Chunyan Ye

Persistent Sin Nombre Virus Infection in the Deer Mouse (Peromyscus maniculatus) Model: Sites of Replication and Strand-Specific Expression

ABSTRACT 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.

Shedding and Intracage Transmission of Sin Nombre Hantavirus in the Deer Mouse (Peromyscus maniculatus) Model

ABSTRACT 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.

Sin Nombre Viral RNA Load in Patients with Hantavirus Cardiopulmonary Syndrome

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.

Induction of Innate Immune Response Genes by Sin Nombre Hantavirus Does Not Require Viral Replication

ABSTRACT 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 interferon-stimulated 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.

Early Innate Immune Responses to Sin Nombre Hantavirus Occur Independently of IFN Regulatory Factor 3, Characterized Pattern Recognition Receptors, and Viral Entry

Sin Nombre virus (SNV) is a highly pathogenic New World virus and etiologic agent of hantavirus cardiopulmonary syndrome. We have previously shown that replication-defective virus particles are able to induce a strong IFN-stimulated gene (ISG) response in human primary cells. RNA viruses often stimulate the innate immune response by interactions between viral nucleic acids, acting as a pathogen-associated molecular pattern, and cellular pattern-recognition receptors (PRRs). Ligand binding to PRRs activates transcription factors which regulate the expression of antiviral genes, and in all systems examined thus far, IFN regulatory factor 3 (IRF3) has been described as an essential intermediate for induction of ISG expression. However, we now describe a model in which IRF3 is dispensable for the induction of ISG transcription in response to viral particles. IRF3-independent ISG transcription in human hepatoma cell lines is initiated early after exposure to SNV virus particles in an entry- and replication-independent fashion. Furthermore, using gene knockdown, we discovered that this activation is independent of the best-characterized RNA- and protein-sensing PRRs including the cytoplasmic caspase recruitment domain-containing RNA helicases and the TLRs. SNV particles engage a heretofore unrecognized PRR, likely located at the cell surface, and engage a novel IRF3-independent pathway that activates the innate immune response.

Peptide Antagonists That Inhibit Sin Nombre Virus and Hantaan Virus Entry through the β3-Integrin Receptor

ABSTRACT Specific therapy is not available for the treatment of hantavirus cardiopulmonary syndrome caused by Sin Nombre virus (SNV). The entry of pathogenic hantaviruses into susceptible human cells is dependent upon expression of the αvβ3 integrin, and transfection of human β3 integrin is sufficient to confer infectibility onto CHO (Chinese hamster ovary) cells. Furthermore, pretreatment of susceptible cells with anti-β3 antibodies such as c7E3 or its Fab fragment ReoPro prevents hantavirus entry. By using repeated selection of a cyclic nonamer peptide phage display library on purified αvβ3, we identified 70 peptides that were competitively eluted with ReoPro. Each of these peptides was examined for its ability to reduce the number of foci of SNV strain SN77734 in a fluorescence-based focus reduction assay according to the method of Gavrilovskaya et al. (I. N. Gavrilovskaya, M. Shepley, R. Shaw, M. H. Ginsberg, and E. R. Mackow, Proc. Natl. Acad. Sci. USA 95:7074-7079, 1998). We found that 11 peptides reduced the number of foci to a greater extent than did 80 μg/ml ReoPro when preincubated with Vero E6 cells. In addition, 8 of the 70 peptides had sequence similarity to SNV glycoproteins. We compared all 18 peptide sequences (10 most potent, 7 peptides with sequence similarity to hantavirus glycoproteins, and 1 peptide that was in the group that displayed the greatest potency and had significant sequence similarity) for their abilities to inhibit SNV, Hantaan virus (HTNV), and Prospect Hill virus (PHV) infection. There was a marked trend for the peptides to inhibit SNV and HTNV to a greater extent than they inhibited PHV, a finding that supports the contention that SNV and HTNV use β3 integrins and PHV uses a different receptor, β1 integrin. We then chemically synthesized the four peptides that showed the greatest ability to neutralize SNV. These peptides inhibited viral entry in vitro as free peptides outside of the context of a phage. Some combinations of peptides proved more inhibitory than did individual peptides. In all, we have identified novel peptides that inhibit entry by SNV and HTNV via β3 integrins and that can be used as lead compounds for further structural optimization and consequent enhancement of activity.

New World Hantaviruses Activate IFNλ Production in Type I IFN-Deficient Vero E6 Cells

Background Hantaviruses 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 Findings We 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/Significance Herein 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.

Neutralizing Antibodies and Sin Nombre Virus RNA after Recovery from Hantavirus Cardiopulmonary Syndrome

Patients who later have a mild course of hantavirus cardiopulmonary syndrome (HCPS) are more likely to exhibit a high titer of neutralizing antibodies against Sin Nombre virus (SNV), the etiologic agent of HCPS, at the time of hospital admission. Because administering plasma from patients who have recovered from HCPS to those in the early stages of disease may be an advantageous form of passive immunotherapy, we examined the neutralizing antibody titers of 21 patients who had recovered from SNV infection. Even 1,000 days after admission to the hospital, 6 of 10 patients had titers of 800 or higher, with one sample retaining a titer of 3,200 after more than 1,400 days. None of the convalescent-phase serum samples contained detectable viral RNA. These results confirm that patients retain high titers of neutralizing antibodies long after recovery from SNV infection.

Kinetics of Immune Responses in Deer Mice Experimentally Infected with Sin Nombre Virus

ABSTRACT 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.

Genetic vaccines protect against Sin Nombre hantavirus challenge in the deer mouse (Peromyscus maniculatus)

We used a deer mouse (Peromyscus maniculatus) infection model to test the protective efficacy of genetic vaccine candidates for Sin Nombre (SN) virus that were known to provoke immunological responses in BALB/c mice (Bharadwaj et al., Vaccine 17, 2836-2843, 1999 ). Protective epitopes were localized in each of four overlapping cDNA fragments that encoded portions of the SN virus G1 glycoprotein antigen; the nucleocapsid gene also was protective. The protective efficacy of glycoprotein gene fragments correlated with splenocyte proliferation in the presence of cognate antigen, but none induced neutralizing antibodies. Genetic vaccines against SN virus can protect outbred deer mice from infection even in the absence of a neutralizing antibody response.