To address Sin Nombre (SN) virus persistence in deer mice, we sacrificed experimentally infected deer mice at eight time points from day 21 to day 217 postinoculation (p.i.) and examined their tissues for viral nucleocapsid (N) antigen expression and both negative-strand (genomic) and positive-strand (replicative/ mRNA) viral S segment RNA titers. All the animals that we inoculated developed persistent infections, and SN virus could be isolated from tissues throughout the course of infection. The transition from an acute to a persistent pattern of infection appeared to occur between days 60 and 90 p.i. Beginning on day 60 p.i., the heart, brown adipose tissue (BAT), and lung retained antigen expression and genomic viral RNA the most frequently. We found a statistically significant association among the presence of replicative RNA in the heart, lung, and BAT, widespread antigen expression (in >5 tissues), and RNA viremia. Of these three tissues, the heart retained negative-strand RNA and viral N antigen the most consistently (in 25 of 26 animals). During persistence, there were two distinct patterns of infection: restricted versus disseminated tissue involvement. Mice with the restricted pattern exhibited N antigen expression in <3 tissues, an absence of viral RNA in the blood, neutralizing antibody titers of <1:1,280 (P ‫؍‬ 0.01), and no replicative RNA in the heart, lung, or BAT. Those with the "disseminated" pattern showed N antigen expression in >5 tissues, neutralizing antibody titers of 1:160 to 1:20,480, replicative RNA in the heart, lung, and BAT at a high frequency, and RNA viremia. Virus could be isolated consistently only from mice that demonstrated the disseminated pattern. The heart, lung, and BAT are important sites for the replication and maintenance of SN virus during persistent infection.

The mechanism(s) by which Sin Nombre (SN) hantavirus is maintained in deer mouse populations is unclear. Field studies indicate that transmission occurs primarily if not exclusively via a horizontal mechanism. Using an experimental deer mouse infection model in an outdoor laboratory, we tested whether infected rodents shed SN virus in urine, feces, and saliva, whether infected mice transmit infection to naïve cage mates, and whether infected dams are able to vertically transmit virus or antibody to offspring. Using pooled samples of urine, feces, and saliva collected from mice infected 8 to 120 days postinoculation (p.i.), we found that a subset of saliva samples, collected between 15 and 90 days p.i., contained viral RNA. Parallel studies conducted on wild-caught, naturally infected deer mice showed a similar pattern of intermittent positivity, also only in saliva samples. Attempts to isolate virus through inoculation of cells or naïve deer mice with the secreta or excreta of infected mice were uniformly negative. Of 54 attempts to transmit infection by cohousing infected deer mice with seronegative cage mates, we observed only a single case of transmission, which occurred between 29 and 42 days p.i. Dams passively transferred antibodies to neonatal pups via milk, and those antibodies persisted for at least 2 months after weaning, but none transmitted infection to their pups. Compared to other hantavirus models, SN virus is shed less efficiently and transmits inefficiently among cage mates. Transmission of SN virus among reservoir rodents may require factors that are not required for other hantaviruses.Hantaviruses (Bunyaviridae family; Hantavirus genus) are rodent-borne viruses with a worldwide distribution. As with other members of the family Bunyaviridae, hantaviruses are enveloped and have a tripartite, negative-sense RNA genome. Two distinct human illnesses are associated with hantavirus infection: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) (36). HFRS occurs primarily in Europe and Asia, with a case/fatality ratio of 0.1 to 10% (36). Etiologic agents of HFRS include the prototypic Hantaan (HTN) virus, Seoul (SEO) virus, Puumala (PUU) virus, and Dobrava-Belgrade virus. HCPS occurs in North and South America (28). There are at least four etiologic agents of HCPS in North America: Sin Nombre (SN), Bayou, New York, and Black Creek Canal (BCC) viruses. Of these four viruses, the primary etiologic agent is SN virus, which is believed to be responsible for a large majority (Ͼ95%) of the more than 300 reported cases. The case/fatality ratio of HCPS is approximately 40%. The deer mouse (Peromyscus maniculatus) is the predominant carrier of SN virus (12,32).Natural transmission of hantaviruses among wild rodents is poorly understood, but both field and laboratory studies suggest that nearly all, if not all, transmission is via one or more horizontal routes and that male mice are differentially affected.In this study, we tested the hypothesis that deer mice experimenta...

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-β) 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.

Maladaptive immune responses are considered to be important factors in the pathogenesis of the two diseases caused by hantaviruses, hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome (HCPS). While the intensity of adaptive antiviral T-cell responses seems to correlate with the severity of HCPS, there is increasing evidence that innate antiviral responses by endothelial cells, the native targets for hantavirus infection in vivo, are induced within hours of exposure to infectious hantaviruses. To investigate early events in the innate response to Sin Nombre virus (SNV), the principal etiologic agent of HCPS in North America, we treated human endothelial cells with live virus, or virus subjected to inactivation by UV irradiation at minimal doses required to inhibit replication, and assayed host expression of interferonstimulated genes (ISG) by microarray and reverse transcription-PCR. We show herein that a variety of ISG are induced between 4 and 24 h after exposure to both live and killed virus. The levels of such induction at early time points (before 24 h) were generally higher in cells treated with SNV particles that had been killed by exposure to UV irradiation. Additionally, SNV exposed to increasing doses of UV irradiation induced ISG better than live virus despite increased disruption of viral RNA integrity. However, SNV replication was required for continued ISG overexpression by 3 days posttreatment. These results suggest that hantavirus particles may themselves be capable of early induction of ISG and that ongoing production of viral particles during infection could contribute to the pathogenic process.

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To address the role that viral load plays in pathogenesis in patients with hantavirus cardiopulmonary syndrome (HCPS), we quantified Sin Nombre virus S segment viral RNA in plasma samples from 27 acutely ill patients. For 6 patients, we examined viral load in matched plasma, urine, and/or tracheal aspirate throughout the time when the patients were in intensive care. Peak titers in plasma reached 1.8 x 106 copies/mL; none of the patients had viral RNA in urine. Titers in tracheal aspirates did not exceed 8 x 104 copies/mL. We found a statistically significant association (P < .005) between plasma viral RNA levels at admission to the hospital and the severity of disease. Of those with plasma viral RNA titers above the threshold for assay sensitivity (5000 copies/mL), those with mild-moderate and severe disease had an average of 27,800 and 438,545 copies/mL, respectively. These results suggest that patients with high viral loads on admission are more likely to have severe disease.

BackgroundHantaviruses indigenous to the New World are the etiologic agents of hantavirus cardiopulmonary syndrome (HCPS). These viruses induce a strong interferon-stimulated gene (ISG) response in human endothelial cells. African green monkey-derived Vero E6 cells are used to propagate hantaviruses as well as many other viruses. The utility of the Vero E6 cell line for virus production is thought to owe to their lack of genes encoding type I interferons (IFN), rendering them unable to mount an efficient innate immune response to virus infection. Interferon λ, a more recently characterized type III IFN, is transcriptionally controlled much like the type I IFNs, and activates the innate immune system in a similar manner.Methodology/Principal FindingsWe show that Vero E6 cells respond to hantavirus infection by secreting abundant IFNλ. Three New World hantaviruses were similarly able to induce IFNλ expression in this cell line. The IFNλ contained within virus preparations generated with Vero E6 cells independently activates ISGs when used to infect several non-endothelial cell lines, whereas innate immune responses by endothelial cells are specifically due to viral infection. We show further that Sin Nombre virus replicates to high titer in human hepatoma cells (Huh7) without inducing ISGs.Conclusions/SignificanceHerein we report that Vero E6 cells respond to viral infection with a highly active antiviral response, including secretion of abundant IFNλ. This cytokine is biologically active, and when contained within viral preparations and presented to human epithelioid cell lines, results in the robust activation of innate immune responses. We also show that both Huh7 and A549 cell lines do not respond to hantavirus infection, confirming that the cytoplasmic RNA helicase pathways possessed by these cells are not involved in hantavirus recognition. We demonstrate that Vero E6 actively respond to virus infection and inhibiting IFNλ production in these cells might increase their utility for virus propagation.

The urgent need for a cure for early phase COVID-19 infected patients critically underlines drug repositioning strategies able to efficiently identify new and reliable treatments by merging computational, experimental, and pharmacokinetic expertise. Here we report new potential therapeutics for COVID-19 identified with a combined virtual and experimental screening strategy and selected among already approved drugs. We used hydroxychloroquine (HCQ), one of the most studied drugs in current clinical trials, as a reference template to screen for structural similarity against a library of almost 4000 approved drugs. The top-ranked drugs, based on structural similarity to HCQ, were selected for in vitro antiviral assessment. Among the selected drugs, both zuclopenthixol and nebivolol efficiently block SARS-CoV-2 infection with EC 50 values in the low micromolar range, as confirmed by independent experiments. The anti-SARS-CoV-2 potential of ambroxol, amodiaquine, and its active metabolite ( N -monodesethyl amodiaquine) is also discussed. In trying to understand the “hydroxychloroquine” mechanism of action, both p K a and the HCQ aromatic core may play a role. Further, we show that the amodiaquine metabolite and, to a lesser extent, zuclopenthixol and nebivolol are active in a SARS-CoV-2 titer reduction assay. Given the need for improved efficacy and safety, we propose zuclopenthixol, nebivolol, and amodiaquine as potential candidates for clinical trials against the early phase of the SARS-CoV-2 infection and discuss their potential use as adjuvant to the current (i.e., remdesivir and favipiravir) COVID-19 therapeutics.