Publications

Central nervous system (CNS) infection of C57BL/6 mice with the TE strain of Sindbis virus (SINV) provides a valuable animal model for studying the pathogenesis of alphavirus encephalomyelitis. While SINV TE inoculated intracranially causes little mortality, 20-30 % of mice inoculated intranasally (IN) died 8 to 11 days after infection, the period during which immune cells typically infiltrate the brain and clear infectious virus. To examine the mechanism behind the mortality, mice infected IN with SINV TE were monitored for evidence of neurological disease, and those with signs of severe disease (moribund) were sacrificed and tissues collected. Mice showing the usual mild signs of encephalomyelitis were concurrently sacrificed to serve as time-matched controls (sick). Sixty-eight per cent of the moribund mice, but none of the sick mice, showed upper gastrointestinal bleeding due to gastric ulceration. Clinical disease and gastrointestinal pathology could not be attributed to direct viral infection of tissues outside of the CNS, and brain pathology and inflammation were comparable in sick and moribund mice. However, more SINV antigen was present in the brains of moribund mice, and clearance of infectious virus from the CNS was delayed compared to sick mice. Lower levels of SINV-specific IgM and fewer B220+ B cells were present in the brains of moribund mice compared to sick mice, despite similar levels of antiviral IgM and IgG in serum. These findings highlight the importance of the local antibody response in determining the outcome of viral encephalomyelitis and offer a model system for understanding individual variation in this response.

Sindbis virus (SINV) infection of neurons in the brain and spinal cord in mice provides a model system for investigating recovery from encephalomyelitis and antibody-mediated clearance of virus from the central nervous system (CNS). To determine the roles of IgM and IgG in recovery, we compared the responses of immunoglobulin-deficient activation-induced adenosine deaminase-deficient (AID-/-), secretory IgM-deficient (sIgM-/-), and AID-/- sIgM-/- double-knockout (DKO) mice with those of wild-type (WT) C57BL/6 mice for disease, clearance of infectious virus and viral RNA from brain and spinal cord, antibody responses, and B cell infiltration into the CNS. Because AID is essential for immunoglobulin class switch recombination and somatic hypermutation, AID-/- mice produce only germ line IgM, while sIgM-/- mice secrete IgG but no IgM and DKO mice produce no secreted immunoglobulin. After intracerebral infection with the TE strain of SINV, most mice recovered. Development of neurologic disease occurred slightly later in sIgM-/- mice, but disease severity, weight loss, and survival were similar between the groups. AID-/- mice produced high levels of SINV-specific IgM, while sIgM-/- mice produced no IgM and high levels of IgG2a compared to WT mice. All mice cleared infectious virus from the spinal cord, but DKO mice failed to clear infectious virus from brain and had higher levels of viral RNA in the CNS late after infection. The numbers of infected cells and the amount of cell death in brain were comparable. We conclude that antibody is required and that either germ line IgM or IgG is sufficient for clearance of virus from the CNS.IMPORTANCE Mosquito-borne alphaviruses that infect neurons can cause fatal encephalomyelitis. Recovery requires a mechanism for the immune system to clear virus from infected neurons without harming the infected cells. Antiviral antibody has previously been shown to be a noncytolytic means for alphavirus clearance. Antibody-secreting cells enter the nervous system after infection and produce antiviral IgM before IgG. Clinical studies of human viral encephalomyelitis suggest that prompt production of IgM is associated with recovery, but it was not known whether IgM is effective for clearance. Our studies used mice deficient in production of IgM, IgG, or both to characterize the antibody necessary for alphavirus clearance. All mice developed similar signs of neurologic disease and recovered from infection. Antibody was necessary for virus clearance from the brain, and either early germ line IgM or IgG was sufficient. These studies support the clinical observation that prompt production of antiviral antibody is a determinant of outcome.

Alphaviruses, members of the positive-sense, single-stranded RNA virus family Togaviridae, represent a re-emerging public health concern worldwide as mosquito vectors expand into new geographic ranges. Members of the alphavirus genus tend to induce clinical disease characterized by rash, arthralgia, and arthritis (chikungunya virus, Ross River virus, and Semliki Forest virus) or encephalomyelitis (eastern equine encephalitis virus, western equine encephalitis virus, and Venezuelan equine encephalitis virus), though some patients who recover from the initial acute illness may develop long-term sequelae, regardless of the specific infecting virus. Studies examining the natural disease course in humans and experimental infection in cell culture and animal models reveal that host genetics play a major role in influencing susceptibility to infection and severity of clinical disease. Genome-wide genetic screens, including loss of function screens, microarrays, RNA-sequencing, and candidate gene studies, have further elucidated the role host genetics play in the response to virus infection, with the immune response being found in particular to majorly influence the outcome. This review describes the current knowledge of the mechanisms by which host genetic factors influence alphavirus pathogenesis and discusses emerging technologies that are poised to increase our understanding of the complex interplay between viral and host genetics on disease susceptibility and clinical outcome.

Chikungunya virus (CHIKV), an Old World alphavirus, is transmitted to humans by infected mosquitoes and causes acute rash and arthritis, occasionally complicated by neurologic disease and chronic arthritis. One determinant of alphavirus virulence is nonstructural protein 3 (nsP3) that contains a highly conserved MacroD-type macrodomain at the N terminus, but the roles of nsP3 and the macrodomain in virulence have not been defined. Macrodomain is a conserved protein fold found in several plus-strand RNA viruses that binds to the small molecule ADP-ribose. Prototype MacroD-type macrodomains also hydrolyze derivative linkages on the distal ribose ring. Here, we demonstrated that the CHIKV nsP3 macrodomain is able to hydrolyze ADP-ribose groups from mono(ADP-ribosyl)ated proteins. Using mass spectrometry, we unambiguously defined its substrate specificity as mono(ADP-ribosyl)ated aspartate and glutamate but not lysine residues. Mutant viruses lacking hydrolase activity were unable to replicate in mammalian BHK-21 cells or mosquito Aedes albopictus cells and rapidly reverted catalytically inactivating mutations. Mutants with reduced enzymatic activity had slower replication in mammalian neuronal cells and reduced virulence in 2-day-old mice. Therefore, nsP3 mono(ADP-ribosyl)hydrolase activity is critical for CHIKV replication in both vertebrate hosts and insect vectors, and for virulence in mice.

Infection of weanling C57BL/6 mice with the TE strain of Sindbis virus (SINV) causes nonfatal encephalomyelitis associated with hippocampal-based memory impairment that is partially prevented by treatment with 6-diazo-5-oxo-l-norleucine (DON), a glutamine antagonist (Potter et al., J Neurovirol 21:159, 2015). To determine the mechanism(s) of protection, lymph node and central nervous system (CNS) tissues from SINV-infected mice treated daily for 1 week with low (0.3mg/kg) or high (0.6mg/kg) dose DON were examined. DON treatment suppressed lymphocyte proliferation in cervical lymph nodes resulting in reduced CNS immune cell infiltration, inflammation, and cell death compared to untreated SINV-infected mice. Production of SINV-specific antibody and interferon-gamma were also impaired by DON treatment with a delay in virus clearance. Cessation of treatment allowed activation of the antiviral immune response and viral clearance, but revived CNS pathology, demonstrating the ability of the immune response to mediate both CNS damage and virus clearance.

Zika virus (ZIKV) crosses the placenta and causes congenital disease. Here we develop an animal model utilizing direct ZIKV inoculation into the uterine wall of pregnant, immunocompetent mice to evaluate transplacental transmission. Intrauterine inoculation at embryonic day (E) 10, but not E14, with African, Asian or American strains of ZIKV reduces fetal viability and increases infection of placental and fetal tissues. ZIKV inoculation at E10 causes placental inflammation, placental dysfunction and reduces neonatal brain cortical thickness, which is associated with increased activation of microglia. Viral antigen localizes in trophoblast and endothelial cells in the placenta, and endothelial, microglial and neural progenitor cells in the fetal brain. ZIKV infection of the placenta increases production of IFNβ and expression of IFN-stimulated genes 48 h after infection. This mouse model provides a platform for identifying factors at the maternal-fetal interface that contribute to adverse perinatal outcomes in a host with an intact immune system.

Measles is an acute viral disease associated both with immune suppression and development of life-long immunity. Clearance of measles virus (MeV) involves rapid elimination of infectious virus during the rash followed by slow elimination of viral RNA. To characterize cellular immune responses during recovery, we analyzed the appearance, specificity and function of MeV-specific T cells for 6 months after respiratory infection of rhesus macaques with wild type MeV. IFN-γ and IL-17-producing cells specific for the hemagglutinin and nucleocapsid proteins appeared in circulation in multiple waves approximately 2-3, 8 and 18-24 weeks after infection. IFN-γ-secreting cells were most abundant early and IL-17-secreting cells late. Both CD4+ and CD8+ T cells were sources of IFN-γ and IL-17, and IL-17-producing cells expressed RORγt. Therefore, the cellular immune response evolves during MeV clearance to produce functionally distinct subsets of MeV-specific CD4+ and CD8+ T cells at different times after infection.

Aspiculuris tetraptera continues to be a problem in rodent vivaria, in part due to difficulties in parasite detection. Although PCR testing is highly sensitive, it is expensive and does not always provide immediate results. Consequently, many institutions rely on passive fecal flotation as a quick inhouse exam for diagnosing A. tetraptera infections. To increase the sensitivity of this test, we examined multiple parameters to determine the optimal test protocol. A 30-min soaking period prior to fecal flotation for 15 min allowed fecal pellets to soften and facilitated efficient egg isolation. We also evaluated the effect of time of day, sample size, age, sex, and housing status on egg isolation. No evidence of cyclical egg shedding was found, and although larger fecal sample sizes did not result in more eggs isolated, their use reduced the incidence of false-negative exams. The most eggs were isolated from 8- and 12-wk-old mice, and as mice aged, the number of eggs isolated declined. Overall, neither sex nor housing status influenced the number of eggs isolated. Finally, examination of multiple diagnostic tests (fecal flotation exam, direct examination of cecal and colonic contents, and fecal PCR) revealed that no single test was definitive, thus indicating that multiple tests might be required to successfully screen mice with low pinworm burdens. These findings provide guidance regarding sample selection, collection, and processing to efficiently detect A. tetraptera.

Immunocompetent weanling mice infested with Myocoptes musculinus harbor high mite loads, yet burdens decrease with age. The development of immunity to the parasite may explain this observation. In this study, we followed M. musculinus burdens in Rag1(-/-) mice and immunocompetent C57BL/6 controls from 4 to 36 wk of age and compared the clinical signs and body weights of noninfested and infested mice of both strains over time. In addition, histopathology of skin lesions and expression of cytokines and transcription factors associated with Th1- and Th2-type immune responses were assessed. Myocoptes burdens decreased and remained low in B6 mice over time, whereas Rag1(-/-) mice showed an initial decrease in burdens after 4 wk of age followed by an increase from 24 to 36 wk. In addition, Rag1(-/-) mice had higher burdens than B6 mice over time. Both strains of infested mice exhibited clinical signs of fur mite infestation-including alopecia, poor weight gain, mite-associated debris, and pruritus-and clinical signs positively correlated with the severity of the Myocoptes burden. Histopathology of skin from both strains of infested mice showed decreased lesion severity with age, likely a result of declining mite populations. Finally, compared with noninfested controls, infested B6 mice had increased expression of markers associated with the Th2-type immune response, which increased in magnitude with increasing age and duration of infestation. These results suggest that development of adaptive immunity plays a role in control of fur mite populations and that heavier infestations may result in more severe clinical signs and skin lesions.

UNLABELLED: Inflammation is a necessary part of the response to infection but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system (CNS). A neurovirulent strain of Sindbis virus (NSV) causes fatal paralysis in adult C57BL/6 mice during clearance of infectious virus from the CNS, and the virus-specific immune response is implicated as a mediator of neuronal damage. Previous studies have shown that survival is improved in T-cell-deficient mice and in mice with pharmacological inhibition of the inflammatory response and glutamate excitotoxicity. Because glutamine metabolism is important in the CNS for the generation of glutamate and in the immune system for lymphocyte proliferation, we tested the effect of the glutamine antagonist DON (6-diazo-5-oxo-l-norleucine) on the outcome of NSV infection in mice. DON treatment for 7 days from the time of infection delayed the onset of paralysis and death. Protection was associated with reduced lymphocyte proliferation in the draining cervical lymph nodes, decreased leukocyte infiltration into the CNS, lower levels of inflammatory cytokines, and delayed viral clearance. In vitro studies showed that DON inhibited stimulus-induced proliferation of lymphocytes. When in vivo treatment with DON was stopped, paralytic disease developed along with the inflammatory response and viral clearance. These studies show that fatal NSV-induced encephalomyelitis is immune mediated and that antagonists of glutamine metabolism can modulate the immune response and protect against virus-induced neuroinflammatory disease.

IMPORTANCE: Encephalomyelitis due to infection with mosquito-borne alphaviruses is an important cause of death and of long-term neurological disability in those who survive infection. This study demonstrates the role of the virus-induced immune response in the generation of neurological disease. DON, a glutamine antagonist, inhibited the proliferation of lymphocytes in response to infection, prevented the development of brain inflammation, and protected mice from paralysis and death during treatment. However, because DON inhibited the immune response to infection, clearance of the virus from the brain was also prevented. When treatment was stopped, the immune response was generated, brain inflammation occurred, virus was cleared, and mice developed paralysis and died. Therefore, more definitive treatment for alphaviral encephalomyelitis should inhibit virus replication as well as neuroinflammatory damage.