Astrovirus presentation

1. Astroviruses
By-SanjuSah
St.Xavier’scollege,Maitighar,Kathmandu
DepartmentofMicrobiology

2. Astroviruses
 family Astroviridae includes human and animal astroviruses
- icosahedral morphology;
- they are nonenveloped and
- genome is composed of plus-sense, single stranded RNA
(ssRNA), with three openreading frames, whose organization
distinguishes them from other virus families.
 Astroviruses (AstV) have been isolated from a variety of animal
species.
associated with
 In most mammals, astrovirus infections are
gastroenteritis.

3. Astroviruses
• Second or third most common cause of viral diarrhea in young
children and cause of sporadic gastroenteritis outbreaks.
• Avian AstV, on the other hand, have been linked with more
severe intestinal and extra-intestinal manifestations of disease.

4. Astroviruses
• The term astrovirus - coined by Madeley and
Cosgrove in 1975
• to describe small, round viruses with a
distinctive five or six-pointed, star-like
appearance (astron, star in Greek) of about
28 to 30 nm in diameter.
• They were observed by direct electron microscopy (EM) in the
stools of infants hospitalized with diarrhea and in outbreaks of
gastroenteritis in newborn nurseries.
• Subsequently, viral particles of similar size and morphology - in
association with gastroenteritis in a wide variety of young
mammals and birds.

5. 5
• Astroviruses- originally classified into genera and species based
only on the host of origin;
• however, recent characterization of novel AstV has shown that
isolates from different animal species can be genetically similar,
while genetically diverse viruses can be isolated from the same
animal species.
• new classification scheme is based on the amino acid sequence
of ORF2 (Astroviruses Study Group, 9th Report ICTV, 2010),
which encodes the capsid polyprotein and represents the most
variable region of the genome.
• Two genera are distinguished within the Astroviridae family:
Mamastrovirus and Avastrovirus
Classification

6. Adapted from: David M. Knipe et al., 2007
The Astroviridae family includes two genera with two genogroups each.
Virus species are classified based on the ORF2 amino acid sequence distances.
(Classification proposed by the Astroviruses Study Group, 9th Report ICTV,
2010).
6

7. 7
• genus Mamastrovirus include isolates from a number of
mammals, including humans, pigs (PAstV), cats (FeAstV), minks
(MAstV), sheep (OAstV), calfs (BoAstV), dogs (CaAstV), bats
(BAstV), rats (RAstV), deer (CcAstV), and marine mammals, such
as sea lions (CSlAstV) and bottlenose dolphins (BdAstV), among
others.
• This genus includes two genogroups, GI and GII, with 10 and 9
genotype species, respectively.

8. 8
• genus Avastrovirus include isolates from turkeys (TAstV), ducks
(DAstV), chicken (CAstV), and guinea fowl.
• This genus includes two proposed species in genogroup GI (GIA,
GIB) and one in genogroup II (GIIA). Similarly to canonical HAstV,
members of some of these species can be distinguished by
serology, indicating the existence of viral serotypes in some,
such as in TAstV-2 and ANV.
• In general, avastroviruses show higher diversity than
mamastroviruses.

9. Structure
• icosahedral particles of 41 nm, with spikes protruding from the
surface.
• The star-like form of the particles was observed only after
alkaline treatment.
• Recent studies by cryo-EM and image processing of trypsin
treated and untreated HAstV particles confirmed the spiked
icosahedral structure of virions - main difference between the
two types of particles is the number of spikes observed
9

10. 1
0
Virion Composition
genome surrounded by an
• Astrovirus particles - viral
icosahedral capsid formed by
• single protein of 70 to 90 kd, or by at least three proteins
in the range of 25 to 34 kd, depending on the extent of
proteolytic processing of the virion. (proteins of different
size (between 24 and 90 kd), probably representing final
and intermediate cleavage products, can be found in
particles of HAstV).

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Virion Composition
identified in animal viruses and in human
• Similar proteins
strains;
• HAstV particles may contain a small protein of 5.2 kd, but its
nature was not established.
• In addition to proteins derived from ORF2, proteins coded by
ORF1a have been suggested to be present in viral particles, since
antibodies to purified HAstV-1 virions recognize a recombinant
protein containing amino acids 757 to 899 of nsp1a.

12. GENOME STRUCTURE AND ORGANIZATION
Genome organization of human astrovirus. The genomic RNA, approximately
6.8 kb, contains three recognized open reading frames (ORF1a, ORF1b, and
ORF2). ORF-X has been proposed as functional, given its conservation among
AstVs.
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• Astroviruses have an ssRNA genome of positive polarity
[ssRNA(+)] that varies in length from 6.17 kb (for the human
strain MLB-1) to 7.72 kb (for DAstV-2), excluding the poly(A) tail
at the 3 end.
• The viral genome includes 5’ and 3’ untranslated regions (UTRs),
and three ORFs of variable length in different isolates.
• The two ORFs located towards the 5’ end of the genome,
designated ORF1a and ORF1b, encode nonstructural proteins
that are presumed to be involved in transcription and replication
of the virus genome.

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• Variation of the ORF1a length in HAstV is mainly due to
insertions or deletions (in/del regions) present near the 3’ end
of ORF1a.
• The third ORF, found at the 3’ end of the genome and
designated ORF2, encodes the capsid polyprotein.
• ORF1a and ORF1b overlap in 10 to 148 nucleotides (nt) in the
genome of mammalian viruses, and between 10 and 45 nt in
avian viruses.
• The overlapping region contains an essential signal for
translation of the viral RNA polymerase (encoded in ORF1b)
through a frameshift mechanism.

15. Nonstructural proteins predicted from the nucleotide sequence of ORF1a and
ORF1b.
NBM, nucleotide binding motif; CC, coiled-coil; v-Pro, viral protease; VPg, viral
protein attached to the genome; in/del, insertion/deletion; DD, putative death
domain; IRE, immune reactive epitope; RdRp, RNA-dependent RNA polymerase
15

16. Translation of the RdRp occurs through a ribosomal-1 frameshift
mechanism in the overlapping region between ORF1a and ORF1b.
The signal that modulates this event has two key features,
conserved among all astroviruses: a heptameric sequence
(AAAAAAC) and the potential to form a downstream stem-loop
structure
astroviruses is composed
16
The frameshift signal (fss) of
by
a slippery sequence (underlined )
and a stem-loop structure.

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• The polypeptide encoded by ORF1a (nsp1a) is 874 to 936
amino acids (aa) in length in most mammalian viruses, although
it can be as short as 787 in a human isolate and can reach 1,240
in avian viruses
• The largest sequence variability in the astrovirus genome is
found in ORF2, which codes for the virus structural polyprotein.
This polypeptide varies from 672 to 851 aa in length in TastV-1
and a porcine strain, respectively.
• In general, the ORF2 of avian viruses codes for shorter
polyproteins.
• The N-terminal half of the polyprotein is more conserved than
the C-terminal domain, encompasses a region of basic
character that is conserved among astroviruses,67,142 and is
thought to interact with the genomic RNA in the virion.

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• The conserved domain of the structural protein forms the
capsid core of the particle, whereas the hypervariable C--
terminal domain forms the spikes of the virion; thus, this last
domain is predicted to participate in the early interactions of
the virus with the host cell.
• An alternative ORF (named ORFX) of 91 to 122 codons
overlapping ORF2 in a +1 reading frame has been described in
all HAstV and some other mammalian viruses.
• Its initiation codon, usually located 41–50 nt downstream of
the ORF2 AUG, is placed in a better Kozak’s consensus
sequence than that of ORF2, and might be translated through
a leaky scanning mechanism. It remains to be determined
whether ORFX is actually translated and, if it is, its significance
for virus replication.

19. Features of the structural protein.
• The primary ORF2 product (named VP90 in HAstV-8), contains
two domains that can be distinguished by their degree of
conservation: the N-terminal domain is highly conserved and
forms the core of the capsid, while the hypervariable C-terminal
domain forms the spikes of the virus particle.
• VP90 contains basic and acidic regions that are highly conserved
among all AstVs characterized.
• The diamond in the conserved domain indicates a lethal
mutation identified in HAstV-1 and the black arrows indicate
motifs recognized by caspases, important for virus maturation
and cell egress. 18

20. Replication
Replication cycle of HAstV. 19

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Attachment and Entry
• Studies directed toward understanding the early interactions of
astroviruses with their host cells have been limited
• Cell receptor molecules for these viruses have not been identified.
• Different HAstV strains show different tropism in cultured cells;
thus, it is likely that their initial interactions with the host cells
might be different
• The infectivity of human astroviruses requires, the treatment of
the virus particles with trypsin.
• the proteolytic pathway of VP70 processing present in the virion
has been elucidated for HAstV-8,
• the mechanism by which this treatment enhances virus
infectivity is still unknown.

22. • Trypsin
drastic
cleavage of the precursor polyprotein induces
structural changes in the particles and the
generation in HAstV-8 of three final products: VP34, VP25,
and VP27
22

23. • VP34 represents the conserved
domain and forms the capsid
core, while both VP25 and VP27
form the spikes on the virion
surface.
• In spite of its high variability, VP25 contains two conserved
structural motifs that have been suggested to be involved in the
interaction of the virus with the host cell,
• which includes residues Lys455, Ser554, Thr575 and Glu610,
predicted to interact with oligosaccharide moieties that could
act as cell receptors or co-receptors
• Antibodies that recognize the spike proteins VP25 and VP27
neutralize virus infectivity, probably by blocking virus binding.
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• The interaction of HAstV with the host cell provokes
activation of the ERK1/2 signaling pathway within the first 15
minutes after virus attachment (the mechanism is unknown),
- this pathway is triggered during virus binding or entry into
the cell.
• ERK1/2 seems to be required at early times to establish a
productive infection

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Uncoating
The mechanism through which the viral genomic RNA is released
from the infecting virus particle into the cytoplasm for
translation, the cell site where it occurs, and the cellular and viral
factors involved in this event are unknown.
(Knipe & Howley, 2007)

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Translation
• No detectable changes in cellular protein synthesis are
observed upon HAstV infection.
• As do most cellular mRNAs, astroviral RNA contains a polyA tail
at the 3′ end, but the presence of a cap structure at its 5′ end
has not been described.
• Since a VPg has been suggested to be encoded in astrovirus
ORF1a,3 this protein could modulate the translation of viral
mRNAs by interacting with translation initiation factors, as has
been described for other viruses, such as calicivirus.

27. Nonstructural Polyproteins Synthesis and Processing
• After uncoating, the gRNA is translated into nonstructural
proteins that are produced as polyprotein precursors and
subsequently proteolytically processed into smaller proteins.
• ORF1a directs the synthesis of protein nsp1a (of about 100 kd),
whereas protein nsp1ab (160 kd) is derived from both ORF1a
and ORF1b, through a frameshift mechanism of translation
• Proteins nsp1a and nsp1ab are apparently processed co-
translationally at their amino terminus, so that the expected
full-length proteins are not, or very rarely, observed in HAstV-
infected cells
• in vitro translation for study includes- transient expression of
cDNA clones, and analysis of HAstV-infected cells,
• Suggested for requirement of cellular factors for
translation
specific
26

28. • Translation of the RdRp occurs through a ribosomal -1
frameshift mechanism in the overlapping region between
ORF1a and ORF1b.
• The signal that modulates this event has two key features,
conserved among all astroviruses: a heptameric sequence
(AAAAAAC) and the potential to form a downstream stem-loop
structure.
The frameshift signal (fss) of
astroviruses is composed by a
slippery sequence (underlined ) and a
stem-loop structure
28

29. Processing of the nonstructural proteins.
29

30. Processing of the nonstructural proteins.
• Nsp1a & nsp1ab are processed by cellular & viral (closed &open
triangles) proteases. (cleavages around aa residues 410 and 654
have been confirmed to be due to v-Pro).
• indirect evidence of protein processing products observed suggests
that the downstream cleavages (dashed triangles) are also due to
viral protease.
• No products from the hydrophobic region have been identified and
the smaller products of 5.5 and 6.5 kd have not been mapped into
nsp1a (dotted boxes) 29

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Structural Polyprotein Synthesis and Processing
• The structural proteins of HAstV, encoded in ORF2, are
synthesized from the sgRNA as a polyprotein precursor of 87 to 90
kd.
• Studies with HAstV8 have revealed that the 90 kd primary
translation product is intracellularly cleaved at Asp657 to yield a
product of 70 kd (named VP70) through intermediates of 75 to 85
kd,94 whose biological relevance, if any, is unknown.
• Processing of VP90 to VP70 is carried out by cellular enzymes
(caspases) that are involved in apoptotic processes, and whose
activity is triggered during viral infection by an unknown
mechanism.

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Structural Polyprotein Synthesis and Processing
• some pro-apoptotic factors—such as TNF-related apoptosis-
inducing ligand (TRAIL) and staurosporine, triggers different
apoptotic pathways—enhance the cleavage of VP90 to VP70.
• Extracellular particles of HAstV-8 formed by VP70 are weakly
infectious, but its infectivity is strongly enhanced by treatment of
the viral particles with trypsin

33. Processing of the structural protein VP90
• The pr. product of ORF2 is sequentially processed at its C-
terminus by caspases to generate VP70, protein present in
the extracellular particles 32

34. Processing of the structural protein VP90
34
• These particles are processed by trypsin to generate protein
intermediates of variable size, and the final products VP34, VP27,
and VP25.
• Closed arrowheads represent cleavages carried out by caspases;
open, long arrowheads represent cleavages by trypsin.
• The cleavage sites indicated by dashed arrowheads have not
been precisely determined, therefore the C-terminus of the
intermediate and final products is unknown.

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Transcription/Replication
• Astrovirus RNA synthesis has been poorly studied.
• it is believed that these viruses follow a strategy of alphaviruses
to replicate and transcribe their genome.
• Based on this assumption, the gRNA would be used as a template
to synthesize a full-length negative-sense RNA, gRNA(-), which in
turn would be used as a template to produce both the full length
gRNA and the sgRNA.
• Synthesis of gRNA(-) and accumulation of gRNA require cell
protein synthesis, but not cellular DNA transcription.

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Transcription/Replication
• v-Pro, RdRp, VP90, gRNA(-), and viral particles have all been found
to be associated with internal cell membranes.
• This suggests that RNA replication and the first steps of
morphogenesis are carried out associated with the observed
membranous structures that probably derive from the ER, since
viral phosphoproteins of 21 to 27 Kd, which interact with the
RdRp, and are likely involved in RNA replication, localize to this
organelle.

37. Assembly and Release
• The expression of ORF2 in cultured cells using recombinant vaccinia
virus or baculovirus as vectors leads to the assembly of virus-like
particles, indicating that the encoded protein is able to assemble in
the absence of viral RNA; however, these particles were unstable and
showed atypical morphology when purified, indicating a defective
assembly.
Model of VP90 processingand
virus maturation 36

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• Virus assembly tolerates some deletion or changes in 70 N-terminal
basic amino acids of VP90/VP70, which are thought to interact with
the gRNA in the particles
• Proteolytic processing by caspases of the VP90 assembled into
particles, to yield VP70, is required for cell egress of the virus
• The ORF2 primary product of HAstV-8, VP90, forms intracellular
particles that can be found associated with membranes or in the
cytosol.
• VP90 assembles into particles associated with membranous structures,
where viral nonstructural proteins and the sense and antisense
genomic RNA are also present.
• Proteolytic processing by caspases of the VP90 assembled into
particles, to yield VP70, is required for cell egress of the virus, since
inhibitors of these proteases prevents it, while it is promoted by pro-
apoptotic factors, such as TRAIL (TNF-related apoptosis-inducing
ligand (TRAIL).

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Pathogenesis
• As a gastrointestinal pathogen,
• HAstV - transmitted to a host through the fecal–oral route, also
person to person, (outbreak of gastroenteritis in California).
• in adults - exposure to a larger dose of astrovirus - through
fomites, and contaminated food or water.
• avian viruses transmission is also through a fecal–oral route,
vertical transmission from parents to their offspring.
• Mammalian astroviruses affect mainly epithelial cells of the
intestinal tract.

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• Histopathologic studies of an immunocompromised child
persistently infected, having a pronounced diarrhea, showed
that astrovirus infection was limited to the small intestine.
• Infection involves the mature epithelial cells near the microvilli
tips - more extensive in the jejune than in the duodenum, but
not in the stomach
• Morphologic abnormalities in the intestine suggested that,
despite severe diarrhea, an inflammatory response does not
occur.
• can infect epithelial cells (OAstV and BoAstV) as well as sub-
epithelial macrophages (OAstV) and M cells (BoAstV) of the
small intestine.
• Astrovirus was recently found as the only pathogen in the central
nervous system (CNS) of an immunodeficient patient who died
of encephalitis

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DIAGNOSIS
• Electron Microscopy
• Enzyme Immunoassays
• Molecular Techniques
PREVENTION AND CONTROL
Gastroenteritis caused by Astrovirus is generally a mild, self-
limiting illness does not require specific therapy.

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