convert

-VE STRAND RNA VIRUSES

References

Flint. Principles of Virology, Pp 45-46, 178-179, 351-352, 538-540, 756-759

Fields Virology, 4

Edition, Chapters 38 thru 45

http://www.who.int/csr/don/

(WHO Disease Outbreak News)

http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/ebola.htm

(CDC - Ebola)

http://www.unicef.org/measles/index.html

(UNICEF - Measles)

http://www.measlesinitiative.org/index3.asp

(Measles Initiative)

The

Mononegavirales

(

mono

- single;

nega

- negative;

virales

- viruses) are a taxomonic order, which

includes several families of viruses with similar genomic organization and replicate strategies -- the

Filoviridae

Paramyxoviridae

and

Rhabdoviridae

, plus Borna disease virus

These viruses probably diverged from a single

common ancestor as recently as the last ice age. They are also frequently associated with

emerging infections

and/or cross-species transmission events (eg, Ebola).

Use of negative sense (-) RNA genomes means, by definition, that the viral genome is of opposite polarity to
mRNA. Thus, the viral genome cannot be used to make proteins until it has first been transcribed to produce
mRNAs. This has the following implications:
(I)

purified virion RNA is not infectious (as noted above, it cannot encode protein)

(II)

the viruses must bring their own RNA polymerase into the cell in order to make mRNA (ie, the viral
polymerase must be incorporated into the viral particle, or virion)

The other key feature of these viruses is that they make

gene-unit length mRNAs

(ie, each mRNA encodes

only a single protein). This is achieved by the use of transcriptional stop and start signals, which are located at
the boundaries of all of the viral genes.

Stop/start transcription

has two major results:

1).

Since there is only a single promoter, located at the 3’ end of the viral genome, the polymerase can only
load onto its RNA template at one site. As it moves along the viral RNA, the polymerase encounters
stop/start signals at the boundaries of each of the viral genes. This results in pausing of the enzyme,
which often falls off the template.  The result is that more mRNA is made from genes that are located
close to the promoter, and less mRNA is made from genes located far from the promoter.  This means
that there is  a

polarity of transcription

(see Figure below). The viruses use this to regulate the

expression of their genes, since highly expressed proteins are encoded close to the promoter (eg,
structural proteins such as the nucleocapsid protein, N), while proteins that are needed in only small
amounts (eg, enzymes such as the RNA polymerase, L) are encoded far away from the promoter.

2).

The other major consequence of stop/start transcription is that it complicates genome replication.  The
only way that the complete viral RNA genome can be copied is if the transcriptional stop/start signals
can be ignored or over-ridden. This means that the critical decision during viral RNA synthesis occurs
very early on -- at the first gene boundary (located between the leader RNA and the N gene).   If  the
stop/start signals here are obeyed, then only subgenomic mRNAs will be produced.   However, if  the
stop/start signal here is ignored or over-ridden, then a complete copy of the viral genome can be made.

Transcriptional polarity

(Flint. Fig 6.11A)

Mononegavirales

mono

- single;

nega

- negative;

virales

- viruses

Included within this order are:

Bornaviridae, Filoviridae

Paramyxoviridae

and

Rhabdoviridae.

Common Features:
1)

Genome: linear monopartite (-) RNA

Genome organization: 3'-[untrans. leader]-[CORE]-[ENVELOPE]-[POL]-[untranslated]-5'

Virion: helical nucleocapsid containing a viral RNA-dependent RNA-polymerase
•

competent for transcription on entry; protein synthesis required for replication

Transcription: make 6-10 discrete RNAs by stop/start synthesis from one promoter
•

leader transcripts are different from others: no polyA, no cap

•

transcriptional signals delineate genes: initiate at 3', terminate at 5' (plus polyA)

Replication: make a full-length (+) RNA that acts as a template for progeny genomes
•

decision to replicate made at leader/core boundary (read-thru)

Divergent Features

Family

Genome

Morphology

Hosts

Disease

Filo-

7 proteins; 19 kb

Filamentous

Reservoir = ?;

can

infect primates

Hemorrhagic fevers

• Genus: Marburg
• Genus: Ebola - 4 subtypes: EBO-Z [Zaire], EBO-CI [Cote d’Ivoire], EBO-R [Reston], EBO-S [Sudan]

Paramyxo-

10-12 proteins; 15-18 kb

Pleomorphic

Vertebrates

Mainly respiratory

Subfamily: Paramyxovirinae
• Genus: Morbillivirus (e.g., measles virus, canine distemper)
• Genus: Respirovirus (e.g., parainfluenzaviruses [Sendai = PIV-1])
• Genus: Rubulavirus (e.g., mumps virus)

• Possible new future genus: Henipavirus (e.g., Hendra virus, Nipah virus); largest of paramyxoviruses

Subfamily: Pneumovirinae
• Genus: Pneumovirus (e.g., respiratory syncytial virus)

Rhabdo-

5 proteins; 11-15 kb

Bullet shape

Animals, plants

Fever, neurologic

• Genus: Lyssavirus (e.g., rabies virus)
• Genus: Vesiculovirus (e.g., vesicular stomatitis virus)

Borna-

5 proteins; 9 kb

Animals

Neurologic

•

similar to member of plant virus genus, Nucleorhabdovirus

Generic mononegaviral replication scheme

Filoviridae

History/Outbreaks.

In 1967 simultaneous outbreaks of hemorrhagic fever occurred in Yugoslavia and in Germany, in lab workers
who were processing kidneys from African green monkeys. There were 31 cases and 7 deaths. The virus was
first characterized in

Marburg

, Germany and traced to a single shipment of Ugandan monkeys. Sporadic

additional cases showed up in 1975, 1980, 1982 and 1987.
In  1976  there  were  epidemics  of  severe  hemorrhagic  fever  in  Zaire  and  Sudan.    In  Zaire,  there  were
approximately 300 cases with an 80% fatality rate (due to Ebola-Zaire; EBO-Z).   In  Sudan,  there  were  a
roughly similar number of cases, with a fatality rate of roughly 50% (due to Ebola-Sudan; EBO-S).
As of 7 March 2003, there is an active outbreak on the Republic of the Congo.   A total of 5 laboratory-
confirmed  and  105  probable  cases,  including  89  deaths  have  been

reported

(

http://www.who.int/csr/don/2003_03_7/en/

)..

Ebola

virus was originally isolated in Zaire (now Democratic Republic of the Congo), and it was named after a

small river in N.W. Zaire. Ultrastructurally the virus resembled Marburg virus but it was antigenically (and
genetically) distinct. It now appears that at least three and probably four EBO viruses exist -- EBO-Z (Zaire),
EBO-S (Sudan), EBO-CI (Côte d’Ivoire) and EBO-R (Reston). The first two are known to be highly lethal in
humans and are spread via bodily fluids and by close (nonsexual) contact. The Reston virus

appears

to be less

lethal in humans (0 deaths in 6 cases), although it is lethal in nonhuman primates.
Major outbreaks of Ebola occurred in 1995 in the Kikwit area of Zaire (over 315 cases, with 80% fatality; due
to EBO-Z) and in the Gulu region of Uganda in 2000 (over 400 cases, but with roughly 50% fatality; due to
EBO-S). It is uncertain how the Kikwit and Gulu outbreaks started.  However, a smaller outbreak in 1996 in
Gabon was traced to a group of 20 young Gabonese who trapped and caught a Chimpanzee that was sick.  It is
believed that exposure to Ebola occured during the  preparation of  the  Chimpanzee, prior  to  cooking  and
consumption of the animal. Interestingly, Ebola was  isolated only  from  meat-eating Chimps, and  not  from
strictly vegetarian members of the same troupe of animals.
Outbreaks of EBO-Reston have occurred in US primate colonies in the Washington area (Reston, 1989) and in
Texas (1990, 1996). These outbreaks were contained by destruction of all animals within the affected area of
the facility. The outbreaks all appear to trace back to shipments of macaques from a single Philippine exporter.
A total of 6 humans have become infected by EBO-Reston, but none has died.
Finally, while the major route of Ebola transmission is clearly close contact with bodily fluids and blood (eg,
during health care, preparation for burial, etc), it is

possible

that some Ebola viruses

might

be transmissable via

an aerosol route in some cases. One piece of evidence to support this idea is the fact that EBO-Zaire has been
shown to infect rhesus monkeys that did not have direct contact with experimentally inoculated monkeys held
in the same room

(Jaax et al. Lancet 346:1669, 1995)

Filoviruses are classic emerging infections.

Filoviruses are Biosafety Level 4 agents (cf. HIV is only 2+).

They are filamentous with a linear ~13-19kb genome. They can infect mice, hamsters, guinea pigs and
monkeys -- although the

viral reservoir in the wild is not known

. Human epidemics seem to be related to

blood-born nosocomial spread (often due to re-use of needles in hospitals;

nosocomial = hospital infection

)

and to close contact with infected persons (since this is a hemorrhagic disease, this presumably would involve
exposure to large amounts of blood). Primary infections with Marburg and Ebola are 25-90% fatal. Death is
thought to be due to visceral organ necrosis (eg, liver) due to viral infection of tissue parenchymal cells. It is
uncertain what role hemorrhage has in death.

Ebola virus vaccine.

The first successful vaccination against this virus was reported in 1998, by Gary

Nabel's group at the University of Michigan. In this report, a DNA vaccine encoding the Ebola virus
glycoprotein was able to elicit a T-cell based immune response in guinea pigs, which was sufficient to protect
the animals against infection with a live-Ebola virus (

Xu et al. Nature Medicine 4:37, 1998

). Subsequent

studies in nonhuman primates have confirmed that a DNA vaccine can represent an important component of an
effective Ebolav irus vaccine. Specifically, a combination of DNA immunization and boosting with adenovirus
vectors encoding viral proteins resulted in the protection of cynomolgus macaques from an otherwise lethal
dose of highly pathogenic, wild-type Ebola Zaire virus

(Sullivan et al. Nature 408:2000).

This advance has led

to the NIH (

http://www.niaid.nih.gov/ttb/profile02.htm#ebola

) forming a partnership with Crucell in May 2002

(

www.crucell.com

), to develop rAd-vectored Ebola virus vaccines, using Crucell’s proprietary PER.C6 cell line

for the propagation of E1-deleted rAd vectors (the major advantage of this cell line is that the E1 sequence it
contains is smaller than the region that is missing from the E1-deleted adenovirus vectors; thus, it cannot
recombine with the rAd vector and so there is no possibility of generating replication-competent adenovirus;
RCA).

Ebolavirus sGP and GP have different functional properties, which may be important in disease pathogenesis.
The functional subdomains of these molecules are shown below.

sGP:

The soluble sGP molecule is secreted as a trimer, and is identical at its N-terminus to the homologous

region of the transmembrane glycoprotein (GP). sGP interacts with neutrophils through CD16b, the
neutrophil-specific form of the Fc

receptor III, whereas the transmembrane glycoprotein (GP) interacts with

endothelial cells but not with neutrophils

(Yang et al. Science 279:1034, 1998).

It is possible that interaction

of sGP with neutrophils results in the blockade of early events in the activation of these cells, thereby inhibiting
inflammatory responses which might contribute to innate protection against viral infection. sGP may also act
as a "decoy" for antiviral antibodies.

GP:

The transmembrane glycoprotein is produced as a long precursor, which undergoes cleavage by a cellular

protease (furin), to produce GP1 and GP2. Ebolavirus GP2 remains in the membrane (due to its
transmembrane domain) and is responsible for mediating fusion between the virus and the plasma membrane,
via its fusion domain. The GP1 component is attached to GP2 via a non-covalent linkage, and is thought to
mediate virus attachment to its host cell(s), which include vascular endothelial cells.

Ebolavirus GP is also cytotoxic for vascular endothelial cells

in vitro

, and this is thought to contribute to the

virus’ ability to trigger vascular leakage (hemorrhage)

in vivo

Ebolavirus glycoproteins

Legend:

GP: transmembrane glycoprotein (subsequently cleaved into GP1 and GP2 subunits)

sGP: soluble glycoprotein

GP/sGP identity: region shared by sGP, GP

Mucin-like domain: highly glycosylated domain of GP that is essential for cytotoxicity

Fusion domain: responsible for membrane fusion; located within GP2

Trimerization domain: allows GP2 to form stable trimers, like other viral fusion proteins

TM: transmembrane domain: anchors GP2 in the membrane

BORNA DISEASE VIRUS

Pathogenesis:

Borna disease virus (BDV) is a

neurotropic

agent that naturally infects horses and sheep, and

which is capable of infecting primates. The disease induced by BDV resembles neuropsychiatric illnesses
(

schizophrenia

). It is uncertain whether the virus has any relationship to human neurologic disease.

RHABDOVIRIDAE

Greek "rhadbo": rod-shaped

Over 100 rhadboviruses exist & they infect almost all animals. Two genera affect mammals:
Genus

Features

Example

Lyssa-

invade CNS;

(fr. Greek "lyssa": frenzy)

Rabies virus

Vesiculo-

invade epithelial cells (usu. tongue) & cause vesicles

Vesicular stomatitis virus (VSV)

Rabies:

Causes encephalitis in animals and in humans they bite

Rabies virus can infect all warm-blooded

animals, but some are more susceptible than others (eg, foxes, wolves > dogs, skunks, raccoons > opposum). It
is spread to humans via animal bites. In the US it is most prevalent in skunks, but also found in raccoons and
sometimes in bats. Elsewhere rabies is more common in humans, due to its presence in dogs.

Bats and rabies:

In the U.S. about 1-2 cases of rabies occur each year. Since 1990, 20 of 22 domestically

acquired human rabies infections in the United States have resulted from infection with bat rabies variants, and
in only one of these cases was there a clearly documented bat bite (

http://www.wadsworth.org/rabies/bat.htm

Many of these bat rabies strains were silver-haired bat (SHB) rabies virus. SHBRV is carried both by silver-
haired bats (relatively rare and solitary) and also by other strains of bats

(overall, much less than 1% of all bats

test positive for rabies, and the bats most often found around humans -- brown bats -- have never been shown
to have cause human disease).

The rarity of SHBRV is strongly suggestive that something unusual is going

on here. In addition, data show that the SHBRV variant replicates with unusually high efficiency in cultured
epithelial cells, particularly at low temperatures (34

C). This may allow the virus to replicate more efficiently in

the skin

(Morimoto et al. PNAS 93:5653, 1996)

CLINICAL PICTURE: Whether disease results reflects the location and severity of bites (typically ~15% rate
of infection). Disease onset is slow, with an unusually variable incubation period (can be over a year) during
which virus replicates in muscle near the entry site. Thereafter, the virus enters peripheral nerves. From here, it
travels to spinal ganglia & enters the brain. It is then disseminated to all tissues (including salivary gland).
Death is inevitable if the virus enters nerves, but post-exposure intervention before this is generally successful.
Control of rabies: is achieved by controlling its animal reservoir -- ie, by vaccinating domestic animals and also
by the use of vaccine-containing bait to target wild animals. For exposed humans, there is a vaccine.

VSV:

Causes epidemic but self-limiting vesicular disease of cattle. Also infects swine, horses, humans & even

insects (

very broad host range

). In humans, it causes a mild flu-like illness that's fairly common in lab

workers. In keeping with its broad host range, the VSV receptor is not a protein (prolonged trypsinization of
cultured cells doesn't block infection). It may be phosphatidyl serine.

VSV Virion (left) and Genome (below)

Flint et al., Appendix Fig 5 (p757)

Molecular Biology of VSV

Overall, the molecular biology of VSV is considerably better understood than that of rabies virus

The morphology and structure of VSV is similar to that of rabies virus. The particles are bullet-shaped and are
composed of two major structures -- a

nucleocapsid

or ribonucleoprotein (RNP) core and a lipoprotein

envelope

which surrounds that core.

VIRAL RNP CORE:

The nucleocapsid or RNP core is the infectious component of VSV and all other

rhadboviruses. As shown in the diagram, this core includes the viral genomic

RNA

which is tightly associated

with the highly abundant

nucleocapsid protein (N)

. The RNP core also contains less abundant proteins --

the

phosphoprotein (P)

, and the

viral RNA polymerase (L)

N protein:

The function of the N protein appears to be (1) to promote RNA encapsidation or packaging and

(2) to allow genome replication, by favoring antitermination of transcription (ie, by allowing the viral
polymerase to read-through the stop/start signals located between the viral genes).

L protein:

This is the viral RNA-directed RNA polymerase. It is not active on its own, however, since P

protein is needed for catalytic activity.

VIRAL ENVELOPE:

The major components of the VSV envelope are (1) the membrane-anchored viral

glycoprotein (G)

and (2) the

matrix protein (M)

. Roughly equivalent amounts of the two protein are found

in each virion (approx. 1500 molecules per virion).

G protein:

The glycoprotein, G, forms trimeric spikes on the surface of the viral particle and it forms both the

major antigenic determinant on the virus, as well as the major receptor-binding molecule on the virus. G
protein undergoes a conformational shift at mildly acidic pH (< 6.0), which stabilizes the trimer and exposes a
hydrophobic domain that can insert into cellular membranes and allow membrane fusion to occur. Thus, VSV
fusion is activated in the endocytic vesicle, in response to acidic pH.

Viral gene expression

After entry into its host cell, and uncoating of the RNP core, VSV begins to express its genes. Since the viral
genome is of negative sense (ie, of opposite polarity to mRNA), the very first step is transcription of viral
mRNAs.

Note that the RNP core is transcriptionally active, and that is not inhibited by actinomycin-D (unlike

cellular RNA polymerases, which use a DNA template to direct mRNA synthesis).

Viral transcription begins at the 3’ end of the viral genome, at a

single promoter element

, and proceeds

sequentially across the genome. It is generally believed that the individual gene-unit-length mRNAs are
produced by a

stop-start

trancription mechanism (see below). One result of this is that the transcriptase

pauses and transcription is attenuated about 30% at each gene junction. This in turn produces  a  gradient of
mRNA production, such that N>P>M>G>L.
Stop/start transcription is acheived by the presence of transcriptional signals at gene boundaries.  There is a 5'-
initiation signal, as well as 3'- polyA and termination signals, which are  ordered:   [polyAsignal/terminator]--
[intergenic region]--[initiator].

Note that the intergenic region (2 nucleotides) is not transcribed during viral

mRNA synthesis.

Viral RNA replication

Unlike viral mRNA transcription, viral RNA replication requires the virus to form a single complete copy of its
genome. Thus, replication differs from mRNA transcription in that transcriptional "start/stop" signals must be
ignored somehow.
The decision to replicate the viral genome must therefore be made when the first intergenic region is
encountered (this is located between the region that encodes the short untranslated leader RNA and the gene
encoding the N-protein). This

intergenic region must be read-through

in order for viral RNA replication to

occur.
Interestingly, viral RNA replication

requires active translation

(this was proved experimentally, since viral

RNA  replication,  but  not  viral  mRNA  synthesis,  was  blocked  by  inhibition  of  protein  synthesis  using
cycloheximide). This observation is consistent with a model in which newly formed viral N  protein selectively
binds to the viral leader RNA.  By doing so, N  prevents the recognition of transcriptional termination signals.
Thus, the switch from mRNA synthesis to RNA replication is regulated principally by the

anti-termination

activity of the N protein

Molecular Genetics and Vectors

Replication  of  VSV  has  been  difficult  to  study  using  recombinant  DNA  methods.   This  is  because
deproteinized RNA is not infectious. Likewise, RNA transcribed from cDNA clones is not by itself competent
to initiate infection. The virion RNA-polymerase is needed for infectivity, and in addition the RNA  must  be
encapsidated to be a functional template for the polymerase. Finally, constant synthesis of N  protein is needed
for replication. Recently, great progress has been made in this area.  Methods for the production of infectious
virus from cDNA clones of both VSV and Rabies virus have been developed. This is allowing the development
of novel vector systems based on VSV and Rabies virus.

KEY:

The dark circles represent cell monolayers, and the white areas are plaques in these monolayers, which were caused by

infectious virus. You can see that infectious virus was produced ONLY when an intact cDNA copy of the VSV genome was

introduced into cells together with support plasmids encoding the viral RNP constituents (ie, N, P and L). The bottom panel
proves that the plaques seen were due to VSV, since plaque formation was blocked by the addition of a neutralizing antiserum

directed against VSV (anti-VSV Ab).

VSV G-protein is widely used to generate

retrovirus pseudotypes

. Pseudotypes are the result of phenotypic

mixing of different viruses, and contain a core (and genome) from one virus, combined with the envelope (and
receptor-binding activity) of the second virus.

VSV-G Pseudotyped Retrovirus Vectors

In  general,  pseudotypes  can  be  generated  only  between
fairly closely related  viruses  (such  as  VSV  and  Ebola
virus), but  exceptions  exist.    One  notable  exception are
retroviruses.   It  has  long  been  known  that  VSV  can
generate  pseudotypes  with  a  variety  of  retroviruses,
including HIV-1.

Retrovirus pseudotypes

bearing the VSV G-protein in

place  of  the  natural  retrovirus  envelope  have  several
features which make them useful for gene therapy.  These
include:
1. Extended  host  cell range.    The  VSV G-protein allows

one to deliver the retrovirus "payload" (i.e., the
recombinant genome) to a wide array of mammalian &
animal cells, including fish. It also allows one to deliver
genes to certain human cell types, such as hematopoietic
progenitor cells, which are otherwise difficult to target.

2. Increased physical stability. The VSV G-protein is

much more stable than the natural retrovirus envelope.
This allows one to concentrate the viral particles, and to
generate high-titer stocks which are more useful for
gene transfer.

PARAMYXOVIRIDAE

Chua et al. Science 288:1432, 2000; Goh et al. N. Engl. J. Med. 342:1229, 2000 (Nipah)

Wang et al. J. Virol. 74:9972, 2000 (Hendra virus)

Subfamily

Paramyxo- virinae (Genera: Paramyxo-, Morbilli-, Rubula-)

Respiro-:

eg, Parainfluenza viruses 1 and 3: have H and N (single molecule: HN)

Rubula-:

eg, mumps virus: have H and N (single molecule: HN); has extra gene (SH)

Morbilli-:

eg, measles virus: have H but no N

Henipa-:

eg, Nipah, Hendra viruses: have H but no N

(this genus has been proposed but is not yet

official)

Subfamily: Pneumo- virinae (Genus: Pneumovirus)
Pneumo-:

eg, respiratory syncytial virus (RSV): has neither H nor N; more divergent

RNA editing during Measles virus mRNA synthesis

The P gene mRNA of Measles virus is cotranscriptionally edited at a specific site. This

RNA editing

event

involves the addition of a nontemplated G residue at this position during mRNA synthesis

(this occurs

because the RNA polymerase slips or “stutters” when it encounters a run of C residues at this site on the
template RNA strand)

MV RNA Editing

Flint et al. Fig 10.13B (p352)

The extra G introduced at the editing site results in a
change in the translational reading frame, to one which
contains a translational stop codon. Thus, the edited RNA
encodes the

V protein

, which is identical to the P protein

at its N-terminus, but different at its C-terminus

Measles Virus: Envelope proteins

All paramyxoviridae possess two membrane or envelope proteins. One is involved in cell

attachment

and

the other mediates

fusion

with the host cell membrane, in a pH-independent manner.

Attachment Protein.

The attachment proteins of the paramyxovirinae bind to sialic-acid containing

receptors on cells, and these viruses are therefore able to agglutinate red blood cells (hemagglutination). In
the case of viruses in the genera respirovirus and rubulavirus, these viral hemagglutinins also possess
neuraminidase activity and are thus refered to as

HN proteins

(hemagglutinin-neuraminidase). In the case

of viruses in the genus morbillivirus, the hemagglutinin lacks neuraminidase activity (thus,

measles virus

encodes an H protein

and not an HN protein).

Role of Neuraminidase. It is believed that neuraminidase prevents aggregation of viral particles to the plasma
membrane during viral budding, and thus  facilitates virus  release  from  infected cells.    This  means  that
neuraminidase must be inhibited during the early steps of virus entry and that it must become activated during
the late stages of virus exit. This may occur in part because the activities of hemagglutinin and neuraminidase
are regulated by pH and by halide ion concentration. Specifically, the pH and halide ion concentration of the
extracellular environment is optimal for hemagglutination, while neuraminidases function best  at  acidic pH
(such as can be found within the Golgi network inside cells).
Note that measles virus (MV) does not in fact use  sialic acid as  its  receptor --  presumably because its  H
protein has a relatively low affinity for sialic acid. As a result, MV does not need a neuraminidase.  In stead,

measles virus H protein is thought to bind to a specific receptor, SLAM

(signalling lymphocyte-

activation molecule; CDw150). SLAM is present on some T cells and B cells (Tatsuo et al. Nature 406:893,
2000).

The Pneumovirus, respiratory syncytial virus (

RSV

) does not hemagglutinate and its receptor is unknown.

In this case, viral attachment to the host cell is mediated by the

G (glycoprotein) protein

Note that some highly passaged vaccine strains of MV bind to

CD46

-- a molecule which is a member of the

immunoglobulin gene superfamily; clinical isolates of MV do not, however, bind CD46.

Paramyxovirus replication

In general terms, paramyxovirus replication is broadly similar to that of rhabdoviruses.

One important and unique feature of

measles virus infection

, in particular, is the virus’ ability to

persistently infect brain cells, which has been implicated in SSPE. Persistent MV infection appears to involve
at least two methods for

specific attenuation of M gene expression

. First, specific downregulation of M

gene expression can occur as the result of inefficient transcriptional termination and polyadenylation at the
upstream ORF (which is expressed normally). Second, biased hypermutation of the M gene region has been
described. This is due to the action of a cellular enzyme that converts adenosines to inosines in dsRNA
(double-stranded-RNA-adenosine deaminase, dsRAD).

Note that dsRAD is also responsible for site-specific

editing of Hepatitis Delta virus RNA.

Pathogenesis of MV infection

MV is a classic emerging pathogen.

MV is a relatively new disease of humans which evolved from an

animal morbillivirus (MV most closely resembles a pathogen of cattle known as rinderpest virus).  Measles
was first described in the tenth century.  Since it causes a highly contagious acute infection which results in
lifelong immunity, and it has no animal reservoir, it requires an urban setting to survive (200,000+ people).
Cities this size first emerged ~3000 BC in Egypt and  Sumeria.   This  is  likely when measles and  mumps
emerged. These cities remained isolated until trade began. Epidemics of disease (measles, smallpox) then
began around 200-400 AD. Measles was carried to the New World  by Europeans and caused many deaths
among native Americans, who had not encountered MV previously.

MV is a major killer of the world’s children.

Worldwide, measles kills almost 800,000 million children

per year (

http://www.unicef.org/measles/disease.htm

). As a result, WHO and UNICEF are working hard to

further raise immunization rates in all villages and cities, including poor areas. Ultimately, the WHO and
UNICEF intend to eradicate measles completely. This is possible because (1) there is no animal reservoir for
MV, (2) there is only one serotype of the virus, (3) most cases are clinically identifiable and (4) an effective
live-attenuated vaccine is available.

Pathogenesis.

Measles is a childhood infection that is spread by a respiratory route. Following infection,

the virus replicates in lymphoid tissues. It then enters the  blood  (viremia) and  spreads  through  the  body,
reaching its target tissues (principally, the lungs), where replication occurs.  This initial period of infection is
asymptomatic and lasts about 10-14 days, at the end of which clinical signs of disease become apparent --
notably, fever, cough  and  conjunctivitis.   Roughly  2-3  days  after  the  onset  of  these  symptoms,  the
characteristic measles rash appears. This coincides with the  appearance of  an  antiviral immune response.
Recovery results in lifelong immunity to infection.

Measles Pathogenesis

Flint et al., Fig 15-10

Immune responses.

Recovery from MV infection is mediated in large part by a cellular immune response

to the virus (cytotoxic T cells, CTLs). However, one of the striking features of MV is its ability to cause

immune suppression

in vivo

and

in vitro

. Production of cellular responses to new antigens is significantly

inhibited, which predisposes MV infected children to concurrent infection by other pathogens (such as
bacteria, which can cause fatal pneumonia in MV-infected children). The mechanism(s) by which MV causes
immune suppression is believed to involve virus infection of dendritic cells (which are involved in antigen
presentation) as well as infection of other immune cells.

MV infection of dendritic cells

(DC) leads to

apoptosis, and to inhibition of the ability of DCs to stimulate T cell proliferation (which is important for the
generation of immune responses.

Autoimmunity.

In addition to immune suppression, MV infection can be associated with autoimmunity.

Specifically, an autoimmune demyelinating disease,

postinfectious encephalomyelitis

(PIE) is an

important complication of measles which occurs within 14-28 days of infection in about 1 in 1000 cases.  It
is associated with an immune response to  myelin basic  protein.   It  is  not  clear how  this  autoimmunity is
initiated, although it is possible that MV antigens may resemble myelin (molecular mimicry).  Thus, an anti-
MV immune response may lead to attack on a self antigen -- myelin.

Persistent infection.

MV can establish a persistent infection in brain cells

in vitro

and

in vivo

. As noted

above, this is often associated with suppression of expression of the viral M protein. Persistent MV infection
of the brain can be associated with a very rare disease, Subacute Sclerosing PanEncephalitis (SSPE), which
occurs several years after initial MV infection in about 1 in 1,000,000 children.

Clinical features and complications of measles.

Serious symptoms of measles include:

(1)

Respiratory disease

. Pneumonia can be caused by MV itself (giant cell pneumonia), particularly in

immune suppressed persons OR (more commonly) by secondary bacterial or viral infections.

(2)

Gastrointestinal disease

. Diarrhea is a very common complication of measles. This can be a major

problem in children who are already malnourished or at risk for malnutrition. Also, the severity of MV
infection has been shown to be much worse in children who are deficient for

vitamin A

This is one of

the major reasons why WHO and UNICEF support vitamin A supplementation programs in the
developing world (vitamin A can be administered very cheaply in megadose capsules)

(3)

Neurologic disease

. Postinfectious encephalomyelitis (PIE) occurs in about 1 in 1,000 cases, usually

within 14-28 days of infection. Slowly progressive neurologic disease can occur in immunosuppressed
persons (measles inclusion body encephalitis, or MIBE) and SSPE occurs at a very low frequency in
immunologically normal children, usually several years after initial MV infection.

(4)

Eye disease.

MV is an important cause of blindness due to corneal lesions. Again, vitamin A deficiency

has also been implicated in blindness, and may exacerbate MV induced eye damage.

(5)

Atypical measles.

A severe form of measles, with more prolonged fever, worse skin lesions and more

serious pneumonitis, has been observed in individuals who received the inactivated MV vaccine used in
the US from 1963 - 1967. This formalin-inactivated vaccine provoked an unbalanced immune response,
with high level antibodies to the viral H protein but little reactivity to the F or N proteins.

Other paramyxoviruses

Respiratory syncytial virus (RSV).

RSV infects infants from

6 weeks to 6 months

of age

and causes

90,000 hospitalizations and

4500 deaths each year in the US

. It usually causes upper respiratory infection,

but in 25-40% of cases, lower respiratory symptoms occur.

RSV is the most important viral cause of lower

respiratory disease in infants and children.

In the elderly, severe pneumonia can occur. RSV is a major

cause of nosocomial (hospital) infections. Aerosolized ribavirin can be helpful, as can passive antibodies
(RSV immune globulin). Two antigenic subtypes exist, but there is no effective vaccine, in part because (1)
reinfection is common; (2) immunity is incomplete (infection occurs in infants, despite the presence of
maternal antibodies).

ParaInfluenza viruses.

PIV-1, 2 and 3 are second only to RSV as causes of serious respiratory tract

disease in infants and children.

Mumps virus.

Mumps usually causes a benign systemic febrile illness

with swelling of salivary glands. It can, however, infect the CNS and can cause meningitis, encephalitis,
deafness plus orchitis. A live attenuated vaccine has been used in the US since 1967; mumps is now rare.

New and emergent paramyxoviruses

A recent example of an emerging morbillivirus infection occured in Australia in 1994. 14 horses died as a
result of infection with

Hendra virus

in Queensland. Two people who had close contact with these horses

also became infected and one developed a fatal respiratory illness. A second fatal case of human Hendra
virus infection was subsequently described in another region of Queensland, some 800 kilometers away from

the first outbreak. Followup studies have revealed that Hendra virus infection of horses is rare. However, the
virus has been found in numerous bats -- suggesting that these animals may be the virus' natural host.

Note

that Hendra virus was initially known as equine morbillivirus, but subsequently renamed to reflect its
somewhat distant genetic relationship to the morbilliviruses.

Research into Hendra revealed two previously unknown diseases associated with bats in Australia
(

http://www.csiro.au/page.asp?type=faq&id=HendraVirus

). The

Australian bat lyssavirus

was identified

in 1996, and is closely related to the rabies virus; it has been associated with at least two human fatalities.
The

Menangle virus

was isolated in 1997 from pigs, and has been associated with a flu-like illness in

humans; it is thought that Menangle is a member of the

Rubulavirus

genus. Like Hendra virus, Menangle

virus is also carried by fruit bats.

The isolation of the Hendra virus in 1994, and the Menangle virus in 1997, presaged the identification of

Nipah virus

in 1999. Nipah was isolated from pigs and from humans with encephalitis in Malaysia; it was

a spread by a respiratory route through the pig population and caused the death of over 100 Malaysians from
encephalitis. All those killed had close contact with pigs (pig farmers and workers) and the outbreak was
contained by slaughter of one million pigs. Like Menangle, it is thought that Nipah was transmitted initially
by bats.

Pigs are thought to have played a crucial role in the emergence of Nipah, since these mammals are unique in
being maintained in very high concentrations, in situations where epidemic disease can  easily occur.    It  has
been suggested that Nipah was able to initially establish itself in these pigs, following transmission from fruit
bats, and that the virus was able to become adapted to mammals (pigs) over the course of perhaps a couple of
years in Malaysian pig farms.  Infection of the pigs then gave the virus ready access to humans (pig farmers,
pig workers). The linkage of the Nipah virus outbreak to livestock production has important implications, and
highlights the potential risks that can be associated with the  high  intensity farming of  hogs  and  chicken, in
particular.

The Nipah and Hendra viruses are genetically related more closely related to each other (70-78% nucleotide
homology) than to any other member of the family

Paramyxoviridae

(maximum of 49% homology to any

other virus in this family). Furthermore, the genomes of Nipah and Hendra viruses
are considerably larger than the other

Paramyxoviridae

(> 18 kb, compared to 15-16 kb for the other family

members). Nipah and Hendra also share a number of other unique genetic features which mark them as
separate members of the

Paramyxoviridae

. As a result, it has been proposed that these viruses be classified

into a new genus in the family

Paramyxoviridae; a proposed new for this group of viruses is the

Henipavirus Genus (Wang et al. J. Virol. 74:9972, 2000).

Nipah and Hendra also share the common feature of being

zoonotic paramyxoviruses

with an expanded

host range that includes humans and other animals (horses, pigs); both are thought to be transmitted from a
natural reservoir in

fruit bats

Phylogenetic analysis of Nipah and Hendra viruses