Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, April 2001, p. 1216-1224, Vol. 45, No. 4
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.4.1216-1224.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Efficacy of Zanamivir against Avian Influenza A
Viruses That Possess Genes Encoding H5N1 Internal Proteins and Are
Pathogenic in Mammals
Irina A.
Leneva,1,2
Olga
Goloubeva,3
Robert J.
Fenton,4
Margaret
Tisdale,4 and
Robert
G.
Webster1,5,*
Department of Virology and Molecular
Biology1 and Department of Biostatistics
and Epidemiology,3 St. Jude Children's
Research Hospital, and Department of Pathology, University of
Tennessee,5 Memphis, Tennessee 38105;
Department of Chemotherapy of Infectious Diseases, Russian
Chemical and Pharmaceutical Institute, Moscow,
Russia2; and Glaxo Wellcome Research and
Development, Stevenage, Hertfordshire, United
Kingdom4
Received 31 July 2000/Returned for modification 25 October
2000/Accepted 24 January 2001
 |
ABSTRACT |
In 1997, an avian H5N1 influenza virus, A/Hong Kong/156/97
(A/HK/156/97), caused six deaths in Hong Kong, and in 1999, an avian
H9N2 influenza virus infected two children in Hong Kong. These viruses
and a third avian virus [A/Teal/HK/W312/97 (H6N1)] have six highly
related genes encoding internal proteins. Additionally, A/Chicken/HK/G9/97 (H9N2) virus has PB1 and PB2 genes that are highly
related to those of A/HK/156/97 (H5N1), A/Teal/HK/W312/97 (H6N1), and
A/Quail/HK/G1/97 (H9N2) viruses. Because of their similarities with the
H5N1 virus, these H6N1 and H9N2 viruses may have the potential for
interspecies transmission. We demonstrate that these H6N1 and H9N2
viruses are pathogenic in mice but that their pathogenicities are less
than that of A/HK/156/97 (H5N1). Unadapted virus replicated in lungs,
but only A/HK/156/97 (H5N1) was found in the brain. After three
passages (P3) in mouse lungs, the pathogenicity of the
viruses increased, with both A/Teal/HK/W312/97 (H6N1) (P3)
and A/Quail/HK/G1/97 (H9N2) (P3) viruses being found in the
brain. The neuraminidase inhibitor zanamivir inhibited viral
replication in Madin-Darby canine kidney cells in virus yield assays
(50% effective concentration, 8.5 to 14.0 µM) and inhibited viral
neuraminidase activity (50% inhibitory concentration, 5 to 10 nM).
Twice daily intranasal administration of zanamivir (50 and 100 mg/kg of
body weight) completely protected infected mice from death. At a dose
of 10 mg/kg, zanamivir completely protected mice from infection with
H9N2 viruses and increased the mean survival day and the number of
survivors infected with H6N1 and H5N1 viruses. Zanamivir, at all doses
tested, significantly reduced the virus titers in the lungs and
completely blocked the spread of virus to the brain. Thus,
zanamivir is efficacious in treating avian influenza viruses that can
be transmitted to mammals.
| |
INTRODUCTION |
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 |
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).
The percent inhibition of virus replication by the antiviral compound
was calculated after correction for the background (cell control)
values with the following formula: percent inhibition = 100 × (1 
OD450 of the treated sample/OD450
of the virus control sample), where OD450 is the optical
density at 450 nm. The concentrations of the compound that effectively
inhibited virus replication by 50% (EC50) were determined
by plotting inhibition of virus replication as a function of compound concentration.
An MDCK plaque assay was used to study the sensitivity of the virus
isolated from mouse lungs at the end of therapy. MDCK
monolayers
(six-well plates) were infected with viruses from lungs
of mice treated
or not treated with zanamivir and then overlaid
with agar containing
the antiviral compound at concentrations
ranging from 0.03 to 10 µM.
After 4 days of incubation, the agar
was removed and the plaques were
visualized by crystal violet
staining.
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 |
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).
Titration of the H6N1 and H9N2 viruses in eggs showed that both viruses
replicated to high titers in chicken eggs; the EID
50 ranged
from 10
8.0 to 10
9.0 and were similar for the
viruses studied (Table
1). Our
experiments
confirmed that A/HK/156/97 (H5N1) virus is highly
pathogenic to
mice. All mice infected with virus diluted
10
1 to 10
6 lost up to 10 to 15% of their
body weight and died by day 6 after
infection. The dose that was lethal
for 50% of the mice (MLD
50)
infected with A/HK/156/97
(H5N1) was estimated to be 7.7 log
10 EID
50 and
contained 1.16 EID
50. All mice showed signs of infection,
including huddling and ruffled fur, and the mice in the group
infected
with 1 MLD
50 lost 15% of their body weight.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Replication of avian influenza viruses containing the
internal genes of 1997 H5N1 viruses in chicken embryos and mice
|
|
A/Teal/HK/W312/97 (H6N1), which had not been serially passaged in mouse
lungs, was less pathogenic, but all mice inoculated
with the undiluted
virus died of infection. The MLD
50 for this
virus was
estimated to be 1.0 log
10 EID
50 and contained
8.5 EID
50.
All mice infected with 1 to 10 MLD
50s of A/Teal/HK/W312/97 (H6N1)
lost 20 to 24% of their
body weight. After three passages of A/Teal/HK/W312/97
(H6N1) virus in
the lungs of mice, the pathogenicity increased;
the MLD
50
increased by almost 1,000-fold, and 1 MLD
50 contained
2.3 EID
50.
A/Quail/HK/G1/97 (H9N2) virus was the least pathogenic virus of those
studied, and when undiluted, the virus did not kill
all mice in the
group. When this virus was serially subcultured
three times in mouse
lungs, the MLD
50 increased from 0.4 to 2.5
log
10 MLD
50, and the amount of infectious virus
units in 1 MLD
50 decreased from 20.0 to 3.2 EID
50. Mice infected with A/Quail/HK/G1/97
(H9N2)
(P
3) virus that was diluted 10-fold and 100-fold lost
approximately
18 to 20% of their body weight, whereas mice infected
with undiluted
and unadapted A/Quail/HK/G1/97 (H9N2) virus lost only
10%.
When mice were infected with undiluted and unadapted A/Chicken/HK/G9/97
(H9N2), all mice died. The pathogenicity of the virus
increased after
three passages in mice; the MLD
50 increased from
1.0 to 2.3 log
10 MLD
50. The MLD
50 of the
unadapted virus contained
8 EID
50, and 1 MLD
50
of the serially passaged virus contained
3.2 EID
50.
To further examine the pathogenicity of these avian viruses, we
compared their levels of growth in the lungs and brains of
infected
mice. Mice were infected with 5 MLD
50s of each virus,
and
three mice in each group were killed 1, 2, 3, 4, 5, and 7
days after
infection. The titers of virus in the lungs and brains
were determined
(Table
2).
On day 1, mice infected with A/HK/156/97 (H5N1) virus had high titers
in the lungs (6.0 to 6.5 log
10 EID
50) and
reached a
peak on day 3 (log
10 8.5). High titers continued
to be present
through to day 5 (7.5 log
10
EID
50). A/HK/156/97 (H5N1) virus (titer,
log
10
2.0) was found in the brain on days 3 and 5 after infection.
Similar
results had previously been obtained by Gubareva et al.
(
14), Gao et al. (
7), and Lu et al.
(
20). Although the
viral titer in the brain did not reach
the level of that in the
lungs, the presence of A/HK/156/97 (H5N1)
virus in the brain confirms
that this virus can spread systemically in
mice.
A/Teal/HK/W312/97 (H6N1) influenza virus replicated in mouse lungs to
high titers, similar to those of A/HK/156/97 (H5N1)
virus, but was not
detected in brains of mice on any day after
infection. After three
passages in mouse lungs, A/Teal/HK/W312/97
(H6N1) (P
3)
continued to replicate in the lungs to high titers
and virus was found
in the brains of mice 3 days after
infection.
Titers of A/Quail/HK/G1/97 (H9N2) virus in the lungs were lower than
those of A/HK/156/97 (H5N1) and A/Teal/HK/W312/97 (H6N1)
viruses, but
after three passages in the lungs of mice, the titer
increased and was
comparable to those found in the lungs of mice
infected with
A/HK/156/97 (H5N1) and A/Teal/HK/W312/97 (H6N1)
viruses. Although we
did not detect unadapted A/Quail/HK/G1/97
virus in the brains of mice,
this virus, after three passages
in mice, replicated in the brain to a
titer of 2.0 log
10 EID
50 and continued to
replicate at the same level on day 5 after
infection.
The unadapted A/Chicken/HK/G9/97 (H9N2) virus replicated in the lungs
of mice to titers of 4.5 log
10 EID
50 on the
first day
after infection and to 4.8 log
10
EID
50 by the third day. On day
5, the titer had decreased
to 2.5 log
10 EID
50. After three passages
in
mice, this virus replicated to high titers in the lungs of
mice. We did
not detect virus in the brains of mice infected with
either the
unadapted or the adapted (serially passaged in mouse
lungs)
A/Chicken/HK/G9/97 (H9N2)
virus.
Our study of the pathogenicity of the avian H6N1 and H9N2 influenza
viruses possessing H5N1 genes showed that A/Teal/HK/W312/97
(H6N1), A/Quail/HK/G1/97 (H9N2), and A/Chicken/HK/G9/97 (H5N1)
viruses are clearly less pathogenic in mice than is the A/HK/156/97
virus. Viruses passaged in mouse lungs had increased pathogenicity,
but
this pathogenicity did not reach the level seen for the A/HK/156/97
virus.
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.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 3.
In vitro effect of zanamivir on replication of influenza
viruses of avian origin in MDCK cells and inhibition of their NA
activities
|
|
Because the target of zanamivir is the active site of NA, we estimated
the efficacies of zanamivir against the H6N1 and H9N2
viruses in NA
inhibition tests. The NA inhibition assays demonstrated
that zanamivir
efficiently inhibited the enzyme activities of
the H6N1 and H9N2
viruses (Table
3). The IC
50 were 5.0 nM against
A/HK/156/97
(H5N1), 7 ± 1 nM against A/Quail/HK/G1/97 (H9N2),
10 ± 2 nM
against A/CK/HK/G9/97 (H9N2), and 7.5 ± 2.5 nM against
A/Teal/HK/W312/97. Thus, zanamivir inhibited both NA activity
and virus
replication in MDCK cells for each virus, with the levels
of inhibition
for the different viruses being comparable (Table
3).
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.
Similar results were obtained when we studied the effect of zanamivir
on A/Teal/HK/W312/97 infection in mice. Zanamivir at
doses of 1 mg/kg
did not prevent the weight loss and death of
mice infected with
A/Teal/HK/W312/97, but doses of 50 mg/kg provided
complete protection
from weight loss and death. When compared
against values for the
control group, the number of survivors
and the MSD of mice treated with
the 10-mg/kg dose
increased.
Because undiluted A/Quail/HK/G1/97 (H9N2) virus was not 100% lethal in
mice, we tested the efficacy of zanamivir against virus
that had been
serially passaged in the lungs of mice three times.
At doses of 10, 50, and 100 mg/kg, zanamivir administered intranasally
prevented the deaths
of mice infected with A/Quail/HK/G1/97 (P
3)
virus. A dose
of 1 mg of zanamivir per kg did not protect mice
from weight loss and
death.
Zanamivir administered intranasally at doses of 10, 50, and 100 mg/kg
fully protected mice infected with A/Chicken/HK/G9/97
(H9N2) influenza
virus from death. Nevertheless, mice infected
with A/Chicken/HK/G9/97
(H9N2) and treated with 10 mg of zanamivir
per kg lost more weight on
days 3 and 7 after infection than did
those treated with 50 or 100 mg/kg. When infected mice were treated
with 1 mg of zanamivir per kg,
the number of survivors and MSD
increased compared with those of mice
in the control
group.
Our findings show that A/Quail/HK/G1/97 (H9N2) and A/Chicken/HK/G9/97
(H9N2) viruses appear to be more sensitive to zanamivir
than
A/Teal/HK/W312/97 (H6N1) and A/HK/156/97 (H5N1) viruses.
Because we
used 5 MLD
50s of virus in each challenge dose, the
difference in antiviral effect was not due to differences in virus
challenge
doses.
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.
View this table:
[in this window]
[in a new window]
|
TABLE 5.
Effects of zanamivir on high and low challenge doses of
A/HK/156/97 (H5N1) and A/Quail/HK/G1/97 (H9N2) in mice
|
|
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.
|
|
On day 7 after treatment with 50 mg of zanamivir per kg, the residual
virus in the lungs of mice infected with A/HK/156/97
(H5N1),
A/Teal/HK/W312/97 (H6N1), and A/Quail/HK/G1/97 (H9N2)
(P
3)
was examined for sensitivity to zanamivir by both ELISA and
NA
inhibition assay. Each of the residual viruses examined was
as
sensitive to zanamivir as the original virus (results not shown);
these
findings indicate that the viruses were not resistant to
zanamivir.
Experiments were also done to determine the presence of virus in the
brains of mice infected with A/HK/156/97 (H5N1) and A/Quail/HK/G1/97
(H9N2) (P
3) and treated with different doses of zanamivir.
Virus
was not detected in brain after treatment with 1, 10, or 50 mg/kg
on any day after infection (data not
shown).
| |
DISCUSSION |
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.
| |
ACKNOWLEDGMENTS |
This work was supported by Public Health Service research grants
AI08831 and AI95357 from the National Institute of Allergy and
Infectious Diseases, by Cancer Center Support (CORE) grant CA-21765,
and by the American Lebanese Syrian Associated Charities (ALSAC).
Mikhail N. Matrosovich and Yi Guan provided valuable consultation; the
viruses were provided by Kennedy Shortridge from Hong Kong University.
We thank Glaxo Wellcome Research and Development for providing the NA
inhibitor zanamivir, Scott Krauss and Melissa Norwood for technical
assistance, Julia Cay Jones for editing the manuscript, and Alice
Herren for typing the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Virology and Molecular Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Phone: (901) 495-3400. Fax:
(901) 523-2622. E-mail: Robert.Webster{at}stjude.org.
| |
REFERENCES |
| 1.
|
Aymard-Henry, M.,
M. T. Coleman,
W. R. Dowdle,
W. G. Laver,
G. C. Schild, and R. G. Webster.
1973.
Influenza virus neuraminidase-inhibition test procedures.
Bull. W. H. O.
48:199-202[Medline].
|
| 2.
|
Belshe, R. B.,
M. H. Smith,
C. B. Hall,
R. Betts, and A. J. Hay.
1988.
Genetic basis of resistance to rimantadine emerging during treatment of influenza virus infection.
J. Virol.
62:1508-1512[Abstract/Free Full Text].
|
| 3.
|
Centers for Disease Control and Prevention.
1998.
Update: isolation of avian influenza A (H5N1) viruses from humans Hong Kong, 1997-1998.
Morb. Mortal. Wkly. Rep.
46:1245-1247[Medline].
|
| 4.
|
Claas, E. C.,
A. D. Osterhaus,
R. van Beek,
J. C. De Jong,
G. F. Rimmelzwaan,
D. A. Senne,
S. Krauss,
K. F. Shortridge, and R. G. Webster.
1998.
Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus.
Lancet
351:472-477[CrossRef][Medline].
|
| 5.
|
de Jong, J. C.,
E. C. Claas,
A. D. Osterhaus,
R. G. Webster, and W. L. Lim.
1997.
A pandemic warning?
Nature
389:554[Medline].
|
| 6.
|
Dybing, J. K.,
S. Schulz-Cherry,
D. E. Swayne,
D. L. Suarez, and M. L. Perdue.
2000.
Distinct pathogenesis of Hong Kong-origin H5N1 viruses in mice compared to that of other highly pathogenic H5 avian influenza viruses.
J. Virol.
74:1443-1450[Abstract/Free Full Text].
|
| 7.
|
Gao, P.,
S. Watanabe,
T. Ito,
H. Goto,
K. Wells,
M. McGregor,
A. J. Cooley, and Y. Kawaoka.
1999.
Biological heterogeneity, including systemic replication in mice, of H5N1 influenza A virus isolates from humans in Hong Kong.
J. Virol.
73:3184-3189[Abstract/Free Full Text].
|
| 8.
|
Grunert, R. R.,
J. W. McGahen, and W. L. Davies.
1965.
The in vivo antiviral activity of 1-adamantadine (amantadine).
Virology
26:262-269.
|
| 9.
|
Guan, Y.,
K. F. Shortridge,
S. Krauss, and R. G. Webster.
1999.
Molecular characterization of H9N2 influenza viruses: were they the donors of the "internal" genes of H5N1 viruses in Hong Kong?
Proc. Natl. Acad. Sci. USA
96:9363-9367[Abstract/Free Full Text].
|
| 10.
|
Guan, Y.,
K. F. Shortridge,
S. Krauss,
P. S. Chin,
K. C. Dyrting,
T. M. Ellis,
R. G. Webster, and M. Peiris.
2000.
H9N2 influenza viruses possessing H5N1-like internal genomes continue to circulate in poultry in southeastern China.
J. Virol.
74:9372-9380[Abstract/Free Full Text].
|
| 11.
|
Gubareva, L. V.,
M. N. Matrosovich,
M. K. Brenner,
R. C. Bethell, and R. G. Webster.
1999.
Evidence for zanamivir resistance in an immunocompromised child infected with influenza B virus.
J. Infect. Dis.
178:1257-1262.
|
| 12.
|
Gubareva, L. V.,
C. R. Penn, and R. G. Webster.
1995.
Inhibition of replication of avian influenza viruses by the neuraminidase inhibitor 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid.
Virology
212:323-330[CrossRef][Medline].
|
| 13.
|
Gubareva, L. V.,
R. Bethell,
G. J. Hart,
K. G. Murti,
C. R. Penn, and R. G. Webster.
1996.
Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en.
J. Virol.
70:1818-1827[Abstract].
|
| 14.
|
Gubareva, L. V.,
J. A. McCullers,
R. C. Bethell, and R. G. Webster.
1998.
Characterization of influenza A/Hong Kong/156/97 (H5N1) virus in a mouse model and protective effect of zanamivir on H5N1 infection in mice.
J. Infect. Dis.
178:1592-1596[CrossRef][Medline].
|
| 15.
|
Guo, Y. J.,
J. W. Li, and I. Cheng.
1999.
Discovery of humans infected by avian influenza A (H9N2) virus.
Chin. J. Exp. Clin. Virol.
15:105-108.
|
| 16.
|
Hayden, F. G.,
J. J. Treanor,
R. F. Betts,
M. Lobo,
J. D. Esinhart, and E. K. Hussey.
1996.
Safety and efficacy of the neuraminidase inhibitor GG167 in experimental human influenza.
JAMA
275:295-299[Abstract/Free Full Text].
|
| 17.
|
Hayden, F. G.,
B. S. Rollins, and A. J. Hay.
1990.
Anti-influenza virus activity of the compound LY253963.
Antivir. Res.
14:25-38[CrossRef][Medline].
|
| 18.
|
Hoffmann, E.,
J. Stech,
I. Leneva,
S. Krauss,
C. Scholtissek,
P. S. Chin,
M. Peiris,
K. F. Shortridge, and R. G. Webster.
2000.
Characterization of the influenza A gene pool in avian species in southern China: was H6N1 a derivative or a precursor of H5N1?
J. Virol.
74:6309-6315[Abstract/Free Full Text].
|
| 19.
|
Lin, Y. P.,
M. Shaw,
V. Gregory,
K. Cameron,
W. Lim,
A. Klimov,
K. Subbarao,
Y. Guan,
S. Krauss,
K. Shortridge,
R. Webster,
N. Cox, and A. Hay.
2000.
Avian-to-human transmission of H9N2 subtype influenza A virusesrelationship between H9N2 and H5N1 human isolates.
Proc. Natl. Acad. Sci. USA
97:9654-9658[Abstract/Free Full Text].
|
| 20.
|
Lu, X.,
T. M. Tumpey,
T. Morken,
S. Zaki,
N. Cox, and J. Katz.
1999.
A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans.
J. Virol.
73:5903-5911[Abstract/Free Full Text].
|
| 21.
|
Matrosovich, M.,
N. Zhou,
Y. Kawaoka, and R. G. Webster.
1999.
The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties.
J. Virol.
73:1146-1155[Abstract/Free Full Text].
|
| 22.
|
McCauley, J. W.,
L. A. Pullen,
M. Forsyth,
C. R. Penn, and G. P. Thomas.
1995.
4-Guanidino-Neu5Ac2en fails to protect chickens from infection with pathogenic avian influenza virus.
Antivir. Res.
27:179-186[CrossRef][Medline].
|
| 23.
|
McCullers, J.,
S. Facchini,
P. J. Chesney, and R. G. Webster.
1999.
Influenza B virus encephalitis.
Clin. Infect. Dis.
28:898-900[Medline].
|
| 24.
|
Paisley, J. W.,
F. W. Bruhn,
B. A. Lauer, and K. McIntosh.
1978.
Type A2 influenza viral infections in children.
Am. J. Dis. Child.
132:34-36[Abstract/Free Full Text].
|
| 25.
|
Patriarca, P. A., and N. J. Cox.
1997.
Influenza pandemic preparedness plan for the United States.
J. Infect. Dis.
176(Suppl. 1):S4-S7.
|
| 26.
|
Peiris, M.,
K. Y. Yuen,
C. W. Leung,
K. H. Chan,
P. L. S. Ip,
R. W. M. Lai,
W. K. Orr, and K. F. Shortridge.
1999.
Human infection with influenza H9N2.
Lancet
354:916-917[CrossRef][Medline].
|
| 27.
|
Rott, R.,
M. Orlich, and C. Scholtissek.
1979.
Correlation of pathogenicity and gene constellation of influenza A viruses. III. Non-pathogenic recombinants derived from highly pathogenic parent strains.
J. Gen. Virol.
44:471-477[Abstract/Free Full Text].
|
| 28.
|
Ryan, D. M.,
J. Ticehurst,
M. H. Dempsey, and C. R. Penn.
1994.
Inhibition of influenza virus replication in mice by GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is consistent with extracellular activity of viral neuraminidase (sialidase).
Antimicrob. Agents Chemother.
38:2270-2275[Abstract/Free Full Text].
|
| 29.
|
Scholtissek, C.,
I. Koennecke, and R. Rott.
1978.
Host range recombinants of fowl plague (influenza A) virus.
Virology
91:79-85[CrossRef][Medline].
|
| 30.
|
Shortridge, K. F.,
N. N. Zhou,
Y. Guan,
P. Gao,
T. Ito,
Y. Kawaoka,
S. Kodihalli,
S. Krauss,
D. Markwell,
K. G. Murti,
M. Norwood,
D. Senne,
L. Sims,
A. Takada, and R. G. Webster.
1998.
Characterization of avian H5N1 influenza viruses from poultry in Hong Kong.
Virology
252:331-342[CrossRef][Medline].
|
| 31.
|
Subbarao, K.,
A. Klimov,
J. Katz,
H. Regnery,
W. Lim,
H. Hall,
M. Perdue,
D. Swayne,
C. Bender,
J. Huang,
M. Hemphill,
T. Rowe,
M. Shaw,
X. Xu,
K. Fukuda, and N. Cox.
1998.
Characterization of an avian influenza A (H5N1) virus isolated from child with fatal respiratory illness.
Science
279:393-396[Abstract/Free Full Text].
|
| 32.
|
Sweet, C., and H. Smith.
1980.
Pathogenicity of influenza virus.
Microbiol. Rev.
44:303-330[Free Full Text].
|
| 33.
|
Thomas, G. P.,
M. Forsyth,
C. R. Penn, and J. W. McCauley.
1994.
Inhibition of the growth of influenza viruses in vitro by 4-guanidino-2,4-dideoxy-N-acetylneuraminic acid.
Antivir. Res.
24:351-356[CrossRef][Medline].
|
| 34.
|
Tisdale, M.
2000.
Monitoring of viral susceptibility: new challenges with the development of influenza NA inhibitors.
Rev. Med. Virol.
10:45-55[CrossRef][Medline].
|
| 35.
|
Togashi, T. Y.,
M. Matsuzono,
M. Anakura, and K. Nerome.
1997.
Acute encephalitis and encephalopathy at the height of influenza in childhood.
Nippon Rinsho
55:2699-2705[Medline].
|
| 36.
|
von Itzstein, M.,
W.-Y. Wu,
G. B. Kok,
M. S. Pegg,
J. C. Dyason,
B. Jin,
T. Van Phan,
M. L. Smythe,
H. F. White,
S. W. Oliver,
P. M. Colman,
J. N. Varghese,
D. M. Ryan,
J. M. Woods,
R. C. Bethell,
V. J. Hotham,
J. M. Cameron, and C. R. Penn.
1993.
Rational design of potent sialidase-based inhibitors of influenza virus replication.
Nature
363:418-423[CrossRef][Medline].
|
| 37.
|
Ward, A. C.
1997.
Virulence of influenza A virus for mouse lung.
Virus Genes
14:187-194[CrossRef][Medline].
|
| 38.
|
Webster, R. G.
1997.
Predictions for future human influenza pandemics.
J. Infect. Dis.
176(Suppl. 1):S14-S19.
|
| 39.
|
Woods, J. M.,
R. C. Bethell,
J. A. Coates,
N. Healy,
S. A. Hiscox,
B. A. Pearson,
D. M. Ryan,
J. Ticehurst,
J. Tilling,
S. M. Walcott, and C. R. Penn.
1993.
4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid is a highly effective inhibitor both of the sialidase (neuraminidase) and of growth of a wide range of influenza A and B viruses in vitro.
Antimicrob. Agents Chemother.
37:1473-1479[Abstract/Free Full Text].
|
| 40.
|
Zhou, N. N.,
K. F. Shortridge,
E. C. Claas,
S. L. Krauss, and R. G. Webster.
1999.
Rapid evolution of H5N1 influenza viruses in chickens in Hong Kong.
J. Virol.
73:3366-3374[Abstract/Free Full Text].
|
Antimicrobial Agents and Chemotherapy, April 2001, p. 1216-1224, Vol. 45, No. 4
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.4.1216-1224.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Peiris, J. S. M., de Jong, M. D., Guan, Y.
(2007). Avian Influenza Virus (H5N1): a Threat to Human Health. Clin. Microbiol. Rev.
20: 243-267
[Abstract]
[Full Text]
-
Sidwell, R. W., Barnard, D. L., Day, C. W., Smee, D. F., Bailey, K. W., Wong, M.-H., Morrey, J. D., Furuta, Y.
(2007). Efficacy of Orally Administered T-705 on Lethal Avian Influenza A (H5N1) Virus Infections in Mice. Antimicrob. Agents Chemother.
51: 845-851
[Abstract]
[Full Text]
-
Thomas, J. K., Noppenberger, J.
(2007). Avian influenza: A review. Am J Health Syst Pharm
64: 149-165
[Abstract]
[Full Text]
-
Wong, S. S. Y., Yuen, K.-y.
(2006). Avian Influenza Virus Infections in Humans. Chest
129: 156-168
[Abstract]
[Full Text]
-
Moscona, A.
(2005). Neuraminidase inhibitors for influenza.. NEJM
353: 1363-1373
[Full Text]
-
Beigel, J. H.
(2005). Avian influenza A (H5N1) infection in humans.. NEJM
353: 1374-1385
[Full Text]
-
Bartlett, J. G., Hayden, F. G.
(2005). Influenza A (H5N1): Will It Be the Next Pandemic Influenza? Are We Ready?. ANN INTERN MED
143: 460-462
[Full Text]
-
McSharry, J. J., McDonough, A. C., Olson, B. A., Drusano, G. L.
(2004). Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors. CVI
11: 21-28
[Abstract]
[Full Text]
Top
Abstract
Introduction
