Persistent infection of macrophages with bovine herpesvirus 4 (BoHV-4) has been proposed to play ... more Persistent infection of macrophages with bovine herpesvirus 4 (BoHV-4) has been proposed to play a secondary causal role, along with bacterial infection, in bovine post-partum metritis. Mechanisms of maintenance of BoHV-4 persistent infection are not understood. We previously generated in vitro models of BoHV-4 persistent infection in human rhadomyosarcoma and bovine macrophage cell lines by drug selection of cells infected with BoHV-4 carrying a drug-resistance marker, and demonstrated circular episomal BoHV-4 genomes. In the present study, we used fluorescent in situ hybridization (FISH) to demonstrate BoHV-4 genomes also integrated into the genomes of these persistently infected cells. Findings Bovine herpesvirus 4 (BoHV-4), a member of the Gammaherpesvirinae subfamily, was first isolated in Europe from respiratory and ocular diseases by Bartha and colleagues [1] and later in the United States by Mohanty and colleagues [2]. BoHV-4 has been isolated from a variety of samples and c...
We previously demonstrated that chickens primed with a recombinant Newcastle disease virus LaSota... more We previously demonstrated that chickens primed with a recombinant Newcastle disease virus LaSota (rLS) expressing the S2 gene of infectious bronchitis virus (IBV) and boosted with an attenuated IBV Massachusetts (Mass)-type vaccine were protected against IBV Arkansas (Ark)-type virulent challenge. A possible basis for the reported ability of IBV 4/91 (serotype 793/B) vaccine to protect against divergent IBV strains (e.g., QX, Q1, and D1466) in a prime-boost approach with an IBV Mass vaccine is that an immune response against the S2 protein of IBV 4/91 is cross-protective. Therefore, we evaluated the protective capabilities of the S2 protein of IBV 4/91 expressed from rLS. The level of S2 amino acid sequence identity between 4/91 and the Ark challenge strain used in this study (90.7%) is within the range of S2 amino acid sequence identities between 4/91 and Q1 (91%-94%) and QX (89%-94%) strains. Chickens primed with attenuated Mass IBV at 1 day of age and boosted with rLS/IBV.S2-4/9...
The function of the chicken's major histocompatibility complex (MHC or B complex) class I maj... more The function of the chicken's major histocompatibility complex (MHC or B complex) class I major (BF2) and minor (BF1) glycoproteins is compared for their expression, ability to present viral antigens to cytotoxic T lymphocytes (CTLs), and interaction with natural killer (NK) cells. MHC-restricted CTLs recognized virus antigen in the context of the BF2*21 major glycoprotein but not the BF1*21 minor glycoprotein. Marek's disease virus (MDV), a large DNA virus known to reduce the cell surface expression of class I glycoprotein, reduced the expression of BF2 glycoprotein while BF1glycoprotein expressions are remained as no change or slight increase. In addition, the expression of BF1*21 class I glycoprotein protected target cells from NK cell lysis while the expression of the BF2*21 class I glycoprotein enhanced NK cell lysis of target cells. Therefore, BF1 and BF2 provide two different cellular immune functions; BF1 negatively regulates the NK cell killing activity and BF2 rest...
The function of the chicken's major histocompatibility complex (MHC or B complex) class I maj... more The function of the chicken's major histocompatibility complex (MHC or B complex) class I major (BF2) and minor (BF1) glycoproteins is compared for their expression, ability to present viral antigens to cytotoxic T lymphocytes (CTLs), and interaction with natural killer (NK) cells. MHC-restricted CTLs recognized virus antigen in the context of the BF2*21 major glycoprotein but not the BF1*21 minor glycoprotein. Marek's disease virus (MDV), a large DNA virus known to reduce the cell surface expression of class I glycoprotein, reduced the expression of BF2 glycoprotein while BF1glycoprotein expressions are remained as no change or slight increase. In addition, the expression of BF1*21 class I glycoprotein protected target cells from NK cell lysis while the expression of the BF2*21 class I glycoprotein enhanced NK cell lysis of target cells. Therefore, BF1 and BF2 provide two different cellular immune functions; BF1 negatively regulates the NK cell killing activity and BF2 rest...
The Figure 3 in the original version of this article was incorrectly published. In this article t... more The Figure 3 in the original version of this article was incorrectly published. In this article the top panel of Figure 3 that describes the amino acid sequence alignment is now added. The original article has been corrected.
With cDNA probes and by Northern (RNA) blot analysis, a region containing immediate-early (IE) ge... more With cDNA probes and by Northern (RNA) blot analysis, a region containing immediate-early (IE) genes in the channel catfish virus (CCV) genome was identified. IE transcription in CCV-infected cells appears to be restricted to the terminal repeat region, suggesting that CCV is most closely related to the alpha subfamily of herpesviruses. CCV DNA fragments from this region encoding IE transcripts were cloned. Northern analysis with one of these cloned fragments, a 3,927-bp EcoRI-XbaI fragment, indicates that it encodes two IE transcripts. Both transcripts (ie1 and ie2) were characterized by S1 nuclease analysis, primer extension analysis, and analysis of cDNAs. The ie2 transcript is a 1.3-kb bicistronic mRNA containing open reading frame (ORF) 8a and ORF 9. ORF 8a is a 5'-truncated version of ORF 8 which, along with ORF 9, was previously identified (A. J. Davison, Virology 186:9-14, 1992). The ie1 transcript is 0.6 kb in size, contains only ORF 9, and is expressed at a level appro...
Although bovine herpesvirus-4 (BHV-4), a gammaherpesvirus lacking a clear disease association, ha... more Although bovine herpesvirus-4 (BHV-4), a gammaherpesvirus lacking a clear disease association, has been demonstrated in many tissues during persistent BHV-4 infection, a likely site of virus persistence is in cells of the monocyte/macrophage lineage. To establish an in vitro model of persistent infection potentially useful for examining the molecular mechanisms of BHV-4 persistence/latency, we infected the bovine macrophage cell line BOMAC. Following extensive cell death, surviving cells were found to be persistently infected, maintaining the viral genome over many passages and producing low levels of infectious virus. Although selection was unnecessary for the maintenance of the viral genome, cells persistently infected with recombinant BHV-4 containing a neomycin-resistance gene could be selected with geneticin, thus confirming that persistent BHV-4 infection was compatible with cell survival and replication. Furthermore, persistent BHV-4 infection caused no decrease in the growth...
EcoRI, HindIII, and PstI restriction fragments representing the entire genome of the DN-599 isola... more EcoRI, HindIII, and PstI restriction fragments representing the entire genome of the DN-599 isolate, the American prototype strain, of bovine herpesvirus 4 (BHV-4) were cloned. These cloned restriction fragments were analyzed by restriction enzyme digestion, hybridization to Southern blots of BHV-4 (DN-599) DNA, and cross-hybridization of cloned fragments. The resulting data were used to construct restriction maps of the unique region of BHV-4 (DN-599) DNA for restriction enzymes EcoRI, HindIII, and PstI. The EcoRI and HindIII restriction maps confirm those previously deduced for the DN-599 isolate by hybridization of cloned fragments of a European strain of BHV-4 to Southern blots of DN-599 DNA. The PstI restriction map is the first reported for BHV-4.
We have begun to identify immediate-early (IE), early (E), and late (L) genes of BHV-4 by analysi... more We have begun to identify immediate-early (IE), early (E), and late (L) genes of BHV-4 by analysis of cytoplasmic polyadenylated RNA transcribed from the BHV-4 genome over the course of infection and in the presence of cycloheximide or phosphonoacetic acid. Labeled cDNA prepared from RNA isolated at different times was hybridized with Southern blots of viral DNA to show which regions of the genome are transcribed at different times. In a second series of experiments, radiolabeled cloned restriction fragments representing the entire BHV-4 genome were hybridized separately to RNA on Northern blots to determine the number and sizes of transcripts at different times. As expected, with increasing time after infection, more portions of the BHV-4 genome are transcribed and a larger number and a greater abundance of viral transcripts are present. RNA transcribed from terminal repeats was not detectable at any time. However, similarity in size of RNA transcribed from opposite ends of the unique region of the genome late in infection suggests that RNA is transcribed over the fused ends of the genome, and terminal repeats are removed during RNA processing. Identification of IE, E, and L transcripts by this analysis lays the foundation for further study of specific BHV-4 transcripts and genes.
Persistent infection of macrophages with bovine herpesvirus 4 (BoHV-4) has been proposed to play ... more Persistent infection of macrophages with bovine herpesvirus 4 (BoHV-4) has been proposed to play a secondary causal role, along with bacterial infection, in bovine post-partum metritis. Mechanisms of maintenance of BoHV-4 persistent infection are not understood. We previously generated in vitro models of BoHV-4 persistent infection in human rhadomyosarcoma and bovine macrophage cell lines by drug selection of cells infected with BoHV-4 carrying a drug-resistance marker, and demonstrated circular episomal BoHV-4 genomes. In the present study, we used fluorescent in situ hybridization (FISH) to demonstrate BoHV-4 genomes also integrated into the genomes of these persistently infected cells. Findings Bovine herpesvirus 4 (BoHV-4), a member of the Gammaherpesvirinae subfamily, was first isolated in Europe from respiratory and ocular diseases by Bartha and colleagues [1] and later in the United States by Mohanty and colleagues [2]. BoHV-4 has been isolated from a variety of samples and c...
We previously demonstrated that chickens primed with a recombinant Newcastle disease virus LaSota... more We previously demonstrated that chickens primed with a recombinant Newcastle disease virus LaSota (rLS) expressing the S2 gene of infectious bronchitis virus (IBV) and boosted with an attenuated IBV Massachusetts (Mass)-type vaccine were protected against IBV Arkansas (Ark)-type virulent challenge. A possible basis for the reported ability of IBV 4/91 (serotype 793/B) vaccine to protect against divergent IBV strains (e.g., QX, Q1, and D1466) in a prime-boost approach with an IBV Mass vaccine is that an immune response against the S2 protein of IBV 4/91 is cross-protective. Therefore, we evaluated the protective capabilities of the S2 protein of IBV 4/91 expressed from rLS. The level of S2 amino acid sequence identity between 4/91 and the Ark challenge strain used in this study (90.7%) is within the range of S2 amino acid sequence identities between 4/91 and Q1 (91%-94%) and QX (89%-94%) strains. Chickens primed with attenuated Mass IBV at 1 day of age and boosted with rLS/IBV.S2-4/9...
The function of the chicken's major histocompatibility complex (MHC or B complex) class I maj... more The function of the chicken's major histocompatibility complex (MHC or B complex) class I major (BF2) and minor (BF1) glycoproteins is compared for their expression, ability to present viral antigens to cytotoxic T lymphocytes (CTLs), and interaction with natural killer (NK) cells. MHC-restricted CTLs recognized virus antigen in the context of the BF2*21 major glycoprotein but not the BF1*21 minor glycoprotein. Marek's disease virus (MDV), a large DNA virus known to reduce the cell surface expression of class I glycoprotein, reduced the expression of BF2 glycoprotein while BF1glycoprotein expressions are remained as no change or slight increase. In addition, the expression of BF1*21 class I glycoprotein protected target cells from NK cell lysis while the expression of the BF2*21 class I glycoprotein enhanced NK cell lysis of target cells. Therefore, BF1 and BF2 provide two different cellular immune functions; BF1 negatively regulates the NK cell killing activity and BF2 rest...
The function of the chicken's major histocompatibility complex (MHC or B complex) class I maj... more The function of the chicken's major histocompatibility complex (MHC or B complex) class I major (BF2) and minor (BF1) glycoproteins is compared for their expression, ability to present viral antigens to cytotoxic T lymphocytes (CTLs), and interaction with natural killer (NK) cells. MHC-restricted CTLs recognized virus antigen in the context of the BF2*21 major glycoprotein but not the BF1*21 minor glycoprotein. Marek's disease virus (MDV), a large DNA virus known to reduce the cell surface expression of class I glycoprotein, reduced the expression of BF2 glycoprotein while BF1glycoprotein expressions are remained as no change or slight increase. In addition, the expression of BF1*21 class I glycoprotein protected target cells from NK cell lysis while the expression of the BF2*21 class I glycoprotein enhanced NK cell lysis of target cells. Therefore, BF1 and BF2 provide two different cellular immune functions; BF1 negatively regulates the NK cell killing activity and BF2 rest...
The Figure 3 in the original version of this article was incorrectly published. In this article t... more The Figure 3 in the original version of this article was incorrectly published. In this article the top panel of Figure 3 that describes the amino acid sequence alignment is now added. The original article has been corrected.
With cDNA probes and by Northern (RNA) blot analysis, a region containing immediate-early (IE) ge... more With cDNA probes and by Northern (RNA) blot analysis, a region containing immediate-early (IE) genes in the channel catfish virus (CCV) genome was identified. IE transcription in CCV-infected cells appears to be restricted to the terminal repeat region, suggesting that CCV is most closely related to the alpha subfamily of herpesviruses. CCV DNA fragments from this region encoding IE transcripts were cloned. Northern analysis with one of these cloned fragments, a 3,927-bp EcoRI-XbaI fragment, indicates that it encodes two IE transcripts. Both transcripts (ie1 and ie2) were characterized by S1 nuclease analysis, primer extension analysis, and analysis of cDNAs. The ie2 transcript is a 1.3-kb bicistronic mRNA containing open reading frame (ORF) 8a and ORF 9. ORF 8a is a 5'-truncated version of ORF 8 which, along with ORF 9, was previously identified (A. J. Davison, Virology 186:9-14, 1992). The ie1 transcript is 0.6 kb in size, contains only ORF 9, and is expressed at a level appro...
Although bovine herpesvirus-4 (BHV-4), a gammaherpesvirus lacking a clear disease association, ha... more Although bovine herpesvirus-4 (BHV-4), a gammaherpesvirus lacking a clear disease association, has been demonstrated in many tissues during persistent BHV-4 infection, a likely site of virus persistence is in cells of the monocyte/macrophage lineage. To establish an in vitro model of persistent infection potentially useful for examining the molecular mechanisms of BHV-4 persistence/latency, we infected the bovine macrophage cell line BOMAC. Following extensive cell death, surviving cells were found to be persistently infected, maintaining the viral genome over many passages and producing low levels of infectious virus. Although selection was unnecessary for the maintenance of the viral genome, cells persistently infected with recombinant BHV-4 containing a neomycin-resistance gene could be selected with geneticin, thus confirming that persistent BHV-4 infection was compatible with cell survival and replication. Furthermore, persistent BHV-4 infection caused no decrease in the growth...
EcoRI, HindIII, and PstI restriction fragments representing the entire genome of the DN-599 isola... more EcoRI, HindIII, and PstI restriction fragments representing the entire genome of the DN-599 isolate, the American prototype strain, of bovine herpesvirus 4 (BHV-4) were cloned. These cloned restriction fragments were analyzed by restriction enzyme digestion, hybridization to Southern blots of BHV-4 (DN-599) DNA, and cross-hybridization of cloned fragments. The resulting data were used to construct restriction maps of the unique region of BHV-4 (DN-599) DNA for restriction enzymes EcoRI, HindIII, and PstI. The EcoRI and HindIII restriction maps confirm those previously deduced for the DN-599 isolate by hybridization of cloned fragments of a European strain of BHV-4 to Southern blots of DN-599 DNA. The PstI restriction map is the first reported for BHV-4.
We have begun to identify immediate-early (IE), early (E), and late (L) genes of BHV-4 by analysi... more We have begun to identify immediate-early (IE), early (E), and late (L) genes of BHV-4 by analysis of cytoplasmic polyadenylated RNA transcribed from the BHV-4 genome over the course of infection and in the presence of cycloheximide or phosphonoacetic acid. Labeled cDNA prepared from RNA isolated at different times was hybridized with Southern blots of viral DNA to show which regions of the genome are transcribed at different times. In a second series of experiments, radiolabeled cloned restriction fragments representing the entire BHV-4 genome were hybridized separately to RNA on Northern blots to determine the number and sizes of transcripts at different times. As expected, with increasing time after infection, more portions of the BHV-4 genome are transcribed and a larger number and a greater abundance of viral transcripts are present. RNA transcribed from terminal repeats was not detectable at any time. However, similarity in size of RNA transcribed from opposite ends of the unique region of the genome late in infection suggests that RNA is transcribed over the fused ends of the genome, and terminal repeats are removed during RNA processing. Identification of IE, E, and L transcripts by this analysis lays the foundation for further study of specific BHV-4 transcripts and genes.
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