Autonomic nervous dysfunction in hamsters infected with West Nile virus

Clinical studies and case reports clearly document that West Nile virus (WNV) can cause respiratory and gastrointestinal (GI) complications. Other functions controlled by the autonomic nervous system may also be directly affected by WNV, such as bladder and cardiac functions. To investigate how WNV can cause autonomic dysfunctions, we focused on the cardiac and GI dysfunctions of rodents infected with WNV. Infected hamsters had distension of the stomach and intestines at day 9 after viral challenge. GI motility was detected by a dye retention assay; phenol red dye was retained more in the stomachs of infected hamsters as compared to sham-infected hamsters. The amplitudes of electromygraphs (EMGs) of intestinal muscles were significantly reduced. Myenteric neurons that innervate the intestines, in addition to neurons in the brain stem, were identified to be infected with WNV. These data suggest that infected neurons controlling autonomic function were the cause of GI dysfunction in WNV-infected hamsters. Using radiotelemetry to record electrocardiograms and to measure heart rate variability (HRV), a well-accepted readout for autonomic function, we determined that HRV and autonomic function were suppressed in WNV-infected hamsters. Cardiac histopathology was observed at day 9 only in the right atrium, which was coincident with WNV staining. A subset of WNV infected cells was identified among cells with hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4) as a marker for cells in the sinoatrial (SA) and atrioventricular (AV) nodes. The unique contribution of this study is the discovery that WNV infection of hamsters can lead to autonomic dysfunction as determined by reduced HRV and reduced EMG amplitudes of the GI tract. These data may model autonomic dysfunction of the human West Nile neurological disease.     

Respiratory insufficiency correlated strongly with mortality of rodents infected with West Nile virus

West Nile virus (WNV) disease can be fatal for high-risk patients. Since WNV or its antigens have been identified in multiple anatomical locations of the central nervous system of persons or rodent models, one cannot know where to investigate the actual mechanism of mortality without careful studies in animal models. In this study, depressed respiratory functions measured by plethysmography correlated strongly with mortality. This respiratory distress, as well as reduced oxygen saturation, occurred beginning as early as 4 days before mortality. Affected medullary respiratory control cells may have contributed to the animals' respiratory insufficiency, because WNV antigen staining was present in neurons located in the ventrolateral medulla. Starvation or dehydration would be irrelevant in people, but could cause death in rodents due to lethargy or loss of appetite. Animal experiments were performed to exclude this possibility. Plasma ketones were increased in moribund infected hamsters, but late-stage starvation markers were not apparent. Moreover, daily subcutaneous administration of 5% dextrose in physiological saline solution did not improve survival or other disease signs. Therefore, infected hamsters did not die from starvation or dehydration. No cerebral edema was apparent in WNV- or sham-infected hamsters as determined by comparing wet-to-total weight ratios of brains, or by evaluating blood-brain-barrier permeability using Evans blue dye penetration into brains. Limited vasculitis was present in the right atrium of the heart of infected hamsters, but abnormal electrocardiograms for several days leading up to mortality did not occur. Since respiratory insufficiency was strongly correlated with mortality more than any other pathological parameter, it is the likely cause of death in rodents. These animal data and a poor prognosis for persons with respiratory insufficiency support the hypothesis that neurological lesions affecting respiratory function may be the primary cause of human WNV-induced death.     

Gamma delta T cells facilitate adaptive immunity against West Nile virus infection in mice

West Nile (WN) virus causes fatal meningoencephalitis in laboratory mice, and gammadelta T cells are involved in the protective immune response against viral challenge. We have now examined whether gammadelta T cells contribute to the development of adaptive immune responses that help control WN virus infection. Approximately 15% of TCRdelta(-/-) mice survived primary infection with WN virus compared with 80-85% of the wild-type mice. These mice were more susceptible to secondary challenge with WN virus than the wild-type mice that survived primary challenge with the virus. Depletion of gammadelta T cells in wild-type mice that survived the primary infection, however, does not affect host susceptibility during secondary challenge with WN virus. Furthermore, gammadelta T cells do not influence the development of Ab responses during primary and at the early stages of secondary infection with WN virus. Adoptive transfer of CD8(+) T cells from wild-type mice that survived primary infection with WN virus to naive mice afforded partial protection from lethal infection. In contrast, transfer of CD8(+) T cells from TCRdelta(-/-) mice that survived primary challenge with WN virus failed to alter infection in naive mice. This difference in survival correlated with the numeric and functional reduction of CD8 memory T cells in these mice. These data demonstrate that gammadelta T cells directly link innate and adaptive immunity during WN virus infection.     

CD8+ T cells require perforin to clear West Nile virus from infected neurons

Injury to neurons after West Nile virus (WNV) infection is believed to occur because of viral and host immune-mediated effects. Previously, we demonstrated that CD8+ T cells are required for the resolution of WNV infection in the central nervous system (CNS). CD8+ T cells can control infection by producing antiviral cytokines (e.g., gamma interferon or tumor necrosis factor alpha) or by triggering death of infected cells through perforin- or Fas ligand-dependent pathways. Here, we directly evaluated the role of perforin in controlling infection of a lineage I New York isolate of WNV in mice. A genetic deficiency of perforin molecules resulted in higher viral burden in the CNS and increased mortality after WNV infection. In the few perforin-deficient mice that survived initial challenge, viral persistence was observed in the CNS for several weeks. CD8+ T cells required perforin to control WNV infection as adoptive transfer of WNV-primed wild-type but not perforin-deficient CD8+ T cells greatly reduced infection in the brain and spinal cord and enhanced survival of CD8-deficient mice. Analogous results were obtained when wild-type or perforin-deficient CD8+ T cells were added to congenic primary cortical neuron cultures. Taken together, our data suggest that despite the risk of immunopathogenesis, CD8+ T cells use a perforin-dependent mechanism to clear WNV from infected neurons.     

Alterations in the developmental patterns of enzymes in chicken embryos infected with West Nile virus.

Infection of chicken embryos with West Nile (WN) virus, a group B togavirus containing structural lipids, caused a rapidly developing hypertriglyceridemia. Changes in the activity of several hepatic regulatory enzymes in glycolytic and lipogenic pathways occurred during infection. Compared to control values in embryos of the same age (16 days), an 8.8-fold increase in the specific activity of ATP-citrate lyase and a 5.6-fold increase in that of hexokinase were observed on the third day of WN virus infection. Hexose monophosphate shunt dehydrogenase specific activities were elevated twofold in virus-infected livers. Activities of malic enzyme and phosphofructokinase were also elevated in WN virus-infected livers. Malate dehydrogenase and NADP-linked isocitrate dehydrogenase levels showed little or no change during infection. The levels of pyruvate kinase and lactate dehydrogenase were decreased in virus-infected livers. Hepatic acetyl-CoA carboxylase activity was at least twofold higher in virus-infected embryos; however, following removal of low-molecular-weight compounds, the specific activities of this enzyme from infected and control embryos were virtually identical. The results of mixing experiments suggest that the low levels of carboxylase activity in control embryos may be due to the presence of enzyme inhibitor(s) which can be removed by gel filtration.The incorporation of radiolabeled precursors into cellular lipids by liver minces from virus-infected and uninfected embryos was measured. There was a twofold increase in carbohydrate incorporation in virus-infected liver as compared to uninfected liver; [¹⁴C]pyruvic acid was incorporated into lipids to the greatest extent. [1-¹⁴C]acetic acid, [U-¹⁴C]alanine, and [U-¹⁴C]leucine were incorporated very poorly in both infected and control livers. Twice as much [1-¹⁴C]oleic acid or [1-¹⁴C oleic]triolein was incorporated in WN-infected livers as in control. The relative distribution of neutral and polar lipids formed from each precursor was generally similar in infected and uninfected livers as determined by thin-layer chromatography of radiolabeled lipids. Except for a threefold increase in oxidation of [¹⁴C]glucose by virus-infected livers, the oxidations of carbohydrates and fatty acids were similar in infected and uninfected livers. The pentose phosphate pathway appears to be the major pathway utilized in glucose oxidation for both control and virus-infected livers. The results indicate that enhanced flux of metabolites into lipids reflects a virus-induced alteration in embryonic development: The enzyme patterns of infected embryos are more characteristic of older embryos or even newly hatched chicks.     

Immunization of mice against West Nile virus with recombinant envelope protein.

West Nile (WN) virus is a mosquito-borne flavivirus that emerged in the United States in 1999 and can cause fatal encephalitis. Envelope (E) protein cDNA from a WN virus isolate recovered from Culex pipiens in Connecticut was expressed in Escherichia coli. The recombinant E protein was purified and used as Ag in immunoblot assays and immunization experiments. Patients with WN virus infection had Abs that recognized the recombinant E protein. C3H/HeN mice immunized with E protein developed E protein Abs and were protected from infection with WN virus. Passive administration of E protein antisera was also sufficient to afford immunity. E protein is a candidate vaccine to prevent WN virus infection.     

Mosquito saliva causes enhancement of West Nile virus infection in mice

West Nile virus (WNV) is transmitted to vertebrate hosts primarily by infected Culex mosquitoes. Transmission of arboviruses by the bite of infected mosquitoes can potentiate infection in hosts compared to viral infection by needle inoculation. Here we examined the effect of mosquito transmission on WNV infection and systematically investigated multiple factors that differ between mosquito infection and needle inoculation of WNV. We found that mice infected with WNV through the bite of a single infected Culex tarsalis mosquito exhibited 5- to 10-fold-higher viremia and tissue titers at 24 and 48 h postinoculation and faster neuroinvasion than mice given a median mosquito-inoculated dose of WNV (10(5) PFU) by needle. Mosquito-induced enhancement was not due to differences in inoculation location, because additional intravenous inoculation of WNV did not enhance viremia or tissue titers. Inoculation of WNV into a location where uninfected mosquitoes had fed resulted in enhanced viremia and tissue titers in mice similar to those in mice infected by a single infected mosquito bite, suggesting that differences in where virus is deposited in the skin and in the virus particle itself were not responsible for the enhanced early infection in mosquito-infected mice. In addition, inoculation of mice with WNV mixed with salivary gland extract (SGE) led to higher viremia, demonstrating that mosquito saliva is the major cause of mosquito-induced enhancement. Enhanced viremia was not observed when SGE was inoculated at a distal site, suggesting that SGE enhances WNV replication by exerting a local effect. Furthermore, enhancement of WNV infection still occurred in mice with antibodies against mosquito saliva. In conclusion, saliva from C. tarsalis is responsible for enhancement of early WNV infection in vertebrate hosts.     

Induction of mucosal homing virus-specific CD8(+) T lymphocytes by attenuated simian immunodeficiency virus

Induction of virus-specific T-cell responses in mucosal as well as systemic compartments of the immune system is likely to be a critical feature of an effective AIDS vaccine. We investigated whether virus-specific CD8(+) lymphocytes induced in rhesus macaques by immunization with attenuated simian immunodeficiency virus (SIV), an approach that is highly effective in eliciting protection against mucosal challenge, express the mucosa-homing receptor alpha4beta7 and traffic to the intestinal mucosa. SIV-specific CD8(+) T cells expressing alpha4beta7 were detected in peripheral blood and intestine of macaques infected with attenuated SIV. In contrast, virus-specific T cells in blood of animals immunized cutaneously by a combined DNA-modified vaccinia virus Ankara regimen did not express alpha4beta7. These results demonstrate the selective induction of SIV-specific CD8(+) T lymphocytes expressing alpha4beta7 by a vaccine approach that replicates in mucosal tissue and suggest that induction of virus-specific lymphocytes that are able to home to mucosal sites may be an important characteristic of a successful AIDS vaccine.     

Reconciling West Nile virus with the autophagic pathway

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus responsible for recurrent outbreaks of meningitis and encephalitis. Several studies analyzing the interactions of this pathogen with the autophagic pathway have reported opposite results with evidence for and against the upregulation of autophagy in infected cells. In this regard, we have recently reported that minimal genetic changes (single amino acid substitutions) in nonstructural proteins of WNV can modify the ability of the virus to induce autophagic features such as LC3 modification and aggregation in infected cells. We think that these results could help explain some of the previously reported discrepancies. These findings could also aid in deciphering the interactions of this pathogen with the autophagic pathway at the molecular level aimed to develop feasible antiviral strategies to combat this pathogen, and other related flaviviruses.     

Beta interferon controls West Nile virus infection and pathogenesis in mice

Studies with mice lacking the common plasma membrane receptor for type I interferon (IFN-αβR(-)(/)(-)) have revealed that IFN signaling restricts tropism, dissemination, and lethality after infection with West Nile virus (WNV) or several other pathogenic viruses. However, the specific functions of individual IFN subtypes remain uncertain. Here, using IFN-β(-)(/)(-) mice, we defined the antiviral and immunomodulatory function of this IFN subtype in restricting viral infection. IFN-β(-)(/)(-) mice were more vulnerable to WNV infection than wild-type mice, succumbing more quickly and with greater overall mortality, although the phenotype was less severe than that of IFN-αβR(-)(/)(-) mice. The increased susceptibility of IFN-β(-)(/)(-) mice was accompanied by enhanced viral replication in different tissues. Consistent with a direct role for IFN-β in control of WNV replication, viral titers in ex vivo cultures of macrophages, dendritic cells, fibroblasts, and cerebellar granule cell neurons, but not cortical neurons, from IFN-β(-)(/)(-) mice were greater than in wild-type cells. Although detailed immunological analysis revealed no major deficits in the quality or quantity of WNV-specific antibodies or CD8(+) T cells, we observed an altered CD4(+) CD25(+) FoxP3(+) regulatory T cell response, with greater numbers after infection. Collectively, these results suggest that IFN-β controls WNV pathogenesis by restricting infection in key cell types and by modulating T cell regulatory networks.     

Rotavirus-specific protein synthesis is not necessary for recognition of infected cells by virus-specific cytotoxic T lymphocytes.

We found that rotavirus-specific protein synthesis was not necessary for recognition by virus-specific cytotoxic T lymphocytes (CTLs). In addition, CTLs lysed rotavirus-infected target cells prior to production of infectious virus. Target cell processing of rotavirus antigens for presentation to CTLs was enhanced by treatment of rotavirus with trypsin prior to infection; trypsin-induced cleavage of the viral hemagglutinin (vp4) has previously been found to facilitate rotavirus entry into target cells by direct penetration of virions through the plasma membrane. We conclude that sufficient quantities of exogenous viral proteins may be introduced into the cytoplasm for processing by target cells. The mechanism by which rotavirus proteins are processed for presentation to the target cell surface remains to be determined.     

High frequency of virus-specific CD8+ T cells in the central nervous system of macaques chronically infected with simian immunodeficiency virus SIVmac251

Infection with human immunodeficiency virus or simian immunodeficiency virus (SIV) induces virus-specific CD8(+) T cells that traffic to lymphoid and nonlymphoid tissues. In this study, we used Gag-specific tetramer staining to investigate the frequency of CD8(+) T cells in peripheral blood and the central nervous system of Mamu-A*01-positive SIV-infected rhesus macaques. Most of these infected macaques were vaccinated prior to SIVmac251 exposure. The frequency of Gag(181-189) CM9 tetramer-positive cells was consistently higher in the cerebrospinal fluid and the brain than in the blood of all animals studied and did not correlate with either plasma viremia or CD4(+)-T-cell level. Little or no infection in the brain was documented for most animals by nucleic acid sequence-based amplification or in situ hybridization. These data suggest that this Gag-specific response may contribute to the containment of viral replication in this locale.     

JC virus-specific cytotoxic T lymphocytes in individuals with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by a reactivation of the polyomavirus JC (JCV) within a setting of immunosuppression. The nature of the immune response that contains replication of this virus is unknown. We have explored JCV-specific cellular immune responses in patients with PML and control subjects. JCV antigen-stimulated peripheral blood mononuclear cells (PBMC) of four human immunodeficiency virus (HIV)-infected patients who were survivors of PML and one HIV-uninfected patient recently diagnosed with PML lysed autologous B-lymphoblastoid cell lines expressing either the JCV T regulatory protein or the VP1 major capsid protein. This lysis was mediated by CD8(+) T lymphocytes and was major histocompatibility complex class I restricted. These cells were therefore cytotoxic T lymphocytes (CTL). JCV-specific CTL could not be detected in PBMC of three HIV-infected PML patients who had progressive neurologic disease and an eventual fatal outcome. These data suggest that the JCV-specific cellular immune response may play a crucial role in the containment of PML. This finding may also prove useful as a favorable prognostic marker in the clinical management of these patients.     

西尼罗病毒的RT-PCR检测与鉴定

建立西尼罗病毒敏感、特异、快速的RT-PCR检测方法用于实验室诊断和流行病学监测。采用一步RT-PCR和套式PCR法对西尼罗病毒感染的乳鼠脑和细胞培养上清进行扩增,并对扩增产物进行序列测定。两种方法均可分别从两种组织中扩增出与预期大小相一致的片段,套式PCR法比一步RT-PCR法更为敏感,该扩增片段与西尼罗病毒埃及Eg101株相应序列的同源性为99％。