INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Since 1997, two avian H5N1 and H9N2 influenza viruses have been transmitted directly from birds to humans. A/Hong Kong/156/97 (A/HK/156/97) (H5N1) influenza virus caused 18 confirmed infections, with six deaths, in Hong Kong (3, 4, 5, 31). In 1998 and 1999, H9N2 influenza viruses infected five humans in China and two in Hong Kong (15, 19). The H5N1 and H9N2 influenza viruses that were transmitted to humans possess six genes encoding internal proteins that are closely related to each other (19) and to A/Quail/HK/G1/97 (H9N2), the likely source of the H9N2 viruses that were transmitted to children in Hong Kong. The increased prevalence of A/Quail/HK/G1/97 in poultry in China, together with serological evidence of infection in up to 60% of quail and up to 16% of quail shedding this virus in cages in Hong Kong poultry markets in 1999 to 2000 (10), increases the likelihood of transmission to mammals, including humans. Additionally, viruses of the H6N1 subtype that are similar to A/Teal/HK/W312/97, which possesses seven gene segments (PB2, PB1, PA, NA, NP, M, and NS), are highly related genetically to those of the H5N1 influenza viruses isolated from humans in 1997 and continue to circulate in domestic poultry (18). There is concern that these H6N1 viruses containing H5N1 genomes will also be transmitted to humans.

Because the H9N2 and H6N1 influenza viruses containing H5N1 genes encoding internal proteins continue to circulate in poultry in southeast Asia (10), there is a potential risk of these avian influenza A viruses infecting and causing disease in mammals. It is important to determine the pathogenicities of these viruses, for they have the potential for interspecies transmission and hence the potential to cause epidemics and pandemics.

The most cost-effective approach in controlling epidemic and pandemic influenza is immunization; however, it takes at least 6 months to prepare a new vaccine, even under ideal conditions. When a vaccine is available, many people remain unvaccinated, despite widespread promotion of immunization. In the face of an emerging pandemic, antiviral drugs have enormous potential for preventing the deaths of infected persons and the further spread of disease. Mice are useful experimental animals for vaccine development, and previous studies have shown that H5N1 viruses are highly pathogenic in mice since they replicate without adaptation, cause systemic infection, and spread to the brain (7, 14, 20, 30).

Zanamivir (Relenza) is a neuraminidase (NA) inhibitor that was recently approved for use in humans in Europe, the United States, and Australia. It has significant antiviral activity in cell culture, in animals, and in humans (12, 16, 17, 33, 36, 39). However, although it was shown that zanamivir failed to protect chickens from infection with highly pathogenic avian influenza viruses (22), zanamivir effectively protected mice from death when they were infected with A/HK/156/97 (14). It is therefore important to determine if zanamivir is efficacious against the H6N1 and H9N2 subtypes when it is presented on an H5N1 internal gene complex. To facilitate this aim, it was necessary to determine the pathogenicities of these emerging avian influenza viruses in mice for in vivo evaluation of zanamivir. These viruses included A/Teal/HK/W312/97 (H6N1), A/Quail/HK/G1/97 (H9N2), and A/Chicken/HK/G9/97 (H9N1). We compared the pathogenicities of these viruses in mice with that of A/HK/156/97 (H5N1) and determined the efficacies of zanamivir against these viruses both in vitro and in vivo.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Compounds. Zanamivir (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuramic acid) was provided by Glaxo Wellcome Research and Development. The compound was dissolved in minimal essential medium (MEM) for in vitro experiments and in distilled water for in vivo experiments.

Cells and viruses. Madin-Darby canine kidney (MDCK) cells were grown in MEM containing 10% fetal bovine serum (FBS) and antibiotics. Influenza A/HK/156/97 (H5N1) virus was isolated from humans in Hong Kong in 1997 (5). A/Teal/HK/W312/97 (H6N1), A/Quail/HK/G1/97 (H9N2), and A/Chicken/HK/G9/97 (H9N2) viruses were isolated in Hong Kong from a teal duck, quail, and chicken, respectively. The viruses were serially passaged three times (P₃) in mouse lungs and were then propagated in embryonated chicken eggs. The resulting viruses were designated A/Teal/HK/W312/97 (H6N1) (P₃), A/Quail/HK/G1/97 (H9N2) (P₃), and A/Chicken/HK/G9/97 (H9N2) (P₃). All experiments were conducted at St. Jude Children's Research Hospital in a biosafety level 3 containment facility.

Mice. Female BALB/c mice were maintained under specific-pathogen-free conditions until they were used at 8 to 12 weeks of age. In these experiments, infected mice were killed 2, 4, and 7 days after infection and their lungs and brains were removed under sterile conditions. Organs were homogenized, suspended in a total volume of 1 ml of phosphate-buffered saline (PBS), serially diluted 10-fold, and assayed for virus infectivity in embryonated chicken eggs. Titers of infectious virus are presented as log₁₀ 50% egg infectious doses (EID₅₀).

Antiviral assay. A modified enzyme-linked immunosorbent assay (ELISA) (2, 17) was used to determine the inhibitory effects of the anti-NA inhibitor zanamivir. This assay detected expression of viral NP protein in infected cells. Briefly, MDCK cells were seeded in 96-well culture plates at a density of 3,000 cells per well in MEM containing 10% FBS, 100 U of penicillin per ml, 100 µg of streptomycin sulfate per ml, and 100 µg of kanamycin sulfate per ml. Cells were incubated at 37°C with 5% CO₂ to reach 90% confluence. Cells were washed twice with serum-free MEM, and residual medium was removed. Each microtiter plate included uninfected control cultures, virus-infected control wells, and virus-infected cultures to which antiviral compounds were added. The cultures were overlaid with MEM containing 2.5 µg of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin per ml and twice the concentration of the antiviral drug being studied (100 µl). After incubation for 30 min at 37°C, 100 µl of virus containing allantoic fluid (0.1 to 1.0 PFU/cell) was added to all wells, except the wells with the cell control. After incubation for 18 h at 37°C in a humidified atmosphere of 5% CO₂, cells were fixed by addition of 100 µl of cold acetone-PBS (80:20). The extent of viral replication was assessed by ELISA, as described previously (2).

NA inhibition assay. NA activity was measured by the colorimetric assay (1), with fetuin as a substrate. The inhibitory effect of zanamivir on viral NAs was determined by assaying for enzyme activity in the presence of different concentrations of compound. Viruses used in NA inhibition tests were diluted in PBS to give a standard level of enzyme activity (0.5 OD₅₄₀ unit). Tenfold dilutions of compound ranging from 0.0001 to 100 nM were incubated with viruses for 30 min at room temperature and then with fetuin overnight. Assays were performed in triplicate. The drug concentrations of zanamivir required to reduce enzyme activity to 50% (IC₅₀) were determined against all tested viruses.

Infection and drug administration in mice. Mice were anesthetized by inhalation of metofane and were inoculated by intranasal administration of virus in a volume of 100 µl. Zanamivir was administered intranasally twice daily for 5 days, with the first dose being given 4 h before the mice were exposed to virus (14). Groups of 5 or 10 mice were used for each dose. For all mice, the changes in weight and the number of mice that died were recorded daily. The mean survival day (MSD), i.e., the mean number of days mice survived, was calculated by the following formula: MSD = [f(d  1)]/n, where f is the number of mice recorded on day d (the number of survivors on day 16 was included in f for that day) and n is the number of mice in a group (8).

Statistical analysis. The effects of different doses of drug in mice infected with different viruses on survival rate were evaluated using logistic regression and Fisher's exact test. Differences in lung and brain virus titers were compared with control values using a multiple-comparison method (Dunnett's two-tailed t test) available with the Analysis of Variance procedure.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Characterization of influenza viruses of avian origin in the mouse model. Mice are not a natural host for influenza viruses; unadapted influenza viruses usually replicate in the lungs of mice and cause asymptomatic infection of the respiratory tract (32, 37). In contrast, some unadapted H5N1 isolates, including A/HK/156/97 (H5N1), are highly pathogenic and neurotropic in mice (6, 7, 14, 20, 30). A/Quail/HK/G1/97 (H9N2) and A/Teal/HK/W312/97 (H6N1) possess genes encoding internal proteins that are highly related genetically to those of A/HK/156/97 (H5N1), whereas A/Chicken/HK/G9/97 (H9N2) has two genes encoding internal proteins, PB1 and PB2, that are highly related genetically to those of A/HK/156/97 (H5N1) (9). Therefore, we wished to establish the pathogenicities of A/Chicken/HK/G9/97 (H9N2), A/Quail/HK/G1/97 (H9N2), and A/Teal/HK/W312/97 (H6N1) for mice, in comparison with that of A/HK/156/97 (H5N1).

1

6

In vitro activity of zanamivir against influenza viruses of avian origin. Because the H6N1 and H9N2 viruses contain gene segments encoding internal proteins similar to those of A/HK/156/97 (H5N1), replicate in mice, and are in some cases neurotropic, these viruses have the potential to spread to humans. We therefore wished to determine the efficacies of zanamivir against these viruses. Zanamivir inhibited the replication of A/Quail/HK/G1/97 (H9N2), A/Chicken/HK/G9/97 (H9N2), and A/Teal/HK/W312/97 (H6N1) in MDCK cells, and the level of inhibition increased with increasing concentration (Fig. 1). The mean EC₅₀ were similar for all of the viruses tested and ranged from 8.5 to 14.0 µM (Table 3).

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Inhibition of H5N1, H6N1, and H9N2 influenza viruses in MDCK cells by zanamivir. MDCK cells were grown in 96-well plates and infected with viruses in the presence of zanamivir as described in Materials and Methods. The levels of NP protein expression were measured by ELISA. Each point is the average of results from four replicate wells ± standard deviations (SD) calculated for three independent experiments.

Efficacies of zanamivir against A/Quail/HK/G1/97 (H9N2), A/Chicken/HK/G9/97 (H9N2), A/Teal/HK/W312/97 (H6N1), and A/HK/156/97 (H5N1) virus infection in mice. We used weight changes and death caused by H6N1 and H9N2 viruses in mice to determine the efficacy of zanamivir administered intranasally (Table 4). At doses of 50 mg/kg, zanamivir completely protected mice infected with A/HK/156/97 (H5N1) from death, and at doses of 10 mg/kg, zanamivir increased the number of survivors and mean survival time to 10.6 days, compared to 6.0 days for the control group of mice. When doses of 1 mg/kg were used, weight loss and mean survival time for the treatment group did not differ from those of the control groups.

Influence of challenge dose on the efficacy of zanamivir in mice infected with the A/HK/156/97 (H5N1) and A/Quail/HK/G1/97 (H9N2) viruses. Because differences among the sensitivities of influenza viruses to zanamivir cannot be due to differences in the low doses of virus administered, we next wished to determine whether high challenge doses would also reveal differences in efficacy. It had been reported previously that zanamivir failed to protect chickens against high doses of a systemic influenza virus (12, 22), so we wished to determine whether this finding also applied in the mouse model system. Groups of mice were infected with low (5 MLD₅₀s) and high (100 MLD₅₀s) doses of A/HK/156/97 (H5N1) and A/Quail/HK/G1/97 (P₃) (H9N2) influenza viruses and then treated with different doses of zanamivir (Table 5). When mice were infected with low challenge doses of A/Quail/HK/G1/97 (P₃) (H9N2), zanamivir at 10 mg/kg completely protected mice from death, whereas a dose of 50 mg of zanamivir per kg was required to protect all mice infected with a low challenge dose of A/HK/156/97 (H5N1). At high challenge doses of A/Quail/HK/G1/97 (P₃) (H9N2), 50 mg/kg was needed to prevent the deaths of all mice. At a high challenge of A/HK/156/97 (H5N1) virus, 100 mg of zanamivir per kg was needed to protect 8 of 10 mice from death. Thus, the differences between the sensitivities of influenza viruses to zanamivir are also found when mice are challenged with high doses of virus. In the mouse, zanamivir was effective against a high challenge dose of a systemic influenza virus, albeit at higher doses.

Effect of zanamivir administered intranasally on lung and brain infection in mice. The above-described experiments demonstrated the efficacy of zanamivir in preventing the weight loss and death of mice infected with H5N1, H6N1, and H9N2 viruses. We also evaluated the extent of reduction of virus titers in the lungs of mice and showed that zanamivir significantly reduced the levels of virus for each of the four influenza viruses tested (Fig. 2). Doses of 1 mg/kg significantly reduced the level of A/Quail/HK/G1/97 (H9N2) virus in the lungs of mice on day 4 (P < 0.01), though this dose of inhibitor was less effective on the other viruses tested (Fig. 2). At higher doses (10 and 50 mg/kg), zanamivir significantly reduced titers, but the only virus for which the levels were reduced to undetectable levels was A/Chicken/HK/G9/97 (H9N2), the virus that grew to the lowest titer in mouse lungs.

View larger version (33K): [in this window] [in a new window]   FIG. 2.   Effects of different doses of zanamivir on titers of H5N1, H6N1, and H9N2 viruses from lungs of infected mice. Groups of mice were infected with viruses and treated with different doses of zanamivir or PBS (control group). Three mice from each dose group were sacrificed 2, 4, and 7 days after infection, and lungs were assayed separately for virus infectivity in eggs. *, the difference between values for control and drug-treated groups was not statistically significant; **, P < 0.05 compared to the value for the control group; ***, P < 0.01 compared to the value for the control group. In these experiments we used A/Quail/HK/G1/97 that was passaged in mouse lungs three times.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Since 1997, two avian influenza viruses have been transmitted directly from birds to humans. H5N1 influenza virus caused 18 confirmed infections, with six deaths, in Hong Kong (3, 4, 5, 31). An influenza virus genetically highly related to A/Quail/HK/G1/97 (H9N2) virus infected two persons in Hong Kong (19, 26) and five persons in China (15). Examination of the consensus amino acid sequences of the internal virion proteins of A/HK/156/97 (H5N1) revealed that the PB2, PA, NP, and M2 proteins possess amino acids previously found in human strains (40). It has been proposed that the presence of human-specific amino acids in these H5N1 viruses permitted transmission of avian influenza viruses to humans. Because A/Quail/HK/G1/97 (H9N2), A/Chicken/HK/G9/97 (H9N2), and A/Teal/HK/W312/97 (H6N1) contain gene segments highly related genetically to those of A/HK/156/97 (H5N1), they have the potential to be transmitted to mammals.

Our findings established that the H9N2 and H6N1 viruses in this study can replicate and cause signs of disease in mice. It has already been shown that A/HK/156/97 (H5N1) virus causes lethal neurotropic infections of mice without adaptation (7, 14, 20). The present study established that A/Quail/HK/G1/97 (H9N2) and A/Teal/HK/W312/97 (H6N1) are much less pathogenic than A/HK/156/97 (H5N1) and that adaptation of the viruses in mice was necessary to increase their pathogenicities. Experiments with A/Chicken/HK/G9/97 (H9N2) gave similar results, in that the virus was much less pathogenic than A/HK/156/97 (H5N1), and even after three passages in mice, virus was not detectable in the brains of infected mice. Thus, the H9N2 and H6N1 influenza viruses possessing six or seven gene segments similar to those of the H5N1 Hong Kong viruses contain the necessary group of genes for systemic spread in mice, but the absence of the H5 hemagglutinin (HA) gene may reduce the level of pathogenicity. The failure of A/Chicken/HK/G9/97 (H9N2) to infect the brain indicates that the PB1 and PB2 genes are insufficient for systemic spread to the brain. It is well established that host-range transmission and pathogenicity are polygenic traits (27, 29). Thus, the A/HK/156/97 (H5N1) virus has a better group of genes for both efficient systemic spread and the killing of mice than do the A/Quail/HK/G1/97 (H9N2) and A/Teal/HK/W312/97 (H6N1) viruses. However, the groups of genes in the latter two viruses are clearly sufficient for systemic spread in mice. These groups of genes are apparently associated with interspecies transmission, including transmission to humans. Additional studies are needed to characterize the other viral and cellular genes involved in transmission.

Because influenza viruses possessing H5N1 gene segments have the capacity to be transmitted to mammals, they have the potential to cause pandemics. We therefore determined the efficacy of the newly approved NA inhibitor zanamivir against these viruses in vitro and in vivo. Because previous studies have established that zanamivir is efficacious against the A/HK/156/97 (H5N1) virus in vitro and in the mouse, we used A/HK/156/97 (H5N1) as a reference strain in establishing the efficacies of zanamivir against the H6N1 and H9N2 influenza viruses. Our experiments established that the viral replication and NA activities of A/Quail/HK/G1/97 (H9N2), A/Teal/HK/W312/97 (H6N1), and A/Chicken/HK/G9/97 (H9N2) are all inhibited in vitro by zanamivir. The results of the ELISAs indicated that the EC₅₀ were 8.5 to 14 µM, and the results of the NA inhibition assays indicated that the IC₅₀ were 5 to 10 nM. The susceptibilities of these avian isolates are within the ranges reported previously from NA assays and plaque reduction assays for human strains and clinical isolates (39). In this study, ELISA-based yield reduction assays were used rather than plaque assays to determine IC₅₀. In yield reduction assays the IC₅₀ determined are virus input dependent and tend to be higher than values determined by plaque reduction (34). Therefore, these data indicate that all five avian isolates are highly susceptible in vitro to zanamivir.

Comparison of the in vivo efficacies of zanamivir in this study with those of previous studies with human influenza strains is difficult because in vivo efficacy is dependent on virus input, which would be different between studies. Overall efficacy appears lower for these avian strains, but this could relate to the relatively high pathogenicities of avian strains in the mouse model compared with those of human strains (30). For comparisons of efficacies between the different avian isolates, dosing was initiated before infection, since this ensures greater reproducibility. Differences in viral sensitivity to zanamivir were detected in the mouse model; A/Quail/HK/G1/97 (H9N2) and A/Chicken/HK/G1/97 (H9N2) were more sensitive to zanamivir than A/Teal/HK/W312/97 (H6N1) and A/HK/156/97 (H5N1). This difference may be due to the interplay between the HAs and NAs of these viruses or to the affinity of binding of the HA (21). In addition, both the A/HK/156/97 (H5N1) and A/Teal/HK/W312/97 (H6N1) viruses have a 19-amino-acid deletion in the NA stalk that results in a decreased ability of viruses to escape from cells (18). Possibly, this deletion in vivo may have some unknown advantage in the presence of NA inhibitors. Zanamivir completely abolished the transmission of influenza virus to the brains of mice even at the lowest doses tested (1 mg/kg); this finding indicates that systemic spread may be related to the level of viral replication in the lungs. At high levels of replication, there is possibly sufficient pulmonary damage for virus to reach the circulation and, if levels are high enough, to reach the brain. The mechanism(s) for spread through the blood-brain barrier is not clearly understood and is probably related to a specific viral protein(s). Although neurologic disorders associated with influenza in humans are rare, there are reports of neurologic disorders associated with H3N2 and B strains of human influenza virus (23, 24, 32, 35). Although the Spanish influenza virus of 1918 has not been causally associated with encephalitis lethargica, the question remains open until further evidence is accumulated.

Although zanamivir significantly reduced the levels of A/Quail/HK/G1/97 (H9N2) and A/Teal/HK/W312/97 (H6N1) in the lungs of mice, it did not completely inhibit virus replication. Similar findings were reported previously when zanamivir was tested against A/HK/156/97 (H5N1) and other influenza viruses in the mouse system (11, 13, 28). These results may relate to the high levels of virus replication in this model overwhelming the inhibitor in experiments in which zanamivir produced up to a 4-log₁₀ EID₅₀ reduction in virus growth. One possible explanation is that zanamivir-resistant mutants were appearing, but the present and previous studies indicate that the viruses sampled late in infection were as sensitive to zanamivir as the original parental virus. This result may also suggest that the virus can evade contact with the drug by some mechanism, possibly by cell-to-cell spread.

A new pandemic of influenza is considered to be inevitable (26, 38). Because of this inevitability and because of the efficacy of zanamivir against H5N1, H6N1, and H9N2 avian viruses, it is now important to determine the long-term stability of both zanamivir and oseltamivir so that consideration can be given to stockpiling these agents to prepare for future pandemics.