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Comparative proteome analyses of host protein expression in


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Comparative proteome analyses of host protein expression in response to Enterovirus 71 and Coxsackievirus A16 infections
Jia Jun Lee a , Joey Bing Kai Seah b , Vincent Tak Kwong Chow d , Chit Laa Poh e , Eng Lee Tan a, b, c,?
a

Department of Paediatrics, University Children's Medical Institute, National University Hospital, Singapore, Singapore School of Chemical and Life Sciences, Singapore Polytechnic, Singapore, Singapore c Centre for Biomedical and Life Sciences, Singapore Polytechnic, Singapore, Singapore d Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore e Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Malaysia
b

AR TIC LE I N FO
Article history: Received 19 February 2011 Accepted 10 May 2011 Available online 19 May 2011 Keywords: Hand, Foot and Mouth Disease Fatal Enterovirus 71 Non fatal Coxsackievirus A16 Comparative proteome analyses

ABS TR ACT
Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are the main etiological agents of Hand, Foot and Mouth Disease (HFMD), a common disease among children and had caused several outbreaks in the Asia-Pacific region. Although being genetically close to each other, EV71 infection can cause serious and fatal neurological complications like encephalitis, myocarditis, acute flaccid paralysis (AFP) and aseptic meningitis, but not in CA16 infections. In this study, the cellular response of host cells infected with EV71 and CA16 was characterized and compared by 2-dimensional proteome analyses. A total of 16 proteins were identified to be differentially expressed in EV71 and CA16-infected host cells. Desmin and HSP27, both indirectly regulate the contraction of muscle cells, were significantly downregulated as a result of EV71 infection, suggesting a link to acute flaccid paralysis. The ability of EV71 to evade host immune system may be due to the downregulation of MHC-I synthesis proteins like protein disulfide isomerase A3 and calreticulin. Proteins such as nucleophosmin, nuclear ribonucleoprotein C, and eukaryotic translation initiation factor 2 were all downregulated significantly, suggesting the rapid shutting down of host translation machinery by EV71. These findings provide insight into the nature of high virulent EV71 infection as compared to CA16. ? 2011 Elsevier B.V. All rights reserved.

1.

Introduction

Hand, Foot and Mouth Disease (HFMD) is a common disease among children and had caused several large epidemics in the Asian-Pacific region [1–6]. The disease is characterized by prolonged fever, poor appetite, vomiting and lethargy, accompanied by development of vesicular exanthem in the mouth, as well as papulovesicular exanthem on the hands, feet and buttocks [7]. HFMD is caused by enteroviruses, which are classified under the Picornaviridae family. The route of

transmission is mainly by fecal–oral route, although transmission via respiratory droplets has been reported in developing countries [8]. The virus is known to be able to survive the acidic pH of the stomach, entering the host through the intestines and spread to other regions of the body via the circulatory system [9]. Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are the main etiological agents for HFMD. The clinical symptoms caused by EV71 and CA16 are similar but EV71 infections are potentially fatal whereas CA16 infections are self-limiting.

? Corresponding author at: Centre for Biomedical and Life Sciences, Singapore Polytechnic, Singapore, Singapore. E-mail addresses: englee@sp.edu.sg, paetel@nus.edu.sg (E.L. Tan). 1874-3919/$ – see front matter ? 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2011.05.022

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Fatalities caused by EV71 are usually found in children less than 5 years old and are often associated with neurological complications like encephalitis, myocarditis, acute flaccid paralysis (AFP) and aseptic meningitis, which are not observed in CA16 infections [10,11]. The reason to the potentially fatal prognosis of EV71 infections but not in CA16 infections is generally unknown, as genetics comparison of EV71 and CA16 genomes showed that they shared a relatively high 77% nucleotide and 89% amino acid homologies [12]. In recent years, focus on proteomic analysis of host cell proteins had become a popular approach to elucidate the interplay of host and viral proteins so as to devise novel ways to combat viral infections [13–16]. Proteome analysis had been used to demonstrate and characterize the cellular proteins in response to bursal disease virus (IBDV) and the membrane proteins of Mycobacterium tuberculosis [14,17]. A comparison between the proteomes of non-virulent Mycobacterium bovis strains and virulent M. tuberculosis strains was also done to identify protein candidates of therapeutic values for further developments [18]. In a study reported by Leong et al. [19], differentially expressed transcripts of EV71-infected RD cells were analyzed by DNA microarrays, providing an overview of the mRNA expression profiles of infected cells. In order to probe into potential cellular proteins involved directly or indirectly in an EV71 viral infection, 2-D proteome analyses were also employed to better understand the host responses to EV71 viral infection [19], since it has been reported that viral infections can lead to post-translational modifications (PTM) such as ubiquitination, phosphorylation and glycosylation without affecting their transcription rates [20–22]. In this study, we characterized and compared the cellular responses to EV71 and CA16 infections. Differential proteomes of cellular proteins, with and without EV71 and CA16 infections, were investigated at different time points, using 2-D PAGE followed by MALDI-TOF identification. The objective is to investigate the underlying reasons for the difference in prognosis between EV71 and CA16 infection by identifying differentially regulated host proteins in the early phase of infection.

Essential Medium supplemented with 10% fetal bovine serum and 2% penicillin–streptomycin at 37 °C with 5% CO2 in a humidified incubator. To carry out the viral infections, RD cells were first seeded at a density of 4 × 105 cells/well in four 6-well plates. After overnight incubation at 37 °C, the cells were infected with either EV71 Strain 41 or CA16 G-10 strain at a MOI of 1. As a negative control, one plate was mock infected with MEM. At 1 h, 2 h and 6 h post infection, one plate of infected cells was harvested by scraping and washing with the wash buffer (Merck, Darmstadt, Germany). Cells were then pelleted by centrifuging at 150 g for 10 min.

2.3.

Protein extraction

Proteins were extracted from the cell pellets with the ProteoExtract Complete Mammalian Proteome Extraction Kit (Merck, Darmstadt, Germany) according to the manufacturer's instruction. In brief, cell pellets were resuspended in ice-cold resuspension buffer and lysed in the extraction buffer. Reducing agents was added to disrupt disulfide bonds and Benzonase? Nuclease was added to degrade DNA and RNA. The solution was then incubated with gentle agitation for 30 min and the protein extract supernatant was collected by centrifuging at 16,000 g for 30 min. The proteins were stored at ? 70 °C until further analysis.

2.4.

Two-dimensional gel electrophoresis

2.
2.1.

Materials and methods
Virus strains

The CA16 strain (CA16-G-10) was kindly provided by Dr. M.A. Pallansch, CDC, Atlanta, GA. The EV71 strain used in this study was the fatal Enterovirus 5865/sin/000009 strain (accession number 316321; designated as Strain 41). This strain was isolated from a fatal HFMD case from a patient during the outbreak in October 2000. Both the viral strains were cultivated in tissue cultures.

The concentrations of the extracted proteins were first measured with the NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, USA). Samples were then diluted to 300 ng with rehydration buffer (Bio-Rad Laboratories, CA, USA), and applied to 11 cm (pH4-7) ReadyStrip IPG Strips (Bio-Rad Laboratories, CA, USA). The strips were first rehydrated for 12 h in a passive mode, and then focused on the Protean IEF cell (Bio-Rad Laboratories, CA, USA) with the following conditions; 250 V for 20 min with linear ramp, 8,000 V for 2? h with linear ramp and finally 8,000 V for 25,000 v-hours with rapid ramping. After equilibration with equilibration buffers (Bio-Rad Laboratories, CA, USA), the gel strips were applied to second-dimensional SDS-PAGE with 12.5% Tris–HCl Criterion gel (Bio-Rad Laboratories, CA, USA) for 65 min at 200 V. The gel was then stained with 0.1% Coomassie Brilliant Blue R-250 (Sigma-Aldrich, St. Louis, USA) for 1 h and subsequently de-stained until background staining was negligible. The gels were captured with a GS-800 calibrated densitometer (Bio-Rad Laboratories, CA, USA). The spot intensities were determined by MELANIE 2D gel analysis software (version 7.05). All the experiments were done in triplicates to ensure reproducibility.

2.5.

In-gel digestion and MALDI-TOF–TOF-MS/MS

2.2.

Cell culture and cell infection

Human Rhabdomyosarcoma (RD) cells (ATCC? catalog no. CCL-136?) were cultured in T75 culture flasks using Minimum

Protein spots of interest were excised from the gel and washed twice with water. The gels were then destained in 100 μl of 50 mM ammonium bicarbonate/50% (v/v) acetonitrile for 5 min. Destaining was repeated two more times followed by addition of 50 μl acetonitrile. After drying down with a vacuum centrifuge, reduction was done by covering the gel in freshly prepared 100 mM ammonium bicarbonate containing 10 mM

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DTT. After incubation at 56 °C for 1 h, 55 mM iodoacetamide (Sigma Aldrich, St Louis, USA) in 100 mM ammonium bicarbonate was used for alkylation. The gel was then incubated at room temperature for 1 h and then washed three times with 100 mM ammonium bicarbonate. The gel was subsequently dehydrated with acetonitrile and dried with a vacuum centrifuge. Digestion was done with 12.5 ng/μl of sequencing grade modified trypsin (Promega, Wisconsin, USA) in 50 mM ammonium bicarbonate and incubated for 30 min at 4 °C. The peptides were extracted three times using 25 μl of 5% formic acid in 50% aqueous acetonitrile. The extract were incubated for 10 min and concentrated by centrifugation. The digested peptides were mixed with freshly prepared matrix solution (10 mg of CHCA in 1 ml of 0.5% TFA and 50% acetonitrile) in a 1:1 (v/v) ratio. The peptides were then analyzed on the ABI 4700 Proteomics Analyzer with TOF/TOF? optics (Applied Biosystems, CA, USA). Peptide tolerance was set at 100 ppm with fixed modification of cysteine carbamidomethyl, variable modification of methionine oxidated and permitted missed cleavage of up to 1. Trypsin cleavage of the protein is at the C-terminal side of KR unless next residue is P. The proteins were identified by searching in the National Center for Biotechnology Information nonredundant (NCBInr) database using MASCOT program (http://www.matrixscience.com). The significance of the change in spot intensities was analyzed by χ2 test with 2 degrees of freedom (α = 0.05).

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3.

Results and discussion

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Here, we studied the comparative proteome analyses of the host cells infected with the more fatal EV71 and the non-fatal CA16 at different time courses of infection. The proteome of EV71 strain 41 (Fig. 1) and CA16 (Fig. 2) infected cells showed significant differences starting from 1 h post infection (hpi). A total of 27 host proteins were identified to be altered to different levels between the two types of infections, but 16 of them were showing highly significant changes. It was observed that generally almost all the host proteins in EV71 infection were downregulated at starting from 1 hpi with a few proteins upregulated at 6 hpi (Table 1). This could be due to the virus shutting down the host translation machinery immediately after cell entry, and later direct the host cell transcription and translation to generate a favorable condition for its replication [23,24].In contrast, host proteins were steadily upregulated in CA16 infection, suggesting that the shutdown of host translation machinery employed by EV71 was more efficient then CA16 (Table 2). Heat shock proteins (HSPs) (spots 1 to 2) were found to be downregulated at 1 hpi and 2 hpi, and then upregulated at 6 hpi in the host cells infected with EV71, whereas in CA16 infection, upregulation was observed as early as 1 hpi. HSPs are one of the early proteins which are produced in response to cellular stress, where they function as chaperones to prevent misfolding of host proteins [25]. In this study, the delay in heat shock response in EV71-infected RD cells may cause more misfolded proteins to be formed, thus disrupting normal cellular function. HSP27 and desmin (spots 2 and 3) were observed to be downregulated at 1 hpi and 2 hpi. It has been reported that a

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Fig. 1 – Proteome of RD cells infected with EV71 strain 41, separated by 2-DE. Infected cells were harvested at 1 h, 2 h and 6 hpi. Protein spot intensities were analyzed with the MELANIE 2D gel analysis software (version 7.05) and their significance in intensity level was determined by χ2 test with 2 degree of freedom (α = 0.05). Sixteen significantly downregulated protein spots were observed as early as 1 hpi with most of them continued on to 6 hpi. These spots were excised for identification using ABI 4700 Proteomics Analyzer with TOF/TOF? optics. NEG denotes proteome of mock infected cells.

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Fig. 2 – Proteome of RD cells infected with CA16, separated by 2-DE. Infected cells were harvested at 1 h, 2 h and 6 hpi. The corresponding spots identified in EV71 infection were analyzed with the MELANIE 2D gel analysis software (version 7.05) and their significance in intensity level was determined by χ2 test with 2 degree of freedom (α = 0.05). Most of the 16 protein spots were found to be continuously upregulated from 1 hpi to 6 hpi. These spots were excised for identification using ABI 4700 Proteomics Analyzer with TOF/TOF? optics. NEG denotes proteome of mock infected cells.

decrease in HSP27 may hinder the association of actin and myosin [26]. The delayed upregulation of HSP27 at 6 hpi supported the previous findings by Leong et al. when RD cells were infected with another fatal EV71 MS/7423/87 strain [19]. Desmin, a structural protein was downregulated from 1 hpi to 6 hpi. Desmin is found in abundance in the Z-line and costameres, and together with actin, are indispensable in sarcomere contraction [27]. The rapid downregulation of both HSP27 and desmin suggested a direct impact on the contractile ability of muscles cells, resulting in loss of muscle tone as observed in cases of acute flaccid paralysis (AFP), a symptom which was observed in serious cases of EV71 infections [1]. On the other hand, in the case of CA16 infections, both HSP27 and desmin were moderately upregulated from 1 hpi, suggesting that the host cells were able to increase the expression of these proteins to prevent the lost of contractile function. Vimentin (spot 4), β-tubulin (spot 5) and β-actin (spot 6) were observed to be upregulated only before 2 hpi in EV71 infection. Upregulating these three proteins may be a strategy used by the virus to maintain the integrity of the host so as to facilitate maturation of its progeny in the early phase of infection [28]. Many viruses including adenovirus and HIV enter host cell in an actin-dependent manner and tubulin may also favor viral genome transport into nucleus via dynein [29]. The subsequent decrease in these proteins suggests EV71 is actively shutting down host protein synthesis whereas continued increase in level was observed in CA16 infection. Another significant protein which showed differential expression was disulfide isomerase A3 (spot 7). Disulfide isomerase A3 is a subunit of the transporter associated with antigen processing (TAP) complex and together with calreticulin (spot 8), are proteins involved in the synthesis and viral peptide presentation of major histocompatibility complex Class I (MHC-I) [30]. Previous studies have suggested that the 3A protein of EV71 may be responsible for the downregulation of these two proteins [31]. The consequence of preventing peptide-MHC-I presentation will deter immune cells from recognizing infected cells and thus unable to clear the infection effectively. This is further supported by the significant downregulation of HSP gp96 (spot 1), in which the reduction compromise the expression of toll-like receptors in the innate immunity [32]. In this study, CA16 infection caused these proteins to be upregulated at 1 hpi to 2 hpi, thus allowing the infected cells to present viral peptide to immune cells and stimulate the adaptive immunity efficiently [30]. Glutathione S-transferase (spot 9), proteins which are responsible for free radical elimination were moderately downregulated as well in EV71 infection, in contrast to the upregulation in the case of CA16 infection. The lost of protection from these potentially dangerous insults increased the cells susceptibility to apoptosis [32]. Rho GDP-dissociation inhibitor 1 (spot 10) and glucose regulated protein 78 (GRP78; spot 11), which have antiapoptotic activity were also observed to be downregulated in EV71 infection. This supported the previous study by Chen et al. [33], who reported that EV71 was able to induce FasL expression and apoptosis in infected T-cell. Although induction of apoptosis may prevent dissemination of viral progeny, it was also a means for virus shedding and not yet triggering inflammation which is not favorable for virus propagation

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Table 1 – Mean change in spot intensities and mass spectrometry data of 16 differentially regulated proteins of EV71-infected RD cells. Spot Mean change (%) 1 hpi 2 hpi 6 hpi Protein name (Homo sapiens)
Heat shock proteins HSP gp96 precursor HSP27 Structural Desmin Vimentin β-Tubulin β-Actin Immune defense Protein disulfide isomerase A3 Calreticulin Free radical protection Glutathione S-transferase Anti-apoptotic Rho GDP-dissociation inhibitor GRP78 precursor Anti-proliferation Stathmin Protein synthesis Nucleophosmin Nuclear ribonucleoprotein C Others Mitochondrial ATP synthase Calumenin

Mascot score

Peptides identified

Sequence coverage (%)
40 33 60 55 66 68 60 65 31 9 30 35 15 33 18 14

Mr (kDa)

Calculated pI

NCBI ID

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

? 41 ? 28 ? 14 21 1 4 ? 11 ? 11 ? 39 ? 20 6 ? 11 ?8 ? 16 ?1 ? 16

? 15 ? 59 ?3 ? 11 ?7 ?5 ? 27 ? 19 ? 33 ? 44 ?4 ? 48 ? 19 ? 40 ? 13 ? 37

13 6 ?9 ? 13 ?8 ?8 ? 22 ? 13 ?3 ?4 ? 15 ?6 5 13 ? 19 8

271 237 799 821 435 418 763 1220 155 90 307 82 53 94 114 55

32 11 33 35 35 29 35 33 8 4 21 8 10 8 10 6

90309 22429 53560 53676 50096 40536 54454 48097 23573 23237 72187 17320 25149 19080 48083 26421

4.73 7.83 5.21 5.06 4.75 5.55 6.78 4.30 5.43 5.02 5.03 5.76 4.72 10.22 4.95 4.49

gi|15010550 gi|662841 gi|55749932 gi|62414289 gi|18088719 gi|15277503 gi|119597640 gi|119604736 gi|4504183 gi|36038 gi|386758 gi|197692339 gi|62913985 gi|119586801 gi|89574029 gi|295848261

Mean change in spot intensities was the difference compared to negative control. Positive values denote upregulation and negative values denote downregulation. χ2 test with a degree of freedom of 2 were used for statistical analysis of the data. Differences in expression level for all proteins were of significance (α = 0.05, p-value >5.991).

[23]. However, it was observed that the host cells infected with CA16 were able to induce upregulation of anti-apoptotic proteins, which is likely a strategy of the host response to discourage viral dissemination. Host cell proliferation was observed to be favored in this study. Stathmin (spot 12), which function to destabilize microtubules and prevent its assembly was observed to be downregulated, reducing microtubules dynamics and favoring its assembly and mitosis [34]. This observation was consistent with the previous study by Leong et al. [19]. Encouraging proliferation may be a strategy similar to poliovirus to program uninfected cells to divide and generate more host cells for rapid multiplication of viral progeny [19,35]. Shutting off the host translation machinery was evident in this study through the downregulation of nucleophosmin (spot 13) and nuclear ribonucleoprotein C (spot 14). Nucleophosmin function to drive ribosomal protein export from the nucleus to the cytosol. Depletion of nucleophosmin thus reduced the availability of functional ribosome for protein translation [36]. Nuclear ribonucleoprotein C stabilizes RNA from degradation and its decreased implies a corresponding decrease in available mRNA [37]. All these proteins were moderately downregulated until 2 hpi and then slightly upregulated at 6 hpi, suggesting that EV71 may have converted host-regulated protein synthesis to a viral-regulated mechanism. EV71 may upregulate nuclear ribonucleoprotein C, which might have binding properties to the 5′UTR of the viral genome, to favor its replication and dissemination as well [38]. The opposite events were seen

with CA16 infection, which further strengthen our explanation that CA16 do not shut down host translation machinery as rapid as EV71, thus allowing the host to have counteractive response to the infection. Lastly, expression of mitochondria ATP synthase (protein spot 15) and calumenin (protein spot 16) was altered as well. A decrease in mitochondria ATP synthase caused the cells to be lack of ATP which may prevent many intracellular signaling and macromolecular synthesis. Although it may block apoptosis, lack of ATP will cause cellular necrosis [39]. Calumenin is a calcium binding protein which regulates SERCA2. Therefore, changes in calumenin level will alter calcium ion homeostasis in RD cells, affecting muscle contraction [40].

4.

Conclusions

In summary, a total of 16 differentially regulated host proteins between EV71 and CA16 infection were identified. Most proteins were downregulated between 1 h to 2 hpi in EV71 infected cells whereas in CA16 infected cells, these proteins were upregulated. The alteration of host protein levels like stathmin and HSP27 by EV71 stain 41 was similar to the previous study by Leong et al. [19]. Furthermore, protein disulfide isomerase A3 and calreticulin were downregulated, preventing MHC-I presentation in infected cells. Observations in this study suggest that the shutdown of host translation machinery and host defense

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Table 2 – Mean change in spot intensities and mass spectrometry data of 16 differentially regulated proteins of CA16 infected RD cells. Spot Mean change (%) 1 hpi 2 hpi 6 hpi Protein name (Homo sapiens)
Heat shock proteins HSP gp96 precursor HSP27 Structural Desmin Vimentin β-Tubulin β-Actin Immune defense Protein disulfide isomerase A3 Calreticulin Free radical protection Glutathione S-transferase Anti-apoptotic Rho GDP-dissociation inhibitor GRP78 precursor Anti-proliferation Stathmin Protein synthesis Nucleophosmin Nuclear ribonucleoprotein C Others Mitochondrial ATP synthase Calumenin

Mascot score

Peptides identified

Sequence coverage (%)
7 30 62 75 61 44 9 30 38 30 14 24 32 10 44 26

Mr (kDa)

Calculated pI

NCBI ID

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

59 6 15 19 59 13 41 33 300 11 39 53 36 25 69 87

40 45 44 30 57 10 56 26 213 48 56 37 37 ?3 70 100

48 ?4 17 13 69 24 ? 17 28 288 50 34 40 30 14 56 83

135 89 654 567 668 368 57 731 137 271 487 152 151 86 518 193

10 10 34 44 40 19 8 15 7 8 14 6 12 6 23 10

90312 22429 53561 53677 50104 42114 54460 48100 19581 23237 72187 17320 29620 33636 48083 37166

4.73 7.83 5.21 5.06 4.75 5.31 6.78 4.30 4.84 5.02 5.03 5.76 4.47 4.99 4.95 4.47

gi|15010550 gi|662841 gi|55749932 gi|62414289 gi|18088719 gi|4501887 gi|119597640 gi|119604736 gi|119595056 gi|36038 gi|386758 gi|197692339 gi|40353734 gi|13937888 gi|89574029 gi|2809324

Mean change in spot intensities was the difference compared to negative control. Positive values denote upregulation and negative values denote downregulation. χ2 test with a degree of freedom of 2 were used for statistical analysis of the data. Differences in expression level for all proteins were of significance (α = 0.05, p-value >5.991).

evasion in EV71 infection was more efficient than CA16. These might be the factors leading to faster onset and higher mortality rate of EV71 infection as compared to CA16 infection. In particular, the downregulation of desmin and HSP27 may be the cause of AFP as seen in some EV71 infected patients. The shutting down of protein translation could be due to the downregulation of nucleophosmin and nuclear ribonucleoprotein C. All these findings provide insight into the nature of highly virulent EV71 as compared to the milder CA16.

Acknowledgments
We thank Dr M.A. Pallansch, CDC, Atlanta, USA, for providing CA16 G-10 strain for this study. We would also like to thank Mrs Phoon Meng Chee from the Department of Microbiology, National University of Singapore for providing the EV71 strain 5865/SIN/00009 as well as her kind support and advice on technical aspects of viral cultures. This research was supported by a grant funded by National Medical Research Council (Grant number R-178-000-176-275).

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