Tracing Transmission of Sin Nombre Virus and Discovery of Infection in Multiple Rodent Species.

Sin Nombre orthohantavirus (SNV), a negative-sense, single-stranded RNA virus that is carried and transmitted by the North American deer mouse Peromyscus maniculatus, can cause infection in humans through inhalation of aerosolized excreta from infected rodents. This infection can lead to hantavirus cardiopulmonary syndrome (HCPS), which has an ∼36% case-fatality rate. We used reverse transcriptase quantitative PCR (RT-qPCR) to confirm SNV infection in a patient and identified SNV in lung tissues in wild-caught rodents from potential sites of exposure. Using viral whole-genome sequencing (WGS), we identified the likely site of transmission and discovered SNV in multiple rodent species not previously known to carry the virus. Here, we report, for the first time, the use of SNV WGS to pinpoint a likely site of human infection and identify SNV simultaneously in multiple rodent species in an area of known host-to-human transmission. These results will impact epidemiology and infection control for hantaviruses by tracing zoonotic transmission and investigating possible novel host reservoirs. IMPORTANCE Orthohantaviruses cause severe disease in humans and can be lethal in up to 40% of cases. Sin Nombre orthohantavirus (SNV) is the main cause of hantavirus disease in North America. In this study, we sequenced SNV from an infected patient and wild-caught rodents to trace the location of infection. We also discovered SNV in rodent species not previously known to carry SNV. These studies demonstrate for the first time the use of virus sequencing to trace the transmission of SNV and describe infection in novel rodent species.

Choclo virus (CHOV) recovered from deep metatranscriptomics of archived frozen tissues in natural history biorepositories.

BACKGROUND: Hantaviruses are negative-stranded RNA viruses that can sometimes cause severe disease in humans; however, they are maintained in mammalian host populations without causing harm. In Panama, sigmodontine rodents serve as hosts to transmissible hantaviruses. Due to natural and anthropogenic forces, these rodent populations are having increased contact with humans. METHODS: We extracted RNA and performed Illumina deep metatranscriptomic sequencing on Orthohantavirus seropositive museum tissues from rodents. We acquired sequence reads mapping to Choclo virus (CHOV, Orthohantavirus chocloense) from heart and kidney tissue of a two-decade old frozen museum sample from a Costa Rican pygmy rice rat (Oligoryzomys costaricensis) collected in Panama. Reads mapped to the CHOV reference were assembled and then validated by visualization of the mapped reads against the assembly. RESULTS: We recovered a 91% complete consensus sequence from a reference-guided assembly to CHOV with an average of 16X coverage. The S and M segments used in our phylogenetic analyses were nearly complete (98% and 99%, respectively). There were 1,199 ambiguous base calls of which 93% were present in the L segment. Our assembled genome varied 1.1% from the CHOV reference sequence resulting in eight nonsynonymous mutations. Further analysis of all publicly available partial S segment sequences support a clear relationship between CHOV clinical cases and O. costaricensis acquired strains. CONCLUSIONS: Viruses occurring at extremely low abundances can be recovered from deep metatranscriptomics of archival tissues housed in research natural history museum biorepositories. Our efforts resulted in the second CHOV genome publicly available. This genomic data is important for future surveillance and diagnostic tools as well as understanding the evolution and pathogenicity of CHOV.

Global landscape of SARS-CoV-2 genomic surveillance and data sharing.

Genomic surveillance has shaped our understanding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. We performed a global landscape analysis on SARS-CoV-2 genomic surveillance and genomic data using a collection of country-specific data. Here, we characterize increasing circulation of the Alpha variant in early 2021, subsequently replaced by the Delta variant around May 2021. SARS-CoV-2 genomic surveillance and sequencing availability varied markedly across countries, with 45 countries performing a high level of routine genomic surveillance and 96 countries with a high availability of SARS-CoV-2 sequencing. We also observed a marked heterogeneity of sequencing percentage, sequencing technologies, turnaround time and completeness of released metadata across regions and income groups. A total of 37% of countries with explicit reporting on variants shared less than half of their sequences of variants of concern (VOCs) in public repositories. Our findings indicate an urgent need to increase timely and full sharing of sequences, the standardization of metadata files and support for countries with limited sequencing and bioinformatics capacity.

Genomic characterization of Staphylococcus aureus isolates causing osteoarticular infections in otherwise healthy children.

BACKGROUND: Pediatric osteoarticular infections are commonly caused by Staphylococcus aureus. The contribution of S. aureus genomic variability to pathogenesis of these infections is poorly described. METHODS: We prospectively enrolled 47 children over 3 1/2 years from whom S. aureus was isolated on culture-12 uninfected with skin colonization, 16 with skin abscesses, 19 with osteoarticular infections (four with septic arthritis, three with acute osteomyelitis, six with acute osteomyelitis and septic arthritis and six with chronic osteomyelitis). Isolates underwent whole genome sequencing, with assessment for 254 virulence genes and any mutations as well as creation of a phylogenetic tree. Finally, isolates were compared for their ability to form static biofilms and compared to the genetic analysis. RESULTS: No sequence types predominated amongst osteoarticular infections. Only genes involved in evasion of host immune defenses were more frequently carried by isolates from osteoarticular infections than from skin colonization (p = .02). Virulence gene mutations were only noted in 14 genes (three regulating biofilm formation) when comparing isolates from subjects with osteoarticular infections and those with skin colonization. Biofilm results demonstrated large heterogeneity in the isolates' capacity to form static biofilms, with healthy control isolates producing more robust biofilm formation. CONCLUSIONS: S. aureus causing osteoarticular infections are genetically heterogeneous, and more frequently harbor genes involved in immune evasion than less invasive isolates. However, virulence gene carriage overall is similar with infrequent mutations, suggesting that pathogenesis of S. aureus osteoarticular infections may be primarily regulated at transcriptional and/or translational levels.

Random transposon mutagenesis of Verrucomicrobium spinosum DSM 4136(T).

The Verrucomicrobia are a bacterial group of growing interest due to their environmental ubiquity as free-living and host-associated microbes. They also exhibit an unusual compartmentalized cell plan, shared with members of neighboring phyla that include the Planctomycete bacteria. However, Verrucomicrobia are currently difficult to study, due to a lack of available genetic tools that would permit robust testing of hypotheses formulated from ecological and genomic data. To our knowledge, there are no published studies describing the transformation of exogenous DNA into any members of the Verrucomicrobia (or the neighboring phylum containing Planctomycetes). Here, we present a procedure for the transformation of DNA into Verrucomicrobium spinosum DSM 4136(T) via electroporation and the first description of a random transposon mutant library in this organism. We anticipate that this approach could be applied successfully to other Verrucomicrobia, providing opportunities to test the role of predicted gene function in ecological interactions and identify genes associated with the distinctive Planctomycete-Verrucomicrobial cell plan.

Emergence in late 2020 of multiple lineages of SARS-CoV-2 Spike protein variants affecting amino acid position 677.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) plays critical roles in host cell entry. Non-synonymous substitutions affecting S are not uncommon and have become fixed in a number of SARS-CoV-2 lineages. A subset of such mutations enable escape from neutralizing antibodies or are thought to enhance transmission through mechanisms such as increased affinity for the cell entry receptor, angiotensin-converting enzyme 2 (ACE2). Independent genomic surveillance programs based in New Mexico and Louisiana contemporaneously detected the rapid rise of numerous clade 20G (lineage B.1.2) infections carrying a Q677P substitution in S. The variant was first detected in the US on October 23, yet between 01 Dec 2020 and 19 Jan 2021 it rose to represent 27.8% and 11.3% of all SARS-CoV-2 genomes sequenced from Louisiana and New Mexico, respectively. Q677P cases have been detected predominantly in the south central and southwest United States; as of 03 Feb 2021, GISAID data show 499 viral sequences of this variant from the USA. Phylogenetic analyses revealed the independent evolution and spread of at least six distinct Q677H sub-lineages, with first collection dates ranging from mid-August to late November 2020. Four 677H clades from clade 20G (B.1.2), 20A (B.1.234), and 20B (B.1.1.220, and B.1.1.222) each contain roughly 100 or fewer sequenced cases, while a distinct pair of clade 20G clusters are represented by 754 and 298 cases, respectively. Although sampling bias and founder effects may have contributed to the rise of S:677 polymorphic variants, the proximity of this position to the polybasic cleavage site at the S1/S2 boundary are consistent with its potential functional relevance during cell entry, suggesting parallel evolution of a trait that may confer an advantage in spread or transmission. Taken together, our findings demonstrate simultaneous convergent evolution, thus providing an impetus to further evaluate S:677 polymorphisms for effects on proteolytic processing, cell tropism, and transmissibility.