Ultrastructure and immuno-cytochemistry of BHK-21 cells infected with a modified Bucyrus strain of equine arteritis virus

Morphogenesis of a modified Bucyrus strain of equine arteritis virus (EAV) in BHK-21 cells was studied. Bacillary tubules were first detected in the cytoplasm 8 h after infection, and mature virions 79 to 122 nm in diameter, 101 nm on average, were mostly observed in the cisternae of the rough endoplasmic reticulum (RER) at 12 h or later. They had isometrical cores and morphological subunits in the outer layer. Budding occurred from the RER and the outer nuclear membrane, but not from the cell surface. Structural linkage was detected between the tubule and the virus core. Aberrant strands were occasionally demonstrated within the nucleus 12 h after infection, and immunofluorescence and immunogold labeling revealed viral antigen also in the nucleus.     

Genetic variation of envelope gp90 gene of equine infectious anemia virus isolated from an experimentally infected horse

Six strains of equine infectious anemia virus (EIAV) were recovered from febrile and non-febrile stages of a horse experimentally infected with the P337-V70 strain given once to a horse. The env gp90 genes of the isolates, the P337-V70 and P337-V26, avirulent virus derived from the P337-V70 strain, were sequenced. A comparison of the gp90 gene sequences revealed that amino acid variations among the viruses tested showed as high as 8.2 to 11.5%. In addition, the comparison also indicated that the isolates that recovered from the non-febrile stage were contained in nucleotide insertions in the principal neutralizing domain (PND) region. The insertions were arranged regularly with smaller segments. The nucleotide sequence of the P337-V26 gp90 gene was found to contain a six-nucleotides insertion and seven nucleotide substitutions outside the PND region, when compared with that of the P337-V70 strain.     

Evaluation of serological diagnosis of Borna disease virus infection using recombinant proteins in experimentally infected rats

We produced two recombinant Borna disease virus (BDV) proteins, p40 and p24, by using a baculovirus vector as a diagnostic antigen. Antigenicities of these recombinant proteins were evaluated by immune rabbit sera. Recombinant p40 was a more sensitive antigen than p24 for the detection of antibodies in infected rats. Rats inoculated with BDV within 24 hr after birth showed higher detection rates of viral RNA and viral proteins from the brain than rats inoculated at 4 weeks-old. Depending on the age of infection and the time postinfection, the detection of BDV RNA, protein, or anti-BDV antibody did not always correlate in individuals. We suggest both serological and molecular biological methods are needed in the diagnosis of BDV infection.     

Hepatitis GB virus C infection in Japanese hemophiliacs with persistent hepatitis C virus infection

We investigated the prevalence of infection of GBV-C, which has been cloned recently and is considered a parenterally transmissible virus. Ninety-one Japanese hemophiliacs who were persistently infected with HCV were evaluated. The presence of GBV-C RNA was measured by nested RT-PCR. We analyzed the prevalence and the association with subtypes of coinfected HCV. 20.9% of hemophiliacs were infected with GBV-C. The distribution of HCV subtypes of patients who are coinfected with GBV-C was similar to that of patients who are coinfected with HIV, and the prevalence of GBV-C infection of patients with HCV subtype la was significantly higher than that of patients without HCV subtype la. High prevalence of GBV-C infection was observed in Japanese hemophiliacs, and most were thought to be imported isolates from foreign origins, as well as HIV infection in these patients.     

Acute pulmonary lesions in sheep experimentally infected with bovine viral diarrhoea virus

Recombinant antibody-cytokine fusion proteins are immunocytokines that achieve high cytokine concentrations in the tumor microenvironment and thereby effectively stimulate cellular immune responses against malignancies. The activation and expansion of immune effector cells, such as CD8+ T lymphocytes, by interleukin-2 immunocytokines resulted in the eradication of established pulmonary and hepatic metastases of murine melanoma and colorectal carcinoma in syngeneic mouse models. These immunocytokines were equally effective in eliminating established bone marrow and liver metastases of murine neuroblastoma by activating natural killer cells. The effective eradication of metastases by immunocytokines resulted in significant prolongation in life span of mice over that of controls receiving equivalent mixtures of antibody and interleukin-2, which failed to reduce the growth of disseminated metastases. Proof of concept was established, indicating that immunocytokine-induced activation and expansion of immune effector cells in the tumor microenvironment can effectively eradicate established tumor metastases. This promising new approach to cancer immunotherapy may lead to clinical applications that improve treatment of cancer patients with minimal residual disease in an adjuvant setting.     

Enzyme-linked immunosorbent assay for serological survey of equine arteritis virus in racehorses

To examine antibodies against equine arteritis virus (EAV), an enzyme-linked immunosorbent assay (ELISA) using purified virus antigen was developed. The results of ELISA were compared with those of serum neutralization (SN) tests. The ELISA absorbance values and the SN titers in sera collected weekly from EAV-infected horses showed a similar pattern. The ELISA could detect antibody to EAV in horses experimentally infected with not only a homologous virus strain, which was used as the ELISA antigen, but also a heterologous strain. Using the ELISA, serum samples collected in 1996 from racehorses in three prefectures (Hokkaido, Ibaraki, and Shiga) were examined and there was no evidence of recent EAV infection among these racehorse populations in Japan. The ELISA should be a simple and highly specific method for rapid screening of EAV infection in racehorses.     

Immune response of calves experimentally infected with non-cell-culture-passaged bovine respiratory syncytial virus

The immune response of calves was studied following infection with non-cell-passaged Bovine respiratory syncytial virus (BRSV). Two groups of 6 specific pathogen free (SPF) calves were housed in separate isolation rooms. One group was inoculated intranasally with a non-cell-passaged BRSV strain and the control group was mock-infected. A BRSV specific antibody response was observed for all the BRSV infected calves. These antibodies were shown to have neutralizing activity. No lymphocyte proliferation response was detected in the mock-infected group whereas three animals in the infected group were positive three weeks after the infection. All BRSV-infected calves, except one, produced interferon-gamma (IFN-gamma) one week post-infection and IFN-gamma was observed in all six infected calves after three weeks. The control group showed no IFN-gamma synthesis. In spite of the limits of the BRSV infection model, humoral and cellular immune responses were actively developed by all the calves against this pathogen.     

Proteolytic processing of the replicase ORF1a protein of equine arteritis virus

To study the proteolytic processing of the equine arteritis virus (EAV) replicase open reading frame 1a (ORF1a) protein, specific antisera were raised in rabbits, with six synthetic peptides and a bacterial fusion protein as antigens. The processing of the EAV ORF1a product in infected cells was analyzed with Western blot (immunoblot) and immunoprecipitation techniques. Additional information was obtained from transient expression of ORF1a cDNA constructs. The 187-kDa ORF1a protein was found to be subject to at least five proteolytic cleavages. The processing scheme, which covers the entire ORF1a protein, results in cleavage products of approximately 29, 61, 22, 31, 41, and 3 kDa, which were named nonstructural proteins (nsps) 1 through 6, respectively. Pulse-chase experiments revealed that the cleavages at the nsp1/2 and nsp2/3 junctions are the most rapid processing steps. The remaining nsp3456 precursor is first cleaved at the nsp4/5 site. Final processing of the nsp34 and nsp56 intermediates is extremely slow. As predicted from previous in vitro translation experiments (E. J. Snijder, A. L. M. Wassenaar, and W. J. M. Spaan, J. Virol. 66:7040-7048, 1992), a cysteine protease domain in nsp1 was shown to be responsible for the nsp1/2 cleavage. The other processing steps are carried out by the putative EAV serine protease in nsp4 and by a third protease, which remains to be identified.     

Longitudinal studies of immune function in cattle experimentally infected with bovine immunodeficiency-like virus and/or bovine leukemia virus

The effects of single or dual infection with bovine immunodeficiency-like virus (BIV) and/or, bovine leukemia virus (BLV) on bovine immune function were examined over a 4 year period. Holstein calves were infected with BIV (four calves), BLV (five calves), BIV and BLV (five calves), or sham inoculated (three calves). Lymphocyte blastogenesis to mitogens, seven tests of neutrophil function, and mononuclear cell subset analysis by flow cytometry (BoCD4, BoCD8, BoCD2, BoWC1, sIgM+, and monocytes) were performed at regular intervals to 49 months post-infection. These data were analyzed for main effects of each virus and interaction as a 2 x 2 factorial. BIV infected cattle had lower neutrophil antibody-dependent cell-mediated cytotoxicity and iodination responses during 2 of the 4 years post-infection (P < 0.05). BIV infection was not associated with any long-term significant changes in lymphocyte blastogenesis to mitogens or changes in mononuclear cell subset numbers in blood. There was a tendency for animals infected with BIV alone to have decreased lymphocyte blastogenic responses to mitogens, but this was not statistically significant. BLV infection caused an increase in total mononuclear cells with no dramatic shift in the relative proportions of the various subsets. Co-infection with BIV and BLV did not consistently cause a different response than either virus did individually. One BIV infected animal died of non-BLV lymphosarcoma 7 months after infection. All other animals had no unusual clinical signs. In summary, infection with BIV caused a significant, temporary decrease in neutrophil function with no consistent statistically significant alteration in lymphocyte blastogenesis or mononuclear cell numbers during the first 4 years after infection. BLV infection caused an increase in lymphocyte numbers, and there appeared to be no synergism between the viruses.     

Expression cloning and humoral immune response to the nucleocapsid and membrane proteins of equine arteritis virus

To provide a convenient and sensitive method for the detection of equine arteritis virus (EAV)-specific serum antibodies, we developed an immunoblot assay employing the EAV nucleocapsid (N) and membrane (M) proteins expressed in a procaryotic expression vector (pMAL-c2) for the production of recombinant maltose-binding (MBP) fusion proteins (MBP-N and MBP-M). The antigenic reactivity of the recombinant fusion proteins and their Xa factor cleavage EAV products was confirmed by immunoblot using horse antisera to EAV. Some horse sera, however, showed immune reactivity to the MBP fusion partner protein. Based on a total of 32 horse sera analyzed for the presence of EAV antibodies by immunoblot, using the MBP-N or -M fusion proteins and the Xa factor cleavage EAV products, and in the serum neutralization test, there was 100% concordance between the assays. Sera from horses experimentally infected with EAV were reactive in the immunoblot test with both the MBP-N and the MBP-M fusion proteins by day 14 after EAV exposure. The reactivity continued to the end of the experiment at day 145 after infection. This immune reactivity correlated with the detection of neutralizing antibodies in the serum samples. Based on these findings, the recombinant N and M proteins might be useful for serodetection of EAV-infected animals.     

Experimental infection with Babesia divergens in cattle persistently infected with bovine virus diarrhoea virus

Nine Norwegian Red cattle, aged 7-14 months, persistently infected with bovine virus diarrhoea virus (BVDV) were inoculated with a Swedish strain of Babesia divergens. Six persistently infected cattle of the same age and breed were kept as controls. Blood and serum samples were collected regularly during the observation period. Rectal temperatures were recorded every morning for 25 days post infection, and the animals were examined clinically on a daily basis. Sera were examined for antibodies to B. divergens by indirect immunofluorescence antibody test (IFAT). Eight of the infected animals developed fever of 2-5 days duration. Babesia divergens organisms appeared in the erythrocytes of all infected animals on the day after inoculation. The parasitaemia lasted for 4-11 days. One animal had a transient haemoglobinuria. Compared with the control group, there was a 20% decrease in the haematocrit. There was a transient lymphopenia and thrombocytopenia during the period of fever. There were no differences in mean numbers of neutrophils between the two persistently infected groups. Compared with cattle free of BVDV, the persistently infected cattle had consistently lower total leucocyte count that was mainly due to decreased mean numbers of neutrophils and monocytes. All infected animals develop antibodies > or = 1:1280 between day 7 and 10 post infection. The magnitude of the antibody response was considerably lower than that of BVDV-free animals inoculated with the same strain and dosage of B.divergens.     

Characterization of surface markers present on cells infected by chicken anemia virus in experimentally infected chickens

Specific-pathogen-free chickens were infected with chicken anemia virus (CAV) at 1 day of age and killed after 6 days. Using a double-antibody staining procedure, spleen, thymus, and bone-marrow cells containing CAV antigen were stained for presence of T-cell antigens and chicken major histocompatibility complex (MHC) Class 1 and 2 antigens. The results demonstrated CAV infection of precursor T-cells in the thymus and of mature T-lymphocytes in the spleen. A significant proportion of the cells infected in bone marrow expressed MHC Class 2 antigens but did not exhibit the characteristics of T-lymphocytes. Since CAV grows in MDCC-MSB1 cells, the staining characteristics of these cells were also studied. MSB1 cells possessed all of the characteristics of mature, helper T-lymphocytes.     

Lymphadenopathy and non-suppurative meningo-encephalitis in calves experimentally infected with bovine immunodeficiency-like virus (FL112)

At least 10% of spontaneous chromosomal antibiotic resistant mutants of bacteria express a strain-dependent graded reduction of virulence; this correlates linearly with a prolonged generation time. Occasionally, these mutants are temperature sensitive or/and auxotrophe. The work described in this paper provides evidence that in such strains the resistance and the accompanying markers exist only as a functional genetic unit. In a series of transduction experiments with a pathogenic strain of Salmonella typhimurium, it was found that without exception, the resistance and the additional markers were 100% simultaneoulsy transferred. Furthermore, antibiotic-resistant Escherichia coli mutants with prolonged generation time, were isolated from faecal samples; it is thus indicated that, such innocuous mutants occur at any time in the intestine. It is concluded that concerns connecting such mutants to the possibility of resistance dissemination are unfounded; furthermore, even if transfer of resistance occurred, only attenuated strains would be disseminated.     

Feline immunodeficiency virus is shed in semen from experimentally and naturally infected cats

Although a laboratory isolate of feline immunodeficiency virus (FIV), FIV-NCSU1, has been transmitted by artificial insemination in domestic cats, transmission by naturally infected males during mating has not been reported. In order to determine whether virus shedding in semen is unique to the NCSU1 isolate, we analyzed electroejaculates from four specific-pathogen-free males infected with another laboratory strain, FIV-Petaluma, and eight random source males with naturally acquired infections. Seminal cell lysates from the cats infected with the Petaluma isolate were screened by nested polymerase chain reaction amplification for FIV gag DNA. Seminal cells and seminal plasma from these FIV-Petaluma cats were further analyzed for the presence of virus by cocultivation with a feline CD4+ T cell line and Crandell feline kidney cells. Electroejaculates from the naturally infected cats were cocultivated with the T cell line. Our results demonstrated that cell-free FIV was present in seminal plasma from two FIV-Petaluma cats and two naturally infected cats. Cell-associated seminal virus was detected in all of the FIV-Petaluma infected cats and one naturally infected cat. Secretion of viral gag p26 antigen, an indication of active viral replication, was evident in cocultures containing motile sperm purified by a swim-up procedure from a FIV-Petaluma cat. These results confirm that FIV shedding in semen is not restricted to a specific virus isolate. Furthermore, swim-up sperm from FIV-infected cats may be infectious in vitro.     

Hepatitis B virus occult infection in subjects with persistent isolated anti-HBc reactivity

The aim of this study was to investigate the presence of hepatitis B virus occult infection in asymptomatic subjects with persistent anti-HBc reactivity but no other hepatitis B virus serological markers, including HBsAg, anti-HBs, IgM anti-HBc and HBV-DNA. For this purpose we used both polymerase chain reaction assays in sera and immunohistochemistry for HBsAg and HBcAg in liver biopsy specimens. Twenty-four cases were studied: 15 were drug abusers or homosexuals (eight with normal alanine aminotransferase levels) and nine were heterosexuals with raised alanine aminotransferase levels (> 45 U/l) but with no history of blood transfusion or ethanol intake (< 80 g daily). In all but five cases, liver biopsy was performed in subjects with persistent elevated alanine aminotransferase levels. In 10 out of 24 cases (41.66%) hepatitis B virus infection was demonstrated by polymerase chain reaction or immunohistochemistry, and when results from both procedures were available (n = 11) hepatitis B virus infection was detected in 63.63% of the subjects. The only clinical feature associated with HBV infection was the presence of persistent elevated alanine aminotransferase levels (p < 0.05). In conclusion, persistent isolated anti-HBc reactivity may be a relatively common serologic pattern for hepatitis B virus occult infection, at least in patients with chronic liver disease.     

Serologic and molecular characterization of an abortigenic strain of equine arteritis virus isolated from infective frozen semen and an aborted equine fetus

A virus isolated from an aborted equine fetus was determined to be antigenically distinct from several other strains of equine arteritis virus (EAV) by use of a neutralization assay with a large panel of neutralizing monoclonal antibodies. The virus was readily neutralized by polyclonal equine anti-EAV serum. Comparative nucleotide and amino acid sequence analyses indicated that the virus (WA97) isolated from the aborted fetus was virtually identical to a virus (S1971) isolated from imported semen used to inseminate another mare on the farm. Phylogenetic analysis indicated that the WA97/S1971 virus was more related to European than to North American strains of EAV. These sensitive molecular procedures may be useful for epidemiologic investigations of EAV infections. Screening and certification of stallions and frozen equine semen would prevent dissemination of pathogenic strains of EAV.