Treatment with the reactive oxygen species scavenger EUK-207 reduces lung damage and increases survival during 1918 influenza virus infection in mice.

The 1918 influenza pandemic caused over 40 million deaths worldwide, with 675,000 deaths in the United States alone. Studies in several experimental animal models showed that 1918 influenza virus infection resulted in severe lung pathology associated with dysregulated immune and cell death responses. To determine if reactive oxygen species produced by host inflammatory responses play a central role in promoting severity of lung pathology, we treated 1918 influenza virus-infected mice with the catalytic catalase/superoxide dismutase mimetic, salen-manganese complex EUK-207 beginning 3 days postinfection. Postexposure treatment of mice infected with a lethal dose of the 1918 influenza virus with EUK-207 resulted in significantly increased survival and reduced lung pathology without a reduction in viral titers. In vitro studies also showed that EUK-207 treatment did not affect 1918 influenza viral replication. Immunohistochemical analysis showed a reduction in the detection of the apoptosis marker cleaved caspase-3 and the oxidative stress marker 8-oxo-2'-deoxyguanosine in lungs of EUK-207-treated animals compared to vehicle controls. High-throughput sequencing and RNA expression microarray analysis revealed that treatment resulted in decreased expression of inflammatory response genes and increased lung metabolic and repair responses. These results directly demonstrate that 1918 influenza virus infection leads to an immunopathogenic immune response with excessive inflammatory and cell death responses that can be limited by treatment with the catalytic antioxidant EUK-207.

Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice.

Highly pathogenic H5N1 influenza shares the same neuraminidase (NA) subtype with the 2009 pandemic (H1N1pdm09), and cross-reactive NA immunity might protect against or mitigate lethal H5N1 infection. In this study, mice were either infected with a sublethal dose of H1N1pdm09 or were vaccinated and boosted with virus-like particles (VLP) consisting of the NA and matrix proteins, standardized by NA activity and administered intranasally, and were then challenged with a lethal dose of HPAI H5N1 virus. Mice previously infected with H1N1pdm09 survived H5N1 challenge with no detectable virus or respiratory tract pathology on day 4. Mice immunized with H5N1 or H1N1pdm09 NA VLPs were also fully protected from death, with a 100-fold and 10-fold reduction in infectious virus, respectively, and reduced pathology in the lungs. Human influenza vaccines that elicit not only HA, but also NA immunity may provide enhanced protection against the emergence of seasonal and pandemic viruses.

Obese mice have increased morbidity and mortality compared to non-obese mice during infection with the 2009 pandemic H1N1 influenza virus.

BACKGROUND: Obesity has been identified as an independent risk factor for severe or fatal infection with 2009 pandemic H1N1 influenza (2009 pH1N1), but was not previously recognized for previous pandemic or seasonal influenza infections. OBJECTIVES: Our aim was to evaluate the role of obesity as an independent risk factor for severity of infection with 2009 pH1N1, seasonal H1N1, or a pathogenic H1N1 influenza virus. METHODS: Diet-induced obese (DIO) and their non-obese, age-matched control counterparts were inoculated with a 2009 pH1N1, A/California/04/2009 (CA/09), current seasonal H1N1, A/NY/312/2001 (NY312), or highly pathogenic 1918-like H1N1, A/Iowa/Swine/1931 (Sw31), virus. RESULTS: Following inoculation with CA/09, DIO mice had higher mortality (80%) than control mice (0%) and lost more weight during infection. No effect of obesity on morbidity and mortality was observed during NY312 or Sw31 infection. Influenza antigen distribution in the alveolar regions of the lungs was more pronounced in DIO than control mice during CA/09 infection at 3 days post-inoculation (dpi), despite similar virus titers. During CA/09 infection, localized interferon-ß and proinflammatory cytokine protein responses in the lungs were significantly lower in DIO than control mice. Conversely, serum cytokine concentrations were elevated in DIO, but not control mice following infection with CA/09. The effect of obesity on differential immune responses was abrogated during NY312 or Sw31 infection. CONCLUSIONS: Together, these data support epidemiologic reports that obesity may be a risk factor for severe 2009 pandemic H1N1 influenza infection, but the role of obesity in seasonal or highly virulent pandemic influenza infection remains unclear.

Immunization with 1976 swine H1N1- or 2009 pandemic H1N1-inactivated vaccines protects mice from a lethal 1918 influenza infection.

BACKGROUND: Zoonotic infections with H1N1 influenza viruses that evolved initially from the 1918 virus (1918) and adapted to swine threatened a pandemic in 1976 (1976 swH1N1) and a novel reassortant H1N1 virus caused a pandemic in 2009-2010 (2009 pH1N1). Epidemiological and laboratory animal studies show that protection from severe 2009 pH1N1 infection is conferred by vaccination or prior infection with 1976 swH1N1 or 1918. OBJECTIVES: Our aim was to demonstrate cross-protection by immunization with 2009 pH1N1 or 1976 swH1N1 vaccines following a lethal challenge with 1918. Further, the mechanisms of cross-protective antibody responses were evaluated. METHODS: Mice were immunized with 1976 swH1N1, 2009 pH1N1, 2009 seasonal trivalent, or 1918 vaccines and challenged with 1918. Cross-reactive antibody responses were assessed and protection monitored by survival, weight loss, and pathology in mice. RESULTS AND CONCLUSIONS: Vaccination with the 1976 swH1N1 or 2009 pH1N1 vaccines protected mice from a lethal challenge with 1918, and these mice lost no weight and had significantly reduced viral load and pathology in the lungs. Protection was likely due to cross-reactive antibodies detected by microneutralization assay. Our data suggest that the general population may be protected from a future 1918-like pandemic because of prior infection or immunization with 1976 swH1N1 or 2009 pH1N1. Also, influenza protection studies generally focus on cross-reactive hemagglutination-inhibiting antibodies; while hemagglutinin is the primary surface antigen, this fails to account for other influenza viral antigens. Neutralizing antibody may be a better correlate of human protection against pathogenic influenza strains and should be considered for vaccine efficacy.

Estrogen and progesterone affect responses to malaria infection in female C57BL/6 mice.

BACKGROUND: Previous data from our laboratory suggest that gonadally intact C57BL/6 male mice are more likely than their female counterparts to die from Plasmodium chabaudi infection, to recover more slowly from weight loss and hematocrit loss, and to have reduced interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) responses. Removal of the ovaries, and hence, the primary production of sex steroids in females, reverses these differences. OBJECTIVE: We hypothesized that sex differences in response to P chabaudi may be mediated by differential synthesis of IFN-gamma and IL-10 that is influenced by estrogen, progesterone, or both. METHODS: C57BL/6 female mice (n = 200; n = 10/time point/treatment/experiment) were ovariectomized and implanted with a 21-day controlled-release pellet containing either 0.1 mg of 17beta-estradiol (E(2)), 10 mg of progesterone (P(4)), 0.1 mg of E(2) plus 10 mg of P(4), or cholesterol (placebo). Females were inoculated with 10(6)P chabaudi-infected erythrocytes. Body mass, body temperature, hematocrit, parasitemia, cytokine production, and antibody responses were monitored 0, 3, 5, 7, 10, 14, and 21 days postinoculation. RESULTS: Administration of E(2), either alone or in combination with P(4), mitigated infection-induced weight loss, hematocrit loss, and hypothermia, compared with females receiving placebo pellets (P < 0.05 in each case). Hormone treatment did not affect levels of parasitemia. Females administered E(2) alone or in combination with P(4) produced 4 to 7 times higher IFN-gamma and IL-10 during peak parasitemia than did females implanted with pellets containing either P(4) alone or placebo (P < 0.05 in each case). Exposure to E(2), either alone or in combination with P(4), increased anti-P chabaudi immunoglobulin G (IgG1) responses and the ratio of IgG1 to IgG2c (P < 0.05 in each case). CONCLUSION: This animal study suggests that physiological levels of estrogen, rather than progesterone, enhance immunity and, possibly, protect females from disease symptoms during malaria infection.

Immunological mechanisms mediating hantavirus persistence in rodent reservoirs.

Hantaviruses, similar to several emerging zoonotic viruses, persistently infect their natural reservoir hosts, without causing overt signs of disease. Spillover to incidental human hosts results in morbidity and mortality mediated by excessive proinflammatory and cellular immune responses. The mechanisms mediating the persistence of hantaviruses and the absence of clinical symptoms in rodent reservoirs are only starting to be uncovered. Recent studies indicate that during hantavirus infection, proinflammatory and antiviral responses are reduced and regulatory responses are elevated at sites of increased virus replication in rodents. The recent discovery of structural and non-structural proteins that suppress type I interferon responses in humans suggests that immune responses in rodent hosts could be mediated directly by the virus. Alternatively, several host factors, including sex steroids, glucocorticoids, and genetic factors, are reported to alter host susceptibility and may contribute to persistence of hantaviruses in rodents. Humans and reservoir hosts differ in infection outcomes and in immune responses to hantavirus infection; thus, understanding the mechanisms mediating viral persistence and the absence of disease in rodents may provide insight into the prevention and treatment of disease in humans. Consideration of the coevolutionary mechanisms mediating hantaviral persistence and rodent host survival is providing insight into the mechanisms by which zoonotic viruses have remained in the environment for millions of years and continue to be transmitted to humans.

Corticosteroids modulate Seoul virus infection, regulatory T-cell responses and matrix metalloprotease 9 expression in male, but not female, Norway rats.

Human hantaviral disease is mediated by excessive proinflammatory and CD8+ T-cell responses, which can be alleviated by administration of corticosteroids. In contrast with humans, male rats that are infected with their species-specific hantavirus, Seoul virus (SEOV), have reduced proinflammatory and elevated regulatory T-cell responses in tissues where virus persists. To determine the effects of glucocorticoids on SEOV persistence and immune responses during infection, male and female Norway rats received sham surgeries (sham) or were adrenalectomized (ADX0), in some of which corticosterone was replaced at low (ADX10) or high (ADX80) doses. Rats were inoculated with SEOV and serum corticosterone, SEOV RNA, gene expression and protein production were measured at different time points post-inoculation. We observed that SEOV infection suppressed corticosterone in sham males to concentrations seen in ADX0 males. Furthermore, males with low corticosterone had more SEOV RNA in the lungs than either females or males with high corticosterone concentrations during peak infection. Although high concentrations of corticosterone suppressed the expression of innate antiviral and proinflammatory mediators to a greater extent in females than in males, these immunomodulatory effects did not correlate with SEOV load. Males with low corticosterone concentrations and high viral load had elevated regulatory T-cell responses and expression of matrix metalloprotease (MMP)-9. MMP-9 is a glycogenase that disrupts cellular matrices and may facilitate extravasation of SEOV-infected cells from circulation into lung tissue. Suppression of glucocorticoids may thus contribute to more efficient dissemination of SEOV in male than in female rats.

Seoul virus enhances regulatory and reduces proinflammatory responses in male Norway rats.

Zoonotic pathogens, including hantaviruses, are maintained in the environment by causing persistent infection in the absence of disease in their reservoir hosts. Spillover of hantaviruses to humans can cause severe disease that is mediated by excessive proinflammatory responses. The mechanisms mediating hantaviral persistence in rodent reservoirs remain largely unknown. Male Norway rats were inoculated with their species-specific hantavirus, Seoul virus (SEOV), and viral RNA, cytokine, and chemokine responses were evaluated in spleen and lung tissue. More viral RNA was detectable in the lungs than spleen, with copies of SEOV peaking 15-30 days post-inoculation (p.i.) and persisting for 60 days p.i. In the lungs, the expression and production of proinflammatory mediators (i.e., IL-1beta, IL-6, TNF-alpha, IFN-gamma, CCL5, CCL2, CX3CL1, CXCL10, VCAM, VEGF, and NOS2) remained at or below baseline throughout SEOV infection; whereas, regulatory factors, including TGF-beta and FoxP3 were elevated. Conversely, in the spleen, proinflammatory responses were induced while regulatory responses remained unchanged during infection. To determine whether reduced proinflammatory responses mediate hantavirus persistence in the lungs, male rats were administered rIL-1beta or vehicle for 30 days during SEOV infection. SEOV persistence and shedding were not affected by IL-1beta treatment. Proinflammatory responses were elevated in rIL-1beta-treated rats, but remained within physiological levels, suggesting that supra-physiological concentrations may be necessary for viral clearance at the cost of causing disease. Elevated regulatory responses may suppress excessively high proinflammatory responses at a site of elevated SEOV replication to contribute to viral persistence and prevent proinflammatory-mediated disease in reservoir hosts.

A survey of rodent-borne pathogens carried by wild-caught Norway rats: a potential threat to laboratory rodent colonies.

Unintentional infection of laboratory rodents can compromise scientific research as well as the health of the animals and animal handlers. The source of contamination often is unknown, but may be introduced by wild rats from surrounding environments. To determine whether rats in Baltimore, Maryland, USA carry infectious agents commonly found in laboratory rodent colonies, we live-trapped 162 rats during 2005 to 2006 and screened them for a panel of viruses, bacteria and parasites. Antibodies against rat coronavirus/sialodacryoadenitis virus (91.7%), Mycoplasma pulmonis (72.9%), cilia-associated respiratory bacillus (52.1%), rat parvovirus/rat minute virus (29.2%), Kilham rat virus (10.4%), Toolan's H-1 virus (10.4%), Sendai virus (4.2%) and Theiler's mouse encephalomyelitis virus (4.2%), were detected in wild-caught Norway rats. Antibodies against reovirus and pneumonia virus of mice were not detected in wild Norway rats. Endoparasites, including Nippostrongylus braziliensis (71.6%), Rodentolepis nana or Hymenolepis diminuta (34.4%), Hetarakis spumosa (24.1%) and Trichuris muris (14.8%), as well as ectoparasites (14.8%), were identified in wild-caught rats. The risk of pathogen transmission from wild-caught rats to laboratory colonies needs to be mitigated by minimizing exposures rather than assuming wild animals represent a minimal hazard.

Regulatory T cells enhance persistence of the zoonotic pathogen Seoul virus in its reservoir host.

Hantaviruses are zoonotic pathogens that maintain a persistent infection in their reservoir hosts, yet the mechanisms mediating persistence remain unknown. Regulatory T cell responses cause persistent infection by suppressing proinflammatory and effector T cell activity; hantaviruses may exploit these responses to cause persistence. To test this hypothesis, male Norway rats were inoculated with Seoul virus and regulatory T cells were monitored during infection. Increased numbers of CD4(+)CD25(+)Forkhead box P3(+) T cells and expression of Forkhead box P3 and TGF-beta were observed in the lungs of male rats during persistent Seoul virus infection. To determine whether regulatory T cells modulate Seoul virus persistence, regulatory T cells were inactivated in male rats by using an anti-rat CD25 monoclonal antibody (NDS-63). Inactivation of regulatory T cells reduced the amount of Seoul virus RNA present in the lungs and the proportion of animals shedding viral RNA in saliva. Because regulatory T cells suppress proinflammatory-induced pathogenesis, pathologic observations in the lungs were evaluated during infection. Subclinical acute multifocal areas of hemorrhage and edema were noted in the lungs during infection; inactivation of regulatory T cells reduced the amount of pathologic foci. Expression of TNF was suppressed during the persistent phase of infection; inactivation of regulatory T cells eliminated the suppression of TNF. Taken together, these data suggest that regulatory T cells mediate Seoul virus persistence, possibly through elevated transcription and synthesis of TGF-beta and suppression of TNF. These data provide evidence of regulatory T cell involvement in the persistence of a zoonotic pathogen in its natural reservoir host.

Elevated testosterone and reduced 5-HIAA concentrations are associated with wounding and hantavirus infection in male Norway rats.

Among rodents that carry hantaviruses, males are more likely to engage in aggression and to be infected than females. One mode of hantavirus transmission is via the passage of virus in saliva during wounding. The extent to which hantaviruses cause physiological changes in their rodent host that increase aggression and, therefore, virus transmission has not been fully documented. To assess whether steroid hormones and neurotransmitters contribute to the correlation between aggression and Seoul virus infection, Norway rats were trapped in Baltimore, Maryland and wounding, infection status, steroid hormones, and concentrations of neurotransmitters, including norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenol acetic acid (DOPAC), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5-HIAA) in select brain regions were examined. Older males and males with high-grade wounds were more likely to have anti-Seoul virus IgG and viral RNA in organs than either juveniles or adult males with less severe wounds. Wounded males had higher circulating testosterone, lower hypothalamic 5-HIAA, and lower NE in the amygdala than males with no wounds. Infected males had higher concentrations of testosterone, corticosterone, NE in the hypothalamus, and DOPAC in the amygdala than uninfected males, regardless of wounding status. In the present study, wounded males that were infected with Seoul virus had elevated testosterone and reduced 5-HIAA concentrations, suggesting that these neuroendocrine mechanisms may contribute to aggression and the likelihood of transmission of hantavirus in natural populations of male Norway rats.

Serologic evidence for Rickettsia typhi and an ehrlichial agent in Norway rats from Baltimore, Maryland, USA.

We screened serum from 90 Norway rats trapped in East Baltimore, Maryland, USA, from April to November 2005 for antibodies against Rickettsia typhi and Ehrlichia chaffeensis. Six rats had positive titers of > or = 1:64 against R. typhi and did not react with R. akari. In addition, four rats had cross-reactive antibodies with titers of > or = 1:64 against Ehrlichia chaffeensis. Sera from these rats also cross-reacted with Anaplasma phagocytophilum or Ehrlichia muris. Our data indicate that the agent of murine typhus and ehrlichial agents are circulating in the Norway rat population in Baltimore.

Norway rat population in Baltimore, Maryland, 2004.

Norway rats are reservoirs for several zoonotic agents, including hantaviruses, and are implicated in the transmission of pathogens to humans in urban environments. The rat population of Baltimore, Maryland was estimated from surveys in 1949 and again in 1952, but has not been evaluated for more than 50 years. Previously identified sociodemographic risk factors for rat infestation, including median income, human density, and percentage of rental properties, were used to categorize census block groups in Baltimore. Rat infestation risk factors, including median income and human density, have improved over the last 50 years in Baltimore. Rat infestation was determined both by observation and trapping of rats in alleys that were representative of the different strata of risk factors. Despite improvements in risk factors, the outdoor, residential rat population of Baltimore in 2004 was estimated to be approximately 48,420 +/- 14,883 rats, which is comparable to the 1949 and 1952 estimates. Approximately half of the rats trapped in Baltimore City had detectable antibody against Seoul virus. The failure to substantially impact rat population levels in the past 50 years indicate that alternative control strategies for rat infestation are needed to reduce the risk of rat-borne pathogen spillover to the human population.