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Flow Cytometric Determination for Dengue Virus-Infected Cells: Its Application for -Dependent Enhancement Study

Kao-Jean Huang*, Yu-Ching Yang*, Yee-Shin Lin*, Hsiao-Sheng Liu*, Trai-Ming Yeh**, Shun-Hua Chen*, Ching-Chuan Liu*** and Huan-Yao Lei*!

*Departments of and , College of , National Cheng Kung University, Tainan, Taiwan **Department of Medical Technology, College of Medicine, National Cheng Kung University, Tainan, Taiwan ***Department of Pediatrics, College of Medicine, National Cheng Kung University, Tainan, Taiwan

Abstract

The theory of antibody-dependent enhancement plays an important role in the dengue virus . However, its molecular mechanism is not clearly studied partially due to lack of a sensitive to determine the dengue virus-infected cells. We developed a flow cytometric assay with anti-dengue antibody intracellular on dengue virus-infected cells. Both anti-E and anti-prM Abs could enhance the dengue virus infection. The anti-prM Ab not only enhanced the dengue virus infected mass, but also increased the dengue virus synthesis within the cells. The effect of anti-prM Ab- mediated enhancement on dengue virus infection is serotype-independent. We concluded that the target cell-based flow with anti-dengue antibody intracellular staining on dengue virus- infected cells is a sensitive assay to detect the dengue virus infected cells and to evaluate the effect of enhancing antibody on dengue virus infection on cell lines or human primary monocytes.

Keywords: Dengue, enhancing antibody, ADE, .

Introduction characterized by abnormalities of haemostasis and increased vascular permeability, which in Dengue is an acute infectious caused some instances results in DSS. DSS is a form of by the dengue virus, of which four serotypes hypovolemic shock that is associated clinically have been identified. It is characterized by with haemoconcentration and frequently leads biphasic fever, headache, pain in various parts to death. We have reported the involvement of of the body, rash, lymphadenopathy and anti-platelet and anti-endothelial cell leukopenia[1,2]. In most cases, the disease of autoantibodies, in addition to immune deviation dengue fever is self-limited. However, there is and over-production in the a risk of progression to dengue haemorrhagic development of DHF[3-6]. An immuno- fever (DHF) or dengue shock syndrome (DSS), has been proposed to explain the especially when cross-infection of different progression of DF to DHF/DSS.[7-8] However, a serotypes occurs. DHF is a severe febrile disease major issue in the DHF/DSS pathogenesis is not

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142 Dengue Bulletin – Vol 29, 2005 Anti-dengue Antibody Staining on Dengue Virus-Infected Cells solved. That is the antibody-dependent Production of anti-dengue virus enhancement theory. Antibody-dependent monoclonal antibody enhancement (ADE) of infection has long been thought to play a central role in the dengue Breeder mice of BALB/c strain were purchased virus infection. The ADE theory has been from either The Jackson Laboratory, Bar Harbor, formulated to explain the finding that the more ME, USA, or Charles River Japan, Inc. (Atsugi, severe manifestations of DHF/DSS seem to Japan). They were maintained in the animal occur predominantly, although not exclusively, facility of the Medical College, National Cheng in children experiencing a second dengue virus Kung University, Tainan, Taiwan. The animals infection that has a different serotype from the were raised and cared for following the first one. It is reasoned that the non-neutralizing guidelines set up by the National Science Council of the Republic of China. Six- to twelve- anti-dengue antibody bound to a dengue virion week-old mice were used in all experiments. would enhance the virus entrance into target Groups of BALB/c mice were inoculated cells via the Fc receptor. However, the intravenously with DENV-2 PL046 (1 x 108 molecular mechanism of ADE has not been PFU)[11]. The mice were sensitized clearly illustrated, and is still a mystery since intraperitoneally 3 or 4 times at two-week Halstead proposed it in the early 1970s[9,10]. interval with 1 x 107 PFU. Before the fusion, The traditional assay for ADE is done by plaque the mouse was boosted intravenously with 1 x assay which is time-consuming and influenced 106 PFU for three days. The splenocytes were by various parameters such as the virus strains, then fused with FO myeloma using 1% PEG as cell type and the multiplicity of infection. In described previously[12]. Several methods this study we reported a sensitive flow including cellular ELISA, immunohistochemical cytometry to detect the dengue virus-infected staining and flow cytometric assay on dengue cells and the ADE effect was evaluated on the virus-infected BHK cells were used to screen target cell-based assay. the . The isotypes of each monoclonal antibody were identified using mouse monoAb ID kit (Zymed Laboratories, Materials and Methods San Francisco, CA). The antigenicity of each clone was further characterized by Western Cells, viruses and reagents blot or immunoprecipitation of dengue- infected C6/36 total cell lysate. Cells of P388D1, K562, 206-35 and BHK were maintained in Dulbecco’s Modified Eagle (DME) Monoclonal antibody biotinylation medium supplemented with 10% fetal bovine serum (FBS). All four dengue virus serotype Various monoclonal antibodies were purified strains were obtained from the Center for from mouse ascites by Montage Prosep-G kit Disease Control (CDC) in Taiwan. Unless (Millipore, Bedford, MA). Purified antibodies otherwise specified, the DENV-2 strain PL046 were dialysed against 0.1 M phosphate buffer was used as the source for most experiments. saline (PBS), pH7.4 and their concentration Viruses were propagated in mosquito C6/36 determined by measuring the absorbance at 280 cell line which was incubated in Eagle’s minimal nm. A 1 mg/ml solution will have an absorbance essential medium containing 2% heat- of 1.4 in a curvet with a 1 cm path length. inactivated FBS at 28 °C for 5 days.[11] The PL046 2 mg/ml antibody solution was reacted with virus can reach a titre of 1x108 PFU/ml by NHS-LC-Biotin (Pierce, Rockford, IL) and then standard quantification methods on BHK cells. incubated for 30 minutes at room temperature.

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Excessive or un-reacted biotin reagents were Results removed by extensively dialysing against PBS. A flow cytometry to detect dengue virus-infected cells Infection and preparation of virus-antibody complexes To study the mechanism of enhancing antibody on dengue virus infection, a flow cytometric The infection was performed by co-incubating analysis can be employed with anti-dengue cells with dengue viruses at the multiplicity of antibody intracellular staining, in which the infection (moi) of 1~10 for 90 min at 37 °C. For percentage of dengue virus-infected cell mass antibody-enhancing experiments, the virus- can be quantitated. Using the monoclonal anti- antibody complexes were prepared by mixing dengue E, NS-1, or core antibody, the intracellular monoclonal antibodies (10 µg/ml) with viruses dengue protein can be stained per cell. Either for 30 minutes at 37 °C before addition to the the adherent BHK cells (Figure 1A) or suspension cell suspension (2 x 105/sample) for infection. K562 cells (Figure 1B), the intracellular staining After infection, cells were washed twice and of the dengue virus envelope, prM, NS-1 or core resuspended in complete medium for further with biotinylated anti-NS1, anti-E, anti- culture. At various time points, cells were assayed prM or anti-core monoclonal antibodies followed for virus infection by flow cytometric analysis, by streptavidin-FITC can be detected. The and the supernatants were assayed for virus proportion of dengue virus-infected cells was production by plaque assay on BHK cells. determined. Forty-one percentage of cells have detectable E protein at moi of 1 at 24 h post- infection on BHK cells. The proportion of cells Flow cytometric detection of dengue expressing NS-1 or prM is equivalent to that of E virus-infected cells protein, but less cells expressed core protein. For K562 cells, a similar observation was found, Various dengue virus-infected cells were twenty-seven percentages, twenty-four, and harvested and fixed with 2% paraformaldehyde twenty-five percentages of cells expressing E, in PBS for 20 minutes on ice. After fixation, NS-1, and prM , respectively. The low they were washed with PBS twice and then percentage of core protein positive cells stained with appropriate amounts of different comparing with that of the E/NS1/prM-expressing biotinylated anti-dengue monoclonal antibodies cells was probable due to the low affinity of the (anti-E, anti-prM, anti-NS1 and anti-Core anti-core antibody used. By testing on antibodies) in the permeabilization buffer (2% -like P388D1 cell lines, we found FBS, 0.1% saponin, 0.1% sodium azide in PBS) that different monoclonal anti-E and prM for 30 minutes on ice. These cells were then antibodies had different effects on the washed with permeabilization buffer twice and enhancement of dengue virus infection. Anti- stained with streptavidin-FITC in the same prM Ab was found to enhance dengue virus- buffer for another 30 minutes. Finally, these cells infected cell mass. Some anti-E antibodies, for were washed again with the permeabilization example 185-10, enhanced the dengue virus- buffer and resuspended in staining buffer infected cell percentage, but anti-E Ab 137-22 (2% FBS, 0.1% sodium azide in PBS) for flow abolished the DENV-2 infection (Figure 2A). cytometric analysis by FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose, The anti-prM Ab 70-21 also enhanced the CA). Flow cytometric data were re-analysed DENV-2 infection. The anti-dengue NS1 or anti- using the software WinMDI version 2.8. core antibodies did not have any effect on

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Figure 1. Flow cytometric assay to quantiate the dengue virus-infected BHK (A) or K562 cells (B) [BHK (A) and K562 (B) cells were infected by dengue virus at moi = 1 and 0.1, respectively. After 24 hours post-infection, dengue virus-infected cells were determined by intracellular antibody staining. The antibodies used included biotinylated anti-dengue monoclonal antibodies (anti-core, anti-prM, anti-E, or anti-NS1 antibody) followed by streptavidin-FITC. The percentage of dengue antigen positive cells was shown. Mock infection was used as the negative control]

A

B

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Figure 2. The enhancement of dengue virus infection by anti-E or anti-prM antibodies [P388D1 cells were infected by dengue virus at the moi = 1 in the presence of monoclonal anti-prM Ab 70-21, anti-E Ab 185-10 and 137-22 (10 µg/ml). The dengue virus infected cells were determined by intracellular antibody staining on flow cytometry. The effect of anti-prM or anti-E Ab on dengue virion production was shown in the bottom. At 24 or 48 hours after dengue virus infection on P388D1 cells, the virus production in the culture supernatant was determined by the standard quantification methods on BHK cells. An asterisk (*) denotes the significant difference from virus control group]

dengue virus infection (data not shown). This comparable to the traditional plaque assay, it flow cytometric assay of dengue virus-infected has the advantages of being simple, fast, cells was compared with the traditional plaque sensitive, time-saving and target cell-based. The assay (Figure 2B). The anti-E Ab 185-10 and flow cytometric assay not only provides the anti-prM Ab 70-21 enhanced the DENV-2 virion information on the percentage of dengue virus- production, but anti-E Ab 137-22 neutralized infected cells, but also the relative amount of the DENV-2 infection at both 24 and 48 h post- synthesized dengue virus protein per cells, infection. whereas the plaque-forming unit determined by the plaque assay is a combination of increased We went further to check the enhancement infected mass and virus replication. by anti-prM Ab on various moi of dengue virus infection. As shown in Figure 3A, the anti-prM Ab enhanced the DENV-2-infected cell mass Antibody-dependent enhancement was observed on moi from 0.001 to 1; the lower of dengue virus infection on the moi, the higher the fold of enhancement. various cells When the moi is high as 10, no enhancement was observed. Furthermore, the mean To further understand the role of antibody in fluorescent intensity of the NS-1 protein per the dengue virus infection, a hybridoma cell line infected cell was also higher in the anti-prM Ab- (206-35) and primary cells were tested. Anti- treated group than non-treated control, prM Ab 70-21 enhanced the dengue virus suggesting that the viral protein synthesis in the infection at the doses of 1~40 µg/ml, the anti- DENV-2-infected cells was also enhanced by E Ab 185-10 enhanced the dengue virus the anti-prM Ab (Figure 3B). Although the flow infection at doses of 10~40 µg/ml (Figure 4A). cytometric method was demonstrated to be The enhancement seems to be dose-

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Figure 3. Enhancement of dengue virus infected cell mass and dengue virus replication [K562 cells were infected by various dose of dengue virus (moi = 0.001-10) in the presence 10 µg/ml of anti-prM Ab for 24 hours, then stained with biotinylated anti-NS1antibody and streptavidin-FITC intracellularly. The percentage of dengue virus-infected cells (A), and the mean fluorescent intensity of NS1 protein within cells (B) were determined. An asterisk (*) denotes P<0.05 from virus control group]

Figure 4. Antibody-dependent enhancement of dengue virus infection on human monocytes [(A). Hybridoma cell line (206-35) were infected with DENV-2 at moi = 10 for 24 h in the presence of various concentration of anti-prM Ab 70-21 and anti-E Ab 185-10, then stained with biotinylated anti-NS1antibody and streptavidin-FITC intracellularly. (B). Human adherent monocytes were infected with dengue virus at the moi of 10 in the presence of 10 µg/ml of anti-prM Ab 70-21 and anti-E Ab 185-10, then stained with biotinylated anti- NS1antibody and streptavidin-FITC intracellularly. An asterisk (*) denotes P<0.05 from virus control group]

dependent. For the human peripheral blood four serotypes of dengue virus by monocytes, anti-prM Ab 70-21 and anti-E Ab immunohistochemical staining of dengue- 185-10 could enhance the DENV-2-infected infected cells (data not shown). This anti-prM CD14 positive cells (Figure 4B). Furthermore, Ab 70-21 could enhance the dengue virus the monoclonal anti-prM Ab 70-21 was derived infection of all four serotypes (Figure 5). Based from DENV-2-infected mice, it cross-reacts with on the above results, we concluded that this

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Figure 5. Enhanced dengue virus infection of four serotypes by anti-prM Ab [P388D1 cells were infected by four different serotypes of dengue virus at the moi of 0.1 in the presence of anti-prM antibody (10 µg/ml) for 24 hours, then stained with biotinylated anti-NS1antibody and streptavidin-FITC intracellularly. An asterisk (*) denotes P<0.05 from virus control group]

target cell-based flow cytometry with anti- DSS. Viral replication plays an important role in dengue antibody intracellular staining in dengue the disease pathogenesis. Severe DHF is virus-infected cells was a sensitive assay to associated with a higher virus load than mild detect the dengue virus infection and could be DF[13]. ADE is a central hypothesis to explain used for the evaluation of antibody-dependent the increased dengue-infected cell mass[9,10]. The enhancement on dengue virus infection. ADE theory proposes that non-neutralizing antibodies from a previous infection would trigger DHF/DSS in secondary infection if it Discussion involves a different serotype of the virus. Newborn babies less than 1 year of age with Dengue disease is an acute infection caused by maternal anti-dengue IgG antibodies were the dengue virus that has four different known to be susceptible to DHF/DSS post serotypes. Most dengue are subclinical primary infection[14,15]. The enhancing antibody or very mild, as manifested by undifferentiated is previously presumed to act against the dengue fever with or without rash that are seen in infants virus envelope protein. Using a sensitive flow and young children. On the other hand, older cytometry for the detection of the dengue virus- children and adults may develop dengue fever infected cells, we reported in this study that (DF). In general, DF is self-limited, but there is anti-prM antibody could also work as an a risk of progressive development to DHF or enhancing antibody. It enhanced the dengue

148 Dengue Bulletin – Vol 29, 2005 Anti-dengue Antibody Staining on Dengue Virus-Infected Cells virion binding to the Fc receptor-bearing cells Dengue virus is icosahedral, ~500 A in resulting in a larger infected cell mass. The diameter[20-22]. It contains three structural dengue virus replication was also enhanced proteins: an anchored capsid consisting of 113 because the mean fluorescent intensity of residues, a pre-membrane consisting of 166 dengue antigen recognized by anti-dengue residues and a glycoprotein consisting of 495 antibody on infected cells from the enhancing residues. There is a maturation step for an antibody-treated group was higher than that of immature prM dengue particle to become a the dengue virus alone group. mature infectious virion. The prM-containing ADE is known as an in vitro observation dengue particle has 60 icosahedrally organized when a non-neutralizing antibody or sub- trimeric spikes on the particle surface. Each neutralization amount of antibody is present in spike consists of three prM:E heterodimers. the culture with the virus. The virus will enter The cleavage of the prM glycoprotein by furin the cells through the Fc receptor and the or a furin-like protease creates an N-terminal replication of the virus in the cells will be pre-peptide 91 amino acid long and an M enhanced. Takada and Kawaoka have reviewed protein 75 residues long. This cleavage leads the possibility that ADE can involve the to the dissociation of prM:E heterodimers and complement C3 fragment receptor, C1q and the formation of E:E homodimers. The its receptor C1qR, in addition to the Fc glycosylated pre-peptide is released from the receptor[16]. Antibody bound to a receptor- maturing particle, altering the accessibility of binding site of the viral protein induces a domain II of the E glycoprotein to fusion. The conformational change, which facilitates infectivity is increased 100-fold in the prM- membrane fusion with HIV. Viral replication via containing tick-borne encephalitis virus after ADE entry to suppress cellular anti-viral furin treatment[23]. The pre-peptide response occurs in Ross River virus[17]. For dengue components of the prM protein in each spike virus infection of Fc receptor-bearing cover the fusion peptide to prevent the E or B cells, ADE is mediated protein from prematurely undergoing primarily by the Fc receptor pathway. conformational changes that trigger fusion in Complement CR and C1qR were not involved endosomal vesicles. The mature virion has a because we used heat-inactivated FCS to culture smooth surface, which is different from the the target cells. Since the receptor for dengue immature virion that had prM-containing virus is not clearly identified, whether anti-prM spikes. Our anti-prM antibody can bind to the antibody binds to dengue virion to the dengue virion produced in the mosquito C6/ conformational change and increase the entry 36 cell line. The immunoprecipitated dengue of dengue virus requires the demonstration of a virion by anti-prM Ab showed E (50 kD) and dengue virus receptor. Although DC-SIGN has prM (20 kD) (unpublished data) by Western been reported as a dengue receptor, no ADE blot, indicating that there are immature prM phenomenon was observed on immature particles in our dengue virus preparation. dendritic cells[18]. The possibility of IL-10 production post dengue virus infection via FcR Currently, we are determining the epitopes signalling to suppress the further dengue virus recognized by our enhancing antibodies. The replication is intriguing. We have found that FcRII possibility that anti-prM Ab binds to a prM- was involved because anti-FcRII Ab blocked the containing particle, causing a conformation dengue virion binding and infection on K562 change that increases the exposure of a fusion cells (unpublished observation). The immune peptide on domain II of E, thereby causing it complex binding to FcRII was reported to to become fusogenic to target cells, and enhance the IL-10 production[19]. We are hence resulting in increased infection, is not currently testing this possibility. excluded.

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References [13] Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S, Endy TP, Raengsakulrach B, Rothman AL, Ennis FA and [1] Bhamarapravati N, Tuchinda P and Nisalak A. Dengue viremia titer, antibody response Boonyapaknavik V. of Thailand pattern, and virus serotype correlate with disease haemorrhagic fever: a study of 100 cases. severity. J Infect Dis. 2000; 181(1): 2-9. Ann Trop Med Parasitol. 1967; 61(4): 500-510. [14] Kliks SC, Nimmanitya S, Nisalak A and Burke DS. [2] Burke DS, Nisalak A, Johnson DE and Scott RM. A Evidence that maternal dengue antibodies are prospective study of dengue infections in Bangkok. important in the development of dengue Am J Trop Med Hyg. 1988; 38(1): 172-180. hemorrhagic fever in infants. Am J Trop Med Hyg. [3] Lin CF, Lei HY, Liu CC, Liu HS, Yeh TM, Wang ST, 1988; 38(2): 411-419. Yang TI, Sheu FC, Kuo CF and Lin YS. Generation [15] Kliks SC, Nisalak A, Brandt WE, Wahl L and Burke of IgM anti-platelet autoantibody in dengue DS. Antibody-dependent enhancement of dengue patients. J Med Virol. 2001; 63(2): 143-149. virus growth in human monocytes as a risk factor [4] Lin CF, Lei HY, Shiau AL, Liu CC, Liu HS, Yeh TM, for dengue hemorrhagic fever. Am J Trop Med Hyg. 1989; 40(4): 444-451. Chen SH and Lin YS. Antibodies from dengue patient sera cross-react with endothelial cells and [16] Takada A and Kawaoka Y. Antibody-dependent induce damage. J Med Virol. 2003; 69(1): 82-90. enhancement of viral infection: molecular mechanisms and in vivo implications. Rev Med [5] Lin CF, Lei HY, Shiau AL, Liu HS, Yeh TM, Chen SH, Virol. 2003; 13(6): 387-398. Liu CC, Chiu SC and Lin YS. Endothelial cell induced by antibodies against dengue [17] Lidbury BA and Mahalingam S. Specific ablation virus nonstructural protein 1 via production of nitric of antiviral gene expression in macrophages by oxide. J Immunol. 2002; 169(2): 657-664. antibody-dependent enhancement of Ross River virus infection. J Virol. 2000; 74(18): 8376-8381. [6] Nguyen TH, Lei HY, Nguyen TL, Lin YS, Huang KJ, Le BL, Lin CF, Yeh TM, Do QH, Vu TQ, Chen LC, [18] Wu SJL, Grouard-Vogel G, Sun W, Mascola JR, Huang JH, Lam TM, Liu CC and Halstead SB. Brachtel E, Putvatana R, Louder MK, Filgueira L, Dengue hemorrhagic fever in infants: a study on Marovich MA, Wong HK, Blauvelt A, Murphy GS, clinical and cytokine profiles. J Infect Dis. 2004; Robb ML, Innes BL, Birx DL, Hayes CG and Frankel 189(2): 221-232. SS. Human skin Langerhans cells are targets of dengue virus infection. Nat Med. 2000; 6(7): 816- [7] Lei HY, Yeh TM, Liu HS, Lin YS, Chen SH and Liu 820. CC. Immunopathogenesis of dengue virus infection. J Biomed Sci. 2001; 8(5): 377-388. [19] Gerber JS and Mosser DM. Reversing lipopolysaccharide toxicity by ligating the [8] Lin YS, Lin CF, Lei HY, Liu HS, Yeh TM, Chen SH macrophage Fcg receptors. J Immunol. 2001; and Liu CC. Antibody-mediated endothelial cell 166(11): 6861-6868. damage via nitric oxide. Curr Pharm Des. 2004; 10(2): 213-221. [20] Kuhn RJ, Zhang W, Rossmann MG, Pletnev SV, Corver J, Lenches E, Jones CT, Mukhopadhyay S, [9] Halstead SB. Observations related to pathogenesis Chipman PR, Strauss EG, Baker TS and Strauss JH. of dengue hemorrhagic fever. VI. Hypotheses and Structure of dengue virus: implications for discussion. Yale J Biol Med. 1970; 42(5): 350-362. flavivirus organization, maturation, and fusion. Cell. 2002; 108(5): 717-725. [10] Halstead SB. Pathogenesis of dengue: challenges to molecular . Science. 1988; 239(4839): [21] Zang Y, Corver J, Chipman PR, Zang W, Pletnev S, 476-481. Sedlak D, Baker TS, Strauss J, Kuhn RJ and Rossmann MG. Structures of immature flavivirus [11] Huang KJ, Li SY, Chen SC, Liu HS, Lin YS, Yeh TM, particles. EMBO Journal. 2003; 22(11): 2604-2613. Liu CC and Lei HY. Manifestation of thrombocytopenia in dengue-2-virus-infected [22] Rey FA, Heinz FX, Mandl C, Kunz C and Harrison mice. J Gen Virol. 2000; 81(9): 2177-2182. S.C. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature. 1995; [12] Lei HY, Dorf ME, and Waltenbaugh C. Regulation 375(6529): 291-298. of immune responses by I-J gene products. II. Presence of both I-Jb and I-Jk suppressor factors in [23] Stadler K, Allison SL, Schalich J and Heinz FX. (nonsuppressor x nonsuppressor) F1 mice. J Exp Proteolytic activation of tick-borne encephalitis Med. 1982; 155(4): 955-967. virus by furin. J Virol. 1997; 71(11): 8475-8481.

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