Distribution and prevalence of Sin Nombre hantavirus in rodent species in eastern New Mexico.

Orthohantaviruses are diverse zoonotic RNA viruses. Small mammals, such as mice and rats are common chronic, asymptomatic hosts that transmit the virus through their feces and urine. In North America, hantavirus infection primarily causes hantavirus cardiopulmonary syndrome (HCPS), which has a mortality rate of nearly 36%. In the United States of America, New Mexico (NM) is leading the nation in the number of HCPS-reported cases (N = 129). However, no reported cases of HCPS have occurred within eastern NM. In this study, we assessed the prevalence of Sin Nombre virus (SNV) in rodent assemblages across eastern NM, using RT-qPCR. We screened for potential rodent hosts in the region, as well as identified areas that may pose significant infection risk to humans. We captured and collected blood and lung tissues from 738 rodents belonging to 23 species. 167 individuals from 16 different species were positive for SNV RNA by RT-qPCR, including 6 species unreported in the literature: Onychomys leucogaster (Northern grasshopper mouse), Dipodomys merriami (Merriam's kangaroo rat), Dipodomys ordii (Ord's kangaroo rat), Dipodomys spectabilis (Banner-tailed kangaroo rat), Perognathus flavus (Silky pocket mouse), and Chaetodipus hispidus (Hispid pocket mouse). The infection rates did not differ between sexes or rodent families (i.e., Cricetidae vs. Heteromyidae). Generalized linear model showed that disturbed habitat types positively influenced the prevalence of SNV at sites of survey. Overall, the results of this study indicate that many rodent species in east New Mexico have the potential to maintain SNV in the environment, but further research is needed to assess species specific infectivity mechanisms and potential risk to humans.

Structural and functional characterization of the Sin Nombre virus L protein.

The Bunyavirales order is a large and diverse group of segmented negative-strand RNA viruses. Several virus families within this order contain important human pathogens, including Sin Nombre virus (SNV) of the Hantaviridae. Despite the high epidemic potential of bunyaviruses, specific medical countermeasures such as vaccines or antivirals are missing. The multifunctional ~250 kDa L protein of hantaviruses, amongst other functional domains, harbors the RNA-dependent RNA polymerase (RdRp) and an endonuclease and catalyzes transcription as well as replication of the viral RNA genome, making it a promising therapeutic target. The development of inhibitors targeting these key processes requires a profound understanding of the catalytic mechanisms. Here, we established expression and purification protocols of the full-length SNV L protein bearing the endonuclease mutation K124A. We applied different biochemical in vitro assays to provide an extensive characterization of the different enzymatic functions as well as the capacity of the hantavirus L protein to interact with the viral RNA. By using single-particle cryo-EM, we obtained a 3D model including the L protein core region containing the RdRp, in complex with the 5' promoter RNA. This first high-resolution model of a New World hantavirus L protein shows striking similarity to related bunyavirus L proteins. The interaction of the L protein with the 5' RNA observed in the structural model confirms our hypothesis of protein-RNA binding based on our biochemical data. Taken together, this study provides an excellent basis for future structural and functional studies on the hantavirus L protein and for the development of antiviral compounds.

Experimental Infection of Peromyscus Species Rodents with Sin Nombre Virus.

We demonstrate that 6 distinct Peromyscus rodent species are permissive to experimental infection with Sin Nombre orthohantavirus (SNV). Viral RNA and SNV antibodies were detected in members of all 6 species. P. leucopus mice demonstrated markedly higher viral and antibody titers than P. maniculatus mice, the established primary hosts for SNV.

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.

New Exposure Location for Hantavirus Pulmonary Syndrome Case, California, USA, 2018.

We describe a case of hantavirus pulmonary syndrome in a patient exposed to Sin Nombre virus in a coastal county in California, USA, that had no previous record of human cases. Environmental evaluation coupled with genotypic analysis of virus isolates from the case-patient and locally trapped rodents identified the likely exposure location.

Novel Focus of Sin Nombre Virus in Peromyscus eremicus Mice, Death Valley National Park, California, USA.

The deer mouse (Peromyscus maniculatus) is the primary reservoir for Sin Nombre virus (SNV) in the western United States. Rodent surveillance for hantavirus in Death Valley National Park, California, USA, revealed cactus mice (P. eremicus) as a possible focal reservoir for SNV in this location. We identified SNV antibodies in 40% of cactus mice sampled.

Transcriptome markers of viral persistence in naturally-infected andes virus (bunyaviridae) seropositive long-tailed pygmy rice rats.

Long-tailed pygmy rice rats (Oligoryzomys longicaudatus) are principal reservoir hosts of Andes virus (ANDV) (Bunyaviridae), which causes most hantavirus cardiopulmonary syndrome cases in the Americas. To develop tools for the study of the ANDV-host interactions, we used RNA-Seq to generate a de novo transcriptome assembly. Splenic RNA from five rice rats captured in Chile, three of which were ANDV-infected, was used to generate an assembly of 66,173 annotated transcripts, including noncoding RNAs. Phylogenetic analysis of selected predicted proteins showed similarities to those of the North American deer mouse (Peromyscus maniculatus), the principal reservoir of Sin Nombre virus (SNV). One of the infected rice rats had about 50-fold more viral burden than the others, suggesting acute infection, whereas the remaining two had levels consistent with persistence. Differential expression analysis revealed distinct signatures among the infected rodents. The differences could be due to 1) variations in viral load, 2) dimorphic or reproductive differences in splenic homing of immune cells, or 3) factors of unknown etiology. In the two persistently infected rice rats, suppression of the JAK-STAT pathway at Stat5b and Ccnot1, elevation of Casp1, RIG-I pathway factors Ppp1cc and Mff, and increased FC receptor-like transcripts occurred. Caspase-1 and Stat5b activation pathways have been shown to stimulate T helper follicular cell (TFH) development in other species. These data are also consistent with reports suggestive of TFH stimulation in deer mice experimentally infected with hantaviruses. In the remaining acutely infected rice rat, the apoptotic pathway marker Cox6a1 was elevated, and putative anti-viral factors Abcb1a, Fam46c, Spp1, Rxra, Rxrb, Trmp2 and Trim58 were modulated. Transcripts for preproenkephalin (Prenk) were reduced, which may be predictive of an increased T cell activation threshold. Taken together, this transcriptome dataset will permit rigorous examination of rice rat-ANDV interactions and may lead to better understanding of virus ecology.

Pathophysiology of hantavirus pulmonary syndrome in rhesus macaques.

The pathophysiology of hantavirus pulmonary syndrome (HPS) remains unclear because of a lack of surrogate disease models with which to perform pathogenesis studies. Nonhuman primates (NHP) are considered the gold standard model for studying the underlying immune activation/suppression associated with immunopathogenic viruses such as hantaviruses; however, to date an NHP model for HPS has not been described. Here we show that rhesus macaques infected with Sin Nombre virus (SNV), the primary etiological agent of HPS in North America, propagated in deer mice develop HPS, which is characterized by thrombocytopenia, leukocytosis, and rapid onset of respiratory distress caused by severe interstitial pneumonia. Despite establishing a systemic infection, SNV differentially activated host responses exclusively in the pulmonary endothelium, potentially the mechanism leading to acute severe respiratory distress. This study presents a unique chronological characterization of SNV infection and provides mechanistic data into the pathophysiology of HPS in a closely related surrogate animal model. We anticipate this model will advance our understanding of HPS pathogenesis and will greatly facilitate research toward the development of effective therapeutics and vaccines against hantaviral diseases.

Twenty-year summary of surveillance for human hantavirus infections, United States.

In the past 20 years of surveillance for hantavirus in humans in the United States, 624 cases of hantavirus pulmonary syndrome (HPS) have been reported, 96% of which occurred in states west of the Mississippi River. Most hantavirus infections are caused by Sin Nombre virus, but cases of HPS caused by Bayou, Black Creek Canal, Monongahela, and New York viruses have been reported, and cases of domestically acquired hemorrhagic fever and renal syndrome caused by Seoul virus have also occurred. Rarely, hantavirus infections result in mild illness that does not progress to HPS. Continued testing and surveillance of clinical cases in humans will improve our understanding of the etiologic agents involved and the spectrum of diseases.

Increased detection of Sin Nombre hantavirus RNA in antibody-positive deer mice from Montana, USA: evidence of male bias in RNA viremia.

Hantaviruses are widespread emergent zoonotic agents that cause unapparent or limited disease in their rodent hosts, yet cause acute, often fatal pulmonary or renal infections in humans. Previous laboratory experiments with rodent reservoir hosts indicate that hantaviruses can be cleared from host blood early in the infection cycle, while sequestered long term in various host organs. Field studies of North American deer mice (Peromyscus maniculatus), the natural reservoir of Sin Nombre hantavirus, have shown that viral RNA can be transiently detected well past the early acute infection stage, but only in the minority of infected mice. Here, using a non-degenerate RT-PCR assay optimized for SNV strains known to circulate in Montana, USA, we show that viral RNA can be repeatedly detected on a monthly basis in up to 75% of antibody positive deer mice for periods up to 3-6 months. More importantly, our data show that antibody positive male deer mice are more than twice as likely to have detectable SNV RNA in their blood as antibody positive females, suggesting that SNV-infected male deer mice are more likely to shed virus and for longer periods of time.

A novel Sin Nombre virus DNA vaccine and its inclusion in a candidate pan-hantavirus vaccine against hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS).

Sin Nombre virus (SNV; family Bunyaviridae, genus Hantavirus) causes a hemorrhagic fever known as hantavirus pulmonary syndrome (HPS) in North America. There have been approximately 200 fatal cases of HPS in the United States since 1993, predominantly in healthy working-age males (case fatality rate 35%). There are no FDA-approved vaccines or drugs to prevent or treat HPS. Previously, we reported that hantavirus vaccines based on the full-length M gene segment of Andes virus (ANDV) for HPS in South America, and Hantaan virus (HTNV) and Puumala virus (PUUV) for hemorrhagic fever with renal syndrome (HFRS) in Eurasia, all elicited high-titer neutralizing antibodies in animal models. HFRS is more prevalent than HPS (>20,000 cases per year) but less pathogenic (case fatality rate 1-15%). Here, we report the construction and testing of a SNV full-length M gene-based DNA vaccine to prevent HPS. Rabbits vaccinated with the SNV DNA vaccine by muscle electroporation (mEP) developed high titers of neutralizing antibodies. Furthermore, hamsters vaccinated three times with the SNV DNA vaccine using a gene gun were completely protected against SNV infection. This is the first vaccine of any kind that specifically elicits high-titer neutralizing antibodies against SNV. To test the possibility of producing a pan-hantavirus vaccine, rabbits were vaccinated by mEP with an HPS mix (ANDV and SNV plasmids), or HFRS mix (HTNV and PUUV plasmids), or HPS/HFRS mix (all four plasmids). The HPS mix and HFRS mix elicited neutralizing antibodies predominantly against ANDV/SNV and HTNV/PUUV, respectively. Furthermore, the HPS/HFRS mix elicited neutralizing antibodies against all four viruses. These findings demonstrate a pan-hantavirus vaccine using a mixed-plasmid DNA vaccine approach is feasible and warrants further development.

Kinetics of immune responses in deer mice experimentally infected with Sin Nombre virus.

Deer mice are the principal reservoir hosts of Sin Nombre virus, the etiologic agent of most hantavirus cardiopulmonary syndrome cases in North America. Infection of deer mice results in persistence without conspicuous pathology, and most, if not all, infected mice remain infected for life, with periods of viral shedding. The kinetics of viral load, histopathology, virus distribution, and immune gene expression in deer mice were examined. Viral antigen was detected as early as 5 days postinfection and peaked on day 15 in the lungs, hearts, kidneys, and livers. Viral RNA levels varied substantially but peaked on day 15 in the lungs and heart, and antinucleocapsid IgG antibodies appeared in some animals on day 10, but a strong neutralizing antibody response failed to develop during the 20-day experiment. No clinical signs of disease were observed in any of the infected deer mice. Most genes were repressed on day 2, suggesting a typical early downregulation of gene expression often observed in viral infections. Several chemokine and cytokine genes were elevated, and markers of a T cell response occurred but then declined days later. Splenic transforming growth factor beta (TGF-ß) expression was elevated early in infection, declined, and then was elevated again late in infection. Together, these data suggest that a subtle immune response that fails to clear the virus occurs in deer mice.

Temporal and geographic evidence for evolution of Sin Nombre virus using molecular analyses of viral RNA from Colorado, New Mexico and Montana.

BACKGROUND: All viruses in the family Bunyaviridae possess a tripartite genome, consisting of a small, a medium, and a large RNA segment. Bunyaviruses therefore possess considerable evolutionary potential, attributable to both intramolecular changes and to genome segment reassortment. Hantaviruses (family Bunyaviridae, genus Hantavirus) are known to cause human hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. The primary reservoir host of Sin Nombre virus is the deer mouse (Peromyscus maniculatus), which is widely distributed in North America. We investigated the prevalence of intramolecular changes and of genomic reassortment among Sin Nombre viruses detected in deer mice in three western states. METHODS: Portions of the Sin Nombre virus small (S) and medium (M) RNA segments were amplified by RT-PCR from kidney, lung, liver and spleen of seropositive peromyscine rodents, principally deer mice, collected in Colorado, New Mexico and Montana from 1995 to 2007. Both a 142 nucleotide (nt) amplicon of the M segment, encoding a portion of the G2 transmembrane glycoprotein, and a 751 nt amplicon of the S segment, encoding part of the nucleocapsid protein, were cloned and sequenced from 19 deer mice and from one brush mouse (P. boylii), S RNA but not M RNA from one deer mouse, and M RNA but not S RNA from another deer mouse. RESULTS: Two of 20 viruses were found to be reassortants. Within virus sequences from different rodents, the average rate of synonymous substitutions among all pair-wise comparisons (pis) was 0.378 in the M segment and 0.312 in the S segment sequences. The replacement substitution rate (pia) was 7.0 x 10-4 in the M segment and 17.3 x 10-4 in the S segment sequences. The low pia relative to pis suggests strong purifying selection and this was confirmed by a Fu and Li analysis. The absolute rate of molecular evolution of the M segment was 6.76 x 10-3 substitutions/site/year. The absolute age of the M segment tree was estimated to be 37 years. In the S segment the rate of molecular evolution was 1.93 x 10-3 substitutions/site/year and the absolute age of the tree was 106 years. Assuming that mice were infected with a single Sin Nombre virus genotype, phylogenetic analyses revealed that 10% (2/20) of viruses were reassortants, similar to the 14% (6/43) found in a previous report. CONCLUSION: Age estimates from both segments suggest that Sin Nombre virus has evolved within the past 37-106 years. The rates of evolutionary changes reported here suggest that Sin Nombre virus M and S segment reassortment occurs frequently in nature.

Increased host species diversity and decreased prevalence of Sin Nombre virus.

Emerging outbreaks of zoonotic diseases are affecting humans at an alarming rate. Until the ecological factors associated with zoonoses are better understood, disease emergence will continue. For Lyme disease, disease suppression has been demonstrated by a dilution effect, whereby increasing species diversity decreases disease prevalence in host populations. To test the dilution effect in another disease, we examined 17 ecological variables associated with prevalence of the directly transmitted Sin Nombre virus (genus Hantavirus, etiologic agent of hantavirus pulmonary syndrome) in its wildlife host, the deer mouse (Peromyscus maniculatus). Only species diversity was statistically linked to infection prevalence: as species diversity decreased, infection prevalence increased. The increase was moderate, but prevalence increased exponentially at low levels of diversity, a phenomenon described as zoonotic release. The results suggest that species diversity affects disease emergence.

Oligomerization of hantaviral nucleocapsid protein: charged residues in the N-terminal coiled-coil domain contribute to intermolecular interactions.

The nucleocapsid (N) protein of hantaviruses (family Bunyaviridae) is the most abundant component of the virion; it encapsidates genomic RNA segments and participates in viral genome transcription and replication, as well as in virus assembly. During RNA encapsidation, the N protein forms intermediate trimers and then oligomers via 'head-to-head, tail-to-tail' interactions. In previous work, using Tula hantavirus (TULV) N protein as a model, it was demonstrated that an intact coiled-coil structure of the N terminus is crucial for the oligomerization capacity of the N protein and that the hydrophobic 'a' residues from the second alpha-helix are especially important. Here, the importance of charged amino acid residues located within the coiled-coil for trimer formation and oligomerization was analysed. To predict the interacting surfaces of the monomers, the previous in silico model of TULV coiled-coils was first upgraded, taking advantage of the recently published crystal structure of the N-terminal coiled-coil of the Sin Nombre virus N protein. The results obtained using a mammalian two-hybrid assay suggested that conserved, charged amino acid residues within the coiled-coil make a substantial contribution to N protein oligomerization. This contribution probably involves (i) the formation of interacting surfaces of the N monomers (residues D35 and D38, located at the tip of the coiled-coil loop, and R63 appear particularly important) and (ii) stabilization of the coiled-coil via intramolecular ionic bridging (with E55 as a key player). It is hypothesized that the tips of the coiled-coils are the first to come into direct contact and thus to initiate tight packing of the three structures.

Selection and characterization of scFv antibodies against the Sin Nombre hantavirus nucleocapsid protein.

Rodent-borne hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in the old world and hantavirus cardio-pulmonary syndrome (HCPS) in the new. Most cases of HCPS in North America are caused by Sin Nombre Virus (SNV). Current viral detection technologies depend upon the identification of anti-viral antibodies in patient serum. Detection of viral antigen may facilitate earlier detection of the pathogen. We describe here the characterization of two single-chain Fv antibodies (scFvs), selected from a large naïve phage antibody library, which are capable of identifying the Sin Nombre Virus nucleocapsid protein (SNV-N), with no cross reactivity with the nucleocapsid protein from other hantaviruses. The utility of such selected scFvs was increased by the creation of an scFv-alkaline phosphatase fusion protein which was able to directly detect virally produced material without the need for additional reagents.

Recombinant adenovirus vector vaccine induces stronger cytotoxic T-cell responses than recombinant vaccinia virus vector, plasmid DNA, or a combination of these.

The efficiency of prime-boost vaccinations on the induction of T-cell responses to Sin Nombre virus nucleocapsid protein expressed by recombinant vaccinia virus, replication-deficient adenovirus, and plasmid DNA in mice was quantitated by the number of epitope-specific interferon-gamma-producing T cells and cytotoxic T-lymphocyte activity induced. In prime-boost immunizations, all combinations that included the recombinant adenovirus induced a much higher number of epitope-specific interferon-gamma-producing T cells than did other combinations. A single immunization of the recombinant adenovirus was able to induce similarly high levels of epitope-specific interferon-gamma-producing cells, despite the fact that the recombinant adenovirus produces less amount of the Sin Nombre virus nucleocapsid protein.

A preliminary study of the patterns of Sin Nombre viral infection and shedding in naturally infected deer mice (Peromyscus maniculatus).

Deer mice (Peromyscus maniculatus) were trapped in southern Manitoba, Canada and tested for evidence of Sin Nombre virus infection. Viral genome was amplified from tissues as well as saliva/oropharyngeal fluid, and urine samples were collected from seropositive animals. Detection of viral RNA in tissue samples and excreta/secreta from mice suggest that differences may exist between naturally infected rodents with respect to viral shedding.

Development of reassortant viruses between pathogenic hantavirus strains.

Segment reassortment of negative strand viruses is an important mechanism for the development of new virus strains with altered pathogenicity. This study reports on in vitro generation of reassortants between Andes (ANDV) and Sin Nombre (SNV) viruses. Although they both cause hantavirus pulmonary syndrome (HPS), ANDV is the only hantavirus that has been transmitted from person to person (). Following dual infection of cells with ANDV and SNV, 8.9% of 337 progeny plaques contained reassortants, of which 66% were diploid, and 34% were monoploid. The monoploid reassortants contained the S and L segments of SNV and ANDV M segment. Analysis of replication of the monoploid reassortant indicated its efficiency was similar to ANDV rather than SNV. Results described in this study illustrate the ability to rapidly generate new hantavirus genotypes between genetically unrelated viruses by gene reassortment and provide a tool to dissect the pathogenesis of these important viruses.

Andes virus M genome segment is not sufficient to confer the virulence associated with Andes virus in Syrian hamsters.

Sin Nombre virus (SNV) and Andes virus (ANDV), members of the genus Hantavirus, in the family Bunyaviridae, are causative agents of hantavirus pulmonary syndrome (HPS) in North and South America, respectively. Although ANDV causes a lethal HPS-like disease in hamsters, SNV, and all other HPS-associated hantaviruses that have been tested, cause asymptomatic infections of laboratory animals, including hamsters. In an effort to understand the pathogenicity of ANDV in the hamster model, we generated ANDV/SNV reassortant viruses. Plaque isolation of viruses from cell cultures infected with both parental viruses yielded only one type of stable reassortant virus: large (L) and small (S) segments of SNV and M segment of ANDV. This virus, designated SAS reassortant virus, had in vitro growth and plaque morphology characteristics similar to those of ANDV. When injected into hamsters, the SAS reassortant virus was highly infectious and elicited high-titer, ANDV-specific neutralizing antibodies; however, the virus did not cause HPS and was not lethal. These data indicate that the ANDV M genome segment is not sufficient to confer the lethal HPS phenotype associated with ANDV.