H. F. Maassab

Cold adapted variants of influenza A

SummaryThe genetic and biological properties of 13 recombinant influenza A clones derived at 25°C from the A/AA/6/60-cold variant (by crosses with 4 different wild type strains) were compared with a set of 5-FU inducedts-mutants. The 5-FU mutants had previously been placed into 7 complementation-recombination groups; the A/AA/6/60-cold parent (PI-7) and the 12 cold recombinant clones which werets were shown to share a lesion with only one of these groups. The parental strain and 5 recombinant clones were evaluated for replication in the lungs and nasal turbinates of hamsters. Each virus appeared to be attenuated; genetic stability correlated with the level of viral replication in the hamster lung, i.e., viruses which grew best showed a tendency to revert to thets+ phenotype. Characterization of thets+ revertants for the presence of the cold adaptation property revealed that these viruses exhibited a spectrum of cold adaptation properties. Two viruses, PI-7 (the parental cold variant) and the CR6 recombinant (A/Queensland/6/72) did not revert in either the lungs or nasal turbinates of hamsters.

Creation of amantadine resistant clones of influenza type A virus using a new transfection procedure

M2, the spliced segment of the matrix (M) gene of influenza A virus, is an integral membrane protein which functions as an ion channel both when the virus is in the host endosome and during protein processing in the trans-Golgi network. Amantadine inhibits replication of influenza A virus by blocking the activity of this ion channel. Reverse genetics were used to generate amantadine resistant virus mutants by introducing mutations into the M gene of cold adapted (ca) A/AA/6/60, an amantadine sensitive virus. The site directed mutagenesis involved substitutions at amino acids 27, 30 and 31, sites hypothesized to be responsible for resistance to this drug in several other influenza A viruses. This M gene was then transfected into wt A/AA/6/60, an amantadine sensitive virus, via electroporation. The desired transfectants were selected for replication in the presence of amantadine. Using this newly devised reverse genetics system to rescue a mutated gene in its homologous wild type background not only establishes the identity of amino acid mutations necessary for the establishment of amantadine resistance but will also allow us to study other mutations in the M gene without gene constellation effects. Resistance to amantadine in wt A/AA/6/60 can also occur naturally if the viruses are grown in the presence of amantadine. These spontaneously generated resistant clones contained point mutations at amino acid 30 or 31 of M2.

The cell receptor level is reduced during persistent infection with influenza C virus

SummaryPersistent influenza C virus infection of MDCK cells perpetuates the viral genome in a cell-associated form. Typically, virus production remains at a low level over extended periods, in the absence of lytic effects of replication. In this study, we demonstrate that persistently infected cells are very restricted in permissiveness for superinfection. By reconstitution experiments, using bovine brain gangliosides as artificial receptors, the degree of super-infection was markedly increased. Analysis of cellular receptor expression revealed reduced concentrations of sialoglycoproteins in general and a limited presentation of the major receptor gp40. Cocultures of persistently infected and uninfected cells (the latter carrying normal receptor levels) initiated a transient rise in virus titers. This kind of induction of virus synthesis appeared to be mainly receptor-linked, since a receptor-deprived subline, MDCK II, did not give rise to a similar effect. Susceptibility of MDCK II cocultures could be partly restored by ganglioside treatment. In accordance to related virus systems, these findings on influenza C virus suggest a role of cell receptor concentrations in the regulation of long-term persistence.

DEVELOPMENT OF COLD RECOMBINANTS OF INFLUENZA VIRUS AS LIVE VIRUS VACCINES

ABSTRACT Cold-adapted (ca) recombinants of influenza viruses were derived, characterized and evaluated in vitro , and in an animal model (ferrets) before use in man as live virus vaccines. The vaccine candidates were found to be attenuated and immunogenic. A set of genetic markers, cold-adaptation (ca) and temperature-sensitivity (ts) were identified which correlated with the level of attenuation. Genetic stability of cold recombinants was proven after administration to ferrets and man. The gene compositions of the vaccine strains were determined, related to the parental types and to the degree of attenuation. Identification of the attenuating lesions has not been fully realized. However, repeatedly the data from in vitro and in vivo studies have shown that cold recombinants (CR) with six genes derived from the cold mutant and the two surface genes from the wild type parent, were predictably attenuated. In these cold recombinants, the ca marker was generally a more reliable indicator of attenuation. Thus, clones having different cut-off temperatures from 37°-39° were equally avirulent in man. In addition, clones which appear to have reverted to (ts + ) in the MDCK cell line are still attenuated and retained the ca and ts markers when evaluated in primary chick kidney cells. Thus, it can be stated that laboratory criteria are at hand to screen the level of attenuation of candidate live influenza virus vaccine for man. Cold recombinant vaccines of influenza virus with six genes from the cold mutants, appear to be the ideal vaccine candidates for use in man.

Influenza Virus Infection of Newborn Rats: Virulence of Recombinant Strains Prepared From a Cold-Adapted, Attenuated Parent

SummaryInfant rats were infected with one of a series of influenza A viruses. The growth of viruses in the turbinates or lungs, and the ability of virus infection to potentiate a subsequent bacterial infection by Haemophilus influenzae (HIb), were measured. The three virus strains known to be virulent for man grew to relatively high titres-of 105.0–106.8 EBID50/ml in the turbinates of infant rats at 48 hours post-infection, and virus infection enhanced subsequent systemic infection following intranasal inoculation of rats with HIb. In contrast, influenza virus A/Ann Arbor/6/60—P17 and the three recombinant viruses prepared from this strain, all of which are attenuated for man, replicated to significantly lower titres of 102.6–104.1 EBID50/ml in infant rats turbinates, and failed to promote systemic infection by HIb to the same degree. The results, together with those of previous studies, suggest that the behaviour of influenza viruses in infant rats may be an indication for virus virulence for man, and thus provide a test which could facilitate the development of live, attenuated virus vaccines.

IN VITRO AND IN VIVO PROPERTIES OF AN INFLUENZA A HOST RANGE VIRUS

Publisher Summary CR43-3 is an influenza A cold-adapted reassortant virus that has a limited host range when compared to either parent virus or to its congenic reassortant, CR31-10. It grows well in primary chick kidney (PCK) cells but very little, if at all, in Madin-Darby canine kidney (MDCK) cells or in ferrets after intranasal inoculation. This host range virus has an aberrant NS gene that migrates differently from the NS gene of either parent on polyacrylamide and agarose gels used for genotyping the reassortants. Reassortants made with experiments using A/Cal/10/78 H1N1 virus and CR43-3 virus showed that the host range property and the aberrant NS gene co-segregated with one another. Sequence studies on this NS gene have demonstrated that the CR43-3 NS gene is a deletion mutant of the NS gene of the A/Alaska/6/77 wt parent. The deletion is 36 bases long and occurs from nucleotides 222–257 of the A/Alaska/6/77 NS gene in the region coding for NS1 protein.