Taqman Real-Time PCR Detects Avipoxvirus DNA in Blood of Hawaìi `Amakihi (Hemignathus virens)

Margaret E. M. Farias et al...Background Avipoxvirus sp. is a significant threat to endemic bird populations on several groups of islands worldwide, including Hawaìi, the Galapagos Islands, and the Canary Islands. Accurate identification and genotyping of Avipoxvirus is critical to the study of this disease and how it interacts with other pathogens, but currently available methods rely on invasive sampling of pox-like lesions and may be especially harmful in smaller birds. Methodology/Principal Findings Here, we present a nested TaqMan Real-Time PCR for the detection of the Avipoxvirus 4b core protein gene in archived blood samples from Hawaiian birds. The method was successful in amplifying Avipoxvirus DNA from packed blood cells of one of seven Hawaiian honeycreepers with confirmed Avipoxvirus infections and 13 of 28 Hawaìi `amakihi (Hemignathus virens) with suspected Avipoxvirus infections based on the presence of pox-like lesions. Mixed genotype infections have not previously been documented in Hawaìi but were observed in two individuals in this study. Conclusions/Significance We anticipate that this method will be applicable to other closely related strains of Avipoxvirus and will become an important and useful tool in global studies of the epidemiology of Avipoxvirus.Funding for this study was provided by: U.S. Geological Survey, Pacific Island Ecosystems Research Center (biology.usgs.gov/pierc/); U.S. Geological Survey Wildlife (biology.usgs.gov/wter/) and Invasive Species (biology.usgs.gov/invasive/) Programs; National Science Foundation (DEB0083944, www.nsf.gov); NIH/NCRR IDeA Networks of Biomedical Research Excellence (INBRE), P20RR016467 (http://www.ncrr.nih.gov/research_infrast​ructure/institutional_development_award/​idea_networks_of_biomedical_research_exc​ellence/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Different roles for CD4+ and CD8+ T lymphocytes and macrophage subsets in the control of a generalized virus infection

The importance of T-lymphocyte subsets in the control of poxvirus infections is controversial. To determine the relative contribution of lymphocyte subsets important for recovery from infection with ectromelia virus (EV), a natural murine poxvirus pathogen, C57BL/6 (B6) mice lacking functional CD8+ T cells because of disruption of the beta2-microglobulin gene or lacking functional CD4+ T cells because of disruption of the I-(A)beta gene, acutely depleted of CD8+ or CD4+ T cells with monoclonal antibody, or depleted of macrophage subsets by the macrophage suicide technique were used. Recovery from infection was strictly dependent on the effector functions of CD8+ T cells, in the absence of which 100% mortality resulted. This lymphocyte population had demonstrable antiviral activity early in the infection process even before class I major histocompatibility complex (MHC)-restricted CD8+ cytotoxic T-lymphocyte (CTL) activity was detectable. CD4+ T cells were found to be necessary for the generation of an optimal virus-specific, class I MHC-restricted CD8+ CTL response and contributed to virus clearance not involving cytolytic mechanisms. In both models of CD4+ T-cell deficiency, virus clearance was incomplete and persisted at low levels in most organs and at very high levels in the skin, but the animals did not die. The elimination of macrophage subpopulations impeded virus clearance, impaired the generation of class I MHC-restricted antiviral CTL response, and resulted in 100% mortality. These findings establish an absolute requirement for CD8+ and CD4+ T lymphocytes and macrophage subsets in the elimination of a natural murine poxvirus infection and support the idea that macrophages may be essential accessory cells for the generation of class I MHC-restricted antiviral CTL responses

Protective effect of exogenous recombinant mouse interferon-gamma and tumour necrosis factor-alpha on ectromelia virus infection in susceptible BALB/c mice

The resistance to mousepox is correlated with the production of type I cytokines: interleukin (IL)-2, IL-12, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. We intend to describe the modulation of generalized ectromelia virus (EV) infection with exogenous administration of mrIFN-γ and mrTNF-α separately and in combination using susceptible BALB/c mice. The treatment schemes presented resulted in the localization of the generalized EV infection and its development into non-fatal sloughing of the infected limb. This was accompanied by low virus titres in the treated mice due to control of systemic virus replication and virus clearance. The balance of type I versus type II cytokines was dominated by a type I response in the treated groups. The group treated with the combination of IFN-γ and TNF-α exhibited the best survival with Th1-dominant (IFN-γ and IL-12) cytokine profiles, whereas the TNF-α-treated group of mice was less successful in clearance of virus and demonstrated the lowest survival rate. The successful cytokine treatment schemes in this orthopoxvirus model system may have important implications in the treatment of viral diseases in humans and, in particular, of variola virus infection

Polarized type 1 cytokine response and cell-mediated immunity determine genetic resistance to mousepox

Ectromelia virus (ECTV), a natural mouse pathogen and an orthopoxvirus, has been used to investigate the correlation between polarized type 1 or type 2 immune responses and resistance to disease in poxvirus infections by using well defined resistant and susceptible mouse strains. Our data show that distinct differences exist in the cytokine profiles expressed in resistant and susceptible mice infected with ECTV. Resistant C57BL/6 mice generate a type 1 cytokine response [IFN-y, IL-2, and tumor necrosis factor (TNF)], within the first few days of infection, which is associated with strong cytotoxic T lymphocyte response (CTL) and recovery from ECTV infection. Susceptible strains of mice (BALB/c and A/J) on the other hand generate a type 2 cytokine response (IL-4 but little or no IFN-y and IL-2), which is associated with a weak or an absent CTL response, resulting in uncontrolled virus replication and death. Although deletion of IL-4 function alone did not change the outcome of infection in susceptible mice, the loss of IFN-y function in resistant mice abrogated natural killer (NK) cell and CTL effector functions resulting in fulminant disease and 100% mortality. Therefore, a clear link exists between the early production of specific type 1 cytokines, in particular, IFN-y, the nature of the cellular immune response, and disease outcome in this virus model. This finding in the mousepox model raises the possibility that inappropriate cytokine responses may result in increased susceptibility to smallpox in humans