Japhet Robert Lyaku | University of Namibia (original) (raw)
Papers by Japhet Robert Lyaku
Ticks present a major challenge in livestock production, given the increased demand for animal pr... more Ticks present a major challenge in livestock production, given the increased demand for animal protein worldwide. In addition to being vectors of numerous diseases in livestock, ticks cause blood loss, worry and damage hides. Anaplasmosis, Theileriosis, Babesiosis and cowdriosis are amongst the critical tickborne diseases causing havoc across the world in the livestock industry. Global economic losses caused by these ectoparasites amount to billions of dollars annually. Although there are different methods of tick control, they all have shortcomings which frustrate the efforts of farmers in controlling ticks and tick-borne diseases. This has motivated researchers to search for sustainable alternative methods of tick control which include the genetic selection of naturally resistant breeds. The Major Histocompatibility complex (MHC) also known as the Bovine Leukocyte Antigen (BoLA) in cattle has been associated with tick-borne disease resistance in some cattle breeds. Different breeds have different levels of resistance to ticks and tick-borne diseases. The Bos indicus breeds and their crosses are known to be more resistant to ticks and tick-borne diseases than the Bos Taurus. Given the downsides of acaricides, vaccines and other tick-control methods, the use of tick and tick-borne disease-resistant cattle breeds is a promising choice for tick and tick-borne disease control. This review summarizes the role of the MHC in resistance to ticks and tick-borne diseases in indigenous cattle breeds of Sub-Saharan Africa.
Journal of Virological Methods, Dec 1, 1999
A total of 20 hybridoma cell lines secreting monoclonal antibodies (MAbs) against E. coli express... more A total of 20 hybridoma cell lines secreting monoclonal antibodies (MAbs) against E. coli expressed bovine herpesvirus-1 (BHV-1) gD fusion protein were produced following the fusion of Sp2/0 myeloma cells with splenocytes from BALB/c mice immunized previously with immunoaffinity purified BHV-1 gD fusion protein. An indirect fluorescent antibody test (IFAT) using BHV-1 infected MDBK cells was used for the selection
Journal of Virological Methods, May 1, 1999
Infection with bovine herpesvirus 1 (BHV 1) occurs worldwide and causes serious economic losses d... more Infection with bovine herpesvirus 1 (BHV 1) occurs worldwide and causes serious economic losses due to loss of animals, abortions, decreased milk production, and loss of body weight. There is a real need for sensitive diagnostic procedures for detection of the presence of virus in order to achieve effective control of BHV 1-induced diseases. BHV 1 is frequently found in bovine semen and can be widely transmitted through artificial insemination. Thus the detection of BHV 1 in artificial insemination centers and semen banks is of crucial importance in the control of its dissemination to the cattle industry, worldwide. In the present study, a protein amplification assay following polymerase chain reaction (PCR) of the highly conserved BHV 1 glycoprotein D gene was used in order to improve the sensitivity of direct virus detection in bovine semen. This method of BHV 1 detection is at least 200 orders of magnitude more sensitive than traditional PCR and would have direct clinical applications in antigen-based detection tests. In this method, amplification of the BHV 1 gD gene by PCR is followed by a coupled in vitro transcription translation of a small aliquot from the reaction. When the transcription translation was carried out in the presence of [35S]methionine and the products analyzed by SDS PAGE and autoradiography, 0.0014 TCID50 of virus could be detected in raw bovine semen in contrast to 0.28 TCID50 of virus detected using traditional PCR. Given the limitations in the method used for protein detection, this 'in vitro protein amplification' has the potential of attaining superior sensitivity for direct virus detection in clinical samples.
Journal of Virology, Apr 1, 1997
This study reports that in bovine herpesvirus 4, glycoprotein B (gB) is a heterodimer and a major... more This study reports that in bovine herpesvirus 4, glycoprotein B (gB) is a heterodimer and a major component of the virion, unlike gBs of Epstein-Barr virus (gp110) and murine gammaherpesvirus 68, two other gammaherpesviruses. These are new characteristics with regard to the general features of gB in the Gammaherpesvirinae subfamily.
Virology, Nov 1, 1995
Because several observations have suggested that replication of the gammaherpesvirus bovine herpe... more Because several observations have suggested that replication of the gammaherpesvirus bovine herpesvirus 4 (BHV-4) is influenced by the physiological state of the host cell, a study was carried out to determine the relationship between BHV-4 infection and the cell cycle. The temporal expression of BHV-4 late (L) proteins in unsynchronized cell cultures was first investigated by flow cytometry. Interestingly, L protein expression occurred in a limited number of cells infected with a high multiplicity of infection, and a reciprocal correlation between the percentage of positive cells and the cell density at the time of infection was demonstrated. Moreover, the finding that a BHV-4 early-late protein was expressed in nearly all the cells suggested that a blockage in the viral replication cycle occurred in some infected cells at the stage of viral DNA synthesis or L protein expression. Because this blockage could be the consequence of the dependence of one or both of these events on the cell cycle, they were investigated after infection of synchronized cell cultures. The following findings were made. (i) Cell transition through the S phase quantitatively increased the rate of BHV-4 DNA replication. (ii) BHV-4 DNA synthesis could not be detected in cells arrested in G 0. (iii) Synchronization of MDBK cells with Lovastatin before infection increased the percentage of cells expressing L proteins. (iv) In contrast, infection of cells arrested in G 0 led to few positive cells. Taken together these results showed that BHV-4 DNA replication and consequently the expression of L proteins are dependent on the S phase of the cell cycle. This dependence could be of importance for several biological properties of BHV-4 infection in vitro and might have implications for the biology of the virus in vivo.
Bioorganic & Medicinal Chemistry Letters, Sep 1, 2000
The objective of this study was to test the effect of lignin as a feed additive under Namibian en... more The objective of this study was to test the effect of lignin as a feed additive under Namibian environmental conditions. A total of 871 chickens (482 experimental and 489 control groups), weighing an average mass of 2 kg/head, were subjected to an experiment under Neudamm Campus (UNAM) environmental conditions (32 • C average temperature and 22% relative humidity). All chickens were fed with ordinary balanced ration earmarked for egg layers for 8 days and subjected to stress for 10 minutes per day. The experimental group was given purified lignin (Lignohumate KD) 60 mg/kg diluted in a litter of drinking water, as an anti-stressor feed additive and metabolic activity stimulator. Results of this study revealed an increase in egg production, reduction in feed intake, resistance to stress, and production of eggs of bigger sizes (graded as Extra-large), with strong shells as compared to those produced by the control group. At a certain stage, some chickens from the experimental group were unable to release eggs freely, a fact possibly related to egg size and possible deficient lubrication of cloacal environment. Studies are ongoing with the objective of identifying accurate amounts of lignin/kg necessary to feed egg-layer chickens for triggering an improvement of egg
Tropical Animal Health and Production, Jun 1, 1991
Journal of General Virology, 1998
The relationship between the two biotypes of bovine viral diarrhoea virus (BVDV) and the biologic... more The relationship between the two biotypes of bovine viral diarrhoea virus (BVDV) and the biological responses they induce was studied in 3-to 6-month-old calves inoculated intranasally with a homologous pair of non-cytopathic and cytopathic strains. Marked differences in virological and serological events occurred following exposure to a specific BVDV strain. The non-cytopathic biotype was frequently recovered from nasal secretions and blood cells during the first 28 days post-inoculation whereas the cytopathic counterpart was detected infrequently in nasopharyngeal swabs only. There was no correlation of the recovery of infectious virus in vivo with the biotype-specific neutralizing humoral immune response. Furthermore, seroconversion did not correlate with resistance to reinfection as judged by the transient viraemia and/or shedding of virus observed in a challenge experiment. Bovine viral diarrhoea virus (BVDV) belongs to the genus Pestivirus in the family Flaviviridae (Wengler, 1991). The strains of BVDV are grouped into two biotypes, non-cytopathic (ncp) and cytopathic (cp), according to their behaviour in cell culture (Baker, 1987) and the rearrangements in the non-structural NS23\NS3 coding gene (reviewed in Meyers & Thiel, 1996). The ncp biotype is commonly isolated from cases of acute infection and is invariably present in animals born persistently
Veterinary Microbiology, Jul 1, 1996
This study was conducted to evaluate the suitability of the rabbit as a model for bovine, herpesv... more This study was conducted to evaluate the suitability of the rabbit as a model for bovine, herpesvirus 5 (BHV-5) acute infection. In a preliminary experiment, a total of 24 one-month old New Zealand white rabbits were inoculated with BHV-5 or bovine herpesvirus 1 (BHV-1) by the intraconjunctival, intracerebral or intranasal routes. BHV-5 or BHV-1 inoculated in the conjunctiva induced virus proliferation in the eye mucosae and the nasal cavity of rabbits without meningo-encephalitis. On the other hand, only BHV-5 infection by intranasal or intracerebral routes produced a fatal meningo-encephalitis. The intranasal route was used in a further experiment for the establishment of a rabbit model for BHV-5 infection. A total of 45 rabbits were inoculated intranasally with BHV-5 or BHV-1. The results showed that intranasal inoculation of BHV-5 strain N569 in rabbits was followed by the development of a lethal meningo-encephalitis for 66% of rabbits while all BHV-1 infected rabbits remained healthy throughout this experiment (28 days). Analysis between the mortalities of rabbits infected with BHV-5 and BHV-1 were highly significant (p < 0.001). The presence of BHV-5 in the central nervous system (CNS) was confirmed by virus isolation (essentially the cerebrum, midbrain and pons) and by immunohistochemical staining of BHV-5 antigen (essentially in the neurons of the cerebrum) only in BHV-5 infected rabbits showing clinical signs of meningo-encephalitis. The findings obtained confirmed the suitability of a rabbit model for the establishment of BHV-5 neurological acute infection and also as a valuable tool for the comparative study of BHV-5 and BHV-1 neuropathogenicity.
Biologicals, Jul 1, 1990
A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure anti... more A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure antibody to bovine herpesvirus type 1 (BHV-1) in cattle sera. The optical density produced from a single dilution of test serum was compared with a standard curve and the results were read and printed out from a computer interfaced to a multichannel ELISA reader. The printed results were expressed in ELISA units. The ELISA results obtained on 370 cattle sera were compared with those of the serum neutralisation test (SNT). An agreement of 90.5% was obtained when reciprocal SNT titres equal to or greater than 4 and IgG ELISA units equal to or greater than 50 were taken as indicative of a specific reaction. Of the 370 sera, 35 gave discrepant results of which 21 were SNT positive/lgG ELISA negative and 14 were SNT negative/lgG ELISA positive. When the SNT positive sera negative in the IgG ELISA were tested in an IgM ELISA, 19 were found to be positive. Thus, when the IgG and IgM ELISA results were combined the overall agreement between the ELISA and SNT increased to 957%. The IgG ELISA had a sensitivity of 82.4% and specificity of 94.4% relative to the SNT, whereas the combined IgG and IgM ELISA results gave a sensitivity and specificity of 98.3% and 94.4% respectively. There was a good positive correlation between the two tests (r = 0.86).
Microbiology, 2000
Infectious salmon anaemia virus (ISAV) is a new orthomyxovirus-like virus. Thirteen isolates of I... more Infectious salmon anaemia virus (ISAV) is a new orthomyxovirus-like virus. Thirteen isolates of ISAV (11 from Canada, one from Norway and one from Scotland) were studied for their replication in the CHSE-214 cell line compared with that in the SHK-1 cell line. All isolates replicated in SHK-1 cells, producing CPE between 3 and 12 days post-inoculation (p.i.). Six Canadian isolates also replicated in CHSE-214 cells, with production of CPE between 4 and 17 days p.i. Analysis of a one-step growth curve of ISAV in CHSE-214 cells showed that progeny virions remained predominantly cell-associated, accounting for the focalized nature of the CPE in the cell monolayer. One isolate (HKS 36) replicated in CHSE-214 cells, as shown by positive RT-PCR results of blind passages, but was non-cytopathic. All of the isolates were analysed for genetic heterogeneity by RT-PCR and RFLP with EcoRI and XhoI in a fraction of genome segment 2. The Canadian isolates showed a different RFLP profile to those of isolates Glesvaer/2/90 from Norway and 390/98 from Scotland. Structural proteins of four isolates, 'Back Bay 98', RPC/NB-877, RPC/NB-049 and Glesvaer/2/90, were examined further by SDS-PAGE. All viruses showed four major polypeptides, designated here as VP1-VP4, in Coomassie blue-stained gels. In isolates Glesvaer/2/90 and RPC/NB-877, these viral proteins had estimated molecular masses of 74, 53, 46 and 26.5 kDa, respectively. Viral proteins in isolates 'Back Bay 98' and RPC/NB-049 were of similar sizes, except that VP3 was 43 kDa. Taken together, these results show that there are phenotypic differences among strains of ISAV.
Isolates of bovine herpesvirus-1 subtype 1 (BHV-1.1) and bovine herpesvirus-1 subtype 2 (BHV-1.2)... more Isolates of bovine herpesvirus-1 subtype 1 (BHV-1.1) and bovine herpesvirus-1 subtype 2 (BHV-1.2), the cause of infectious bovine rhinotracheitis-infectious pustular vulvovaginitis were compared with three other distinct ruminant alphaherpesviruses isolated from a goat (CapHV-2), a red deer (CerHV-1) and a reindeer (RanHV-1). Restriction endonuclease profiles produced by Bgin differentiated the five ruminant alphaherpesviruses. Amplification of a 468 bp fragment of each of the five alphaherpesviruses by the polymerase chain reaction (PCR) was achieved using 22 bp primers selected from the BHV-1 glycoprotein gl DNA sequence. All the amplified products contained Bgll and Hinfl cleavage sites. Only RanHV-1 did not contain Smal or Aval sites. Serological comparison by ELISA and serum neutralisation (SN) tests using rabbit hyperimmune sera, and cattle, goat, red deer and reindeer convalescent sera revealed a close serological cross-reactivity between all 5 viruses. Analysis of their relationships confirmed that: 1) the two BHV-1 viruses were most closely related, 2) CerHV-1 and CapHV-2 were more closely related to BHV-1 than they were to each other, and 3) RanHV-1 was more related to CerHV-1 than to BHV-1 and CapHV-2. Western blotting using polyclonal sera identified at least seven major proteins common to all the viruses with molecular weights of 130kDa, 108 kDa, 90 kDa, 87 kDa, 74 kDa, 64 kDa and 45 kDa. With a view to identifying epitopes which would allow the differentiation of these viruses, a panel of 14 monoclonal antibodies (Mabs) against CerHV-1 were derived from BALB/c mice previously immunised with gradient purified CerHV-1. Four Mabs which had neutralising activity against CerHV-1 reacted in i i i Western blots with polypeptides of Mr 68-70 kDa and 74 kDa. The other 10 Mabs reacted in Western blots with polypeptides of Mr 68-70 kDa and/or 64 kDa. Radioimmunoprecipitation (RIP) results indicated that the 74 kDa protein detected in CerHV-1 was analogous to the 74 kDa cleavage product of the gl glycoprotein of BHV-1. The reactivity of each Mab against the five ruminant alphaherpesviruses was tested by ELISA. Two Mabs cross-reacted with all the five ruminant alphaherpesviruses while 10 others showed variable reactivity with the 5 viruses. Two Mabs reacted exclusively with CerHV-1 antigens and these may be suitable for developing a diagnostic test to distinguish antibodies between BHV-1 and CerHV-1 in their natural hosts.
Archives of Virology, Sep 1, 1992
Using enzyme-linked immunosorbent assays the cross reactivity of bovine herpesvirus-l.l, bovine h... more Using enzyme-linked immunosorbent assays the cross reactivity of bovine herpesvirus-l.l, bovine herpesvirus-l.2, caprine herpesvirus-2, cervine (red deer) herpesvirus-1 and rangiferine (reindeer) herpesvirus-1 has been examined using rabbit hyperimmune antisera and convalescent cattle and red deer field sera. Significant cross-reactivity among all the five viruses was demonstrated. A detailed analysis showed that: (1) the two bovine herpesviruses are most closely related, (2) the cervine, caprine and rangiferine viruses are more closely related to the bovine viruses than they are to each other, (3) the cervine herpesvirus is more related to the bovine herpesvirus than to the rangiferine or caprine herpesviruses and (4) the rangiferine virus is more related to the cervine virus than to the bovine and caprine viruses. Cattle and red deer sera reacted most strongly with the bovine and cervine viruses respectively.
Journal of General Virology, Aug 1, 1997
Genes encoding glycoprotein gH and gL homologues were localized in the genome of the gammaherpesv... more Genes encoding glycoprotein gH and gL homologues were localized in the genome of the gammaherpesvirus bovine herpesvirus-4 (BHV-4). Both genes were sequenced and glutathione S-transferase fusion proteins were produced and used to immunize rabbits against the translation products of the two genes. The anti-gH serum recognized a protein with an apparent molecular mass (MM) of 110 kDa both in infected cells and in virions. This protein was sensitive to endo-β-N-acetylglucosaminase-H (endoH) and endoglycosidase F-N-glycosidase F (endoF-PNGaseF) digestion. A protein with the same relative mobility was immunoprecipitated from infected cells radiolabelled with [ 3 H]glucosamine which confirmed that this product (gp110), now designated BHV-4 gH, was glycosylated. Western blotting with the anti-gL serum detected in infected cells a product with an apparent
Biochemical Society Transactions, 2000
Tropical Animal Health and Production, Sep 1, 1988
Inactivated bovine herpesvirus 1 induces apoptotic cell death of mitogen-stimulated
Ticks present a major challenge in livestock production, given the increased demand for animal pr... more Ticks present a major challenge in livestock production, given the increased demand for animal protein worldwide. In addition to being vectors of numerous diseases in livestock, ticks cause blood loss, worry and damage hides. Anaplasmosis, Theileriosis, Babesiosis and cowdriosis are amongst the critical tickborne diseases causing havoc across the world in the livestock industry. Global economic losses caused by these ectoparasites amount to billions of dollars annually. Although there are different methods of tick control, they all have shortcomings which frustrate the efforts of farmers in controlling ticks and tick-borne diseases. This has motivated researchers to search for sustainable alternative methods of tick control which include the genetic selection of naturally resistant breeds. The Major Histocompatibility complex (MHC) also known as the Bovine Leukocyte Antigen (BoLA) in cattle has been associated with tick-borne disease resistance in some cattle breeds. Different breeds have different levels of resistance to ticks and tick-borne diseases. The Bos indicus breeds and their crosses are known to be more resistant to ticks and tick-borne diseases than the Bos Taurus. Given the downsides of acaricides, vaccines and other tick-control methods, the use of tick and tick-borne disease-resistant cattle breeds is a promising choice for tick and tick-borne disease control. This review summarizes the role of the MHC in resistance to ticks and tick-borne diseases in indigenous cattle breeds of Sub-Saharan Africa.
Journal of Virological Methods, Dec 1, 1999
A total of 20 hybridoma cell lines secreting monoclonal antibodies (MAbs) against E. coli express... more A total of 20 hybridoma cell lines secreting monoclonal antibodies (MAbs) against E. coli expressed bovine herpesvirus-1 (BHV-1) gD fusion protein were produced following the fusion of Sp2/0 myeloma cells with splenocytes from BALB/c mice immunized previously with immunoaffinity purified BHV-1 gD fusion protein. An indirect fluorescent antibody test (IFAT) using BHV-1 infected MDBK cells was used for the selection
Journal of Virological Methods, May 1, 1999
Infection with bovine herpesvirus 1 (BHV 1) occurs worldwide and causes serious economic losses d... more Infection with bovine herpesvirus 1 (BHV 1) occurs worldwide and causes serious economic losses due to loss of animals, abortions, decreased milk production, and loss of body weight. There is a real need for sensitive diagnostic procedures for detection of the presence of virus in order to achieve effective control of BHV 1-induced diseases. BHV 1 is frequently found in bovine semen and can be widely transmitted through artificial insemination. Thus the detection of BHV 1 in artificial insemination centers and semen banks is of crucial importance in the control of its dissemination to the cattle industry, worldwide. In the present study, a protein amplification assay following polymerase chain reaction (PCR) of the highly conserved BHV 1 glycoprotein D gene was used in order to improve the sensitivity of direct virus detection in bovine semen. This method of BHV 1 detection is at least 200 orders of magnitude more sensitive than traditional PCR and would have direct clinical applications in antigen-based detection tests. In this method, amplification of the BHV 1 gD gene by PCR is followed by a coupled in vitro transcription translation of a small aliquot from the reaction. When the transcription translation was carried out in the presence of [35S]methionine and the products analyzed by SDS PAGE and autoradiography, 0.0014 TCID50 of virus could be detected in raw bovine semen in contrast to 0.28 TCID50 of virus detected using traditional PCR. Given the limitations in the method used for protein detection, this 'in vitro protein amplification' has the potential of attaining superior sensitivity for direct virus detection in clinical samples.
Journal of Virology, Apr 1, 1997
This study reports that in bovine herpesvirus 4, glycoprotein B (gB) is a heterodimer and a major... more This study reports that in bovine herpesvirus 4, glycoprotein B (gB) is a heterodimer and a major component of the virion, unlike gBs of Epstein-Barr virus (gp110) and murine gammaherpesvirus 68, two other gammaherpesviruses. These are new characteristics with regard to the general features of gB in the Gammaherpesvirinae subfamily.
Virology, Nov 1, 1995
Because several observations have suggested that replication of the gammaherpesvirus bovine herpe... more Because several observations have suggested that replication of the gammaherpesvirus bovine herpesvirus 4 (BHV-4) is influenced by the physiological state of the host cell, a study was carried out to determine the relationship between BHV-4 infection and the cell cycle. The temporal expression of BHV-4 late (L) proteins in unsynchronized cell cultures was first investigated by flow cytometry. Interestingly, L protein expression occurred in a limited number of cells infected with a high multiplicity of infection, and a reciprocal correlation between the percentage of positive cells and the cell density at the time of infection was demonstrated. Moreover, the finding that a BHV-4 early-late protein was expressed in nearly all the cells suggested that a blockage in the viral replication cycle occurred in some infected cells at the stage of viral DNA synthesis or L protein expression. Because this blockage could be the consequence of the dependence of one or both of these events on the cell cycle, they were investigated after infection of synchronized cell cultures. The following findings were made. (i) Cell transition through the S phase quantitatively increased the rate of BHV-4 DNA replication. (ii) BHV-4 DNA synthesis could not be detected in cells arrested in G 0. (iii) Synchronization of MDBK cells with Lovastatin before infection increased the percentage of cells expressing L proteins. (iv) In contrast, infection of cells arrested in G 0 led to few positive cells. Taken together these results showed that BHV-4 DNA replication and consequently the expression of L proteins are dependent on the S phase of the cell cycle. This dependence could be of importance for several biological properties of BHV-4 infection in vitro and might have implications for the biology of the virus in vivo.
Bioorganic & Medicinal Chemistry Letters, Sep 1, 2000
The objective of this study was to test the effect of lignin as a feed additive under Namibian en... more The objective of this study was to test the effect of lignin as a feed additive under Namibian environmental conditions. A total of 871 chickens (482 experimental and 489 control groups), weighing an average mass of 2 kg/head, were subjected to an experiment under Neudamm Campus (UNAM) environmental conditions (32 • C average temperature and 22% relative humidity). All chickens were fed with ordinary balanced ration earmarked for egg layers for 8 days and subjected to stress for 10 minutes per day. The experimental group was given purified lignin (Lignohumate KD) 60 mg/kg diluted in a litter of drinking water, as an anti-stressor feed additive and metabolic activity stimulator. Results of this study revealed an increase in egg production, reduction in feed intake, resistance to stress, and production of eggs of bigger sizes (graded as Extra-large), with strong shells as compared to those produced by the control group. At a certain stage, some chickens from the experimental group were unable to release eggs freely, a fact possibly related to egg size and possible deficient lubrication of cloacal environment. Studies are ongoing with the objective of identifying accurate amounts of lignin/kg necessary to feed egg-layer chickens for triggering an improvement of egg
Tropical Animal Health and Production, Jun 1, 1991
Journal of General Virology, 1998
The relationship between the two biotypes of bovine viral diarrhoea virus (BVDV) and the biologic... more The relationship between the two biotypes of bovine viral diarrhoea virus (BVDV) and the biological responses they induce was studied in 3-to 6-month-old calves inoculated intranasally with a homologous pair of non-cytopathic and cytopathic strains. Marked differences in virological and serological events occurred following exposure to a specific BVDV strain. The non-cytopathic biotype was frequently recovered from nasal secretions and blood cells during the first 28 days post-inoculation whereas the cytopathic counterpart was detected infrequently in nasopharyngeal swabs only. There was no correlation of the recovery of infectious virus in vivo with the biotype-specific neutralizing humoral immune response. Furthermore, seroconversion did not correlate with resistance to reinfection as judged by the transient viraemia and/or shedding of virus observed in a challenge experiment. Bovine viral diarrhoea virus (BVDV) belongs to the genus Pestivirus in the family Flaviviridae (Wengler, 1991). The strains of BVDV are grouped into two biotypes, non-cytopathic (ncp) and cytopathic (cp), according to their behaviour in cell culture (Baker, 1987) and the rearrangements in the non-structural NS23\NS3 coding gene (reviewed in Meyers & Thiel, 1996). The ncp biotype is commonly isolated from cases of acute infection and is invariably present in animals born persistently
Veterinary Microbiology, Jul 1, 1996
This study was conducted to evaluate the suitability of the rabbit as a model for bovine, herpesv... more This study was conducted to evaluate the suitability of the rabbit as a model for bovine, herpesvirus 5 (BHV-5) acute infection. In a preliminary experiment, a total of 24 one-month old New Zealand white rabbits were inoculated with BHV-5 or bovine herpesvirus 1 (BHV-1) by the intraconjunctival, intracerebral or intranasal routes. BHV-5 or BHV-1 inoculated in the conjunctiva induced virus proliferation in the eye mucosae and the nasal cavity of rabbits without meningo-encephalitis. On the other hand, only BHV-5 infection by intranasal or intracerebral routes produced a fatal meningo-encephalitis. The intranasal route was used in a further experiment for the establishment of a rabbit model for BHV-5 infection. A total of 45 rabbits were inoculated intranasally with BHV-5 or BHV-1. The results showed that intranasal inoculation of BHV-5 strain N569 in rabbits was followed by the development of a lethal meningo-encephalitis for 66% of rabbits while all BHV-1 infected rabbits remained healthy throughout this experiment (28 days). Analysis between the mortalities of rabbits infected with BHV-5 and BHV-1 were highly significant (p < 0.001). The presence of BHV-5 in the central nervous system (CNS) was confirmed by virus isolation (essentially the cerebrum, midbrain and pons) and by immunohistochemical staining of BHV-5 antigen (essentially in the neurons of the cerebrum) only in BHV-5 infected rabbits showing clinical signs of meningo-encephalitis. The findings obtained confirmed the suitability of a rabbit model for the establishment of BHV-5 neurological acute infection and also as a valuable tool for the comparative study of BHV-5 and BHV-1 neuropathogenicity.
Biologicals, Jul 1, 1990
A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure anti... more A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure antibody to bovine herpesvirus type 1 (BHV-1) in cattle sera. The optical density produced from a single dilution of test serum was compared with a standard curve and the results were read and printed out from a computer interfaced to a multichannel ELISA reader. The printed results were expressed in ELISA units. The ELISA results obtained on 370 cattle sera were compared with those of the serum neutralisation test (SNT). An agreement of 90.5% was obtained when reciprocal SNT titres equal to or greater than 4 and IgG ELISA units equal to or greater than 50 were taken as indicative of a specific reaction. Of the 370 sera, 35 gave discrepant results of which 21 were SNT positive/lgG ELISA negative and 14 were SNT negative/lgG ELISA positive. When the SNT positive sera negative in the IgG ELISA were tested in an IgM ELISA, 19 were found to be positive. Thus, when the IgG and IgM ELISA results were combined the overall agreement between the ELISA and SNT increased to 957%. The IgG ELISA had a sensitivity of 82.4% and specificity of 94.4% relative to the SNT, whereas the combined IgG and IgM ELISA results gave a sensitivity and specificity of 98.3% and 94.4% respectively. There was a good positive correlation between the two tests (r = 0.86).
Microbiology, 2000
Infectious salmon anaemia virus (ISAV) is a new orthomyxovirus-like virus. Thirteen isolates of I... more Infectious salmon anaemia virus (ISAV) is a new orthomyxovirus-like virus. Thirteen isolates of ISAV (11 from Canada, one from Norway and one from Scotland) were studied for their replication in the CHSE-214 cell line compared with that in the SHK-1 cell line. All isolates replicated in SHK-1 cells, producing CPE between 3 and 12 days post-inoculation (p.i.). Six Canadian isolates also replicated in CHSE-214 cells, with production of CPE between 4 and 17 days p.i. Analysis of a one-step growth curve of ISAV in CHSE-214 cells showed that progeny virions remained predominantly cell-associated, accounting for the focalized nature of the CPE in the cell monolayer. One isolate (HKS 36) replicated in CHSE-214 cells, as shown by positive RT-PCR results of blind passages, but was non-cytopathic. All of the isolates were analysed for genetic heterogeneity by RT-PCR and RFLP with EcoRI and XhoI in a fraction of genome segment 2. The Canadian isolates showed a different RFLP profile to those of isolates Glesvaer/2/90 from Norway and 390/98 from Scotland. Structural proteins of four isolates, 'Back Bay 98', RPC/NB-877, RPC/NB-049 and Glesvaer/2/90, were examined further by SDS-PAGE. All viruses showed four major polypeptides, designated here as VP1-VP4, in Coomassie blue-stained gels. In isolates Glesvaer/2/90 and RPC/NB-877, these viral proteins had estimated molecular masses of 74, 53, 46 and 26.5 kDa, respectively. Viral proteins in isolates 'Back Bay 98' and RPC/NB-049 were of similar sizes, except that VP3 was 43 kDa. Taken together, these results show that there are phenotypic differences among strains of ISAV.
Isolates of bovine herpesvirus-1 subtype 1 (BHV-1.1) and bovine herpesvirus-1 subtype 2 (BHV-1.2)... more Isolates of bovine herpesvirus-1 subtype 1 (BHV-1.1) and bovine herpesvirus-1 subtype 2 (BHV-1.2), the cause of infectious bovine rhinotracheitis-infectious pustular vulvovaginitis were compared with three other distinct ruminant alphaherpesviruses isolated from a goat (CapHV-2), a red deer (CerHV-1) and a reindeer (RanHV-1). Restriction endonuclease profiles produced by Bgin differentiated the five ruminant alphaherpesviruses. Amplification of a 468 bp fragment of each of the five alphaherpesviruses by the polymerase chain reaction (PCR) was achieved using 22 bp primers selected from the BHV-1 glycoprotein gl DNA sequence. All the amplified products contained Bgll and Hinfl cleavage sites. Only RanHV-1 did not contain Smal or Aval sites. Serological comparison by ELISA and serum neutralisation (SN) tests using rabbit hyperimmune sera, and cattle, goat, red deer and reindeer convalescent sera revealed a close serological cross-reactivity between all 5 viruses. Analysis of their relationships confirmed that: 1) the two BHV-1 viruses were most closely related, 2) CerHV-1 and CapHV-2 were more closely related to BHV-1 than they were to each other, and 3) RanHV-1 was more related to CerHV-1 than to BHV-1 and CapHV-2. Western blotting using polyclonal sera identified at least seven major proteins common to all the viruses with molecular weights of 130kDa, 108 kDa, 90 kDa, 87 kDa, 74 kDa, 64 kDa and 45 kDa. With a view to identifying epitopes which would allow the differentiation of these viruses, a panel of 14 monoclonal antibodies (Mabs) against CerHV-1 were derived from BALB/c mice previously immunised with gradient purified CerHV-1. Four Mabs which had neutralising activity against CerHV-1 reacted in i i i Western blots with polypeptides of Mr 68-70 kDa and 74 kDa. The other 10 Mabs reacted in Western blots with polypeptides of Mr 68-70 kDa and/or 64 kDa. Radioimmunoprecipitation (RIP) results indicated that the 74 kDa protein detected in CerHV-1 was analogous to the 74 kDa cleavage product of the gl glycoprotein of BHV-1. The reactivity of each Mab against the five ruminant alphaherpesviruses was tested by ELISA. Two Mabs cross-reacted with all the five ruminant alphaherpesviruses while 10 others showed variable reactivity with the 5 viruses. Two Mabs reacted exclusively with CerHV-1 antigens and these may be suitable for developing a diagnostic test to distinguish antibodies between BHV-1 and CerHV-1 in their natural hosts.
Archives of Virology, Sep 1, 1992
Using enzyme-linked immunosorbent assays the cross reactivity of bovine herpesvirus-l.l, bovine h... more Using enzyme-linked immunosorbent assays the cross reactivity of bovine herpesvirus-l.l, bovine herpesvirus-l.2, caprine herpesvirus-2, cervine (red deer) herpesvirus-1 and rangiferine (reindeer) herpesvirus-1 has been examined using rabbit hyperimmune antisera and convalescent cattle and red deer field sera. Significant cross-reactivity among all the five viruses was demonstrated. A detailed analysis showed that: (1) the two bovine herpesviruses are most closely related, (2) the cervine, caprine and rangiferine viruses are more closely related to the bovine viruses than they are to each other, (3) the cervine herpesvirus is more related to the bovine herpesvirus than to the rangiferine or caprine herpesviruses and (4) the rangiferine virus is more related to the cervine virus than to the bovine and caprine viruses. Cattle and red deer sera reacted most strongly with the bovine and cervine viruses respectively.
Journal of General Virology, Aug 1, 1997
Genes encoding glycoprotein gH and gL homologues were localized in the genome of the gammaherpesv... more Genes encoding glycoprotein gH and gL homologues were localized in the genome of the gammaherpesvirus bovine herpesvirus-4 (BHV-4). Both genes were sequenced and glutathione S-transferase fusion proteins were produced and used to immunize rabbits against the translation products of the two genes. The anti-gH serum recognized a protein with an apparent molecular mass (MM) of 110 kDa both in infected cells and in virions. This protein was sensitive to endo-β-N-acetylglucosaminase-H (endoH) and endoglycosidase F-N-glycosidase F (endoF-PNGaseF) digestion. A protein with the same relative mobility was immunoprecipitated from infected cells radiolabelled with [ 3 H]glucosamine which confirmed that this product (gp110), now designated BHV-4 gH, was glycosylated. Western blotting with the anti-gL serum detected in infected cells a product with an apparent
Biochemical Society Transactions, 2000
Tropical Animal Health and Production, Sep 1, 1988
Inactivated bovine herpesvirus 1 induces apoptotic cell death of mitogen-stimulated