PubMed Health⌕ Search

Biomedical subjects

N J MacLachlan

Publications and source records attributed to N J MacLachlan.

At least 55 records · Page 3Linked to original sources
PubMed1996

Isolation of a poxvirus from a black-tailed deer (Odocoileus hemionus columbianus).

A poxvirus was isolated during the latter half of 1993 from a black-tailed deer (Odocoileus hemionus columbianus) that died of fulminant adenovirus infection in California (USA). The poxvirus was isolated from a pooled tissue homogenate, after repeated serial blind passages in primary black-tailed deer testicular cells. Based on electron microscopic examination of the virus, we observed morphologic features typical of the genus Orthopoxvirus, although definitive characterization was not done.

Adenoviridae Infections↗
PubMed1995

Phylogenetic analysis of open reading frame 5 of field isolates of equine arteritis virus and identification of conserved and nonconserved regions in the GL envelope glycoprotein.

The variation and phylogenetic relationship of open reading frame 5 (ORF5) of 3 different laboratory strains of the original prototype Bucyrus strain of equine arteritis virus (EAV), the modified live virus vaccine (ARVAC, Fort Dodge Laboratories), and 18 field isolates of EAV from North America and Europe were determined by comparison of their gene sequences. The viruses differed from the published sequence by between 3 (99.6% homology) and 94 (87.8%) nucleotides and by between 3 (98.8%) and 24 (90.6%) amino acids. The field isolates differed from each other by between 2 (99.7%) and 110 (85.7%) nucleotides and by between 1 (99.6%) and 26 (89.8%) amino acids. Comparison of the nucleotide sequences of these viruses indicates that although they are very closely related, the ORF5 of each virus is distinct. The ORF5 of EAV encodes the GL envelope glycoprotein which expresses the neutralization determinants of the virus. Comparative analysis of the deduced amino acid sequence of the GL protein of the viruses identified three distinct variable regions (V1 [aa 61-121], V2 [141-178], and V3 [aa 202-222]), a putative signal sequence (S [aa 1-18]), and four conserved regions (C1 [aa 19-60], C2 [aa 122-140], C3 [aa 179-201], and C4 [aa 223-255]). Amino acid substitutions in the V1 region of the GL protein of EAV field isolates had significant effects on the predicted hydrophobicity and secondary structure of the protein, which is potentially important because this region contains a major neutralization site. Estimation of genetic distances and phylogenetic tree analysis of these viruses identified four distinct groups of EAV isolates, including two North American (NA1 and NA2) and two European (E1 and E2) groups. The sequence data obtained from individual European and North American isolates suggest movement of viruses between the two continents.

Amino Acid Sequence↗
PubMed1995

Homotypic and heterotypic neutralization determinants of bluetongue virus serotype 17.

Homotypic and heterotypic neutralization determinants of bluetongue virus serotype 17 (BTV-17) were investigated with a panel of five neutralizing monoclonal antibodies (MAbs). One MAb (MAb 034) was originally raised to BTV serotype 10 (BTV-10) but also neutralizes BTV-17 (P. V. Rossitto, and N. J. MacLachlan, 1992, J. Gen. Virol. 73, 1947-1952). Competitive binding studies indicate that the MAbs recognize at least two epitopes on the neutralizing outer capsid protein VP2 of BTV-17. The MAbs were used to select neutralization-resistant variant [escape mutant (EM)] viruses and to determine the phenotypic characteristics of these EM viruses by immunoprecipitation and neutralization assays. Sequencing of the L2 gene, which encodes VP2, identified mutations responsible for the altered phenotypic properties exhibited by each EM virus. Four amino acids in three regions of VP2 are critical to the expression of the epitopes recognized by the panel of neutralizing MAbs. Amino acid 199 affects the binding of MAbs 17.82, 17.83, and 17.813; amino acid 213 affects the binding of MAb 17.85; and amino acids 327 and 582 synergistically affect the binding of MAb 034. Similarly, amino acids 327 and 402 synergistically affect the binding of MAb 034 to BTV-10 (C. D. DeMaula, H. W. Heidner, P. V. Rossitto, C. M. Pierce, and N. J. MacLachlan, 1993, Virology 195, 292-296), suggesting that the neutralizing epitope common to BTV-10 and BTV-17 has a similar location in VP2 of these two antigenically distinct viruses.

Animals↗
PubMed1995

Response of the regional lymph node to bluetongue virus infection in calves.

Bluetongue virus (BTV) infection of cattle is characterized by prolonged cell-associated viremia. In an effort to further evaluate the antiviral response of BTV-infected cattle, the role of the regional lymph node (LN) in the immune response of calves to BTV was characterized. Calves were inoculated with BTV in the skin of the neck in an area drained by the superficial cervical LN. Calves were euthanized at regular intervals after inoculation and both BTV-challenged and contralateral (control) superficial cervical LNs were harvested. In addition, some calves had cannulation of the superficial cervical efferent lymphatics prior to inoculation. Lymphocyte subpopulation analysis was done on LN cell suspensions and lymph cells using a panel of cell-specific monoclonal antibodies. There was a significant increase in the proportion of B cells in the challenged LN after inoculation as compared with the control LN. In addition, BTV-specific antibodies were detected in efferent lymph plasma from the challenged LN in one cannulated calf by 9 days after inoculation (DAI), as determined by competitive enzyme-linked immunosorbent assay, whereas BTV-specific antibodies were not detected in serum from this calf through 12 DAI. Analysis of lymph cells revealed a sustained increase in cell output from the challenged LN due to an increase in lymphoblasts and CD8+ T cells. In contrast, the cell output from the control LN dropped markedly by 8 DAI and there was no significant increase in any specific cell population. Double label analysis characterized lymphoblasts as activated CD8+ cells, as determined by expression of MHC Class II antigens (CD8+ MHC II+). These cells were only transiently present as CD8+ MHC II+ cells were not identified in lymph from the challenged LN at 14 DAI. Few CD8+ MHC II+ cells were identified at any time in lymph from the control LN or in lymph from a mock infected calf. The data indicate that B cell proliferation in the challenged LN and release of activated CD8+ cells from this LN were specific responses to BTV infection. The rapid expansion of activated CD8+ T cells indicates that these cells may limit early viral spread. It is concluded that the regional LN draining inoculated skin is critical to the immune response of calves to BTV infection.

Animals↗
PubMed1994

Evolution of the L2 gene of strains of bluetongue virus serotype 10 isolated in California.

The genetic characteristics and phylogenetic relationships between the U.S. prototype strain of bluetongue virus serotype 10 (BTV 10), the modified live virus vaccine currently used in California, and three field isolates of BTV 10 obtained in 1980 and three in 1990 in California were determined by comparison of their L2 gene sequences. The L2 genes of the 1980 field isolates were very closely related to the L2 genes of the prototype strain and the vaccine strain, differing by only 0.1 to 0.5%. The 1990 field isolates diverged from all the other viruses by an average of 4.8%. They showed a high degree of genetic similarity that ranged from 98.2 to 99.7% and formed a separate group. All BTV 10 viruses derived from a common ancestor (bootstrap value 100%) from which two different lineages have diverged giving rise to two monophyletic groups, one including all the 1990 viruses and the other the prototype, the vaccine, and all 1980 field strains. The bootstrap analyses placed a 100% confidence value at each of these two nodes. These results indicate that two different lineages of BTV 10 circulated in California between 1953 and 1990. The effect of the vaccine on the evolutionary pathways of the BTV 10 population present in California in 1980 was not clearly established, but it did not influence the evolution of the BTV 10 field isolates obtained in 1990.

Base Sequence↗
PubMed1994

The pathogenesis of bluetongue virus infection of bovine blood cells in vitro: ultrastructural characterization.

Cattle are proposed to be reservoir hosts of bluetongue virus (BTV) because infected animals typically have a prolonged cell-associated viremia. Enriched populations of bovine monocytes, erythrocytes and lymphocytes were inoculated with BTV serotype 10 (BTV 10) and the infected cells then were examined by transmission electron microscopy to characterize the interaction of BTV with bovine blood cells. Replication of BTV 10 in monocytes and stimulated (replicating) lymphocytes was morphologically similar to that which occurred in Vero cells, with formation of viral inclusion bodies and virus-specific tubules. In contrast, BTV 10 infection of unstimulated (non-replicating) lymphocytes and erythrocytes did not progress beyond adsorption, after which virus particles persisted in invaginations of the cell membrane. Studies with core particles and neutralizing monoclonal antibodies established that outer capsid protein VP2 is necessary for attachment of BTV 10 to erythrocytes. These in vitro virus-cell interactions provide a cogent explanation for the pathogenesis of BTV infection of cattle, especially the prolonged cell associated viremia that occurs in BTV-infected cattle.

Animals↗
PubMed1994

Detection of bluetongue virus in the blood of inoculated calves: comparison of virus isolation, PCR assay, and in vitro feeding of Culicoides variipennis.

The interval after infection when bluetongue virus (BTV) was present in the blood of calves inoculated with BTV serotype 10 (BTV 10) was evaluated by virus isolation (VI) in embryonated chicken eggs (ECE), BTV-specific polymerase chain reaction (PCR), and in vitro blood feeding of vector Culicoides variipennis (C.v.) sonorensis. BTV nucleic acid was detected by PCR in blood cells for 16 to 20 weeks after infection whereas infectious virus was detected by VI in ECE for 2 to 8 weeks. BTV was detected in calf blood by in vitro feeding of C.v. sonorensis for only 0 to 2 weeks after inoculation of calves with BTV 10. Selected bloods which were positive by PCR analysis but not by VI in ECE were not infectious for sheep. The data are consistent with the hypothesis that prolonged viremia in BTV-infected cattle results from association of the virus with blood cells, especially erythrocytes. The fact that calf blood that contained viral nucleic acid as determined by PCR analysis, but not infectious virus as determined by VI in ECE, was not infectious for either the insect vector or sheep suggests that cattle whose blood contains BTV nucleic acid but not infectious virus are unimportant to the epidemiology of BTV infection.

Animals↗
PubMed1994

Heterogeneity of the L2 gene of field isolates of bluetongue virus serotype 17 from the San Joaquin Valley of California.

Genome segment 2 (L2) from six field isolates of bluetongue virus (BTV) serotype 17 was sequenced by cycling sequencing after the amplification of the viral cDNA by the polymerase chain reaction. The viruses were isolated from sheep, cattle and a goat in the San Joaquin Valley of California during the years 1981 and 1990. These viruses exhibit divergent patterns of neutralization with BTV 17-specific monoclonal antibodies. The six L2 genes of the BTV 17 field isolates all encode a protein of 955 amino acids. Similarity of the nucleotide sequences of the L2 genes with respect to the prototype strain ranges between 93.8% and 95.1%, whereas the similarity between the field isolates ranges from 96.8% to 99.1%. Although very closely related, the L2 gene of each virus is distinct. Furthermore, mutations in the L2 gene of field isolates of BTV do not consistently follow a linear pattern of accumulation over time. Some amino acid changes in the VP2 protein of field strains were conserved over time, whereas others were not correlated with the year of isolation and some substitutions were unique to individual viruses. The predicted VP2s constitute a group of non-identical, but closely related proteins. Phylogenetic analyses suggest that the viral variants which co-circulate in the San Joaquin Valley could evolve by different evolutionary pathways.

Amino Acid Sequence↗
PubMed1994

Dynamics of viral spread in bluetongue virus infected calves.

The kinetics of viremia and sites of viral replication in bluetongue virus (BTV) infected calves were characterized by virus isolation, serology and immunofluorescence staining procedures. In addition, the role of the regional lymph node and lymphatics draining inoculated skin in the pathogenesis of BTV infection was determined by analyzing efferent lymph collected from indwelling cannulas. Viremia persisted for 35 to 42 days after inoculation (DAI) and virus co-circulated with neutralizing antibodies for 23 to 26 days. Virus was first isolated from peripheral blood mononuclear (PBM) cells at 3 DAI, after stimulation of PBM cells with interleukin 2 and mitogen. BTV was frequently isolated from erythrocytes, platelets and stimulated PBM cells but never from granulocytes and rarely from plasma during viremia. Virus was consistently isolated from erythrocytes late in the course of viremia. Interruption of efferent lymph flow by cannulation delayed the onset of viremia to 7 DAI. BTV was infrequently isolated from lymph cells, and few fluorescence positive cells were observed after lymph and PBM cells were labelled with a BTV-specific monoclonal antibody. Virus was isolated from spleen by 4 DAI and most tissues by 6 DAI, whereas virus was isolated from bone marrow only at 10 DAI. Virus was not isolated from any tissue after termination of viremia. It is concluded that primary viral replication occurred in the local lymph node and BTV then was transported in low titer to secondary sites of replication via infected lymph and PBM cells. We speculate that virus replication in spleen resulted in release of virus into the circulation and non-selective infection of blood cells which disseminated BTV to other tissues. Virus association with erythrocytes likely was responsible for prolonged viremia, although infected erythrocytes eventually were cleared from the circulation and persistent BTV infection of calves did not occur.

Animals↗
PubMed1994

Evidence of natural bluetongue virus infection among African carnivores.

Bluetongue is an International Office of Epizootics List A disease described as the century's most economically devastating affliction of sheep. Bluetongue (BLU) viruses were thought to infect only ruminants, shrews, and some rodents, but recently, inadvertent administration of BLU virus-contaminated vaccine resulted in mortality and abortion among domestic dogs. We present evidence of natural BLU virus infection among African carnivores that dramatically widens the spectrum of susceptible hosts. We hypothesize that such infection occurred after ingestion of meat and organs from BLU virus-infected prey species. The effect of BLU virus on endangered carnivores such as the cheetah and African wild dog requires urgent investigation. Also, the role of carnivores in the epizootiology of this disease needs elucidation.

Africa↗
PubMed1993

Induction of type I diabetes by interferon-alpha in transgenic mice.

Type I diabetes is an autoimmune disease involving an interaction between an epigenetic event (possibly a viral infection), the pancreatic beta cells, and the immune system in a genetically susceptible host. The possibility that the type I interferons could mediate this interaction was tested with transgenic mice in which the insulin-producing beta cells expressed an interferon-alpha. These mice developed a hypoinsulinemic diabetes associated with a mixed inflammation centered on the islets. The inflammation and the diabetes were prevented with a neutralizing antibody to the interferon-alpha. Thus, the expression of interferon-alpha by the beta cells could be causal in the development of type I diabetes, which suggests a therapeutic approach to this disease.

Animals↗
PubMed1993

Neutralization determinants of United States bluetongue virus serotype ten.

A panel of five neutralization-resistant escape mutant (EM) viruses was used to investigate the neutralization determinants of the U.S. prototype strain of bluetongue virus serotype 10 (BTV-10). The phenotypic properties of each EM virus were characterized by neutralization and immuneprecipitation assays with a panel of four monoclonal antibodies (MAbs). These MAbs were used to select the various EM viruses and together the MAbs define four distinct neutralizing epitopes on the prototype strain of BTV-10 (Heidner, H.W., Rositto, P.V., and MacLachlan, N.J., Virology 176, 658-661 (1990)). Sequencing of the L2 gene identified mutations responsible for the altered phenotypic properties exhibited by each EM virus. The L2 gene encodes BTV outer capsid protein VP2 which is responsible for virus neutralization. Four amino acids in three distinct regions of VP2 are critical to expression of the epitopes recognized by the MAb panel. Both amino acid 208 and 211 can affect the binding of MAb 039 and MAb 045, amino acid 327 affects binding of MAb 041, and amino acids 327 and 402 cooperatively interact to affect binding by MAb 034. The location of two of these critical regions on VP2 of BTV-10 is identical to two of those which affect neutralization of Australian BTV-1, despite the fact that these two viruses are antigenically distinct and have divergent L2 gene sequences (Gould, A.R., and Eaton, B.T., Virus Res. 17, 161-172 (1990)). The four individual neutralizing epitopes on VP2 of BTV-10 are interactive (Heidner, H.W., Rositto, P.V., and MacLachlan, N.J., Virology 176, 658-661 (1990)) and at least two are conformationally dependent.

Antibodies, Monoclonal↗
PubMed1993

A 29K envelope glycoprotein of equine arteritis virus expresses neutralization determinants recognized by murine monoclonal antibodies.

A panel of six neutralizing murine monoclonal antibodies (MAbs) to equine arteritis virus (EAV) was produced. The MAbs were characterized by Western immunoblotting assay and competitive ELISA. The six MAbs identify a single neutralization site on a 29K envelope glycoprotein. Deglycosylation of viral proteins prior to immunoblotting showed that the 29K protein is the glycosylated form of a 20K protein. Equine anti-EAV serum also strongly bound the 29K glycoprotein, as well as an unglycosylated protein of 17K. The equine antisera to EAV blocked the binding of a selected MAb to EAV, whereas normal equine serum did not. Two neutralization-resistant escape mutant (EM) variants of the EAV prototype were produced using MAb 6D10. The phenotypic properties of the EM viruses were characterized by neutralization and immunoblotting assays with two MAbs (6D10 and 5G11). The two MAbs failed to neutralize either EM virus, and they did not react in an immunoblot assay with any proteins of the EM viruses. In contrast, binding of the equine antiserum to viral proteins was equivalent with prototype and EM virus strains. These data clearly indicate that a 29K envelope glycoprotein expresses at least one neutralization determinant of EAV.

Animals↗
PubMed1993

Prevalence of bluetongue virus expression in leukocytes from experimentally infected ruminants.

Replication of bluetongue virus (BTV) in leukocytes from the blood of sheep, cattle, elk, and mule deer inoculated with BTV serotype 10 or 17 was assessed by immunocytochemical staining and dot blot northern hybridization to determine if differences in the prevalence of infection in this blood fraction might account for the differences in clinical disease among these species. Viremia was confirmed by virus isolation in all inoculated animals. Analysis of leukocytes with monoclonal antibodies specific for BTV proteins revealed low numbers of infected leukocytes in only 2 sheep 8 days after inoculation with BTV serotype 10. Most of the cells expressing BTV were identified morphologically as monocytes; approximately 10% of infected cells were lymphocytes. Bluetongue virus was not detected by use of dot-blot hybridization on samples of blood. Our results suggest that differential infection of leukocytes does not account for the pronounced differences in clinical signs and pathologic changes among ruminants.

Animals↗
PubMed1992

Variation amongst the neutralizing epitopes of bluetongue viruses isolated in the United States in 1979-1981.

Neutralizing epitopes present on field isolates of bluetongue virus (BTV) serotypes 10, 11, 13 and 17 were evaluated with a panel of polyclonal and neutralizing monoclonal antibodies (MAbs). A total of 91 field isolates were evaluated, including 15 isolates of BTV-10, 29 isolates of BTV-11, 26 isolates of BTV-13, and 21 isolates of BTV-17. The viruses were isolated from cattle, goats, sheep, elk and deer in Idaho, Louisiana, Nebraska and, predominantly, California, in the years 1979, 1980 and 1981. The isolates were analyzed and compared using a panel of neutralizing MAbs which included five MAbs raised against BTV-2, seven against BTV-10, five against BTV-13, and six against BTV-17. Neutralization patterns obtained with the MAb panel and individual field isolates were compared to those obtained with prototype viruses of each serotype. All field isolates were neutralized by at least some of the MAbs raised against the prototype virus of the same serotype. All field isolates of BTV-10 were neutralized by the seven MAbs raised to BTV-10, whereas the field isolates of BTV-11, BTV-13 and BTV-17 were not consistently neutralized by all of the MAbs raised against the prototype virus of the same serotype. Variation in neutralizing epitopes recognized by the MAb panel was most pronounced amongst the field isolates of BTV-17. A one-way cross neutralization was evident between BTV-10 and BTV-17 as all field isolates of BTV-17 were neutralized by four of the MAbs raised against BTV-10. In contrast, no BTV-10 isolates were neutralized by the MAbs raised against BTV-17. Differences in the MAb neutralization patterns of field isolates of BTV-11, BTV-13 and BTV-17 suggest that the immunogenic domain responsible for their neutralization is plastic, such that individual epitopes within the domain may vary in their significance to the neutralization of different viruses, even of the same serotype. The apparent conservation of neutralizing epitopes on field isolates of BTV-10 suggests that the field isolates may be derived from the modified-live vaccine strain of BTV-10.

Animals↗
PubMed1992

The detection of intracellular bluetongue virus particles within ovine erythrocytes.

1. We report here a simplified method for detecting viruses and other antigenic agents in red blood cells (RBCs). Using a nonionic detergent to prepare cytoskeletons, the interior of RBCs can be scanned rapidly using immunoelectron microscopy. 2. In this study, RBCs from bluetongue (BLU) virus-infected sheep were adsorbed directly onto Formvar-coated, gold electron microscope grids. 3. Cytoskeletons were prepared and then probed using a monoclonal antibody to VP 7, a structural BLU-virus protein and Protein-A gold. 4. Of the ca 32,000 RBCs that were examined from BLU virus-infected sheep, 34 (0.106%) contained labelled BLU virus particles. 5. No labelled particles were observed in any of ca 8000 RBCs taken prior to BLU virus inoculation of sheep. 6. If the antigenic BLU virus particles (which may be viral cores) are in fact infectious, this method of sequestration of virus within RBCs could contribute to the prolonged viremia typical of this arboviral disease, which is known to occur concurrently with circulating neutralizing antibody.

Animals↗