Reassortant virus derived from avian and human influenza A viruses is attenuated and immunogenic in monkeys.

An influenza A reassortant virus that contained the hemagglutinin and neuraminidase genes of a virulent human virus, A/Udorn/72 (H3N2), and the six other influenza A virus genome segments from an avirulent avian virus, A/Mallard/New York/6750/78 (H2N2), was evaluated for its level of replication is squirrel monkeys and hamsters. In monkeys, the reassortant virus was as attenuated and as restricted in its level of replication in the upper and lower respiratory tract as its avian influenza virus parent. Nonetheless, infection with the reassortant induced significant resistant to challenge with virulent human influenza virus. In hamsters, the reassortant virus replicated to a level intermediate between that of its parents. These findings suggest that the nonsurface antigen genes of the avian parental virus are the primary determinants of restriction of replication of the reassortant virus in monkeys. Attenuation of the reassortant virus for primates is achieved by inefficient functioning of the avian influenza genes in primate cells, while antigenic specificity of the human influenza virus is provided by the neuraminidase and hemagglutinin genes derived from the human virus. This approach could lead to the development of a live influenza A virus vaccine that is attenuated for man if the avian influenza genes are similarly restricted in human cells.

Virulence of avian influenza A viruses for squirrel monkeys.

Ten serologically distinct avian influenza A viruses were administered to squirrel monkeys and hamsters to compare their replication and virulence with those of human influenza A virus, A/Udorn/307/72 (H3N2). In squirrel monkeys, the 10 avian influenza A viruses exhibited a spectrum of replication and virulence. The levels of virus replication and clinical response were closely correlated. Two viruses, A/Mallard/NY/6874/78 (H3N2) and A/Pintail/Alb/121/79 (H7N8), resembled the human virus in their level and duration of replication and in their virulence. At the other end of the spectrum, five avian viruses were restricted by 100- to 10,000-fold in replication in the upper and lower respiratory tract and were clearly attenuated compared with the human influenza virus. In hamsters, the 10 viruses exhibited a spectrum of replication in the nasal turbinates, ranging from viruses that replicated as efficiently as the human virus to those that were 8,000- fold restricted. Since several avian viruses were closely related serologically to human influenza viruses, studies were done to confirm the avian nature of these isolates. Each of the avian viruses plaqued efficiently at 42 degrees C, a restrictive temperature for replication of human influenza A viruses. Avian strains that had replicated either very efficiently or very poorly in squirrel monkeys still grew to high titer in the intestinal tracts of ducks, a tropism characteristic of avian, but not mammalian, influenza viruses. These observations indicate that some avian influenza A viruses grow well and cause disease in a primate host, whereas other avian viruses are very restricted in this host. These findings also provide a basis for determining the gene or genes involved in the restriction of replication that is observed with the attenuated avian viruses. Application of such information may allow the preparation of reassortant viruses derived from a virulent human influenza virus and an attenuated avian virus for possible use in a live attenuated vaccine for prevention of influenza in humans.

Production and level of genetic stability of an influenza A virus temperature-sensitive mutant containing two genes with ts mutations.

Temperature-sensitive (ts) reassortant vaccine strains derived from the A/Udorn/72 ts-1A2 donor virus were not sufficiently stable genetically in humans. We therefore sought to produce a new, more stable donor virus. We had previously identified a stable ts virus with a ts P3 gene and in the current study identified another relatively stable single-lesion ts virus with a ts mutation in the NP gene. A new ts reassortant virus was constructed by mating these two single mutants and by isolating three reassortant progeny, clones 20, 53, and 55, that contained both a ts P3 and a ts NP gene. These reassortant progeny possessed a 37 to 38 degrees C shutoff temperature and were as restricted in their replication in hamster lungs as the A/Udorn/72 ts-1A2 virus. All isolates from the lungs and nasal turbinates of hamsters were temperature sensitive. An in vitro stress test was used to determine whether the new ts P3 ts NP reassortant virus would undergo loss of its ts phenotype after replication at semipermissive temperature. Clone 20 and 55 reassortants underwent progressive loss of their ts phenotype in vitro, although at a rate slightly less than that of the A/Udorn/72 ts-1A2 virus. The level of genetic stability after replication in vivo was assessed in cyclophosphamide-treated hamsters in which virus replication continued for up to 15 days. Again, both the A/Udorn/72 ts-1A2 and the new ts P3 ts NP reassortant clone 55 manifested a progressive loss of temperature sensitivity after 7 days of replication. Clone 55 virus lost temperature sensitivity significantly less rapidly than the A/Udorn/72 ts-1A2 virus. These results indicated that, although the new ts P3 ts NP reassortant virus was more stable than the A/Udorn/72 ts-1A2 virus, it nevertheless underwent progressive loss of temperature sensitivity after replication in vitro and in vivo. Therefore, it does not appear to be a satisfactory donor virus. This experience plus that gained earlier with other ts mutants of influenza A virus suggest that influenza A virus mutants that rely solely upon their ts phenotype for attenuation are unlikely to exhibit the phenotypic stability required of a vaccine virus. Other genetic techniques are needed to produce more stable influenza A virus strains.

Live temperature-sensitive equine influenza virus vaccine: generation of the virus and efficacy in hamsters.

Temperature-sensitive (ts) reassortants of an equine influenza virus, subtype A-1, were produced by mating a human influenza ts donor virus with an equine influenza A/Cornell/16/74 wild-type virus and by isolating a ts reassortant virus possessing the equine hemagglutinin and neuraminidase surface antigens. Two equine its reassortant clones, 8B1 and 71A1, were produced which had an in vitro shutoff temperature for plaque formation of 38 and 37 C, respectively. The human ts donor virus had ts mutation(s) on the polymerase 3 (P3) and nucleoprotein genes so that a ts equine reassortant virus could have either or both of these ts genes. It was found by complementation analysis that reassortant clone 8B1 had a ts lesion on the P3 gene and clone 71A1 had ts lesions on the nucleoprotein and P3 genes. An analysis of the parental origin of the genes in each ts equine reassortant virus indicated that clone 8B1 received 6 of its 8 genes and clone 71A1, 3 of its 8 from the equine parent virus, the remainder genes being from the human ts donor virus. The growth of both clones was restricted in the lungs of hamsters, but similar to that of the equine wild-type virus in the nasal turbinates. Each virus isolate obtained from the hamster's lungs or nasal turbinates retained the ts phenotype. These findings form the basis for further evaluation of the equine ts reassortant viruses for their level of attenuation and immunogenicity in horses.

Evaluation of three strains of influenza A virus in humans and in owl, cebus, and squirrel monkeys.

The virulence of three cloned influenza A viruses was compared in humans and in three readily available species of nonhuman primates (owl, squirrel, and cebus monkeys) in an attempt to identify a species of monkey that could be used to investigate the genetic basis of attenuation of influenza A viruses for humans. Three influenza A viruses from two subtypes, i.e., the A/Udorn/72 (H3N2), A/Alaska/77 (H3H2), and A/Hong Kong/77 (H1H1) viruses, produced febrile influenzal illness in humans. Squirrel monkeys developed mild upper respiratory tract illness in response to each of the three viruses. Illness was accompanied by a high level of virus shedding; each of nine squirrel monkeys that shed equal to or greater than 10(5.0) 50% tissue culture infective doses of virus became ill, whereas those that shed less remained well. In contrast, the cebus and owl monkeys remained clinically well despite infection with each of the three viruses. Thus, squirrel monkeys appear to be moderately permissive primate hosts in which to investigate the genetic basis of virulence of human influenza A viruses.

Temperature-sensitive mutants of influenza A virus: transfer of the two temperature-sensitive lesions in the Udorn/72-ts-1A2 virus to the A2Hong Kong/123/77 (H1N1) wild-type virus.

The influenza A/Udorn/72-ts-1A2 virus possesses temperature-sensitive mutations in the genes coding for the P1 and P3 polymerase proteins. It is being evaluated as a donor of its attenuating temperature-sensitive genes to produce recombinant live vaccine strains of epidemic variants of influenza A virus. Transfer of the P1 and P3 genes to two viruses within the H3N2 subtype of influenza A virus (i.e., the A/Victoria/3/75 and A/Alaska/6/77 viruses) conferred on each variant the following properties: (i) 37 degrees C shutoff temperature for plaque formation, (ii) almost complete restriction of viral replication in the lungs, (iii) a 100-fold restriction of viral replication in the nasal turbinates, and (iv) genetic stability after replication in hamsters. This study was undertaken to determine whether the transfer of the two ts-1A2 temperature-sensitive genes into a virus belonging to the H1N1 subtype (i.e., the A/Hong Kong/123/77 virus) would result in a restriction of replication in vitro and in vivo comparable to that observed with the previously studied H3N2 recombinant viruses in hamsters. This was found to be the case. In addition, infection of hamsters with the A/Hong Kong/77-ts-1A2 virus induced significant resistance to infection with wild-type A/Hong Kong/77 virus. Thus, the two ts-1A2 temperature-sensitive genes attenuated influenza A viruses belonging to two distinct subtypes to a specific and predictable level. An unexpected genetic interaction was observed between several A/Hong Kong/77-ts-1A2 segregants bearing the group 5 (P1) temperature-sensitive lesion. One interpretation of these results is that intracistronic complementation occurred between these segregants.

Temperature-sensitive mutants of influenza A virus: production and characterization of A/Victoria/3/75-ts-1[E] recombinants.

The Hong Kong/68-ts-1[E] virus, which has a 38 degrees C shutoff temperature for plaque formation, has been proposed as a donor of its two ts lesions to new variants of influenza A virus that pose an epidemic threat. To further examine whether the acquisition of the two ts-1[E] lesions will predictably attenuate new influenza A variants, the HK/68-ts-1[E] virus was mated with the A/Vic/3/75 wild-type virus. The Vic/75-ts-[E] recombinants that had the two ts-1[E] lesions also had a 38 degrees C shutoff temperature. Two Vic/75-ts-1[E] recombinants (clones 81 and 113) that had the two ts-1[E] lesions, a 38 degrees C shutoff temperature, and the Vic/75 hemagglutinin and neuraminidase glycoproteins were similar to each other and to their ts-1[E] parent in the pattern of replication and genetic stability in hamsters. These findings support the hypothesis that the acquisition of the two ts-1[E] lesions will predictably attenuate wild-type influenza A virus. Each Vic/75-ts-1[E] recombinant virus that possessed only the group 1 ts-1[E] lesion had a 39 degrees C shutoff temperature. Two of three of the Vic/75-ts-1[E] recombinants that had only the group 2 ts-1[E] lesion had a 39 degrees C shutoff temperature. This suggests that the HK/68-ts-1[E] donor virus contains two ts genes each of which by itself restricts plaque formation at 39 degrees C and above. The HK/68-ts-1[E] parent virus and its Vic/75 recombinant clones 81 and 113 were evaluated in ferret tracheal organ cultures maintained at permissive and restrictive temperatures. The Vic/75-ts-1[E] clone 81 differed from its parent and sister clone 113 in that it replicated readily and caused ciliostasis at 37 degrees C, a temperature restrictive for the replication of other ts-1[E] recombinants with a 38 degrees C shutoff temperature. The genetic basis underlying this difference was not elucidated.

Temperature-sensitive mutants of influenza A virus: evaluation of A/Victoria/3/75-ts-1[E] recombinant viruses in volunteers.

The Hong Kong/68-ts-1[E] virus and its Udorn/72 and Georgia/74 recombinants, which have a 38 degrees C shutoff temperature and a ts lesion(s) on the genes coding for the P3 and NP proteins, were adequately attenuated and immunogenic in adult volunteers who lacked serum hemagglutination-inhibiting antibody (titer,