Presence Of Viral Antigen Research Articles

Western blot detection of infectious bursal disease virus infection.

In order to evaluate the use of a Western blot methodology for the diagnosis of infectious bursal disease virus (IBDV) infection, chickens were experimentally infected with IBDV strains and tested for the presence of viral antigens and antibodies by a blocking Western blot test (bWB). The viral proteins obtained from the bursa of Fabricius (BF) were transferred to a nitrocellulose membrane after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the chicken sera obtained by heart puncture were used for the detection of these proteins. In order to eliminate nonspecific reactions, we used a rabbit anti-chicken serum (blocking tool). By the use of the bWB test, two distinct viral proteins of 43-kDa (VP2) and 32-kDa (VP3) were detected. We suggest the use of this methodology for the detection of IBDV infection in animals suspected of having IBDV reinfection and a chronic subclinical form of the disease. With the use of the rabbit anti-chicken sera for blocking, this method is practical, sensitive and less time consuming.

Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe.

Samples of lung, liver, kidney, pancreas, spleen, and lymph node from pigs with postweaning multisystemic wasting syndrome from California (USA) and samples of mesenteric lymph nodes from similarly diseased pigs from Brittany (France) were examined by light microscopy, in situ hybridization (ISH), and/or virus isolation. Whole genomic probes for porcine circovirus (PCV) and chicken anemia virus (CAV) were used for ISH. Tissue homogenate supernatants were inoculated onto PK/15 cells for virus isolation, and the presence of viral antigen and viral particles was verified by indirect immunofluorescence, ISH, and electron microscopy. Histologic examination of lung from pigs from California revealed interstitial pneumonia, alveolar epithelial hyperplasia, and basophilic nuclear and cytoplasmic inclusions in mononuclear cell infiltrates and various pulmonary epithelial cells. Granulomatous lymphadenitis with syncytial cells typified the lesions seen in the pigs from France. PCV-like nucleic acid was detected by ISH in lung, pancreas, lymph node, kidney, and liver in pigs from California. Positive signal was also obtained in lymph node sections from pigs from France. Probes for CAV were consistently negative. PK/15 cell cultures inoculated with lung preparations from diseased California pigs and mesenteric lymph node preparations from pigs from France had positive fluorescence by indirect staining for PCV using pooled polyclonal pig sera and hyperimmune rabbit serum and had variable staining with a panel of 7 monoclonal antibodies specific for cell culture contaminant PCV. PCV-like nucleic acid was also detected by ISH in cell cultures. Cytopathic effect was not observed. Electron microscopic examination of inoculated cell cultures revealed 17-nm viral particles morphologically consistent with PCV. No other virus particles were observed. Although genomic analysis for the definitive identification of these viral isolates remains to be done, the evidence provided strongly suggests that these tissue isolates are closely related to, although antigenically distinct from, the original PCV cell culture contaminant.

Dynamics of wheat spindle streak mosaic bymovirus in winter wheat

The dynamics of wheat spindle streak mosaic bymovirus in winter wheat were studied during two crop cycles in a field site with a history of high virus incidence. Individual plants of two susceptible cultivars were sampled from autumn to spring and the presence of virus antigen in roots and leaves was determined by ELISA. Virus incidence was higher in cv. Frankenmuth than in cv. Augusta. During year one, incidence of viral antigen in roots remained very low for four months after sowing, and did not reach maximum levels until the following spring. During year two, incidence of viral antigen in roots rose to maximum levels in autumn, only three months after sowing. These results strongly suggested that root infection occurred in spring as well as in autumn. In both cultivars and in both years, we detected the virus in roots one month prior to its detection in leaves, suggesting that virus moves slowly from roots into leaves. Maximum incidence of virus in leaves occurred in spring of both years, coinciding with the period of symptom development. Typical symptoms (yellow streaks, spindles, and mosaic) were observed in year two, whereas only mild mosaic was observed in year one. Virus antigen was detected in nonsymptomatic leaves from two months after sowing through crop senescence. Because antigen could be detected in roots throughout the crop cycle, and zoosporangia and cystosori of the fungal vector could be detected one and two months, respectively, after sowing, it is possible that wheat spindle streak mosaic bymovirus is acquired and/or spread by the vector during the majority of the crop cycle.

Persistent poliovirus infection of human fetal brain cells

It has been suggested that poliovirus (PV), the causative agent of poliomyelitis, could persist in surviving patients. We have previously shown that PV can persistently infect some human cell lines in vitro, particularly neuroblastoma cell lines. We report here an ex vivo model in which PV can persistently infect primary cultures of human fetal brain cells. Two mutations involving capsid residues 142 of VP2 and 95 of VP1 were repeatedly selected during the persistent infections. These residues are located in capsid regions known to be involved in interactions between PV and its receptor. During the first week after infection, viral antigens were found in cells of both the neuronal and glial lineages. In contrast, 2 weeks after infection, viral antigens were detected almost exclusively in cells of the neuronal lineage. They were detected predominantly in cells expressing a marker of early commitment to the neuronal lineage, MAP-5, particularly in neuroblasts. Viral antigens were also found in immature progenitors expressing a neuroepithelium marker, nestin, and in cells expressing a marker of postmitotic neurons, MAP-2. The presence of viral antigens in postmitotic neurons suggests that PV can persist in neurons of patients who have survived poliomyelitis.

Pathogenesis of an attenuated and a virulent strain of group A human rotavirus in neonatal gnotobiotic pigs.

Gnotobiotic (Gn) pigs were orally inoculated with Wa strain (G1P1A[P8]) human rotavirus (Wa HRV) serially passaged in Gn pigs (virulent) or cell culture (attenuated) to determine the median virus infectious dose (ID50) and to assess the site of infection and type and progression of morphological lesions and clinical responses induced by these two strains in Gn pigs. The ID50 of virulent Wa HRV was = or < 1 f.f.u. whereas the infectivity of attenuated Wa HRV had to be determined by seroconversion and was approximately 1.3 x 1O(6) f.f.u. Diarrhoea developed at 13 h post-inoculation (p.i.) in pigs inoculated with approximately 1O(5) f.f.u. of virulent Wa HRV and correlated with the presence of viral antigen within villous epithelial cells; villous atrophy developed later at 24 h p.i. and correlated with peak faecal viral titres; recovery from disease correlated with the return of morphologically normal villi. Virus, diarrhoea and villous atrophy were not detected in pigs inoculated with approximately 2 x 10(8)f.f.u. attenuated Wa HRV although HRV-specific serum antibodies were present by 7 days p.i. These findings demonstrate that virulent Wa HRV infection in Gn pigs occurs primarily within intestinal villous epithelial cells with villous atrophy developing as a sequela to infection. However, factors other than villous atrophy appear to contribute to the early stages of HRV-associated disease expression in Gn pigs. The ability of the attenuated virus to elicit virus-neutralizing serum antibodies without disease or pathology indicates promise in the use of such strains for oral immunization.

Presence of rabbit haemorrhagic disease virus antigen in rabbit tissues as revealed by a monoclonal antibody dependent capture ELISA.

The production of monoclonal antibodies (mAbs) to a common antigenic region on rabbit haemorrhagic disease virus (RHDV) has enabled the development of a capture ELISA for virus detection. The assay was shown to detect reliably the presence of viral antigen in crude homogenates of a range of rabbit tissues and has provided the first evidence for the presence of RHDV in blood, serum and heart tissue. A limited time course study of the progression of virus infection revealed viral antigen is first detected in the liver at between 18 and 24 h post-infection (p.i.) and in the spleen between 24 and 30 h p.i. The assay displayed a high level of specificity, clearly differentiating between groups of infected (lowest observed optical density (O.D.) 0.45) and uninfected (highest O.D. 0.06) rabbits. No viral antigen was detected in the urine or faeces collected from infected rabbits at post-mortem. Experiments employing the 'spiking' of pools of urine and faecal homogenates collected from uninfected rabbits, with a known amount of virus, indicated that RHDV may not be present in the faeces of infected animals and if present in urine, it appears to undergo substantial degradation.

Prospective study of antigenemia, plasma viremia and lymphocytic viremia in HIV-infected hemophiliacs.

A total of 186 blood samples from 24 HIV-1 seropositive hemophiliac patients, monitored every four months for 29 months, were investigated for the presence of viral antigen in plasma. In addition, peripheral blood mononuclear cells (PBMC) were cultured for HIV-1, using normal PBMC as a target for replication. Antigenemia was detected in 51% of the patients and from PBMC in 87.5% of the patients. The incidence of HIV isolation in asymptomatic patients (42.8%) was similar to that found in symptomatic patients (51.4%). Patients with opportunistic infections had a higher incidence of lymphocytic viremia (p < 0.05). Plasma viremia was closely associated (p < 0.05) with low CD4+ counts and infection progression. The persistence of antigenemia was also a marker of a poor clinical course. In treated patients, plasma viremia was the marker that better correlated with the clinical course, and it did not appear during the first nine months of therapy. Zidovudine doses of > 500 mg/day significantly lowered the appearance of antigenemia and lymphocytic viremia (p < 0.05).

Experimental rabies infection of non-nervous tissues in skunks (Mephitis mephitis) and foxes (Vulpes vulpes).

Non-neural tissues, from three male and four female stripped skunks (Mephitis mephitis), 5 to 7 months old, and one male and two female red foxes (Vulpes vulpes), 12 to 16 months old, experimentally infected with street rabies virus, were examined by light microscopic immunohistochemical and electron microscopic methods. This is the first report of ultrastructural lesions in rabies-infected adrenal medulla, cornea, and nasal glands. Using the streptavidin biotin peroxidase technique, antigen was detected in mucous cells and interstitial neurons and their processes in the submandibular salivary gland, in chromaffin cells of the adrenal medulla, in epidermal cells of the skin, in external root sheath cells of hair follicles, and in corneal epithelial cells. Electron microscopically, matrix (viral nucleocapsid), virions, and anomalous viral products were common in most tissues examined, but their relative proportions varied. The results suggested that replication with minimal accumulation of matrix and anomalous viral growth products was characteristic of growth in tissues (submandibular salivary gland) that frequently produce high titers of virus, whereas replication with large amounts of matrix and anomalous structures occurred in tissues (adrenal gland and nasal gland) that generally contained low or moderate titers of virus. Novel findings included viral budding into secretory granules, increase in microfilaments in infected mucogenic cells, and continuity of viral convoluted membranous profiles with rough endoplasmic reticulum of chromaffin cells and nasal glandular cells. The presence of viral antigen and developing virus in extra-neural tissues constitutes a potential risk of non-bite exposure to people in certain groups/occupations.

Electron microscopic evidence of a viral nature for osteoclast inclusions in Paget's disease of bone.

Circumstantial evidence from electron microscopic and immunological studies support the view that Paget's disease of bone represents a slow virus infection. However, there is only limited information available regarding its electron microscopic, enzyme and immunocytochemical characteristics. Two cases were studied using electron microscopy with particular emphasis on the inclusions in osteoclasts. Detailed ultrastructural and cytochemical studies including immuno-electron microscopy were performed. Some osteoclasts demonstrated specific virus-like structures composed of aggregations of microtubules in the nucleus and cytoplasm. The structures were easily digested by trypsin or protease, and were sensitive to RNase, which provided substantial evidence of a proteinaceous nature and inclusion of ribonucleic acid. Immunocytochemical examination identified binding of anti-respiratory syncytial virus and anti-measles virus antibodies in the tissue obtained from one of the two cases examined. The presence of viral antigens in structures in the cytoplasm of Pagetic osteoclasts supports the theory of paramyxovirus involvement in this disease.

A study from south India on the association between the papilloma virus and carcinoma of the penis.

Specimens from 39 cases of squamous cell carcinoma of the penis were treated for the papilloma virus using the bovine papilloma virus antibody and the peroxidase-antiperoxidase technique. All 39 cases showed a viral antigen situated intranuclearly, intracytoplasmically or both intranuclearly and intracytoplasmically. No correlation could be seen between the presence of viral antigen and age of patient, duration of lesion, cell morphology and histological grade. The adjacent mucosa and skin were used as controls and showed no viral antigen. The inflammatory infiltrate was dense and predominantly "T" lymphocyte in type.

Pathogenesis of Pichinde virus infection in strain 13 guinea pigs: an immunocytochemical, virologic, and clinical chemistry study.

Pichinde virus has been adapted to produce lethal infection of Strain 13 guinea pigs. Viral replication and presence of viral antigen in frozen tissues stained by immunofluorescence has been previously described. Further investigation into the pathogenesis of this disease has been hampered by the lack of a light microscopic method for correlating histologic lesions and the presence of Pichinde viral antigens. For this purpose, we developed a sensitive immunocytochemical technique for staining Pichinde viral antigens in formalin-fixed, paraffin-embedded tissue. Enhancement of the immunocytochemical staining with nickel chloride markedly improved detection of viral antigens. We examined frozen and formalin-fixed tissues from Strain 13 guinea pigs for viral antigens by light microscopy and immunocytochemistry at various intervals after infection with Pichinde virus. Progressive involvement of different tissues correlated with organ injury measured by serum biochemical abnormalities. Pichinde viral antigen was first detected in splenic macrophages five days after infection and their subsequent destruction facilitated persistent viremia. The inability to clear virus led to multiple organ infection and vascular involvement. Ensuing infections involved particularly the liver, spleen, adrenal glands, lungs, and intestines. Gastroenteritis developed, with extensive involvement of the muscularis mucosa throughout the gastrointestinal tract. Water and food intake decreased rapidly after day 8, leading to marked weight loss. Fatty changes of the liver suggested metabolic derangement that was further exacerbated terminally by adrenal infection and pulmonary impairment.

Antivirally protective cytotoxic T cell memory to lymphocytic choriomeningitis virus is governed by persisting antigen.

The basis of antiviral protection by memory cytotoxic T lymphocytes (CTL) was investigated in vivo and in vitro using lymphocytic choriomeningitis virus (LCMV) and recombinant vaccinia viruses expressing the LCMV-glycoprotein (vacc-GP) or -nucleoprotein (vacc-NP). The widely replicating LCMV with a tendency to persist induced solid long-term protective memory. The poorly replicating vaccinia recombinant viruses revealed in the vaccinated host that the antiviral capacity of the secondary immune T cell response and the protection against lethal LCM was dependent upon the immunizing antigen and its dose. Protection against lethal choriomeningitis is less sensitive to assess memory because it depends upon high levels of CTL precursors (p) and/or on an activated state of memory CTL. In contrast, antiviral protection measured as the capacity of the primed host to reduce virus titers after challenge infection correlated with elevated CTLp frequencies after immunization with live LCMV or recombinant vaccinia virus-expressing the major LCMV epitope. CTLp frequencies were constantly increased up to 70 d for LCMV immune mice, but rapidly decreased a few weeks after immunization with low dose vaccinia recombinant virus. For example, mice primed with 2 x 10(6) plaque-forming units (PFU) of vacc-NP, or 2 x 10(2) PFU, or 2 x 10(6) PFU of vacc-GP were antivirally protected on day 7 but not after day 30 when CTLp could not be measured any longer in vitro. However, greater priming doses of vacc-NP (10(4) or 2 x 10(6) PFU) as well as LCMV (2 x 10(2) PFU) induced elevated levels of CTLp and antiviral protection for 60 d or longer. Adoptive transfer experiments of immune spleen cells into syngeneic recipients without addition of antigen demonstrated that maintenance of the antiviral protective capacity of the transferred cells depended on the presence of viral antigen. Thus, antiviral protection by memory CTL may be rather short-lived since it is based on activated T cells continuously stimulated by persisting antigen. This is best achieved by high immunizing antigen doses yielded either by widely replicating viruses or high doses of poorly replicating recombinant vaccines.

Effect of “in vitro” exposure of bovine alveolar macrophages to different strains of bovine respiratory syncytial virus

A vaccine strain of respiratory syncytial (RS) virus and an isolate from pneumonic calves (AC2) were inoculated onto cultures of bovine alveolar macrophages recovered by lung lavage, and the functional properties of the cells observed over a period of 10 days. In most cultures no infectious virus was produced although immunofluorescence indicated the presence of virus antigens in some cells. No significant difference was noted between infected and control macrophage cultures in their capacity to phagocytose latex particles (neutral phagocytosis), although the ability to phagocytose complement-coated Candida krusei cells was affected, particularly with the AC2 strain after 6 days. Killing of C. krusei cells was slightly affected by infection of macrophages with the vaccine strain and was dramatically affected by infection with strain AC2. C3b and Fc receptor expression was adversely affected by both virus strains. Production of neutrophil chemotactic factors was increased in cultures infected with both strains, but was greater with AC2, suggesting that some properties of the cells were activated.

Pathogenesis of early and late disease in mice infected with Theiler's virus, using intratypic recombinant GDVII/DA viruses

Intratypic recombinant Theiler's viruses prepared between GDVII and DA strains were used to identify genomic sequences important in neurovirulence, virus persistence, and demyelination and to clarify the mechanisms involved in disease induction. The coding region between 1B and 2C of the highly virulent GDVII strain contains a determinant partly responsible for neurovirulence (early paralysis and death) which correlates with elevated levels of infectious virus and the presence of virus antigen within neurons of the brain stem and gray matter of the spinal cord. Both the GDVII and the DA strains of virus contain genetic determinants for late demyelination in spinal cord. However, quantitative analysis of demyelination produced by recombinant GDVII/DA viruses suggest that multiple gene segments influence the number and extent of demyelinating lesions.

Detection of viral antigen following exposure of one‐day‐old chickens to the Holland 52 strain of infectious bronchitis virus

One-day-old specific-pathogen-free single comb White Leghorn chickens were inoculated by eyedrop with either 0.1 ml of phosphate buffered saline containing 10(4.3) EID50 of the Holland 52 strain of infectious bronchitis virus or normal allantoic fluid. Trachea, caecal tonsils and kidneys were removed from randomly selected birds at 0, 3, 5, 7 and 10 days post-inoculation (pi) and the presence or absence of viral antigen was detected utilizing virus isolation (VI), an indirect fluorescent antibody technique (IFA), or a streptavidin-biotin immunohistochemical (IH) technique. The presence of viral antigen as detected by the IH technique was also compared to histopathological changes in serial sections stained with haematoxylin and eosin. Detection of viral antigen occurred more frequently with VI than with IFA or IH. The IFA and IH techniques detected viral antigen with about the same frequency. Viral antigen was detected in the mucosa and submucosa of the trachea as early as 3 days pi, reached maximum levels at 5 days pi, and could still be detected in the mucosa at 10 days pi. In the kidney, viral antigen was not detectable by IFA or IH at 3 days pi, but could be visualized in distal convoluted tubules and collecting tubules at 5 days pi. At 7 days pi, antigen was detectable in the proximal convoluted tubules also. The presence of antigen in the caecal tonsils was sporadic, but it was detected in histiocytic cells and, occasionally, lymphoid cells of that organ.

The infection of mouse preimplantation embryos to sendai virus (parainfluenza I)

To provide information on the susceptibility of mouse embryos to Sendai virus, it was investigated if viral replication occurs in the preimplantation embryo at different stages of development, with or without the zona pellucida (ZP). Mice were induced to superovulate, and embryos were collected on Days 2, 3 and 4 after mating. The ZP was removed by digestion with 0.5% pronase. Embryos were exposed to Sendai virus, washed, and allowed to develop in fresh culture medium. The presence of viral antigen in the embryonic cells was examined by the fluorescent antibody test (FAT). Specific immunofluorescence was demonstrated in the ZP-free morula and ZP-intact blastocyst. However, viral antigen was not detected in the ZP-intact two-cell, four-cell, eight-cell or morula stage embryos. Infected embryos developed normally to expanded blastocysts. These findings show that mouse embryonic cells are permissive hosts to Sendai virus replication and that the ZP played the role of a barrier against the virus.

Variations in the Replication and Antigenic Properties of Human Herpesvirus 6 Strains

The Z29 and U1102 strains of human herpesvirus 6 (HHV-6) were compared for their ability to replicate in fresh peripheral blood lymphocytes (PBL) and in continuous T cell lines. The replication of both strains in PBL was enhanced by mitogenic activation of cell growth. U1102 replicated in the continuous T cell lines, J JHAN and HSB-2, whereas no Z29 replication was observed in these cell lines as judged by infectious virus yields, the presence of viral antigens, and viral DNA replication. The two strains were compared with respect to their ability to react in immunofluorescence assays with monoclonal antibodies (MAbs) prepared against the GS strain of HHV-6. These MAbs are directed against six different polypeptides including three glycoproteins. All MAbs reacted with cells infected with the U1102 strain. The Z29-infected cells reacted with four MAbs but failed to react with MAbs specific for an 82- to 105-kDa major surface glycoprotein and with one MAb reactive with a nonglycosylated 180-kDa protein. Taken together, the two strains of HHV-6 exhibit variations with regard to their growth and antigenic properties.

Development and application of an improved infectivity assay for the standardization of avian encephalomyelitis vaccines

An improved infectivity assay for avian encephalomyelitis viruses (AEVs) is described in which susceptible 7-day-old chicken embryos were inoculated with dilutions of a particular AEV strain and, after incubation for 12 days, the presence of viral antigen in brains was detected by an enzyme-linked immunosorbent assay (ELISA). The assay (the brain antigen ELISA), which may be used for the titration of both egg-adapted and non-egg-adapted strains, can be completed within 12 days. For the conventional essays of non-egg-adapted vaccine strains, embryos are infected in ovo and hatched chickens examined for neurological signs after 3-4 weeks. No animal handling facilities are required with the brain antigen ELISA and results of comparable sensitivity to the hatch-out assay were obtained.

Antiviral antibodies stimulate production of reactive oxygen species in cultured canine brain cells infected with canine distemper virus.

Canine distemper is characterized mainly by respiratory, enteric, and nervous symptoms. Infection of the central nervous system results in demyelination, to which inflammation has been shown to contribute significantly. It has been proposed that macrophages play a major role as effector cells in this process. We report that cultured dog brain cells contain a population of macrophages capable of producing reactive oxygen species as measured by luminol-dependent chemiluminescence. In cultures infected with canine distemper virus, a burst of reactive oxygen is triggered by antiviral antibody. This response depends on the presence of viral antigens on the surfaces of infected cells and is mediated by the interaction of antigen-bound antibody with Fc receptors on the macrophages. Since there is no evidence in vitro or in vivo that oligodendrocytes, the cells forming myelin, are infected, our observation supports the hypothesis that "innocent bystander killing" is important in demyelination caused by canine distemper virus. Reactive oxygen species released from macrophages may contribute to destruction of myelin.

Theiler's virus infection in nude mice: viral RNA in vascular endothelial cells.

Infection of athymic (nu/nu) mice with Theiler's murine encephalomyelitis virus results in an acute encephalitis which resembles poliomyelitis. Immunohistochemistry and in situ hybridization were used to delineate the presence of viral proteins and RNA in the nervous systems of nude mice infected with the Daniels strain of Theiler's virus. This system permits the analysis of a viral infection in the absence of an effective immune response. By immunohistochemistry, viral antigen was found in the processes and cell bodies of neurons and glial cells. Besides the presence of viral antigen in these cell types, by in situ hybridization, Theiler's virus RNA was also found in cells associated with vascular endothelium in the brains and spinal cords of these infected mice. Theiler's virus RNA-positive endothelial cells were observed not only near the primary lesions but also away from demonstrable lesions in normal-appearing regions in the central nervous system. Earlier work had suggested an intra-axonal dissemination for this virus (M. C. Dal Canto and H. L. Lipton, Am. J. Pathol. 106:20-29, 1982). Our findings are consistent with this model but also suggest an additional mechanism for virus spread within the central nervous system, i.e., by infecting vascular cells and crossing the blood-brain barrier. Lastly, after Theiler's murine encephalomyelitis virus infection, not only glial cells but also endothelial cells express major histocompatibility complex class II (la) antigen on their surface (M. Rodriguez, M. L. Pierce, and E. A. Howie, J. Immunol. 138:3438-3442, 1987). Our demonstration of Theiler's virus-infected endotheliumlike cells may explain interactions of virus products in stimulating antigen presentation.