Elucidation of Nipah virus morphogenesis and replication usingultrastructural 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, USAb 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

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 cere-

brospinal fluid from human patients and identified as

belonging to the family Paramyxoviridae (Chua et al.,

1999, 2000).

Members of the family Paramyxoviridae are nonseg-

mented, 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 Para-

myxovirinae and Pneumovirinae . The subfamily Para-

myxovirinae has been divided into three genera:

Rubulavirus (prototype, mumps virus), Respirovirus

(prototype, human parainfluenza virus 1), and Morbil-

livirus (prototype, measles virus). An additional fourth

* Corresponding author. Tel.: �/1-404-639-3306; fax: �/1-404-639-

1377.

E-mail address: cgoldsmith@cdc.gov (C.S. Goldsmith).1 Current address: Consortium for Conservation Medicine,

Lamont-Doherty Earth Observatory, 61 Route 9W, Palisades, NY

10964-8000, USA.

Virus Research 92 (2003) 89�/98

www.elsevier.com/locate/virusres

0168-1702/02/$ - see front matter. Published by Elsevier Science B.V.

doi:10.1016/S0168-1702(02)00323-4

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 severerespiratory 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, negativestain, 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 identi-fication of several novel features of this newly recog-

nized virus.

2. Methods

2.1. Virus isolation and cell infections

Nipah virus was isolated at the Department ofMedical 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 mMglutamine, 100 mg/ml streptomycin, and 100 U/ml

penicillin (Life Technologies). Harvesting of infected

and uninfected cells occurred at 35 and 50 h postinocu-

lation.

2.2. Standard EM

Infected and uninfected Vero E6 cells and formalin-fixed 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 Epon-

substitute and Araldite (Mollenhauer, 1964). Thinsections 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 concen-

trated at 100 000�/g for 2 h. Pellets were resuspended in

small amounts of dH2O or 2.5% buffered glutaralde-

hyde. Copper mesh grids coated with formvar andcarbon (Electron Microscopy Sciences) were glow-dis-

charged 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, gamma-

irradiated, 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 paraformal-dehyde.

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 anti-

mouse conjugated to colloidal gold particles (Jackson

ImmunoReagents; Electron Microscopy Sciences) di-

luted in wash buffer with 1% fish gelatin added, andthen 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 Nipah-

infected cells to check for non-specific staining. All

antibodies were pre-absorbed against uninfected VeroE6 cells prior to their use (Goldsmith et al., 1995).

2.4. Ultrastructural in situ hybridization (ISH)

Reverse transcription-polymerase chain reaction pro-

ducts from portions of nucleoprotein (N), glycoprotein

(G) or fusion protein (F) genes of Nipah virus were

cloned into the dual promoter plasmid pCRII (Invitro-gen) (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�/9890

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 negative-

sense riboprobes were generated to detect viral genRNA

and messenger RNA (mRNA), respectively. The nega-

tive-sense riboprobe would also hybridize with the anti-

genomic 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 trans-

ferred onto ice. Sections were allowed to hybridize on

drops of cocktail in a moist, closed chamber at 37 8Covernight. 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 anti-

digoxigenin or goat anti-biotin conjugated to colloidal

gold (Electron Microscopy Sciences).

Negative controls for Nipah virus experiments in-

cluded hybridizations using Nipah virus probes with

uninfected and Hendra virus-infected Vero E6 cells.

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 specificityof 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 double-

label experiments to detect nucleic acid and proteinwithin 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 appro-

priate combination (anti-digoxigenin, anti-biotin, or

anti-mouse) of colloidal gold-conjugated antibodies.

These antibodies were tagged with varying sizes ofcolloidal 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, extracel-lular virions appeared as tangled collections of filamen-

tous, 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 anaverage of 21 nm in diameter with a 5 nm periodicity,

and up to 1.67 mm in length. In thin section prepara-

tions, nucleocapsids averaged 18 nm in diameter, and

Table 1

Riboprobes used for ISH studies of Nipah and Hendra viruses

Virus, GenBank accession

no., gene

Nucleotide posi-

tions

Riboprobe size (nu-

cleotides)

Nipah-AF212302

Nucleocapsid 1292�/1515 223

Glycoprotein 8943�/9333 390

Fusion 6654�/6954 300

Hendra-AFO17149

Nucleocapsid 1292�/1515 223

C.S. Goldsmith et al. / Virus Research 92 (2003) 89�/98 91

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-

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.

C.S. Goldsmith et al. / Virus Research 92 (2003) 89�/9892

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 Paramyx-

oviridae , 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 nucleo-

capsids were variously oriented. Occasionally, budding

profiles had very organized (fingerprint-like) arrange-

ments of nucleocapsids as they juxtaposed along the

viral envelope (Fig. 3B).

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.

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.

C.S. Goldsmith et al. / Virus Research 92 (2003) 89�/98 93

Additional noteworthy features were associated with

Nipah virus-infected cells. First, an unusual cytoplasmicinclusion was found, composed of a network of mem-

brane-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 inclu-

sion (RI) was morphologically distinct from the typical

nucleocapsid inclusions (NCIs) and absent from unin-fected 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 incor-

porated 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 less-

intense label was also observed over the RIs

(Fig. 6A) and the long cytoplasmic tubules. No IEM

labeling was observed by reacting anti-Nipah or anti-

Hendra 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 RNA-

protein 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).

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.

C.S. Goldsmith et al. / Virus Research 92 (2003) 89�/9894

The specificity of the ISH reactions was also con-

firmed 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. 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.

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.

C.S. Goldsmith et al. / Virus Research 92 (2003) 89�/98 95

endothelial cells, and were immunogold-labeled by using

specific antisera (Fig. 8C). No mature virus particles

were identified in the limited number of tissues exam-

ined.

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 epidemiologicfeatures were not consistent with this disease. Ultra-

structural observations of a virus isolated from cerebral

spinal fluids were instrumental in identifying the etiolo-

gic agent as a member of the family Paramyxoviridae .

Serologic, molecular, and immunohistochemical ap-

proaches 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 Paramyx-

ovirinae , 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 virus-

infected 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.

C.S. Goldsmith et al. / Virus Research 92 (2003) 89�/9896

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 replica-tion in vivo. This is, to our knowledge, a previously

undescribed feature for members of the family Para-

myxoviridae .

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 bedue 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 beenreported 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 andRNA 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 towarrant 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 Para-

myxovirinae . These include RIs, cytoplasmic tubules, a

longer length of viral nucleocapsid, and the highly

pleomorphic nature of the virus particles. Studies onNipah 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 Har-

court, for primer information, virus purification, and

review of the manuscript; Larry Anderson and Brian

Mahy, for guidance; and Claudia Chesley for editorial

review.

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