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Metabolic bone disease (MBD) is an important cause of morbidity in premature, very low birth weight (VLBW) and sick infants and, if left undiagnosed, may lead to structural deformities and spontaneous fractures. MBD is defined as impaired bone mineralization in a neonate with lower than expected bone mineral levels in either a fetus or a neonate of comparable gestational age and/or weight, coupled with biochemical abnormalities with or without accompanying radiological manifestations. MBD has been reported to occur in 16% to 40% of extremely low birth weight neonates and presents by 6-16 weeks after birth. Insufficient calcium and phosphorous stores during the phase of accelerated growth predispose to MBD in neonates along with the use of some medications such as caffeine or steroids, prolonged parenteral nutrition and chronic immobilization. Enhanced physical activity in preterm infants facilitates bone mineralization and weight gain. Biochemical abnormalities tend to worsen significantly, as the severity of disease progresses. These abnormalities may include hypocalcemia, hypophosphatemia, hyperphosphatasia and secondary hyperparathyroidism. In addition, urinary phosphate wasting and hypovitaminosis D can be additional complications. Conversely, biochemical abnormalities may not be accompanied by rachitic changes. Newer diagnostic modalities include non-invasive bone densitometry by quantitative ultrasound over the mid-tibial shaft. The management of MBD includes adequate calcium, phosphorous and vitamin D supplementation, along with optimum nutrition and physical activity. Similarly, preventive strategies for MBD should target nutritional enhancement in combination with enhanced physical activity. MBD usually results in preventable morbidity in preterm and VLBW neonates. Treatment consists of optimum nutritional supplementation and enhanced physical activity.

Despite a substantial understanding of the factors regulating oligodendrocyte differentiation, the signaling mechanisms involved in this process are not well-understood. This study elaborates on the findings (Bhat NR, Zhang P (1997) FASEB J 11:A925; Baron W, Metz B, Bansal R, Hoekstra D, de Vries H (2000) Mol Cell Neurosci 15:314-329) of a role for p38 MAP kinase signaling in oligodendrocyte differentiation and myelin gene expression. When proliferating oligodendrocyte progenitors were switched to a growth factor-free differentiation medium, there was a rapid activation of p38 kinase that correlated with an increased phosphorylation of CREB, a down-stream target and a factor involved in oligodendrocyte differentiation. Addition of forskolin, a known inducer of intracellular c-AMP and of oligodendrocyte differentiation, also stimulated CREB phosphorylation in a p38 kinase dependent way. Pharmacological inhibition of p38 interfered with the morphological and antigenic changes associated with differentiating oligodendrocytes as well as with the developmental and forskolin-induced expression of myelin basic protein, thereby supporting an essential role for p38 MAPK pathway in oligodendrocyte differentiation.

The nucleocapsid (N) protein of bovine respiratory syncytial virus (BRSV) in the baculovirus expression system was evaluated as a source of antigen in an enzyme-linked immunosorbent assay (ELISA) for the detection of respiratory syncytial virus (RSV) antibodies. The recombinant N protein was purified from infected-cell extracts by sucrose gradient centrifugation and used in the ELISA for the detection of antibodies to various RSV strains. The ELISA was compared with the virus neutralization (VN) test for determining BRSV antibodies in 10 consecutive serum samples from four calves vaccinated with a live modified BRSV vaccine and from two nonvaccinated control calves. The ELISA compared favorably with the VN test for detecting serological responses. All serum samples which were positive in the VN test were also positive in the ELISA. None of the serum samples collected from the two nonvaccinated calves reacted in the ELISA. To determine the usefulness of the ELISA for epidemiological studies, 58 cattle serum samples were tested in the ELISA and the VN test. Approximately 94% (42 of 45) of field serum samples which were positive in the ELISA were also positive in the VN test. No case was found in which the ELISA result was negative and the VN test result was positive. Thirteen of the serum samples were negative in both methods. Our results indicate that the ELISA with the baculovirus-expressed N protein as an antigen is an efficient, sensitive, and specific method for detecting serum antibodies to RSV.

The coding region of the gene for the nucleocapsid (N) protein of bovine respiratory syncytial virus has been inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) using transfer vector pVL 1393. Infection of Spodoptera frugiperda cells with recombinant virus resulted in the synthesis of high levels of N protein. This protein was indistinguishable from the authentic bovine respiratory syncytial virus (BRSV) N protein by SDS-gel electrophoresis and immunoprecipitation with anti-BRSV serum. [35S]methionine-labeled N protein synthesized in S. frugiperda cells was used in a protein-blotting protein overlay assay for its interactions with BRSV-infected cell proteins. The N protein synthesized in S. frugiperda cells was found to specifically interact with the phosphoprotein (P) and M2 protein of BRSV but only with the P protein of human RSV. Our results suggest that the recombinant N protein produced in S. frugiperda cells could be used to determine the domains on N protein required for binding to P and M2 proteins.

The nucleotide sequence of the gene encoding the major nucleocapsid (N) protein of bovine respiratory syncytial virus (BRSV) has been determined. The N mRNA is 1196 nucleotides long with a single, large open reading frame. The derived polypeptide has 391 amino acids corresponding to a calculated molecular weight of 42,600 Da. This is in agreement with the molecular weight of 43,000 Da determined for the BRSV N protein by SDS-polyacrylamide gel electrophoresis (PAGE). Comparison of the nucleotide sequence of BRSV N gene with the sequence of the N gene of human respiratory syncytial virus (HRSV) revealed a homology of 80.7%. There is a 93.3% homology at the amino acid level between the N proteins of BRSV and HRSV. The 5'- and 3'-terminal untranslated sequences that are conserved among HRSV mRNAs were also identified in the N mRNA of BRSV. The results indicate that the N genes are highly conserved in the bovine and human strains of respiratory syncytial virus.

Biochemical characteristics of five rotavirus-like viruses isolated from striped bass (Morone saxatilis), turbot (Scophthalmus maximus), smelt (Osmerus mordax) and Atlantic salmon (Salmo salar) in North America and Europe were compared. The genome of each isolate was composed of 11 segments of dsRNA and each isolate had a unique electropherotype in polyacrylamide gels. Agarose gel electrophoresis showed similar RNA profiles for all four isolates from North America, whereas the RNA profile of the isolate from Europe was different. Analysis of virion proteins revealed that each virus had five structural proteins ranging in Mr from 130,000 to 34,000. Each isolate had a unique polypeptide profile but their overall polypeptide patterns were similar. Reciprocal RNA-RNA blot hybridization demonstrated that all these rotavirus-like viruses cross-hybridized with each other except for the isolate from Europe which did not hybridize with the RNA from any of the other isolates. No genetic relationship was found between these rotavirus-like viruses of fish and a true group A rotavirus (SA11).

The characteristics of a rotaviruslike (SBR) virus isolated from striped bass (Morone saxatilis) were examined following purification of viruses from infected cell cultures. Virions had a double-layered capsid of icosahedral symmetry and a diameter of 75 nm. Purified viruses contained five polypeptides ranging in molecular mass from 130 to 35 kDa. None of the structural proteins were glycosylated. Treatment with EDTA did not remove the outer capsid. By using enzymes and a chaotropic agent, it was shown that VP5 was the most external polypeptide. The genome of SBR virus was composed of 11 segments of double-stranded RNA (dsRNA). The electrophoretic pattern of the dsRNA of SBR virus was different from that of reovirus type 1 (Lang) and rotavirus (SA11) dsRNA. The SBR virus was compared with reovirus type 1 and SA11 virus by RNA-RNA blot hybridization. There was no cross-hybridization between any of the genome segments of the SBR, reovirus type 1, or SA11 viruses. Antigenic comparison of SBR virus and SA11 virus by cross-immunoprecipitation and cross-immunofluorescence tests did not show any relationship. These results suggest that SBR virus could represent a new genus within the family Reoviridae.

Ten virus-specific polypeptides ranging in molecular weight from approximately 200k to 11k were identified in bovine respiratory syncytial virus (BRSV-)infected cells. Time course analysis of the induction of the viral polypeptides indicated that they could be detected as early as 30 min post-infection and their synthesis reached a plateau 12 h after infection. Cell free translation of total infected-cell mRNA in a rabbit reticulocyte system yielded 7 proteins corresponding in size to virus-specific proteins synthesized in BRSV-infected cells. The P protein was highly phosphorylated; G and F were identified as glycoproteins by [3H]glucosamine labeling. Glycosylation of G protein was largely resistant to tunicamycin, suggesting that the majority of the carbohydrate residues are attached via O-glycosidic bonds, whereas the F protein was N-linked glycosylated. Tunicamycin caused a drastic reduction in the yield of infectious virus titer indicating that the carbohydrate moieties serve a critical role in the infectious cycle of BRSV.

The effect of levamisole in calves experimentally infected with bovine viral diarrhea virus was evaluated in a double-blind study. The infection was mild and there was no difference in severity of infection or speed of recovery between levamisole-treated and 0.9% NaCl solution-treated (control) calves. Also, the serum antibody titers and viral recovery data of these calves were comparable. The white blood cell counts were consistently higher in the treated group than in the control group, with the difference peaking on postinoculation day 15. The detection of marked lymphopenia in control calves but not in levamisole-treated calves indicated a potential use of levamisole in bovine viral diarrhea.

Calves exposed by intranasal (IN) or intratracheal (IT) or both routes of inoculation to bovine respiratory syncytial (BRS) virus had a transient increase in body temperature for 2 days. Histopathologic changes indicative of pneumonia, along with multinucleated giant cells, were observed in alveolar lumen. The virus was recovered from infected calves, and their serologic response was poor. In a limited serologic survey with 100 serums from cattle in Maryland, low levels of serum-neutralizing (SN) antibody to the virus were prevalent in 38% of the serums.

Pretreatment of bovine turbinate (BTu) cells with cloned human interferon (IFN)-alpha 2a reduced the yield of infectious bovine parainfluenza-3 virus (PI-3 V). Intracellular synthesis of HN glycoprotein was reduced on postinfection day 1 (PID 1), but it recovered to normal levels subsequently. However, reduction of this protein persisted in the released virus through PID 2. Thin section electron microscopy demonstrated a drastically reduced release of mature virions and an accumulation of viral nucleocapsids inside the cytoplasm on PID 2. These results suggest that cloned human IFN-alpha 2a affects the glycoprotein synthesis, and morphogenesis of bovine PI-3 V, and thus inhibits the release of viral particles from treated cells.

Calves exposed to a bovine respiratory syncytial virus showed mild clinical signs of respiratory illness and responded serologically. The disease occurred in the presence or absence of circulating antibodies, but there was no evidence of exacerbation of the disease due to preexisting serum antibody. Nasal secretory antibody appeared to protect the calves against the disease. Calves previously exposed to the virus were immune to challenge. The virus was recovered at a high frequency when specimens of nasal secretion (not subjected to freezing and thawing) were inoculated into susceptible cells within 1 hr after collection.

Reciprocal cross-titrations using constant concentrations of bovine antiserum (1: 100) to either bovine herpesvirus (BHV) 1 or BHV 4 (obtained from controlled infection status) against varying concentrations of both viral antigens in enzyme-linked immunosorbent assay (ELISA) indicated a subtle difference between the antigenic composition of these 2 agents. Although control antiserum consistently yielded higher ELISA titers against homologous viral antigen, a considerable cross-reactivity between the 2 viruses was observed. Based on control sera results, serologic profiling of field bovine serum samples against both BHVs 1 and 4 antigen preparations indicated that the ELISA was unsatisfactory for distinguishing between previous BHV 1 or 4 exposure. The ELISA, however, was more sensitive than the virus neutralization test for detecting antibodies to BHVs 1 and 4 in cattle sera.

An antibody-complement-mediated cytotoxicity assay was employed to determine whether susceptible host cells infected with bovine parainfluenza-3 virus could be destroyed by the humoral immune mechanism. The cytotoxicity assay yielded 21.5% to 76.9% specific release of 51Cr with sera collected from calves 28 days after inoculation with the virus. A multiplicity of infection of 5, a postinfection period of 2 days, an antibody dilution of 1:10, and an incubation period of 12 hours with antibody and complement were found to be optimal for this assay.

From Aug 1, 1979 to Aug 15, 1980, a total of 624 samples were collected from the vaginas, cervices, and uteri of 208 dairy cows 30 to 37 days after parturition. An additional 90 samples were collected from the reproductive tracts from 30 of the 208 cows. The 30 cows had aborted or were repeat breeders. Samples from cows that had aborted were collected within a week after clinical abortion or immediately after rectal palpation revealed subclinical abortion. All samples were tested for the presence of cytopathogenic viruses. All samples were negative.

The inconsistency in naming and labeling bovine herpesviruses (BHVs), other than BHV types 1 and 2 (BHV-1 and BHV-2), found in the literature is reviewed. To resolve the confusion and misunderstanding caused by the use of BHV-3, BHV-4 and BHV-5 for the same kind of BHVs, the most used label BHV-4 is proposed for designating Movar-type BHVs (which also were named 'orphan viruses' or 'cytomegaloviruses').

Earlier we reported a reduction to 1/30th-1/100th of the original number of infectious particles in the infectious vesicular stomatitis virus (VSV) released from L cells treated with 10 or 30 reference units of interferon per ml. However, in these cultures virus particle production, as measured by VSV particle-associated viral RNA, virus nucleocapsid protein, and viral transcriptase, was inhibited by less than 10%. Data reported in this paper show that there was a significant reduction in glycoprotein and membrane protein of VSV particles released from interferon-treated cells. Evidence supporting the deficiency of glycoprotein in VSV released from interferon-treated cells was derived from electron microscopic studies. Under conditions where glycoprotein spikes or projections were clearly detectable on the surface of VSV released from cells not treated with interferon, very few spikes were observed on VSV released from interferon-treated cells. These results suggested that interferon-treated cells produced VSV particles with low infectivity and that this low infectivity may be related to the reduced amount of glycoprotein and membrane protein incorporated into such particles.

Daily injection of 2-deoxy-D-glucose (2-dG) had no protective effect against respiratory tract infection in calves caused by infectious bovine rhinotracheitis virus. It also did not reduce the severity of this infection. Ocular instillation of the drug, however, markedly reduced the severity of viral-induced conjunctivitis and keratoconjunctivitis. The drug was effective when given at the time of ocular infection or after clinical conjunctivitis developed.

The effect of a single therapeutic dose of levamisole in calves experimentally infected with bovine viral diarrhea virus (BVDV) was evaluated in 2 separate double-blind experiments. The infection was mild and there was no difference in severity of infection or speed of recovery between levamisole-treated and 0.9% Nacl solution-treated (control) calves. There were no significant differences between drug-treated and control calves in respect to total white blood cell, lymphocyte, and neutrophil counts, and the viral recovery data from these calves were comparable. The differences in blastogenic response of lymphocytes to the mitogen phytohemagglutinin and serum antibody titers of the 2 groups of calves were also insignificant. An increased lymphocytic hyperplasia was observed in a larger number of lymphoid tissues of drug-treated calves than in those of control calves. It appeared that a single therapeutic dose of levamisole was ineffective in altering the response of calves infected with BVDV.

The effect of tunicamycin (TM) treatment on the production, glycosylation, morphology and infectivity of vesicular stomatitis virus (VSV) released from mouse (LB), monkey (Cos-1 and VERO), bovine (MDBK), hamster (BHK) and human (HeLa, U and GM2504) cells was investigated. The yield of VSV particles released from TM treated cells, as measured by total viral proteins, was inhibited only 2-10 fold while the infectivity of these particles was greatly reduced (10-1000 fold). The morphology of VSV particles formed in the presence of TM appeared similar to that of VSV particles released from untreated cells as demonstrated by electronmicroscopy. Analysis of intracellular and extracellular virus specific proteins by SDS polyacrylamide gel electrophoresis revealed that TM blocks the glycosylation of VSV glycoprotein G in all cells tested. These results suggest that inhibition of glycosylation of VSV G protein could affect the biological infectivity of the VSV particle released from the treated cells.

Human lymphoblastoid interferon (IFN) had an antiviral activity in bovine embryonic kidney cells that resulted in the release of vesicular stomatitis virus (VSV) particles with decreased infectivity. The inhibition was dose dependent and the cells were highly sensitive to human IFN. Examination of the proteins of VSV released from bovine cells after IFN treatment showed a reduction in the glycoprotein. Electron microscopic studies revealed a large number of VSV particles with characteristic spike-like surface projections released from nontreated cells. There was a reduction in the number of mature virions produced in IFN-treated cells and the virions lacked the characteristic surface projections.

Six structural proteins of bovine parainfluenza-3 virus (PI-3V) labeled with [35S]-methionine could be resolved by polyacrylamide gel electrophoresis (PAGE). Five structural proteins of this virus had been previously reported. The 6 proteins found in this study were: L, a 180,000 (180 kD) molecular weight (MW) large protein; P, 83 kD phosphoprotein; HN, 69 kD hemagglutinin-neuraminidase glycoprotein; NP, 66 kD nucleocapsid protein; F, 55 kD fusion glycoprotein; and M, 38 kD matrix protein. Selective labeling with [2-3H]-mannose revealed only HN and F glycoprotein bands. A cellular actin protein (43 kD), associated with many enveloped viruses, was also found as a seventh protein in bovine PI-3V.

Microcultures of swine whole blood, purified blood lymphocytes (PBL), spleen cells, and lymph node (LN) cells were tested for stimulation by concanavalin A (conA) and phytohemagglutinin (PHA). A cell concentration of 2 x 10(6)/ml and a 1:20 dilution of whole blood were needed for optimal stimulation of conA and PHA. Optimal stimulation of whole blood lymphocytes (WBL) and LN cells was obtained by 20 micrograms of conA/ml, but 10 micrograms/ml of this mitogen was adequate for spleen cells. The PBL and LN cells were optimally stimulated by 20 microliters of PHA/ml, but 80 microliters and 40 microliters/ml of this mitogen were required for stimulating the whole blood and spleen cells, respectively. Four days' incubation was needed for maximal blastogenesis of WBL with conA. This could be achieved after 3 days' incubation with PBL and spleen and LN cells. Whole blood and spleen cells were stimulated by PHA after 3 days' incubation, but PBL and LN cells required 4 days' incubation. Reproducibility, sensitivity, and comparison of various cultures were discussed.

Fertilized two-celled mouse eggs deprived of their zona pellucida were susceptible to infection with minute virus of mice. The virus had no deleterious effect on development of embryos cultivated in vitro.

Intracellular antigens of strain DN-599 bovine herpesvirus were detected in the cytoplasm and the nucleus of infected bovine embryonic kidney cells by the indirect immunoferritin (IF) technique. Specific tagging was observed in viral envelope and capsids. Aggregates of viral particles heavily coated with antibody were seen by immune electron microscopy (IEM).

Hemagglutinating DNA viruses of 20 nm diameter were isolated from bovine adenovirus types 1 and 2. The isolates were heat stable, chloroform resistant, and defective. Their densities were 1.38 to 1.39 g/cm3, and they were found to be serologically identical to the bovine adeno-associated virus strain X7. A partial antigenic relationship was found between these and the canine adeno-associated virus.

A defective transport of bovine parainfluenza-3 virus (PI-3V) hemagglutinin-neuraminidase (HN) glycoprotein was evidenced in interferon (IFN)-treated bovine turbinate (BTu) cells. Indirect immunofluorescence performed with monoclonal antibody to PI-3 HN glycoprotein demonstrated accumulation of this protein in the perinuclear cytoplasm of IFN-treated cells. Untreated, infected control cells had a generalized widespread fluorescence. Unfixed control cells showed a uniform surface fluorescence in contrast to a few specs of fluorescence on the plasma membrane of IFN-treated cells. Electron microscopic localization of HN protein was done by immuno-gold ultrastructural cytochemistry. Untreated cells had uniform gold label on the plasma membrane and around the budding virus particles with no label in the cytoplasm. In IFN-treated cells, however, there was an accumulation of gold particles in the cytoplasm with only a few particles on the cell surface. Quantitative analysis of HN protein on the cell surface by solid phase radioimmune-assay revealed a greater amount of this protein on the surface of control cells, than those on the IFN-treated cells.

2-Deoxy-D-glucose (2-dG) and glucosamine reversibly inhibited the replication and cytopathic effects of bovine respiratory syncytial virus in bovine turbinate cell cultures. The inhibitors were effective when added up to 12 hours after cell cultures were inoculated. Their effectiveness decreased as the time between inoculation of cells and drug treatment lengthened. The sugars did not inactivate the virus directly, and inoculation of drug-treated cells in drug-free medium produced normal yield of virus. Mannose fully reversed the inhibitory effects of 2-dG, but was ineffective for glucosamine. Protein synthesis was necessary for production and release of infective virus after removal of 2-dG and glucosamine blocks. Electron microscopic studies revealed a large amount of bovine respiratory syncytial virus production with characteristic spike-like projections on the viral envelopes in the absence of these inhibitors. There was a drastic reduction in the number of mature virions produced in inhibitor-treated bovine turbinate cells, and the virions lacked the characteristic surface projections.

Calves experimentally infected with bovine rhinovirus type 1 developed a mild respiratory disease and exhibited a cell mediated immune (CMI) response, as determined by leukocyte migration inhibition (LMI) and lymphocyte blastogenesis tests. The CMI response to the virus was detected by day 3 post-inoculation using direct and indirect LMI tests. It persisted through day 28, with maximal responses occurring between days 4 and 14. Measurement of cellular immunity by blast transformation, however, was not evidenced until day 12. The humoral antibody response was minimal with serum neutralizing titers ranging from 1:4 to 1:16 and this response was not evident until 3 weeks after inoculation.