Mark H. Snyder

The B allele of the NS gene of avian influenza viruses, but not the A allele, attenuates a human influenza A virus for squirrel monkeys

The nonstructural (NS) genes of avian influenza A viruses have been divided into two groups on the basis of nucleotide sequence homology, which we have referred to here as alleles A and B. We sequenced the NS genes of eight additional avian influenza A viruses in order to define the differences between these two alleles more thoroughly. Four of the viruses had NS gene sequences which resembled that of A/FPV/Rostock/34 and belonged to allele A while the other four viruses had NS gene sequences more similar to that of A/Duck/Alberta/76 and belonged to allele B. There was approximately 90% sequence homology within alleles and 72% homology between alleles. As previously reported the NS genes of human influenza A viruses belong to allele A. We constructed single gene avian-human reassortant influenza A viruses containing an allele A or B NS gene segment from an avian influenza A virus and all other genes from a human influenza A virus and tested these reassortants for their ability to grow in the respiratory tract of a nonhuman primate. Reassortants containing an avian NS gene segment of allele B were significantly restricted in growth in the respiratory tract of squirrel monkeys while reassortants with an allele A NS gene segment were not. The divergent evolution of the B NS allele in birds may have resulted in gene products which do not function optimally in cooperation with genes from a human virus in viral replication in primate respiratory epithelium.

A 36 nucleotide deletion mutation in the coding region of the NS1 gene of an influenza A virus RNA segment 8 specifies a temperature-dependent host range phenotype

Previously a spontaneous 36 nucleotide deletion in the coding region of NS1 was detected in the NS gene of a reassortant virus (CR43-3) recovered from a dual infection by the influenza A/Ann Arbor/6/60 cold-adapted (ca) mutant and wild-type (wt) influenza A/Alaska/6/77 (H3N2). The hemagglutinin, neuraminidase and NS genes were derived from the wild type virus parent while the other 5 genes were derived from the ca parent. The CR43-3 reassortant virus exhibited: (i) a host range (hr) phenotype, i.e. the reassortant replicated efficiently in avian cells in tissue culture but failed to grow in mammalian (MDCK) cell culture and (ii) an attenuation (att) phenotype, i.e., the reassortant was restricted in replication in the upper and lower respiratory tract of ferrets and hamsters. Since the CR43-3 reassortant possessed 5 genes from the ca parent which are each known to contain one or more mutations, it was not possible to assign the hr and att phenotypes solely to the NS deletion mutant gene. In order to determine the phenotype(s) specified solely by the mutant NS gene, it was transferred into a reassortant virus (143-1) which derived its seven other genes from the homologous wild type A/Alaska/6/77 virus. The deletion mutant NS gene specified only a partial hr phenotype manifested by a reduction in plaque size in MDCK tissue, but not a reduction in plaque number. Thus, the complete hr manifested by the CR43-3 parent virus is specified by the mutant NS1 gene acting in concert with one or more genes derived from the ca virus. The clone 143-1 virus exhibited the ts phenotype and was restricted in plaque formation at 37 degrees C in MDCK cells, a level of temperature sensitivity previously shown with other ts mutants to correlate with significant restriction of viral replication in the lower respiratory tract of hamsters. However, the clone 143-1 virus grew almost as well as the wt virus in the upper and lower respiratory tracts of hamsters and chimpanzees and thus did not possess the att phenotype. The finding that the ts phenotype was not manifest in vivo in animals with a 37 degrees C core temperature indicates that the mutated NS1 gene specifies a host dependent ts phenotype with replication restricted in vitro (MDCK tissue culture) at 37 degrees C but not in vivo in the lungs of hamsters and chimpanzees. ts+ virus was readily recovered from infected hamsters and chimpanzees indicating that the ts phenotype specified by the 36-base deletion was not stable following replication in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the M protein and nucleoprotein genes of an avian influenza A virus which are involved in host range restriction in monkeys.

A reassortant virus possessing RNA segment 7, which codes for the M1 and M2 proteins, of the avian influenza A/Mallard/New York/6750/78 (H2N2) virus and the other seven RNA segments of the human influenza A/Udorn/307/72 (H3N2) virus had been shown previously to be markedly restricted in replication in the respiratory tract of squirrel monkeys. In contrast, a reassortant possessing segment 7 of another avian influenza virus, A/Pintail/Alberta/119/79 (H4N6), and the seven other RNA segments from the A/Udorn/72 virus was not restricted. The nucleotide and deduced amino acid sequence of the RNA segment 7 of each virus was determined to identify the structural basis for the attenuation phenotype specified by RNA segment 7 of the A/Mallard/78 virus. Analysis of the deduced amino acid sequences revealed only two amino acid differences in the M1 protein and one difference in the M2 protein, suggesting that the attenuation phenotype of a reassortant virus possessing segment 7 of the A/Mallard/78 virus may be specified by one to three amino acids. Reassortant viruses possessing RNA segment 6, which codes for the nucleoprotein, of either avian influenza virus and the other seven RNA segments of a human influenza virus were also restricted in replication in squirrel monkeys. A comparison of the deduced amino acid sequences of the two avian nucleopeoteins demonstrated only three amino acid differences indicating that these two avian viruses possess NP genes that are highly related. The high degree of relatedness of both the NP and M proteins of these two avian viruses contrasts with their divergent surface antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Live viral vaccines for respiratory and enteric tract diseases

In its programme for accelerated development of vaccines for viral respiratory and enteric tract diseases the WHO has assigned a very high priority to respiratory syncytial virus (RSV), parainfluenza viruses and rotaviruses. There is also some interest in alternative approaches to immunization against influenza viruses because of the failure of inactivated vaccines to provide complete and reasonably durable immunity. Current attempts to develop satisfactorily attenuated viruses for use in prevention of disease caused by the above viral pathogens are described.

SAFETY AND IMMUNOGENICITY OF LIVE ATTENUATED INFLUENZA VACCINES IN INFANTS AND CHILDREN

Inactivated influenza vaccines confer incomplete and transient protection from disease, and the whole-virus inactivated vaccines cause high rates of reactions in children. Live, attenuated vaccines have been developed from cold-adapted (CA) and avian-human (AH) influenza virus reassortants, and are safe and effective in adults. We compared the infectious dose50, immunogenicity, reactogenicity and transmissability of CA and AH influenza A/Bethesda/85 (H3N2) reassortant vaccines in young children. Fifty-eight seronegative 6 to 36 months old children were studied in groups of 6 to 10 in close contact in a day-care-like setting for 2 days before and 9 days after receiving a placebo or 103 to 106 TCID50 of either vaccine. Seroconversion occurred in >50% of children who received a dose of 106 TCID50 of either vaccine. There were no differences in rates of febrile or respiratory illness between vaccinees and placebo recipients. Vaccine virus was shed in low titers (5.6 to 27.5 TCID50/ml) for 1 to 5 days by vaccinees, and no transmission to placebo contacts occurred. Both vaccines appear to be safe and immunogenic in young children and infants.