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T M Chambers

Publications and source records attributed to T M Chambers.

At least 19 recordsLinked to original sources
PubMed2001

The involvement of a stress-activated pathway in equine influenza virus-mediated apoptosis.

We have shown elsewhere that equine-2 influenza virus (EIV; subtype H3N8) induced pronounced cell death in infected cells through apoptosis as demonstrated by DNA fragmentation assay and a combined TUNEL and immunostaining scheme. In this study, we investigated the mechanism of EIV-mediated cytotoxicity on a permissive mammalian epithelial cell line, Madin-Darby canine kidney (MDCK) cells. EIV infection increased the cellular levels of oxidative stress and c-Jun/AP-1 protein (which is known to be affected by oxidative stress), as well as its DNA binding activity. Increased production of TGF-beta1, an inducer of c-Jun N-terminal kinase or stress-activated protein kinase (JNK/SAPK) activation, was also detected in EIV-infected MDCK cells. It has been reported that TGF-beta may initiate a signaling cascade leading to JNK/SAPK activation. Addition of c-Jun antisense oligodeoxynucleotide, antioxidant N-acetyl-cysteine (NAC), JNK/SAPK inhibitor carvedilol, or TGF-beta-neutralizing antibody effectively blocked c-Jun/AP-1 upregulation and TGF-beta1 production mediated by EIV infection. These treatments also attenuated EIV-induced cytopathogenic effects (CPE) and apoptosis. Our results suggest that a stress-activated pathway is involved in apoptosis mediated by EIV infection. It is likely that EIV infection turns on the JNK/SAPK cascade, which modulates the activity of apoptosis-promoting regulatory factor c-Jun/AP-1 and epithelial growth inhibitory cytokine TGF-beta.

Animals↗
PubMed2001

Safety, efficacy, and immunogenicity of a modified-live equine influenza virus vaccine in ponies after induction of exercise-induced immunosuppression.

OBJECTIVE: To determine safety, efficacy, and immunogenicity of an intranasal cold-adapted modified-live equine influenza virus vaccine administered to ponies following induction of exercise-induced immunosuppression. DESIGN: Prospective study. ANIMALS: Fifteen 9- to 15-month old ponies that had not had influenza. PROCEDURE: Five ponies were vaccinated after 5 days of strenuous exercise on a high-speed treadmill, 5 were vaccinated without undergoing exercise, and 5 were not vaccinated or exercised and served as controls. Three months later, all ponies were challenged by nebulization of homologous equine influenza virus. Clinical and hematologic responses and viral shedding were monitored, and serum and nasal secretions were collected for determination of influenza-virus-specific antibody isotype responses. RESULTS: Exercise caused immunosuppression, as indicated by depression of lymphocyte proliferation in response to pokeweed mitogen. Vaccination did not result in adverse clinical effects, and none of the vaccinated ponies developed clinical signs of infection following challenge exposure. In contrast, challenge exposure caused marked clinical signs of respiratory tract disease in 4 control ponies. Vaccinated and control ponies shed virus after challenge exposure. Antibody responses to vaccination were restricted to serum IgGa and IgGb responses in both vaccination groups. After challenge exposure, ponies in all groups generated serum IgGa and IgGb and nasal IgA responses. Patterns of serum hemagglutination inhibition titers were similar to patterns of IgGa and IgGb responses. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that administration of this MLV vaccine to ponies with exercise-induced immunosuppression was safe and that administration of a single dose to ponies provided clinical protection 3 months later.

Administration, Intranasal↗
PubMed2001

Cultures of equine respiratory epithelial cells and organ explants as tools for the study of equine influenza virus infection.

Equine nasal turbinate epithelial cells and tracheal rafts were maintained with sustained viability in culture. Both types of culture supported productive replication of equine influenza virus (equine-2, subtype H3N8) and cell death occurred through apoptosis following viral infection. Thus, primary respiratory epithelial cell and organ cultures of equine origin may be valuable as alternatives to the intact animal for studying the virus-host interaction of equine respiratory viruses including influenza.

Animals↗
PubMed2001

Diverged evolution of recent equine-2 influenza (H3N8) viruses in the Western Hemisphere.

We reported previously that equine-2 influenza A virus (H3N8) had evolved into two genetically and antigenically distinct "Eurasian" and "American" lineages. Phylogenetic analysis, using the HA1 gene of more recent American isolates, indicated a further divergence of these viruses into three evolution lineages: A South American lineage, a Kentucky lineage, and a Florida lineage. These multiple evolution pathways were not due to geographic barriers, as viruses from different lineages co-circulated. For the Kentucky lineage, the evolution rate was estimated to be 0.89 amino acid substitutions per year, which agreed with the previously estimated rate of 0.8. For the South American lineage, the evolution rate was estimated to be only 0.27 amino acid substitutions per year. This low evolution rate was probably due to a unique alternating Ser138 to Ala138 substitutions at antigenic site A. For the Kentucky lineage, there was a preference for sequential nonsynonymous substitutions at antigenic site B, which was also a "hot spot" for amino acid substitutions. Convalescent sera had minimal cross-reactivity to viruses of different lineages, indicating antigenic distinctions among these viruses. In contrast to human H3N2 viruses, our results suggested that the evolution of equine-2 influenza virus resembled the multiple evolution pathways of influenza B virus.

Amino Acid Sequence↗
PubMed2001

Derivation and characterization of a live attenuated equine influenza vaccine virus.

OBJECTIVE: To develop and characterize a cold-adapted live attenuated equine-2 influenza virus effective as an intranasal vaccine. ANIMALS: 8 ponies approximately 18 months of age. PROCEDURES: A wild-type equine-2 virus, A/Equine/Kentucky/1/91 (H3N8), was serially passaged in embryonated chicken eggs at temperatures gradually reduced in a stepwise manner from 34 C to 30 C to 28 C to 26 C. At different passages, infected allantoic fluids were tested for the ability of progeny virus to replicate in Madin-Darby canine kidney (MDCK) cells at 34 C and 39.5 C. Virus clones that replicated at 26 C in eggs and at 34 C in MDCK cells, but not at 39.5 C in MDCK cells, were tested for stability of the cold-adapted, temperature-sensitive (ts), and protein synthesis phenotypes. A stable clone, P821, was evaluated for safety, ability to replicate, and immunogenicity after intranasal administration in ponies. RESULTS: Randomly selected clones from the 49th passage were all ts with plaquing efficiencies of < 10(-6) (ratio of 39.5 C:34 C) and retained this phenotype after 5 serial passages at 34 C in either embryonated eggs or MDCK cells. The clone selected as the vaccine candidate (P821) had the desired degree of attenuation. Administered intranasally to seronegative ponies, the virus caused no adverse reactions or overt signs of clinical disease, replicated in the upper portion of the respiratory tract, and induced a strong serum antibody response. CONCLUSION AND CLINICAL RELEVANCE: A candidate live attenuated influenza vaccine virus was derived by cold-adaptation of a wild-type equine-2 influenza virus, A/Equine/Kentucky/1/91, in embryonated eggs.

Animals↗
PubMed2001

A new modified live equine influenza virus vaccine: phenotypic stability, restricted spread and efficacy against heterologous virus challenge.

Flu Avert IN vaccine is a new, live attenuated virus vaccine for equine influenza. We tested this vaccine in vivo to ascertain 1) its safety and stability when subjected to serial horse to horse passage, 2) whether it spread spontaneously from horse to horse and 3) its ability to protect against heterologous equine influenza challenge viruses of epidemiological relevance. For the stability study, the vaccine was administered to 5 ponies. Nasal swabs were collected and pooled fluids administered directly to 4 successive groups of naïve ponies by intranasal inoculation. Viruses isolated from the last group retained the vaccine's full attenuation phenotype, with no reversion to the wild-type virus phenotype or production of clinical influenza disease. The vaccine virus spread spontaneously to only 1 of 13 nonvaccinated horses/ponies when these were comingled with 39 vaccinates in the same field. For the heterologous protection study, a challenge model system was utilised in which vaccinated or naïve control horses and ponies were exposed to the challenge virus by inhalation of virus-containing aerosols. Challenge viruses included influenza A/equine-2/Kentucky/98, a recent representative of the 'American' lineage of equine-2 influenza viruses; and A/equine-2/Saskatoon/90, representative of the 'Eurasian' lineage. Clinical signs among challenged animals were recorded daily using a standardised scoring protocol. With both challenge viruses, control animals reliably contracted clinical signs of influenza, whereas vaccinated animals were reliably protected from clinical disease. These results demonstrate that Flu Avert IN vaccine is safe and phenotypically stable, has low spontaneous transmissibility and is effective in protecting horses against challenge viruses representative of those in circulation worldwide.

Administration, Intranasal↗
PubMed2000

Sialic acid species as a determinant of the host range of influenza A viruses.

The distribution of sialic acid (SA) species varies among animal species, but the biological role of this variation is largely unknown. Influenza viruses differ in their ability to recognize SA-galactose (Gal) linkages, depending on the animal hosts from which they are isolated. For example, human viruses preferentially recognize SA linked to Gal by the alpha2,6(SAalpha2,6Gal) linkage, while equine viruses favor SAalpha2,3Gal. However, whether a difference in relative abundance of specific SA species (N-acetylneuraminic acid [NeuAc] and N-glycolylneuraminic acid [NeuGc]) among different animals affects the replicative potential of influenza viruses is uncertain. We therefore examined the requirement for the hemagglutinin (HA) for support of viral replication in horses, using viruses whose HAs differ in receptor specificity. A virus with an HA recognizing NeuAcalpha2,6Gal but not NeuAcalpha2,3Gal or NeuGcalpha2,3Gal failed to replicate in horses, while one with an HA recognizing the NeuGcalpha2,3Gal moiety replicated in horses. Furthermore, biochemical and immunohistochemical analyses and a lectin-binding assay demonstrated the abundance of the NeuGcalpha2,3Gal moiety in epithelial cells of horse trachea, indicating that recognition of this moiety is critical for viral replication in horses. Thus, these results provide evidence of a biological effect of different SA species in different animals.

Animals↗
PubMed2000

Comparison of the phenotypes of Streptococcus zooepidemicus isolated from tonsils of healthy horses and specimens obtained from foals and donkeys with pneumonia.

OBJECTIVE: To determine whether streptococcal pneumonia is caused by strains of Streptococcus zooepidemicus similar to those obtained from the tonsils of healthy horses. SAMPLE POPULATION: 5 tonsils from healthy horses, 8 tracheal washes and 6 lung specimens from foals with pneumonia, and 5 nasopharyngeal swab specimens from donkeys with acute bronchopneumonia. PROCEDURE: Variable M-like protectively immunogenic SzP proteins of 5 isolates of S. zooepidemicus from each tonsil and clinical specimen were compared, using immunoblots. The SzP gene of 13 isolates representative of various SzP immunoblot phenotypes from 1 healthy horse and 9 horses and donkeys with pneumonia were sequenced and compared. Cell-associated hyaluronic acid concentration and resistance to phagocytosis of some isolates were measured. RESULTS: Tonsils of each healthy horse were colonized by several SzP phenotypes similar to those of foals or donkeys with pneumonia. In contrast, multiple isolates from animals with pneumonia had the same SzP phenotype, indicating infection by a single strain or clone. Analysis of the SzP sequence confirmed that differences in immunoblot phenotype were associated with sequence differences and that several SzP genotypes were in healthy horses and animals with pneumonia. Isolates with high concentrations of cell-associated hyaluronic acid were more resistant to phagocytosis. CONCLUSIONS AND CLINICAL RELEVANCE: An SzP-specific immunoblot is a useful, sensitive measure of diversity among strains of S. zooepidemicus. Single strains with SzP phenotypes similar to those found in tonsils of healthy horses cause pneumonia. Because of the diversity of SzP phenotype and genotype among isolates from animals with pneumonia, SzP phenotype is not an important determinant of invasiveness or epizootic capabilities.

Animals↗
PubMed1999

Pharmacokinetics and therapeutic efficacy of rimantadine in horses experimentally infected with influenza virus A2.

OBJECTIVE: To determine pharmacokinetics of single and multiple doses of rimantadine hydrochloride in horses and to evaluate prophylactic efficacy of rimantadine in influenza virus-infected horses. ANIMALS: 5 clinically normal horses and 8 horses seronegative to influenza A. PROCEDURE: Horses were given rimantadine (7 mg/kg of body weight, i.v., once; 15 mg/kg, p.o., once; 30 mg/kg, p.o., once; and 30 mg/kg, p.o., q 12 h for 4 days) to determine disposition kinetics. Efficacy in induced infections was determined in horses seronegative to influenza virus A2. Rimantadine was administered (30 mg/kg, p.o., q 12 h for 7 days) beginning 12 hours before challenge-exposure to the virus. RESULTS: Estimated mean peak plasma concentration of rimantadine after i.v. administration was 2.0 micrograms/ml, volume of distribution (mean +/- SD) at steady-state (Vdss) was 7.1 +/- 1.7 L/kg, plasma clearance after i.v. administration was 51 +/- 7 ml/min/kg, and beta-phase half-life was 2.0 +/- 0.4 hours. Oral administration of 15 mg of rimantadine/kg yielded peak plasma concentrations of < 50 ng/ml after 3 hours; a single oral administration of 30 mg/kg yielded mean peak plasma concentrations of 500 ng/ml with mean bioavailability (F) of 25%, beta-phase half-life of 2.2 +/- 0.3 hours, and clearance of 340 +/- 255 ml/min/kg. Multiple doses of rimantadine provided steady-state concentrations in plasma with peak and trough concentrations (mean +/- SEM) of 811 +/- 97 and 161 +/- 12 ng/ml, respectively. Rimantadine used prophylactically for induced influenza virus A2 infection was associated with significant decreases in rectal temperature and lung sounds. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of rimantadine to horses can safely ameliorate clinical signs of influenza virus infection.

Administration, Oral↗
PubMed1997

Amantadine and equine influenza: pharmacology, pharmacokinetics and neurological effects in the horse.

Amantadine is an antiviral agent effective against influenza A viruses. We investigated 1) the antiviral efficacy, 2) analytical detection, 3) bioavailability and disposition, 4) pharmacokinetic modelling and 5) adverse reactions of amantadine in the horse. In vitro, amantadine and its derivative rimantadine suppressed the replication of recent isolates of equine-2 influenza virus with effective doses (EDs) of less than 30 ng/ml. Rimantadine was more effective than amantadine against most viral isolates; we suggest a minimum plasma concentration of 300 ng/ml of amantadine for therapeutic efficacy. In vivo an i.v. dose of amantadine 15 mg/kg bwt produced mild, transient CNS signs which were no longer apparent after 30 min. Amantadine administered at a dose of 15 mg/kg bwt was established as the maximum safe single i.v. dose. However, if repeated i.v. administration of amantadine is required no more than 10 mg/kg bwt t.i.d. should be used. The maximal safe plasma concentration of amantadine was not evaluated but is probably greater than 2000 ng/ml and possibly greater than 4000 ng/ml. On the other hand, horses with lower seizure thresholds, or those on medications that lower seizure thresholds, may be at increased risk of amantadine-induced seizures, which show few premonitory signs and are rapidly fatal. After i.v. administration of amantadine 10 mg/kg bwt, the disposition kinetics were well fitted by a 2-compartment open model. The estimated peak plasma concentration after this dose was about 4500 ng/ml, the volume of distribution at steady-state (Vdss) was (mean +/- s.d.) 4.9 +/- 1.9 l/kg bwt and the beta phase half-life was 1.83 +/- 0.87 h. Computer projections of plasma amantadine concentrations after i.v. administration of amantadine at a dose of 10 mg/kg bwt t.i.d. at 8 h intervals suggest peak plasma concentrations of 4000-5000 ng/ml and troughs of less than 300 ng/ml will be achieved. Amantadine administered orally at 10 mg/kg bwt and 20 mg/kg bwt showed mean oral bioavailability of about 40-60% and a plasma half life of 3.4 +/- 1.4 h; however, there was substantial inter-animal variation in bioavailability. Projections based on the kinetics observed in individual animals suggest that some animals readily maintain effective plasma concentrations of amantadine after oral administration of 20 mg/kg bwt t.i.d. On the other hand, animals in which amantadine is poorly bioavailable may require up to a 6-fold (120 mg/kg bwt) increase in the oral dose to achieve effective blood concentrations. Withholding food for 15 h did not reduce these inter-animal differences in bioavailability. Our results showed that simple dosing with oral amantadine will not yield effective plasma concentrations in all animals. While i.v. administration yielded more reproducible plasma concentrations, care should be taken to see that the seizure threshold is not exceeded. In acute situations, i.v. administration (5 mg/kg bwt) every 4 h should maintain safe and effective plasma and respiratory tract concentrations of amantadine.

Administration, Oral↗
PubMed1996

Antigenic and genetic evolution of equine H3N8 influenza A viruses.

Evolution of equine influenza a H3N8 viruses was examined by antigenic and genetic analysis of a collection isolates from around the world. It was noted that antigenic and genetic variants of equine H3N8 viruses cocirculate, and in particular that variants currently circulating in Europe and the USA are distinguishable from one another both in terms of antigenic reactivity and genetic structure of the HA1 portion of the haemagglutinin (HA) molecule. Whilst the divergent evolution of American and European isolates may be due to geographical isolation of the two gene pools, some mixing is believed to occur as 'American-like' viruses have been isolated during outbreaks of equine influenza in the UK. The cocirculation of two antigenically and genetically distinct lineages of equine influenza H3N8 viruses has serious implications for vaccine strain selection.

Amino Acid Sequence↗
PubMed1994

Genetic and antigenic analysis of the influenza virus responsible for the 1992 Hong Kong equine influenza epizootic.

An outbreak of influenza occurred among thoroughbred racehorses in Hong Kong in November-December 1992, with morbidity of 37%. All horses involved had been vaccinated against equine-1 and equine-2 influenza viruses but not against the virus responsible for the 1989 equine influenza outbreak in northern China (influenza A/equine/Jilin/89, subtype H3N8). Therefore the source and nature of the virus causing the Hong Kong outbreak was investigated. Virus isolated from a horse infected during the outbreak was used for genetic analysis. All the viral gene segments were similar to those of equine-2 (H3N8) influenza viruses and unrelated to those of equine/Jilin/89 virus. The nucleotide sequence of the viral hemagglutinin gene showed high homology (99.4%) to that of influenza A/equine/Suffolk/89 (H3N8) virus which has circulated extensively in Europe. However, these viruses differed in their antigenic reactivity to a panel of monoclonal antibodies. Preliminary epizootiological information plus the concordance of amino acid sequence between hemagglutinins of the Hong Kong isolate and a contemporaneous equine-2 influenza virus isolate from the United Kingdom indicated that the probable source of the Hong Kong outbreak was horses recently imported from England or Ireland.

Amino Acid Sequence↗
PubMed1994

Rapid diagnosis of equine influenza by the Directigen FLU-A enzyme immunoassay.

The Directigen FLU-A enzyme immunoassay was tested for its ability to detect equine-2 influenza viruses in nasopharyngeal fluids from horses and ponies. A total of 125 swabs from experimental infections and from different sources of natural infection in the USA and Hong Kong were examined. The assay results were compared with the results of standard virus culture in embryonated chicken eggs or Madin-Darby canine kidney cells, and with the serology of the horses sampled. In comparison with virus culture the enzyme immunoassay exhibited 83 per cent sensitivity, 78 per cent specificity, 70 per cent positive predictive value and 88 per cent negative predictive value. The test appeared to be more sensitive than haemagglutination for the detection of low levels of virus in embryonated egg cultures. It also detected equine-1 influenza virus in culture. The test is rapid (15 minutes), simple, and should be a convenient method for the rapid diagnosis and screening of horses for equine influenza infection.

Animals↗
PubMed1994

Sequence specificity of furin, a proprotein-processing endoprotease, for the hemagglutinin of a virulent avian influenza virus.

The virulence of avian influenza viruses correlates with the sensitivity of their hemagglutinin (HA) to cellular proteases. Furin, a proprotein-processing subtilisin-related endoprotease, is a leading candidate for the enzyme that cleaves the HA of virulent avian viruses. We therefore compared the specificity of furin with those of proteases in a variety of cultured cells and in a rat Golgi fraction, using the HA cleavage mutants of a virulent avian influenza virus, A/Turkey/Ireland/1378/85 (H5N8). The results indicated similar sequence specificities among the endoproteases when purified furin was used. In experiments with the vaccinia virus expression system, overexpressed furin cleaved mutant HAs that were not recognized by the endogenous proteases, resulting in an apparent broader specificity of furin. These findings authenticate the proposed role of furin as an HA-activating protease in vivo and caution against the use of expression vectors to study protease sequence specificity.

Amino Acid Sequence↗
PubMed1994

Diagnosis of equine influenza by the polymerase chain reaction.

Influenza A is a common respiratory infection of horses, and rapid diagnosis is important for its detection and control. Sensitive detection of influenza currently requires viral culture and is not always feasible. The polymerase chain reaction (PCR) was used to detect DNA produced by reverse transcription of equine influenza in stored nasal secretions, vaccines, and allantoic fluids. Primers directed at a target of 212 bp on conserved segment 7 (matrix gene) of human influenza A/Bangkok/1/79(H3N2) produced amplification products of appropriate size with influenza A/Equine/Prague/1/56 (H7N7), A/Equine/Miami/63 (H3N8), A/Equine/Kentucky/79 (H3N8), and A/Equine/Kentucky/2/91 (H3N8) in infected frozen allantoic fluids and in frozen extracts of nasal swabs of 2 horses with naturally acquired influenza. The products bound a 32P-labeled hybridization probe to an inner region of the target. Control samples, including nasal secretions from a horse infected with herpesvirus, were negative. In a prospective study, 2 ponies inhaled aerosols of influenza A/Equine/Kentucky/2/91 (H3N8), and thereafter supernatants of nasal swabs in transport medium were obtained daily for 10 days for culture and PCR. Amplification products were evaluated by size and binding of a 32P-labeled probe and also by dot-blotting and binding of a biotin-labeled probe. Culture detected influenza more consistently than did PCR in the first 2 days of infection, but PCR detected virus more often later in infection. Gels were the most sensitive, but radiometric and biotin-labeled probes gave specific results and were consistently positive from days 3-6. PCR is suitable for detection of equine influenza in clinical samples.

Animals↗
PubMed1992

Cross-reactivity of existing equine influenza vaccines with a new strain of equine influenza virus from China.

A novel strain of equine influenza virus, influenza A/equine/Jilin (China)/1/89, has emerged which is genetically distinct from all earlier strains of equine influenza. It is therefore possible that the vaccines against equine influenza may be unable to protect horses against disease caused by this virus strain. In vitro serological assays established that there were low levels of immunological cross-reactivity between the new virus, the current vaccine strains and the strains of equine-2 influenza virus now in circulation.

Animals↗