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Biomedical subjects

J Brownlie

Publications and source records attributed to J Brownlie.

At least 19 recordsLinked to original sources
PubMed2006

Real-time RT-PCR detection of Bovine Viral Diarrhoea virus in whole blood using an external RNA reference.

A novel two-step real-time RT-PCR assay using SYBR Green I was developed for the detection of acute Bovine Viral Diarrhoea virus (BVDV) infection in whole blood from cattle. During infection animals experience a characteristic transient leucopenia and the number of cells per volume of blood changes over time; so quantitation of viral load by reference to a cellular housekeeping gene is not ideal as this may hide significant animal to animal variation. Therefore, to facilitate comparison of different samples, an external RNA reference was used for normalisation whereby each sample was spiked with the RNA virus, Canine Enteric Coronavirus (CECov), prior to RNA extraction, for comparative purposes. Real-time RT-PCR was carried out with two primer sets designed to amplify either a 156 bp region of the BVDV 5'-UTR or a 280 bp region of the CECov nucleocapsid protein gene. Linearity and efficiency of the assay was established and the method assessed using samples from BVDV-challenged calves. Viral RNA was quantified on days 6 and 14 post-challenge by real-time RT-PCR. Infectious virus isolation by traditional cell culture was negative after day 7. This study demonstrates encouraging results for rapid, sensitive and reliable detection of acute BVDV infection and provides an alternative real-time RT-PCR method for use on whole blood samples or samples where suitable housekeeping genes are not available.

5' Untranslated Regions↗
PubMed2005

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) in NS3 DNA vaccinated and naturally infected cattle.

Immune responses to non-structural protein 3 (NS3) of bovine viral diarrhoea virus (BVDV) were investigated. cDNA encoding NS3 from type 1a BVDV was used to vaccinate five calves, another five calves remained unvaccinated. Three weeks after final vaccination animals were challenged intranasally with heterologous type 1a BVDV. Anti-NS3 antibodies were detected in only one animal post-vaccination. Partial protection from virus challenge was observed in the vaccinates. Virus was not isolated from nasal mucosa of two vaccinates, and virus clearance from nasal mucosa was faster in the vaccinates compared to the controls. While elevated rectal temperatures were evident in both groups 7 days post-challenge, the mean increase in the controls was twice that observed in the vaccinates. In conclusion, NS3 DNA vaccination induced humoral immunity in one calf, and prevented fever and virus establishment in the nasal mucosa in 2/5 calves, demonstrating the efficacy of NS3 vaccination, which may benefit future development of pestivirus and flavivirus vaccines.

Animals↗
PubMed2005

Investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations.

Two training centres for working dogs were monitored for one year to determine the presence of viruses and viral antibodies and their association with canine infectious respiratory disease (CIRD). Tonsillar swabs and serum were obtained from dogs on entry into the kennels and in regular intervals thereafter. Additional samples were collected during outbreaks of CIRD. The swabs were examined by virus culture and PCR for canine parainfluenza virus, canine adenovirus, canine herpesvirus (CHV) and canine respiratory coronavirus (CRCoV). Furthermore the prevalence of antibodies to CHV and CRCoV was determined. During this study CIRD was reported mainly in one of the two kennels investigated. In that kennel antibody responses to CRCoV indicated a seasonal occurrence of the virus, which coincided with two outbreaks of respiratory disease. CHV antibody responses were detected throughout the year. In the other kennel, which reported few cases of CIRD a high prevalence of antibodies to CRCoV was detected on entry but only sporadic seroconversions to CRCoV or CHV. By PCR three dogs were found positive for CRCoV in one kennel whereas all PCR tests for other viruses were negative for both kennels. Virus culture failed to detect any viruses in either kennel.

Animals↗
PubMed2004

Analysis of variation of bovine viral diarrhoea virus E2 sequence following transplacental infection of cattle.

The genetic and antigenic diversity observed in field isolates of bovine viral diarrhoea virus (BVDV) is thought to occur during acute infection because of the genetic stability observed in BVDV throughout the lifetime of persistently infected (PI) cattle. In this study, 15 cows in early pregnancy were inoculated with identical challenge doses obtained from a single infectious inoculum of the virologically cloned isolate Pe515nc. In order to examine the diversity that may develop in utero in the PI foetus, the variable E2 sequence of the virus isolated directly from the serum of each PI calf was compared. A high degree of sequence similarity was demonstrated, with 0-4 nucleotide differences out of 608 bases compared. Thus, the virus showed relatively few genomic changes in any of the PI calves, although we observed that the in utero environment did provide some opportunity for genetic variation to become established.

Amino Acid Sequence↗
PubMed2003

The role of the defective interfering particle DI9c in mucosal disease in cattle.

Mucosal disease occurs in cattle persistently infected with a noncytopathogenic strain of bovine viral diarrhoea virus (BVDVnc) following in utero infection. The disease can be initiated by superinfection with a cytopathogenic biotype (BVDVc) of the virus with antigenic "homology" to the persisting virus. A BVDVc isolated from a clinical case of mucosal disease has been discovered to consist of a defective interfering particle, DI9, and an associated BVDVnc helper virus. A defective virus corresponding to DI9 was recently recovered from an infectious cDNA clone and was named DI9c. To evaluate the role of DI9 in the pathogenesis of mucosal disease a two-part experimental study was carried out which included clinical, haematological, pathological and virological investigations. Eight of nine calves persistently infected with BVDVnc were experimentally inoculated with DI9c. The defective virus was propagated in cells preinfected with the same strain of virus used to persistently infect the calves in utero. The calves were euthanased on days 4, 7, 14, 21, 28, 40, 40 or 87 post inoculation. None of the inoculated animals developed classical mucosal disease, neither clinically nor pathologically. DI9c was not found in serum, nasal swab or tissue samples from the calves by observing cytopathogenic effect and/or using a polymerase chain reaction after reverse transcription (RT-PCR) of viral RNA. DI9c did not replicate to a detectable extent in these assays, and its participation in the pathogenesis of mucosal disease could not be proven.

Animals↗
PubMed2002

Tat protein expression in MDBK cells does not confer susceptibility to bovine immunodeficiency virus.

The ability of BIV strain R29 to infect bovine cell lines in the presence or absence of a functional lentiviral Tat protein is described. Jembrana disease virus (JDV) Tat protein was stably expressed in MDBK cells. No viral replication could be detected in this cell line after infection with BIV R29. Transfection of MDBK cells and MDBK Tat expressing cells with BIV R29 proviral DNA established that BIV R29 could not replicate in MDBK cells. Whether viral entry into MDBK cells is also a block to BIV R29 infection of MDBK cells has yet to be established.

Animals↗
PubMed2001

Cytokine adjuvancy of BVDV DNA vaccine enhances both humoral and cellular immune responses in mice.

The effect of cytokine adjuvancy on a bovine viral diarrhoea virus (BVDV) DNA vaccine expressing the major glycoprotein E2 was investigated in mice. Murine interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were chosen for their potential ability to enhance the humoral and cellular immune responses involved in protection against BVDV. Both cytokines, co-administered as separate plasmid constructs, had a marked effect on ELISA and neutralising antibody titres, improving the spectrum of neutralisation induced by the E2 DNA vaccine, as demonstrated in heterologous neutralisation assays. The predominance of IgG2a isotypes, in sera from all DNA injected groups, indicated a Th1 biased immune response. Antigen specific proliferation of murine splenocytes from immunised mice was enhanced by cytokine co-administration, with the highest stimulation indexes observed in the group co-injected with the GM-CSF construct. These results obtained in the mouse (Balb/c; H2-kd) animal model demonstrate the value of the two cytokines as adjuvants for the E2 DNA vaccine. The need for an adjuvant in this system was emphasised by the MHC restriction observed when C57BL/6 mice (H2-kb) were immunised with the E2 DNA construct. Antibody levels were dramatically lower in this mouse strain.

Animals↗
PubMed2001

Evidence for the presence of two bovine lentiviruses in the cattle population of Bali.

Antibodies directed against two bovine lentiviruses, Jembrana disease virus (JDV) and bovine immunodeficiency virus (BIV), were detected in Balinese cattle sera using two new enzyme-linked immunosorbent assays (ELISAs) based on the combination of capsid (CA) protein and transmembrane (TM) peptides derived from JDV or BIV sequences. Twenty eight of the 77 sera tested on the JDV ELISA showed anti-JDV antibodies with an unequal distribution of seropositive animals throughout the different districts of Bali. Furthermore, when 17 of the JDV positive sera were tested on Western blot, using the same JDV CA antigen, only 13 were judged positive confirming that the ELISA was a more sensitive technique for the detection of seropositive animals. Finally, 9 of the 49 JDV seronegative animals showed anti-BIV antibodies when tested on BIV-specific ELISA. These two ELISAs appeared to be highly sensitive for the detection of anti-JDV and anti-BIV antibodies. Moreover, for the first time, animals showing antibodies against BIV were identified on the main island of Bali and on the JDV-free Nusa Penida island.

Animals↗
PubMed2001

An immunosensor with potential for the detection of viral antigens in body fluids, based on surface second harmonic generation.

Field methods of assessing the immune status of animals are required to optimise vaccination programmes to control bovine viral diarrhoea (BVD) virus. An optoelectronic immunosensor was evaluated for the detection of viral antigens in a crude cell lysate in a pilot study. Binding of (BVD) virus antigen by two monoclonal antibodies immobilised on two different media (ELISA plate wells, and glass coverslips) was detected and quantified using the laser induced surface second harmonic generation (SSHG) technique. The results for both assays were correlated with an enzyme-linked immunoassay (ELISA) used for the diagnosis of BVD virus infection in cattle (ELISA plate; R(2)=0.86, coverslips; Exp. 1; R(2)=0.75, Exp. 2; R(2)=0.67). The method will allow rapid detection of antigens in the body fluids of farm animals.

Animals↗
PubMed2000

Co-administration of IL-2 enhances antigen-specific immune responses following vaccination with DNA encoding the glycoprotein E2 of bovine viral diarrhoea virus.

Induction of effective immunity requires the delivery of a protective antigen with appropriate co-stimulatory signals. For bovine viral diarrhoea virus (BVDV) this antigen is the major viral glycoprotein E2. Neutralising antibodies are directed towards the E2 protein and passive transfer of antibodies in serum or colostrum can completely protect against viral infection. DNA vaccination of mice with a construct encoding the E2 glycoprotein induced neutralising antibody levels that were potentially sufficient to prevent virus replication in a challenge system. The co-delivery of interleukin-2 (IL-2) further enhanced the levels of antibody raised. The strong IgG2a component of the antigen-specific antibody suggests a Th1 bias to the immune response induced following vaccination.

Adjuvants, Immunologic↗
PubMed1999

Bovine viral diarrhea virus quasispecies during persistent infection.

Analysis of viral genome sequences from two calves persistently infected with bovine viral diarrhea virus revealed a quasispecies distribution. The sequences encoding the glycoprotein E2 were variable, translating to a number of changes in predicted amino acid sequences. The NS3 region was found to be highly conserved in both animals. The number of E2 clones showing variant amino acids increased with the age of the animal and comparison of the consensus sequences at the different time points confirmed differences in the predicted E2 sequences over time. The immune tolerance that allows the lifelong persistence of this viral infection is highly specific. It is likely that some of the variant viruses generated within these animals will differ antigenically from the persisting virus and be recognized by the immune system. Evidence of an immune response to persisting virus infection was gathered from a larger sample of cattle. Serum neutralizing antibodies were found in 4 of 21 persistently infected animals. Accumulations of viral RNA in the lymph nodes of all animals examined, particularly in the germinal center light zone, may represent antigenic variants held in the form of immune complexes on the processes of follicular dendritic cells.

Amino Acid Sequence↗
PubMed1998

Noncytopathogenic bovine viral diarrhea virus (BVDV) reduces cleavage but increases blastocyst yield of in vitro produced embryos.

The growing application of in vitro embryo production systems that utilize slaughterhouse tissues of animals of unknown health status conveys the risk of disease transmission. One pathogen of concern in this regard is bovine viral diarrhea virus (BVDV), and the objective of this study was to investigate the effect of BVDV on in vitro embryonic development. A bovine in vitro embryo production system was experimentally infected with BVDV at 2 stages: prior to in vitro maturation by incubating cumulus-oocyte complexes (COC) with virus (strain Pe515; titer 10(6.2) tissue culture infective dose (TCID)50/mL) or vehicle for 2 h, and then during in vitro culture by the use of BVDV infected granulosa cells. Exposure to BVDV throughout in vitro production reduced cleavage rates (P = 0.01) but increased (P = 0.05) the number of embryos that reached the 8-cell stage when expressed as a percentage of cleaved oocytes. Blastocyst yield was increased by the presence of virus when expressed as a proportion of oocytes (P = 0.0034) or of those cleaved (P < 0.0001). The percentage of total blastocyst yield on Days 7, 8 and 9 for the control and virus treatments was 20, 51, 29 and 29, 41, and 29%, respectively, indicating that the rate of blastocyst development was nonsignificantly faster in the virus-treated group (P = 0.06). These results indicate that the presence of non-cytopathogenic BVDV in an in vitro production system may reduce cleavage rates but allow those cleaved to develop to blastocysts at a higher rate.

Animals↗
PubMed1998

Maternal recognition of foetal infection with bovine virus diarrhoea virus (BVDV)--the bovine pestivirus.

BACKGROUND: Pestiviruses are the veterinary viruses with genome homology to human hepatitis C virus (HCV). This group includes classical swine fever virus (CSFV), border disease virus of sheep (BDV) and bovine virus diarrhoea virus (BVDV). There are some similarities in the pathology of all three virus infections; in utero transmission to the foetus can cause early embryonic losses, severe congenital abnormalities and, particularly with BVDV, lifelong persistent infections. In situ hybridisation studies have demonstrated virus within reproductive tissues and the germinal centres of lymphoid tissue. OBJECTIVES: To examine the immune response of cattle throughout their pregnancy following infection with bovine pestivirus (BVDV) during the first trimester (before 110 days). STUDY DESIGN: In two experimental studies, heifers were infected with BVDV before 98 days gestation. Their antibody response was monitored during the remainder of the pregnancy. In another study, the antibody response of pregnant cattle was monitored following a natural infection of BVDV on a farm. Calves of the dams from all these three studies were examined, following birth, for persistent BVDV infection. RESULTS: It was observed that in dams carrying persistently infected foetuses, the immune response was markedly higher (13811 + 1273 U ELISA antibody) than in those dams carrying uninfected foetuses (2542+/-588 U ELISA antibody). These results were used to establish an antibody threshold (10000 U ELISA antibody) to predict the virus status of unborn calves during a farm outbreak of BVDV infection. The combined results of experimental and farm studies showed that in dams with low antibodies, 5/15 calves were infected whereas in dams with high antibodies, 17/19 calves were infected. CONCLUSIONS: The predictive reliability of the assay appeared valuable but not secure. The ability of BVDV to infect the foetus with consequent maternal recognition, whilst remaining inaccessible to maternal immune exclusion, is a novel finding.

Abortion, Veterinary↗
PubMed1998

Genome instability in BVDV: an examination of the sequence and structural influences on RNA recombination.

The cytopathogenic biotype of the pestivirus, bovine viral diarrhea virus, is frequently a product of nonhomologous recombination in the region of the genome encoding the viral NS2-NS3 proteins. The possibility that sequences or structures in this region contributed to a hotspot for RNA recombination was examined. A PCR-based strategy was used to examine viral genomic RNA isolated from tissue samples of cattle persistently infected with the noncytopathogenic biotype of the virus. Analysis of two different regions of the viral genome revealed that recombination was not restricted to particular sequences. Alignment of the genomic sequences undergoing recombination and examination of the predicted secondary structures of the participating RNAs revealed that the dissociation of partial, newly synthesized negative strand RNAs from the positive strand template occurred at many different sites on the molecule. Similarly, it appeared that the reassociation of the RNA polymerase complex with a second positive strand template was frequently influenced by short regions of homology between the nascent RNA strand and open secondary structures in the template molecule.

Animals↗
PubMed1998

Detection of Jembrana disease virus in spleen, lymph nodes, bone marrow and other tissues by in situ hybridization of paraffin-embedded sections.

Jembrana disease virus (JDV) is a lentivirus that causes an acute, severe disease syndrome in infected Bali cattle in Indonesia. An in situ hybridization technique was developed that detected JDV genomic RNA in formalin-fixed paraffin-embedded tissue sections, using a digoxigenin-labelled riboprobe. Large numbers of JDV-infected cells were demonstrated in many tissue sections from experimentally infected animals early in the disease course, which was consistent with the extremely high circulating viraemia previously reported to occur during the febrile phase. The number of infected cells was consistently highest in sections of spleen, followed by many other tissues including lymph nodes, lungs, bone marrow, liver and kidney. Infected cells were also identified in the general circulation and within unusual intravascular lesions in lung sections. The relatively high level of infection found in bone marrow suggested that its involvement may be important in the disease pathogenesis, as it is with other lentiviruses.

Animals↗