiotemporal analysis showed significantly different time to the most recent ancestor (TMRA) estimates between the M and S segments, but a common geographic origin. Our estimates suggest the additional geographic diversity of the Juquitiba virus within the Interior Atlantic Forest and highlight the need for more extensive sampling across this biome.

Dissecting Phenotype from Genotype with Clinical Isolates of SARS-CoV-2 First Wave Variants.

The emergence and availability of closely related clinical isolates of SARS-CoV-2 offers a unique opportunity to identify novel nonsynonymous mutations that may impact phenotype. Global sequencing efforts show that SARS-CoV-2 variants have emerged and then been replaced since the beginning of the pandemic, yet we have limited information regarding the breadth of variant-specific host responses. Using primary cell cultures and the K18-hACE2 mouse, we investigated the replication, innate immune response, and pathology of closely related, clinical variants circulating during the first wave of the pandemic. Mathematical modeling of the lung viral replication of four clinical isolates showed a dichotomy between two B.1. isolates with significantly faster and slower infected cell clearance rates, respectively. While isolates induced several common immune host responses to infection, one B.1 isolate was unique in the promotion of eosinophil-associated proteins IL-5 and CCL11. Moreover, its mortality rate was significantly slower. Lung microscopic histopathology suggested further phenotypic divergence among the five isolates showing three distinct sets of phenotypes: (i) consolidation, alveolar hemorrhage, and inflammation, (ii) interstitial inflammation/septal thickening and peribronchiolar/perivascular lymphoid cells, and (iii) consolidation, alveolar involvement, and endothelial hypertrophy/margination. Together these findings show divergence in the phenotypic outcomes of these clinical isolates and reveal the potential importance of nonsynonymous mutations in nsp2 and ORF8.

Targeted Hybridization Capture of SARS-CoV-2 and Metagenomics Enables Genetic Variant Discovery and Nasal Microbiome Insights.

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic variants that may alter viral fitness highlights the urgency of widespread next-generation sequencing (NGS) surveillance. To profile genetic variants of the entire SARS-CoV-2 genome, we developed and clinically validated a hybridization capture SARS-CoV-2 NGS assay, integrating novel methods for panel design using double-stranded DNA (dsDNA) biotin-labeled probes, and built accompanying software. This test is the first hybrid capture-based NGS assay given Food and Drug Administration (FDA) emergency use authorization for detection of the SARS-CoV-2 virus. The positive and negative percent agreement (PPA and NPA, respectively) were defined in comparison to the results for an orthogonal real-time reverse transcription polymerase chain reaction (RT-PCR) assay (PPA and NPA, 96.7 and 100%, respectively). The limit of detection was established to be 800 copies/ml with an average fold enrichment of 46,791. Furthermore, utilizing the research-use-only analysis to profile the variants, we identified 55 novel mutations, including 11 in the functionally important spike protein. Finally, we profiled the full nasopharyngeal microbiome using metagenomics and found overrepresentation of 7 taxa and evidence of macrolide resistance in SARS-CoV-2-positive patients. This hybrid capture NGS assay, coupled with optimized software, is a powerful approach to detect and comprehensively map SARS-CoV-2 genetic variants for tracking viral evolution and guiding vaccine updates. IMPORTANCE This is the first FDA emergency-use-authorized hybridization capture-based next-generation sequencing (NGS) assay to detect the SARS-CoV-2 genome. Viral metagenomics and the novel hybrid capture NGS-based assay, along with its research-use-only analysis, can provide important genetic insights into SARS-CoV-2 and other emerging pathogens and improve surveillance and early detection, potentially preventing or mitigating new outbreaks. Better understanding of the continuously evolving SARS-CoV-2 viral genome and the impact of genetic variants may provide individual risk stratification, precision therapeutic options, improved molecular diagnostics, and population-based therapeutic solutions.

Common Themes in Zoonotic Spillover and Disease Emergence: Lessons Learned from Bat- and Rodent-Borne RNA Viruses.

Rodents (order Rodentia), followed by bats (order Chiroptera), comprise the largest percentage of living mammals on earth. Thus, it is not surprising that these two orders account for many of the reservoirs of the zoonotic RNA viruses discovered to date. The spillover of these viruses from wildlife to human do not typically result in pandemics but rather geographically confined outbreaks of human infection and disease. While limited geographically, these viruses cause thousands of cases of human disease each year. In this review, we focus on three questions regarding zoonotic viruses that originate in bats and rodents. First, what biological strategies have evolved that allow RNA viruses to reside in bats and rodents? Second, what are the environmental and ecological causes that drive viral spillover? Third, how does virus spillover occur from bats and rodents to humans?

Prevalence of Hantaviruses Harbored by Murid Rodents in Northwestern Ukraine and Discovery of a Novel Puumala Virus Strain.

In Europe, two species of hantaviruses, Puumala orthohantavirus (PUUV) and Dobrava orthohantavirus (DOBV), cause hemorrhagic fever with renal syndrome in humans. The rodent reservoirs for these viruses are common throughout Ukraine, and hence, the goal of this study was to identify the species and strains of hantaviruses circulating in this region. We conducted surveillance of small rodent populations in a rural region in northwestern Ukraine approximately 30 km from Poland. From the 424 small mammals captured, we identified nine species, of which the most abundant were Myodes glareolus, the bank vole (45%); Apodemus flavicollis, the yellow-necked mouse (29%); and Apodemus agrarius, the striped field mouse (14.6%) Using an indirect immunofluorescence assay, 15.7%, 20.5%, and 33.9% of the sera from M. glareolus, A. glareolus, and A. flavicollis were positive for hantaviral antibodies, respectively. Additionally, we detected antibodies to the hantaviral antigen in one Microtus arvalis, one Mus musculus, and one Sorex minutus. We screened the lung tissue for hantaviral RNA using next-generation sequencing and identified PUUV sequences in 25 small mammals, including 23 M. glareolus, 1 M. musculus, and 1 A. flavicollis, but we were unable to detect DOBV sequences in any of our A. agrarius specimens. The percent identity matrix and Bayesian phylogenetic analyses of the S-segment of PUUV from 14 M. glareolus lungs suggest the highest similarity (92-95% nucleotide or 99-100% amino acid) with the Latvian lineage. This new genetic information will contribute to future molecular surveillance of human cases in Ukraine.

Corrigendum: Amplicon-Based, Next-Generation Sequencing Approaches to Characterize Single Nucleotide Polymorphisms of Orthohantavirus Species.

[This corrects the article DOI: 10.3389/fcimb.2020.565591.].

Seroprevalence of Old World Hantaviruses and Crimean Congo Hemorrhagic Fever Viruses in Human Populations in Northwestern Ukraine.

In Ukraine, a retrospective review of clinical case reports by public health officials suggest that human cases of febrile illnesses associated with hemorrhage may be due to infections of Crimean-Congo hemorrhagic fever virus (CCHFV) and Old World hantaviruses. In a serosurvey of 966 healthy individuals in the Lviv Oblast, Ukraine, bordering Poland, we found that 1.6% showed cross-reactivity to hantaviral antigens by an immunofluorescence assay (IFA) and 1.7% of the study participants had antibodies cross-reactive to CCHFV by enzyme-linked immunosorbent assay (ELISA). Demographic variables and history of exposures obtained through questionnaires were assessed by logistic regression models for association with seroprevalence for both viruses with no significant risk factors found. Analysis of spatial distribution identified two clusters of samples positive for antibodies to both hantaviruses and CCHFV, which, however, were not statistically significant (p > 0.05). In general, the study results suggest that the population of the study area is exposed to hantaviruses and CCHFV. Further surveillance for respective pathogens in Ukraine is warranted and prospective surveillance of febrile patients with unidentified febrile illness.

Amplicon-Based, Next-Generation Sequencing Approaches to Characterize Single Nucleotide Polymorphisms of Orthohantavirus Species.

Whole-genome sequencing (WGS) of viruses from patient or environmental samples can provide tremendous insight into the epidemiology, drug resistance or evolution of a virus. However, we face two common hurdles in obtaining robust sequence information; the low copy number of viral genomes in specimens and the error introduced by WGS techniques. To optimize detection and minimize error in WGS of hantaviruses, we tested four amplification approaches and different amplicon pooling methods for library preparation and examined these preparations using two sequencing platforms, Illumina MiSeq and Oxford Nanopore Technologies MinION. First, we tested and optimized primers used for whole segment PCR or one kilobase amplicon amplification for even coverage using RNA isolated from the supernatant of virus-infected cells. Once optimized we assessed two sources of total RNA, virus-infected cells and supernatant from the virus-infected cells, with four variations of primer pooling for amplicons, and six different amplification approaches. We show that 99-100% genome coverage was obtained using a one-step RT-PCR reaction with one forward and reverse primer. Using a two-step RT-PCR with three distinct tiling approaches for the three genomic segments (vRNAs), we optimized primer pooling approaches for PCR amplification to achieve a greater number of aligned reads, average depth of genome, and genome coverage. The single nucleotide polymorphisms identified from MiSeq and MinION sequencing suggested intrinsic mutation frequencies of ~10-5-10-7 per genome and 10-4-10-5 per genome, respectively. We noted no difference in the coverage or accuracy when comparing WGS results with amplicons amplified from RNA extracted from infected cells or supernatant of these infected cells. Our results show that high-throughput diagnostics requiring the identification of hantavirus species or strains can be performed using MiSeq or MinION using a one-step approach. However, the two-step MiSeq approach outperformed the MinION in coverage depth and accuracy, and hence would be superior for assessment of genomes for epidemiology or evolutionary questions using the methods developed herein.

Cocirculation of Two Orthohantavirus Species in Small Mammals of the Northwestern Ukraine.

Rodent-borne hantaviruses have been reported in many of the countries surrounding Ukraine; however, to date we have no knowledge of the viral strains circulating in Ukraine within reservoirs such as the striped field mouse (Apodemus agrarius), the yellow-necked field mouse (Apodemus flavicollis), and the bank vole (Myodes glareolus). To determine the prevalence of hantaviruses in Ukraine, we captured 1,261 mammals, of which 1,109 were rodents, in 58 field sites within the province of Volyn in western Ukraine. Foci of the striped field mouse tended to occur in the eastern and southern parts of the province, whereas the bank vole were clustered in western and northern regions. The striped field mouse and bank vole had detectable serum antibodies to Puumala virus (PUUV) or Dobrava virus (DOBV) antigens at 7% or 2%, respectively, using an indirect immunofluorescence assay. Antibody prevalence among the bank vole males and females was equivalent, whereas for the striped field mouse, the prevalence among males was 5% versus 1% for females. In two bank vole specimens, we were able to detect partial nucleotide sequences that showed identity to PUUV. In summary, this study suggests that two human pathogens, PUUV and DOBV, cocirculate in the bank vole and the striped field mouse, respectively, in Ukraine. Future studies will focus on new rodent collections that will enable obtaining the complete genome sequences of the PUUV and DOBV strains circulating in Ukraine to provide guidance on the design of optimal molecular diagnostics that can enable insight into the potential contribution of hantaviruses to human disease in Ukraine.

Diverse Morphology and Structural Features of Old and New World Hantaviruses.

To further understanding of the structure and morphology of the Orthohantavirus, family Hantaviridae, we have employed cryo-electron microscopy (cryo-EM) for three New World hantaviruses: Andes (ANDV), Sin Nombre (SNV), and Black Creek Canal (BCCV). Building upon our prior cryo-EM and cryo-tomography study of the Old World hantavirus, Hantaan virus (HTNV), we have expanded our studies to examine the entire virion population present in cell culture supernatant. Hence, in contrast to the prior cryo-EM/ET studies in which we used a polyethylene precipitation, a sucrose gradient, and a sucrose cushion, we used two sucrose cushions. We inactivated the material after the first cushion. We tested the method using HTNV which has a known cryo-EM structure and observed equivalent results. Therefore, we used this method to assess the particle distribution of the New World hantaviruses by cryo-EM. Cryo-EM images showed a diverse range of sizes and morphologies for the New World viruses that we classified as round, tubular, and irregular. Strikingly, BCCV virions were mostly tubular. These first cryo-EM images of the New World Orthohantavirus confirm prior EM observations that noted tubular projections of SNV at the plasma membrane during virion morphogenesis but were not confirmed. These findings underscore the need for further investigation of virion morphogenesis of the Orthohantavirus.

Rapid Sequencing of Multiple RNA Viruses in Their Native Form.

Long-read nanopore sequencing by a MinION device offers the unique possibility to directly sequence native RNA. We combined an enzymatic poly-A tailing reaction with the native RNA sequencing to (i) sequence complex population of single-stranded (ss)RNA viruses in parallel, (ii) detect genome, subgenomic mRNA/mRNA simultaneously, (iii) detect a complex transcriptomic architecture without the need for assembly, (iv) enable real-time detection. Using this protocol, positive-ssRNA, negative-ssRNA, with/without a poly(A)-tail, segmented/non-segmented genomes were mixed and sequenced in parallel. Mapping of the generated sequences on the reference genomes showed 100% length recovery with up to 97% identity. This work provides a proof of principle and the validity of this strategy, opening up a wide range of applications to study RNA viruses.

Habitat, species richness and hantaviruses of sigmodontine rodents within the Interior Atlantic Forest, Paraguay.

Four of the nine sigmodontine tribes have species that serve as reservoirs of rodent-borne hantaviruses (RBO-HV), few have been studied in any depth. Several viruses have been associated with human cases of hantavirus pulmonary syndrome often through peridomestic exposure. Jabora (JABV) and Juquitiba (JUQV), harbored by Akodon montensis and Oligoryzomys nigripes, respectively, are endemic and sympatric in the Reserva Natural de Bosque Mbaracayú (RNBM), Paraguay, a protected area of the Interior Atlantic Forest. Rodent communities were surveyed along a 30 km stretch of the RNBM in eight vegetation classifications (Low, High, Bamboo, Riparian and Liana Forests, Bamboo Understory, Cerrado, and Meadow/Grasslands). We collected 417 rodents from which 11 species were identified; Akodon montensis was the predominant species (72%; 95%CI: 64.7%-76.3%), followed by Hylaeamys megacephalus (15% (11.2%-18.2%)) and Oligoryzomys nigripes (9% (6.6%-12.4%)). We examined the statistical associations among habitat (vegetation class) type, rodent species diversity, population structure (age, sex, and weight), and prevalence of RBO-HV antibody and/or viral RNA (Ab/RNA) or characteristic Leishmania tail lesions. Ab/RNA positive rodents were not observed in Cerrado and Low Forest. A. montensis had an overall Ab/RNA prevalence of 7.7% (4.9%-11.3%) and O. nigripes had an overall prevalence of 8.6% (1.8%-23.1%). For A. montensis, the odds of being Ab/RNA positive in High Forest was 3.73 times of the other habitats combined. There was no significant difference among age classes in the proportion of Ab/RNA positive rodents overall (p = 0.66), however, all 11 RNA-positive individuals were adult. Sex and habitat had independent prognostic value for hantaviral Ab/RNA in the study population; age, presence of tail scar/lesion (19% of the rodents) and weight did not. Adjusting for habitat, female rodents had less risk of becoming infected. Importantly, these data suggest habitat preferences of two sympatric rodent reservoirs for two endemic hantaviruses and the importance of including habitat in models of species diversity and habitat fragmentation.