Localization of West Nile Virus in monkey brain: double staining antigens immunohistochemically of neurons, neuroglia cells and West Nile Virus

West Nile virus (WNV) can cause encephalitis or meningitis that affects brain tissue, which can also lead to permanent neurological damage that can be fatal. To our knowledge, no consistent double immunohistochemical staining of neurons, neuroglia cells, and WNV has yet been reported. To establish a method for performing double-label immunohistochemical detection of neurons, neuroglia cells and WNV, examining the pathological characteristics of WNV-infected neurons, neuroglia cells, and investigating distribution of WNV in monkey brain, paraffin-embedded monkey brain tissue were retrospectively studied by immunohistochemical staining of neurons, neuroglia cells and WNV. Antibodies against neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and WNV were used to develop the method of double-label immunohistochemical staining, which allowed independent assessment of neuron status and WNV distribution. A range of immunohistochemical WNV infection in monkey brain was observed in both neurons and neuroglia cells in terms of the thickness of lesion staining, and the WNV staining was slightly higher in neuroglia cells than in neurons. All these findings suggest that WNV invasion in the brain plays a crucial role in neurological damage by inducing central nervous system (CNS) cell dysfunction or cell death directly.     

Evaluation of Newcastle disease virus immunoassays for waterfowl using a monoclonal antibody specific for the duck immunoglobulin light chain

In the present study a monoclonal antibody (mAb 14A3) was tested for its reactivity against serum immunoglobulin Y (IgY) of several waterfowl species, and subsequently for its applicability as anti-species antibody in common immunoassays. Western blot analyses demonstrated its broad cross-reactivity with the serum IgY light chain of different duck species: Muscovy duck (Cairina moschata), Mallard (Anas platyrhynchos), white-winged wood duck (Asarcornis scutulatus), common pintail (Dafila acuta). Reactivity was also evident with IgY of two swan species—mute swan (Cygnus olor) and black-necked swan (Sthenelides melanocoryphus)—and two goose species—domestic goose (Anser anser var. domestica) and red-breasted goose (Rufibrenta ruficollis). Applying the mAb for Newcastle disease virus (avian paramyxovirus serotype 1 [APMV-1]) test systems, its functionality within indirect immunoassays was evaluated. Using APMV-1-positive sera of domestic geese and Muscovy ducks, mAb 14A3 facilitated specific staining of APMV-1-infected cells in an immunofluorescence test. In addition, it proved to be functional in an indirect enzyme-linked immunosorbent assay (ELISA) and a western blot assay. Thus, the analysed mAb represents an attractive and versatile reagent that offers the opportunity to develop serological tests for waterfowl, allowing a high sample throughput using the ELISA technique or the fine analysis of humoral immune responses using the western blot.     

Virus hepatitis of geese. 3. Properties of the causal agent

Characteristics of the goose hepatitis virus strain SHM 319 were studied in goose embryos and tissue cultures of avian origin. The virus was found to be resistant to both ether and sodium deoxycholate (0.25%). The growth of the virus was inhibited by 5-iodo-2-deoxyuridine. Virus infectivity was not affected by heating at 56 degrees C for 3 hours, exposure to pH 3.0, formalin (1:1000), and other inactivating chemicals. Cell culture systems of three avian species were inoculated with GVH-SHM 319. Extensive cytopathic effect was produced only in goose cell cultures. No virus replication was observed in chicken or White Pekin duck cells. Fluorescent antibody staining revealed granular nuclear staining in infected susceptible tissue cultures. May-Grunwald-Giemsa-staining of infected tissue culture cells showed formation of mainly intranuclear inclusion bodies. Millipore filtration procedures revealed a particle size less than 50 nm. Hemagglutination was not observed. Precipitating antibodies could not be detected in GHV hyperimmunized birds. Neutralization tests could not demonstrate a relationship to known viruses of waterfowl, including duck virus enteritis and duck virus hepatitis. A certain discrepancy in the neutralization test with Hungarian goose sera is discussed.     

Complex adenovirus-mediated expression of West Nile virus C, PreM, E, and NS1 proteins induces both humoral and cellular immune responses

West Nile Virus (WNV), a member of the family Flaviviridae, was first identified in Africa in 1937. In recent years, it has spread into Europe and North America. The clinical manifestations of WNV infection range from mild febrile symptoms to fatal encephalitis. Two genetic lineages (lineages I and II) are recognized; lineage II is associated with mild disease, while lineage I has been associated with severe disease, including encephalitis. WNV has now spread across North America, significantly affecting both public and veterinary health. In the efforts to develop an effective vaccine against all genetic variants of WNV, we have studied the feasibility of inducing both neutralizing and cellular immune responses by de novo synthesis of WNV antigens using a complex adenoviral vaccine (CAdVax) vector. By expressing multiple WNV proteins from a single vaccine vector, we were able to induce both humoral and cellular immune responses in vaccinated mice. Neutralization assays demonstrated that the antibodies were broadly neutralizing against both lineages of WNV, with a significant preference for the homologous lineage II virus. The results from this study show that multiple antigens synthesized de novo from a CAdVax vector are capable of inducing both humoral and cellular immune responses against WNV and that a multiantigen approach may provide broad protection against multiple genetic variants of WNV.     

Detection of enterovirus capsid protein VP1 in myocardium from cases of myocarditis or dilated cardiomyopathy by immunohistochemistry: further evidence of enterovirus persistence in myocytes

The association of enteroviruses with myocardial disease has been investigated extensively by molecular biological techniques to detect viral RNA, but remains controversial. This retrospective study investigated the involvement of enterovirus in myocarditis or dilated cardiomyopathy (DCM) by detection of viral antigens in myocardial samples from a new patient series using an optimized immunohistochemical technique. Formalin-fixed, paraffin-embedded biopsy, autopsy or explanted myocardial tissue samples were obtained from 136 subjects. These comprised histologically proven cases of acute fatal myocarditis (n=10), DCM (n=89, including 10 patients with healing/borderline myocarditis) and a comparison group of samples from 37 unused donor hearts and cases with other conditions. A monoclonal antibody 5-D8/1 directed against a conserved, non-conformational epitope in capsid protein VP1 was employed for broad detection of different enterovirus serotypes. Investigations were performed blindly. Histological sections from 7 of 10 fatal myocarditis cases, 47 of 89 patients (52.8%) with DCM were positive for the viral capsid protein VP1 by immunohistochemical staining. Consecutive sections of positive samples were negative when the antibody was omitted or replaced with subclass- and concentration-matched normal mouse IgG. In contrast, only 3 of 37 samples (8.1%) in the comparison group were positive (Yates corrected ²=19.99, P<0.001: odds ratio =12.68). VP1 staining was distributed in individual or grouped myofibers and localized in the cytoplasm of myocytes. In some cases, VP1 was detected in only a few myofibers within an entire section. These results provide further evidence of enterovirus involvement in a high proportion of DCM cases and demonstrate that VP1 is present in disease stages from acute myocarditis, healing myocarditis to end-stage DCM requiring cardiac transplantation, indicating translation of viral protein during persistent enterovirus infection.     

Nek6在心力衰竭家兔心脏中的表达增加

目的：通过刺破主动脉瓣造成反流加重心脏前负荷联合缩窄腹主动脉加重心脏后负荷制作心力衰竭模型,研究Nek6在心力衰竭家兔心脏的心肌细胞中含量表达的改变,初步探讨Nek6在心力衰竭发生发展中的作用。方法将日本大耳白兔分为心衰组和对照组各10只,通过刺破主动脉瓣及缩窄腹主动脉两次手术制作心衰组模型,对照组仅行假手术。2月后使用超声检测心脏各腔室大小及室壁厚度,并计算射血分数;处死家兔后,计算心脏重量与身体重量比值,使用HE染色分析比较心肌细胞面积,使用PSR染色分析比较基质胶原含量,使用Nek6免疫组化染色分析Nek6蛋白含量变化。结果与对照组相比,心衰组家兔左室收缩末期内径及舒张末期内径均增加,射血分数降低,身心比增加,细胞明显增大,胶原增多, Nek6蛋白含量增加。结论 Nek6在心力衰竭家兔心脏的心肌细胞中表达明显上升。     

Reversal of West Nile virus-induced blood-brain barrier disruption and tight junction proteins degradation by matrix metalloproteinases inhibitor

Though compromised blood-brain barrier (BBB) is a pathological hallmark of WNV-associated neurological sequelae, underlying mechanisms are unclear. We characterized the expression of matrix metalloproteinases (MMP) in WNV-infected human brain microvascular endothelial cells (HBMVE) and human brain cortical astrocytes (HBCA), components of BBB and their role in BBB disruption. Expression of multiple MMPs was significantly induced in WNV-infected HBCA cells. Naïve HBMVE cells incubated with the supernatant from WNV-infected HBCA cells demonstrated loss of tight junction proteins, which were rescued in the presence of MMP inhibitor, GM6001. Further, supernatant from WNV-infected HBCA cells compromised the in vitro BBB model integrity. Our data suggest astrocytes as one of the sources of MMP in the brain, which mediates BBB disruption allowing unrestricted entry of immune cells into the brain, thereby contributing to WNV neuropathogenesis. Because of the unavailability of WNV antivirals and vaccines, use of MMP inhibitors as an adjunct therapy to ameliorate WNV disease progression is warranted.     

Immunohistochemical and in situ hybridization studies of influenza A virus infection in human lungs

Influenza viruses are responsible for acute febrile respiratory disease. When deaths occur, definitive diagnosis requires viral isolation because no characteristic viral inclusions are seen. We examined the distribution of influenza A virus in tissues from 8 patients with fatal infection using 2 immunohistochemical assays (monoclonal antibodies to nucleoprotein [NP] and hemagglutinin [HA]) and 2 in situ hybridization (ISH) assays (digoxigenin-labeled probes that hybridized to HA and NP genes). Five patients had prominent bronchitis; by immunohistochemical assay, influenza A staining was present focally in the epithelium of larger bronchi (intact and detached necrotic cells) and in rare interstitial cells. The anti-NP antibody stained primarily cell nuclei, and the anti-HA antibody stained mainly the cytoplasm. In 4 of these cases, nucleic acids (ISH) were identified in the same areas. Three patients had lymphohistiocytic alveolitis and showed no immunohistochemical or ISH staining. Both techniques were useful for detection of influenza virus antigens and nucleic acids in formalin-fixed paraffin-embedded tissues and can enable further understanding of fatal influenza A virus infections in humans.     

Serial right ventricular endomyocardial biopsy in rapid-onset severe heart failure due to giant cell myocarditis.

Giant cell myocarditis (GCM) is a serious condition that warrants immediate diagnosis and treatment. It often presents as rapidly progressive heart failure and/or malignant ventricular arrhythmias. Here, we describe a 34-year-old patient with myasthenia gravis who presented with GCM 2 weeks after resection of a thymoma. A cardiac biopsy confirming the diagnosis was done within 3 days after admission. After institution of an aggressive immunosuppressive drug regimen, implantation of an implantable cardioverter defibrillator, and intensive cardiac rehabilitation, the patient recovered dramatically. In control biopsies after 4 weeks and 6 months, no more giant cells were found. We conclude that, in the case of nonischemic acute heart failure in young patients, a biopsy should be performed as soon as possible to prevent an unfavourable outcome of this often fatal disease.     

Pro-inflammatory cytokines derived from West Nile virus (WNV)-infected SK-N-SH cells mediate neuroinflammatory markers and neuronal death

Background  

WNV-associated encephalitis (WNVE) is characterized by increased production of pro-inflammatory mediators, glial cells activation and eventual loss of neurons. WNV infection of neurons is rapidly progressive and destructive whereas infection of non-neuronal brain cells is limited. However, the role of neurons and pathological consequences of pro-inflammatory cytokines released as a result of WNV infection is unclear. Therefore, the objective of this study was to examine the role of key cytokines secreted by WNV-infected neurons in mediating neuroinflammatory markers and neuronal death.     

The effects of acetylcholinesterase inhibitors on the heart in acute myocardial infarction and heart failure: From cells to patient reports

Cardiovascular diseases remain a major cause of morbidity and mortality worldwide. Cardiovascular diseases such as acute myocardial infarction, ischaemia/reperfusion injury and heart failure are associated with cardiac autonomic imbalance characterized by sympathetic overactivity and parasympathetic withdrawal from the heart. Increased parasympathetic activity by electrical vagal nerve stimulation has been shown to provide beneficial effects in the case of cardiovascular diseases in both animals and patients by improving autonomic function, cardiac remodelling and mitochondrial function. However, clinical limitations for electrical vagal nerve stimulation exist because of its invasive nature, costly equipment and limited clinical validation. Therefore, novel therapeutic approaches which moderate parasympathetic activities could be beneficial for in the case of cardiovascular disease. Acetylcholinesterase inhibitors inhibit acetylcholinesterase and hence increase cholinergic transmission. Recent studies have reported that acetylcholinesterase inhibitors improve autonomic function and cardiac function in cardiovascular disease models. Despite its potential clinical benefits for cardiovascular disease patients, the role of acetylcholinesterase inhibitors in acute myocardial infarction and heart failure remediation remains unclear. This article comprehensively reviews the effects of acetylcholinesterase inhibitors on the heart in acute myocardial infarction and heart failure scenarios from in vitro and in vivo studies to clinical reports. The mechanisms involved are also discussed in this review.     

Cardiomyopathy in adult Holstein Friesian cattle in Britain

The clinical signs, pathology and breeding data of two cases of cardiomyopathy of an unusual kind in adult Holstein Friesian cattle in Britain are reported and compared with those in similar entities in Switzerland, Japan and Canada. The principal and primary lesions affect the heart and these produce secondary changes, particularly in the liver, and result in fatal congestive heart failure. The cardiac lesions consist of extensive myocyte vacuolation, endomysial and perimysial fibrosis and focal cardiac myocyte degeneration, atrophy and hypertrophy resulting in an extended range of myocyte size. Lesions affected all four heart chambers but were most severe in the ventricles. Vascular lesions, particularly moderate medial hypertrophy and intimal thickening of arterioles and arteries, occurred in heart, lung, kidney and lymph nodes. The liver showed severe fibrosis, chronic congestion and hepatocyte loss. There was a chronic multifocal nephritis. The cause is unknown, but the affected animals were full brother and sister and have a common ancestor in the male and female line five generations earlier.     

Virus neutralizing antibodies to arboviruses in birds of the order Anseriformes in Czechoslovakia.

Sera from birds of the order Anseriformes in Czechoslovakia were examined for virus neutralizing (VN) antibodies to arboviruses. VN antibodies to Sindbis, Calovo and Tahyna viruses were found in 15, 5 and 6 out of 106 greylag goose (Anser anser) sera. Out of 38 ducks, 6 mallards (Anas platyrhynchos) and 1 garganey (Anas querquedula) contained VN antibodies to Sindbis virus, 6 mallards to Calovo virus, 4 mallards and 1 garganey to Tahyna virus, 2 mallards and 1 garganey to tick-borne encephalitis (TE) virus and 1 mallard to West Nile (WN) virus.     

IgG anti-cardiomyocyte antibodies in giant cell myocarditis

Giant cell myocarditis, a rare, fatal, and poorly understood cause of myocarditis, requires pathological examination for diagnosis. It is considered to be an autoimmune disease and is frequently associated with other conditions, in particular thymoma and myasthenia gravis. The typical patient with giant cell myocarditis is young and has severe, progressive congestive cardiac failure that is unresponsive to standard medical therapy and ultimately requires cardiac transplantation. Hence giant cell myocarditis is the most dangerous form of myocarditis. Here we report an unusual presentation of giant cell myocarditis, which mimicked acute myocardial infarction in an elderly woman with myasthenia gravis and a previous diagnosis of thymoma. This patient had evidence of anti-myocyte antibodies, consistent with an autoimmune mechanism.     

Cardiac autoantibodies to myosin and other heart-specific autoantigens in myocarditis and dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is characterized by dilation and impaired contraction of the left ventricle or both; it is a relevant cause of heart failure and a common indication for heart transplantation. It may be idiopathic, familial/genetic, viral, autoimmune or immune-mediated, associated with a viral infection. Myocarditis is an inflammatory disease of the myocardium; it may be idiopathic, infectious or autoimmune and may heal or lead to DCM. Thus, in a patient subset, myocarditis and DCM are thought to represent the acute and chronic stages of an organ-specific autoimmune disease of the myocardium. In keeping with this hypothesis, autoimmune features in patients with myocarditis/DCM include: familial aggregation, a weak association with HLA-DR4, abnormal expression of HLA class II on cardiac endothelium on endomyocardial biopsy, detection of organ- and disease-specific cardiac autoantibodies in the sera of affected patients and of symptom-free relatives. The organ-specific cardiac autoantibodies detected by immunofluorescence are directed against multiple antigens. One of these, first identified using immunoblotting and confirmed by ELISA, is the cardiac-specific alpha-myosin isoform. Myosin fulfils the expected criteria for organ-specific autoimmunity, in that immunization with cardiac but not skeletal myosin reproduces, in susceptible mouse strains, the human disease phenotype of myocarditis/DCM; in addition, alpha-myosin is entirely cardiac-specific. The organ-specific cardiac autoantibodies detected by immunofluorescence in symptom-free relatives were associated with echocardiographic features suggestive of early disease. Short-term follow-up is in keeping with this interpretation, although extended follow-up is necessary to define better the role of the antibody as predictor of disease susceptibility in healthy subjects at risk of myocarditis/DCM, such as first-degree relatives.     

West Nile virus: a new challenge?

West Nile Virus (WNV), a member of the family Flaviviridae, was first isolated in 1937. Since the original isolation of the WNV outbreaks have occurred with increase in frequency of cases in humans and horses, apparent increase in severe human disease and high avian death rates. In 1999, 2000 and 2002 outbreaks of the WNV encephalitis were reported in horses, birds and humans from New York and Canada. Ornithophilic mosquitoes are the principal vectors of the WNV and birds of several species chiefly migrants appear to be the major introductory or amplifying host. The pattern of outbreaks in the old and new world suggests that viremic migratory birds may also contribute to movement of the virus. If so, Central America, Caribbean Islands and countries of South America including Venezuela, are in potential risk for suffering a severe outbreak for WNV, since several species of birds have populations that pass trough New York and cross the western north Atlantic or Caribbean Sea. It is important the knowledge of the ecology of WNV as well of the efficacy of control efforts in order to minimize the public health impact in these countries, where all population is susceptible to this infection.     

Cardiac biomarkers: a review

Cardiac injury can develop secondary to compromise due to disease or drug toxicity, among other causes. The clinical signs of cardiac problems can be confused with those of other conditions, such as respiratory disease. Therefore, specific, sensitive, rapid and inexpensive blood tests for cardiac injury are desirable. Cardiac troponins are uniquely expressed in the myocardium and increase with injury. Changes in serum concentrations of these have been found to be specific and sensitive indicators of such injury. Troponins are phylogenetically highly conserved proteins with > 95% homology between mammals. Thus assays developed for human serum can be used in other species. Natriuretic peptides have been more recently used as indicators of cardiac injury in humans. They are becoming more available for animals but homology is lower, therefore many animal species-specific assays need to be developed. These peptides are sensitive to changes in vasoconstriction and dilation within the heart and are used for the diagnosis and prognosis of heart failure in humans. Assays for endothelins and cardiotrophins have similar potential and are under development and evaluation for human use. Combinations of these tests can be followed by more specialised tests (e.g. echocardiography) to confirm the severity and type of injury that has occurred.Abbreviations AMI acute myocardial infarction - CK-MB creatine kinase muscle/brain - ANP atrial natriuretic peptide - AST asparatate aminotransferase - BNP brain natriuretic peptide - CHF chronic heart failure - CK creatine kinase - CNP C-type natriuretic     

Molecular changes in the heart of a severe case of arrhythmogenic right ventricular cardiomyopathy caused by a desmoglein-2 null allele

IntroductionArrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder caused by mutations in desmosomal genes. It is often associated with life-threatening arrhythmias. Some affected individuals develop progressive heart failure and may require cardiac transplantation.MethodsThe explanted heart of a young adult with end-stage heart failure due to a null allele in desmoglein-2 was studied at macroscopic, microscopic, and molecular level. Myocardial samples were probed for junctional localization of desmosomal components and the gap junction protein connexin43 by immunohistochemical staining. In addition, the protein content of desmosomal and adherens junction markers as well as connexin43 was assessed by Western blotting.ResultsHistological analysis confirmed ARVC. Despite the loss of specific immunoreactive signal for desmosomal components at the cardiac intercalated disks (shown for plakoglobin, desmoplakin, and plakophilin-2), these proteins could be detected by Western blotting. Only for desmoglein-2, desmocollin-2, and plakoglobin were reduced protein levels observed. Adherens junction proteins were not affected. Lower phosphorylation levels were observed for connexin43; however, localization of the gap junction protein displayed regional differences. At the molecular level, disease progression was more severe in the right ventricle compared to the left ventricle.ConclusionOur data suggest that, in the ARVC heart, plakoglobin is mainly redistributed from the junctions to other cellular pools and that protein degradation only plays a secondary role. Homogenous changes in the phosphorylation status of connexin43 were observed in multiple ARVC samples, suggesting that this might be a general feature of the disease.     

Clinicopathologic study and laboratory diagnosis of 23 cases with West Nile virus encephalomyelitis

The differences in pathologic findings of fatal cases of West Nile virus (WNV) encephalitis in the context of underlying conditions and illness duration are not well known. During 2002, we studied central nervous system (CNS) tissue samples from 23 patients who had serologic and immunohistochemical (IHC) evidence of a recent WNV infection. Fifteen patients had underlying medical conditions (5 malignancies, 3 renal transplants, 3 with diabetes or on dialysis, 2 with AIDS, and 2 receiving steroids). WNV serology was positive for 18 patients, negative for 2, and not available for 3. Perivascular lymphocytic infiltrates, microglial nodules, and loss of neurons were predominantly observed in the brainstem and anterior horns in the spinal cord. IHC using antibodies against flaviviruses and WNV showed viral antigens in 12 (52%) of 23 patients. Viral antigens were found inside neurons and neuronal processes predominantly in the brainstem and anterior horns. In general, the antigens were focal and sparse; however, in 4 severely immunosuppressed patients, extensive viral antigens were seen throughout the CNS. Positive IHC staining was observed in tissues of 7 of 8 patients who died within 1 week after illness onset, compared with 4 of 14 with more than 2 weeks' illness duration. WNV causes an encephalomyelitis by primarily affecting brainstem and spinal cord. Differences in the amount of viral antigen may be related to underlying medical conditions and length of survival. IHC can be an important diagnostic method, particularly during the 1st week of illness, when antigen levels are high.