J. Banks, E. C. Speidel, D. J. Alexander.

VLA Weybridge, Addlestone, UK.

Two H5N1 viruses were isolated from a single turkey during an outbreak of highly pathogenic avian influenza (HPAI) in Norfolk, England in 1991. One (A/ty/Eng/50-92/91) is highly pathogenic for domestic poultry, has multiple basic amino acids at the haemagglutinin (HA) cleavage site and grows well in MDCK cells without the addition of trypsin, characteristics typical of HPAI viruses. The other (A/ty/Eng/87-92/91) has the same HA cleavage site, but is not pathogenic and does not grow in MDCK cells even with the addition of trypsin. The two isolates do not have any sequence differences in the haemagglutinin (HA) gene which correlate with virulence (1). This study investigates the growth and molecular characteristics of a limiting dilution passage clone of A/ty/Eng/87-92/91 (LDP3).

Two groups of ten 3-4-week old specific pathogen free leghorn chickens were inoculated with 10⁶ EID50 of freshly harvested LDP3 virus, one group intranasally and the other group by the intravenous route. One-day post infection (PI) six uninoculated birds were placed in contact. Cloacal and tracheal swabs were taken daily for 10 days, and one bird from each inoculated group was killed from days 3-11. Eight organ samples were taken from each bird at sacrifice (kidney, pancreas, lung, heart, brain, spleen, intestine, and trachea). Virus isolation was attempted from the samples by three passages in 9 or 10-day-old embryonating fowls’ eggs. Brain tissues were tested by nested RT/PCR for evidence of H5 influenza virus. The experiment was repeated using 3-4-week old turkeys.

Fourteen-day-old embryonating fowls’ eggs were inoculated with the avian influenza isolate LDP3. Allantoic fluids were harvested after 48 hours incubation at 37^oC and screened for growth on confluent MDCK monolayers without the addition of trypsin. Parental virus LDP3 was included as a control. Each culture was sampled daily and tested for HA activity. Selected cultures positive by HA were passaged in MDCK cells and 11-day-old embryonating fowls’ eggs. Two isolates (G33 and B22) were selected for sequencing of the entire HA gene. The entire PB2 gene for isolates G33, LDP3 and clone 2L was sequenced and partially sequenced for A/ty/Eng/50-92/91 clones 3L, 5L, and 87v from A/ty/Eng/87-92/91. The neuraminidase (NA) gene was sequenced for LDP3 and 2L. An IVPI was determined for clone G33 in 6-week-old white leghorn chickens

Chickens and turkeys infected with clone LDP3 by the intranasal or by the intravenous routes excreted very little virus. No clinical signs were seen and there was no transmission to ‘in contact’ birds. Virus was only recovered from the kidneys of sacrificed chickens and nested RT/PCR analysis of the brains for evidence of virus was negative. This is in contrast to A/ty/Eng/50-92/91, which can be easily isolated or detected by RT/PCR from tracheal or cloacal swabs, or from organs, including the lungs, kidney and brain. All sentinel birds become infected with this virus.

Of the 128 14-day-old embryonating fowls’ eggs inoculated with LDP3 55 (43%) gave positive HA results in the MDCK screening assay. All MDCK isolates inoculated into 11-day-old embryonating fowls’ eggs were lethal to the embryos and the allantoic fluids were positive for HA activity. The second passage of 32 MDCK isolates yielded 19 HA positive cultures, the parental virus LDP3 did not initiate a productive infection on primary or second passage. An IVPI index for isolate G30 was 0 and no clinical signs were observed. Nucleotide sequencing of the HA gene for isolates G33 and B22 showed no differences from the parental LDP3 sequence. Nucleotide sequencing of the entire PB2 gene for clones LDP3, G33 and 2L and partial sequencing of clones 5L from A/ty/Eng/50-92/91and 87v from A/ty/Eng/87-92/91 did not show any amino acid changes that correlate with pathogenicity

There are no nucleotide differences between the NA genes of LDP3 and 2L but there is a deletion in the stalk region of 23 amino acids as compared with A/parrot/NI/73.

The H5N1 isolate A/ty/Eng/87-92/91 was isolated from the brain of a turkey that had died of HPAI. However, clone LDP3 derived from this isolate is unable to disseminate throughout the body or to replicate in the brains of chickens and turkeys, as would be expected for a virus with multiple basic amino acids at the HA cleavage site. Passage in 14-day-old embryonating fowls’ eggs readily selects mutants from the avirulent clone LDP3 of A/ty/Eng/87-92 that can grow in MDCK cells in the absence of trypsin. This is in contrast to passage of LDP3 in chickens or 10 to 11-day-old embryonating fowls’ eggs, which fails to produce mutants capable of growth in MDCK. This increased measure of virulence however, did not translate to virulence in chickens, clone G30 had an IVPI of 0. No changes were seen in the HA nucleotide sequences relative to the parental LDP3 virus and therefore the selective pressure giving rise to growth in MDCK cells of mutants B22 and G33 must lie elsewhere in the genome. Reassortant experiments have led to speculation that the PB2 and Matrix genes may play a role in the modulation of pathogenicity (2). Therefore the entire PB2 gene for clones 2L G33 and LDP3 was nucleotide sequenced highlighting three deduced amino acid changes that appeared to correlate with pathogenicity. However, partial nucleotide sequencing of clones 5L, and 87v showed that there was no correlation with the deduced amino acid changes and IVPI.

Recently it has been suggested that additional glycosylation of the HA together with a shortened NA stalk are characteristic features of the H5 and H7 chicken viruses (3). Interestingly the NA genes of LDP3 and A/ty/Eng/50-92/91 have a predicted 23 amino acid deletion as compared with A/parrot/NI/73. However neither of these isolates have additional glycosylation sites on the HA thus proving to be exceptions for this rule.

This work was funded by MAFF UK.