Previous Article | Next Article 
Journal of Virology, April 1999, p. 3184-3189, Vol. 73, No. 4
Department of Pathobiological Sciences,
School of Veterinary Medicine, University of Wisconsin
Received 25 September 1998/Accepted 23 December 1998
An H5N1 avian influenza A virus was transmitted to humans in Hong
Kong in 1997. Although the virus causes systemic infection and is
highly lethal in chickens because of the susceptibility of the
hemagglutinin to furin and PC6 proteases, it is not known whether it
also causes systemic infection in humans. The clinical outcomes of
infection in Hong Kong residents ranged widely, from mild respiratory
disease to multiple organ failure leading to death. Therefore, to
understand the pathogenesis of influenza due to these H5N1 isolates, we
investigated their virulence in mice. The results identified two
distinct groups of viruses: group 1, for which the dose lethal for 50%
of mice (MLD50) was between 0.3 and 11 PFU, and group 2, for which the MLD50 was more than 103 PFU. One
day after intranasal inoculation of mice with 100 PFU of group 1 viruses, the virus titer in lungs was 107 PFU/g or 3 log
units higher than that for group 2 viruses. Both types of viruses had
replicated to high titers (>106 PFU/g) in the lungs by day
3 and maintained these titers through day 6. More importantly, only the
group 1 viruses caused systemic infection, replicating in
nonrespiratory organs, including the brain. Immunohistochemical
analysis demonstrated the replication of a group 1 virus in brain
neurons and glial cells and in cardiac myofibers. Phylogenetic analysis
of all viral genes showed that both groups of Hong Kong H5N1 viruses
had formed a lineage distinct from those of other viruses and that
genetic reassortment between H5N1 and H1 or H3 human viruses had not
occurred. Since mice and humans harbor both the furin and the PC6
proteases, we suggest that the virulence mechanism responsible for the
lethality of influenza viruses in birds also operates in mammalian
hosts. The failure of some H5N1 viruses to produce systemic infection
in our model indicates that multiple, still-to-be-identified, factors contribute to the severity of H5N1 infection in mammals. In addition, the ability of these viruses to produce systemic infection in mice and
the clear differences in pathogenicity among the isolates studied here
indicate that this system provides a useful model for studying the
pathogenesis of avian influenza virus infection in mammals.
An H5N1 avian influenza A virus was
transmitted from birds to humans in 1997 in Hong Kong, infecting 18 humans, 6 of whom died (3, 4). This outbreak was unique in
that the virus that was transmitted to humans is lethal in chickens
(20, 22). Although the virulence of avian influenza viruses
is polygenic, the susceptibility of the hemagglutinin (HA) to host
proteases is the major determinant for this property. That is,
influenza virus HA must be cleaved into HA1 and HA2 subunits for the
virus to be infectious, as this event generates the amino terminus of HA2, which mediates the fusion of the viral envelope with the endosomal
membrane (13, 15). Lethal and nonlethal avian viruses differ
in this mode of activation: the HA of the former is cleaved by the
ubiquitous proteases furin and PC6 (9, 19), whereas the HA
of the latter is not susceptible to these proteases but rather is
cleaved by proteases localized in the respiratory or intestinal organs
or both (12). We have shown that pathogenic avian viruses
replicate in the capillary endothelial cells of a variety of organs,
leading to the hemorrhagic manifestations found in infected chickens
(14). Similarly, the H5N1 Hong Kong virus replicated in the
capillary endothelial cells of chickens (20, 22). Despite
the high mortality rate (33%) associated with H5N1 virus infection, it
is still unclear whether the virus has the potential to cause systemic
infection in humans.
The clinical outcomes of H5N1 virus infection in apparently healthy
humans ranged from mild respiratory symptoms to death (26).
Epidemiologic studies indicate that there has been no human-to-human
transmission of the H5N1 virus, suggesting that the human cases in Hong
Kong originated from independent transmissions of the virus from birds.
An H5N1 virus that is genetically related to that isolated from the
patients was isolated from chickens in April 1997 in Hong Kong
(5). In fact, H5N1 viruses cocirculating among birds in Hong
Kong in December 1997 were genetically heterogeneous (4).
Therefore, biologic heterogeneity among infecting strains of the H5N1
virus could have accounted for the different clinical outcomes seen in patients.
In this study, we examined the extent of biologic and genetic
heterogeneities among the human H5N1 isolates in an attempt to
explain the differences in clinical manifestations seen in infected
patients. To this end, we investigated the virulence and
pathobiological features of human H5N1 isolates in mice and chickens
and established the phylogenetic relationships among these viruses.
Viruses and cells.
H5N1 influenza A viruses isolated from
patients during the Hong Kong outbreak in 1997 were obtained from the
Centers for Disease Control and Prevention (CDC) through the courtesy
of Nancy Cox and are listed in Table 1.
In the text, these viruses are designated with "HK" plus the field
number; for example, HK156 represents A/Hong Kong/156/97 (H5N1). They
were isolated and propagated in Madin-Darby canine kidney (MDCK) cells.
A/Udorn/307/72 (H3N2) (Udorn) was obtained from Robert G. Webster, St.
Jude Children's Research Hospital, and had been isolated and grown in
embryonated eggs. MDCK cells were cultured in minimal essential medium
with 5% newborn calf serum. All of the experiments with live H5N1
viruses isolated in Hong Kong were done in a biosafety level 3 containment laboratory approved for such use by the CDC and the U.S.
Department of Agriculture.
Experimental infections.
To determine the dose lethal for
50% of mice (MLD50), we infected 6-week-old female BALB/c
mice, anesthetized with methoxyflurane, intranasally with 50 µl of
serial 10-fold dilutions of virus and observed them for 2 weeks. Virus
titers in organs were determined by use of MDCK cells 1, 3, 6, and 10 days after intranasal inoculation of the mice with 100 PFU. To
determine the dose lethal for 50% of chickens (CLD50), we
infected 2-week-old specific-pathogen-free chickens (SPAFAS)
intranasally and orally with 100 µl of serial 10-fold dilutions of
virus and observed them for 2 weeks. Virus titers in the organs of
chickens infected with 105 PFU were determined as described above.
Immunohistochemistry.
Formalin-fixed and paraffin-embedded
sections were deparaffinized, hydrated, and treated with 0.1% Triton
X-100 in phosphate-buffered saline-bovine serum albumin. Endogenous
peroxidases were quenched with 0.3% peroxide in methanol. Sections
were incubated with a monoclonal antibody (NP5/1) specific for the
nucleoprotein of influenza virus at a dilution of 1:400 for 1 h at
room temperature. Bound antibody was detected by the peroxidase-labeled
streptavidin-biotin staining method (Vectastain ABC kit; Vector
Laboratories) with diaminobenzidine as the substrate. Sections were
counterstained with hematoxylin.
Sequencing analysis.
Viral RNA was isolated from the
virus-containing culture fluid of MDCK cells as previously described
(1). The cDNA was synthesized by use of reverse
transcriptase and an oligonucleotide complementary to the conserved 3'
end of the viral RNA as described by Katz et al. (10). Genes
were amplified by PCR with this cDNA, gene-specific oligonucleotide
primers, and LA Taq polymerase (Panvera). PCR products were
cloned into a plasmid and sequenced with an Autosequencer (Applied
Biosystems Inc.) in accordance with the protocol recommended by the
manufacturer. The sequences of the oligonucleotide primers will be
supplied upon request. At least three independent cDNA clones were
sequenced for each gene. When one of the cDNA clones contained a
different nucleotide at a given position, it was considered to be an
error introduced by the polymerase during PCR, unless otherwise
stated. Phylogenetic analysis of the sequence data was performed with
Clustal W software, version 1.6 (23), which relies on the
neighbor-joining method (17) to generate phylogenetic trees.
Replication in tissue cultures and eggs.
The virus titers of
stocks measured as PFU in MDCK cells and 50% egg infectious
doses were similar to each other. There was an appreciable difference
in plaque size among the H5N1 viruses in MDCK cells. Five
viruses Human H5N1 isolates differ in virulence for mice.
One
explanation for the differences in clinical manifestations among the
patients in Hong Kong may be that the H5N1 viruses differ in their
ability to replicate in mammalian hosts. Thus, we compared the
virulence of 10 Hong Kong viruses in mice. HK156, HK483, HK485, HK491,
HK514, and HK516 (group 1) were highly pathogenic, with an
MLD50 of less than 11 PFU, whereas the remaining four viruses (group 2) were clearly less pathogenic, as indicated by an
MLD50 of greater than 103 PFU. There was, in
addition, a correlation between virulence and plaque size (Table 1),
with the less pathogenic viruses tending to produce pinpoint plaques,
while the pathogenic viruses produced medium to large plaques. Only the
HK156 virus, which was highly lethal for mice, produced a mixed
population of plaques. Regardless of their pathogenicity, all of the
viruses induced disease symptoms within 24 h, including rapid
breathing, ruffled fur, and hunched posture. In contrast, other
mouse-adapted human viruses, such as A/WSN/33 (H1N1) and
A/PR/8/34 (H1N1), usually do not induce these effects until 3 days after infection (11a), even though they are lethal for mice.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Biological Heterogeneity, Including Systemic
Replication in Mice, of H5N1 Influenza A Virus Isolates from Humans
in Hong Kong
Madison,
Madison, Wisconsin 53706,1 and
Laboratory of Microbiology,
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Clinical and laboratory features of the human H5N1
viruses used in this study
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
HK483, HK485, HK491, HK514, and HK516produced medium to
large plaques, whereas four produced pinpoint plaques (Table 1).
HK156 displayed a mixed population with respect to plaque size:
~20% pinpoint and ~80% medium plaques. These results suggested
biologic differences among these viruses.
TABLE 2.
Kinetics of virus replication
in micea
Immunohistochemical analysis of virus replication in the brains and hearts of mice. To test whether HK483 indeed replicated in the mouse brain or whether its recovery from this organ was simply due to viremia, we performed immunohistochemical analysis of organs from HK483-infected mice by using a monoclonal antibody specific for the nucleoprotein of influenza A virus. On day 5 (Fig. 1A and B) and day 6 (data not shown), tissue specimens from the brain stem at the metencephalon showed multiple foci that stained intensely with this antibody in both the nucleus and the cytoplasm of neurons as well as glial cells. Because lesions were also apparent by histopathologic examination in the hearts of mice infected with HK483 on day 6 (data not shown), we also elected to examine hearts by immunohistochemical methods. Necrotic cardiac myofibers were positive for viral antigen in both the nucleus and the cytoplasm (Fig. 1C). These results prove that the H5N1 virus causes systemic infection in mice.
|
Comparison of virulence for chickens.
Because the index human
isolate of the Hong Kong H5N1 virus, HK156, retained its virulence in
chickens (22), we compared the virulence of representative
viruses from group 1 and group 2 in chickens. As shown by
CLD50 values, the three viruses testedHK156, HK483, and
HK486were equally pathogenic for chickens (Table 1). Interestingly,
the two group 1 viruses (HK156 and HK483) were less pathogenic for
chickens than they were for mice (MLD50, <1 PFU;
CLD50, ~200 PFU).
|
Phylogenetic analysis. It is possible that the difference in mouse virulence among the human H5N1 isolates is attributable to genetic reassortment with other avian or human viruses. We therefore sequenced portions of the PA, PB1, PB2, NP, NA, and NS genes and the entire HA and M genes from two group 1 and two group 2 viruses. We then analyzed these sequences phylogenetically, together with the published sequences of HK156 and A/chicken/Hong Kong/258/97 (H5N1), which was isolated in April 1997 and is closely related to HK156 (5). Regardless of the genes tested, all of the Hong Kong H5N1 viruses examined formed a lineage that was distinct from those of other viruses. As an example, the phylogenetic tree of the HA genes is shown in Fig. 2. Although the H5N1 viruses were divided into two sublineages, they did not correlate with mouse virulence. Instead, each of the two sublineages contained viruses from both group 1 (highly pathogenic) and group 2 (less pathogenic). Interestingly, HK483 was most closely related to A/chicken/Hong Kong/258/97; only 4 nucleotides were different in the HA genes of these viruses. HK486 and HK488 were also highly similar; there were only 4 nucleotide differences between the two viruses among 6,321 nucleotides compared.
|
Amino acid comparison of HA, PB2, and M1 proteins.
Because
previous work had suggested that the HA and M1 proteins can affect the
virulence of influenza A viruses in mice (18), we sought to
compare the deduced amino acid sequences of group 1 (HK156, HK483, and
HK485) and group 2 (HK486 and HK488) viruses. In the HA, one amino acid
of HK156 differed from the published sequence (at the fourth position
in the signal peptideIle in our sequence versus Thr in the sequence
reported by Subbarao et al. [22]); all other amino
acids were identical. None of the amino acid differences correlated
with mouse virulence. Claas et al. (5) reported that
A/chicken/Hong Kong/258/97 (H5N1), which is closely related to the
human H5N1 isolates, contains a glycosylation site at position
156, whereas the human isolate HK156 does not. We found
that HK483 and HK485 also possess this glycosylation site
(N156ST for HK483 and N156SS for HK485), in
contrast to HK486 and HK488 (N156SA for both viruses).
Thus, the presence or absence of the glycosylation site cannot account
for the observed differences in mouse virulence.
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
We demonstrate here that the H5N1 viruses isolated from Hong Kong residents are biologically heterogeneous with respect to their replicative potential in mammalian hosts. Although some of the viruses were relatively benign, others were extremely pathogenic for mice (MLD50, <1 PFU). Moreover, the virulent HK483 strain caused systemic infection, replicating in the brains and hearts of mice. Influenza A viruses have also been isolated from the brains of seals (25), but it is not clear whether they replicated in that tissue or were present there due to viremia. Thus, our report presents the first direct evidence of systemic infection of a mammal with an avian influenza virus. Since mice possess the same proteases (i.e., furin and PC6) that allow systemic replication of influenza viruses in chickens, we suggest that the susceptibility of the HA to these proteases is critical for influenza viruses to cause systemic infection in both avian and mammalian hosts, but other viral factors also contribute to this ability.
Although the majority of human influenza A viruses do not replicate systemically in mice (11, 11a), A/WSN/33 (H1N1) (6), which was derived by repeated passage of A/WS/33 (H1N1) in mouse brain, replicates in this organ upon intracerebral inoculation. A/WSN/33 (H1N1) replicates in systemic organs, including the brain, even upon intranasal inoculation of mice (2). Interestingly, A/WSN/33 does not have multiple basic amino acids at the HA cleavage site, a property required for cleavage by the ubiquitous furin and PC6 proteases (9, 19). We have recently demonstrated that NA binds and sequesters plasminogen, leading to higher local concentrations of this ubiquitous protease precursor and thus to increased cleavage of the HA (8). Therefore, regardless of the mechanism (recognition of multiple basic amino acids by furin and PC6 proteases or sequestration of plasminogen by NA), HA cleavage by ubiquitous proteases seems a prerequisite for systemic influenza virus infection of animals.
Titration of viruses in mouse organs revealed that the pathogenic H5N1 viruses differed in replicative ability from the less pathogenic viruses. The former group caused systemic infection, whereas the latter remained in the respiratory tract. This outcome is inconsistent with the properties of the HK486 HA, which possesses multiple basic amino acids at the cleavage site and can be cleaved by the ubiquitous proteases (as indicated by plaque formation without trypsin [data not shown]). Thus, viral properties other than HA cleavage must contribute to the difference in virulence among these H5N1 viruses in mice. Comparison of the amino acid sequences of the HA and M1 proteins failed to disclose changes that might account for differences in replicative capacity.
The amino acid residue at position 627 in the PB2 protein is associated with plaque formation in MDCK cells (21). A single mutation from Glu to Lys at this position converts a non-plaque-forming virus to a plaque producer in these cells. All avian influenza A viruses contain Glu at position 627, whereas human influenza A viruses possess Lys (7). Interestingly, we found that the Hong Kong H5N1 viruses differed in the amino acid residue at this position, although this difference was not directly correlated with plaque size in MDCK cells or with virulence in mice. Nonetheless, because all viruses that produced pinpoint plaques in this study had Glu at position 627, as opposed to Lys in three of the six viruses that produced medium to large plaques, it is possible that amino acid substitutions in PB2 or other viral proteins allowed the remaining medium- to large-plaque producers with 627-Glu in PB2 (i.e., HK156, HK491, and HK516) to increase their replicative potential in these cells. It will be important to perform reassortment studies to identify the viral gene products that determine the difference in mouse virulence between the two groups of H5N1 viruses.
Despite their clustering in phylogenetic trees, the human H5N1 isolates were genetically divergent enough to form sublineages (4). One explanation for this genetic diversity is that the H5N1 virus had been circulating among Hong Kong poultry as early as March 1997 (when the outbreak of highly pathogenic avian H5N1 virus infections was identified on Hong Kong chicken farms), leaving adequate time for the generation of multiple sublineages. Two of the viruses (i.e., HK486 and HK488), isolated from two cousins, are genetically and biologically closely related, albeit not identical. This finding suggests that either a virus was transmitted from one cousin to the other or that both patients acquired the virus from the same avian source.
It is premature to conclude that the human H5N1 isolates differ in their virulence potential, as mouse pathogenicity may not extrapolate directly to humans. However, we find it intriguing that four of the six viruses in group 1 (highly virulent in mice) were isolated from patients who eventually died, whereas three of the four viruses in group 2 (less virulent in mice) were isolated from patients who eventually recovered (Table 1). Definitive studies for determining virulence heterogeneity will have to be conducted in primates. The disease outcomes of patients cannot be used as a criterion for evaluating virus virulence because of the heterogeneous nature of the genetic background and health conditions of patients. However, the ability of the viruses to produce systemic infection in mice and the clear differences in pathogenicity among the isolates studied indicate that our system provides a useful model for molecular analysis of avian influenza virus infection in mammals.
| |
ACKNOWLEDGMENTS |
|---|
We gratefully acknowledge Nancy Cox for the Hong Kong H5N1 viruses and Susan Watson and John Gilbert for editing the manuscript.
Support for this work was provided by National Institute of Allergy and Infectious Diseases Public Health Service research grants AI-29599 and AI-33898.
| |
FOOTNOTES |
|---|
*
Corresponding author. Mailing address: Department of
Pathobiological Sciences, School of Veterinary Medicine, University of WisconsinMadison, 2015 Linden Dr. West, Madison, WI 53706. Phone: (608) 265-4925. Fax: (608) 265-5622. E-mail:
kawaokay{at}svm.vetmed.wisc.edu.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Bean, W. J., G. Sriram, and R. G. Webster. 1980. Electrophoretic analysis of iodine-labeled influenza virus RNA segments. Anal. Biochem. 102:228-232[Medline]. |
| 2. |
Castrucci, M. R., and Y. Kawaoka.
1993.
Biologic importance of neuraminidase stalk length in influenza A virus.
J. Virol.
67:759-764 |
| 3. |
Centers for Disease Control and Prevention.
1997.
Isolation of avian influenza A (H5N1) viruses from humansHong Kong, May-December 1997.
Morbid. Mortal. Weekly Rep.
46:1204-1207[Medline].
|
| 4. |
Centers for Disease Control and Prevention.
1998.
Update: isolation of avian influenza A (H5N1) viruses from humansHong Kong, 1997-1998.
Morbid. Mortal. Weekly Rep.
46:1245-1247[Medline].
|
| 5. | Claas, E. C. J., A. D. M. E. 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[Medline]. |
| 6. | Francis, T., and A. E. Moore. 1940. A study of the neurotropic tendency in strains of the virus of epidemic influenza. J. Exp. Med. 72:717-728[Abstract]. |
| 7. |
Gorman, O. T.,
R. O. Donis,
Y. Kawaoka, and R. G. Webster.
1990.
Evolution of influenza A virus PB2 genes: implications for evolution of the ribonucleoprotein complex and origin of human influenza A virus.
J. Virol.
64:4893-4902 |
| 8. |
Goto, H., and Y. Kawaoka.
1998.
A novel mechanism for the acquisition of virulence by a human influenza A virus.
Proc. Natl. Acad. Sci. USA
95:10224-10228 |
| 9. |
Horimoto, T.,
K. Nakayama,
S. P. Smeekens, and Y. Kawaoka.
1994.
Proprotein-processing endoproteases PC6 and furin both activate hemagglutinin of virulent avian influenza viruses.
J. Virol.
68:6074-6078 |
| 10. |
Katz, J. M.,
M. Wang, and R. G. Webster.
1990.
Direct sequencing of the hemagglutinin gene of influenza (H3N2) virus in original clinical samples reveals sequence identity with mammalian cell-grown virus.
J. Virol.
64:1808-1811 |
| 11. |
Kawaoka, Y.
1991.
Equine H7N7 influenza A viruses are highly pathogenic in mice without adaptation: potential use as an animal model.
J. Virol.
65:3891-3894 |
| 11a. | Kawaoka, Y. Unpublished data. |
| 12. | Klenk, H.-D., and W. Garten. 1994. Host cell proteases controlling virus pathogenicity. Trends Microbiol. 2:39-43[Medline]. |
| 13. | Klenk, H. D., R. Rott, M. Orlich, and J. Blodorn. 1975. Activation of influenza A viruses by trypsin treatment. Virology 68:426-439[Medline]. |
| 14. | Kobayashi, Y., T. Horimoto, Y. Kawaoka, D. J. Alexander, and C. Itakura. 1996. Pathological studies of chickens experimentally infected with two highly pathogenic avian influenza viruses. Avian Pathol. 25:285-304[Medline]. |
| 15. | Lazarowitz, S. G., and P. W. Choppin. 1975. Enhancement of the infectivity of influenza A and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology 68:440-454[Medline]. |
| 16. |
Myers, E. W., and W. Miller.
1988.
Optimal alignments in linear space.
Comput. Appl. Biosci.
4:11-17 |
| 17. | Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425[Abstract]. |
| 18. |
Smeenk, C. A., and E. G. Brown.
1994.
The influenza virus variant A/FM/1/47-MA possesses single amino acid replacements in the hemagglutinin, controlling virulence, and in the matrix protein, controlling virulence as well as growth.
J. Virol.
68:530-534 |
| 19. | Stieneke-Grober, A., M. Vey, H. Angliker, E. Shaw, G. Thomas, C. Roberts, H. D. Klenk, and W. Garten. 1992. Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO J. 11:2407-2414[Medline]. |
| 20. |
Suarez, D. L.,
M. L. Perdue,
N. Cox,
T. Rowe,
C. Bender,
J. Huang, and D. E. Swayne.
1998.
Comparisons of highly virulent H5N1 influenza A viruses isolated from humans and chickens from Hong Kong.
J. Virol.
72:6678-6688 |
| 21. |
Subbarao, E. K.,
W. London, and B. R. Murphy.
1993.
A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.
J. Virol.
67:1761-1764 |
| 22. |
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 a child with a fatal respiratory illness.
Science
279:393-396 |
| 23. |
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680 |
| 24. | Ward, A. C. 1996. Neurovirulence of influenza A virus. J. Neurovirol. 2:139-151[Medline]. |
| 25. | Webster, R. G., V. S. Hinshaw, W. J. Bean, K. L. Van Wyke, J. R. Geraci, D. J. St. Aubin, and G. Peturson. 1981. Characterization of an influenza A virus from seals. Virology 113:712-724[Medline]. |
| 26. | Yuen, K. Y., P. S. Chan, M. Peiris, D. C. Tsang, T. L. Que, K. F. Shortridge, P. T. Cheung, W. K. To, E. F. Ho, R. Sung, A. B. Cheng, I. Mena, and the H5N1 Study Group. 1998. Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancet 351:467-471[Medline]. |
Journal of Virology, April 1999, p. 3184-3189, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Tate, M. D., Deng, Y.-M., Jones, J. E., Anderson, G. P., Brooks, A. G., Reading, P. C.
(2009). Neutrophils Ameliorate Lung Injury and the Development of Severe Disease during Influenza Infection. J. Immunol.
183: 7441-7450
[Abstract] [Full Text] -
Fornek, J. L., Gillim-Ross, L., Santos, C., Carter, V., Ward, J. M., Cheng, L. I., Proll, S., Katze, M. G., Subbarao, K.
(2009). A Single-Amino-Acid Substitution in a Polymerase Protein of an H5N1 Influenza Virus Is Associated with Systemic Infection and Impaired T-Cell Activation in Mice. J. Virol.
83: 11102-11115
[Abstract] [Full Text] -
Belser, J. A., Wadford, D. A., Xu, J., Katz, J. M., Tumpey, T. M.
(2009). Ocular Infection of Mice with Influenza A (H7) Viruses: a Site of Primary Replication and Spread to the Respiratory Tract. J. Virol.
83: 7075-7084
[Abstract] [Full Text] -
Szretter, K. J., Gangappa, S., Belser, J. A., Zeng, H., Chen, H., Matsuoka, Y., Sambhara, S., Swayne, D. E., Tumpey, T. M., Katz, J. M.
(2009). Early Control of H5N1 Influenza Virus Replication by the Type I Interferon Response in Mice. J. Virol.
83: 5825-5834
[Abstract] [Full Text] -
Van Hoeven, N., Belser, J. A., Szretter, K. J., Zeng, H., Staeheli, P., Swayne, D. E., Katz, J. M., Tumpey, T. M.
(2009). Pathogenesis of 1918 Pandemic and H5N1 Influenza Virus Infections in a Guinea Pig Model: Antiviral Potential of Exogenous Alpha Interferon To Reduce Virus Shedding. J. Virol.
83: 2851-2861
[Abstract] [Full Text] -
Yen, H.-L., Aldridge, J. R., Boon, A. C. M., Ilyushina, N. A., Salomon, R., Hulse-Post, D. J., Marjuki, H., Franks, J., Boltz, D. A., Bush, D., Lipatov, A. S., Webby, R. J., Rehg, J. E., Webster, R. G.
(2009). Changes in H5N1 influenza virus hemagglutinin receptor binding domain affect systemic spread. Proc. Natl. Acad. Sci. USA
106: 286-291
[Abstract] [Full Text] -
Gillim-Ross, L., Santos, C., Chen, Z., Aspelund, A., Yang, C.-F., Ye, D., Jin, H., Kemble, G., Subbarao, K.
(2008). Avian Influenza H6 Viruses Productively Infect and Cause Illness in Mice and Ferrets. J. Virol.
82: 10854-10863
[Abstract] [Full Text] -
Korteweg, C., Gu, J.
(2008). Pathology, Molecular Biology, and Pathogenesis of Avian Influenza A (H5N1) Infection in Humans. Am. J. Pathol.
172: 1155-1170
[Abstract] [Full Text] -
Zeng, H., Goldsmith, C., Thawatsupha, P., Chittaganpitch, M., Waicharoen, S., Zaki, S., Tumpey, T. M., Katz, J. M.
(2007). Highly Pathogenic Avian Influenza H5N1 Viruses Elicit an Attenuated Type I Interferon Response in Polarized Human Bronchial Epithelial Cells. J. Virol.
81: 12439-12449
[Abstract] [Full Text] -
Mukhtar, M. M., Rasool, S. T., Song, D., Zhu, C., Hao, Q., Zhu, Y., Wu, J.
(2007). Origin of highly pathogenic H5N1 avian influenza virus in China and genetic characterization of donor and recipient viruses. J. Gen. Virol.
88: 3094-3099
[Abstract] [Full Text] -
Belser, J. A., Lu, X., Maines, T. R., Smith, C., Li, Y., Donis, R. O., Katz, J. M., Tumpey, T. M.
(2007). Pathogenesis of Avian Influenza (H7) Virus Infection in Mice and Ferrets: Enhanced Virulence of Eurasian H7N7 Viruses Isolated from Humans. J. Virol.
81: 11139-11147
[Abstract] [Full Text] -
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] -
Szretter, K. J., Gangappa, S., Lu, X., Smith, C., Shieh, W.-J., Zaki, S. R., Sambhara, S., Tumpey, T. M., Katz, J. M.
(2007). Role of Host Cytokine Responses in the Pathogenesis of Avian H5N1 Influenza Viruses in Mice. J. Virol.
81: 2736-2744
[Abstract] [Full Text] -
Shinya, K., Watanabe, S., Ito, T., Kasai, N., Kawaoka, Y.
(2007). Adaptation of an H7N7 equine influenza A virus in mice. J. Gen. Virol.
88: 547-553
[Abstract] [Full Text] -
Mase, M., Tanimura, N., Imada, T., Okamatsu, M., Tsukamoto, K., Yamaguchi, S.
(2006). Recent H5N1 avian Influenza A virus increases rapidly in virulence to mice after a single passage in mice. J. Gen. Virol.
87: 3655-3659
[Abstract] [Full Text] -
Li, Z., Jiang, Y., Jiao, P., Wang, A., Zhao, F., Tian, G., Wang, X., Yu, K., Bu, Z., Chen, H.
(2006). The NS1 Gene Contributes to the Virulence of H5N1 Avian Influenza Viruses. J. Virol.
80: 11115-11123
[Abstract] [Full Text] -
Gillim-Ross, L., Subbarao, K.
(2006). Emerging Respiratory Viruses: Challenges and Vaccine Strategies. Clin. Microbiol. Rev.
19: 614-636
[Abstract] [Full Text] -
Chen, H., Li, Y., Li, Z., Shi, J., Shinya, K., Deng, G., Qi, Q., Tian, G., Fan, S., Zhao, H., Sun, Y., Kawaoka, Y.
(2006). Properties and Dissemination of H5N1 Viruses Isolated during an Influenza Outbreak in Migratory Waterfowl in Western China.. J. Virol.
80: 5976-5983
[Abstract] [Full Text] -
Gabriel, G., Dauber, B., Wolff, T., Planz, O., Klenk, H.-D., Stech, J.
(2005). The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc. Natl. Acad. Sci. USA
102: 18590-18595
[Abstract] [Full Text] -
Maines, T. R., Lu, X. H., Erb, S. M., Edwards, L., Guarner, J., Greer, P. W., Nguyen, D. C., Szretter, K. J., Chen, L.-M., Thawatsupha, P., Chittaganpitch, M., Waicharoen, S., Nguyen, D. T., Nguyen, T., Nguyen, H. H. T., Kim, J.-H., Hoang, L. T., Kang, C., Phuong, L. S., Lim, W., Zaki, S., Donis, R. O., Cox, N. J., Katz, J. M., Tumpey, T. M.
(2005). Avian Influenza (H5N1) Viruses Isolated from Humans in Asia in 2004 Exhibit Increased Virulence in Mammals. J. Virol.
79: 11788-11800
[Abstract] [Full Text] -
Li, Z., Chen, H., Jiao, P., Deng, G., Tian, G., Li, Y., Hoffmann, E., Webster, R. G., Matsuoka, Y., Yu, K.
(2005). Molecular Basis of Replication of Duck H5N1 Influenza Viruses in a Mammalian Mouse Model. J. Virol.
79: 12058-12064
[Abstract] [Full Text] -
Shinya, K., Hatta, M., Yamada, S., Takada, A., Watanabe, S., Halfmann, P., Horimoto, T., Neumann, G., Kim, J. H., Lim, W., Guan, Y., Peiris, M., Kiso, M., Suzuki, T., Suzuki, Y., Kawaoka, Y.
(2005). Characterization of a Human H5N1 Influenza A Virus Isolated in 2003. J. Virol.
79: 9926-9932
[Abstract] [Full Text] -
Nguyen, D. C., Uyeki, T. M., Jadhao, S., Maines, T., Shaw, M., Matsuoka, Y., Smith, C., Rowe, T., Lu, X., Hall, H., Xu, X., Balish, A., Klimov, A., Tumpey, T. M., Swayne, D. E., Huynh, L. P. T., Nghiem, H. K., Nguyen, H. H. T., Hoang, L. T., Cox, N. J., Katz, J. M.
(2005). Isolation and Characterization of Avian Influenza Viruses, Including Highly Pathogenic H5N1, from Poultry in Live Bird Markets in Hanoi, Vietnam, in 2001. J. Virol.
79: 4201-4212
[Abstract] [Full Text] -
Lee, C.-W., Suarez, D. L., Tumpey, T. M., Sung, H.-W., Kwon, Y.-K., Lee, Y.-J., Choi, J.-G., Joh, S.-J., Kim, M.-C., Lee, E.-K., Park, J.-M., Lu, X., Katz, J. M., Spackman, E., Swayne, D. E., Kim, J.-H.
(2005). Characterization of Highly Pathogenic H5N1 Avian Influenza A Viruses Isolated from South Korea. J. Virol.
79: 3692-3702
[Abstract] [Full Text] -
de Jong, M. D., Cam, B. V., Qui, P. T., Hien, V. M., Thanh, T. T., Hue, N. B., Beld, M., Phuong, L. T., Khanh, T. H., Chau, N. V. V., Hien, T. T., Ha, D. Q., Farrar, J.
(2005). Fatal Avian Influenza A (H5N1) in a Child Presenting with Diarrhea Followed by Coma. NEJM
352: 686-691
[Abstract] [Full Text] -
Govorkova, E. A., Rehg, J. E., Krauss, S., Yen, H.-L., Guan, Y., Peiris, M., Nguyen, T. D., Hanh, T. H., Puthavathana, P., Long, H. T., Buranathai, C., Lim, W., Webster, R. G., Hoffmann, E.
(2005). Lethality to Ferrets of H5N1 Influenza Viruses Isolated from Humans and Poultry in 2004. J. Virol.
79: 2191-2198
[Abstract] [Full Text] -
Puthavathana, P., Auewarakul, P., Charoenying, P. C., Sangsiriwut, K., Pooruk, P., Boonnak, K., Khanyok, R., Thawachsupa, P., Kijphati, R., Sawanpanyalert, P.
(2005). Molecular characterization of the complete genome of human influenza H5N1 virus isolates from Thailand. J. Gen. Virol.
86: 423-433
[Abstract] [Full Text] -
Karasin, A. I., West, K., Carman, S., Olsen, C. W.
(2004). Characterization of Avian H3N3 and H1N1 Influenza A Viruses Isolated from Pigs in Canada. J. Clin. Microbiol.
42: 4349-4354
[Abstract] [Full Text] -
Lipatov, A. S., Govorkova, E. A., Webby, R. J., Ozaki, H., Peiris, M., Guan, Y., Poon, L., Webster, R. G.
(2004). Influenza: Emergence and Control. J. Virol.
78: 8951-8959
[Full Text] -
Chen, H., Deng, G., Li, Z., Tian, G., Li, Y., Jiao, P., Zhang, L., Liu, Z., Webster, R. G., Yu, K.
(2004). The evolution of H5N1 influenza viruses in ducks in southern China. Proc. Natl. Acad. Sci. USA
101: 10452-10457
[Abstract] [Full Text] -
Shinya, K., Fujii, Y., Ito, H., Ito, T., Kawaoka, Y.
(2004). Characterization of a Neuraminidase-Deficient Influenza A Virus as a Potential Gene Delivery Vector and a Live Vaccine. J. Virol.
78: 3083-3088
[Abstract] [Full Text] -
Tumpey, T. M., Garcia-Sastre, A., Taubenberger, J. K., Palese, P., Swayne, D. E., Basler, C. F.
(2004). Pathogenicity and immunogenicity of influenza viruses with genes from the 1918 pandemic virus. Proc. Natl. Acad. Sci. USA
101: 3166-3171
[Abstract] [Full Text] -
Matsuda, K., Park, C. H., Sunden, Y., Kimura, T., Ochiai, K., Kida, H., Umemura, T.
(2004). The Vagus Nerve is One Route of Transneural Invasion for Intranasally Inoculated Influenza A Virus in Mice. Vet Pathol
41: 101-107
[Abstract] [Full Text] -
Hatta, M., Kawaoka, Y.
(2003). The NB Protein of Influenza B Virus Is Not Necessary for Virus Replication In Vitro. J. Virol.
77: 6050-6054
[Abstract] [Full Text] -
Kuiken, T., Rimmelzwaan, G. F., Van Amerongen, G., Osterhaus, A. D. M. E.
(2003). Pathology of Human Influenza A (H5N1) Virus Infection in Cynomolgus Macaques (Macaca fascicularis). Vet Pathol
40: 304-310
[Abstract] [Full Text] -
Lipatov, A. S., Krauss, S., Guan, Y., Peiris, M., Rehg, J. E., Perez, D. R., Webster, R. G.
(2003). Neurovirulence in Mice of H5N1 Influenza Virus Genotypes Isolated from Hong Kong Poultry in 2001. J. Virol.
77: 3816-3823
[Abstract] [Full Text] -
Tumpey, T. M., Garcia-Sastre, A., Mikulasova, A., Taubenberger, J. K., Swayne, D. E., Palese, P., Basler, C. F.
(2002). Existing antivirals are effective against influenza viruses with genes from the 1918 pandemic virus. Proc. Natl. Acad. Sci. USA
99: 13849-13854
[Abstract] [Full Text] -
Tumpey, T. M., Suarez, D. L., Perkins, L. E. L., Senne, D. A., Lee, J.-g., Lee, Y.-J., Mo, I.-P., Sung, H.-W., Swayne, D. E.
(2002). Characterization of a Highly Pathogenic H5N1 Avian Influenza A Virus Isolated from Duck Meat. J. Virol.
76: 6344-6355
[Abstract] [Full Text] -
Zitzow, L. A., Rowe, T., Morken, T., Shieh, W.-J., Zaki, S., Katz, J. M.
(2002). Pathogenesis of Avian Influenza A (H5N1) Viruses in Ferrets. J. Virol.
76: 4420-4429
[Abstract] [Full Text] -
Govorkova, E. A., Leneva, I. A., Goloubeva, O. G., Bush, K., Webster, R. G.
(2001). Comparison of Efficacies of RWJ-270201, Zanamivir, and Oseltamivir against H5N1, H9N2, and Other Avian Influenza Viruses. Antimicrob. Agents Chemother.
45: 2723-2732
[Abstract] [Full Text] -
Hatta, M., Gao, P., Halfmann, P., Kawaoka, Y.
(2001). Molecular Basis for High Virulence of Hong Kong H5N1 Influenza A Viruses. Science
293: 1840-1842
[Abstract] [Full Text] -
Rimmelzwaan, G. F., Kuiken, T., van Amerongen, G., Bestebroer, T. M., Fouchier, R. A. M., Osterhaus, A. D. M. E.
(2001). Pathogenesis of Influenza A (H5N1) Virus Infection in a Primate Model. J. Virol.
75: 6687-6691
[Abstract] [Full Text] -
Tumpey, T. M., Renshaw, M., Clements, J. D., Katz, J. M.
(2001). Mucosal Delivery of Inactivated Influenza Vaccine Induces B-Cell-Dependent Heterosubtypic Cross-Protection against Lethal Influenza A H5N1 Virus Infection. J. Virol.
75: 5141-5150
[Abstract] [Full Text] -
Massin, P., van der Werf, S., Naffakh, N.
(2001). Residue 627 of PB2 Is a Determinant of Cold Sensitivity in RNA Replication of Avian Influenza Viruses. J. Virol.
75: 5398-5404
[Abstract] [Full Text] -
Yao, Y., Mingay, L. J., McCauley, J. W., Barclay, W. S.
(2001). Sequences in Influenza A Virus PB2 Protein That Determine Productive Infection for an Avian Influenza Virus in Mouse and Human Cell Lines. J. Virol.
75: 5410-5415
[Abstract] [Full Text] -
Brown, E. G., Liu, H., Kit, L. C., Baird, S., Nesrallah, M.
(2001). Pattern of mutation in the genome of influenza A virus on adaptation to increased virulence in the mouse lung: Identification of functional themes. Proc. Natl. Acad. Sci. USA
10.1073/pnas.111165798v1
[Abstract] [Full Text] -
Leneva, I. A., Goloubeva, O., Fenton, R. J., Tisdale, M., Webster, R. G.
(2001). Efficacy of Zanamivir against Avian Influenza A Viruses That Possess Genes Encoding H5N1 Internal Proteins and Are Pathogenic in Mammals. Antimicrob. Agents Chemother.
45: 1216-1224
[Abstract] [Full Text] -
Perkins, L. E. L., Swayne, D. E.
(2001). Pathobiology of A/Chicken/Hong Kong/220/97 (H5N1) Avian Influenza Virus in Seven Gallinaceous Species. Vet Pathol
38: 149-164
[Abstract] [Full Text] -
Horimoto, T., Kawaoka, Y.
(2001). Pandemic Threat Posed by Avian Influenza A Viruses. Clin. Microbiol. Rev.
14: 129-149
[Abstract] [Full Text] -
Katz, J. M., Lu, X., Tumpey, T. M., Smith, C. B., Shaw, M. W., Subbarao, K.
(2000). Molecular Correlates of Influenza A H5N1 Virus Pathogenesis in Mice. J. Virol.
74: 10807-10810
[Abstract] [Full Text] -
O’Neill, E., Krauss, S. L., Riberdy, J. M., Webster, R. G., Woodland, D. L.
(2000). Heterologous protection against lethal A/HongKong/156/97 (H5N1) influenza virus infection in C57BL/6 mice. J. Gen. Virol.
81: 2689-2696
[Abstract] [Full Text] -
Karasin, A. I., Brown, I. H., Carman, S., Olsen, C. W.
(2000). Isolation and Characterization of H4N6 Avian Influenza Viruses from Pigs with Pneumonia in Canada. J. Virol.
74: 9322-9327
[Abstract] [Full Text] -
Nishimura, H., Itamura, S., Iwasaki, T., Kurata, T., Tashiro, M.
(2000). Characterization of human influenza A (H5N1) virus infection in mice: neuro-, pneumo- and adipotropic infection. J. Gen. Virol.
81: 2503-2510
[Abstract] [Full Text] -
Kunzelmann, K., Beesley, A. H., King, N. J., Karupiah, G., Young, J. A., Cook, D. I.
(2000). Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia. Proc. Natl. Acad. Sci. USA
10.1073/pnas.160041997v1
[Abstract] [Full Text] -
Hoffmann, E., Stech, J., Leneva, I., Krauss, S., Scholtissek, C., Chin, P. S., Peiris, M., Shortridge, K. F., Webster, R. G.
(2000). Characterization of the Influenza A Virus Gene Pool in Avian Species in Southern China: Was H6N1 a Derivative or a Precursor of H5N1?. J. Virol.
74: 6309-6315
[Abstract] [Full Text] -
Cauthen, A. N., Swayne, D. E., Schultz-Cherry, S., Perdue, M. L., Suarez, D. L.
(2000). Continued Circulation in China of Highly Pathogenic Avian Influenza Viruses Encoding the Hemagglutinin Gene Associated with the 1997 H5N1 Outbreak in Poultry and Humans. J. Virol.
74: 6592-6599
[Abstract] [Full Text] -
Tumpey, T. M., Lu, X., Morken, T., Zaki, S. R., Katz, J. M.
(2000). Depletion of Lymphocytes and Diminished Cytokine Production in Mice Infected with a Highly Virulent Influenza A (H5N1) Virus Isolated from Humans. J. Virol.
74: 6105-6116
[Abstract] [Full Text] -
Hiromoto, Y., Yamazaki, Y., Fukushima, T., Saito, T., Lindstrom, S. E., Omoe, K., Nerome, R., Lim, W., Sugita, S., Nerome, K.
(2000). Evolutionary characterization of the six internal genes of H5N1 human influenza A virus. J. Gen. Virol.
81: 1293-1303
[Abstract] [Full Text] -
Dybing, J. K., Schultz-Cherry, S., Swayne, D. E., Suarez, D. L., Perdue, M. L.
(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] [Full Text] -
Kato, A., Kiyotani, K., Hasan, M. K., Shioda, T., Sakai, Y., Yoshida, T., Nagai, Y.
(1999). Sendai Virus Gene Start Signals Are Not Equivalent in Reinitiation Capacity: Moderation at the Fusion Protein Gene. J. Virol.
73: 9237-9246
[Abstract] [Full Text] -
Brown, E. G., Liu, H., Kit, L. C., Baird, S., Nesrallah, M.
(2001). Pattern of mutation in the genome of influenza A virus on adaptation to increased virulence in the mouse lung: Identification of functional themes. Proc. Natl. Acad. Sci. USA
98: 6883-6888
[Abstract] [Full Text] -
Kunzelmann, K., Beesley, A. H., King, N. J., Karupiah, G., Young, J. A., Cook, D. I.
(2000). From the Cover: Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia. Proc. Natl. Acad. Sci. USA
97: 10282-10287
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»