Mark W. Ambrose

Measles Virus Replication in Lungs of Hispid Cotton Rats after Intranasal Inoculation

Abstract Hispid cotton rats were inoculated intranasally with either measles virus (MV) Edmonston, a multipassaged, tissue culture-adapted strain of MV, or with one of three clinical MV isolates that had limited passages (three to five times) in tissue culture cells. MV Edmonston was recovered from the lungs of every (n = 37) hispid cotton rat inoculated with this virus for at least 7 days after virus inoculation. Peak pulmonary titers occurred on Day +4 (3.3–4.4 log10/g lung). Scattered areas of inflammation were observed interstitially in lung sections from infected animals stained with hematoxylin and eosin, and a similar pattern of diffuse fluorescence was seen in cryostat sections stained with an indirect fluorescent antibody procedure specific for virus antigens. Fluorescent antibody and virus isolation studies on lung lavage cells both suggested that lung leukocytes were a primary target of the virus. In contrast to these findings, virus was isolated only sporadically from hispid cotton rats inoculated with any of the clinical measles virus isolates. Despite the restricted growth of MV in these animals, cotton rats may be useful for studying certain aspects of measles virus pathogenesis and for screening potential antiviral compounds in vivo.

The antiviral activity of SP-303, a natural polyphenolic polymer, against respiratory syncytial and parainfluenza type 3 viruses in cotton rats

SP-303, a naturally occurring polyphenolic polymer (average M.W. = 2100 Da), was tested in cotton rats (Sigmoden hispidus) for antiviral activity against respiratory syncytial (RSV) and parainfluenza type 3 (PIV3) viruses, and for acute toxicity. Significant reductions in pulmonary RSV titers, compared to pulmonary RSV titers in comparably treated control animals, were seen in cotton rats given 1-10 mg SP-303/kg/day intraperitoneally (i.p.) on days 1 through to 3, after experimental inoculation with RSV. The minimum efficacious dose of SP-303 against PIV3, when given i.p. for 3 days, was 3 mg/kg/day. Higher doses of SP-303 could not be given i.p., as doses > or = 30 mg/kg/day given once daily by this route for 3 or more consecutive days caused both significant weight loss and death in infected or uninfected animals. Although no toxicity was observed following oral administration of up to 270 mg of SP-303 daily for 3 days, this compound had variable antiviral activity when given by this route.

Evaluation of the immunogenicity and protective efficacy of a candidate parainfluenza virus type 3 subunit vaccine in cotton rats

A parainfluenza virus type 3 (PIV3) subunit vaccine consisting of detergent-solubilized, affinity-purified haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins was tested in cotton rats for immunogenicity, short-term effects on virus-induced immunopathology and protective efficacy. Groups of animals were immunized twice, 4 weeks apart, with graded doses of vaccine administered either alone or with aluminium phosphate (AlPO4). The minimum immunogenic dose of vaccine was 0.1 microgram HN and F when the vaccine was given alone and 0.01 microgram when the vaccine was administered with AlPO4 adjuvant. Antibody responses in animals immunized with 1 microgram HN and F mixed with adjuvant were similar to those in control animals infected with live PIV3 intranasally. Pulmonary and nasal wash PIV3 titres generally were inversely correlated with serum antibody levels. Virus titres were significantly reduced in all groups of animals immunized with greater than or equal to 0.1 microgram HN and F compared with control animals immunized with vehicle only. Four days after virus challenge, there was no evidence of enhanced histopathology in lung sections from animals immunized with the candidate vaccine.

Evaluation of the toxicity and antiviral activity of carbocyclic 3-deazaadenosine against respiratory syncytial and parainfluenza type 3 viruses in tissue culture and in cotton rats

The toxicity and antiviral efficacy of carbocyclic 3-deazaadenosine (Cc3Ado) against respiratory syncytial (RSV) and parainfluenza type 3 (PIV3) virus infections were tested in tissue culture and in cotton rats. The mean median efficacious dose (ED50) of Cc3Ado in HEp2 cells against RSV and PIV3 was 9 and 14 micrograms/ml, respectively. These values were 85- and 55-fold less than the median inhibitory (toxic) dose (ID50) of Cc3Ado in this cell line (750 micrograms/ml), and similar to values obtained for ribavirin. Cc3Ado exhibited no significant antiviral activity against influenza A, influenza B, adeno type 5 or adeno type 7 viruses (all ED50 were greater than 1000 micrograms/ml). In cotton rats, animals given greater than or equal to 1 mg/kg/day Cc3Ado intraperitoneally on days 1, 2 and 3 after experimental challenge with virus, consistently had significant reductions in pulmonary RSV and PIV3 titers compared to pulmonary virus titers in comparably treated control animals. The minimum efficacious dose of ribavirin given under the same conditions was 30 mg/kg/day. Cc3Ado was also efficacious in cotton rats when given orally by gavage, or when different administration schedules were used. The median efficacious dose of Cc3Ado when given orally was 10 mg/kg/day. No significant toxic effects were noted in cotton rats, even in animals given 20 mg/kg daily for eight consecutive days.

Further studies with short duration ribavirin aerosol for the treatment of influenza virus infection in mice and respiratory syncytial virus infection in cotton rats.

Ribavirin aerosol administration has been shown to be effective in the treatment of respiratory syncytial virus (RSV) infections in infants and in influenza A and B virus infections in young adults. Long treatment schedules and potential for environmental contamination have stimulated the search for alternative dosing schedules. Thus, we attempted to determine the length of time of ribavirin aerosol necessary for effective treatment of influenza and RSV. In RSV-infected cotton rats, aerosolization for just 30 min with high-dose ribavirin (HDR:60 mg ribavirin/ml in reservoir), 3 times daily, reduced viral lung titers/gm of tissue by 1.1 log10. In influenza virus-infected mice, 15 min of aerosolized HDR, 3 times daily, was effective in reducing both mortality and pulmonary virus titers (1.1 log10 reduction). When the intervals between aerosol administration each day were equally divided (i.e., q.8 h), the treatments were most effective. Treatment for 45 min, once daily, was not as effective as divided doses. Calculations of ribavirin concentrations in respiratory secretions following 15 min treatment in mice with HDR indicated that drug levels dropped below the ED50 for influenza viruses after about 9 h. A daily dosage of ribavirin, estimated to be 8-15 mg/kg, was effective for the treatment of influenza and RSV infections.

Toxicity and antiviral activity of LY253963 against respiratory syncytial and parainfluenza type 3 viruses in tissue culture and in cotton rats

LY253963, the sodium salt of 1,3,4-thiadiazol-2-ylcyanamide, was evaluated in tissue culture and in cotton rats for toxicity and antiviral efficacy against respiratory syncytial (RSV) and parainfluenza type 3 (PIV3) viruses. The selective index (ratio of the median toxic dose: median efficacious dose) of LY253963 in HEp2 tissue culture cells was greater than 100 against both RSV and PIV3. When given intraperitoneally to cotton rats, the minimum protective dose of LY253963 against both of these viruses was between 1 and 3 mg/kg/day. In contrast, doses of LY253963 as high as 30 mg/kg/day, administered orally after experimental inoculation of virus, did not significantly reduce pulmonary virus titers in treated animals compared to control animals given placebo. No toxic effects were noted in cotton rats, even in those given 20 mg/kg/day for eight consecutive days.

Comparison of the anti-respiratory syncytial virus activity and toxicity of papaverine hydrochloride and pyrazofurin in vitro and in vivo

Based on reports describing their broad antiviral activity, the toxicity and antiviral efficacy of papaverine hydrochloride and pyrazofurin against respiratory syncytial virus (RSV) infection were tested in vitro in tissue culture cells and in vivo in cotton rats. Papaverine inhibited RSV replication in vitro; however, the median minimal toxic dose-median minimal inhibitory concentration ratios (MTD50:MIC50) in vitro and in vivo for papaverine were less than 4. Further work with this compound was discontinued. In contrast, pyrazofurin inhibited RSV replication in vitro (a mean MIC50 of 0.04 microgram/ml was obtained) and in vivo (RSV pulmonary titers were significantly reduced consistently in cotton rats given daily 10 mg/kg doses compared to untreated control animals). However, some toxic effects were observed in both the in vitro and in vivo tests of this compound. The remaining potential of pyrazofurin as an anti-RSV compound is discussed.

Parainfluenza virus type 3 (PIV3)-specific and non-virus-specific delayed type hypersensitivity responses in cotton rats given different PIV3 antigen preparations

Virus-specific, T-lymphocyte-mediated delayed type hypersensitivity (DTH) responses were studied in cotton rats using replicating (wild-type parainfluenza virus type 3 (PIV3) and recombinant vaccinia virus expressing PIV3 haemagglutinin-neuraminidase (HN) or fusion (F) glycoproteins), and non-replicating (detergent-solubilized, affinity chromatography purified HN and F glycoproteins or inactivated whole PIV3) virus preparations. Significant virus-specific DTH responses were observed in all test groups 1-2 weeks after a single antigen inoculation or 5 days after two inoculations given 3 weeks apart. Peak swelling of ear pinnas in these animals generally occurred 24 h after elicitation and was marked by a cellular infiltration consisting of mono- and polymorphonuclear leucocytes. A considerable non-virus-specific inflammatory response, presumably due to tissue culture or media components in the priming antigen preparations, was observed 3 weeks after priming. No significant differences in DTH responses were observed in cotton rats primed with any of the PIV3 preparations. The possible roles and significance of both the virus-specific and non-virus-specific DTH responses in paramyxovirus-induced disease in humans and cotton rats are discussed.