Distribution Of Viral Antigen Research Articles

Lesions and Distribution of Viral Antigen in Birds Infected with the Pathogenic Strain of H5N1 Avian Influenza Virus

Lesions and Distribution of Viral Antigen in Birds Infected with the Pathogenic Strain of H5N1 Avian Influenza Virus

Avian Bornavirus is Present in Many Tissues of Psittacine Birds with Histopathologic Evidence of Proventricular Dilatation Disease

Proventricular dilatation disease (PDD) is a neurologic disease of psittacine birds suspected to be caused by a recently identified Avian bornavirus (ABV). In the current report, data supporting the causal association of ABV with PDD are presented. Immunohistochemistry (IHC) with rabbit polyclonal antiserum raised against ABV nucleocapsid protein was used to identify cell and organ distribution of viral antigen. The ABV antigen was most consistently detected in brain, spinal cord, adrenal gland, pancreas, and kidney. Histopathologic evaluation was correlated with ABV-specific polymerase chain reaction (PCR) and immunohistochemical tests in multiple tissues from 16 psittacine birds with and without PDD. Using histopathologic diagnosis as the gold standard, the sensitivity and specificity of IHC for ABV antigens were found to be 100% and 100%, respectively. Many more tissues were positive for ABV RNA by reverse transcription PCR than were positive for pathologic changes or viral antigens by IHC, indicating the presence of subclinical or asymptomatic infection at many sites.

Pathology and Viral Antigen Distribution of Lethal Pneumonia in Domestic Cats Due to Pandemic (H1N1) 2009 Influenza A Virus

A novel swine-origin H1N1 influenza A virus has been identified as the cause of the 2009 influenza pandemic in humans. Since then, infections with the pandemic (H1N1) 2009 influenza virus have been documented in a number of animal species. The first known cases of lethal respiratory disease associated with pandemic (H1N1) 2009 influenza virus infection in house pets occurred in domestic cats in Oregon. A 10-year-old neutered domestic shorthair and an 8-year-old spayed domestic shorthair died shortly after developing severe respiratory disease. Grossly, lung lobes of both cats were diffusely firm and incompletely collapsed. Histologically, moderate to severe necrotizing to pyonecrotizing bronchointerstitial pneumonia was accompanied by serofibrinous exudation and hyaline membranes in the alveolar spaces. Influenza A virus was isolated from nasal secretions of the male cat and from lung homogenate of the female cat. Both isolates were confirmed as pandemic (H1N1) 2009 influenza virus by real-time reverse transcriptase polymerase chain reaction. With immunohistochemistry, influenza A viral antigen was demonstrated in bronchiolar epithelial cells, pneumocytes, and alveolar macrophages in pneumonic areas. The most likely sources of infection were people in the household with influenza-like illness or confirmed pandemic (H1N1) 2009 influenza. The 2 cases reported here provide, to the best of the authors' knowledge, the first description of the pathology and viral antigen distribution of lethal respiratory disease in domestic cats after natural pandemic (H1N1) 2009 influenza virus infection, probably transmitted from humans.

Persistent, triple-virus co-infections in mosquito cells

BackgroundIt is known that insects and crustaceans can carry simultaneous, active infections of two or more viruses without showing signs of disease, but it was not clear whether co-infecting viruses occupied the same cells or different cells in common target tissues. Our previous work showed that successive challenge of mosquito cell cultures followed by serial, split-passage resulted in stabilized cultures with 100% of the cells co-infected with Dengue virus (DEN) and an insect parvovirus (densovirus) (DNV). By addition of Japanese encephalitis virus (JE), we tested our hypothesis that stable, persistent, triple-virus co-infections could be obtained by the same process.ResultsUsing immunocytochemistry by confocal microscopy, we found that JE super-challenge of cells dually infected with DEN and DNV resulted in stable cultures without signs of cytopathology, and with 99% of the cells producing antigens of the 3 viruses. Location of antigens for all 3 viruses in the triple co-infections was dominant in the cell nuclei. Except for DNV, this differed from the distribution in cells persistently infected with the individual viruses or co-infected with DNV and DEN. The dependence of viral antigen distribution on single infection or co-infection status suggested that host cells underwent an adaptive process to accommodate 2 or more viruses.ConclusionsIndividual mosquito cells can accommodate at least 3 viruses simultaneously in an adaptive manner. The phenomenon provides an opportunity for genetic exchange between diverse viruses and it may have important medical and veterinary implications for arboviruses.

A 16-year old male with cortical blindness and focal motor seizures

A 16-year old male with cortical blindness and focal motor seizures

Effect of the viral protein Npro on virulence of bovine viral diarrhea virus and induction of interferon type I in calves

To characterize the influence of the viral protein N(pro) on virulence of bovine viral diarrhea virus (BVDV) and on type I interferon responses in calves. 10 calves, 4 to 6 months of age. BVDV virulence and type I interferon responses of calves (n = 5) infected with a noncytopathic BVDV with a deleted N(pro) were compared with those of calves (5) infected with a noncytopathic BVDV with a functional N(pro). Rectal temperatures, clinical signs, platelet counts, and total and differential WBC counts were evaluted daily. Histologic examinations and immunohistochemical analyses of tissues were conducted to assess lesions and distribution of viral antigens, respectively. Serum type I interferon concentrations were determined. Calves infected with N(pro)-deleted BVDV developed leukopenia and lymphopenia, without developing increased rectal temperatures or lymphoid depletion of target lymphoid organs. There was minimal antigen deposition in lymphoid organs. Calves infected with N(pro) BVDV developed increased rectal temperatures, leukopenia, lymphopenia, and lymphoid depletion with marked BVDV antigen deposition in lymphatic tissues. Interferon type I responses were detected in both groups of calves. Deletion of N(pro) resulted in attenuation of BVDV as evidenced by reduced virulence in calves, compared with BVDV with a functional N(pro). Deletion of N(pro) did not affect induction of type I interferon. The N(pro)-deleted BVDV mutant may represent a safe noncytopathic virus candidate for vaccine development.

Neuroanatomical mapping of rabies nucleocapsid viral antigen distribution and apoptosis in pathogenesis in street dog rabies – an immunohistochemical study

To date, there is no study from Asian countries describing the pathology and topographic distribution of virulent, "street" rabies viral infection in the canine brain. In the present study, neuroanatomical distribution of rabies viral antigen in the brains of rabid street dogs, by immunohistochemical techniques is documented and the role of apoptosis in pathogenesis of rabies in natural hosts especially canines infected with street virus is studied. 10 brains of adult street dogs from urban areas of Bangalore, South Central India, infected with rabies were collected. The diagnosis was confirmed by immunofluorescent study. The pathomorphological features and the neuroanatomic distribution of the viral antigen by immunohistochemistry were studied. The ability of the virus to activate apoptosis in nerve cells if any, was studied by determining the DNA fragmentation and TUNEL technique in infected canine brains. The viral antigen was mostly localized to the neuronal perikaryon extending along the dendrites, while occasional astrocytes were also labeled. In the brain, the limbic areas, thalamus and the reticular formation of the brain stem, the trigeminal and the vagal nuclei were involved, corresponding to areas of cholinergic innervation. It is proposed that the preferential involvement of these cholinergic zones could explain some of the clinical features of rabies in canines. The extensive involvement of thalamus and immunolocalization of the rabies viral antigen in the axons are the unusual features noted in a dog's brain in contrast to murine experimental studies with "fixed virus". Characteristic DNA fragmentation forming 180-200 bp, leading to laddering was not seen, indicating apoptosis is not involved in the evolution of lesions in rabies in adult dogs infected by street virus.

Achados patológicos e imuno-histoquímicos em cães infectados naturalmente pelo vírus da cinomose canina

A cinomose canina é uma doença viral e afeta principalmente os sistemas respiratório, gastrintestinal e nervoso. Neste trabalho foram analisados os achados patológicos e imuno-histoquímicos de 54 cães com cinomose de um total de 760 cães necropsiados no período de julho de 2006 a outubro de 2007. As lesões macroscópicas observadas foram caracterizadas por corrimento ocular e nasal mucopurulentas, hiperqueratose dos coxins digitais, pulmões avermelhados e não colapsados, atrofia do timo, conteúdo intestinal diarréico e placas de Peyer proeminentes. Os achados histológicos caracterizavam-se principalmente por pneumonia intersticial, rarefação linfóide, desmielinização da substância branca, manguitos perivasculares e corpúsculos de inclusão intranucleares e intracitoplasmáticos, que se localizam principalmente na mucosa do estômago, epitélios da bexiga, brônquios e bronquíolos, pelve renal, coxins digitais, pálpebra, orelha e tonsila no sistema nervoso central e em células mononucleares dos linfonodos, baço e tonsilas. Os tecidos foram marcados pela técnica imuno-histoquímica utilizando anticorpo monoclonal anti-cinomose canina. O coxim digital foi o tecido com maior número de casos marcados positivamente (67,4% dos casos), seguido pelo estômago com 62,7%. A imuno-histoquímica mostrou ser uma ferramenta importante para o estudo da distribuição do antígeno em cães infectados pela cinomose bem como indicou o melhor tecido para a confirmação do diagnóstico de casos suspeitos.

Distribution of Bovine Viral Diarrhea Virus Antigen in Aborted Fetal and Neonatal Goats by Immunohistochemistry

Bovine viral diarrhea virus (BVDV) infection in goats can result in severe reproductive losses, with abortion rates reaching 80%. Infection with BVDV in aborted goat fetuses and stillborn kids can result in placentitis, encephalitis, myocarditis, and thymic depletion. This study investigates the distribution of viral antigen within the organ systems of aborted goat fetuses, stillborn kids, and nonviable kids infected with BVDV at various stages of gestation using immunohistochemistry (IHC). Virus antigen was detected within the placenta (8/13), thymus (4/9), heart (4/11), and brain (4/15) of affected goats. Uncommonly, BVDV antigen was detected within the skin (1/14), liver (1/13), kidney (1/12), lung (1/11), and trachea (1/3). BVDV antigen was not detected within the spleen (0/9), nasal turbinate (0/2), or thyroid (0/3). The results of this study indicate that placenta, heart, thymus, and brain are the most reliable tissues for BVDV antigen detection using IHC in aborted goat fetuses.

Pathologic Findings and Immunohistochemical Distribution of Viral Antigen in Experimental Avian Encephalomyelitis of the Chick and Quail

Summary In this study, the differences and diagnostic importances of the pathological and immunohistochemical findings in avian encephalomyelitis were investigated comparatively in chicks and quails by inoculating van Roekel strain (reference strain) via intracerebral and intramuscular routes. Histopathologically, nonpurulent encephalomyelitis characterized with infiltration of perivascular mononuclear cells and gliosis was described in both species. Neuronal lesion in the chicks consisted of central chromatolysis and neuronal shrinkage (neuronal pyknosis), whereas in quails it was composed of only neuronal shrinkage. Lymphoid aggregates and/or infiltration of mononuclear cells were observed in the proventriculus, gizzard, intestines, liver, pancreas, kidneys, myocardium, lungs, spleen and eyes. However, these lesions in quails were restricted only in the proventriculus, intestines, myocardium and eyes. In the immunofluorescein examination, viral antigen was found especially in neurons of the midbrain, cerebral hemispheres, cerebellum, and occasionally in medulla oblongata and medulla spinalis in both species. Viral antigen in the brain and spinal cord samples of the chicks decreased gradually by increasing inflammatory reaction. Viral antigen in both species was continously detected in the proventriculus, gizzard, intestines, myocardium, pancreas, spleen, liver, kidneys, lungs and eyes..

Comparative neuropathogenesis and neurovirulence of attenuated flaviviruses in non-human primates

Based on previous preclinical evaluation in mice and monkeys, the chimeric TBEV/DEN430 virus, carrying the prM and E protein genes from a highly virulent Far Eastern strain of tick-borne encephalitis virus (TBEV) on the backbone of a nonneuroinvasive dengue type 4 virus (DEN4), has been identified as a promising live attenuated virus vaccine candidate against disease caused by TBEV. However, prior to use of this vaccine candidate in humans, its neurovirulence in nonhuman primates needed to be evaluated. In the present study, we compared the neuropathogeneses of the chimeric TBEV/DEN430 virus; Langat virus (LGTV), a former live TBEV vaccine; and yellow fever 17D virus vaccine (YF 17D) in rhesus monkeys inoculated intracerebrally. TBEV/DEN430 and YF 17D demonstrated remarkably similar spatiotemporal profiles of virus replication and virus-associated histopathology in the central nervous system (CNS) that were high in cerebral hemispheres but progressively decreased toward the spinal cord. In contrast, the neurovirulence of LGTV exhibited the reverse profile, progressing from the site of inoculation toward the cerebellum and spinal cord. Analysis of the spatiotemporal distribution of viral antigens in the CNS of monkeys revealed a prominent neurotropism associated with all three attenuated viruses. Nevertheless, TBEV/DEN430 virus exhibited higher neurovirulence in monkeys than either LGTV or YF 17D, suggesting insufficient attenuation. These results provide insight into the neuropathogenesis associated with attenuated flaviviruses that may guide the design of safe vaccines.

The peptide AF-16 abolishes sickness and death at experimental encephalitis by reducing increase of intracranial pressure

Elevated intracranial pressure (ICP) is strongly aggravating the injury at brain inflammation, resulting in persistent neurological and psychiatric malfunctions. There is no efficient pharmacological treatment to achieve beneficial ICP reduction. Here, the peptide AF-16, comprising the amino terminal part of the endogenous protein Antisecretory Factor (AF), was used to suppress the raised ICP in experimental herpes simplex encephalitis (HSE) in rats. Intranasal instillation of the peptide AF-16 counteracted the ICP elevation and the prevalence of ICP spikes, abrogated the neurological morbidity, and abolished the mortality in a dose-dependent manner. AF-16, 25 μg twice daily intranasally, rescued all animals with HSE and abrogated neurological malfunction. In contrast, only 10% of the rats survived if treated with the vehicle. A single intranasal dose of 25 μg AF-16 to a rat displaying overt HSE symptoms reduced the ICP to normal levels within an hour. No effects on viral replication or antigen distribution were demonstrable. Thus, AF-16 abolished the prevalence of sickness signs, ICP elevation, neurological malfunctions and completely prevented deaths. We advocate use of AF-16 for suppression of elevated ICP.

The Fetal Brain in Bovine Viral Diarrhea Virus-infected Calves: Lesions, Distribution, and Cellular Heterogeneity of Viral Antigen at 190 Days Gestation

Previous studies have shown that the brain is a target of persistent infection with bovine viral diarrhea virus (BVDV) and have demonstrated viral tropism for neurons as well as other endogenous cell types in diverse brain areas. Apart from foci of mild residual inflammation in some postnatal calves, consistent brain lesions, per se, have not been reported. No similar comprehensive studies of the brain have been reported in bovine fetuses. In the current study, 12 BVDV-seronegative heifers were inoculated intranasally with a 2-ml 4.4 log(10) TCID(50)/ml dose of noncytopathic type 2 BVDV at 75 and 175 days of gestation to create persistently and transiently infected fetuses, respectively. In only persistently infected fetuses, encephaloclastic lesions resulting in pseudocysts were observed in the subependymal zone in the region of the median eminence and adjacent corona radiata as well as in the region of the external capsule associated with lenticulostriate arteries. Additionally, areas of rarefaction in white matter were observed at the tips of cerebrocortical gyri and in the external capsule. The distribution of viral antigen was examined by immunohistochemical labeling using the 15C5 anti-BVDV monoclonal antibody. Viral antigen was detected only in calves inoculated at 75 days of gestation, i.e., persistently infected. The pattern of BVDV immunolabeling revealed both similarities and differences compared with previous studies in postnatal calves, suggesting that viral infection in the brain is a dynamic and progressive rather than static process.

Genetic characterisation of a tick-borne encephalitis virus isolated from the brain of a naturally exposed monkey ( Macaca sylvanus)

In a recently published case study, we have described a clinical case of severe tick-borne encephalitis (TBE) in a monkey ( Macaca sylvanus) after natural exposure (tick bite) in a TBE endemic area in Germany. Using histological, immunohistochemical, serological methods, and RT-PCR, the TBE virus infection was confirmed. Here, we describe the isolation of a TBE virus from the brain tissue in Vero-B4 cell cultures and the sequencing of the complete genome of that isolate. The isolated TBE virus strain (named ‘Salem’) is closely related to the Kumlinge strain or strain Neudoerfl, the prototype of the Central European TBE virus subtype. However, a total of 268 nt changes were found in comparison with TBE virus strain Neudoerfl resulting in 28 amino acid changes, none of which affecting any of the known or supposed functional regions of the viral genome. Further investigation of the distribution of viral antigen in brain tissue and characterisation of the host's inflammatory reactions by immunohistology revealed similarities between the course of the TBE virus infection in the macaque and acute to peracute cases of TBE in humans.

Comparative Neuropathogenesis and Neurovirulence of Attenuated Flaviviruses in Nonhuman Primates

Based on previous preclinical evaluation in mice and monkeys, the chimeric TBEV/DEN4Delta30 virus, carrying the prM and E protein genes from a highly virulent Far Eastern strain of tick-borne encephalitis virus (TBEV) on the backbone of a nonneuroinvasive dengue type 4 virus (DEN4), has been identified as a promising live attenuated virus vaccine candidate against disease caused by TBEV. However, prior to use of this vaccine candidate in humans, its neurovirulence in nonhuman primates needed to be evaluated. In the present study, we compared the neuropathogeneses of the chimeric TBEV/DEN4Delta30 virus; Langat virus (LGTV), a former live TBEV vaccine; and yellow fever 17D virus vaccine (YF 17D) in rhesus monkeys inoculated intracerebrally. TBEV/DEN4Delta30 and YF 17D demonstrated remarkably similar spatiotemporal profiles of virus replication and virus-associated histopathology in the central nervous system (CNS) that were high in cerebral hemispheres but progressively decreased toward the spinal cord. In contrast, the neurovirulence of LGTV exhibited the reverse profile, progressing from the site of inoculation toward the cerebellum and spinal cord. Analysis of the spatiotemporal distribution of viral antigens in the CNS of monkeys revealed a prominent neurotropism associated with all three attenuated viruses. Nevertheless, TBEV/DEN4Delta30 virus exhibited higher neurovirulence in monkeys than either LGTV or YF 17D, suggesting insufficient attenuation. These results provide insight into the neuropathogenesis associated with attenuated flaviviruses that may guide the design of safe vaccines.

Evaluation of Hunter-Harvested White-Tailed Deer for Evidence of Bovine Viral Diarrhea Virus Infection in Alabama

Bovine viral diarrhea virus (BVDV) is one of the most relevant pathogens affecting today's cattle industries. Although great strides have been made in understanding this virus in cattle, little is known about the role of wildlife in the epidemiology of BVDV. While persistently infected cattle are the most important reservoir, free-ranging ungulates may become infected with BVDV as demonstrated by serosurveys and experimental infections. Therefore, free-ranging wildlife may maintain BVDV as the result of an independent cycle and may serve as a reservoir for the virus. Systematic studies on prevalence of BVDV-specific antibodies or frequency of persistent BVDV infection in North American wildlife are sparse, and no information is available from the southeastern United States. The objective of this study was to evaluate blood and skin samples from hunter-harvested white-tailed deer (Odocoileus virginianus) for evidence of BVDV infection. Virus-neutralizing antibodies were detected in 2 of 165 serum samples. Skin biopsy immunohistochemistry (IHC) was performed on samples from 406 deer using a BVDV-specific monoclonal antibody (MAb) (15c5), and BVDV antigen was detected in one sample. A similar IHC staining pattern was obtained using a second BVDV MAb (3.12F1). Viral antigen distribution in the skin sample of this deer resembled that found in persistently infected cattle and in a previously described persistently infected white-tailed deer; thus, the deer was presumed to be persistently infected. Evidence of BVDV infection in free-ranging white-tailed deer should encourage further systematic investigation of the prevalence of BVDV in wildlife.

Immunohistochemical Distribution of Viral Antigens in Pigs Naturally Infected with Porcine Teschovirus

A distribution of porcine teschovirus (PTV) antigens in pigs naturally infected with PTV is presented using the method of immunohistochemical examination. In the nervous system, PTV antigens were found in the cytoplasm of neuronal cells and glial cells distributed in the spinal ventral horn and brain stem, and also in the cytoplasm of ganglion cells in the spinal ganglion. No antigens were seen in the cerebral hemisphere. In the nervous system, the distribution of PTV antigens was consistent with lesions characteristic of nonsuppurative encephalomyelitis. In the other examined organ, PTV antigens were observed in bronchiolar epithelial cells in the lung, hepatocytes in the liver, epithelial cells in the tonsils and the myenteric nerve plexus in the small and large intestine.

Encephalitis in a Stone Marten (Martes foina) after Natural Infection with Highly Pathogenic Avian Influenza Virus Subtype H5N1

Recent outbreaks of disease in different avian species, caused by the highly pathogenic avian influenza virus (HPAIV), have involved infection by subtype H5N1 of the virus. This virus has also crossed species barriers and infected felines and humans. Here, we report the natural infection of a stone marten (Martes foina) from an area with numerous confirmed cases of H5N1 HPAIV infection in wild birds. Histopathological examination of tissues from this animal revealed a diffuse nonsuppurative panencephalitis with perivascular cuffing, multifocal gliosis and neuronal necrosis. Additionally, focal necrosis of pancreatic acinar cells was observed. Immunohistochemically, lesions in these organs were associated with avian influenza virus antigen in neurons, glial cells and pancreatic acinar cells. Thus, the microscopical lesions and viral antigen distribution in this stone marten differs from that recently described for cats naturally and experimentally infected with the same virus subtype. This is the first report of natural infection of a mustelid with HPAIV H5N1.

Characterization of protection against systemic infection and disease from experimental bovine viral diarrhea virus type 2 infection by use of a modified-live noncytopathic type 1 vaccine in calves

To evaluate protection resulting from use of a modified-live noncytopathic bovine viral diarrhea virus (BVDV) type 1 vaccine against systemic infection and clinical disease in calves challenged with type 2 BVDV. 10 calves, 5 to 7 months of age. Calves were allocated (n = 5/group) to be nonvaccinated or vaccinated SC on day 0 with BVDV 1 (WRL strain). Calves in both groups were challenged intranasally with BVDV type 2 isolate 890 on day 21. Rectal temperatures and clinical signs of disease were recorded daily, and total and differential WBC and platelet counts were performed. Histologic examinations and immunohistochemical analyses to detect lesions and distribution of viral antigens, respectively, were performed. After challenge exposure to BVDV type 2, nonvaccinated calves developed high rectal temperatures, increased respiratory rates, viremia, leukopenia, lymphopenia, and infection of the thymus. Vaccinated calves did not develop high rectal temperatures or clinical signs of respiratory tract disease. Vaccinated calves appeared to be protected against systemic replication of virus in that they did not develop leukopenia, lymphopenia, viremia, or infection of target organs, and infectious virus was not detected in peripheral blood mononuclear cells or the thymus. The modified-live BVDV type 1 vaccine protected against systemic infection and disease after experimental challenge exposure with BVDV type 2. The vaccine protected calves against infection and viremia and prevented infection of target lymphoid cells.

Novel Strategy for Treatment of Viral Central Nervous System Infection by Using a Cell-Permeating Inhibitor of c-Jun N-Terminal Kinase

Viral encephalitis is a major cause of morbidity and mortality worldwide, yet there is no proven efficacious therapy for most viral infections of the central nervous system (CNS). Many of the viruses that cause encephalitis induce apoptosis and activate c-Jun N-terminal kinase (JNK) following infection. We have previously shown that reovirus infection of epithelial cell lines activates JNK-dependent apoptosis. We now show that reovirus infection resulted in activation of JNK and caspase-3 in the CNS. Treatment of reovirus-infected mice with a cell-permeating peptide that competitively inhibits JNK activity resulted in significantly prolonged survival of intracerebrally infected mice following an otherwise lethal challenge with T3D (100 x 50% lethal dose). Protection correlated with reduced CNS injury, reduced neuronal apoptosis, and reduced c-Jun activation without altering the viral titer or viral antigen distribution. Given the efficacy of the inhibitor in protecting mice from viral encephalitis, JNK inhibition represents a promising and novel treatment strategy for viral encephalitis.