Pathogenesis of visna/maedi and caprine arthritis-encephalitis: new leads on the mechanism of restricted virus replication and persistent inflammation

Lentiviruses are unique retroviruses which cause diseases with long incubation periods and prolonged clinical courses. The prototype lentiviruses, visna/maedi of sheep and arthritis-encephalitis virus of goats (CAEV), infect cells of the monocyte-macrophage system and replicate at a restricted level in these cells. The virus life cycle is closely associated with maturational factors in the cells; monocytes support the early stages of the replication cycle which goes to completion only when the cells mature to macrophages. Virus replication in the monocyte-macrophage results in lesions characterized by mononuclear cell infiltration of the central nervous system (CNS), lungs, synovium and mammary gland and their draining lymph nodes. Co-cultivation of sheep or goat lymphocytes with macrophages infected with visna or CAE viruses results in production of a unique interferon (LV-IFN). LV-IFN is a non-glycosylated protein of 54,000 to 64,000 daltons and has biological properties which have several implications for pathogenesis. Firstly, it retards the rate of maturation of monocytes and thus indirectly slows the rate of virus replication. Second, it restricts the rate of virus replication in mature macrophages by preventing virus maturation. Third, it induces expression of class II (Ia) antigens of the major histocompatibility complex on cells of macrophage lineage. Thus, by curtailing virus replication and enhancing expression of MHC class II antigens, LV-IFN may contribute to the induction and augmentation of the host's lymphoproliferative response to the virus.     

Characteristics of neonatal calf diarrhea virus ribonucleic acid.

The nucleic acids of neonatal calf diarrhea virus were characterized by isopycnic centrifugation in Cs2SO4, electron microscopy, ultraviolet absorbance temperature profiles and polyacrylamide gel electrophoresis. These studies indicated that the neonatal calf diarrhea virus genome consists of 11 segments of double stranded RNA with a total molecular weight of 10.75 million daltons.     

Characterization of deoxyribonucleic acid from cells infected with Aleutian disease virus

Viral DNA was extracted from Crandell feline kidney (CRFK) cells infected with Aleutian disease virus (ADV) and labeled with [ 3H ]thymidine. The sedimentation coefficient in alkaline sucrose gradients was 16S corresponding to a molecular weight of 1.5 X 10(6). The buoyant densities of DNA from infected and control cells were determined by isopyknic sedimentation in CsCl and NaI gradients. Two additional peaks of [ 3H ]DNA were found in infected cells, but not in control cell extracts. Fractionation of this DNA on hydroxylapatite indicated that the new peaks represented a single-stranded component, density 1.728 g/cm3, and a double-stranded component, presumed to be a viral replicative intermediate, density 1.718 g/cm3. The target antigen formation in CRFK cells was measured by gamma-irradiation of ADV and assayed for focus formation. The calculated size of ADV based on these measurements was 1.1 X 10(6). The H-1 parvovirus also was shown to have a size of 1.5 X 10(6) daltons for both antigen and plaque formation. The data indicated similarities existed between ADV and other autonomously replicating parvoviruses in most properties, except that less-than-unit length genome of ADV may be transcribed.     

Structural proteins of bovine viral diarrhea virus.

A procedure for the purification of radioactively labeled bovine viral diarrhea virus was critically evaluated. Purification of virus from artificial mixtures of unlabeled infected and labeled noninfected cells indicated that the extent of purification was approximately 100-fold with respect to host proteins. Residual host proteins were found to contaminate the viral preparation even after extensive purification by differential and isopycnic zonal centrifugation. Co-electrophoresis of 3H-labeled virus with 14C-labeled host cell material in neutral sodium dodecyl sulfate-7.5% polyacrylamide gels provided a means to distinguish viral specific proteins from host cell protein contaminants. Four major electrophoretic components were identified as being of viral origin; molecular weights of the components were estimated from their migration rates relative to protein markers of known molecular weight. Two viral components (VC), VC 1 and VC 3, migrated heterogeneously and had molecular weights of 93,000 to 110,000 and 50,000 to 59,000 daltons, respectively. Molecular weights of VC 2 and VC 4 were 70,000 and 25,000 daltons, respectively.     

Range of viral neutralizing activity and molecular specificity of antibodies induced in cattle by inactivated bovine viral diarrhea virus vaccines

The range of neutralizing activity to bovine viral diarrhea (BVD) virus and viral protein specificity of antibodies induced by 3 inactivated vaccines were evaluated by use of samples of sera obtained from 13 cattle 14 days after vaccination. Viral neutralizing antibodies wee detected in all cattle to each of 10 noncytopathic and 10 cytopathic isolates of BVD virus. A viral-induced polypeptide (53,000 to 56,000 daltons) was detected by radioimmunoprecipitation with serum from all vaccinates. Other viral-induced polypeptides of 115,000, 80,000, 48,000, and 25,000 daltons were precipitated with sera from some vaccinates. Precipitation of those polypeptides was related to the vaccine used. When multiple viral polypeptides were precipitated, the 53,000- to 56,000-dalton polypeptide appeared immunodominant.     

Sporadic congenital transmission of avian leukosis virus in hens discharging the virus into the oviducts.

The efficacy of the albumen test for infectious avian leukosis virus (ALV) was examined in detecting congenitally transmitting hens. Seventy-three White Leghorn non-viremic hens with antibody to ALV were used. Eleven of the hens shed infectious ALV into their egg albumen, whereas only 7 of the 11 ALV-positive hens shed ALV antigens. The egg albumen test for infectious ALV was shown to be more effective in detecting the congenitally transmitting hens than that for ALV antigens. Then, twenty of the 62 hens which shed no infectious ALV into the albumen were studied for transmission of ALV to their embryos and for discharging ALV into the oviduct and vagina. Six of the 50 embryos from 4 hens were found to be infected with ALV but all of the 227 embryos from remaining 16 hens were free from the infection. Discharge of the virus into the oviduct and vagina was found both in the 4 transmitting hens and in 6 of the 16 non-transmitting hens. These results suggest that the hens discharging ALV into the oviduct, even though they do not shed ALV into egg albumen, may transmit the virus sporadically to their embryos.     

Comparative antigenic analysis of extracellular proteins of Bacteroides nodosus isolated from virulent and benign ovine footrot

Antigens in the extracellular protein (ECP) complexes of Bacteroides nodosus, isolated from sheep with either benign or virulent footrot, were studied by immunoelectrophoresis (IEP). Rabbit antisera against ECP from virulent and benign strains, were used in homologous and heterologous crossed IEP. Four precipitin peaks unique to the virulent strain, and five peaks unique to the benign strain were identified. In an attempt to characterize the different antigens in ECP, rabbit antisera were raised against an outer membrane protein (OMP, mol. wt. 35 000 daltons), pili and various proteases of virulent and benign strains of B. nodosus. No precipitin band was observed when ECP from both B. nodosus strains were reacted against anti-OMP and anti-pilus antisera. However, single precipitin bands unique to one protease from the benign strain and one protease from the virulent strain were identified. The results suggest that specific antigens other than proteases or pili are important in determining whether a B. nodosus isolate is virulent or benign.     

Comparative studies on three isolates of Breda virus of calves

Three isolates of Breda virus of calves were compared morphologically and antigenically. The isolates demonstrated similar morphology and shared common antigens, as determined by enzyme-linked immunosorbent assay and immunoelectron microscopy. On the basis of results of the hemagglutination-inhibition test, enzyme-linked immunosorbent assay, and immunoelectron microscopy, the 3 isolates were further subdivided into 2 serotypes: serotype 1 (Breda virus 1) represented by the Iowa isolate 1; and serotype 2 (Breda virus 2), by the Ohio isolate and the Iowa isolate 2. The 3 isolates caused diarrhea in gnotobiotic calves.     

肌旋毛虫免疫亲和层析抗原的制备及其特性

以Sepharose 4B为载体,兔抗旋毛虫多克隆抗体为配体,纯化了旋毛虫的S_3抗原,得到亲和层析抗原.本实验结果表明,存在于S_3抗原中的主要抗原成分,同样存在于亲和层析抗原中,亲和层析抗原的分子量范围在103000～40000 u之间,等电点范围在pH4.7～8.8之间.亲和层析抗原经转印后,在硝酸纤维素膜上至少有7条显色带,其中分子量为48000 u,50000u,58000u和87000u的4种蛋白的抗原性较强,而以分子量为48000u的蛋白抗原性最强.     

Antigenic relationship among strains of ibaraki virus and epizootic haemorrhagic disease virus studied with monoclonal antibodies

Twelve monoclonal antibodies against ibaraki virus (IbV) were established and preliminarily characterised by indirect immunoperoxidase (IIP), haemagglutination inhibition (HI) and neutralisation (NT) tests. Five antibodies reacted in the IIP test with all IbV and epizootic haemorrhagic disease virus (EHDV) strains tested, and five antibodies reacted with IbV and Alberta strain (serotype 2) but not with New Jersey strain (serotype 1) of EHDV. Two of 12 antibodies showed both HI and NT activities. Viral proteins with molecular weights of about 24,000 daltons (24KD) and 78KD were determined by two monoclonal antibodies in Western blot analysis. One of two antibodies with the ability of both HI and NT recognised a viral protein with a molecular weight of about 78KD.     

A feline retrovirus induced T-lymphoblastoid cell-line that produces an atypical alpha type of interferon

A cell-line, designated LSA-1, was derived from a thymic lymphosarcoma that occurred in a cat with experimentally induced feline leukemia virus (FeLV) infection. LSA-1 cells possessed surface receptors and antigens of normal T-lymphocytes, but were unresponsive to interleukin-2 stimulation. The LSA cell-line was found to constitutively produce and release an interferon into the culture supernatants. Production of this interferon was enhanced in certain clones of the original LSA-1 cell lines. The interferon produced by LSA-1 cells and some of its clones was compared to the standard alpha, beta, and gamma interferons of cats. Unlike alpha and beta interferons, which were acid, SDS, and heat stable, LSA interferon was acid labile and SDS and heat stable. In comparison, standard feline gamma interferon was acid, SDS, and heat labile. LSA interferon had a molecular weight of 20,000 daltons, compared to 17-19,000 daltons for gamma, 19-25,000 for beta, and 25-45,000 daltons for alpha interferons. Standard feline interferons were active only on cat cell lines, with the exceptions of alpha interferon, which also reacted with MDCK canine cells. LSA interferon resembled the standard feline alpha interferon because it also reacted with feline and canine cells. It was concluded that LSA interferon was an atypical acid labile alpha interferon, resembling in this respect the abnormal alpha interferon seen in humans with AIDS and SLE, and mice with retrovirus infections. LSA-1 cells produced high levels of FeLV structural proteins but very little infectious virus. This effect was due to endogenously produced interferon; LSA cell clones that were selected for low interferon production produced much higher levels of infectious FeLV than parent cells or clones selected for high interferon production. Cat cells pretreated with LSA or with standard feline alpha and beta interferons, and then infected with FeLV, produced high levels of FeLV proteins but very little infectious virus.     

The porcine humoral response to detergent extracted Aujeszky's disease (pseudorabies) virus antigens

Twenty Aujeszky's disease (AD) virus antigens were demonstrated by crossed immunoelectrophoresis in a Triton-X-100 detergent extract of virus-infected PK-1a cells. Eight of these antigens were shown to be glycosylated based on their ability to be specifically bound by the lectin Ricinus communis agglutinin II. Pigs nasally infected with AD virus showed a significant serum antibody titer to seven of the known glycosylated antigens and to four additional antigens. The antibody titer to these antigens persisted for at least 116 days. Pigs which were vaccinated parenterally with the whole detergent extract survived a nasal challenge of 10(8 . 5) PFU of virulent AD virus. The antibody response of these vaccinated pigs on the day of challenge was essentially identical to the recovery response previously observed in non-vaccinated nasally infected pigs. These results indicate that the optimum components of future AD virus subunit vaccines and their complementary diagnostic reagents should be selected from these 11 antigens.     

Identification of major antigenic proteins of bovine herpesvirus 1 and their correlation with virus neutralizing activity

The polypeptides of an Australian isolate of bovine herpesvirus 1 were analysed by polyacrylamide gel electrophoresis, and Western blotting was used to identify those polypeptides which reacted most strongly with sera from infected animals. Approximately 20 polypeptides ranging in molecular weight from 11,000 to 240,000 daltons (11-240K) were identified by 35S-methionine labelling of virus and approximately half of these classed as glycoproteins using 14C-mannose and 3H-glucosamine incorporation into infected cells. Convalescent sera from cattle all reacted strongly with glycoprotein bands at 85 and 70K, with most sera also recognizing another band at 140-150K. The intensity of bands on the Western blot analyses was found to correlate well with neutralization titres of individual serum samples, indicating the involvement of these proteins in virus neutralization. The importance of the 70K glycoprotein was supported by the finding that, of 12 monoclonal antibodies studied, those 3 with the strongest neutralizing activity, were those which recognized a band at 70K in Western blot experiments.     

Specificity of neutralizing and precipitating antibodies induced in healthy calves by monovalent modified-live bovine viral diarrhea virus vaccines

Serum was obtained at weekly intervals after vaccination of 6 healthy calves with either of 2 commercially available monovalent modified-live bovine viral diarrhea (BVD) virus vaccines. Detectable neutralizing antibodies to each of 10 cytopathic and 10 noncytopathic isolates of BVD virus were produced by 1 or more of the calves by 14 days after vaccination, but no calf produced detectable neutralizing antibodies to all 20 BVD viruses. At that time, precipitating antibodies against viral-induced polypeptides of approximately 115,000; 80,000; 56,000; 48,000; 39,000; and 25,000 daltons were detected in sera from some calves. Also at that time, specificity of the antibodies for polypeptides of certain viruses was detected. At 21 days after vaccination, each calf produced neutralizing antibodies to all 20 BVD viruses. At that time, precipitating antibodies to each of the aforementioned viral induced polypeptides were detected in serum from each calf. Precipitating antibodies to viral induced polypeptides of 61,000 and 37,000 daltons were detected in samples of sera obtained from some calves at 42 days after vaccination.     

Release of immunosuppressive substances from tissue culture cells infected with bovine viral diarrhea virus

Bovine fetal lung tissue culture cells were infected with bovine viral diarrhea virus. The cells released substances into the supernatant fluid that suppressed the proliferative response of bovine peripheral blood mononuclear cells which had been stimulated with concanavalin A. Activity could be found in low molecular weight fractions (less than or equal to 2,000 daltons). During the assay, these fractions were required to inhibit the response of leukocytes to concanavalin A. Addition of indomethacin to the infected tissue culture cells inhibited generation of immunosuppressive supernatants; addition of indomethacin to the suppressive supernatants did not block their activity. The possible involvement of prostaglandins in this immunosuppressive activity is discussed.     

Isolation and identification of chicken infectious anemia virus in Brazil.

Seven chicken infectious anemia virus (CIAV) isolates were obtained from seven broiler flocks with poor performance in two states of Brazil. All isolates induced thymus atrophy, bone-marrow aplasia, and low hematocrit values when inoculated into 1-day-old susceptible chicks. The CIAV isolates were resistant to treatment with chloroform and were able to pass through 50-nm-pore-size filters. CIAV-specific antigens could be demonstrated in tissues of experimentally infected chicks using a monoclonal antibody specific for CIAV. These characteristics of the virus and the virus-induced lesions demonstrate that CIAV is present in Brazil and that the virus is associated with production problems.     

Production and characterization of monoclonal antibodies against formalin-inactivated Nipah virus isolated from the lungs of a pig

Eight clones of monoclonal antibodies (Mabs) to Nipah virus (NV) were produced against formalin-inactivated NV antigens. They reacted positive by indirect immunofluorescent antibody test, and one of them also demonstrated virus neutralizing activity. They were classified into six different types based on their biological properties. These Mabs will be useful for immunodiagnosis of NV infections in animals and further research studies involving the genomes and proteins of NV.     

Structural proteins of equine infectious anemia virus and their antigenic activity

Using purified equine infectious anemia (EIA) virus labeled with 3H-glucosamine or 14C-protein hydrolysate, structural proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a result, 2 glycoproteins and 10 proteins with molecular weights (mol wt) ranging from 12,000 to 115,000 daltons were demonstrated. Of 12 structural proteins, 3 proteins, namely a glycoprotein with mol wt of 76,000 (gp76) and 2 proteins with mol wt of 25,000 (p25) and 12,000 (p12), respectively, had distinct antigenic activity from one another in immunodiffusion. Development of antibodies against gp76 and p25 was compared in infected horses. The antibody to gp76 appeared earlier and stronger than to p25 in horses infected with the homologous virus strain. The fraction with glycoproteins was found to have hemagglutinating activity which was inhibited by the serum sample from horses infected with equine infectious anemia virus.     

Methanol precipitation of transmissible gastroenteritis virus.

Methanol precipitation of transmissible gastroenteritis virus was tested at Ph 4.0, 5.0, 6.0, and 7.0 and at methanol concentrations of 15%, 25%, and 30%. Supernatant and precipitate fractions were tested for complement-fixing and agar-diffusion soluble antigens and plaque-forming units, and were examined by electron microscopy. Virus could be obtained free of detectable agar-diffusion antigens and most of the complement-fixing antigens. Most of the virions were without peplomers after methanol treatment but they retained infectivity.     

Reactivity of anti-Nipah virus monoclonal antibodies to formalin-fixed, paraffin-embedded lung tissues from experimental Nipah and Hendra virus infections

The immunohistochemical reactivity of seven clones of mouse monoclonal antibodies raised to Nipah virus antigens were investigated using formalin-fixed, paraffin embedded porcine and equine lung tissues from experimental Nipah and Hendra virus infection, respectively. Either microwave irradiation or enzymatic digestion effectively unmasked the viral antigens in formalin-fixed, paraffin-embedded tissue sections. Four clones showed positive reaction to both Nipah virus-infected porcine lung tissue and Hendra virus-infected equine lung tissue. Two clones (11F6 and 13A5) reacted with Nipah virus-infected porcine lung tissue, but not with Hendra virus-infected equine lung tissue. These Nipah virus-specific monoclonal antibodies may therefore be useful for immunohistological diagnosis of Nipah virus infection and for further research on Nipah virus pathogenesis.