Relations between Household Livestock Ownership, Livestock Disease, and Young Child Growth.

BACKGROUND: In resource-limited settings in which child malnutrition is prevalent, humans live in close proximity to household livestock. However, the relation between household livestock and child nutrition represents a considerable knowledge gap. OBJECTIVE: We assessed whether household livestock ownership or livestock disease episodes were associated with growth in young children in western Kenya. METHODS: We incorporated monthly anthropometric measurements for children <5 y of age into an ongoing linked human and animal surveillance cohort in rural western Kenya. Using linear mixed models adjusted for age, sex, and household wealth, we tested whether baseline household livestock ownership was related to baseline child height for age or prospective growth rate. We also evaluated whether livestock disease episodes were associated with child growth rate over 11 mo of follow-up. RESULTS: We collected data on 925 children over the course of follow-up. Greater household livestock ownership at baseline was not related to baseline child height-for-age z score (adjusted ß: 0.01 SD; 95% CI: -0.02, 0.04 SD) or child growth rate (adjusted ß: 0.02 cm/y; 95% CI: -0.03, 0.07 cm/y). Livestock disease episodes were not significantly associated with child growth across the entire cohort (adjusted ß: -0.007 cm/mo; 95% CI: -0.02, 0.006 cm/mo). However, children in households with livestock digestive disease between June and November gained less height than did children in households that did not report livestock disease (ß: -0.063 cm/mo; 95% CI: -0.112, -0.016 cm/mo). Children <2 y of age in households with livestock digestive disease gained less weight than did those who did not report disease (ß: -0.033 kg/mo; 95% CI: -0.063, -0.003 kg/mo). CONCLUSION: In this cohort of young children in western Kenya, we did not find an association between ownership of livestock and child growth status. However, disease episodes in household livestock may be related to a lower child growth rate in some groups.

Epidemiology, seasonality, and burden of influenza and influenza-like illness in urban and rural Kenya, 2007-2010.

BACKGROUND: The epidemiology and burden of influenza remain poorly defined in sub-Saharan Africa. Since 2005, the Kenya Medical Research Institute and Centers for Disease Control and Prevention-Kenya have conducted population-based infectious disease surveillance in Kibera, an urban informal settlement in Nairobi, and in Lwak, a rural community in western Kenya. METHODS: Nasopharyngeal and oropharyngeal swab specimens were obtained from patients who attended the study clinic and had acute lower respiratory tract (LRT) illness. Specimens were tested for influenza virus by real-time reverse-transcription polymerase chain reaction. We adjusted the incidence of influenza-associated acute LRT illness to account for patients with acute LRT illness who attended the clinic but were not sampled. RESULTS: From March 2007 through February 2010, 4140 cases of acute LRT illness were evaluated in Kibera, and specimens were collected from 1197 (27%); 319 (27%) were positive for influenza virus. In Lwak, there were 6733 cases of acute LRT illness, and specimens were collected from 1641 (24%); 359 (22%) were positive for influenza virus. The crude and adjusted rates of medically attended influenza-associated acute LRT illness were 6.9 and 13.6 cases per 1000 person-years, respectively, in Kibera, and 5.6 and 23.0 cases per 1000 person-years, respectively, in Lwak. In both sites, rates of influenza-associated acute LRT illness were highest among children <2 years old and lowest among adults ≥50 years old. CONCLUSION: In Kenya, the incidence of influenza-associated acute LRT illness was high in both rural and urban settings, particularly among the most vulnerable age groups.

Tuberculosis risk among staff of a large public hospital in Kenya.

SETTING: In sub-Saharan Africa, high rates of tuberculosis (TB) and human immunodeficiency virus (HIV) infection pose a serious threat for occupationally acquired TB among health care workers. OBJECTIVE: To identify factors associated with TB disease among staff of an 1800-bed hospital in Kenya. DESIGN: We calculated TB incidence among staff and conducted a case-control study where cases (n = 65) were staff diagnosed with TB and controls (n = 316) were randomly selected staff without recent TB. RESULTS: The annual incidence of TB from 2001 to 2005 ranged from 645 to 1115 per 100000 population. Factors associated with TB disease were additional daily hours spent in rooms with patients (adjusted odds ratio [aOR] 1.3, 95%CI 1.2-1.5), working in areas where TB patients received care (aOR 2.1, 95%CI 1.1-4.2), HIV infection (aOR 29.1, 95%CI 5.1-167) and living in a slum (aOR 4.7, 95%CI 1.8-12.5) or hospital-provided low-income housing (aOR 2.6, 95%CI 1.2-5.6). CONCLUSION: Hospital exposures were associated with TB disease among staff at this hospital regardless of their job designation, even after controlling for living conditions, suggesting transmission from patients. Health care facilities should improve infection control practices, provide quality occupational health services and encourage staff testing for HIV infection to address the TB burden in hospital staff.

Evidence of avian pneumovirus spread beyond Minnesota among wild and domestic birds in central North America.

To detect avian pneumovirus (APV) in central North America, nasal turbinates or choanal deft tissues from domestic turkeys and wild birds were examined for the presence of APV RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas serum samples from domestic turkeys were analyzed for APV antibodies by enzyme-linked immunosorbent assay (ELISA). In 2002, the seroprevalence of disease in domestic turkeys in Minnesota remained high (42.3% of the flocks). In addition, there is evidence the disease has spread to turkey flocks in North Dakota (8.2%), South Dakota (7%), Iowa (10%), and Wisconsin (8.6%) as detected by RT-PCR and/or ELISA. House sparrows and ring-billed gulls sampled in Minnesota and snow geese from Saskatchewan, Canada, were found to harbor APV RNA. Sequence analysis of wild bird APV strains showed high amino acid sequence identity among wild bird isolates (<97%) and between wild bird and turkey viral isolates (93.2%-99.3%). This study demonstrated that APV infections were present in domestic turkey flocks and wild birds outside the state of Minnesota; however, the role of wild birds in spreading APV to domestic turkeys remains unclear.

Detection of avian pneumovirus in wild Canada (Branta canadensis) and blue-winged teal (Anas discors) geese.

Choanal cleft swab samples from 770 wild Canada geese (Branta canadensis) and 358 blue-winged teal (Anas discors), captured for relocation or banding, were examined for the presence of avian pneumovirus (APV) RNA by reverse transcription (RT)-polymerase chain reaction (PCR) and for virus isolation. The swab samples were pooled into groups of 5 or 10. Sixty eight of 102 (66.7%) pooled goose samples were RT-PCR positive for APV RNA. Thirteen of 52 (25.0%) pooled blue-winged teal samples were RT-PCR positive for APV RNA. APV RNA-positive samples were inoculated onto chick embryo fibroblasts (CEF) and QT-35 cells. Infectious APV was isolated from five Canada goose pooled samples in CEF and from one Canada goose pool in QT-35 cells but not from blue-winged teal.

Pathogenic and immunosuppressive effects of avian pneumovirus in turkeys.

Avian pneumovirus (APV) causes a respiratory disease in turkeys. The virus has been associated with morbidity and mortality due to secondary infections. Our objective was to determine if APV caused immunosuppression in the T-cell or B-cell compartments and to study the pathogenesis of the disease in APV maternal antibody-lacking 2-wk-old commercial turkeys. APV was administered by the eyedrop/intranasal route. Observations were made for gross lesions, viral genome, and T-cell mitogenesis and cytokine secretion at 3, 5, 7, 14, and 21 days postinoculation (DPI). During the acute phase of the disease that lasted for about 1 wk, the turkeys exposed to APV showed clinical signs characterized by nasal discharge and sinus swelling. Virus genome was detected by in situ hybridization in cells of turbinates and trachea at 3 and 5 DPI. At 3 and 5 DPI, spleen cells of the birds infected with APV markedly decreased proliferative response to concanavalin A (Con A). Con A and lipopolysaccharide stimulation of spleen cells from virus-exposed turkeys resulted in accumulation of nitric oxide-inducing factors (NOIF) in the culture fluid. NOIF were not detected in culture fluids of Con A-stimulated spleen cells of virus-free turkeys. APV did not compromise the antibody-producing ability of turkeys against several extraneous antigens such as Brucella abortus and tetanus toxoid.

Neonatal avian pneumovirus infection in commercial turkeys.

Eleven market turkey flocks developed a respiratory disease characterized by coughing, swollen sinuses and nasal discharge. These symptoms first appeared between 3 and 16 days of age. Avian pneumovirus (APV) RNA was detected by reverse transcriptase (RT)-polymerase chain reaction (PCR) in six of six flocks tested. APV was detected by immunohistochemistry in turbinates of three of three affected flocks tested. Virus isolation attempts were negative. Ten of 11 flocks became seropositive on the APV enzyme-linked immunosorbent assay. Five weeks prior to hatch of these affected market turkeys, several breeder flocks in one geographic area had developed clinical signs and experienced decline in egg production typical of APV infection. In two breeder flocks, acute and convalescent sera indicated APV infection during the period of declining egg production. Attempts to detect APV RNA by RT-PCR from choanal cleft swabs of newly hatched poults were successful. Attempts to isolate the virus from these PCR-positive samples were negative.

Role of intrabursal T cells in infectious bursal disease virus (IBDV) infection: T cells promote viral clearance but delay follicular recovery.

Infectious bursal disease virus (IBDV) induces an acute, highly contagious immunosuppressive disease in young chickens. We examined the role of T cells in IBDV-induced immunopathogenesis and tissue recovery. T cell-intact chickens and birds compromised in their T cell function by a combination of surgical thymectomy and Cyclosporin A treatment (Tx-CsA) were infected with an intermediate vaccine strain of IBDV (Bursine 2, Fort Dodge). Our data revealed that functional T cells were needed to control the IBDV-antigen load in the acute phase of infection at 5 days post infection. The target organ of IBDV, the bursa of Fabricius, of Tx-CsA-birds had a significantly higher antigen load than the one of T cell-intact birds (P < 0.05). Tx-CsA-treatment abrogated the IBDV-induced inflammatory response and significantly (P < 0.05) reduced the incidence of apoptotic bursa cells and the expression of cytokines such as interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) in comparison to T cell-intact birds. T cell-released IL-2 and IFN-gamma may have mediated the induction of inflammation and cell death in T cell-intact birds. The IBDV-induced upregulation of tumor necrosis like-factor (TNF) expression was comparable between T cell-intact and Tx-CsA-birds. Tx-CsA-birds showed a significantly faster resolution of IBDV-induced bursa lesions than T cell-intact birds (P < 0.05). This study suggests that T cells modulate IBDV pathogenesis in two ways: a) they limit viral replication in the bursa in the early phase of the disease at 5 days post infection, and b) intrabursal T cells promote bursal tissue damage and delay tissue recovery possibly through the release of cytokines and cytotoxic effects.

Porcine encephalomyocarditis virus persists in pig myocardium and infects human myocardial cells.

Recent advances toward using pig tissues in human transplantation have made it necessary to determine the risk of transmitting zoonotic viruses from pigs to humans or vice versa. We investigated the suitability of the porcine encephalomyocarditis virus (EMCV) model for such studies by determining its ability to persist in pigs, escape detection by routine serological methods, and infect human cells. Intraperitoneal inoculation of 5-week-old pigs with EMCV-30, a strain isolated from commercial pigs, resulted in acute cellular degeneration, infiltration of lymphocytes, and apoptosis in myocardium in 13 of 15 (86.7%) pigs during the acute phase of disease (3 to 21 days postinfection), followed by less-severe lymphocytic infiltration and apoptosis in 5 of 10 (50%) pigs during the chronic phase of the disease (day 45 to 90 postinfection). In the brain, lymphocytic infiltration, neuronal degeneration, and gliosis were observed in 26 to 33% of pigs in the acute phase of disease whereas perivascular cuffing was the predominant feature during chronic disease. EMCV antigens and RNA were demonstrated in the myocardium and brain during the chronic phase of disease. Analysis of 100 commercial pigs that were negative for EMCV antibodies identified two pig hearts positive for EMCV RNA. Porcine EMCV productively infected primary human cardiomyocytes as demonstrated by immunostaining using a monoclonal antibody specific for EMCV RNA polymerase, which is expressed only in productively infected cells, and by a one-step growth curve that showed production of 100 to 1,000 PFU of virus per cell within 6 h. The findings that porcine EMCV can persist in pig myocardium and can infect human myocardial cells make it an important infectious agent to screen for in pig-to-human cardiac transplants and a good model for xenozoonosis.

Theiler's virus-infected L-selectin-deficient mice have decreased infiltration of CD8(+) T lymphocytes in central nervous system but clear the virus.

Mice with targeted deletion of L-selectin gene (L-sel(-/-)) were used to investigate the role of adhesion molecule in immunologic responses following virus infection in the central nervous system (CNS). L-Sel(-/-) mice from a resistant H-2(b) genetic background and parental wild-type H-2(b) (C57BL/6) mice were infected with Theiler's murine encephalomyelitis virus (TMEV) intracerebrally and the kinetics of virus replication and infiltration of immune cells in the CNS determined. The levels of infectious TMEV, as measured by plaque assay at 3, 7, 14, and 28 days after infection were between 4 and 6 log(10) PFU of virus per gram of CNS tissues at days 3 and 7 post-infection, and then decreased to undetectable levels by day 14 after infection in both strains of mice. The L-sel(-/-) mice had decreased numbers of CD8(+) T lymphocytes (17.72%+/-2.4) infiltrating into the CNS at 7 days post-infection when compared to wild-type mice (31.02%+/-7.5). In addition, the L-sel(-/-) mice had significantly lower levels of TMEV-specific serum IgG resulting in lower virus neutralizing activity of the serum when compared to wild-type mice. However, the L-sel(-/-) mice had 2.5-fold increase in B lymphocytes in the CNS (8.29%+/-1.1) when compared to wild-type mice (3.2%+/-0.4). Taken together, these data indicate that L-selectin plays a role in recruitment of B and CD8(+) T lymphocytes into the CNS following virus infection, which, however, did not affect the ability of the mice to clear TMEV infection.

Susceptibility of ducks to avian pneumovirus of turkey origin.

OBJECTIVE: To determine the susceptibility of ducks to avian pneumovirus (APV) of turkey origin. ANIMALS: 30 Pekin ducks that were 2 weeks old. PROCEDURE: Ducks were assigned to 3 groups (10 ducks/group). Ducks of groups 1 and 2 were inoculated (day 0) with 200 microl of cell-culture fluid containing APV of turkey origin (10(5.5) median tissue-culture infective dose/ml) by the oculonasal (group 1) or oral (group 2) route. Ducks of group 3 served as noninoculated control birds. Two ducks from each group were euthanatized 3, 6, 9, 15, and 21 days after inoculation. Blood samples, tissue samples from the lungs, trachea, nasal turbinates, duodenum, diverticulum vitellinum (Meckel's diverticulum), and cecum, and swab specimens from the choana, cloaca, and trachea were obtained from all birds during necropsy and examined for APV by use of reverse transcriptase-polymerase chain reaction (RT-PCR), virus isolation, and histologic examination. Blood samples also were examined for APV antibodies, using an ELISA. RESULTS: Tissue samples obtained up to 21 days after inoculation had positive results when tested by use of RT-PCR. Virus was isolated from nasal turbinates of birds inoculated via the oculonasal route. Serum samples obtained 15 and 21 days after inoculation had positive results when tested for APV-specific antibody. Clinical signs of disease were not observed in ducks inoculated with APV of turkey origin. CONCLUSIONS AND CLINICAL RELEVANCE: Ducks inoculated with APV of turkey origin may not develop clinical signs of disease, but they are suspected to play a role as nonclinical carriers of APV.

Antigens to viral capsid and non-capsid proteins are present in brain tissues and antibodies in sera of Theiler's virus-infected mice.

Recombinant proteins to the LP, VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, and 3D genes of Theiler's murine encephalomyelitis virus (TMEV) were generated and antibodies were produced against them for use in analysis of the TMEV epitopes responsible for eliciting the antibody responses observed during acute and chronic disease. Antibodies against recombinant VP1, VP2, and VP3 recognized the corresponding proteins from purified TMEV particles. In immunohistochemical analysis, antibodies against recombinant capsid (VP1, VP2, and VP3), and non-capsid (2A, 2C, 3A) proteins were reactive with PO-2D cells (astrocytes) infected with TMEV in vitro and with brain tissues of acutely infected mice. Antibodies against VP4, 2B, and 3D antigens were not reactive with corresponding viral proteins in infected astrocytes cells or brain tissues, but they reacted with TMEV precursor proteins produced during the early viral replication phase. Sera from SJL/J mice infected with TMEV acutely (14 days) and chronically (45 days) reacted with VP1, VP2, VP4, 2A, and 2C proteins. In an in vitro assay for neutralization, only anti-VP1 antibodies neutralized TMEV infection. These findings suggest that both capsid and non-capsid proteins of TMEV play a role in the immunopathology of the TMEV disease in the central nervous system.

Development of a highly sensitive and specific enzyme-linked immunosorbent assay based on recombinant matrix protein for detection of avian pneumovirus antibodies.

The matrix (M) protein of avian pneumovirus (APV) was evaluated for its antigenicity and reliability in an enzyme-linked immunosorbent assay (ELISA) for diagnosis of APV infection, a newly emergent disease of turkeys in United States. Sera from APV-infected turkeys consistently contained antibodies to a 30-kDa protein (M protein). An ELISA based on recombinant M protein generated in Escherichia coli was compared with the routine APV ELISA that utilizes inactivated virus as antigen. Of 34 experimentally infected turkeys, 33 (97.1%) were positive by M protein ELISA whereas only 18 (52.9%) were positive by routine APV ELISA 28 days after infection. None of the serum samples from 41 uninfected experimental turkeys were positive by M protein ELISA. Of 184 field sera from turkey flocks suspected of having APV infection, 133 (72.3%) were positive by M protein ELISA whereas only 99 (53.8%) were positive by routine APV ELISA. Twelve serum samples, which were negative by M protein ELISA but positive by routine APV ELISA, were not reactive with either recombinant M protein or denatured purified APV proteins by Western analysis. This indicates that the samples had given false-positive results by routine APV ELISA. The M protein ELISA was over six times more sensitive than virus isolation (11.5%) in detecting infections from samples obtained from birds showing clinical signs of APV infection. Taken together, these results show that ELISA based on recombinant M protein is a highly sensitive and specific test for detecting antibodies to APV.

Avian pneumovirus (APV) RNA from wild and sentinel birds in the United States has genetic homology with RNA from APV isolates from domestic turkeys.

Nasal turbinates or swabs were collected from wild ducks, geese, owls, sparrows, swallows, and starlings and from sentinel ducks placed next to turkey farms experiencing avian pneumovirus (APV) infections and were analyzed for APV genome and infectious particles. APV RNA was detected in samples examined from geese, sparrows, and starlings. APV RNA and antibodies were also detected in two different groups of sentinel ducks. Infectious APV was recovered from sentinel duck samples. The APV M gene isolated from the wild birds had over 96% predicted amino acid identity with APV/Minnesota 2A, which was isolated earlier from domestic turkeys showing respiratory illness, suggesting that wild birds may be involved in spreading APV infection.

Short-term treatment with interferon-alpha/beta promotes remyelination, whereas long-term treatment aggravates demyelination in a murine model of multiple sclerosis.

The mechanisms by which type I interferons (IFN) reduce the rate and severity of exacerbations in multiple sclerosis are unknown. We utilized a model of multiple sclerosis to determine the extent of demyelination and remyelination in Theiler's murine encephalomyelitis virus (TMEV)-infected SJL/J mice treated with mouse IFN-alpha/beta for a short (5 weeks) or a long (16 weeks) period. All mice were chronically infected with TMEV to simulate the clinical situation in multiple sclerosis. Short-term IFN-alpha/beta treatment increased the percent of remyelinated spinal cord white matter by threefold when compared with phosphate-buffered saline (PBS) treatment (P < 0.02), but it did not affect the extent of demyelination. In contrast, long-term IFN-alpha/beta treatment increased the extent of demyelination by twofold (P < 0.03). Long-term treatment increased the absolute area of remyelination, but the percent remyelination as a function of area of demyelination was not changed because of increased demyelination. An immunomodulatory mechanism may have contributed to the effect of IFN-alpha/beta on white matter pathology because treated mice had higher anti-TMEV IgGs in serum and demonstrated decreased numbers of B and T lymphocytes infiltrating the central nervous system (CNS). There was no correlation between the level of anti- IFN-alpha/beta antibodies and the extent of demyelination or remyelination. These results indicate that the length of type I IFN treatment may have paradoxical effects on demyelination and remyelination.

Prevalent class I-restricted T-cell response to the Theiler's virus epitope Db:VP2121-130 in the absence of endogenous CD4 help, tumor necrosis factor alpha, gamma interferon, perforin, or costimulation through CD28.

C57BL/6 mice mount a cytotoxic T-lymphocyte (CTL) response against the Daniel's strain of Theiler's murine encephalomyelitis virus (TMEV) 7 days after infection and do not develop persistent infection or the demyelinating syndrome similar to multiple sclerosis seen in susceptible mice. The TMEV capsid peptide VP2121-130 sensitizes H-2Db+ target cells for killing by central-nervous-system-infiltrating lymphocytes (CNS-ILs) isolated from C57BL/6 mice infected intracranially. Db:VP2121-130 peptide tetramers were used to stain CD8(+) CNS-ILs, revealing that 50 to 63% of these cells bear receptors specific for VP2121-130 presented in the context of Db. No T cells bearing this specificity were found in the cervical lymph nodes or spleens of TMEV-infected mice. H-2(b) mice lacking CD4, class II, gamma interferon, or CD28 expression are susceptible to persistent virus infection but surprisingly still generate high frequencies of CD8(+), Db:VP2121-130-specific T cells. However, CD4-negative mice generate a lower frequency of Db:VP2121-130-specific T cells than do class II negative or normal H-2(b) animals. Resistant tumor necrosis factor alpha receptor I knockout mice also generate a high frequency of CD8(+) CNS-ILs specific for Db:VP2121-130. Furthermore, normally susceptible FVB mice that express a Db transgene generate Db:VP2121-130-specific CD8(+) CNS-ILs at a frequency similar to that of C57BL/6 mice. These results demonstrate that VP2121-130 presented in the context of Db is an immunodominant epitope in TMEV infection and that the frequency of the VP2121-130-specific CTLs appears to be independent of several key inflammatory mediators and genetic background but is regulated in part by the expression of CD4.

Absence of spontaneous central nervous system remyelination in class II-deficient mice infected with Theiler's virus.

We previously showed that Theiler's murine encephalomyelitis virus (TMEV)-infected major histocompatibility complex (MHC) class II-deficient mice develop both demyelination and neurologic deficits, whereas MHC class I-deficient mice develop demyelination but no neurologic deficits. The absence of neurologic deficits in the class I-deficient mice was associated with preserved sodium channel densities in demyelinated lesions, a relative preservation of axons, and extensive spontaneous remyelination. In this study, we investigated whether TMEV-infected class II-deficient mice, which have an identical genetic background (C57BL/6 x 129) as the class I-deficient mice, have preserved axons and spontaneous myelin repair following chronic TMEV-infection. Both class I- and class II-deficient mice showed similar extents of demyelination of the spinal cord white matter 4 months after TMEV infection. However, the class I-deficient mice demonstrated remyelination by oligodendrocytes, whereas class II-deficient mice showed minimal if any myelin repair. Demyelinated lesions, characterized by inflammatory infiltrates in both mutants, revealed disruption of axons in class II- but not class I-deficient mice. Further characterization revealed that even though class II-deficient mice lacked TMEV-specific IgG, they had virus-specific IgM, which, however, did not neutralize TMEV in vitro. In addition, class II-deficient mice developed TMEV-specific cytotoxic T-lymphocytes in the CNS during the acute (7 days) disease, but these cytotoxic lymphocytes were not present in the chronic stage of disease, despite a high titer of infectious virus throughout the disease. We envision that the presence of demyelination, high virus titer, absence of remyelination, and axonal disruption in chronically infected class II-deficient mice contributes to the development of paralytic disease.

Perforin-dependent neurologic injury in a viral model of multiple sclerosis.

In this study we demonstrate perforin-mediated cytotoxic effector function is necessary for viral clearance and may directly contribute to the development of neurologic deficits after demyelination in the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. We previously demonstrated major histocompatability complex (MHC) class I-deficient (beta2m-deficient) mice with an otherwise resistant genotype develop severe demyelination with minimal neurologic disease when chronically infected with TMEV. These studies implicate CD8(+) T cells as the pathogenic cell in the induction of neurologic disease after demyelination. To determine which effector mechanisms of CD8(+) T cells, granule exocytosis or Fas ligand expression, play a role in the development of demyelination and clinical disease, we infected perforin-deficient, lpr (Fas mutation), and gld (Fas ligand mutation) mice with TMEV. Perforin-deficient mice showed viral persistence in the CNS, chronic brain pathology, and demyelination in the spinal cord white matter. Perforin-deficient mice demonstrated severely impaired MHC class I-restricted cytotoxicity against viral epitopes, but normal MHC class II-restricted delayed-type hypersensitivity responses to virus antigen. Despite demyelination, virus-infected perforin-deficient mice showed only minimal neurologic deficits as indicated by clinical disease score, activity monitoring, and footprint analysis. Perforin- and MHC class II-deficient mice (with functional CD8(+) T cells and perforin molecules and an H-2(b) haplotype) had comparable demyelination and genotype, however, only the latter showed severe clinical disease. Gld and lpr mice demonstrated normal TMEV-specific cytotoxicity and maintained resistance to TMEV-induced demyelinating disease. These studies implicate perforin release by CD8(+) T cells as a potential mechanism by which neurologic deficits are induced after demyelination.

The immune system preferentially clears Theiler's virus from the gray matter of the central nervous system.

Infection of susceptible strains of mice with Daniel's (DA) strains of Theiler's murine encephalomyelitis virus (DAV) results in virus persistence in the central nervous system (CNS) white matter and chronic demyelination similar to that observed in multiple sclerosis. We investigated whether persistence is due to the immune system more efficiently clearing DAV from gray than from white matter of the CNS. Severe combined immunodeficient (SCID) and immunocompetent C.B-17 mice were infected with DAV to determine the kinetics, temporal distribution, and tropism of the virus in CNS. In early disease (6 h to 7 days postinfection), DAV replicated with similar kinetics in the brains and spinal cords of SCID and immunocompetent mice and in gray and white matter. DAV RNA was localized within 48 h in CNS cells of all phenotypes, including neurons, oligodendrocytes, astrocytes, and macrophages/microglia. In late disease (13 to 17 days postinfection), SCID mice became moribund and permitted higher DAV replication in both gray and white matter. In contrast, immunocompetent mice cleared virus from the gray matter but showed replication in the white matter of their brains and spinal cords. Reconstitution of SCID mice with nonimmune splenocytes or anti-DAV antibodies after establishment of infection demonstrated that both cellular and humoral immune responses decreased virus from the gray matter; however, the cellular responses were more effective. SCID mice reconstituted with splenocytes depleted of CD4+ or CD8+ T lymphocytes cleared virus from the gray matter but allowed replication in the white matter. These studies demonstrate that both neurons and glia are infected early following DAV infection but that virus persistence in the white matter is due to preferential clearance of virus from the gray matter by the immune system.

Interferon alpha/beta mediates early virus-induced expression of H-2D and H-2K in the central nervous system.

Cells of the central nervous system (CNS) normally do not express detectable levels of major histocompatibility complex (MHC) Class I antigens. However, MHC Class I expression can be induced after virus infection. We tested the hypothesis that virus-induced Class I expression is mediated by lymphocytes or cytokines using lymphocyte- and cytokine-deficient mice. We used Theiler's murine encephalomyelitis virus (TMEV), which induces CNS demyelination that maps genetically to the D region of MHC Class I and is associated with high levels of Class I products. TMEV infection of severe combined immunodeficiency (SCID) and recombination activation gene-1-deficient mice, which lack B and T lymphocytes, resulted in equivalent H-2D and H-2K expression in brain and spinal cord, according to analysis of the area and intensity of immunoperoxidase staining. Class I antigens were demonstrated as early as 6 hours after infection, and they were more widely distributed than viral RNA, indicating that expression was induced indirectly via a soluble factor. To determine whether cytokines induced the expression, we infected mice lacking receptors for interferon-alpha/beta (IFN-alpha/beta R (-/-)), interferon-gamma (IFN-gamma R(-/-)), and tumor necrosis factor-alpha (TNFRp55(-/-)). TMEV-infected IFN-gamma R(-/-) and TN-FRp55(-/-) mice expressed Class I antigens in the CNS, whereas IFN-alpha/beta R(-/-) mice did not, establishing that IFN-alpha/beta mediated the expression. In contrast to the equivalent expression in SCID mice, we observed greater area and higher intensity of H-2D versus H-2K antigens in infected SCID mice reconstituted with normal spleen cells. Collectively, the data indicate that after TMEV infection, early generalized MHC Class I expression is mediated by IFN-alpha/beta independently of lymphocytes, but the differential regulation of H-2D over H-2K may be controlled by B and/or T lymphocytes.