Thesis
Reference
Recombinaison expérimentale intra-et interespèce chez les Rhinovirus et Quantification d'ARN de Rhinovirus par RT-PCR en
temps réel
SCHIBLER, Manuel
Abstract
Les rhinovirus sont des petits virus à ARN positif appartenant au genre Enterovirus de la famille des Picornaviridae, caractérisée par une importante variabilité génétique acquise par mutation et recombinaison. La première partie de cette thèse est consacrée à l'étude de la recombinaison des rhinovirus en utilisant des génomes chimériques synthétisés in vitro ainsi que la recombinaison non réplicative, résultant de la co-transfection de génomes défectifs complémentaires. Nous avons ainsi observé que la région 5'non codante est interchangeable entre membres d'espèces differentes, alors que seule la recombinaison intraespèce semble viable dans la région codante. Les sites de recombinaison obtenus sont décrits. La seconde partie de ce travail consiste en une analyse critique des erreurs possibles liées à la quantification d'ARN de rhinovirus dans des échantillons cliniques par RT-PCR en temps réel.
Différentes expériences de validation montrent qu'une quantification relative est possible avec une marge d'erreur de plus ou moins 10%.
SCHIBLER, Manuel. Recombinaison expérimentale intra-et interespèce chez les
Rhinovirus et Quantification d'ARN de Rhinovirus par RT-PCR en temps réel. Thèse de doctorat : Univ. Genève, 2013, no. Sc. Méd. 13
URN : urn:nbn:ch:unige-311007
DOI : 10.13097/archive-ouverte/unige:31100
Available at:
http://archive-ouverte.unige.ch/unige:31100
Disclaimer: layout of this document may differ from the published version.
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Section de médecine fondamentale Département de médecine génétique et de laboratoire
Service de médecine de laboratoire
Thèse préparée sous la direction du Docteur Caroline Tapparel et du Professeur Laurent Kaiser
Recombinaison Expérimentale Intra- et Interespèce chez les Rhinovirus et Quantification d’ARN de Rhinovirus par RT-PCR
en Temps Réel
Thèse
présentée à la Faculté de Médecine de l'Université de Genève
pour obtenir le grade de
Docteur en Sciences médicales « MD-PhD » par
Manuel David SCHIBLER de
Biberist (SO)
Thèse n° 13 Genève
2013
Aknowledgments
I would like to sincerely thank the following people who directly or indirectly contributed to this thesis:
Laurent Kaiser, for your trust, support, guidance and generosity throughout this long-lasting process
Caroline Tapparel, for your precious expertise in the rhinovirus field, and for your patience and commitment in making an MD do biology
Lara Turin and Sandra Van Belle, for your technical help
Samuel Cordey, for your constant availability and willing to help, especially when experiments became tricky
Yves Thomas and Pascal Cherpillod, for your advice and the nice atmosphere in the office Lorena Sacco and Michael Bel, for the nice breaks
Sabine Yerly, for your analytical assistance, particularly during the rhinovirus RNA quantification project
All other members of the Virology Laboratory, for the good times and nice working atmosphere
Dominique Garcin and Laurent Roux, for your availability and all the discussions we have had concerning my projects
Manel Essaidi, Carole Bampi and Geneviève Mottet-Osman for the fun we have had while you were teaching me the secrets of Western Blots
Mylène Docquier, for your useful advices regarding RNA quantification by real-time RT- PCR
Jérôme Pugin, Dominique Garcin, Francis Delpeyroux, Caroline Tapparel and Laurent Kaiser, for constituting such a great thesis Jury
My wife Lamarana, my parents Monika and Ueli, my sister Muriel and my friends, for their precious presence during this important period of my life
1
UNIVERSITY OF GENEVA FACULTY OF MEDICINE
Doctor Caroline Tapparel Professor Laurent Kaiser
Experimental intra- and interspecies Rhinovirus Recombination
and
Rhinovirus RNA quantification by real-time RT-PCR
THESIS
Presented to the Faculty of Medicine of the University of Geneva for the MD-PhD Doctorate in Medical Sciences
by
Manuel David SCHIBLER
from
Biberist (Switzerland)
Jury members:
Prof Jérôme Pugin - Faculty of Medicine of Geneva and Geneva University Hospitals (President)
Dr Caroline Tapparel - Faculty of Medicine of Geneva and Geneva University Hospitals (Thesis Director)
Prof Laurent Kaiser - Faculty of Medicine of Geneva and Geneva University Hospitals (Thesis Co-Director)
Dr Dominique Garcin - Faculty of Medicine of Geneva, University of Geneva (Local Expert) Prof Francis Delpeyroux - Pasteur Institute, France (External Expert)
2 Table of contents
Introduction ... 5
RHINOVIRUS CLASSIFICATION ... 5
VIRION STRUCTURE AND GENOMIC ORGANIZATION ... 7
RHINOVIRUS LIFE CYCLE ... 12
Viral entry ... 12
HRV translation and polyprotein processing ... 13
Replication ... 16
Autossembly and release. ... 19
MECHANISMS UNDERLYING HRV GENETIC VARIABILITY ... 22
High mutation rate ... 22
Recombination ... 23
HRV VERSUS HEV PHENOTYPES in cell culture ... 28
HRV TRANSMISSION, IMMUNE RESPONSE AND PATHOGENESIS ... 29
Route of transmission and site of infection ... 29
Immediate host defense mechanisms: innate immunity ... 30
Cellular and humoral immune responses ... 33
Pathogenesis ... 34
CLINICAL MANIFESTATIONS RELATED TO HRV INFECTIONS ... 34
Rhinosinusitis and otitis ... 35
Lower respiratory tract infection ... 35
Exacerbation of underlying airways diseases ... 35
Asymptomatic patients ... 36
EPIDEMIOLOGY ... 36
RHINOVIRUS DETECTION ... 37
3
HRV isolation in cell culture ... 37
HRV antigen detection ... 38
HRV RNA detection ... 38
ANTIVIRAL DRUGS AGAINST HRVs ... 39
ANTI-HRV VACCINES ... 41
Objectives... 42
EXPERIMENTAL INTRA- AND INTERSPECIES RHINOVIRUS RECOMBINATION ... 42
RHINOVIRUS RNA QUANTIFICATION BY REAL-TIME RT-PCR IN RESPIRATORY SPECIMENS ... 43
Materials and methods ... 43
PLASMIDS AND CONSTRUCTS ... 43
Construction of the chimeric HRV P1-2A genomes ... 45
Construction of the deleted HRV “parental” genomes used in non replicative recombination experiments ... 50
RNA EXTRACTION AND REVERSE TRANSCRIPTION ... 52
IN VITRO TRANSCRIPTION AND TRANSFECTION ... 52
CELL CULTURE ... 52
VIRAL CULTURE ... 53
IMMUNOFLUORESCENCE ... 53
SEQUENCING AND MAPPING OF RECOMBINATION SITES ... 54
Results ... 55
EXPERIMENTAL INTRA- AND INTERSPECIES RHINOVIRUS RECOMBINATION ... 55
Chimeric HRV genomes generated by molecular cloning methods ... 55
Non replicative recombination ability of artificially engineered defective viral genomes ... 60
4
RHINOVIRUS RNA QUANTIFICATION BY REAL-TIME RT-PCR IN RESPIRATORY SPECIMENS:
ARTICLE 2 ... 65
Discussion ... 66
EXPERIMENTAL INTRA- AND INTERSPECIES RHINOVIRUS RECOMBINATION ... 66
Artificially engineered chimeric HRV genomes ... 67
Experimental non replicative HRV RNA recombination ... 68
HRV RNA QUANTIFICATION BY REAL-TIME RT-PCR IN RESPIRATORY SPECIMENS ... 71
CONCLUSIONS AND PROSPECTS ... 72
References ... 74
Appendix ... 90
ARTICLE 3 ... 90
5
Introduction
RHINOVIRUS CLASSIFICATION
Although taxonomic criteria are under constant debate and evolution, the International Committee on Taxonomy of Viruses (ICTV) developed a reference strain for virus classificiation (http://ictvonline.org/). Based on this system, the Picornaviridae family, belonging to the Picornavirales order, is currently divided into 12 genera: Enterovirus, Cardiovirus, Hepatovirus, Parechovirus, Erbovirus, Kobuvirus, Teschovirus, Sapelovirus, Senecavirus, Tremovirus and Avihepatovirus (Figure 1, http://ictvonline.org/ and http://www.picornaviridae.com). Recently, five new genera were proposed: "Aquamavirus",
"Cosavirus", "Dicipivirus", "Megrivirus" and "Salivirus".
Human pathogens are found among the Enterovirus, Cardiovirus, Hepatovirus, Parechovirus and Kobuvirus genera [1].
The Enterovirus genus, the largest of the family, is constituted of 10 human species: Human enterovirus A, Human enterovirus B, Human enterovirus C, Human enterovirus D, Simian enterovirus A, Bovine enterovirus, Porcine enterovirus B, Human rhinovirus A, Human rhinovirus B and Human rhinovirus C (Figure 1). According to the International ICTV, two members of the Enterovirus genus are considered to belong to the same species if they share more than 70 % amino acid (aa) identity in P1; if they share greater than 70 % aa identity in the non-structural proteins 2C and 3CD; if they share a limited range of host cell receptors; if they share a limited natural host range; if they have a genome base composition (GC) that varies by no more than 2.5 %; and if they share a significant degree of compatibility in proteolytic processing, replication, encapsidation, and genetic recombination (International
6
Committee on Taxonomy of Viruses,
http://talk.ictvonline.org/files/ictv_official_taxonomy_updates_since_the_8th_report/m/verteb rate-official/1201.aspx). Each species is further divided in several types. Concerning the rhinovirus species, 100 Rhinovirus (HRV) serotypes were historically defined by cross- neutralization properties in vitro [2, 3]. This classification obviously concerned only cultivable viruses. Later, a correlation between phylogenetic relationships of capsid proteins encoding sequences in the VP4/VP2 region or VP1 and serotypes has been established [4, 5]
and HRVs are now classified by sequence similarities. Accordingly, the term „serotype‟ tends to be replaced by the terms „genotype‟ or simply „type‟. A nucleotide sequence divergence in the VP1 gene higher than 12% for HRV-A and -B and than 13% for HRV-C separates two strains into different types inside the same species [6]. This classification system also allows type assignment for viruses that are not cultivable and thus cannot be submitted to serological characterization, such as HRV-Cs.
It is noteworthy that the 5‟UTR region, used as a PCR target to detect HRV, is not suited for reliable HRV typing, notably because HRV-A and HRV-Ca 5‟UTR sequences cluster together [7] (see “HRV recombination” below).
HRVs are currently divided into 153 types (77 HRV-A types, 25 HRV-B types and 51 HRV- C types) (Figure 1 and Nick Knowles, 2012, picornaviridae.com, http://www.picornaviridae.com/enterovirus/enterovirus.htm). This number will likely increase rapidly with the ongoing sequencing of previously undetected strains.
7
Figure 1. Picornavirus genera and enterovirus species
The picornavirus genera are indicated in bold and the enterovirus species in normal characters. Black, grey, and white panels indicate genera and species with viruses infecting respectively animals without humans, animals including humans, and humans only. The proposed new genera or species are indicated in italics. The number of genotypes according to the host is indicated in brackets for each enterovirus species.
SAFV, saffold Attachment Virus; EV, enterovirus; BEV, bovine enterovirus; PEV, porcine enterovirus; SEV, simian enterovirus; RV, rhinovirus; swi, swine; sim, simian; hum, human (Adapted from [8]).
VIRION STRUCTURE AND GENOMIC ORGANIZATION
The HRV genome is protected by a non-enveloped icosahedral capsid constituted of four structural proteins, VP1, VP2, VP3 and VP4. Most of the capsid‟s interaction with the environment resides in the VP1 protein. VP1, and to a lesser extent VP2 and VP3 proteins are the most exposed viral proteins and the targets of the immune response [9-11]. VP4 is localized on the inner surface of the capsid and interacts with genomic RNA [12].
8
Each of the capsid protein assembles to form a protomer, and 60 protomers assemble to form the icosahedral capsid. Five VP1 tips join and form a five-fold symmetry axis (Figure 2).
There are 12 such five-fold axis in an icosahedral capsid. VP2 and VP3 tips join and form a three-fold symmetry axis. There is a valley separating the VP1 surfaces from the VP2 and VP3 surfaces that surrounds each five-fold axis. In HRVs this valley is particularly deep and is referred to as the canyon [13]. The so-called hydrophobic pocket situated at the bottom of that canyon is involved in receptor binding and is an antiviral drug target [14, 15].
Although there is no sequence homology among the structural proteins, VP1, VP2 and VP3 all display an eight-stranded anti-parallel β–barrel structure. These β–strands are separated from each other by loops that protrude out from the viral capsid. These loops, along with the NH2 and COOH ends of the capsid proteins, are variable among the different picornaviruses and are responsible for their different receptor-binding and immunogenic features [16].
Figure 2. A. Schematic HRV icosahedral capsid diagram showing the subunit organization and canyon (shaded).
Thick lines encircle five protomers of VP1-VP4. Two five-fold symmetry axis (black pentagons), two three-fold symmetry axis (black triangles), and three two-fold symmetry axis (black eye shaped symbols) are indicated (adapted from [16]).
9
The HRV genome consists of a single positive-sense RNA whose length is about 7‟200 bases.
It can be subdivided into four parts: a 5‟ untranslated region (5‟UTR); a single open reading frame (ORF); a short 3‟ untranslated region (3‟UTR); and a poly(A) tail [17] (Figure 3A). The open reading frame codes for a polyprotein that is co- and post-translationally cleaved into four structural proteins (VP4, VP2, VP3 and VP1) and seven non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). The genomic RNA also harbours a stem-loop structure called the cis-acting replication element (cre), which is essential for replication initiation. In HRVs, this cre stem-loop structure is part of the ORF, and its location varies in the three HRV species. It lies within the 2A coding region for HRV-A, VP1 for HRV-B and VP2 for HRV-C (Figure 3B). In contrast, all HEV species have their cre situated in the 2C region [18]. A conserved AAAC sequence, found in all currently known picornavirus cre loop sequences (Figure 3B), is implicated in virion protein genome-linked (VPg) urydylation, a process essential to prime picornavirus replication.
The Enterovirus 5‟UTR folds into six structurally distinct domains that can be divided into two functional units: a 5‟ cloverleaf structure (CL) essential for replication (domain I) and a type I internal ribosome entry site (IRES) (domains II-VI) necessary for the cap-independent translation of the polyprotein ORF [19] (Figure 3C). The CL contains four structural domains:
stem A, stem-loop B, stem-loop C and stem-loop D. Stem-loop B is a known binding site for poly(rC)-binding protein 2 (PCBP2), and stem-loop D interacts with the viral 3C and 3CD proteases. The functions of stem A and stem-loop C are currently unknown. The CL, cellular PCBP2 and viral 3C and 3CD are all implicated in the regulation of picornavirus translation and replication. Two different models describe putative mechanisms for the switch from translation to replication regarding the poliovirus life cycle. In the first model, PCBP2 binding to the CL stem-loop B stimulates the PV polyprotein translation, and once a sufficient amount of viral 3CD is present, it binds to stem-loop D to repress translation. The positive strand PV
10
RNA thereby becomes available for minus strand RNA synthesis [20]. Alternatively, according to the second and more recent model, PCBP2 binds to the IRES stem-loop IV and thereby stimulates translation. 3C and 3CD proteinases resulting from viral polyprotein translation then cleave PCBP2. This cleavage releases a specific domain, KH3. The remaining PCBP2∆KH3 protein, whose affinity for stem-loop IV but not for the CL stem-loop B is lost, is released from the IRES, and translation is no longer stimulated, leaving the viral genome accessible for replication [21]. Although replication regulation has been mainly attributed to the CL, sequences located in the 3‟ region of the poliovirus (PV) IRES may also be implicated [22]. The viral protein 3B (or VPg), is covalently bound to the 5‟ extremity of the viral genome. Its role in HRV RNA synthesis is discussed in the Replication section.
The HRV 3‟UTR, whose length ranges from 40 to 60 nuctleotides (nt), includes a 13 to 16 nt long stem, ending with the poly(A) tail. The function of this stem is not elucidated, but it may be implicated in protein binding or RNA:RNA interactions with other parts of the genome during the replication process. It has been shown that the 3‟UTR is not essential for poliovirus RNA replication, but its presence greatly enhances its efficiency [23].
11
Figure 3. A. HRV genomic organization (adapted from [17]). B. Sequence and secondary structure of an HRV- A, an HRV-B and an HRV-C cre stem-loop. The conserved AAAC key-sequence is shown in bold (adapted from [24]). C. Enterovirus 5‟UTR structure depicting the 6 characteristic domains (I-VI). The subdomains of the cloverleaf structure are also shown (A-D) (adapted from [25]).
A.
B.
C.
B A C
I. D II
.
III.
IV.
V.
VI.
12
RHINOVIRUS LIFE CYCLE
Viral entry
Rhinoviruses use different receptors. Most HRV-A and all HRV-B bind to intracellular adhesion molecule 1 (ICAM-1) via the above mentioned canyon region of the capsid. In contrast, 11 HRV-A members [26] use the low density lipoprotein receptor (LDLR), which binds to the five-fold axis. Finally, the HRV-C receptor(s) is(are) currently unknown, and seem(s) to be different from ICAM-I and LDLR, based on bioinformatic comparisons and inhibition assays [27, 28]. In silico studies suggest that an ICAM-I-like molecule may serve as an HRV-C receptor [29].
HRV entry has been studied intensely for HRV-A2 and HRV-B14. However, the precise mechanisms concerning viral particle conformational changes, internalization, uncoating and RNA release into the cytosol remain largely unknown (reviewed in [30]).
Binding of HRV to its specific receptor triggers conformational changes in the capsid. This initial conformational change is necessary for receptor-mediated endocytosis and further structural changes induced by the low-pH endosomal environment that are responsible for uncoating and RNA release into the cytoplasm [30](Figure 4). This likely involves endosomal membrane rupture or pore formation by viral capsid proteins. The fact that empty HRV-14 capsids have been highlighted in the cell cytosol favors the endosomal rupture model [30]. It is hypothesized that the viral RNA might be released into the cytosol concomitantly to this process.
13
Figure 4. Model for HRV entry and uncoating.
The virus binds to its receptor (ICAM-1 or LDLR) at the plasma membrane (1.), is uptaken by various endocytic pathways depending on the cell type (2.) and is delivered to early endosomes (3.). Viral capsid conformational changes occur following receptor binding and exposure to low pH in late endosomes (4.), resulting in the release of viral capsids and HRV RNA release into the cytoplasm (5.). Receptors present in the early endosomes are recycled to the cell surface (6.).
HRV translation and polyprotein processing
The RNA released in the cytoplasm is of positive polarity and thus directly translated by host cell ribosomes. For translation to occur, VPg is cleaved from the 5‟end of the viral genome by
14
a cellular enzyme named VPg-unlinkase. This enzyme has been proposed to serve as a marker to distinguish viral RNAs used for translation (devoid of VPg) from those implicated in replication (VPg-linked) [31]. Picornaviruses ORF translation is cap-independent and mediated via the IRES. The eukaryotic initiation factors eIF4G, eIF4A, and eIF4B are recruited to the 5‟UTR domain V of the IRES. This ribonucleoprotein (RNP) complex in turn recruits the 40S ribosomal subunit associated with eIF3 and eIF2-GTP-met-tRNA. The 40S subunit then scans the 3‟ region of the 5‟UTR until it reaches the authentic start codon where the complete ribosome is assembled [32].
In addition to bypass cap-dependant translation, HRVs efficiently inhibit host cell translation.
As shown for poliovirus, HRV 2A protease cleaves the eIF4G factor (formerly p220), whose integrity is essential for cap-dependant translation [33].
Various cellular proteins called IRES trans-acting factors (ITAFs) are also implicated in HRV translation regulation [34].
Translation results in the synthesis of a viral polyprotein precursor of about 2000 aa which is co- and post-translationally cleaved into smaller protein products. The first cleavage occurs between the P1 and P2 region and is mediated by the viral 2A proteinase. The second cleavage is made by the 3C proteinase at the P2-P3 junction (Figure 5). All subsequent cleavages are carried out by the 3C proteinase, except for the cleavages between VP0 and VP3 and between VP3 and VP1 that are performed by the 3CD precursor proteinase [35].
VP0 cleavage into VP4 and VP2 occurs later in the viral life cycle, during progeny virions maturation, and is probably mediated by an autocatalytic process [36]. The various cleavage products and their functions are shown in Figure 5. Of note, some protein precursors, such as 2BC, 3AB and 3CD, display specific functions in the infected cell (Figure 5).
As shown in Table 1, some polyprotein cleavage sites vary among the different HRV and HEV species. This may limit the interspecies HRV recombination possibilities, as discussed
15
later. The polyprotein cleavage sites of 7 HRV-A genomes, 3 HRV-B genomes, 3 HRV-C genomes, 1 HEV-A genome, 1 HEV-B genome, 2 HEV-C genomes, and 2 HEV-D genomes are shown in Table 1. As the number of HRV and HEV genomes used in this analysis is limited, the existence of additional cleavage sites cannot be excluded.
Figure 5. HRV and HEV polyprotein processing cascade and mature protein functions. The primary and secondary polyprotein cleavages are depicted. The viral proteinases are highlighted in dark blue. The known functions of the various cleavage products are described (adapted from [37]).
16
Table 1. Polyprotein cleavage sequence according to the three HRV species and the four HEV species.
Polyprotein cleavage
site
Protease HRV-A
(HRV-A1, -A2, -A16,
-A39, - A81, -
A89, -A100)
HRV-B
(HRV-B14, -B84, -
B42)
HRV-C
(HRV- C11, -C6,
-C28)
HEV-A
(HEV-A71)
HEV-B
(HEV-B69)
HEV-C
(PV-1, CV- A20)
HEV-D
(HEV-D68, -D94)
VP4-VP2 - Q-S N-S M-S K-S S-P N-S L-S
VP2-VP3 3C Q-G Q-G Q-G Q-G Q-G Q-G Q-G
VP3-VP1 3C Q-N E-G Q-N Q-G Q-N Q-G Q-S
Q-G D-L Q-L
VP1-2A 2A V-G Y-G A-G L-G H-G Y-G T-G
A-G L-G
V-G
2A-2B 3C Q-G Q-G Q-G Q-G Q-G Q-G Q-G
2B-2C 3C Q-S Q-A Q-S Q-S Q-N Q-G Q-G
E-S Q-S Q-G Q-S
2C-3A 3C Q-G Q-G Q-G Q-G Q-G Q-G Q-G
Q-Y
3A-3B 3C Q-G Q-G Q-G Q-G Q-G Q-G Q-G
3B-3C 3C Q-G Q-G Q-G Q-G Q-G Q-G Q-G
3C-3D 3C Q-G Q-G Q-G Q-G Q-G Q-G Q-G
F-E
Replication
Picornavirus replication is a two-step process taking place in membrane-associated replication complexes. The first step consists of the synthesis of a negative-sense full length RNA that is complementary to the viral positive sense genome and the second step implies the synthesis of new positive sense viral genomes from the negative sense RNA templates. As for translation initiation, the precise mechanisms implicated in HRV replication are not fully understood and most of the data derive from research involving poliovirus.
17
To initiate negative sense RNA synthesis, the picornavirus 3D RNA-dependant-RNA- polymerase (RdRp) requires a primer. This primer, VPg-pU-pU, is constituted of the viral VPg protein, to which two uridine residues are covalently linked by the 3D polymerase by a process called VPg-uridylylation. This process is orchestrated by the cre, which serves as template via a specific and conserved AAAC sequence (Figure 3B) to which 3CD binds and thereby stimulates 3D-mediated VPg uridylylation. VPg-pU-pU remains linked to 3D, and acts as a primer, enabling negative sense RNA synthesis initiation (Figure 6) at the 3‟end of the viral poly(A) tail [24].
Figure 6. Cre-dependent VPg uridylylation. The scheme depicts VPg uridylylation by 3D on the cre AAAC template, with 3CD being bound to the cre and promoting the process (adapted from [24]).
In parallel to VPg uridylylation, according to a current model, the 3AB precursor protein associates with cellular membrane elements via its hydrophobic domain, while 3CD precursor protein binds to the D domain of the CL (see above) and PCBP2 binds to the B domain of the CL. The complex formed by the viral positive-strand RNA and these two proteins is recruited to cellular membrane elements via an interaction between membrane-associated 3AB and PCBP2. A cellular protein, poly(A)-binding protein (PABP), in turn binds to the viral genome
18
poly(A) tail, and interacts with 3CD and PCBP2 which are bound to the CL, thereby circularizing the viral RNA. This process is required for negative sense RNA synthesis initiation [38].
In addition to the 5‟UTR and the cre, cis-active RNA structures implicated in replication are included in the 3‟UTR. Although not essential for poliovirus replication, the 3‟UTR has been shown to greatly increase replication efficiency [23, 39]. The poly(A) tail is required for replication and its length influences replication efficiency [40].
The double-stranded RNA helix constituted of a positive strand RNA and the complementary negative strand RNA is called the replicative form (RF) [41].
After synthesis of the minus strand RNA, new positive strand RNAs can be synthesized. In fact, 40 to 70 progeny positive strand RNAs derive from a single negative strand RNA [42].
Positive-strand synthesis is also initiated via VPg-pU-pU priming. In this instance, the two uridine residues hybridize to the two adenosine residues situated at the 3‟end of the negative strand RNA. Partial denaturation of the RF is needed for this process to happen. How the separation of the two RNA strands of the RF occurs is not yet known [41, 43].
The newly synthesized positive RNA strands are either used as progeny genomes encapsidated into new viral particles or as templates for further translation.
To date, it is still not elucidated whether cre-dependant VPg uridylylation is implicated in positive-strand RNA synthesis, in negative-strand RNA synthesis, or in both processes. Some researchers attribute a role of the cre motif only for positive-strand RNA synthesis [44, 45], whereas others have found evidence suggesting that cre-dependant VPg uridylylation is only involved in negative-strand RNA synthesis [46, 47].
As mentioned above, picornaviruses replication does not occur randomly in the infected cell cytoplasm. Like all known plus-strand RNA viruses, picornaviruses replicate in close
19
relationship to intracellular membranes. In the case of picornaviruses, these membranes seem to be derived from the endoplasmic reticulum-golgi apparatus network [48] and autophagosomes [49], and are rearranged to form vesicles, by the viral protein 2C [50]. The HRV 3A protein also seems to be implicated in membranous rearrangements [51]. The association of picornavirus replication to membranous vesicles represents a strategy providing several advantages: it enables the concentration of viral components to a defined compartment, it may promote the spatial organization of replication complexes, and the membranes may protect the viral replication machinery from the cellular innate immunity sensors [48].
Autossembly and release.
Little is known about rhinovirus assembly and release processes. Rhinovirus assembly supposedly occurs in a fashion similar to that happening in polioviruses [52]. The first step is the assembly of VP1, VP3 and VP0 precursor into a 5S protomer, which is thought to form co-translationally. Five protomers then assemble into a 14S pentamer, and 12 pentamers are linked together to form the icosahedral empty 80S capsid. Whether this capsid forms around the viral RNA or the RNA enters pre-assembled capsids is still not elucidated. The final step in the maturation of the viral particle is the autocatalytic cleavage of VP0 into VP4 and VP2 [53]. Release of viral particles, at least concerning poliovirus, is classically thought to occur via cell lysis induced by the viral infection [54]. However, a more recent model suggests that vesicles derived from autophagosomes and induced by replication of poliovirus and HRVs might trap mature viral particles. These could ultimately be delivered into the extracellular medium by membrane fusion, thus avoiding cell lysis [49]. This would be in agreement with
20
the observation of intact respiratory epithelium during the course of in vivo HRV infections [55].
Figure 7 summarizes the key steps involved in the HRV life cycle.
21
Figure 7. Summary of the HRV life cycle (adapted from [54]). The virion recognizes its receptoir on the host cell‟s surface (1). The interaction between the viral capsid and the receptor triggers viral entry by endocytosis (2). Once the viral genome is released into the cytoplasm, it is directly translated by cellular ribosomes, in a CAP-independent manner (3). The viral polyprotein is co-translationnaly and autocatalytically processed into individual viral structural and non structural proteins (4). Once present in sufficient amounts, the viral 3D RNA polymerase initiates negative-strand RNA (5) and then positive-strand RNA (6). The latter is either used for further translation (7) or as a viral genome packed in newly assembled capsids during morphogenesis (8), leading to progeny virions which are released from the infected cell (9).
22
MECHANISMS UNDERLYING HRV GENETIC VARIABILITY
As discussed above, there are more than 150 HRV genotypes (http://www.picornaviridae.com) [6]. The main explanations for such an important genetic variability are the high error rate of the viral RNA-dependant RNA polymerase and recombination [56].
High mutation rate
The RNA-dependant RNA polymerase is characterized by a high mutation rate, estimated around 10-4 mutations per nucleotide. This represents almost one mutation per HRV genome per replication cycle [56]. During an infection, the viral population is thus not genetically homogeneous, but each genome differs by one or several point mutation(s). Such a viral population harbouring a “cloud” of related but slightly different genomes is called a quasispecies (Figure 8). The advantage of such a heterogeneous genetic repertoire is that upon environmental changes, one or several genomes may already be suited to the new environmental conditions. This or these will be selected and further mutate to recreate a diverse population.
23
Figure 8. Generation of a viral quasispecies. This scheme illustrates in a simplified manner how a quasispecies arises starting from a single viral genome. In these trees, each branch links together viral genomes differing by one point mutation, and each circle represents a replication cycle (Adapted from [57]).
Recombination
Viral recombination involves the exchange of genomic fragments between two different viruses. These two viruses need to be sufficiently similar to be compatible and to generate functional recombinant progeny. This classically occurs between two viruses belonging to the same species.
Intraspecies recombination events have been extensively described regarding HEVs and are considered as an evolutive driving force for this virus group [58, 59]. The HEV recombination breakpoints mostly map around the 5‟ (VP4) and 3‟ ends (VP1-2AB junction) of the P1 region, while they are almost absent in the VP2-VP3-VP1 capsid region (Figure 9A)[58, 60].
A frequently observed HEV intraspecies recombination phenomenon concerns circulating vaccine-derived poliovirus (cVDPV) strains. These cVDPVs, whose pathogenicity can be
24
similar to that of wildtype PV, result from recombination between attenuated oral polioviruses and co-circulating non-poliovirus HEV-C members [61-63].
Some recent natural interspecies recombination events have also been described among circulating HEVs [64-66]. The recombination sites in these instances have been mapped to the 5‟UTR and the 3D region (Figure 9B).
Recombination events seem to occur less frequently among circulating HRVs. The recombination breakpoints identified such circulating HRV recombinants are situated at the 3‟
end of the 5‟UTR and at the 5‟ end of the 3C gene (Figure 9C) [67].
Whether the difference in recombination frequency between the two virus groups is related to the type and site of infection, the frequency of co-infection or genomic features remains an open question.
Phylogenetic studies indicate that interspecies HRV recombination occurred in the past. For instance, recombination between the 5‟UTR of HRV-A and the polyprotein of HRV-C was proposed as the mechanism at the origin of the HRV-Ca subgroup that harbours HRV-A-like 5‟UTR sequences [68, 69]. The remainder HRV-C strains, called HRV-Cc, exhibit 5‟UTR sequences divergent from those of HRV-A, HRV-B and HRV-Ca members. Three putative interspecies recombination breakpoints in the 5‟UTR have been mapped for HRV-Ca strains around position 481, 565, in the polypyrimidine tract, and 523, within stem-loop 5 of the IRES (Figure 9D)[69]. In the majority of the sequences analyzed, recombination presumably occurred in either one of the last two recombination hotspots, which are located in highly conserved sequence stretches. These two particular locations may therefore represent preferred sites for other interspecies 5‟UTR recombination within the Enterovirus genus.
Furthermore, some HRV-C strains harbour short HRV-A sequences in their 2A region (Figure 9D) [68, 69]. Analysis of the full-length sequences of all known HRV types in 2009 suggests that some HRV types resulted from ancient intraspecies recombination [70]. The majority of
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the recombination sites revealed by this analysis are situated in the 5‟UTR and the adjacent P1 region (Figure 9E).
Figure 9. Major genomic regions involved in HEV and HRV recombination (in red). HEV intraspecies recombination among circulating strains (A). HEV interspecies recombination sites among circulating strains (B). HRV intraspecies recombination sites among circulating strains (C). Ancient HRV interspecies recombination between HRV-A and HRV-C species (D). Ancient HRV intraspecies recombination resulting in new HRV types (E).
Finally, based on full genome phylogenetic analysis, it was proposed that ancient recombination events between HRV-A and HEV members gave rise to the HRV-B species (Figure 10) [71].
A.
B.
C.
D.
E.
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Figure 10. The HRV-B species might have arisen from recombination between HRV-A and HEV members.
The whole-polyprotein, maximum likelihood phylogenetic tree shows a closer relation between HRV-B and HEV than between HRV-A and HRV-B. The percentage of bootstraps (out of 1000) supporting corresponding clades is indicated. The sequence of simian picornavirus 1 (SV-2) was used as an outgroup. The branch lengths are measured in substitutions per site (adapted from [71]).
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However, based on sequence homology, all the above proposed natural interspecies recombination events likely occurred between ancestors of the current HRV circulating strains.
Two RNA virus recombination models have been proposed. The first is referred to as the
“copy-choice” or “template switch model” (Figure 11A). The viral RNA polymerase synthesizes an RNA fragment complementary to the positive sense RNA template originating from one of the two viruses co-infecting the same cell, before being released from that template and resuming RNA synthesis on a neighbour genome originating from the other infecting virus [72]. Gmyl et al unraveled an alternative poliovirus recombination mechanism in vitro, called “non replicative RNA” recombination (Figure 11B), in which two different viral genomes are cleaved and joined together, resulting in a chimeric genome [73]. In their study, deleted poliovirus genomes were designed to promote non replicative recombination in the spacer region of the poliovirus RNA, situated between the IRES and the coding sequence and known to tolerate sequence variations without altering viability. Poliovirus genomes consisting of intact CL and IRES elements but lacking the polyprotein and 3‟UTR sequences were co-transfected with genomes harbouring intact polyprotein and 3‟UTR sequences but lacking an essential cis-acting element of the 5‟UTR. Rescued polioviruses were sequenced, allowing recombination sites mapping. The vast majority of them were found in the spacer region. The exact molecular reactions underlying this type of recombination are not understood.
While both mechanisms may be implicated in recombination events, it is currently not possible to determine which one is predominant, although it is generally believed that the copy-choice model is responsible for natural recombination.
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Figure 11. RNA viruses recombination models. Template switch model (A, adapted from [74]), and the non replicative RNA recombination model (B, adapted from [75].
HRV VERSUS HEV PHENOTYPES IN CELL CULTURE
In this section, in vitro HRV and HEV different phenotypic characteristics are described.
Two characteristics have been used in the past to differentiate HRVs and HEVs. The first is the optimal growth temperature. While enteroviruses replicate efficiently at 37°C, rhinoviruses were found to have an optimal growth temperature of 33°C, the temperature of the upper respiratory tract. This rule is not absolute however, as some respiratory enteroviruses such as EV-D68 multiply the best at 33°C [76], and several HRVs replicate well at 37°C [77]. The second characteristic that differentiates both virus groups is acid tolerance.
A. B.
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Most HEVs are resistant to acidic pH values of 3 and even lower [54], which is not surprising since they need to survive the acidic environment of the stomach, before reaching their replication site in the intestine. In contrast, HRVs are labile at pH below 6 [54], and completely inactivated at pH 3 [78]. Again, some enteroviruses, such as EV-D68 are acid labile [76].
HRVs and HEVs exhibit very different cell culture tropism. HRVs are mainly cultivated in human diploid fibroblast cell lines such as embryonic lung fibroblast cell lines WI-38 and MRC-5, and HeLa Ohio cells overexpressing ICAM-1 [79, 80]. Of note, the recently discovered HRV-Cs are not cultivable in standard cell lines, and as mentioned earlier, their receptor(s) is(are) not yet identified.
HEVs grow in a wide variety of human and non human cell lines [81]. However a given cell line is susceptible to some HEVs and not to others, depending on receptor usage [82].
Common cell lines used for HEV isolation include human rhabdomyosarcoma cells (RD), human embryonic lung cells (MRC-5), laryngeal cancer cells (Hep-2), human lung cancer cells (A549) and African green monkey kidney cells (Vero), among many others [81].
HRV TRANSMISSION, IMMUNE RESPONSE AND PATHOGENESIS
Route of transmission and site of infection
Experimental data suggest that HRVs are transmitted mainly by hand carriage, and infection occurs mostly via eye or nose, more rarely by inoculation into the mouth [83]. Aerosols appear to be an alternative transmission route [84, 85]. HRV particles deposited on the eye and transported to the nasal cavity via the lachrymal duct, and those deposited into the nose
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are carried via the mucociliary system to the nasopharynx, the major HRV replication site [86].
Immediate host defense mechanisms: innate immunity
Once the HRV genome is released in an infected cell, it is recognized by Toll-like receptors (TLR) and by a cytoplasmic pattern recognition receptor (PRR). The HRV capsid is detected by TLR2 on the epithelial surface, and HRV ssRNA and dsRNA are recognized by TLR3, TLR7 and TLR8 [87]. The cytoplasmic PRR involved in HRV genome detection is melanoma differentiation-associated gene 5 (MDA-5), which recognizes long double-stranded RNA [88, 89]. After having recognized picornaviral double-stranded RNA (i.e. the RF), MDA-5 activates interferon-β promoter simulator-1 (IPS-1) (also called Cardif, MAVS and VISA).
IPS-1 in turn activates the kinases TBK-1 and IKKε, which phosphorylate interferon regulatory factors 3 and 7 (IRF-3 and IRF-7). These transcription factors induce type I interferons (IFN) (IFN-β and IFN-α) gene expression [90] (Figure 12).
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Figure 12. Cytosolic recognition of various RNA viruses. The signaling pathway triggered by picornavirus double-stranded RNA detection is shown on the left (adapted from [91]).
Type I IFNs are secreted and act in an autocrine and paracrine manner by binding cell surface type I interferon receptor, which triggers an intracellular signaling cascade implicating the Jak/STAT pathway [92] (Figure 13). This results in the expression of more than several hundred interferon stimulated genes (ISGs), inducing an antiviral state that inhibits viral replication [93].
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Figure 13. Type I interferon signaling via the Jak/STAT pathway (Adapted from [94]).
However, this cellular defense mechanism, from viral detection to the establishment of the antiviral state, is the target of several viral proteins. For example, regarding viral detection by the host cell, MDA-5 is cleaved in a proteasome- and caspase-dependent manner in poliovirus infected cells [95], and IPS-1 is cleaved by HRV 2A and 3C proteases [96]. Furthermore, the antiviral state induced by type I IFNs can be reversed, at least to some point, as Enterovirus members (PV-1, PV-2, PV-3, EV-70, and HRV-A16) were shown to replicate in IFN-α treated cells. This ability was linked to the viral 2A protease [97].
Thus, a balance seems to exist between the host defense mechanisms that prevent massive tissue damage due to uncontrolled viral replication, and viral countermeasures that ensure sufficient HRV multiplication to allow viral transmission and survival.
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Cellular and humoral immune responses
The innate immune response triggered by rhinovirus infection, is implicated in the establishment of the adaptive cellular and humoral immune response.
T-cells
RANTES and IFN- -inducible protein (IP)-10 secreted by HRV-infected epithelial cells recruit T-cells to the infected airways [98]. It has been shown that a given CD4+ cell clone can be activated by exposure to different HRV serotypes, indicating the existence of shared epitopes [99]. Thus, memory CD4+ cells could be activated upon an HRV infection, and be implicated in a subsequent infection involving a serotype exhibiting common epitopes. The role of CD8+ T-cells in the immune response against HRVs is not yet defined.
B-cells
Anti-HRV antibodies are usually detected one to two weeks after inoculation, and the maximal titer is achieved five weeks after infection, with elevated titers persisting for at least one year [100]. Since the duration of an HRV infection is of a few days, with viral loads in nasal washes peaking at 48 to 72 hours post inoculation [101], the role of these antibodies is to prevent a subsequent infection involving the same HRV type rather than clearing the virus that triggers their synthesis [102, 103]. HRV-neutralizing antibodies protecting against another infection with the same HRV type are probably mucosal secretory IgAs [104].
However, as the number of different HRV types exceeds 150, a human individual can experience numerous HRV infections during his life.
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Pathogenesis
In addition to induce type 1 IFNs, viral detection by the host cell also results in the production of pro-inflammatory cytokines, via NF- B activation [105]. Several inflammatory mediators such as kinins, leucotriens, histamine, interleukins 1, 6 and 8, Tumor Necrosis Factor alpha (TNF-α) and Regulated upon Activation, Normal T-cell Expressed and Secreted (RANTES), were found to be increased in nasal secretions in the course of an HRV infection [106]. The inflammatory reaction induced by these molecules leads to vasodilatation and increased capillary permeability, causing rhinorrhea and nasal obstruction. Tissue damage resulting from HRV replication is almost inexistent, and the integrity of the respiratory epithelium remains intact upon an HRV infection [55]. This suggests that the pathogenic effects are mostly related to the innate immune response triggered by the infection. This hypothesis is supported by the correlation between interleukin-8 (IL-8) levels and symptoms following HRV infection [107].
CLINICAL MANIFESTATIONS RELATED TO HRV INFECTIONS
HRVs are the most frequent agent of the common cold, an infection restricted to the upper respiratory tract. The major symptoms include nasal obstruction and discharge. Sneezing, sore throat and cough are often associated. Although this illness is benign and self-limited, it generates important costs due to medical seeking and absenteeism, and is associated with inappropriate antibiotics use. Furthermore, HRV can cause more severe clinical manifestations in selected populations. Of note, it is not yet clear whether a given HRV genotype or species is associated with a particular clinical presentation [8, 108-111]. Results from a recent publication concerning HRV infections in infants suggest that HRV-A and
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HRV-C species are associated with infections causing similar clinical manifestations that are more severe than those caused by HRV-B strains [112].
Rhinosinusitis and otitis
HRV infections are not exclusively restricted to the nasal cavity and the pharynx, but also involve the para-nasal sinuses and are often associated with rhinosinusitis [106, 113]. In children, HRV infection frequently provokes a Eustachian tube dysfunction, which can lead to otitis media [114, 115].
Lower respiratory tract infection
HRVs have long been considered unable to infect the lower respiratory tract. This belief was based on observations indicating that the optimal HRV growth temperature was 33°C [116].
However, these experiments involved only one HRV serotype. It has been shown since then that the higher temperature found in the lower respiratory tract is not necessarily restrictive for HRV replication [77], and that these viruses do indeed infect this site [117-122].
Furthermore, HRVs have been identified as major pathogens of viral pneumonia among children, representing the most frequent virus group recovered from children with viral pneumonia in one study [123].
Exacerbation of underlying airways diseases
The use of PCR enabled to establish an association between rhinovirus infections and exacerbation of chronic lung conditions, such as chronic obstructive pulmonary disease (COPD) [124], asthma [105, 125, 126] and cystic fibrosis [127-129].
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It has been demonstrated that primary bronchial epithelial cells from asthmatic patients produced less IFN-β when compared to normal individuals [130]. It is therefore believed that viral clearance is decreased in these patients, resulting in increased inflammation and facilitated viral spread to the lower airways, explaining HRV-induced asthmatic exacerbations [98].
Asymptomatic patients
Asymptomatic HRV infections have been documented for all age categories [131-133] but seem to be more frequent among children [134]. However, asymptomatic chronic HRV shedding has not been clearly reported. Prolonged HRV shedding (8 months and longer) has only been documented among immunosuppressed patients [8, 121].
EPIDEMIOLOGY
HRVs are circulating throughout the year in the human population, but HRV detection rates peak in autumn and spring [135]. However, HRV infections acquired during winter months showed to be more severe than those occurring during spring and summer, in an infant population [112]. They are represented worldwide [136], and no particular geographic distribution of the various HRV types has been documented. Various HRV types circulate concomitantly, without following a predictable scheme [135].
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RHINOVIRUS DETECTION
The clinical specimens currently used for HRV detection are nasopharyngeal swabs (NPS), nasopharyngeal aspirates (NPA), nasal washes (NW), tracheal aspirates (TA), bronchial aspirates (BA) and bronchoalveolar lavages (BAL) [137] and the principal diagnostic means are virus isolation in cell culture and molecular assays. Electron microscopy is not used for routine diagnostics, due to the need for specialized equipment, the high costs linked to the technique, the slowness of the procedure and its reduced sensitivity compared to molecular diagnostic tools. Serology is neither used given the large number of different serotypes.
HRV isolation in cell culture
Assessment of viral multiplication in cell cultures via the observation of a cytopathic effect was the classical laboratory diagnostic method for HRV detection. Cell lines initially used for HRV isolation include primary human embryo kidney epithelial cells, human embryo kidney epithelial cells, and human embryo lung fibroblasts (HEL) [80]. These were replaced by semicontinuous strains of HEL cells, such as the WI-38 strain, and selected strains of HeLa cells overexpressing ICAM-1, the major HRV group receptor [79, 80]. An advantage of virus isolation is virus enrichment for phenotypic characterization. Furthermore, this method can rescue divergent strains that might escape molecular diagnostics due to sequence mismatches, provided that the virus is cultivable. However, this laboratory technique is time-consuming, requires expertise and its sensitivity is limited, as some HRV strains cannot be propagated on standard cell lines, and samples with low viral loads or poorly conserved samples will likely be culture-negative.
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HRV antigen detection
The large number of HRV serotypes renders HRV antigen detection difficult. This method is thus not used for HRV diagnosis in many virology laboratories.
HRV RNA detection
The major breakthrough in HRV diagnostics certainly regards the development of molecular techniques involving HRV RNA detection in clinical samples. The first HRV classic reverse- transcription, polymerase chain reaction (PCR) assays were developed in the 1980s and targeted conserved sequences stretches in the 5‟UTR of HRV genomes [138-140]. This technique is far more sensitive than cell-culture, allowing the detection of small amounts of HRV RNA. However, conventional PCR remains time-consuming and tedious.
The advent of real-time RT-PCR applied to HRV RNA detection further increased sensitivity and specificity, in addition to significantly reduce the time of the procedure [141, 142].
Diagnostic HRV real-time RT-PCR assay are designed to detect sequence stretches in the 5‟UTR that are conserved among HRVs. With the ongoing discovery of new HRV strains and even previously unknown HRV species, the design of new real-time RT-PCR assays regularly needs to be adapted in order to detect as much different HRV genomes as possible [143].
However, this implies the use of primers and/or probes with degenerate positions that will inevitably favor the amplification of certain HRV genomes and disfavor the detection of others.
One major advantage of real-time RT-PCR over cell culture is the possibility to detect HRV strains or species unable to be propagated in standard cell lines, as exemplified by the recent discovery of the HRV-C species.
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Attempts are currently made to apply real-time RT-PCR to HRV RNA load quantification in respiratory specimens [144]. Such data are of clinical interest, as they allow studying the possibility of a correlation between viral load and disease severity. Furthermore, HRV RNA quantification could be used to monitor the course of chronic HRV infections in immunocompromised patients such as lung transplant recipients, and to explore possible associations between HRV load and graft rejection in these patients.
In addition to real-time RT-PCR, other molecular techniques used to detect HRV RNA have recently been developed: in situ hybridization, which allows to highlight the presence HRV nucleic acids in various respiratory tissues [143, 145Bardin, 1994 #110, 146]; nucleic acid sequence-based amplification (NASBA), which directly amplifies HRV RNA [147, 148];
DNA microarray called “virochip”, that detects all known respiratory viruses [149]; and a new technology coupling nucleic acid amplification to high-performance electrospray ionization mass spectrometry and base-composition analysis [150]. These new generic detection tools will likely result in the discovery of previously unknown HRV strains in the future.
ANTIVIRAL DRUGS AGAINST HRV
SThe availability of efficient antiviral drugs against rhinoviruses is certainly of interest given the high frequency of common colds and the importance of more severe HRV infections in selected populations (see clinical manifestations).
The first molecule that was studied to fight HRV infections was intra nasal IFN-α,
administred intra-nasally, that diminished symptoms caused by experimentally inoculated HRV in human volunteers [151, 152]. However, as IFN itself causes symptoms similar to those of the common cold, the clinical interest of this agent is limited. Recombinant soluble
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ICAM-1 also somewhat reduced the severity of experimental HRV colds [153]. The costs and limited efficacy related to these agents hampered their development.
The design of molecules able to inhibit receptor binding resulted in the elaboration of
pleconaril, which irreversibly binds the hydrophobic pocket situated in the VP1 canyon [154].
This agent proved to be active in vitro against most HRV types, with variable efficacy
however, some HRV types displaying inherent resistance [155]. In vivo, it was able to reduce the severity and duration of symptoms related to HRV colds [154]. Pleconaril also exhibits activity against HEVs, in vitro and in vivo [156], and was promising in the setting of the treatment of severe HEV infections [157]. However, the drug was shown to select resistant quasispecies. Furthermore, pleconaril was associated with induction of cytochrome P-450 3A enzymes [154], and it was rejected by the US Food and Drug Administration (FDA) for safety issues. VP1 remains a potential antiviral target and research focused on the development of drugs inhibiting receptor binding is ongoing.
Another antiviral target includes the viral 3C protease. A potent 3C protease inhibitor,
rupintrivir, exhibited high activity against both HRVs and HEVs and intranasal administration of this compound reduced symptoms related to HRV infections after inoculation of healthy volunteers [158]. This effect was not reproducible in naturally infected subjects however, which impeded further drug development. An orally administered 3C protease inhibitor, compound 1, was subsequently designed. This molecule displayed broad anti-HRV activity, but its glutamine aldehyde had the tendency to cyclize with a side chain of the compound, resulting in loss of antiviral activity [159]. Similar 3C inhibitors devoid of this flaw are currently being studied.
Other strategies being developed include targeting the viral 2C protein, the viral 3A protein, the viral 3D polymerase, as well as host cell factors [160].
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In summary, no antiviral agent against HRVs is available so far. Factors impeding the
development of such drugs include the necessity for the compound to be active against a large variety of HRV types, the emergence of resistance, and the requirement for the quasi absence of adverse effects. Indeed, the potential adverse effects of an anti-HRV agent should be significantly less harmful than the common cold itself for the drug to elicit interest. Another difficulty resides in the fact that an antiviral drug needs to be administered very early in the course of an HRV infection in order to be clinically effective, as the duration of an HRV infection is short. However, the finding that HRVs are related to asthmatic and COPD exacerbations as well as to chronic infections in immunosuppressed patients raises interest in pursuing research in the field of anti-HRV antivirals.
ANTI-HRV VACCINES
The development of an anti-HRV vaccine would be of high medical interest, given the frequency of HRV infections and the complications resulting from HRV infections in selected populations. However, the development of such a vaccine is limited by the fact that it should protect against all known HRV types. A recent study brought some hope in this area of research. Edlmayr et al [161] were able to create an anti-HRV vaccine by immunizing mice with recombinant HRV-B14 and HRV-A89 VP1 proteins. The resulting antisera were able to neutralize several HRV types in vitro. However, not all HRV types were shown to be neutralized by these experimental vaccines, further illustrating the difficulty related to the elaboration of an effective anti-HRV vaccine. Even if such a vaccine was available, its efficiency would likely be only temporary, given the possibility of an antigenic drift related to the high mutation rate of these viruses.
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Objectives
EXPERIMENTAL INTRA- AND INTERSPECIES RHINOVIRUS RECOMBINATION
Though less frequently than for HEVs, recombination events are observed in HRVs, and probably contribute to their evolution. Such events occur among members of the same HRV species, except for ancient HRV-C/HRV-A recombination at the 5‟UTR-ORF junction. The aim of this work was to analyse to what extent HRVs could recombine among members of the same species or among members of different species, and in which regions of the genome such recombination could generate competent viral genomes. Secondary goals were 1) to map preferential recombination sites and 2) to study the phenotypes of interspecies HRV recombinants.
We used two distinct experimental approaches to achieve these goals. The first involved the design of chimeric HRV genomes generated by molecular cloning methods, whose ability to yield infectious viral particles was assessed by reverse genetics.
The second approach relied on non replicative recombination. Constructs representing defective but complementary viruses were co-transfected in cells. Recovery of infectious viruses from these transfected RNAs implied that viable recombination had occurred, and recombination sites were mapped by genome sequencing.
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RHINOVIRUS RNA QUANTIFICATION BY REAL-TIME RT-PCR IN RESPIRATORY SPECIMENS
As mentioned in the introduction, HRV RNA quantification in respiratory specimens is of clinical interest. We assessed the feasibility of this procedure by testing HRV-positive nasal washes and bronchoalveolar lavages with a one-step real-time RT-PCR assay designed in our laboratory, and experimentally estimated the implication of different parameters in the quantification error margin.
Materials and methods
Materials and methods used for experiments corresponding to results, parts 1.2. and 1.3., are described here whereas material and methods corresponding to results, part 1.1. and part 2., are described in the corresponding publications [162, 163].
PLASMIDS AND CONSTRUCTS
Primers used for the construction of chimeric HRV genomes of partially deleted HRV genomes are listed in Table 2.
