Experimental Sendai virus infection in laboratory rats. II. Pathology and immunohistochemistry

Intranasal inoculation of 5 to 8 week old specific pathogen-free Sprague-Dawley rats with 5 X 10(3) egg infectious doses of Sendai virus resulted in severe rhinitis, bronchiolitis and alveolitis. The most severe rhinitis occurred on postinoculation (PI) days 4-6, and pneumonia on day 4. Rhinitis and pneumonia persisted to PI day 21, with peribronchial lymphoid infiltration detectable at PI day 42. Immunohistochemical studies showed that Sendai virus antigens were present primarily in columnar epithelial cells of the respiratory mucosa of the nasal cavity and in bronchiolar and alveolar epithelium. Antigen was first detectable at PI day 1, was most prominent at days 3-4 and was undetectable after day 7. More antigen could be seen in the nasal mucosa than in the lung at any stage in the infection. These studies show that Sendai virus by itself is capable of evoking severe, although transient, rhinitis and pneumonia in laboratory rats free of other significant pathogens.     

Immunofluorescence and electron microscopy of the cytoplasmic surface of the human erythrocyte membrane and its interaction with Sendai virus

A method was developed for directly observing the inner surfaces of plasma membranes by light and electron microscopy. Human erythrocytes were attached to cover slips (glass or mica) treated with aminopropylsilane and glutaraldehyde, and then disrupted by direct application of a jet of buffer, which removed the distal portion of the cells, thus exposing the cytoplasmic surface (PS) of the flattened membranes. Antispectrin antibodies and Sendai virus particles were employed as sensitive markers for, respectively, the PS and the external surface (ES) of the membrane; their localization by immunofluorescence or electron microscopy demonstrated that the major asymmetrical features of the plasma membrane were preserved. The fusion of Sendai virus particles with cells was investigated using double- labeling immunofluorescence techniques. Virus adsorbed to the ES of cells at 4 degrees C was not accessible to fluorescein-labeled antibodies applied from the PS side. After incubation at 37 degrees C, viral antigens could be detected at the PS. These antigens, however, remained localized and did not diffuse from the site of attachment, as is usually seen in viral antigens accessible on the ES. They may therefore represent internal viral antigens not incorporated into the plasma membrane as a result of virus-cell fusion.     

Prevalence of antibodies to Sendai virus and rotavirus in laboratory rabbits

One hundred and sixty rabbit sera from 10 breeding colonies and 13 laboratory colonies were tested for antibodies to Sendai virus and rotavirus by enzyme-linked immunosorbent assay (ELISA). Antibodies were detected to Sendai virus in 53% and to rotavirus in 81%, indicating the prevalence of these viral infections in laboratory rabbit colonies.     

Analysis of experimental mink enteritis virus infection in mink: in situ hybridization, serology, and histopathology.

Strand-specific hybridization probes were used in in situ hybridization studies to localize cells containing mink enteritis virus (MEV) virion DNA or MEV replicative-form DNA and mRNA. Following the experimental MEV infection of 3-month-old unvaccinated mink, a significant increase in serum antibodies to MEV was detected at postinfection day (PID) 6, 2 days after the onset of fecal shedding of virus. Prior to the appearance of virus in feces, viral DNA could be detected in the mesenteric lymph node and intestine. The largest percentage of cells positive for virion DNA was 10% and was detected in the intestine on PID 6. However, replication of the virus apparently peaked at PID 4. The number of MEV replicative-form DNA and mRNA molecules was found to be approximately 250,000 copies per infected lymph node cell or crypt epithelial cell. The localization, levels, and time course of viral replication have important implications for the pathogenesis of MEV-induced disease. The data presented on MEV are correlated with earlier results on the other mink parvovirus, Aleutian mink disease parvovirus, and a possible explanation for the remarkable differences in pathogenesis of disease caused by these two parvoviruses is discussed.     

Synthesis of viral components in hybrids of differentially permissive cells infected with adenovirus type 12

Sendai virus induced heterokaryocytes of non-permissive BHK21 cells and permissive HEp2 cells were infected with adenovirus Type 12. Neoantigens, virus structural antigens and viral DNA were detected in the BHK21 nuclei as well as the HEp2 nuclei of heterokaryocytes, thus demonstrating positive control of these viral functions by the permissive cell component in the heterokaryocyte.     

Immunoelectron microscopic study on interactions of noninfectious sendai virus and murine cells.

The early interactions of LLC-MK2 cell-grown noninfectious Sendai virus and a murine cell line, P815 mastocytoma ascitic cells, were studied by electron microscopy, using the ferritin-conjugated antibody technique with anti-virus glycoprotein serum. For comparison, the interactions of egg-grown infectious Sendai virus with the same cells were also examined. When noninfectious virus was adsorbed to the cells in the cold, the cell membranes become partially invaginated at the site of contact of adsorbed virions, but ferritin-conjugated antibodies did not penetrate into the areas of envelope-cell membrane association. This pattern of virus attachment was similar to that of infectious virus attachment. Upon subsequent incubation at 37 degrees C, most of the adsorbed noninfectious virions were taken into cytoplasmic vesicles and then degraded, although a few virions remained attached to the cell membrane. No evidence of fusion of envelopes of noninfectious virions was obtained. On the other hand, envelopes of infectious virions fused with the cell membrane, and the transferred viral antigens diffused on the cell surfaces and then decreased in number.     

Protection of mice from respiratory Sendai virus infections by recombinant vaccinia viruses.

Mechanisms of protection of mice from Sendai virus, which is exclusively pneumotropic and causes a typical respiratory disease, by immunization with recombinant vaccinia viruses (RVVs) were investigated. Although the RVV carrying a hemagglutinin-neuraminidase gene of Sendai virus (Vac-HN) propagated in the noses and lungs of mice by either intranasal (i.n.) or intraperitoneal (i.p.) inoculation, no vaccinia virus antigens were detected in the mucosal layer of upper and lower airways of the i.p.-inoculated mice. The mice immunized i.n. with Vac-HN or Vac-F (the RVV carrying a fusion protein gene of Sendai virus) demonstrated the strong resistance to Sendai virus challenge both in the lung and in the nose, whereas the i.p.-immunized mice showed almost no resistance in the nose but showed a partial resistance in the lung. Titration of Sendai virus-specific antibodies in the nasal wash (NW), bronchoalveolar lavage (BAL), and serum collected from the Vac-F-immunized mice showed that the NW from the i.n.-immunized mice contained immunoglobulin A (IgA) antibodies but no IgG and the BAL from the mice contained both IgA and IgG antibodies. On the other hand, neither IgA nor IgG antibodies were detected in the NW from the i.p.-immunized mice and only IgG antibodies were detected in the BAL, although both i.n.- and i.p.-immunized mice exhibited similar levels of serum IgG, IgA, and neutralizing antibodies. The resistance to Sendai virus in the noses of i.n.-immunized mice could be abrogated by the intranasal instillation of anti-mouse IgA but not of anti-IgG antiserum, while the resistance in the lung was not significantly abrogated by such treatments. These results demonstrate that IgA is a major mediator for the immunity against Sendai virus induced by the RVVs and IgG is a supplementary one, especially in the lung, and that the RVV should be intranasally inoculated to induce an efficient mucosal immunity even if it has a pantropic nature.     

Survey of virally mediated permeability changes.

1. Sendai virus causes permeability changes when added to freshly isolated brain cells (cerebellum or ependymal cells) or to a culture of forebrain cells. 2. Sendai virus causes permeability changes when added to organ cultures of ferret lung or nasal turbinate. Influenza virus causes no permeability changes under these conditions. 3. Rabies virus and vesicular-stomatitis virus, in contrast with Sendai virus, do not cause permeability changes in BHK cells or Lettrée cells. 4. Serum from patients suffering from viral hepatitis does not cause permeability changes in human leucocytes; addition to Sendai virus causes permeability changes. 5. It is concluded that permeability changes accompanying viral entry occur only with certain types of paramyxovirus, but that there is little restriction on cell type. 6. MDBK cells infected with Sendai virus show permeability changes during viral release, similar to those that occur during viral entry. Because these changes do not appear to be restricted to paramyxoviruses, they may have considerable clinical significance.     

清洁级实验动物四种病毒抗体玻片酶免疫检测试剂盒的研制

本文报道对清洁级实验动物应排除的四种病毒（淋巴细胞脉络丛脑膜炎病毒、小鼠脱脚病病毒、鼠肝炎病毒和仙台病毒）抗体玻片酶免疫（ＥＩＡ）检测试剂盒的研制。四种病毒感染的细胞和对照细胞经冷丙酮固定于载玻片上制成特异性抗原和对照抗原,此四种病毒的抗血清各１０份和ＳＰＦ小鼠血清２０份分别与四种病毒的特异性抗原和对照抗原进行ＥＩＡ交叉试验,结果显示,抗原只与其相应抗血清发生特异性显色反应,与非特异性小鼠血清和ＳＰＦ小鼠血清不显色。与ＨＩ或ＥＬＩＳＡ方法比较,通过对１１２份普通小鼠血清进行测试,结果表明,ＥＩＡ对仙台病毒抗体的检出率（１９．６％）显著高于（＜０．００５）ＨＩ（６．３％）,对小鼠脱脚病病毒抗体的检出率（２３．３％）与ＨＩ（２１．４％）无显著性差异（Ｐ＞０．０５）。ＥＩＡ对淋巴细胞脉络丛脑膜炎病毒和鼠肝炎病毒抗体的检出率分别为１．８％和７１．２％,ＥＬＩＳＡ对两种病毒抗体的检出率分别为１．８％和６７．６％,两种方法对两种病毒抗体的检出率无显著性差异（Ｐ＞０．０５）。重复性试验表明两批四种病毒抗体试剂盒对１０８份小鼠血清两次测定的符合率为９６～１００％。四种病毒的ＥＩＡ抗原在－１８℃保存１２个月或在２－８℃保存３     

Colonization of rabbits by Pasteurella multocida: serum IgG responses following intranasal challenge with serologically distinct isolates.

Enzyme-linked immunosorbent assays (ELISAs) and immunoblots were used to measure serum IgG responses in rabbits which were intranasally challenged with Pasteurella multocida. The responses to two serologically distinct isolates (isolate 1, serotype 3:A and isolate 10, serotype 1:D) were compared and then correlated with the ability of the isolates to colonize the nasal passages. Five rabbits were challenged with each isolate (10(5) CFU); nasal washings and sera were collected weekly for 8 weeks. Serum IgG levels were measured by ELISA and immunoblots, using bacterial whole cells and lipopolysaccharides (LPSs) as antigens. The serum IgG response to isolate 1 was evident earlier and was significantly stronger than the response to isolate 10 (P less than 0.025). Immunoblots supported this observation and confirmed that both isolates elicited antibodies which reacted with bacterial protein and LPS antigens, with antibody to protein detectable before antibody to LPS. Results of weekly nasal cultures suggested that the antibody response data could be explained by a difference in the ability of the isolates to colonize the nasal passages: isolate 1 was recovered from four of five rabbits for 8 weeks, whereas isolate 10 was recovered for a maximum of 2 weeks, even when the challenge dose was increased tenfold. The strong response elicited by isolate 1 was therefore probably a result of persistent colonization, whereas the weak response to isolate 10 may have resulted from an inability to persistently colonize the nasal passages. The results of this study demonstrate that isolates of P. multocida elicit antibody responses of differing intensities and vary in their ability to colonize the nasal passages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of Immune Responses of Rabbits Infected with Bovine Immunodeficiency-Like Virus

Nine 3-month-old rabbits were inoculated with bovine immunodeficiency-like virus (BIV) to study the pathogenesis of BIV and alteration of the immune responses in experimentally infected rabbits. BIV proviral DNA and anti-BIV antibodies were detected from all rabbits inoculated with BIV-infected bovine embryo spleen (BESP) cells. Rabbits inoculated with spleen cells of the BIV-infected rabbit also converted to proviral DNA-positive and BIV-antibody-positive. The blastogenic responses to concanavalin A of peripheral blood mononuclear cells prepared from BIV-infected rabbits were not significantly different from those from uninfected controls at 2 and 4 months post-inoculation (PI). The humoral immune responses against bovine serum albumin (BSA) were depressed in two of four BIV-infected rabbits at 1 to 3 months PI. The antibody responses against sheep red blood cells (SRBCs) were significantly depressed in all BIV-infected rabbits at 2 to 4 months PI. BIV was rescued by cocultivation of spleen cells of infected rabbits with BESP cells. Distinct development of lymphoid follicle was observed in lymph nodes and spleens of uninfected rabbits which received BSA and SRBCs. In contrast, moderate lymphoid cell depletion was observed in BIV-infected rabbits which received the same immunogens.     

Loss of reactivity of a myeloma tumor with H-2 compatible thymus-derived lymphocytes.

It was previously shown that MOPC-315-EL, a subline of the BALB/c myeloma tumor MOPC-315, reversibly alters its reactivity with T cells that recognize H-2^d, 2,4,6-trinitrophenyl, and minor histocompatibility antigens. This report demonstrates (a) that CTLs directed against vesicular stomatitis virus and Sendai virus are unable to recognize viral antigens associated with the unreactive tumor cell, and (b) that incorporation of Sendai virus antigens into the same membrane as the H-2 gene products is required for effective recognition by cytotoxic T lymphocytes.     

Cooperation between humoral factor(s) and Lyt-2+ T cells in effective clearance of Sendai virus from infected mouse lungs

The mechanism of cooperation between the L3T4+ and Lyt-2+ T cell subsets in effective clearance of Sendai virus from infected mouse lungs was studied by adoptive cell transfer using nude mice. Simultaneous transfer of a long-term-cultured Sendai virus-specific L3T4+ T cell line with L3T4+ cell-depleted immune spleen cell (L3T4-) fraction to infected nude mice could result in viral clearance, although single injection with either of these cells was not effective. Instead of the L3T4+ T cells, culture supernatants of the L3T4- T cell line or concanavalin A-stimulated mouse spleen cells and mouse serum immunized with the virus were also active in the cooperative viral clearance with L3T4- fraction. The role of the Sendai virus-sensitized L3T4- cell fraction in cooperative viral clearance with humoral factors could be replaced by neither T cell-deprived immune spleen cell fraction nor normal spleen cells. The 1,500 units of recombinant mouse interleukin 2 (IL-2), which was more than 12 times the IL-2 activity present in the supernatants of the T cell line or concanavalin A-stimulated spleen cells, failed to clear the virus in combination with the L3T4- fraction. Monoclonal antibodies to Sendai or mouse hepatitis viruses were also effective in the cooperative antiviral activity. IL-2 activity was not detected in these monoclonal antibodies and the mouse immune serum. Single injection of any humoral factors failed to clear the virus. These results indicate that Sendai virus-sensitized Lyt-2+ subset of T cells acts cooperatively with humoral factor(s) other than IL-2 or Sendai virus-specific antibody present in supernatants of the T cell line, of concanavalin A-stimulated spleen cells or hybridomas, and in mouse serum immunized with the virus.     

Subcellular location of the major protein antigens of paramyxoviruses revealed by immunoperoxidase cytochemistry

The major structural proteins of Newcastle disease virus and Sendai virus were localized in infected BHK-21 and MDBK cells by ultrastructural immunoperoxidase cytochemistry using antibodies against the individual viral protein antigens. The intracellular glycoproteins were strictly membrane bound, being localized in the rough endoplasmic reticulum (RER), perinuclear spaces, smooth membrane vesicles, and presumed Golgi apparatus. The nucleocapsid proteins were detected exclusively in membrane free cytosol and accumulated there, forming inclusions. The membrane (M) protein was found both in cytosol and on RER. The viral proteins on RER exhibited a distinct site specificity; the glycoproteins were facing the lumen of RER whereas M protein was present at the outer cytoplasmic surface. All the viral proteins were detectable at the plasma membrane where virus assembly takes place. However, their modes of distribution differed remarkably. The glycoproteins were spread widely over the entire cell surface including the areas of virus budding and those of normal morphology, whereas M protein was localized in restricted areas of the membrane, frequently forming a patch of virus specific membrane. The presence of nucleocapsids was confined to the virus particles budding from the plasma membrane. These results complement and extend the earlier morphological and biochemical data on the assembly or morphogenesis of paramyxoviruses.     

Membrane receptor mobility changes by sendai virus

Treatment with Sendai virus does affect the lateral mobility of cell surface components on cultured human cells (KB) and cultured mouse cells (3T3). Diffusion coefficients and mobile fractions of a fluorescent lipid analog, tetramethyl rhodamine-succinyl concanavalin A (ConA) and tetramethyl rhodamine-anti human β₂ microglobulin antibodies on the cell surface were measured by fluorescence photobleaching recovery (FPR). Diffusion coefficients of receptors for ConA and for antibodies to β₂ microglobulin (presumably histocompatibility antigens) were increased 2- to 3-fold by exposure to the ultraviolet inactivated virus at virus doses from 250 to 2 500 HAU/ml, regardless of whether the particular cells measured had been fused. No mobility enhancement was induced with trypsinized Sendai virus, nor by influenza virus, indicating a probable requirement for the Sendai virus F protein. Virus treatment reduced the diffusion coefficient of a lipid analog in 3T3 cells. The results are compatible with the hypothesis that the Sendai virus disrupts a mobility restraint system, perhaps cytoskeleton-membrane connections.     

Crossed immunoelectrophoretic studies of the solubility immunogenicity of herpes simplex virus antigens.

The nonionic detergent Triton X-100 was capable of solubilizing 90% of the protein content in herpes simplex virus (HSV)-infected rabbit cornea cells. The solubilized HSV antigens formed well-characterized precipitates by crossed immunoelectrophoresis in Triton X-100-containing agarose gel, allowing both identification and relative quantitation. Water-soluble and detergent-requiring HSV antigens were identified by different solubilization procedures in buffer with and without detergent. Five glycoprotein antigens were solubilized only in the presence of detergent, indicating their membrane-bound state. One non-glycosylated antigen was present in both a water-soluble and a membrane-bound form. Based upon the crossed immunoelectrophoretic precipitating patterns of Triton X-100-solubilized HSV antigens, it has been estimated that infected cells yield an amount of virus-specific protein equivalent to 2,000 enveloped virions per cell. Rabbits inoculated intracutaneously with Triton X-100-solubilized HSV antigens developed neutralizing antibodies against HSV almost as effectively as rabbits with an active HSV infection. Precipitins against individual HSV antigens in sera from rabbits infected with HSV and immunized with the Triton X-100-solubilized HSV antigens were assayed by the crossed immunoelectrophoretic technique. Sera from infected rabbits reacted more strongly and with a higher number of HSV antigens than sera from immunized rabbits.