Department of Virology I1, Department of Pathology2 and Department of Viral Diseases and Vaccine Control3, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan
Author for correspondence: Hidekazu Nishimura. Present address: Virus Research Center, Sendai National Hospital, 2-8-8 Miyagino, Miyagino Ward, Sendai 983-8520, Japan. Fax +81 22 293 1173. e-mail nisimurh{at}sendai.hosp.go.jp
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All of the human isolates of the H5N1 virus possessed haemagglutinin (HA) and neuraminidase genes of avian origin and all of the other genes of one of the human isolates, A/Hong Kong/156/97 (HK156), were characterized as of avian origin (Subbarao et al., 1998 ; Claas et al., 1998
; Suarez et al., 1998
), and the isolated H5N1 viruses were divisible into two groups, represented by HK156 and A/Hong Kong/483/97 (HK483), according to the antigenic and phylogenetic properties of HA (Suarez et al., 1998
; Bender et al., 1999
).
Avian influenza viruses had not been thought to be pathogenic in humans, except for reports of mild cases of conjunctivitis caused by the H7N7 subtype (Webster et al., 1981 ; Kurtz et al., 1996
), although they have caused fatal outbreaks of influenza several times in wild and domestic mammalian species (Hinshaw, 1998
). Clinical features of the 18 human cases in this outbreak of H5N1 influenza varied from mild illness to severe illness, including six cases of death (Yuen et al., 1998
), although, epidemiologically, the number of non-apparent cases in the community during the outbreak was not available. Both the apparently high case-fatality rate among the confirmed cases and the novel antigenicity of the emerged viruses for humans suggested a great threat of a large-scale outbreak of highly virulent influenza in humans. In response to this, trials to develop vaccines against H5N1 influenza virus were commenced at several places worldwide, including our laboratory. We employed the technique of reverse genetics and made a recombinant virus as a candidate strain for vaccine manufacture: we modified the HA gene of HK156 so that its deduced HA molecule lacked the basic amino acid array at its protease-cleavage site and introduced the gene into an avirulent, avian influenza A (H3N1) virus (unpublished results).
In assessing safety issues and evaluating the potency of the recombinant as the candidate vaccine strain, we needed some proper animal model with which infection experiments with the recombinant and wild strains and immunization experiments with the candidate vaccine could be performed. HK156 and HK483 caused highly lethal, systemic infection in chickens and pathological features in chickens infected with HK156 have been well studied (Suarez et al., 1998 ). Mice have proved to be a good mammalian model for human H1N1, H2N2 and H3N2 influenza, since the pathology of the lungs of infected mice was similar to that of severe cases of influenza in humans. Mice were therefore used for the H5N1 influenza viruses as well, and it was revealed that some human isolates of H5N1 influenza virus were highly virulent to mice without any adaptation step. Consequently, the mouse model was used in challenge and protection experiments with the H5N1 viruses and candidate vaccines (Li et al., 1999
; Lu et al., 1999
; Takada et al., 1999
). In the H5N1 virusmouse models, live virus and/or viral antigen were recovered in some organs of infected mice, suggesting that the infection was pantropic (Gubareva et al., 1998
; Shortridge et al., 1998
; Gao et al., 1999
; Lu et al., 1999
).
Histopathological analyses of organs are essential in discussing the pathogenicity of infectious agents in vivo, especially when the possibility of pantropic infection is suspected. It has been reported that HK483 caused pneumonia, encephalitis and degeneration of cardiomyofibres (Gao et al., 1999 ; Lu et al., 1999
) and that HK156 caused severe respiratory tract lesions (Dybing et al., 2000
) in BALB/c mice. Nevertheless, information on other organs and from a viewpoint of comparison of pathological features among virus strains, based on histopathological analysis, is not fully available to date.
In this study, we performed infection experiments in mice with two H5N1 influenza viruses, HK156 and HK483, which were isolated from deceased patients in the outbreak and represent the two distinguishable antigenic/phylogenic groups among the isolated H5N1 viruses, and attempted the detailed pathological and virological investigation of infected mice.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HK483 was passed three or four times in MDCK cells and HK156 was passed once in the allantoic cavity of embryonated hens egg and twice in MDCK cells before the final propagation step for seed preparation was performed in MDCK cells. Egg-grown A/PR/8/34 (H1N1) virus seed was kindly provided from Dr Tamura of the Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.
Infection and virus titration.
For infection, mice were anaesthetized with pentobarbital and the virus suspension was dropped into the nostrils. The volume and infectivity titre of the inoculum were set at 20 µl (10 µl per nostril) and 106 p.f.u. in MDCK cells throughout this study unless mentioned otherwise. For virus titration of organs and blood of infected mice, each organ was excised and washed three times in PBS pH 7·2. Blood was drawn and left at room temperature for more than 30 min. Organs and blood were kept at -80 °C without thawing until the titration was commenced. Each organ or blood sample (clot and serum) was ground with PBS to make a 10% (w/v) homogenate. The homogenate was cleared by centrifugation at 3000 r.p.m. for 20 min and the supernatant was subjected to plaque assay using MDCK cells in the presence of 10 µg/ml acetylated trypsin.
Counting and profiling of leukocytes.
Blood drawn from mice into a haematocrit capillary treated with heparin (Terumo, Tokyo, Japan) was collected and mixed with Türk solution and leukocytes were counted on a Neubauer haematocytometer. For profiling of the leukocyte population, blood was smeared on a glass slide, stained with MayGiemsa solution and examined under a microscope.
Pathology and immunohistochemistry.
Organs were fixed in 4% (v/v) formaldehyde in PBS and embedded in paraffin. Serial sections were prepared and stained with haematoxylin and eosin (H&E) solution for light microscopy or subjected to immunohistochemical staining with an antiserum to the nucleoprotein (NP) of influenza A/PR/8/34 virus. The specificity of the anti-NP antibody has been confirmed elsewhere (Iwasaki et al., 1999 ) and the reactivity of the antibody to H5N1 influenza viruses was confirmed by Western blotting analysis (data not shown). The staining was performed by the avidinstreptavidinperoxidase method using diaminobenzidine as the substrate.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mice killed by HK156 in this assay died between day 4 and 13 post-inoculation (p.i.) and those killed by HK483 died between day 5 and 8 p.i., but there was no significant difference between the viruses in the mean time to death at each virus load (data not shown). Severe emaciation and prostration were observed in most mice that subsequently died.
Lymphopenia and/or leukopenia were prominent without exception for the severe human cases in the clinical records (Yuen et al., 1998 ). Therefore, blood was drawn from three mice by venipuncture at the orbital plexus before and 4 days after infection with HK483 to compare the leukocyte count and profile in each mouse at the two dates. The leukocyte count decreased drastically in all the three mice (40, 60 and 70% decrease) and the decrease was caused mainly by depletion of lymphocytes. A similar drastic decrease in the ratio of the lymphocyte population was recognized in the blood of mice infected with HK156 at day 4 p.i. (data not shown).
Pathological and virological findings
In order to determine the basis of the high virulence of the viruses, mice were infected with HK156 or HK483 and their organs were subjected to pathological and immunohistochemical analyses and virus titration on days 1, 4 and 7 using sets of three mice each. Mice infected with HK156 and HK483 showed essentially similar pathological features for all of the organs we examined, except that a quantitative difference was recognized in the brain lesion. Macroscopically, on day 4, lungs of each group showed lobular consolidation; however, no significant change was observed in the heart, liver or kidney (data not shown). Histologically, the pulmonary lesions caused by both viruses were acute bronchopneumonia: desquamation of the bronchial epithelial cells, peribronchial infiltrates and enlargement of the alveolar cells were observed (Fig. 1a). These changes were first noticed on day 1 and became prominent on day 4. The thymus showed involution with depletion of lymphocytes. However, no degeneration/necrosis was observed in the tissue. No degeneration/necrosis was detected in the heart (Fig. 1g
), liver, kidney, spleen, pancreas, gastrointestinal tract or lymph nodes (data not shown). Damage to endothelial cell linings was not recognized in our mouse model, although it was reported as the basis for the pathogenicity in chickens infected with HK156 (Suarez et al., 1998
). The brain showed viral encephalitis on day 4, but the lesions of HK483-infected mice were far more severe than those of HK156-infected mice. Many foci of neuronal degeneration with granulocytic infiltration and with neuronophagia and slight perivascular cuffing were recognized in the brainstem and cerebrum of HK483-infected mice (Fig. 1d
). Even in such mice, however, no marked inflammatory infiltrates were observed in the subarachnoid space.
|
|
|
The titres of brains of HK483-infected mice were more than 100-fold greater than those of HK156-infected mice, consistent with the findings of the immunohistochemical analysis (Table 1). The difference between HK483 and HK156 in the degree of their invasion of the brain was shown more strikingly by an additional infection experiment, in which 105 p.f.u. of virus was inoculated in a smaller volume (1 µl per nostril), with the intention of not allowing the virus to run directly into the lung in order to confine its initial replication site to the nasal cavity. Consequently, the virus was detected on day 4 in the brain of all three HK483-infected mice at high titres (102·4±0·7 p.f.u./ml), whereas the virus was detected in the brain of only one of the HK156-infected mice and the titre was very low (100·8 p.f.u./ml).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The major difference between HK483 and HK156 was in their ability to invade to central nervous system, and this was more prominent in the result of an infection experiment using a smaller inoculum volume (2 µl per mouse), intended to make the infection resemble a natural infection more closely than a larger volume (20 µl per mouse). Either or both lung and brain lesions might be responsible for the lethal outcome in mice. The severity of brain lesions may, however, account for the higher lethality of HK483, since the severity of lesions of the lungs was almost equivalent for the two viruses or even greater for HK156 and no definitive difference was found in the pathological findings on other organs (Table 1).
Gao et al. (1999) separated the H5N1 viruses isolated in Hong Kong into two groups based on the degree of virulence in mice and categorized HK156 and HK483 into the same group of highly virulent strains. However, our results demonstrated great differences in pathogenicity between the two strains: the amount of HK156 virus required to give one LD50 in mice was greater than HK483 virus in our study. Furthermore, it was greater than that reported previously by Gubareva et al. (1998)
and Gao et al. (1999)
but close to that in the report by Lu et al. (1999)
.
The degree of lethality of HK156 might be affected by a single factor or combination of factors in each experiment system such as strains, ages of mice, volumes of inocula or passage histories of virus seeds. We have preliminary observations that, when 7-week-old BALB/c mice were infected with HK156 in 20 and 2 µl inoculum volumes, the amounts of HK156 required to give one LD50 were about 3200 and more than 104·8 p.f.u. (end-point not determined), respectively, which demonstrates the importance of the inoculum volume in this kind of experiment.
The heart showed no pathological change in our study, or showed only slight necrosis at day 7, although the viral antigen was found in myocytes. This was not consistent with an earlier study, where BALB/c mice were used and necrosis of myofibres was described (Gao et al., 1999 ). The difference in the extent of the heart lesion might be attributable to the mouse strains used, since we obtained preliminary results that foci made by HK483 in the hearts of BALB/c mice were larger than those in ddY mice (data not shown).
Viral antigen was detected in adipose tissues, associated with necrotic change of this tissue. Fat necrosis of virus aetiology has been reported for several kinds of viruses: rabies virus in bats and rodents (Baer, 1975 ; Botvinkin et al., 1985
), cytomegalovirus in rats and mice (Bruggeman et al., 1987
; Price et al., 1990
); Ross River virus, coxsackievirus B3 and vaccinia virus in mice (Murphy et al., 1973
; Hashimoto et al., 1985
; Yang et al., 1985
) and Hantaan virus in suckling mice (Kurata et al., 1983
). In the case of rabies virus, the virus was detected in the brown fat of wild species and it was even suggested that the fat might serve as a reservoir tissue for the virus (Baer, 1975
). This is the first report that influenza virus targets adipose tissue in vivo and causes fat necrosis. High titres of virus were recovered in the fat tissue of BALB/c mice as well as ddY mice infected with HK483 and HK156 (data not shown). The pathophysiological significance of this adipotropism remains to be clarified. However, from a practical aspect, one should be reminded that contamination of the virus associated with the fat tissue should be borne in mind when interpreting the results of virus titration of organs of mice infected with the H5N1 viruses. Before this study, we had nearly concluded that HK156 and HK483 caused pantropic infection involving many organs in mice, because the viruses were actually detected in organ homogenates of infected mice in previous reports (Gao et al., 1999
; Lu et al., 1999
). However, it is possible that at least part of the virus detected in the homogenates might be attributed to tiny fragments of adipose tissue that happened to be associated with the excised organs. Careful pathological analyses of tissues are needed in addition to virus titration of organs in order to know whether the viruses are truly pantropic.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bender, C., Hall, H., Huang, J., Klimov, A., Cox, N., Hay, A., Gregory, V., Cameron, K., Lim, W. & Subbarao, K. (1999). Characteristics of the surface proteins of influenza A (H5N1) viruses isolated from humans in 19971998. Virology 254, 115-123.[Medline]
Botvinkin, A. D., Nikiforova, T. A. & Sidorov, G. N. (1985). Experimental rabies in hibernator rodents. Acta Virologica 29, 44-50.[Medline]
Bruggeman, C. A., Bruning, J. H., Grauls, G., van den Bogaard, A. E. J. M. & Bosman, F. (1987). Presence of cytomegalovirus in brown fat. Study in a rat model.Intervirology27, 32-37.[Medline]
Claas, E. C., Osterhaus, A. D., van Beek, R., De Jong, J. C., Rimmelzwaan, G. F., Senne, D. A., Krauss, S., Shortridge, K. F. & Webster, R. G. (1998). Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus.Lancet351, 472-477.[Medline]
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.Journal of Virology74, 1443-1450.
Gao, P., Watanabe, S., Ito, T., Goto, H., Wells, K., McGregor, M., Cooley, A. J. & Kawaoka, Y. (1999). Biological heterogeneity, including systemic replication in mice, of H5N1 influenza A virus isolates from humans in Hong Kong.Journal of Virology73, 3184-3189.
Gubareva, L. V., McCullers, J. A., Bethell, R. C. & Webster, R. G. (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.Journal of Infectious Diseases178, 1592-1596.[Medline]
Hashimoto, I., Tomita, M., Kohara, T., Uchino, I. & Harashima, A. (1985). Steatitis in mice infected with coxsackie virus B3.Microbiology and Immunology29, 371-376.[Medline]
Hinshaw, V. (1998). Influenza in other species (seal, whale and mink). In Textbook of Influenza, pp. 163-167. Edited by K. Nicholson, R. G. Webster & A. Hay. London: Blackwell Science.
Iwasaki, T., Tamura, S., Kumasaka, T., Sato, Y., Hasegawa, H., Asanuma, H., Aizawa, S., Yanagihara, R. & Kurata, T. (1999). Exacerbation of influenza virus pneumonia by intranasal administration of surfactant in a mouse model.Archives of Virology144, 675-685.[Medline]
Kurata, T., Tsai, T. F., Bauer, S. P. & McCormick, J. B. (1983). Immunofluorescence studies of disseminated Hantaan virus infection of suckling mice.Infection and Immunity41, 391-398.[Medline]
Kurtz, J., Manvell, R. J. & Banks, J. (1996). Avian influenza virus isolated from a woman with conjunctivitis.Lancet348, 901-902.[Medline]
Li, S., Liu, C., Klimov, A., Subbarao, K., Perdue, M. L., Mo, D., Ji, Y., Woods, L., Hietala, S. & Bryant, M. (1999). Recombinant influenza A virus vaccines for the pathogenic human A/Hong Kong/97 (H5N1) viruses.Journal of Infectious Diseases179, 1132-1138.[Medline]
Lu, X., Tumpey, T. M., Morken, T., Zaki, S. R., Cox, N. J. & Katz, J. M. (1999). A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans.Journal of Virology73, 5903-5911.
Murphy, F. A., Taylor, W. P., Mims, C. A. & Marshall, I. D. (1973). Pathogenesis of Ross River virus infection in mice. II. Muscle, heart, and brown fat lesions.Journal of Infectious Diseases127, 129-138.[Medline]
Price, P., Eddy, K. S., Papadimitriou, J. M., Robertson, T. A. & Shellam, G. R. (1990). Cytomegalovirus infection of adipose tissues induces steatitis in adult mice.International Journal of Experimental Pathology71, 557-571.[Medline]
Shortridge, K. F., Zhou, N. N., Guan, Y., Gao, P., Ito, T., Kawaoka, Y., Kodihalli, S., Krauss, S., Markwell, D., Murti, K. G., Norwood, M., Senne, D., Sims, L., Takada, A. & Webster, R. G. (1998). Characterization of avian H5N1 influenza viruses from poultry in Hong Kong.Virology252, 331-342.[Medline]
Suarez, D. L., Perdue, M. L., Cox, N., Rowe, T., Bender, C., Huang, J. & Swayne, D. E. (1998). Comparisons of highly virulent H5N1 influenza A viruses isolated from humans and chickens from Hong Kong.Journal of Virology72, 6678-6688.
Subbarao, K., Klimov, A., Katz, J., Regnery, H., Lim, W., Hall, H., Perdue, M., Swayne, D., Bender, C., Huang, J., Hemphill, M., Rowe, T., Shaw, M., Xu, X., Fukuda, K. & Cox, N. (1998). Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness.Science279, 393-396.
Takada, A., Kuboki, N., Okazaki, K., Ninomiya, A., Tanaka, H., Osaki, H., Itamura, S., Nishimura, H., Enami, M., Tashiro, M., Shortridge, K. F. & Kida, H. (1999). Avirulent avian influenza virus as a vaccine strain against a potential human pandemic.Journal of Virology73, 8303-8307.
Webster, R. G., Geraci, J., Petursson, G. & Skirnisson, K. (1981). Conjunctivitis in human beings caused by influenza A virus of seals.New England Journal of Medicine304, 911.[Medline]
Yang, H. Y., Joris, I., Majno, G. & Welsh, R. M. (1985). Necrosis of adipose tissue induced by sequential infections with unrelated viruses.American Journal of Pathology120, 173-177.[Abstract]
Yuen, K. Y., Chan, P. K. S., Peiris, M., Tsang, D. N. C., Que, T. L., Shortridge, K. F., Cheung, P. T., To, W. K., Ho, E. T. F., Sung, R., Cheng, A. F. B. and members of the H5N1 study group (1998). Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus. Lancet 351, 467471.[Medline]
Received 18 February 2000;
accepted 13 June 2000.