Morphogenesis of Human Enterovirus 71: An Electron Microscopy Analysis Coupled with Immunogold Labeling Techniques.
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Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
______________________________________________
Morphogenesis of Human Enterovirus 71: An Electron Microscopy
Analysis Coupled with Immunogold Labeling Techniques.
Cai Yun CHEN, Hui Min ONG and Justin Jang Hann CHU*
Department of Microbiology, Yong Loo Lin School of Medicine, Block MD4, 5 Science Drive2,
National University Health System, National University of Singapore, Singapore 117597.
*Corresponding author email – miccjh@nus.edu.sg or justin_chu@nuhs.edu.sg
Recently, we have seen increasing epidemic outbreak of the hand, foot and mouth disease (HFMD), and majority of these
HFMD cases were caused by Enterovirus 71 (EV71). However, little is currently known about the replication and
morphogenesis of EV71. In this study, we revealed for the first time by transmission electron microscopy analysis coupled
with immungold-labeling technique on the morphogenesis of EV71 within infected human cell lines. The identity as well
as the involvement of the various cellular membranes in the formation of the EV71 replication complexes was revealed
with immunogold-labeling with specific cellular markers. In addition, a comparison between the araldite and London
white resin (LR white) embedding media on EV71-infected cells was also carried out. The latter was found to be suitable
for immunogold labeling but araldite was better in structural preservation of EV71-infected cells.
Keywords: ultrastructural analysis, biological sample embedding techniques, immuno-electron microscopy.
1. Introduction
Human EV71 was first described in 1974 after it was isolated and characterized from cases of neurological disease in
California in 1969 [15]. It has been associated with the childhood disease, hand, foot and mouth disease (HFMD), and
could be associated with neurological diseases, such as aseptic meningitis, poliomyelitis-like paralysis and brainstem
encephalitis [10, 16].
Since EV71 was first isolated, four epidemic outbreaks with high mortality rates had occurred in Bulgaria in 1975,
Hungary in 1978, Malaysia in 1997, Taiwan in 1998 [2, 10, 16]. The most extensive epidemic of EV71 occurred in
Taiwan in 1998 with 405 cases of severe neurological disease and 78 deaths. The deaths were mainly due to
encephalitis [1]. In year 2000, there was another severe outbreak with 80,677 reports of HFMD and 41 deaths in Taiwan
[18]. Epidemics usually occurred in summer and autumn months [7]. In addition, sporadic cases or smaller outbreaks
with some mortality occurred in the United States, Sweden, Japan, China, Hong Kong, Australia and Singapore [7, 13,
17].
EV71 belongs to the Human Enterovirus A species of Enterovirus genus within the family of Picornaviridae, which
also includes poliovirus and coxsackievirus. The virion is a non-enveloped, positive-sense RNA [8]. Studies have
shown that EV71 and Coxsackievirus A16 (CA16) share a close genetic relationship and could both result in HFMD,
but EV71 could also cause severe neurological disease, which is not observed in CA16 infections [8].
The non-enveloped picornaviruses bind to the receptor on the cell surface for the entry into the cell. Upon entering,
the virus undergoes uncoating and the viral genome is released. The replication is initiated by the presence of IRES
(Internal Ribosomal Entry Site). This is followed by the formation of the viral replication complex, which is formed by
binding of the 5’ UTR of the viral genome to the host proteins for replication. Virus replication mainly occurs in the
replication complex within the cytoplasm of the infected cell [6].
With an increase in the number of cases of EV71 infections in the world recently, it is necessary to equip ourselves
with a better understanding of this medically important virus by deciphering the replication process at the ultrastructural
level.
Since the invention of the first transmission electron microscope in early 1929 [4], there has been a great
improvement in the embedding medium used for sample preparation, from the methacrylates employed at the start, to
the use of the araldite and epoxy resin and lastly the re-introduction of the acrylic resin, such as LR white in the 1980s
[12]. The introduction of the various embedding medium had further inspired the development of more techniques that
enabled more detailed data to be produce from the micrograph. One of the important techniques for processing of
biological sample is immunogold labeling, which enables the identification and cellular localization of specific
biological substance of interest. Thus, this study aims to utilize transmission electron microcopy technique to visualize
the replication process of EV71 at the ultrastructural level within EV71-infected human cell lines. In addition, this
study also seeks to compare the effects of araldite and London Resin (LR) white embedding on the ultrastructural
appearance of the EV71-infected cells, and the suitability of the embedding medium for the immunogold labeling
process.
66 ©FORMATEX 2010Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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2. Material and Methods
Cells and virus. The virus used in this study is the human EV71 strain H (VR-1432TM, ATCC) that was isolated
from an adult patient in China [19]. The virus was propagated on Rhabdomyosarcoma cells (RD cells) and harvested
within 24 hours. The harvested viruses were then used to infect RD cells for 6, 8, 12, 16 and 24 hours.
Electron microscopy with araldite as an embedding medium. At the appropriate time, the cell monolayer was
fixed in 2% paraformaldeyde and 3% glutaraldehyde in PBS. After 20 minutes, the fixed cells were scrapped and placed
at 4ºC for 16 hours. It was then washed with PBS, followed by deionised water twice and centrifuged at 3000rpm for 5
minutes. Following the primary fixation, the cells were post-fixed with 1% osmium tetroxide for 2 hours. Few grains of
potassium ferrocyanide were added to enhance contrast of membranous structure. The samples were then washed in
buffer for 5-10 minutes twice at room temperature before dehydration in ascending graded series of ethanol, starting
with 25%, 50%, 75%, 95%, absolute ethanol and lastly two rounds of absolute acetone. The dehydrated cell pellet then
undergoes series of infiltration with increasing ratio of araldite to acetone at increasing temperature before embedding
in fresh araldite for 24 hours at 60ºC. Ultrathin section were cut and trimmed with an ultramicrotome to the size of
approximately 50nm-70nm. Cut sections were picked up onto a 200 mesh copper grid before staining with 2% uranyl
acetate (8 minutes) and post-fixed with lead cirate (8 minutes). The stained section were viewed under the transmission
electron microscope (Phillips EM208) and captured digitally with a Dual view digital camera (Gatan).
Electron microscopy with London Resin white (LR white) as an embedding medium. At the appropriate time,
the cell monolayer was fixed with 4% paraformaldeyde and 0.25% glutaraldehyde in PBS. After 20 minutes, the fixed
cells were scrapped and placed at 4ºC for 16 hours. The cells were then washed with PBS, followed by deionised water
twice and centrifuged at 3000rpm for 5 minutes. The samples were dehydrated in ascending graded series of ethanol at
room temperature, starting with 25%, 50%, 75%, 95% and lastly three rounds of absolute ethanol. The dehydrated cell
pellet then undergoes series of infiltration with increasing ratio of LR white to ethanol at increasing temperature before
embedding in fresh LR white for at least 48 hours at 50ºC. Ultrathin section were cut and trimmed with an
ultramicrotome to the size of approximately 50nm-70nm. Cut sections were picked up onto a 200 mesh copper grid
before staining with 2% uranyl acetate (8 minutes) and post-fixed with lead cirate (8 minutes). The stained section were
viewed under the transmission electron microscope (Phillips EM208) and captured digitally with a Dual view digital
camera (Gatan).
Immunogold labeling. LR white embedding samples were cut and collected on a 200 mesh grid. The grids collected
were washed twice with PBS for 5 minutes. The grids were then placed in the 0.05M glycine diluted in PBS to
inactivate residual aldehyde in the collected sections. This was followed by blocking with 0.2% Bovine Serum Albumin
(BSA, Gibcobul) in PBS to prevent non-specific binding. After that, the grids were once again washed in PBS before
incubated in primary antibody for an hour. It was followed by another stage of washing of the grids in PBS for another
15 minutes before incubating in 10nm gold probe (Ted Pella) for an hour. For double labeling, a secondary antibody
and different sizes gold particles were used. The grids were washed with PBS, and fixed in 2% glutaraldehyde in PBS
(Merck, Germany) for 5 minutes. More washing was done using PBS, followed by deionised water. Lastly, the grids
were stained with uranyl acetate for 10 minutes.
3. Results
In this study, a detailed analysis of the replication process EV71 in human RD cells was performed using transmission
electron microscopy. A time sequence study was first conducted from 6 to 24 hours p.i to analyze the replication
process of EV71 in RD cells.
The basic ultrastructure of the mock-infected RD cell was first analyzed in details (Figure 1). Besides having spindle
shape morphology and multi-nucleated cell, RD cell was unique for the presence of multiple mitochondria of different
shapes (Figure 1B). In addition, the cytoplasm was filled with lysosomes of irregular shapes (Figure 1C).
©FORMATEX 2010 67Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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A B
N
NM C
M
L
L
Figure 1. Mock-infected RD cells. A) The mocked-infected cell has typical nucleus (N), nuclear membrane (NM), mitochondria (M)
and lysosomes (L) morphology. Bar represents 1µm. B) Higher magnification of Figure 1A, showing mitochondria (arrows) of
various shapes (bar represents 0.5µm). C) Higher magnification of Figure 1A, with lysosomes (arrows) of various shapes and sizes
(bar represents 0.5µm).
The earliest major difference between EV71-infected cells and mock-infected cells was observed at the 6 hours post-
infection (Figure 2A). At this time point, the nucleus was still intact, with little nuclear changes observed. However,
more cytoplasmic alterations were identified. The developing EV71 replication active site was characterized by
proliferation of rough endosplasmic reticulum (rER) with the presence of ribosomes lining its peripheral region as well
as the accumulation of large number of mitochondria in close proximity to the developing EV71 replication active site
(Figure 2B). However, the rER were mostly fragmented and enlarged. At this stage, no virus particle was observed.
At 8 hours post-infection, the majority of cells showed similar virus-induced altered features as the 6 hours post-
infected RD cells. Besides the developing viral replication sites observed, small number of smooth, membrane bound
vesicles was seen (Figure 2D). These virus-induced cellular structures were not observed in the mock-infected RD cell.
The virus-induced vesicles were either single membrane-bound or double membrane vesicles.
68 ©FORMATEX 2010Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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A B rER
N
RS
C D
RS M
MV
L
Figure 2. EV71-infected RD cells at 6-8 hours post infection. A) Low magnification of the 6 hours post-infected RD cells, with intact
nucleus (arrow) and minimal cytopathic changes (bar represents 2µm). B) High magnification of the developing replication active site
(RS) surrounded by mitochondria (arrows) and lined by ribosomes (arrowheads, bar represents 0.2µm). C) High magnification of 8
hours post-infected RD cell. Presence of ribosomes (arrows) lining the cytoplasm for the replication active site (RS) was observed
(bar represents 0.2µm). D) High magnification of 8 hours post-infected RD cell. Single membrane-bound vesicles (MV) were
observed, surrounded by several mitochondria (M), and lysosomes (L) (bar represents 0.5µm).
Nuclear changes were first observed at 12 hours post-infection. The nucleus appeared to be irregular in outline and
the nucleus had shrunken (Figure 3A). Furthermore, chromatin condensation (an initial hallmark of apoptosis) was also
observed within the nucleus of the EV71-infected cells. In addition, accumulations of both single and double
membrane-bound vesicles (arrows) were also more prominent as compared to the earlier stage and it remained as one of
the features of the EV71-infected RD cells. Immunogold detection of EV71 specific antigen on these vesicles was also
carried out to substantiate that these membrane-bound vesicles were induced by EV71 (Figure 3B). This was done using
the gold probe, whereby the gold particles would bind to EV71 antibody, which is specific for EV71 viral protein. This
data indeed revealed the presence of EV71 viral antigens on the membrane bound vesicles within the infected cells.
©FORMATEX 2010 69Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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A B
N
Figure 3. EV71-infected RD cells at 12 hours post infection. A) Low magnification of 12 hours post-infected RD cell. The nucleus
(N) had undergone slight fragmentation with prominent number of vesicles (arrows) in the cytoplasm of the infected cells (bar
represents 2µm). B) Immunostaining of EV71- infected RD cell. The gold particles (arrows) were conjugated with protein A, which
binds to the primary antibody specific for EV71 viral antigens (bar represents 0.2µm).
At 16 hours post-infection, the EV71 replication sites were more prominent compared to the early stage, with the
cytoplasm composing almost exclusively of the viral replication site (Figure 4A). Virus particles were also observed for
the first time in the infected cells. The viral replication sites contained developing individual virus particles of 30nm in
size (Figure 4B). There was also a minority of membrane-bound vesicles, which appeared to contain a few virus-like
particles (Figure 4C). In addition, large crystalline aggregates of virus were also observed in the cytoplasm, these
viruses were of larger arrays as compared to those in the replication sites or bounded by vesicles (Figure 4D).
A B
RS
C D
MV
MV
Figure 4. EV71-infected RD cells at 12 hours post infection. A) High magnification of 16 hours post-infected RD cell. The
replication active sites (RS) occupying almost 75% of the cell, with few cytoplasmic organelles remained (bar represents 0.5µm). B)
High magnification of 16 hours-infected RD cell. EV71 particles of 30nm in size (arrows) were observed in the replication site (bar
represents 200nm). C) High magnification of 16 hours post-infected RD cell. The virus particles surrounded by membrane bound
vesicles (MV). Viruses were aligned on the cytoskeleton filaments in RD cells (arrows, bar represents 200nm). D) High
magnification of 16 hours post-infected RD cell. The densely clustered virus was found outside the replication site, as compared to
Figure 4B, which was less densely clustered together (bar represents 0.2µm).
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By 24 hours post-infection, majority of the EV71-infected cells were rounded, with nucleus fragmentation as one of
the hallmarks of apoptosis (Figure 5A). The nuclear changes were more obvious at this stage, with the chromatin
detached from the nuclear membrane (Figure 5B). The condensed chromatin contained light areas of different sizes
scattered irregularly throughout the nucleus and the perinuclear extensions were more distinct. Apoptosis was also
marked by the presence of numerous cytoplasmic vesicles. The once prominent replication sites were no longer
observed at this last phase, with accumulation of virus in crystalline aggregates observed in the cytoplasm or near the
plasma membrane (Figure 5C). Immunogold staining showed gold particles lining the remnants of the cytoplasm, which
were once the replication sites of the virus (Figure 5D).
A B
C D
Figure 5. EV71-infected RD cells at 24 hours post infection. A) Low magnification of the 24 hours post-infected RD cell. Nuclear
fragmentation (arrows) was observed (bar represents 2µm). B) Low magnification of 24 hours post-infected RD cell. Chromatin
detached from the nuclear membrane (arrow), and perinuclear extensions (arrowhead) were observed (bar represents 2µm). C) Low
magnification of 24 hours post-infected RD cell. Virus particles (arrows) were observed to arrange in crystalline array (bar represents
0.5µm). D) Immunogold labeling of RD cell infected for 24 hours. Gold particles (arrows) lined the cytoplasm, which were once the
replication active sites of the virus (bar represents 0.5µm).
With aid of the LR white as an embedding medium and immunogold labeling technique, the time sequence study of
the EV71 infection was well-documented. In addition, this study has also taken another step to compare araldite and
LR white as an embedding medium used in sample analysis.
Araldite is one of the well-established and commonly used embedding media for transmission electron microscopy,
which is heat-stable and able to preserve the cellular tissue well. On the other hand, LR white was introduced only in
the 1980s as a hydrophilic medium, which is useful for immunogold labeling [5]. When comparing the micrograph of
the two different embedding medium, it was observed that the nuclear and cytoplasmic features in araldite embedded
cells were more defined and homogeneous than that in LR white embedded cells. In addition, the staining contrast in
araldite embedded cells was also much better. Mitochondria in the araldite sample were also more defined than that
embedded in LR white sample. In araldite embedded cells, the membranes of the virus-induced structures, including
that of the EV71 replication active sites (Figure 6A) and membrane-bound vesicles were also more distinguishable as
compared to that of the LR white embedded sample (Figure 6B). For the membrane-bound vesicles, in the LR white
embedded sample (Figure 6D), appeared to be smeary and fine details were not as crisp, on the other hand, the
membrane of the vesicles in the araldite embedded cells were more defined (Figure 6C). Even the shape of the virus
was more defined in araldite embedded cells as compared to that of the LR white embedded cells. The virus particles
found within the different cellular regions, in the replication active site or in the cytoplasm was also easier to distinguish
©FORMATEX 2010 71Microscopy: Science, Technology, Applications and Education
A. Méndez-Vilas and J. Díaz (Eds.)
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in araldite embedded cells than LR white embedded cells due to the much defined membranes. In this study, only the
araldite samples were post-fixed with OsO4. The OsO4 is a type of heavy metal that will bind to the lipids. Together
with the viscous araldite, the samples in this medium would have a better contrast and defined structures than LR white
samples. However, araldite would destroy the antigens in cells that were required for immunogold labeling. Therefore,
LR white, which could preserve the antigenicity of cells, was the choice of embedding medium for immunogold
labeling.
A B
RS
RS
M
C D
VP VP
MV MV
M MV
M
MV
VP
MV
E F
VP VP
VP
VP
Figure 6. Comparison between araldite and LR white resin in embedding EV71-infected samples. The EV71 replication active site in
araldite embedded cells (A) had more defined membranes than that in LR white (B). Bars represent 0.5µm. C and D) High
magnification of the membrane bound vesicles (MV) surrounding the virus particles (VP). The membrane in araldite (arrows)
embedded cells (C) was more defined than that in LR white embedded cells (bar represents 0.2µm and 0.5 µm for C and D,
respectively). E and F) Accumulation of virus particles (crystalline aggregates) can be observed within the cytoplasm of infected
cells. The virus particle in araldite embedded cells (E) had a much defined shape as compared to that in LR white (F) embedded cells.
(bars represent 0.5µm)
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4. Discussion
In understanding the exact mechanism for the replication of viruses, the ultrastructural analysis is one of the main
techniques that could be employed. Although there is an increased epidemic outbreak of EV71, the morphogenesis of
EV71 within the infected cell was not well documented. Thus, this study has taken a step to map out the ultrastructural
changes of EV71-infected cells.
The replication active site in the cytoplasm was the main focus of the events that took place in the infected RD cell
and served as the site for replication of viral RNA. It was usually surrounded by the mitochondria, which provided
energy for the replication of the virus (Figure 2B). The rER appeared to be distorted and formed the viral replication
sites. Ribosomes were observed to attach to the rER and outlining the replication site. Early observations showed that
the virus replication active site includes a rosette-like shell of virus-induced double membrane and single membrane-
bound vesicles. These vesicles became more numerous towards the late stage of infection. These vesicles increased the
efficiency of the virus replication. The proportion of single membrane-bound vesicles was much more in EV71-infected
RD cells than double membrane-bound vesicles. The gold particles conjugated with protein A, which bind to the anti-
EV71 antibody, are present in the vesicles (Figure 3B), thus confirmed these vesicles were probably induced and
associated with the virus.
The origin of the membrane vesicles induced by the virus was a debate whether they are derived from ER or Golgi.
Previous studies have argued that at the early stage, it was a reorganization of the membrane of rER, and it was from
Golgi at the later stage of infection [9]. This was followed by the secretory pathways, whereby the vesicles budded off
from the ER before fusing to the cis-Golgi stacks and budding from the trans-Golgi stacks again. Whereas in some other
studies, these vesicles were formed from the rearrangement of the Golgi stacks and Golgi was no longer observed in
infected cells [14]. However, for some, it was believed the vesicles were produced from the arrangement of the rER [3].
From this study, using the immunogold labeling technique, gold particles that bind to the rER protein marker was
observed to associate with the membrane bound vesicles induced by EV71 (data not shown), but not for the gold
particles that bind to the anti-golgi antibody. Therefore, most of the vesicles could be derived from the rearrangement of
the rER that produced the massive proliferation of the membrane-bound vesicles. Since it was a non-enveloped
positive-sense RNA virus, it would interact with the intracellular membranes, in this case, the rER, which resulted in the
disassembly of the cellular organelle to form the vesicles and replication sites.
At the later stage of the EV71 infection, formation of the virus particles occurred in the replication active site.
Apoptotic cells were also observed, with fragmented nucleus and chromatin condensation. The remnants of the
replication active site were observed and surrounded by empty vesicles. This was once again substantiated by
immunogold labeling from this study that gold particles that specifically bind to the EV71-antibody, were found
surrounding these vesicles, which was once the replication active site. Large crystalline array of virus particles were
released from the replication active sites as early as 16 hours of post-infection. The virus were observed to pack into
large clusters and released as bags of crystalline array of virus particles.
The resolution of the virus-induced structures appeared different in the comparison of the two different embedding
media. Generally, the resolution of the ultrastuctures in the araldite was much better than that of the LR white. This
could be explained by the mechanism of interaction by the different medium. Araldite, a form of epoxy resin, covalently
bond with the cells including the antigenic protein and nucleic acid, thus, minimal shrinkage was shown and sample
will be well preserved [5]. Better preservation was coupled by the use of glutaraldehyde as a fixative. Glutaraldehyde, a
potent protein cross-linker, was able to preserve the sample and prevent shrinkage and distortion of the cells. LR white,
on the other hand, was able to preserve the antigenicity for immunogold labeling. This was due to their ability in
retaining water during polymerization [11]. However, the sample will be less preserved and the ultrastructures of cells
can be distorted and resolution was much lower. However, being able to preserve the antigenicity, enable LR white to
be use as an embedding medium for immunogold labeling. Thus, the samples for immunogold labeling were processed
with LR white in this study.
Together with immunogold labeling, the transmission electron microscope analysis on the replication of the EV71 at
the ultrastructural level has further improved our understanding on the replication cycle of this infectious virus, and its
interaction with the cellular organelles in the cells. By understanding the replication process of the EV71 will enable
subsequent development of specific antiviral strategies against this medically important viral pathogen.
Acknowledgements: This project is funded by Dr Justin Chu’s Academic Research Fund (MOE) - Lee Kuan Yew Fellowship
(R182-000-117-112), DSTA-DIRP Grant (POD0713895), NMRC Grant (NMRC/NIG/0012/2007) and NUS Cross Faculty Grant
(R182-000-133-646).
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A. Méndez-Vilas and J. Díaz (Eds.)
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References
[1] Blomberg J, Lycke E, Ahlfors K, Johnsson T, Wolontis S, and von Zeipel G. Letter: New enterovirus type associated with
epidemic of aseptic meningitis and-or hand, foot, and mouth disease. Lancet. 1974; 2(7872): 112.
[2] Chan LG, Parashar UD, Lye MS, Ong FG, Zaki SR, Alexander JP, Ho KK, Han LL, Pallansch MA, Suleiman AB, Jegathesan M
and Anderson LJ. Deaths of Children during an Outbreak of Hand, Foot, and Mouth Disease in Sarawak, Malaysia: Clinical and
Pathological Characteristics of the Disease. Clin Infect Dis. 2000; 31: 678-683.
[3] Cho MW, Teterina N, Egger D and Bienz K. Membrane Rearrangement and Vesicle Induction by recombinant Poliovirus 2C and
2BC in Human Cells. Virology. 1994; 202: 129-146.
[4] Curry A, Appleton H and Dowsett B. "Application of transmission electron microscopy to the clinical study of viral and bacterial
infections: Present and future." Micron. 2006; 37(2): 91-106.
[5] Glauert AM and Glauert RH. Araldite as an Embedding Medium for Electron Microscopy. Miscrosc Anal. 1957; 25:15-20.
[6] Whitton JL, Cornell CT, and Feuer R. Host and virus determinants of picornavirus pathogenesis and tropism. Nat Rev Microbiol.
2005; 3(10): 765–776.
[7] McMinn PC. An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiology
Reviews. 2002; 26:91-107.
[8] McMinn P, Stratov I, Nagarajan L, and Davis S. Neurological manifestation of enterovirus 71 infection in children during an
outbreak of hand, foot, and mouth diseases in Western Australia. Clin Infect Dis. 2001; 32:236-242.
[9] Monaghan P, Cook H, Jackson T and Ryan M and Wileman T. The ultrastructure of the developing replication site in foot-and-
mouth disease virus-infected BHK-38 cells. J Gen Virol. 2004; 85(4): 933-946.
[10] Nagy G, Takatsy S, Kulan E, Mihaly I, and Domokl I. Virological diagnosis of enterovirus type 71 infections: experience gained
during the epidemic of acute CNS diseases in Hungary in 1978. Arch Virol. 1982; 71:217-227.
[11] Newman GR and Hobot JA. Modern Acrylics for Post-embeding immunostaining Techniques. The Journal of Histchemistry and
Cytochemistry. 1987; 35:971-981.
[12] Newman GR and Hobot JA. Resin for combined light and electron microscopy: A half century of development. The
Histochemical Journal. 1999; 31:495-505.
[13] Tu PV, Thao NT, Perera D, Huu TK, Tien NT, Thuong TC, How OM, Cardosa MJ, McMinn PC. Epidemiologic and Virologic
Investigation of Hand, Foot, and Mouth Disease, Southern Vietnam, 2005. Emerg Infect Dis. 2007; 3(11): 1733-1741.
[14] Schlegel A, Giddings TH Jr, Ladinsky MS and Kiregaard K. Cellular Origin and Ultrastructure of Membranes Induced during
Poliovirus Infection. J Virol. 1996; 7(10): 6576–6588.
[15] Schmidt NJ, Lennette EH and Ho HH. An apparently new enterovirus isolated from patients with disease of the central nervous
system. J Infect Dis. 1974; 129:304-309.
[16] Shindarov LM, Chumakov MP, Voroshilov MK, Bojinov S, Vasilenko SM, Iordanov I, Kirov ID, Kamenov E, Leshchinskaya
EV, Mitov G, Robinson IA, Sivchev S, Staikov S. Epidemiological, clinical, and pathomorphological characteristics of epidemic
poliomyelitis-like disease caused by enterovirus 71. J Hyg Epidemiol Microbiol Immunol. 1979; 23:284-295.
[17] Tagaya I and Tachibana K. Epidemic of hand, foot and mouth disease in Japan, 1972-1973: Difference in epidemiologic and
virologic features from the previous one. Jpn J Med Sci Biol. 1975; 28(4): 231–234.
[18] Wang JR, Tuan YC, Tsai HP, Yan JJ, Liu CC and Su IJ. Change of Major Genotype of Enterovirus 71 in Outbreaks of Hand-
Foot-and-Mouth Disease in Taiwan between 1998 and 2000. J Clin Microbiol. 2002; 4(1): 10–15.
[19] Zheng ZM, He PJ, Cayeffield D, Neumann M, Specter S, Baker CC and Bamkowski MJ. Enterovirus 71 isolated from China is
serologically similar to the prototype EV71 BrCr strain but differs in the 5’-noncoding region. J Med Virol. 1995; 47:161-167.
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