Pathogenicity of influenza virus.

The parent viruses and clones 64c and 6 of the A/Puerto Rico/8/34-A/England/939/69 (H3N2) series were examined. Virulence was assessed from 50% infectious dose, replication in the upper respiratory tract, degree of lung infection, and production of fever. Viral infection of the upper respiratory tract declined with onset of the inflammatory response and pyrexia, and restriction of replication in nasal turbinate tissue at pyrexial temperatures correlated with onset and rapidity of decline of infection in the upper respiratory tract. Compared with the H3N2 strains, the HON1 strain of A/Puerto Rico/8/34 stimulated a lower and later inflammatory response, which correlated with a delayed and low fever and, in turn, a high nasal infection that was slow to decline. The differential ability to infect the lung in vivo (clones 64c and 6 more than A/England/ 939/69 and clone 7a more than A/Puerto Rico/8/34 and clone 64d) was not always explained by differences in ability to replicate in lung tissue at normal or pyrexial temperatures. Neither overall virulence nor any particular facet of it can yet be correlated with particular gene pieces. The results are discussed in relation to the virulence of the strains for humans.

Aspects of Virulence in Ferrets Exhibited by Influenza Virus Recombinants of Known Genetic Constitution

The effect of standard concentrations of lidocaine HC1 and methylparaben (preservative) on the ciliary activity of ferret tracheal rings in organ culture was assessed. Pure lidocaine concentrations as low as 0.5% caused complete ciliostasis which was reversible after removal of the drug. One percent and two percent effects were not completely reversible. Pure methylparaben concentrations as low as 0.06 mg/ml paralyzed cilia; and, at concentrations of 0.5 mg/ml or higher, adverse effects were not completely reversible. When lidoacine and methylparaben were combined at the same ratio used in commercial preparations, their ciliotoxic effects were additive. These data suggest that topical respiratory anesthesia may result in prolonged ciliary paralysis.

Adverse effects of lidocaine and methylparaben on tracheal ciliary activity.

Piglet tracheal organ cultures were infected with respiratory syncytial virus (RSV) and observed for 21 days. Light and immunofluorescence microscopy demonstrated destruction of the ciliated epithelial cells and the presence of viral antigens in the epithelium. Virus was shed in high titer for 12--19 days. Ciliostasis could be quantitated, and it was shown that several strains of RSV grew and damaged tracheal organ cultures in a similar fashion. A temperature-sensitive mutant of RSV, ts-1, was examined at permissive (33 C) and restrictive (37 C) temperatures. This mutant, although somewhat attenuated at 37 C, was still found to cause damage to the ciliated epithelium and to replicate at both temperatures. THIS BEHAVIOR IS SIMILAR TO THAT AFTER INOCULATION OF TS-1 INTO VOLUNTEERS. This in vitro model may prove useful in the study of RSV disease and in the evaluation of candidate live virus vaccines.

Behavior of Respiratory Syncytial Virus in Piglet Tracheal Organ Culture

For development of an in vitro assay of attenuation for virus vaccine candidates, tracheal ring organ cultures from young ferrets were infected with influenza A/Hong Kong/68 and A/Udorn/72 temperature-sensitive (ts) recombinant and wild-type viruses and observed for 21 days at permissive (33 C) and restrictive (37 C) temperatures for effects on ciliated epithelium and viral replication. Results were compared with the known effects of these viruses in humans. Viruses that damaged ferret cilia and grew to high titers at 37 C were insufficiently attenuated for humans, and viruses that failed to damage cilia and grew to low titers at 33 C were excessively attenuated for humans. This system did not completely differentiate the relative virulence of A/Hong Kong/68 ts recombinants for humans but was successful for the A/Udorn/72 ts recombinants. This organ culture system has adjunctive usefulness for detection of most insufficiently attenuated influenza A vaccine candidates and for identification of excessively attenuated candidates that might contain non-ts lesions.

Temperature-Sensitive Mutants of Influenza Virus. XIII. Evaluation of Influenza A/Hong Kong/68 and A/Udorn/72 ts and Wild-Type Viruses in Tracheal Organ Culture at Permissive and Restrictive Temperatures

Influenza strains produced by recombination and tested as possible live vaccine candidates were studied in organ cultures of trachea. Two strains which proved to be too virulent in human volunteers regularly caused damage to the ciliated epithelium and viruses grew to high titre. Two strains which proved to be attenuated for volunteers did not cause appreciable damage, although they replicated to low titre in the epithelium. Similar results were obtained with influenza A virus attenuated by passage in the presence of horse sera. The method may be of value for detecting virulent live influenza vaccine candidates without risking severe illness in volunteers.

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D. A. J. Tyrrell

Papers

The behaviour in vitro of attenuated recombinant influenza viruses.