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Biomedical subjects

H J Field

Publications and source records attributed to H J Field.

At least 37 records · Page 2Linked to original sources
PubMed1997

Detection and distribution of equine herpesvirus 2 DNA in the central and peripheral nervous systems of ponies.

The distribution of equine herpesvirus 2 (EHV-2) DNA within neurological and lymphoid tissues from 12 EHV-2 seropositive Welsh mountain ponies was determined by PCR. The lymphoid sites sampled in this study were almost universally PCR positive, thus confirming the existing virus co-cultivation data which suggest that the lymph nodes draining the respiratory tract are the main reservoirs of EHV-2 DNA. In addition, EHV-2 DNA was also detected, albeit with lower frequency, within both the peripheral and central nervous systems (PNS and CNS) of these animals. Of the CNS sites sampled 11% were PCR-positive and in the PNS the trigeminal ganglion proved PCR-positive in 50% of the animals tested. Since the nasal epithelium is innervated by the maxillary division of the trigeminal nerve, these observations suggest that the trigeminal ganglion may represent a biologically important site for EHV-2 latency.

Animals↗
PubMed1997

Transmission of equine herpesvirus 2 to the mouse: characterization of a new laboratory infection model.

Intranasal inoculation of BALB/c mice with a recent clinical isolate of equine herpesvirus 2 (EHV-2 strain MR) produced a productive infection characterized by clinical signs, including weight-loss and conjunctivitis, but no mortality. Infectious virus was isolated from the lung, trachea and nasal turbinates with the highest titres present in lung tissue; EHV-2 neutralizing antibody was detected in the serum on day 21 post-inoculation. No infectious virus was detected in neural tissue, blood or lymphoid tissues. An EHV-2-specific nested PCR confirmed the presence of EHV-2 DNA in the respiratory tissues. In addition, EHV-2 DNA was detected in the trigeminal ganglia and olfactory bulbs from the first day after inoculation and these tissues remained positive for EHV-2 DNA for at least 30 days, by which time EHV-2 DNA had been cleared from all other tissues.

Animals↗
PubMed1997

The pathogenesis of ED71, a defined deletion mutant of equine herpesvirus-1, in a murine intranasal infection model for equine abortion.

A series of mutants of equine herpesvirus-1 (EHV-1) which contain deletions in non-essential genes was previously characterized in a murine intranasal infection model. One mutant, ED71 which was shown to be attenuated in the model, was further characterized by inoculation into pregnant mice. Despite the attenuation previously reported, intranasal inoculation of pregnant mice resulted in premature parturition and the birth of dead or dying foetuses. Furthermore, mice inoculated before pregnancy with the same mutant, and subsequently challenged 14 days after conception with wild-type virus, were not protected from abortion.

Abortion, Veterinary↗
PubMed1996

Molecular studies of the acute infection, latency and reactivation of equine herpesvirus-1 (EHV-1) in the mouse model.

The murine intranasal (i.n.) infection model was used to study the molecular distribution of equine herpesvirus-1 (EHV-1) during acute infection, latency and following a reactivation stimulus. After inoculation, infectious virus was detected in lungs, nasal turbinates, brains and olfactory bulbs during the acute phase. A nested PCR (nPCR) readily detected virus in these tissues and, in addition, virus was detected in spleens and (in the second round of nPCR) in peripheral blood mononuclear cells (PBMC). A digoxigenin-labelled in situ hybridization probe detected EHV-1 DNA in bronchiolar and vascular endothelium in the lungs and in and around germinal centres in the spleens. One month later, although infectious virus was absent from all tissues, the trigeminal ganglia, olfactory bulb and PBMC remained positive for virus DNA although this was detected only on the second round of nPCR. Furthermore, in situ hybridization, using either DNA or RNA probes, suggested that little or no transcription of virus occurred in neural tissues during the 'latent phase'. Following a reactivation stimulus, infectious virus was not isolated from any tissues, however, EHV-1 DNA was detected on the first round of nPCR in olfactory bulb, trigeminal ganglia and PBMC. This suggested a quantitative increase in EHV-1 DNA occurred following reactivation stimulus. The significance of these results is discussed in relation to the molecular state of EHV-1 in different tissues at various stages of infection and the validity of the murine model for studying latency and reactivation of EHV-1 in the horses.

Acute Disease↗
PubMed1996

Differential effects of famciclovir and valaciclovir on the pathogenesis of herpes simplex virus in a murine infection model including reactivation from latency.

The ability of famciclovir and valaciclovir to affect the establishment and maintenance of latency in mice with a cutaneous herpes simplex type 1 (HSV-1) infection was examined. Mice were treated via drinking water starting at various times between days 1 and 5 and terminating on day 10 after inoculation. Clinical signs and viral replication in the target tissues were monitored. Three to four months later, trigeminal and dorsal root ganglia were explanted from groups of 16 mice and examined for latent virus by cocultivation. The two compounds differed in their effects on the acute neural infection, and ganglia explanted from famciclovir-treated mice were markedly reduced in their ability to reactivate virus, although neither drug affected latency if treatment was delayed for several months. The difference between the compounds is likely to reflect differences in the metabolism of their respective products, penciclovir and acyclovir, in infected neurons.

2-Aminopurine↗
PubMed1996

Comparison of effects of famciclovir and valaciclovir on pathogenesis of herpes simplex virus type 2 in a murine infection model.

The effects of famciclovir (FCV) and valaciclovir (VACV) were compared in a cutaneous infection model for herpes simplex virus type 2 (HSV-2). The compounds were administered orally from day 1 to day 5 postinfection. Both compounds reduced local inflammation and virus replication in the skin. FCV markedly reduced mortality and virus replication in the nervous system. On the cessation of therapy after 5 days, when the levels of infectious virus in the tissues were reduced to below the level of detection, there followed a rebound of virus replication in the ganglia and brain stems of mice that had been treated with VACV. The recurrence of infection in the brain stem occurred on three separate occasions. No such recurrences were observed following FCV treatment. When ganglia were explanted from survivors 6 weeks later, latent virus was shown to be reactivated in all 10 of 10 control, untreated mice. The number of mice whose ganglia yielded virus was reduced to 60% in mice that had been treated with VACV, whereas no mice that had been treated with FCV had evidence of latent infection by this test.

2-Aminopurine↗
PubMed1995

Immunization with glycoprotein C of equine herpesvirus-1 is associated with accelerated virus clearance in a murine model.

The glycoprotein C (gC) gene of equine herpesvirus-1 (EHV-1) was expressed in insect cells by a recombinant baculovirus as several products with apparent molecular weights of 66 kDa-80 kDa. The baculovirus EHV-1 gC products were recognised by monoclonal antibody and by EHV-1 convalescent equine sera, indicating conservation of antigenic determinants and confirming this glycoprotein as a target for the equine immune system. Mice immunized with recombinant EHV-1 gC showed accelerated clearance of EHV-1 from respiratory tissues following intranasal challenge. Virus clearance was accompanied by virus specific antibodies and by cell mediated immune responses measured by a delayed type hypersensitivity reaction and lymphocyte stimulation by killed EHV-1 as antigen.

Animals↗
PubMed1995

EHV-1-induced abortion in mice and its relationship to stage of gestation.

The most important consequence of equine herpesvirus-1 (EHV-1) infection is abortion. The object of the present study was to characteristic further a murine EHV-1 abortion model and to make comparisons with the natural host with particular reference to the stage of gestation during which the infection occurs. BALB/c mice at different stages of pregnancy were infected intranasally with EHV-1 (strain AB4); they suffered respiratory distress, weight loss, and other constitutional signs of infection. When the virus was inoculated in the late second or early third week of gestation dead or dying fetuses were aborted, whereas infection between seven and nine days of pregnancy led to fetal death and resorption. During the process of resorption, complications were observed. Virus was frequently isolated from the placentas and occasionally from the tissues of the aborting fetuses, depending on the severity of the infection of the placentas. In some cases, therefore, the inoculation resulted in abortion although the infection was restricted to the placenta. Virus antigen was detected in the placentas, lungs and occasionally in other tissues of the aborting fetuses. The potential of this murine model for testing methods for the diagnosis and control of equine abortion is discussed.

Abortion, Veterinary↗
PubMed1995

Characteristics of equine herpesvirus 1 glycoproteins expressed in insect cells.

A series of recombinant baculoviruses containing genes for glycoproteins C, D, H and L of equine herpesvirus 1 (EHV-1) have been constructed, and the EHV-1 products characterised by gel electrophoresis and immunoblotting. The EHV-1 glycoproteins expressed in insect cells were similar but not identical in apparent sizes to those expressed in EHV-1 infected mammalian cells. Each of the EHV-1 products was recognised by convalescent equine sera, indicating that they were all targets for an equine immune response. Mice immunised with baculovirus-expressed EHV-1 gD and gC acquired an enhanced ability to clear challenge EHV-1 from respiratory tissues, in association with both neutralising antibody and cell mediated immune responses.

Animals↗
PubMed1995

The detection of latency-associated transcripts of equine herpesvirus 1 in ganglionic neurons.

Neural tissues from specific pathogen-free ponies that had been experimentally infected with equine herpesvirus 1 (EHV-1) were analysed by in situ hybridization. Digoxigenin-labelled EHV-1 BamHI fragments spanning almost the entire EHV-1 genome were hybridized to RNA in tissue sections from latently infected trigeminal ganglia. The BamHI E fragment detected EHV-1 RNA antisense to gene 63 (HSV-1 homologue ICP0) in a small number of neurons. Sixteen other BamHI fragments gave negative results in 20 sections tested with each fragment. Latency associated transcripts (LATs) were localized to the neuronal nuclei. EHV-1 nucleotide sequence data in the region reveals the presence of a putative EHV-1 LAT promoter that shares a similar motifs with the HSV-1 LAT promoter, including the LAT promoter-binding factor, and may have a role in EHV-1 LAT expression.

Amino Acid Sequence↗
PubMed1995

Comparison of efficacies of famciclovir and valaciclovir against herpes simplex virus type 1 in a murine immunosuppression model.

A mouse model of herpes simplex virus type 1 infection in an immunocompromised host was established by using cyclosporin-A to impair T-cell function. Following inoculation of herpes simplex virus type 1 into the skin of the ear pinna, cyclosporin-A prolonged virus replication in the skin and neural tissues compared with that in immunocompetent mice. This model was used to investigate the activity of famciclovir (FCV) and valaciclovir (VACV), which are oral products of the antiherpesvirus agents penciclovir and acyclovir, respectively. Both prodrugs gave similar blood profiles of the antiherpesvirus agents in normal and cyclosporin-treated mice. The compounds were administered by the oral route at 50 mg/kg per dose twice daily for 5 days. Both compounds were very effective at clearing infectious virus from the tissues despite the immunosuppression; FCV-treated animals cleared virus from the ear pinna more rapidly than VACV-treated animals. The areas under the concentration-time curve (AUC) for virus replication with time were reduced to 50 and 30% of control values for ear pinna and brain stem, respectively, with VACV therapy and to < 5% in both tissues by FCV. When treatment was continued to day 10, the reductions in AUC for ear and brain stem, respectively, were to 33 and 26% of control values with VACV and to < 3 and < 5% with FCV. However, on cessation of the antiviral treatment, there was a reproducible recurrence of infectious virus in the tissues obtained from VACV-treated mice. The recurrence of infectious virus was also evident after 10 days of treatment with VACV. In mice which had received FCV for 10 or 5 days, these was no resumption of virus replication in the ear pinna or brain stem. When dosing was reduced to once per day, both compounds were less effective at controlling the infection. Nevertheless, no recurrence of infectious virus was observed on cessation of FCV therapy.

2-Aminopurine↗
PubMed1994

Characterization of immune responses to baculovirus-expressed equine herpesvirus type 1 glycoproteins D and H in a murine model.

A murine intranasal infection model for equine herpesvirus type 1 (EHV-1) was used to evaluate immune responses following immunization with insect cells infected by baculoviruses that express EHV-1 glycoproteins. Baculovirus recombinant glycoprotein D (gD) and gH both induced serum antibodies to EHV-1 when measured by ELISA. The gD recombinant also produced a neutralizing antibody response. Protective immunity, determined by accelerated clearance of virus from the target organs in the respiratory tract, was demonstrated in mice immunized with a baculovirus recombinant expressing gD. In addition to the serological response, evidence is presented which shows that cell-mediated responses also play an important role in protection. Both recombinants induced delayed-type hypersensitivity and lymphoproliferation to EHV-1 antigen. The protective effects of T cells were confirmed by adoptive transfer of spleen cells from baculovirus gD-immunized donors to recipients that were challenged with live EHV-1. Depletion of either CD4- or CD8-bearing cells from the gD-immunized donors reduced the ability of the recipients to clear virus from the target organs, although depletion of CD4 cells had a more marked effect.

Animals↗
PubMed1994

The trigeminal ganglion is a location for equine herpesvirus 1 latency and reactivation in the horse.

Four specific pathogen-free ponies were infected intranasally with equine herpesvirus 1 (EHV-1) and two were similarly infected with an EHV-1 thymidine kinase deletion mutant. The primary infections were characterized by a transient fever accompanied by virus shedding into nasal mucus and viraemia. No virus was detected in clinical specimens after 15 days post-infection. Two months later a reactivation stimulus was administered to all six ponies and only the four that had been previously inoculated with wild-type EHV-1 shed virus into nasal mucus (for 10 days), proving the presence of a latent infection. No recurrence of viraemia was observed. The animals were monitored for a further 6 weeks and were consistently shown to be free from infectious virus. Tissues were then obtained postmortem. Co-cultivation of explanted trigeminal ganglia from two out of the four ponies that carried the wild-type virus yielded cultures positive for infectious virus. Apart from nasal epithelium, no infectious virus was recovered from any other tissue. PCR confirmed the presence of virus DNA in the ganglia from all six ponies. Lymphoid tissues also yielded positive signals using this technique. The relevance of virus detection by PCR in lymphoid and neural tissues is discussed in relation to the potential for reactivation of latent virus in the host. However, evidence is presented to show that EHV-1 is neurotropic and, in common with other members of the alpha-herpesvirus subfamily, establishes latency in sensory ganglia from which virus can be reactivated.

Administration, Intranasal↗
PubMed1993

Sequence analysis of thymidine kinase-defective mutants of equine herpesvirus-1 (EHV-1).

We have amplified, cloned and sequenced the gene encoding the thymidine kinase (TK) of a wild-type strain (Ab4) of equine herpesvirus-1 (EHV-1) and two mutants with defective TK activity isolated for resistance to penciclovir (PCV). One of the mutants, PR1, has suffered a 879-bp deletion which reduces the size of TK to 180 bp. The other mutant, PR3, has an adenine to cytosine mutation resulting in a Lys38-->Thr change. This mutation modifies the amino acid sequence of a domain involved in binding ATP, leading to non-detectable enzymatic activity. Lys38 thus appears to be essential for the activity of the TK of EHV-1.

Amino Acid Sequence↗
PubMed1993

The pathogenesis of wild type and drug resistant mutant strains of bovine herpesvirus-1 (BHV-1) in the natural host.

An experimental infection with bovine herpesvirus-1 was established in calves by means of intranasal inoculation. Three calves were infected with the parental strain BHV-1 w/t, three with the TK-defective strain, B 1 and four with the HPMPA-resistant strain, 3 A. Inoculation with w/t virus resulted in a reproducible clinical disease characterised by respiratory distress, fever and the presence of virus in nasal mucus. Following the acute infection, w/t-inoculated animals became seropositive for BHV-1 specific antibody. The TK-defective mutant (BHV-1 B 1) produced an acute infection similar to the parental virus in all three calves inoculated. The HPMPA-resistant mutant (BHV-1 3 A), however, showed a reduced pattern of infection and virus of lower titre was isolated from three of four calves; the antibody responses were generally lower, and one calf remained seronegative until reactivation. Following stimulation with dexamethasone 72 days after the primary inoculation, virus was re-isolated from all wild type-inoculated calves. In contrast, no evidence of reactivation was obtained from the three B 1-inoculated animals. However, all four animals inoculated with the mutant 3 A showed virus reactivation including the calf which had remained seronegative following primary virus inoculation. Previous studies have suggested that drug-resistance mutations in herpesviruses frequently are associated with reduced pathogenicity on the basis of experiments in laboratory models. The importance of the present study is the demonstration that two different drug-resistant variants of an alpha herpesvirus both have altered pathogenicity in the natural host for that infection. These results also have implications for the design and use of attenuated vaccine strains.

Adenine↗
PubMed1993

Modulation of the serological response of specific pathogen-free (EHV-free) foals to EHV-1 by previous infection with EHV-4 or a TK-deletion mutant of EHV-1.

EHV-1 was inoculated into specific pathogen-free (SPF) foals in order to study uncomplicated primary responses. Infection resulted in a strong serological response recognizing EHV-1-specific antigens; this contrasts with a previous publication where a weak response was recorded in SPF animals. Antibodies to EHV-1 were readily detected by four techniques (virus neutralization, complement fixation, Western blots and immune precipitation), yet there was comparatively little cross-reaction to EHV-4 target antigen. Re-inoculation with the same virus strain stimulated antibodies to EHV-1 but no additional antigens were recognized and antibodies cross-reacting with EHV-4 antigens were not enhanced. Having characterized the uncomplicated primary response to EHV-1 in SPF foals, further animals were exposed to either EHV-4 or a thymidine kinase-deficient mutant of EHV-1 prior to challenge with w/t EHV-1 to investigate how these infections might modulate the immune responses to EHV-1 or 4. Primary inoculation with EHV-4 or with a thymidine kinase-deficient mutant of EHV-1 produced productive infections as evidenced by virus shedding and pyrexia. In both these cases, however, in contrast to that with w/t EHV-1, the serological response was very weak. Re-infection of foals primed with either EHV-4 or TK-deficient EHV-1 with w/t EHV-1 resulted in a strong response to EHV-1 antigens detected by all four methods. In addition, in the foals given a primary inoculation with EHV-4, superinfection with EHV-1 resulted in a strong cross-reactive response to EHV-4 target antigens. The relevance of these observations to the interpretation of previously reported serological responses to EHVs in SPF and naturally reared animals is discussed.

Animals↗
PubMed1993

The effect of (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine (HPMPC) on bovine herpesvirus-1 (BHV-1) infection and reactivation in cattle.

A reproducible pattern of respiratory disease was produced in calves inoculated intranasally with a pathogenic strain of bovine herpesvirus-1 (BHV-1). A latent infection was established which could be reactivated by means of corticosteroid administration. Groups of calves were given a single dose of 20 mg/kg of (S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl)cytosine (HPMPC) either the day before or the day following virus inoculation. The drug markedly reduced clinical signs and virus replication; the therapeutic dose appeared to be more effective than the dose given one day before virus inoculation. The establishment of latency was not prevented and a single dose of HPMPC, the day before a course of dexamethasone (6 weeks after the acute infection), did not prevent virus shedding.

Animals↗