1 Laboratory of Comparative Pathology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
2 Laboratory of Microbiology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Correspondence
Takashi Umemura
umemura{at}vetmed.hokudai.ac.jp
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ABSTRACT |
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INTRODUCTION |
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In the 20th century, many influenza epidemics and pandemics have been caused by H1N1, H2N2 and H3N2 viruses. From 1997, H5N1 infections occurred in Asian countries and resulted in high mortality (Centers for Disease Control & Prevention, 2004; Tran et al., 2004
; Yuen et al., 1998
). Although it is thought that these viruses were derived directly from avian species, and person-to-person spreads were rare and inefficient (Claas et al., 1998
; Mounts et al., 1999
). The emergence of H5 viruses in humans has proved that avian influenza viruses can cross the host species barrier and warns of the threat of the pandemic of new human influenza A viruses resulting from the mutation or reassortment of human influenza viruses with co-infecting avian influenza A viruses.
Previously, we have established mouse models in which intranasally inoculated H5 influenza A viruses afferently infected the CNS via transneural routes (Matsuda et al., 2004; Park et al., 2002
; Shinya et al., 1998
, 2000
; Tanaka et al., 2003
). Here, we demonstrate retrograde axonal transport of a neurotropic H5 influenza virus using neurons in a compartmentalized culture system, and evaluate the effects on viral spread in the presence of drugs interfering with microtubules (MTs), microfilaments (MFs) and intermediate filaments (IFs), making comparisons with pseudorabies virus, a neurotropic alphaherpesvirus which can propagate transaxonally in vitro and in vivo (Card et al., 1990
; Enquist et al., 2002
; Field & Hill, 1974
).
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METHODS |
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Pseudorabies virus, also known as Aujeszky's disease virus or suid herpes virus 1, belongs to the subfamily Alphaherpesvirinae of the family Herpesviridae. Pseudorabies virus strain Yamagata-S 81 (PRV) is the first Japanese isolate from piglets (Itakura et al., 1981). This virus was propagated in cloned porcine kidney (CPK) cells.
Neuron culture.
Sensory neurons from the dorsal root ganglia (DRG) of the spinal cord of newborn BALB/c mice (24 days old) were dissociated by incubation with 1 mg collagenase (Sigma-Aldrich) ml1 at 37 °C for 30 min. Dissociated neurons were resuspended at a concentration of 40 000 neurons (about 80 ganglia) per ml in maintenance medium (MM) comprising Eagle's minimal essential medium (Sigma-Aldrich) supplemented with 10 % heat-inactivated fetal bovine serum (Sigma-Aldrich), 50 U penicillin (Invitrogen) ml1 and 50 µg streptomycin (Invitrogen) ml1. Collagen-coated 35 mm dishes with a central 14 mm glass coverslip (Matsunami Glass) were seeded with 100 µl of the cell suspension. Cells were then incubated in MM with 10 µM 5-fluoro-2'-deoxyuridine (Sigma-Aldrich) and 40 nM 2·5S nerve growth factor (Invitrogen) in a 5 % CO2 atmosphere at 37 °C. For the elimination of dividing cells, 6 µM aphidicolin (Sigma-Aldrich) was added to the medium from days 2 to 3 in addition to fluorodeoxyuridine. The medium was changed every 23 days. After 710 days of incubation, when an axonal network of neurons had formed, dishes were used for the experiments. At this time, over 1600 neurons survived in each dish and less than 3 % of all surviving cells were non-neuronal cells, i.e. Schwann cells and fibroblasts which were identified by morphology and immunocytochemistry (data not shown).
Infectious titre in neurons.
To detect the infectious titre of stock viral suspensions in neurons, both AIV and PRV were diluted 10-fold in MM and overlaid on cultured neurons. After 60 h incubation in a 5 % CO2 atmosphere at 37 °C, cells were screened for infectivity by immunofluorescent staining. Infectivity was calculated as 50 % neuronal tissue culture infective dose (NTCID50). Neuronal infectious titres of stock suspensions of AIV were 105·7 NTCID50 ml1, which was 107·5 50 % egg infectious doses per ml, and that of PRV were 107·1 NTCID50 ml1, 107·7 TCID50 ml1 in CPK cells, respectively.
Infection of neuronal cell bodies with AIV.
Cultured neurons in the 14 mm coverslip were inoculated with AIV at an NTCID50 of 102·7 in 100 µl MM and incubated for 1 h at 37 °C. The viral suspension was removed and cells were washed three times with MM, then incubated in MM for 6, 12, 18, 24 and 36 h. At least three samples were examined for each time point. Mock-infected neurons were used as a negative control.
Compartmentalized neuron culture.
The culture dishes for the compartmentalized culture system were prepared as follows (Fig. 1, Campenot, 1977
; Campenot & Martin, 2001
): 35 mm dishes with a central 27 mm glass coverslip (Matsunami Glass) were coated with collagen. Parallel scratches were made by fine needles on the coverslip. A drop of MM containing 0·4 % methyl cellulose was placed on the scratched region. A silicon divider (Shin-Etsu Chemical) with one end coated with a ribbon of silicon high vacuum grease (Dow Corning) was placed on the coverslip so that each chamber was sealed off. The wet surface of the scratched region with the medium prevented the silicon grease from adhering to the collagen, and neurons seeded in the central chamber could extend their axons to the side chambers through the gap between the silicon divider and collagen bed without any exchange of medium between the chambers. No leakage of the medium from the chambers was checked at each change of medium. Samples of resuspended cells (2000 neurons in 50 µl MM) were seeded into the central chamber. Growing axons penetrated under the divider walls and when the total number of axons in both chambers reached 20, each dish was used for viral inoculation. At this time, over 800 neurons survived in each central chamber and less than 3 % of all surviving cells were non-neuronal cells.
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Cytoskeletal interference and viral infection.
Full-grown neurons with axonal networks were prepared on 14 mm coverslips on 35 mm dishes. Neurons pre-treated and recovered, or following treatment with 10 µM nocodazole (NOC; Sigma-Aldrich), 5 µM cytochalasin D (CYD; Sigma-Aldrich) or 4 mM acrylamide (ACR; Sigma-Aldrich), were inoculated with either AIV at an NTCID50 of 103·7 or PRV at an NTCID50 of 104·1 in 100 µl MM and incubated for 1 h at 37 °C (Fig. 2). The inoculum was then removed by washing with MM three times and MM was applied with or without each drug. NOC was used for the disruption of MTs, CYD for the disruption of MFs and ACR for the perturbation of IFs. Stock solutions of NOC (10 mM) in DMSO, CYD (1 mM) in DMSO and ACR (1 M) in double-distilled water were prepared and diluted to a final concentration in MM. The final concentrations were chosen to maximize the effect without any apparent morphological cytotoxicity (data not shown). Controls were treated with an equal volume of DMSO or double-distilled water. On the withdrawal of each drug, cells were washed with MM three times. At 24 or 36 h p.i., infected neurons were fixed for morphological examination. At least three dishes were used for each treatment. Student's t-test was used to assess the statistical significance of differences between the groups (P<0·01).
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RESULTS |
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DISCUSSION |
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The increase in virus antigen-positive neurons in the central chamber of the compartmentalized culture system suggests that AIV as well as PRV spread transsynaptically, since the medium in the chambers contained methyl cellulose for immobilizing viruses and antiserum for neutralizing extracellular-free viruses, and that viruses spreading neuron-to-neuron via synapses are not effectively neutralized by antibodies (Price et al., 1982). The machinery of the transsynaptic spread of influenza virus is unknown. Dotti et al. (1993)
detected the haemagglutinin antigens at the axonal cell surface but failed to find budding virus from neurons infected with a fowl plague virus by immunoelectron microscopy. A possible explanation might be that the influenza viruses spread as the ribonucleoproteins encompassed in the synaptic vesicles, and that the antiserum cannot prevent the transsynaptic spread of the viruses. The rate of increase in the number of antigen-positive neurons clearly differed between AIV and PRV, and the result suggests different modes of neuronal propagation for the two viruses, i.e. AIV was transported more promptly within the axons than PRV, and proliferated in the neurons less effectively. There is a possibility that coexisting non-neuronal cells play a substantial role for the virus spread in our culture, but, from the fact that the number of non-neuronal cells was rather small and infected neurons were rarely associated with them, we concluded that transsynaptic spread is likely to be the major cause of the infection of neurons in the central chamber.
There have been many reports concerning viruscytoskeleton interactions, and MT and associated motor proteins, kinesin and cytoplasmic dynein, play a major role in the transport of internalized viruses to the nucleus (reviewed by Ploubidou & Way, 2001; Sodeik, 2000
). However, no information is available on the mechanism of axonal transport of influenza viruses. NOC is a reversible mitotic inhibitor that binds the fast-growing ends of MTs and prevents monomer addition (Cheung & Terry, 1980
; Lee et al., 1980
). In the present experiments, propagation of PRV was markedly inhibited by NOC treatment. This result was consistent with a previous report that PRV entered neurons by fusing their envelope with the host-cell plasma membrane and their capsid/tegument structure was transported to the nucleus on the MTs by fast axonal transport (Kaelin et al., 2000
; Tomishima et al., 2001
). The degree of propagation of PRV among the neurons recovered from the NOC treatment was equivalent to the untreated control, indicating that a reconstruction of MTs could afford the effective propagation of the virus. In contrast, the effects of MT disruption by NOC treatment were insignificant for the propagation of AIV. Lakadamyali et al. (2003)
reported that the incoming transport of influenza A virus strain X-31 (H3N2) to the perinuclear region depends on MTs and dynein-directed motilities in Chinese hamster ovary cells. Whereas, Arcangeletti et al. (1997)
described independence of influenza virus strain U/73 (H7N1) infection from MT construction in LLC monkey kidney epithelial cells. In the present study, AIV could spread transneurally independent of MTs, whereas the spread of PRV was sensitive to MT inhibition. These results suggest a difference in the mechanism of neuronal spread between these two viruses. However, the enlarged dots of viral antigens in MT-disrupted neurons suggest a significant, but not crucial, effect of MT on the axonal transport of AIV.
MFs and their constituent actin filaments interact with many viruses at various stages throughout their life cycles (reviewed by Cudmore et al., 1997). The most extensively documented viralactin interactions are those of vaccinia virus, which induces the formation of actin tails that launch viral particles from the cell surface on the tips of microvilli toward neighbouring cells (Cudmore et al., 1995
). CYD destabilizes MFs by binding to the fast-growing end of the filaments (Cooper, 1987
). With the CYD treatment, the propagation of both AIV and PRV was not obviously affected. PRV has been reported not to interact with MFs (Kaelin et al., 2000
; Tomishima et al., 2001
), which is consistent with our results. As for influenza virus infection, alternative theories for dependence on actin filaments have been reported. One study emphasized actin-dependent movements of internalized virus during the first step at the cell periphery (Lakadamyali et al., 2003
), and another reported that CYD-induced modifications of MFs did not significantly affect influenza virus production (Arcangeletti et al., 1997
). From our results, the neuronal spread of AIV was not significantly affected by CYD at the concentration we used. We chose a maximum concentration of CYD not causing detachment of neurons from the floor of the dish based on preliminary experiments. However, it is possible that actin filaments were not sufficiently disrupted at this concentration, and that actin filaments functioned normally for viral propagation.
IFs comprise diverse filamentous proteins characteristically 1012 nm wide and their distribution is closely related to cell differentiation (reviewed by Coulombe & Wong, 2004). NFs are the major components of IFs in neurons. IFs have long been believed to simply form static space filling cytoplasmic networks and their interactions with viruses are not well documented. Several viral infections have been shown to require intact IF networks using ACR as an IF disruption agent (Eckert, 1985
), but very little is known about the roles of the IF network in viral infections (Arcangeletti et al., 1997
; Ashok & Atwood, 2003
; Cordo & Candurra, 2003
). The significant suppression of the spread of AIV and PRV in the ACR-treated cultured neurons infers that intact IF constructions are involved in the transmission of the viruses in the neuronal network. To understand the significant difference in the reduction rate of the infected neurons for both viruses by ACR, however, further experiments for virus-IF interactions are required.
Arcangeletti et al. (1997) attributed the dependence of influenza A virus infection on intact IFs to the potential participation of prosomes in viral protein synthesis. Prosomes are ribonucleoprotein particles accompanying untranslated forms of mRNAs (Scherrer & Bey, 1994
). Based on their close association with prosomes (Arcangeletti et al., 1992
; Olink-Coux et al., 1992
), IFs might serve as a network to guide specific prosomes, and possibly mRNAs in the cytoplasm. ACR is a water-soluble vinyl monomer and a well documented neurotoxicant in both humans and laboratory animals (reviewed by LoPachin, 2004
). Axonal swelling containing an abundant NFs and degenerating mitochondria have long been considered hallmark lesions produced by the direct action of ACR at axonal or perikaryal sites. However, recent advances in the characterization of ACR neurotoxicity suggest that the nerve terminal is the primary site of ACR action and that inhibition of neurotransmission and membrane turnover in nerve terminals resulting from the ACR-induced disruption of membrane-fusion processes, contributes significantly to the pathogenesis of the neurological defects (LoPachin, 2004
; LoPachin et al., 2002
). Based on such information, it is conceivable that the disruption of IFs by ACR suppressed the viral spread by either preventing viral assembly and/or transmisson by IFs, inducing prosome dysfunction or interfering with membrane fusion at synapses.
In conclusion, we demonstrated here, for the first time in vitro, the axonal transport of avian influenza virus and MT-independent neuronal propagation of the influenza virus.
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ACKNOWLEDGEMENTS |
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Received 19 October 2004;
accepted 6 January 2005.
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