Virus Research 92 (2003) 89 /98 www.elsevier.com/locate/virusres
Elucidation of Nipah virus morphogenesis and replication using ultrastructural and molecular approaches Cynthia S. Goldsmith a,*, Toni Whistler a, Pierre E. Rollin a, Thomas G. Ksiazek a, Paul A. Rota a, William J. Bellini a, Peter Daszak a,1, K.T. Wong b, Wun-Ju Shieh a, Sherif R. Zaki a a
Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Mailstop G30, 1600 Clifton Road NE, Atlanta, GA 30333, USA b University of Malaysia, Kuala Lumpur, Malaysia Received 8 September 2002; received in revised form 18 November 2002; accepted 18 November 2002
Abstract Nipah virus, which was first recognized during an outbreak of encephalitis with high mortality in Peninsular Malaysia during 1998 /1999, is most closely related to Hendra virus, another emergent paramyxovirus first recognized in Australia in 1994. We have studied the morphologic features of Nipah virus in infected Vero E6 cells and human brain by using standard and immunogold electron microscopy and ultrastructural in situ hybridization. Nipah virions are enveloped particles composed of a tangle of filamentous nucleocapsids and measured as large as 1900 nm in diameter. The nucleocapsids measured up to 1.67 mm in length and had the herringbone structure characteristic for paramyxoviruses. Cellular infection was associated with multinucleation, intracytoplasmic nucleocapsid inclusions (NCIs), and long cytoplasmic tubules. Previously undescribed for other members of the family Paramyxoviridae , infected cells also contained an inclusion formed of reticular structures. Ultrastructural ISH studies suggest these inclusions play an important role in the transcription process. Published by Elsevier Science B.V. Keywords: Nipah virus; Electron microscopy; Paramyxoviridae ; Henipavirus; In situ hybridization; Replication complex; Zoonotic; Encephalitis; Ultrastructure; Immunogold labeling
1. Introduction An increase in cases of acute febrile encephalitis occurred in Peninsular Malaysia between September 1998 and May 1999, with a similar illness being reported in Singapore in March 1999. Most cases occurred in males who had been exposed to pigs, including abattoir workers, and over 100 deaths were reported. The illness was characterized by fever and headache, followed by drowsiness and disorientation; in severe cases, seizures and coma occurred within 24 /48 h. Concurrently, there
* Corresponding author. Tel.: /1-404-639-3306; fax: /1-404-6391377. E-mail address:
[email protected] (C.S. Goldsmith). 1 Current address: Consortium for Conservation Medicine, Lamont-Doherty Earth Observatory, 61 Route 9W, Palisades, NY 10964-8000, USA. 0168-1702/02/$ - see front matter. Published by Elsevier Science B.V. doi:10.1016/S0168-1702(02)00323-4
was an increase in illness among pigs in the same region (Centers for Disease Control and Prevention, 1999a,b). The etiologic agent for both human and swine diseases, now known as Nipah virus, was isolated from cerebrospinal fluid from human patients and identified as belonging to the family Paramyxoviridae (Chua et al., 1999, 2000). Members of the family Paramyxoviridae are nonsegmented, negative-stranded RNA viruses composed of helical nucleocapsids enclosed within an envelope to form roughly spherical, pleomorphic virus particles (Lamb and Kolakofsky, 2001). Within the family Paramyxoviridae , there are two subfamilies, the Paramyxovirinae and Pneumovirinae. The subfamily Paramyxovirinae has been divided into three genera: Rubulavirus (prototype, mumps virus), Respirovirus (prototype, human parainfluenza virus 1), and Morbillivirus (prototype, measles virus). An additional fourth
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genus, Henipavirus, has been proposed recently, with Hendra virus as the prototype virus, and Nipah virus as the second member (Mayo, 2002). Hendra virus was first recognized in 1994 during an outbreak of severe respiratory illness in horses and humans that occurred in Queensland, Australia (Murray et al., 1995). Serologic, molecular, and immunohistochemical methodologies developed to detect Hendra virus were instrumental in the rapid identification of Nipah virus. In this study, we have examined the morphogenesis of Nipah virus in cell culture and in the human central nervous system by using standard thin section, negative stain, and immunogold ultrastructural techniques. An ultrastructural in situ hybridization (ISH) assay to Nipah virus was also developed to study the cellular localization of viral messenger and genomic RNA (genRNA). Although Nipah virus shares many of its morphologic features with other members of the family Paramyxoviridae, the combined ultrastructural and molecular tools used in this study also allowed identification of several novel features of this newly recognized virus.
2. Methods 2.1. Virus isolation and cell infections Nipah virus was isolated at the Department of Medical Microbiology, University Hospital (Kuala Lumpur, Malaysia) and at the Centers for Disease Control and Prevention (CDC; Atlanta, GA), by cocultivation of patient cerebrospinal fluid with Vero E6 cells (Chua et al., 1999, 2000). Virus was grown within the Biosafety Level 4 laboratory at CDC in Vero E6 cells (ATCC CRLI586) with Basal Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 mg/ml streptomycin, and 100 U/ml penicillin (Life Technologies). Harvesting of infected and uninfected cells occurred at 35 and 50 h postinoculation. 2.2. Standard EM Infected and uninfected Vero E6 cells and formalinfixed brain tissues were washed in 0.1 M phosphate buffer, pH 7.3, fixed in buffered 2.5% glutaraldehyde, gamma-irradiated (2 /106 rad) on ice, and stored in phosphate buffer. Specimens were post-fixed in 1% buffered osmium tetroxide, stained in 4% uranyl acetate, dehydrated through a graded series of alcohols and propylene oxide, and embedded in a mixture of Eponsubstitute and Araldite (Mollenhauer, 1964). Thin sections were stained with 4% uranyl acetate and Reynold’s lead citrate. For negative stain preparations, supernatants from infected cultures were gamma-irra-
diated, banded on a 30% sucrose cushion, and concentrated at 100 000 /g for 2 h. Pellets were resuspended in small amounts of dH2O or 2.5% buffered glutaraldehyde. Copper mesh grids coated with formvar and carbon (Electron Microscopy Sciences) were glow-discharged and placed on drops of the specimen for 10 min, then dried, and stained with 2% phosphotungstic acid, pH 5.7. 2.3. Immunogold electron microscopy (IEM) For thin section preparations, infected and uninfected Vero E6 cells were fixed in phosphate-buffered 1.5% paraformaldehyde and 0.025% glutaraldehyde, gammairradiated, dehydrated in alcohols, and embedded in LR White resin (London Resin Company). Formalin-fixed brain tissues were also dehydrated in alcohols and embedded in LR White resin. For negative stain preparations, supernatants from infected and uninfected cells were gamma-irradiated, banded and concentrated, and resuspended in dH2O or 2% buffered paraformaldehyde. For immunogold procedures, all specimens were placed on nickel mesh grids. The wash buffer, also used for dilution of the antibodies, consisted of 0.01 M phosphate-buffered saline (PBS), 1% bovine serum albumin, 0.2% Tween-20, and 0.1% Triton-X. Grids were sequentially placed on drops of 0.05% ovalbumin in PBS, followed by drops of wash buffer containing 1% normal goat serum. Next, specimens were allowed to react with appropriate dilutions of HMAF raised against Hendra virus or Nipah virus; these antibodies are known to cross-react with either virus. After several washes, the specimens were reacted with goat antimouse conjugated to colloidal gold particles (Jackson ImmunoReagents; Electron Microscopy Sciences) diluted in wash buffer with 1% fish gelatin added, and then rinsed several times in wash buffer and then water. Negative stain preparations were stained with 2% phosphotungstic acid, pH 5.7, and sections were stained with 1% osmium tetroxide and 4% uranyl acetate. Uninfected Vero E6 cells were used as negative controls, and an unrelated HMAF was reacted with Nipahinfected cells to check for non-specific staining. All antibodies were pre-absorbed against uninfected Vero E6 cells prior to their use (Goldsmith et al., 1995). 2.4. Ultrastructural in situ hybridization (ISH) Reverse transcription-polymerase chain reaction products from portions of nucleoprotein (N), glycoprotein (G) or fusion protein (F) genes of Nipah virus were cloned into the dual promoter plasmid pCRII (Invitrogen) (Table 1). Riboprobes used for ISH were generated from the linearized plasmid, using the appropriate RNA polymerase and incorporating either digoxigenin-11-
C.S. Goldsmith et al. / Virus Research 92 (2003) 89 /98 Table 1 Riboprobes used for ISH studies of Nipah and Hendra viruses Virus, GenBank accession no., gene
Nucleotide posi- Riboprobe size (nutions cleotides)
Nipah-AF212302 Nucleocapsid Glycoprotein Fusion
1292 /1515 8943 /9333 6654 /6954
223 390 300
Hendra-AFO17149 Nucleocapsid
1292 /1515
223
dUTP or biotin-11-dUTP (RNA Labeling Kit, Roche Molecular Biochemicals). Optimal lengths of riboprobes were determined in pilot experiments and found to be in the range of 200/400 bases. Both positive- and negativesense riboprobes were generated to detect viral genRNA and messenger RNA (mRNA), respectively. The negative-sense riboprobe would also hybridize with the antigenomic RNA template. Nipah virus probes were pooled into positive- or negative-sense cocktails to increase sensitivity. Several pretreatment methodologies were evaluated in order to permeabilize the ribonucleoprotein complex and increase the efficiency of nucleic acid detection. Pretreatment solutions used included 0.5 /5 N NaOH, 1 N HCl, 1 /10% Triton-X 100, Proteinase K (100 mg/ml), 1 /Citra (Biogenix), 0.01 M EDTA, 10% sodium periodate, 1% IGEPAL, 0.005% saponin, and 0.05 M glycine. Sections of LR White-embedded specimens placed on nickel mesh grids were first floated onto drops of 2 / standard saline citrate (SSC: 1/SSC contains 0.15 M sodium chloride, 0.015 M sodium citrate). Grids were then prehybridized at room temperature for 30 min in hybridization buffer (Roche Molecular Biochemicals) containing 50% formamide, washed in 4 /SSC and blotted dry. Hybridization cocktail was prepared by adding riboprobes to hybridization buffer at a final concentration of 1 ng/ml per probe. The mix was denatured at 95 8C for 5 min and immediately transferred onto ice. Sections were allowed to hybridize on drops of cocktail in a moist, closed chamber at 37 8C overnight. The grids were washed in 6 /SSC for 10 min at room temperature, followed by two washes in 2 / SSC containing 50% formamide at 44 8C for 5 and then 20 min. Two final 2 /SSC washes and a water rinse were performed at room temperature. Immunogold detection of the digoxigenin and biotin markers was performed as described above by using either sheep antidigoxigenin or goat anti-biotin conjugated to colloidal gold (Electron Microscopy Sciences). Negative controls for Nipah virus experiments included hybridizations using Nipah virus probes with uninfected and Hendra virus-infected Vero E6 cells.
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Hendra virus probes and an irrelevant riboprobe of similar length, GC content, and concentration as the Nipah virus riboprobe pool were hybridized with Nipah virus-infected cells as additional controls. The specificity of hybridization was also confirmed by application of RNAse just prior to hybridization in some instances. This was done by incubation of the grids in a solution containing 100 mg/ml DNAse-free RNAse (Roche Molecular Biochemicals) in 2/SSC for 1 h in a humid chamber at 37 8C. Sections were then thoroughly rinsed in PBS, post-fixed in 4% paraformaldehyde for 5 min, and equilibrated to 2 /SSC prior to hybridization. 2.5. Multiple labeling For double-label experiments to detect both genRNA and mRNA on a single section, the positive- and negative-sense riboprobes were added sequentially to avoid self-annealing of probes. Therefore, hybridization and washes were performed in two rounds, the first with the genomic riboprobe pool, and then repeated using a differently labeled messenger riboprobe pool. In doublelabel experiments to detect nucleic acid and protein within a single section, ISH was conducted first and the sections were rinsed in distilled water, followed by the HMAF binding steps of the immunogold protocol. Last, to detect riboprobes and HMAF, grids were processed for gold labeling as described above, using an appropriate combination (anti-digoxigenin, anti-biotin, or anti-mouse) of colloidal gold-conjugated antibodies. These antibodies were tagged with varying sizes of colloidal gold (from 5 to 18 nm) to allow for differential localization.
3. Results 3.1. Morphologic characteristics of Nipah virus in cell culture The morphologic features of Nipah virus isolates were similar to those of other members of the family Paramyxoviridae , subfamily Paramyxovirinae. In both thin section and negative stain preparations, extracellular virions appeared as tangled collections of filamentous, helical nucleocapsids surrounded by the viral envelope (Fig. 1A, B). Particles were pleomorphic and varied greatly in size (Fig. 1C), averaging about 500 nm in diameter (range, 180/1900 nm). Negative stain electron microscopy (EM) revealed nucleocapsids with the typical herringbone appearance that is characteristic for paramyxoviruses (Fig. 1D). These measured an average of 21 nm in diameter with a 5 nm periodicity, and up to 1.67 mm in length. In thin section preparations, nucleocapsids averaged 18 nm in diameter, and
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Fig. 1. Morphologic features of Nipah virus particles grown in Vero E6 cells. (A) In thin section preparations, extracellular particles consist of a compact tangle of filamentous nucleocapsids enclosed within the virus envelope. Note that some nucleocapsids are arranged transversely or tangentially along the virus envelope. (B) Negative staining of particles revealed a dense accumulation of filamentous nucleocapsids within the envelope. A few spikes (arrow) are seen along the surface. (C) Aggregation of extracellular particles, with a variety of shapes and sizes. At times, the virus envelope appears detached from the nucleocapsid core (arrow). Long tubules (arrowhead) are also apparent within some particles (see Fig. 5D). Immunogold labeling of negative stain preparation of Nipah virus nucleocapsid, using HMAF and 12 nm colloidal gold. Note the herringbone appearance of the helical nucleocapsid, characteristic for paramyxoviruses. (E) Prominent spikes on the envelope (arrowhead) were seen only occasionally for Nipah virus particles. Bars, 100 nm.
spikes along the viral envelope, when seen, measured 12 nm in length (Fig. 1E). When thin sections of infected Vero E6 cells were examined, the cytopathic effect observed included the
formation of massive multinucleate giant cells (Fig. 2A), vacuolation, and apoptotic features as evidenced by condensed and fragmented nuclei. Within the cytoplasm of infected cells, inclusions were formed by accumula-
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Fig. 2. (A) Multinucleate giant cell with a large NCI (*) measuring almost 25 mm across and filling most of the cytoplasm. (B) Enlargement of area at arrow in A, showing a typical NCI (*) and a separate RI (encompassed by arrowheads) composed of a network of membrane-like structures. Nu, nucleus; RER, rough endoplasmic reticulum. Bars: (A) 1 mm; (B) 100 nm.
tions of viral nucleocapsids surrounded by ill-defined, ‘fuzzy’ electron-dense material. Inclusions could become quite large, at times filling most of the cytoplasm (Fig. 2A). As with other members of the family Paramyxoviridae , nucleocapsids accumulated and became closely aligned along the plasma membrane as particles pre-
pared to bud (Fig. 3A). Viral budding was associated with darkening at the plasma membrane, where nucleocapsids were variously oriented. Occasionally, budding profiles had very organized (fingerprint-like) arrangements of nucleocapsids as they juxtaposed along the viral envelope (Fig. 3B).
Fig. 3. Budding particles. (A) Nipah virus nucleocapsids (arrow) become tightly aligned along the plasma membrane as particle prepares to bud. (B) Rigid positioning of nucleocapsids sometimes formed fingerprint-like structures. Bars, 100 nm.
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Fig. 4. (A) RI composed of a network of open and closed ring-like structures (arrowheads). Note the continuity between these structures and the membranes of the rough endoplasmic reticulum (RER). (B) Uninfected Vero E6 cell, showing a comparable area of RER as is shown in Fig. 4A. Bars, 100 nm. Fig. 5. Long cytoplasmic tubules (arrowhead), at times continuous with the plasma membrane (arrow), were observed in Nipah virus-infected cells. Bar, 100 nm.
Additional noteworthy features were associated with Nipah virus-infected cells. First, an unusual cytoplasmic inclusion was found, composed of a network of membrane-like reticular structures (Fig. 2B, Fig. 4A). These were formed by open and closed ring-like structures, which averaged 38 nm in diameter, and were observed in close proximity to, and at times connected with, rough endoplasmic reticulum membranes. This reticular inclusion (RI) was morphologically distinct from the typical nucleocapsid inclusions (NCIs) and absent from uninfected Vero E6 cells (Fig. 4B), and its viral origin became evident after ISH labeling experiments (see below). Second, also found within the periphery of Nipah virus-infected cells were long cytoplasmic tubules, 30 /45 nm in diameter (Fig. 5). These occasionally were continuous with the plasma membrane or were incorporated into virus particles (Fig. 1C). 3.2. Localization of viral proteins, messenger RNA, and genomic RNA In both thin section and negative stain preparations, immunogold EM (IEM) labeling employed anti-Nipah or anti-Hendra hyperimmune mouse ascitic fluid (HMAF). Negative stain preparations showed gold label
on the naked nucleocapsids (Fig. 1D) and lightly along the fringe of viral glycoproteins. In thin section preparations, label was evident on nucleocapsids in inclusions and within particles (Fig. 6A, B). A lessintense label was also observed over the RIs (Fig. 6A) and the long cytoplasmic tubules. No IEM labeling was observed by reacting anti-Nipah or antiHendra HMAFs with uninfected Vero E6 cells, or by reacting an unrelated HMAF with Nipah virus-infected cells. In contrast, ultrastructural ISH using positive- or negative-sense riboprobes, or a combination of both, revealed localization of genRNA, anti-genomic RNA template, and mRNA almost exclusively over the RIs (Fig. 7A /C). Only rarely was signal associated with the NCIs, despite several attempts to ‘open up’ the RNAprotein complex. ISH label was detected in only some of the virus particles, and in these cases signal was seen in association with the RIs. Results of double-label experiments where both protein and either mRNA or genRNA were detected (IEM/ISH experiments) underscored the finding that viral nucleic acids were found almost exclusively in the RIs, whereas viral antigens were seen in both NCIs and RIs (Fig. 7D).
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Fig. 6. Immunogold labeling, using HMAF. (A) Detection of Nipah virus proteins (12 nm gold) on the NCI (*) and lightly over the RI (arrow) by using immunolabeling. (B) Localization of Nipah virus proteins (18 nm gold) on nucleocapsids within extracellular virus particles. Bars, 100 nm.
The specificity of the ISH reactions was also confirmed by hybridization with control cells and probes. Nipah virus probes did not react with uninfected or Hendra virus-infected Vero E6 cells. No signal was observed when Nipah virus-infected cells were hybridized with unrelated or Hendra virus probes. Finally, pretreatment of Nipah virus-infected cells with RNAse eliminated the reactivity with homologous probes.
3.3. Human brain tissue During ultrastructural examination of central nervous system tissues from fatal cases of Nipah virus infection, both the typical viral NCIs and the RIs were recognized (Fig. 8A, B). Infected cells were generally difficult to locate and were mostly seen in areas surrounding blood vessels of the brain stem. Inclusions were seen within neurons and neuronal processes and occasionally within
Fig. 7. Localization of Nipah virus RNA by using ultrastructural ISH. (A) Low-power magnification of Nipah virus-infected cell with detection of genRNA (6 nm gold) over the RI (arrow) but not within the NCI (*). (B) Detection of mRNA (10 nm gold) over the RI. (C) Colocalization of Nipah virus mRNA (10 nm gold) and genRNA (6 nm gold) over the region of the RI. (D) Detection of Nipah virus genRNA (6 nm gold) in the RI by using ultrastructural ISH, and of Nipah virus proteins (10 nm gold) on the NCI and lightly over the RI by using immunogold labeling. Bars, 100 nm.
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endothelial cells, and were immunogold-labeled by using specific antisera (Fig. 8C). No mature virus particles were identified in the limited number of tissues examined.
4. Discussion When an outbreak of severe encephalitis with a high mortality rate occurred in Malaysia during 1998/1999, Japanese encephalitis virus was initially suspected as the causative agent. Even so, the clinical and epidemiologic features were not consistent with this disease. Ultrastructural observations of a virus isolated from cerebral spinal fluids were instrumental in identifying the etiologic agent as a member of the family Paramyxoviridae . Serologic, molecular, and immunohistochemical approaches indicated it was closely related to, but distinct from, Hendra virus, another recently recognized virus first detected in Australia in 1994 (Chua et al., 1999, 2000; Goldsmith et al., 2000; Wong et al., 2002). Nipah virus-infected Vero E6 cells had numerous ultrastructural features in common with other members of the family Paramyxoviridae , sub-family Paramyxovirinae , including characteristic herringbone-structured
viral nucleocapsids; cytoplasmic NCIs; nucleocapsids aligned at the plasma membrane during the budding process; pleomorphic, extracellular particles; and the formation of multinucleate giant cells (Compans et al., 1966; Raine et al., 1969; Dubois-Dalcq and Reese, 1975; Choppin and Compans, 1975; Hyatt and Selleck, 1996; Hyatt et al., 2001). The typical cytoplasmic NCIs, which measured up to 28 mm in diameter, displayed strong immunogold labeling of nucleocapsids when reacted with HMAF raised against either Nipah or Hendra viruses. Additional structures present in Nipah virusinfected cells, which have also been reported for Hendra virus-infected cells (Hyatt et al., 2001), were long cytoplasmic tubules that were at times continuous with the plasma membrane. Of interest were the highly pleomorphic virions. These measured up to 1900 nm in diameter, which would allow for the inclusion of multiple copies of the viral nucleocapsids within a single particle. Although not a common finding, polyploid virions have been previously reported for other members of the family Paramyxoviridae (Rager et al., 2002). The technique of ultrastructural ISH has become a powerful tool in the EM laboratory (McFadden et al., 1988; Puvion-Dutilleul and Puvion, 1991; Bienz and Egger, 1995). In the current study, ISH techniques aided
Fig. 8. Central nervous system of Nipah virus patients. (A) Thin section electron micrograph of human brain tissue, showing Nipah virus NCI (arrow) within a neuron. (B) Mixed inclusion of virus nucleocapsids (arrow) and reticular membrane-like structures (arrowheads) within a neuronal process. (C) Antigen-positive staining of virus inclusions, using HMAF and 18 nm colloidal gold. Nu, nucleus; RER, rough endoplasmic reticulum. Bars: (A) 1 mm; (B, C) 100 nm.
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in the recognition of a RI (see Fig. 7) and confirmed its viral nature. The RIs were also seen in autopsy tissues from patients infected with Nipah virus, indicating that this feature may play a significant role in virus replication in vivo. This is, to our knowledge, a previously undescribed feature for members of the family Paramyxoviridae . The long-standing model for paramyxoviruses holds that viral genRNA remains encapsidated during mRNA transcription and genome replication (Sedlmeier and Neubert, 1998). Although ultrastructural ISH did not detect genRNA in the nucleocapsids, this result may be due to the tight packaging of the RNA with viral proteins; similar ISH results have been reported for other RNA viruses (Troxler et al., 1992; Grief et al., 1997). Instead, Nipah virus RNAs, along with viral proteins, were detected within the RIs. These findings raise the possibility that for Nipah virus, these structures are central to the transcription process. The RIs may be analogous to the ‘replication complexes’ that have been reported for positive-stranded RNA viruses (see Egger et al., 2000). This term has been used to describe smooth membrane clusters present within positive-stranded RNA virus-infected cells (e.g. vesicular rosettes in poliovirus, double-membrane vesicles in arteriviruses, and vesiculate inclusions in members of the plant virus family Comoviridae; Francki et al., 1985; Pedersen et al., 1999; Teterina et al., 2001). Ultrastructural ISH and RNA protection assay studies have also shown the presence of viral RNA associated with these structures and strongly suggest their integral involvement with virus RNA initiation and replication (Bienz et al., 1992; Egger et al., 1996; Grief et al., 1997). Phylogenetic and serologic data have established that Nipah and Hendra viruses are sufficiently different from other members of the subfamily Paramyxovirinae to warrant a new genus, Henipavirus, to include these two related, recently emergent viruses (Wang et al., 2000; Harcourt et al., 2001; Mayo, 2002). Here, we report ultrastructural features of Nipah virus that are also different from other members of the subfamily Paramyxovirinae . These include RIs, cytoplasmic tubules, a longer length of viral nucleocapsid, and the highly pleomorphic nature of the virus particles. Studies on Nipah and Hendra viruses have emphasized the unique role of traditional EM techniques in the recognition of emerging pathogens and of the insights into virus replication gained when molecular techniques are adapted to ultrastructural studies.
Acknowledgements We are most thankful to the following people: K.B. Chua, for initial isolation of Nipah virus; Brian Harcourt, for primer information, virus purification, and
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review of the manuscript; Larry Anderson and Brian Mahy, for guidance; and Claudia Chesley for editorial review.
References Bienz, K., Egger, D., 1995. Immunocytochemistry and in situ hybridization in the electron microscope: combined application in the study of virus-infected cells. Histochem. Cell Biol. 103, 325 / 338. Bienz, K., Egger, D., Pfister, T., Troxler, M., 1992. Structural and functional characterization of the poliovirus replication complex. J. Virol. 66, 2740 /2747. Centers for Disease Control and Prevention, 1999a. Outbreak of Hendra-like virus */Malaysia and Singapore, 1998 /1999. MMWR Morb. Mortal. Wkly. Rep. 48, 265 /269. Centers for Disease Control and Prevention, 1999b. Update: outbreak of Nipah virus */Malaysia and Singapore, 1999. MMWR Morb. Mortal. Wkly. Rep. 48, 335 /337. Choppin, P.W., Compans, R.W., 1975. Reproduction of Paramyxoviruses. Plenum Press, New York, pp. 95 /178. Chua, K.B., Goh, K.J., Wong, K.T., Kamarulzaman, A., Tan, P.S., Ksiazek, T.G., Zaki, S.R., Paul, G., Lam, S.K., Tan, C.T., 1999. Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet 354, 1257 /1259. Chua, K.B., Bellini, W.J., Rota, P.A., Harcourt, B.H., Tamin, A., Lam, S.K., Ksiazek, T.G., Rollin, P.E., Zaki, S.R., Shieh, W.J., Goldsmith, C.S., Gubler, D.J., Roehrig, J.T., Eaton, B., Gould, A.R., Olson, J., Field, H., Daniels, P., Ling, A.E., Peters, C.J., Anderson, L.J., Mahy, B.W.J., 2000. Nipah virus: a recently emergent deadly paramyxovirus. Science 288, 1432 /1435. Compans, R.W., Holmes, K.V., Dales, S., Choppin, P.W., 1966. An electron microscopic study of moderate and virulent virus-cell interactions of the parainfluenza virus SV5. Virology 30, 411 /426. Dubois-Dalcq, M., Reese, T.S., 1975. Structural changes in the membrane of Vero cells infected with a paramyxovirus. J. Cell Biol. 67, 551 /565. Egger, D., Pasamontes, L., Bolten, R., Boyko, V., Bienz, K., 1996. Reversible dissociation of the poliovirus replication complex: functions and interactions of its components in viral RNA synthesis. J. Virol. 70, 8675 /8683. Egger, D., Teterina, N., Ehrenfeld, E., Bienz, K., 2000. Formation of the poliovirus replication complex requires coupled viral translation, vesicle production, and viral RNA synthesis. J. Virol. 74, 6570 /6580. Francki, R.I.B., Milne, R.G., Hatta, T., 1985. Comovirus group. In: Atlas of Plant Viruses, vol. 2. CRC Press, Boca Raton, FL, pp. 1 / 22. Goldsmith, C.S., Elliott, L.H., Peters, C.J., Zaki, S.R., 1995. Ultrastructural characteristics of Sin Nombre virus, causative agent of hantavirus pulmonary syndrome. Arch. Virol. 140, 2107 /2122. Goldsmith, C.S., Whistler, T., Rollin, P.E., Chua, K.B., Bellini, W.J., Rota, P.A., Wong, K.T., Daszak, P., Ksiazek, T.G., Zaki, S.R., 2000. Ultrastructural studies of Nipah virus, a newly emergent paramyxovirus, using thin section, negative stain, immunogold, and in situ hybridization electron microscopy. Microsc. Microanal. 6, 644 /645. Grief, C., Galler, R., Cortes, L.M., Barth, O.M., 1997. Intracellular localisation of dengue-2 RNA in mosquito cell culture using electron microscopic in situ hybridisation. Arch. Virol. 142, 2347 /2357. Harcourt, B.H., Tamin, A., Halpin, K., Ksiazek, T.G., Rollin, P.E., Bellini, W.J., Rota, P.A., 2001. Molecular characterization of the
98
C.S. Goldsmith et al. / Virus Research 92 (2003) 89 /98
polymerase gene and genomic termini of Nipah virus. Virology 287, 192 /201. Hyatt, A.D., Selleck, P.W., 1996. Ultrastructure of equine morbillivirus. Virus Res. 43, 1 /15. Hyatt, A.D., Zaki, S.R., Goldsmith, C.S., Wise, T.G., Hengstberger, S.G., 2001. Ultrastructure of Hendra virus and Nipah virus within cultured cells and host animals. Microbes Infect. 3, 297 /306. Lamb, R.A., Kolakofsky, D., 2001. Paramyxoviridae: the viruses and their replication. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology. Lippincott Williams & Wilkins, Philadelphia, pp. 1305 / 1340. Mayo, M.A., 2002. Virus taxonomy */Houston 2002. Arch. Virol. 147, 1071 /1076. McFadden, G.I., Bonig, I., Cornish, E.C., Clarke, A.E., 1988. A simple fixation and embedding method for use in hybridization histochemistry on plant tissues. Histochem. J. 20, 575 /586. Mollenhauer, H.H., 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111 /114. Murray, K., Selleck, P., Hooper, P., Hyatt, A., Gould, A., Gleeson, L., Westbury, H., Hiley, L., Selvey, L., Rodwell, B., 1995. A morbillivirus that caused fatal disease in horses and humans. Science 268, 94 /97. Pedersen, K.W., van der Meer, Y., Roos, N., Snijder, E.J., 1999. Open reading frame la-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex. J. Virol. 73, 2016 /2026. Puvion-Dutilleul, F., Puvion, E., 1991. Ultrastructural localization of defined sequences of viral RNA and DNA by in situ hybridization
of biotinylated DNA probes on sections of herpes simplex virus type 1 infected cells. J. Electron Microsc. Tech. 18, 336 /353. Rager, M., Vongpunsawad, S., Duprex, W.P., Cattaneo, R., 2002. Polyploid measles virus with hexameric genome length. EMBO J. 21, 2364 /2372. Raine, C.S., Feldman, L.A., Sheppard, R.D., Bornstein, M.B., 1969. Ultrastructure of measles virus in cultures of hamster cerebellum. J. Virol. 4, 169 /181. Sedlmeier, R., Neubert, W.J., 1998. The replicative complex of paramyxoviruses: structure and function. Adv. Virus Res. 50, 101 /139. Teterina, N.L., Egger, D., Bienz, K., Brown, D.M., Semler, B.L., Ehrenfeld, E., 2001. Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis. J. Virol. 75, 3841 /3850. Troxler, M., Egger, D., Pfister, T., Bienz, K., 1992. Intracellular localization of poliovirus RNA by in situ hybridization at the ultrastructural level using single-stranded riboprobes. Virology 191, 687 /697. Wang, L.F., Yu, M., Hansson, E., Pritchard, L.I., Shiell, B., Michalski, W.P., Eaton, B.T., 2000. The exceptionally large genome of Hendra virus: support for creation of a new genus within the family Paramyxoviridae . J. Virol. 74, 9972 /9979. Wong, K.T., Shieh, W.J., Kumar, S., Norain, K., Abdullah, W., Guarner, J., Goldsmith, C.S., Chua, K.B., Lam, S.K., Tan, C.T., Goh, K.J., Chong, H.T., Jusoh, R., Rollin, P.E, Ksiazek, T.G, Zaki, S.R., and the Nipah Virus Pathology Working Group, 2002. Nipah virus infection: pathology and pathogenesis of a new, emerging paramyxovirus infection, Am. J. Pathol. 161, 2153 /2167.
