ACTAUNIVERSITATISUPSALIENSISUPPSALA2006

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 168

Molecular Diagnosis of CommonViral Infectious Diseases Based onReal-Time PCR

NAHLA MOHAMED

ISSN 1651-6206ISBN 91-554-6639-7urn:nbn:se:uu:diva-7118

In memory of my grand mother Naaima

and parents Ihsan, Fareed

To my sister Ghada,

my brother Rida

and my husband Ahmed.

List of publications

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals:

I Mohamed N, Elfaitouri A, Fohlman J, Friman G, Blomberg J. A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA. J Clin Virol. 30:150-156 (2004).

II Elfaitouri A, Mohamed N, Fohlman J, Aspholm R, Frisk G, Friman G, Magnius L, Blomberg J. Quantitative PCR-enhanced immunoassay for measurement of enteroviral immunoglobulin M antibody and diagnosis of aseptic meningitis. Clin Diagn Lab Immunol. 12:235-241 (2005).

III Mohamed N, Belak S, Hedlund KO, Blomberg J. Experience from the development of a diagnostic single tube real-time PCR for human caliciviruses, Norovirus genogroups I and II. J Virol Methods. 132:69-76 (2006).

IV Mohamed N, Belak S, Czifra G, Herrmann B, Berencsi G, Blomberg J. Broadly targeted triplex real-time PCR detection of Influenza A, B and C Viruses based on the nucleoprotein gene (Submitted).

V Mohamed N, Escutenaire S, Belak S, Blomberg J. Broadly targeted TaqMan® QPCR method for the detection of corona viruses in humans and animals (Manuscript)

Copies of the articles are included by kind permission of the publishers of the journals.

Supervisors:

Main supervisor:

Jonas Blomberg, Professor of Clinical Virology, Section of Virology, Department of Medical Sciences, Academic Hospital, Uppsala, SwedenPhone: +46 (0)18 511 55 93 mobile: +46 +73 979 63 80 Fax: +46-18-55 1012

Co- Supervisor:Jan Fohlman, Consultant, Asst Prof, R&D-Centre and Dept of Infectious Diseases, Central Hospital, Växjö, Sweden Phone: +46470588280 Mobile: +46708106486 Fax: +46470588265

Contents

Introduction.....................................................................................................9Scope of the thesis......................................................................................9Background ................................................................................................9

Evolutionary basis for viral sequence conservation ............................10Virus classification and taxonomy ...........................................................11RNA Viruses ............................................................................................12

Pathogenesis of RNA viruses ..............................................................12Viral Transmission ..............................................................................12Virus Nucleic Acid Sequence Conservation........................................13Conserved protein structures ...............................................................13Conserved RNA structures ..................................................................14Diagnostic methods for detecting viral Infection ................................17

Aims..............................................................................................................19General aim ..............................................................................................19Specific aims ............................................................................................19

Materials and Methods:.................................................................................20How to measure viral RNA?................................................................20Clinical Specimens used in this study .................................................21Polymerase chain reaction (PCR)........................................................21Real-time PCR.....................................................................................22Broadly targeted PCR..........................................................................25Multiplex real-time PCR......................................................................28Computer program for detection of conserved target sequences.........29Optimisation ........................................................................................291. Primer and Probe design..................................................................302. Carryover contamination containment.............................................313. Recombinant Thermus thermophilus (rTth) DNA polymerase .......314. Touch down quantitative PCR.........................................................32

Results and Discussion .................................................................................35Enterovirus Project “as a model”: Paper I................................................35

The new enterovirus PCR....................................................................38QPIA, Paper II..........................................................................................39

Current serological tests for enteroviruses...........................................39Calicivirus Project, Paper III ....................................................................41

Viral gastroenteritis .............................................................................41Genus Sapovirus ..................................................................................42

Influenza Project, Paper IV ......................................................................45The new influenza PCR.......................................................................45

Corona Virus Project,”Paper V” ..............................................................48The new corona virus PCR..................................................................48

Concluding remarks and future prospectives................................................52

Acknowledgements.......................................................................................53

References:....................................................................................................55

Abbreviations

RNA ribonucleic acid mRNA messenger ribonucleic acid Mn(OAc)2 manganese acetate RT-PCR reverse transcriptase polymerase chain reaction QPCR Quantitative reverse transcriptase polymérase Chain

ReactionUNG Uracil-N-Glycosylase FRET fluorescence resonance energy transfer FAM 6-carboxyfluorescein DABCYL 4-((4-(dimethylamino) phenyl) azo) benzoic acid ester TET tetrachloro-6-carboxyfluorescein ROX 6-carboxy-X-rhodamine Cy5 5'-disulfonatoindodicarbocyanine HuCV Human Calicivirus rTth recombinant Thermus thermophilus polymerase TD-QPCR Touch down quantitative PCR Ct threshold cycle TCID50 50% tissue culture-infective dose UTR untranslated region IRES internal ribosomal entry siteCSF cerebrospinal fluid EV Enterovirus EM electron microscopy NVs NorovirusesSV SapovirusNP NucleoproteinRSV Respiratory Syncytial Virus SIIDC Swedish Institute for Infectious Disease Control HBsAg Hepatitis B surface antigen NQPCR Nuclease based reverse transcription real-time PCR

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Introduction

Scope of the thesis This PhD thesis concerns QPCR methods for diagnosis of infectious RNA viruses, with special regard to Enterovirus, Calicivirus, Influenza A, B, C and Coronavirus. A common theme is the use of evolutionary conservation for diagnostic purposes.

BackgroundA virus is a microorganism smaller than a bacterium, which cannot grow or reproduce apart from a living cell. A virus is a parasite which invades living cells and uses their chemical machinery to produce its building stones and to replicate itself. Cellular life forms divide in binary fashion. Each round of replication doubles the number of cells. In contrast, viruses use a multicopy mechanism of replication. One round of replication can give many, up to thousands of, new viruses. A consequence of this abundance is that minor errors in the viral information backbone, its nucleic acid, can be tolerated. Even if the majority of viral progeny are crippled by mutations, there are enough of replication competent particles to keep the virus infection going. This tendency to mutate is responsible for the ability of some viruses to change slightly in each infected person, making diagnosis and treatment more difficult (Howley 2001).

Viruses cause many common human infections, and are also responsible for a bevy of rare diseases. Examples of viral illnesses range from the common cold, which is usually caused by rhino- or coronaviruses, to acquired immunodeficiency syndrome (AIDS), which is caused by the human immunodeficiency virus (HIV) (Monto 2002; Bono 2006; Nwachuku and Gerba 2006).

Viruses are unique in nature; they are the smallest of the self-replicating nucleic-acid containing structures. Some of them pass through filters that retain even the smallest bacteria. Viruses can be classified in several ways, such as by their geometry, by whether they have envelopes, by the identity of the host organism they can infect, by mode of transmission, or by the type of disease they cause. A useful classification is by the type of nucleic acid

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the virus contains and its mode of expression. They can use either RNA or DNA as their genetic backbone.

A virus is an infectious agent that can be inside or outside cells. It is minimally constructed of two components: 1) a genome consisting of either DNA or RNA, usually not both and 2) a protein –containing structure (the capsid) designed to protect the genome, many viruses have additional structural features, for example an envelope composed of protein containing lipid bilayer, whose presence or absence further distinguishes one virus group from another. Viruses can be rod-shaped, sphere-shaped, or multifaceted. Some look like tadpoles. In 1962, Lwofff, Horne and Tournier put forward criteria to classify viruses: 1- the type of nucleic acid (DNA or RNA). 2-Symmetry of the virus. 3- presence or absence of an envelope. 4- diameter of the nucleocapsid for helical viruses (Howley 2001).

When a virus enters a cell it opens the envelope or capsid, so that their inside content of DNA or RNA is free inside the cell. The host cell then is tricked into making multiple copies of them. Eventually, the cell is often killed. However, some viruses let it live, so is the case for herpes virus and many other enveloped viruses (Howley 2001).

Evolutionary basis for viral sequence conservation Viruses may have existed since the beginning of life on Earth. Currenttheories say that many viruses probably stem from acellular self-replicating biological structures which evolved to parasitize cellular life forms, like animals, plants, fungi and protozoa. Present-day viruses may be either harmless commensals or harmful agents with serious medical consequences. Just as life evolved, developed and adapted to the new and changing environment, so did viruses (Forterre 2006).

According to one of several alternative theories, viral ancestors existed primarily as unprotected genetic strands that carried hereditary information from newly developed life to its offspring. Over time the ever-changing environment of Earth influenced many changes in the method of this transfer of information. The prebiotic self-replicating structures eventually developed cell membranes to protect themselves from the elements. As life became more complex and cells began to self-reproduce, viruses-to-be lost some of their primary functions. They began to infect cells rather than interact directly with other molecules. The newly evolved viruses infected many different life forms. They became obligate intracellular parasites. The viruses of today are highly complex. Over their probable span of existence of at least one billion years, viruses have developed their own protection, means of survival and more efficient ways of infecting their hosts. Mechanisms stemming from the early forms of life are so basic that they cannot be changed, despite of the great variability of many viruses. Today, we can see them as conserved nucleotide and protein sequences. They are primary

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targets for diagnostic methods (and for antiviral therapy) (Lecellier and Voinnet 2004).

The high error rates of the polymerases of many viruses (see below) lead to a high variability (rapid evolution). This is an essential problem for nucleic acid based diagnostic methods, which forces development of a new breed of methods, the broadly targeted ones.

Virus classification and taxonomy Viral taxonomy traditionally uses such characteristics as morphology in

the electron microscope and the nucleic acid type. Viruses can have either RNA or DNA as their genetic backbone (Fig. 1).

Fig: 1 Classification of Viruses. The roman numerals refer to the papers in the thesis.

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RNA Viruses Ribonucleic acid (RNA) is a nucleic acid polymer consisting of covalently bound ribonucleotides. RNA nucleotides contain ribose rings and uracil unlike deoxyribonucleic acid (DNA), which contains Deoxyribose and thymine. In eukaryotic cells, it is transcribed from DNA by enzymes called RNA polymerases and further processed by other enzymes. RNA serves as an intermediate template for translation of genes into proteins, transferring amino acids to the ribosome to form proteins, and also translating the transcript into proteins. The RNA of most RNA viruses is synthesized by viral RNA dependent RNA polymerases.

RNA viruses are important human pathogens. Many acutely infecting enteric (Calici, Entero, Corona, Hepatitis A and E), respiratory (Orthomyxo -influenza, Paramyxo, Corona) viruses, and persistent ones (Retro -HIV, Flavi- hepatitis C) are RNA viruses. A common property of RNA viruses is their sequence variability. This can be explained by the lack of “proof-reading” in RNA dependent RNA/DNA polymerases. In contrast, proof-reading is present in DNA dependent DNA polymerases (Fenner 1994; Morse 1994; Howley 2001).

Pathogenesis of RNA viruses Pathogenicity is the capacity of one organism to cause disease in another. In viral diseases, there are two components involved: the direct effects of the virus replication and the effects of the body responses to the infection. The RNA viruses cause common infectious diseases, in humans and animals. Examples are: Meningitis, gastroenteritis and respiratory infections (Koblet 1988).

Viral Transmission Virus transmission typically begins with the dissemination from the infected host through respiratory, enteric or genitourinary secretions. However, the mode of transmission varies for each virus. The fecal-oral route transmits most of the infections of RNA viruses, aerosols of respiratory secretions and by mechanical transmission. Most virus growth occurs in epithelial cells. Occasionally the liver, kidneys, heart or eyes may be infected. To cause an infection, the virus (like other infectious agents), must gain entry to the body, multiply, and spread either locally or systemically.

Viruses may remain localized to the body surface through which they entered: skin, respiratory tract, intestine, genital tract or conjunctiva. The blood is the most effective and rapid vehicle for the spread of virus through the body (Howley 2001). Knowledge regarding virus localisation in the

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infected body is important for selecting the right sample material for viral diagnosis.

Virus Nucleic Acid Sequence Conservation RNA viruses are more variable than DNA viruses, which in their turn are more variable than pro- and eukaryotic organisms (Drake, Charlesworth et al. 1998). The RNA dependent polymerases of RNA viruses lack proofreading. A misincorporated nucleotide will often not be excised. Some RNA viruses, like retroviruses, caliciviruses and orthomyxoviruses, are especially variable. The viral benefit of being variable is i) a faster adaptation to variable growth conditions (host cells and host organisms) and ii) faster escape from adaptive and innate host defenses. The drawback is a waste of viral progeny. Only a small portion of viral particles are successful. Therefore, a great overproduction of virus is necessary to ensure the transmission of a fit virus.

Viral variability also creates problems for virus detection. The abovementioned highly variable viruses are notorious in this respect. However, some portions of the viral genome cannot vary as much because they have indispensable, almost immutable, functions. They constitute important targets for diagnostic methods based on nucleic acid sequence. In order to create robust methods with a minimum of false negative results it is important to understand the functional basis of sequence conservation. In so far as it is understood, major mechanisms in RNA viruses are either selection for protein function, or selection for RNA function.

Conserved protein structures Selection for protein structure occurs at both primary and higher structural levels. Selection for primary protein structure leads to a particular kind of nucleic acid sequence conservation. In the genetic code, some codons (triplets) can encode the same amino acid. For example, the codons TTA, TTG, CTT, CTC. CTA and CTG all encode the amino acid leucine (L). In contrast, tryptophan (W) is encoded by only one codon (TGG). Thus, the degree of degeneracy of the genetic code is one determinant of the degree of conservation. The third codon position is also called the “wobble” position because it is the most variable of the three positions. If the viral protein has an important function, and needs to interact with other molecules, the amino acids which participate in that function will tend to be evolutionarily conserved. In this situation, the first two codon positions will be rather constant, while the third one can vary more (Drake, Charlesworth et al. 1998; Johnson and Chisholm 2004; Firth and Brown 2006). Selection for secondary or tertiary protein structure may lead to conservation of large sets of amino acids which may or may not reside in a contiguous stretch.

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Conservation of contiguous stretches are most useful for nucleic acid based diagnostic methods. Examples are the polymerases, which interact with a rigid, essentially invariant, nucleic acid substrate in an elaborate three-dimensional mechanism, necessitating conservation of a precise protein structure.

Another type of protein-derived conservation is caused by overlapping reading frames. Viral genomes have to economise with their genetic information. They sometimes use overlapping reading frames. In this case, the selection constraints will be severe. In two overlapping reading frames, the third position in one frame will become the second position in the other. Thus, the entire overlapping region must be encoded by a highly conserved nucleic acid sequence (Johnson and Chisholm 2004; Firth and Brown 2006).

Conserved RNA structures Viral RNA genomes not only code for proteins but in many instances also carry RNA motifs that play a crucial role in the viral life-cycle. This is reflected in a kind of nucleotide conservation different from that of protein-based negative selection. Conservation based on RNA or DNA structure does not allow synonymous mutations, as conservation based on protein structure does. Like in proteins, RNA secondary and tertiary structure is highly coupled and difficult to predict accurately (Martinez-Salas, Regalado et al. 1996; Lopez de Quinto and Martinez-Salas 1997).

RNA Structure RNA molecules are usually single stranded, except in some virus genomes. An RNA molecule can fold back onto itself to form double helical structures consisting mainly of Watson-Crick (GC and AU) base pairs or the slightly less stable pair GU. The stacking energy of these allowed base pairs is the major driving force for RNA structure formation. Other, usually weaker, intermolecular forces and the interaction with the aqueous solvent shape its spatial structure. The list of base pairs of an RNA structure which can be drawn as an outerplanar graph (i.e. all base pairs can be drawn in the half-plane without intersections) forms the secondary structure. The three-dimensional configuration of the molecule is called the tertiary structure (Kim 1976; Chen, Carlini et al. 1999), exemplified in Figure 2.

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Fig: 2 Sequence, secondary structure and schematic representation of the tertiary structure of tRNA.

Secondary RNA structures of single stranded RNA viruses are known to play important roles in the regulation of the viral life cycle. Secondary structure elements that are consistently present in a group of related sequences with less than 95 % mean pairwise identity are likely the result of stabilizing (negative) selection, not a consequence of the high degree of sequence homology. This fact can be exploited to design algorithms that detect conserved RNA secondary structure elements in a small sample of related RNA sequences (Hofacker, Fekete et al. 1998; Hofacker and Stadler 1999; Thurner, Witwer et al. 2004) (Fig 2).

The untranslated regions (5´UTRs) in the 5´end of eukaryotic mRNAs are generally not inert but rather constitute a repository of functional RNA elements, instrumental in controlling mRNA localization, stability, or translation. Even though they do not encode protein, they can be highly conserved. Likewise, the 5´UTRs of viral genes are involved in RNA-RNA and RNA–protein interactions for regulation of gene expression as well as for packaging of viral genomes.

The highly conserved non-coding region in the 5´end of picornaviral genomes (the 5´NCR, also referred to as the 5´UTR) comprises approximately 10% of the entire genome. It is involved in the replication, and possibly packaging, of the virus genome. It contains an internal ribosomal entry site (IRES). IRESs are responsible for translation of many viral mRNAs with clinical relevance in humans, including hepatoviruses, poliovirus, human rhinovirus, other picornaviruses, Hepatitis C, and HIV. These RNA structures may provide targets for drugs against these diseases (Hellen and Pestova 1999; Pickering and Willis 2005).

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The central domain of the enteroviral IRES (Fig. 3) appears to foldindependently of the surrounding IRES nucleotides. It has been proposed that the basal portion of the central domain performs a stand function that contributes to IRES activity by holding the apical region in the appropriate conformation, compatible with its recognition by the translation machinery.Experimental evidence for tertiary structure of IRES elements suggests a dynamic structure indicative of translation efficiency regulation at the level of RNA structure (Martinez-Salas, Lopez de Quinto et al. 2002).

Fig: 3 The Secondary Structure of the part of poliovirus 5´-UTR and IRES. Domains are numbered I–V. RNA structure of the central domain (domain IV) is depicted according to RNA probing analysis. The predicted secondary structure was obtained on-line from the Jena Picornavirus Page of the University of Jena (http://www.uni-jena.de/_i6zero/picorna.htm (Spriggs, Bushell et al. 2005). Positions of the forward primer and probe in domain V are indicated.

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Evolutionarily conserved RNA viral sequences are not only useful for understanding viral function and for understanding viral evolution, but are also important targets for diagnostic methods and therapeutics.

In this thesis, the diagnostic usefulness of evolutionarily conserved RNA viral sequences will be tested in practice, primarily for Picorna, Calici, Orthomyxo, and Corona viruses.

Computer programs for detection of evolutionary conservation will be used.

Diagnostic methods for detecting viral Infection Viral diagnostic methods generally belong to one of four categories: Virus isolation, Virus antigen detection, Virus nucleic acid detection and Virus antibody detection (serology). Virus isolation is a rather cumbersome and slow technique. Virus antigen detection is more rapid, but still is manually intensive and relatively insensitive. Virus serology is an indirect approach with many limitations. There is a great clinical need to develop new virus diagnostic techniques. Rapid, sensitive and rational virus detection and quantification methods are needed. This is true for both human and veterinary medicine. Broadly targeted methods are the major theme of this thesis. They can reduce the time and cost of diagnosis of infectious disease. Once a diagnosis has been reached, appropriate medical action can be taken.

Currently, newly developed diagnostic tests for most RNA viral infections tend to fall into two types:

Serological testing for anti-virus antibodies include indirect fluorescent antibody testing and enzyme-linked immunosorbent assays (ELISA) which detect antibodies against the virus produced in response to infection. The performance of viral serologies is useful in the diagnosis of recent, past or chronic viral infections. Serum allows the detection of infection after clearance of virus. In most viral infections immunoglobulin M (IgM) appears within several days after onset of symptoms, peaks at 7-10 days, and declines to undetectable levels within 1-2 months. IgM methods are useful adjuncts to the direct detection of virus and virus nucleic acid.

Molecular testing consists of reverse transcriptase-polymerase chain reaction (RT-PCR) tests specific for the RNA. It can detect infection within the first few days after the onset of fever in viral infection patients, but the duration of detectable viraemia and virus shedding is unknown, so RT-PCR tests performed too early or late, or from the wrong sample, could give negative results.

The focus of the routine clinical diagnostic virology laboratory, using traditional cell cultures and viral antigen methods, has been confined to detection of a fairly narrow range of viruses such as: herpes viruses, adenovirus; the enteroviruses; influenza virus A and B; parainfluenza virus types 1, 2, 3 and 4 and respiratory syncytial virus

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Methods based on the detection of the viral genome are also commonly known as molecular methods. The role of molecular methods will increase rapidly in the near future. PCR has been widely used for detection of both DNA and RNA viruses in various specimens such as: CSF, serum, feces or nasopharyngeal aspirates.

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Aims

General aim The aim of this study was to develop rapid, sensitive and robust procedures for the qualitative detection of variable RNA viruses, based on evolutionarily conserved viral sequences. The diagnostic usefulness of evolutionary conservation was tested for Picorna, Calici, Orthomyxo, and Corona viruses. To this end, computer programs for detection of evolutionary conservation were used. Primers and probes of varying degree of degeneration and length were tested. The usefulness of modified nucleotides, primarily of locked nucleic acid (LNA) nucleotides, was also evaluated. However, the cost of large systematic evaluations obviated a comprehensive approach.

Specific aims 1. The feasibility of a simple and rapid QPCR protocol for variable RNA

viruses was evaluated. The use of rTth DNA polymerase (Hofmann-Lehmann, Swenerton et al. 2000) under somewhat novel conditions will be evaluated by using enteroviruses in a clinical setting, as a model (Paper I).

2. The development and exploitation of a variant of PIA (PCR-enhanced Immunoassay, a method developed in Uppsala) for detection of antiviral IgM was performed (Paper II).

3. The usefulness of evolutionarily conserved RNA viral sequences in QPCR was tested in clinical samples, primarily for Calici, Orthomyxo, and Corona viruses (Paper III, Manuscripts IV and V).

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Materials and Methods:

How to measure viral RNA? The processing of samples for RNA measurement is demanding (Fig. 4). To obtain a reproducible nucleic acid yield from tissue samples can be difficult, since the yield is affected by several factors such as the type of the original samples, age, the quality of starting material and the time of collection. The best RNA yield and quality is usually achieved from fresh material. To avoid viral RNA degradation before analysis, one must either allow the RNA to remain packaged inside the viral particle (e.g. Caliciviral RNA), or one must extract the RNA under RNAse free conditions and freeze it if the RT-PCR is not performed immediately. Some examples of sample materials for clinical virology that can be used for real-time PCR are:

Swabs for vesicles and wounds (herpes simplex, herpes zoster and enterovirus)Throat / nose swabs for upper respiratory disease (eg: influenza viruses). Stool for enteroviruses and gastroenteritis (eg; calicivirus). Cerebrospinal fluid (CSF) for meningitis/encephalitis (eg: enteroviruses).

Fig. 4. RNA measurement. Items for protection against RNA loss and quality control are shown in italics.

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Specimen collection and delivery to the laboratory without delay and with minimal losses are vital components for reliable RNA measurements in a diagnostic service.

Whole blood samples are good for surveillance of systemic inection. A viremia is often detectable in plasma or serum during a relatively narrow time window, compared to samples taken at the site of local viral replication. Acute and convalescent samples may increase the chance of a diagnosis, including a viral antibody seroconversion.

Internal positive control To confirm the efficiency and the sensitivity of PCR, it is helpful to use standard molecules as indicators of the assay performance. Accurate estimation of recovery efficiency for particular human or animal samples requires analysis of a duplicate sample spiked with a known number of standard molecules as an internal positive control. This is a way to detect false-negative results arising from experimental errors or inhibition of amplification that may occur in complex samples such as feces or blood. A simple way of avoiding inhibition is to purify the viral nucleic acid free from inhibitors. Internal positive controls were not evaluated in this work.

Clinical Specimens used in this study (1) Cerebrospinal fluid (CSF) was obtained from 62 patients with aseptic meningitis visiting the Infectious Diseases Clinic at the Uppsala University Hospital between 1997 and 2001 (Paper I). (2) Serum samples from 43 patients with AM from Uppsala University Hospital (UAS). The sera had been sampled during the autumns of 1995 and 1996 and had been stored at –20°C (Paper II). (3) Seventy-one stool specimens from adults and children involved in outbreaks and sporadic cases of gastroenteritis between 1997 and 2004 were obtained from SIIDC. Stool specimens were prepared as a 10% (v/v) suspension in sterile PBS (phosphate-buffered saline) (Paper III). (4) Two hundred and three nasopharyngeal aspirates were collected from patients with a suspected influenza virus infection during winter 1999-2005 (Manuscript IV). (5) Seventy-seven nasopharyngeal aspirates were collected from patients during winter 1999-2002 admitted to hospital with acute respiratory syndromes (Manuscript V).

Polymerase chain reaction (PCR) The polymerase chain reaction (PCR) was invented by Kary B Mullis in 1985. It is a method that allows logarithmic amplification of short DNA sequences (usually 100 to 600 bases) within a longer double stranded DNA

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molecule, PCR entails the use of a pair of primers, each about 20 nucleotides in length that are complementary to a defined sequence on each of the two strands of the DNA. These primers are extended by a DNA polymerase so that a copy is made of the designated sequence. After making this copy, the same primers can be used again, not only to make another copy of the input DNA strand but also of the short copy made in the first round of synthesis. This leads to logarithmic amplification. Reverse transcriptase-PCR analysis of mRNA is often referred to as "RT-PCR" (Chapman, Tracy et al. 1990; Holland, Abramson et al. 1991; Myers and Gelfand 1991).

Traditional reverse transcriptase-PCR, methods are non-quantitative because ethidium bromide is a rather insensitive stain, and because the amount of amplimer does not accurately reflect the amount of template sequence. Methods such as competitive PCR were developed to make the method more quantitative but they are cumbersome and time-consuming. Thus, real-time PCR makes a significant step forward to the quantitative PCR (Gibson, Heid et al. 1996; Wittwer, Herrmann et al. 1997).

Real-time PCR Real-time PCR assays are powerful techniques for detection and accurate quantitation of specific RNA transcripts or specific DNA in samples. The combination of ease of use and accuracy make them superior to other methods of quantitation.

The development of fluorogenic probes by (Lee, Connell et al. 1993) made it possible to eliminate post-PCR processing for the analysis of probe degradation. The probe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. While the probe is intact, the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer (FRET) through space.

Probe design and synthesis has been simplified by the finding that adequate quenching is observed for probes with the reporter at the 5' end and the quencher at the 3' end.

TaqMan® Real Time PCR A TaqMan® probe is based on the concept of FRET, 5´nuclease assay first described by (Holland, Abramson et al. 1991) which uses the 5´ 3´ exonuclease activity of the Taq DNA polymerase to cleave a dual-labeled probe annealed to a target sequence during PCR amplification.

An ssDNA probe ( 18-35 nucleotides) is labeled in the 5´end with a reporter fluorophore and in the 3´end (or some times internally) with the quencher. The approach uses dual-labeled fluorogenic hybridization probes. One fluorescent dye serves as a reporter. Its emission spectrum is quenched by the second fluorescent dye. A quencher is a molecule that can absorb the energy released by the reporter molecule. The TaqMan® probe is designed

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to match the complementary sequence within the amplicon generated by PCR. If the probe binds to the amplicon during each annealing step of the PCR, when the Taq extends from the primer bound to the amplicon it displaces the 5' end of the probe which is then degraded by the 5' 3'exonuclease activity of the Taq. Cleavage continues until the remaining probe melts off the amplicon. This process releases the fluorophore and quencher into solution, spatially separating them. This leads to an irreversible increase in fluorescence from the reporter. During each cycle of the PCR process the calculated Ct value is proportional to the number of target copies present in the sample; using a charge coupled device (CCD) camera permits the detection of a wide spectrum of emission wavelengths (fig:5) (Bustin, Benes et al. 2005).

Fig: 5 The principle of the 5´ nuclease (TaqMan®) assay

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In a further development of the real-time PCR concept, a cDNA copy of the RNA template is first synthesized in an initial RT step. After denaturation, primers and a fluorogenic probe anneal to their targets. The increase in signal is directly proportional to the number of molecules released during that cycle. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye.

If the target sequence is present, the probe anneals downstream from one of the primer sites and is cleaved by the 5' nuclease activity of Taq DNApolymerase as this primer is extended. This cleavage of the probe separates the reporter dye from quencher dye, increasing the reporter dye signal. Cleavage removes the probe from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process. Additional reporter dye molecules are cleaved from their respective probes with each cycle, causing an increase in fluorescence intensity proportional to the amount of amplicon produced.

The advantage of fluorogenic probes over DNA binding dyes is that specific hybridization between probe and target is required to generate fluorescent signal. Thus, with fluorogenic probes, non-specific amplification due to mis-priming or primer-dimer artifact does not generate signal. Another advantage of fluorogenic probes is that they can be labeled with different, reporter dyes distinguishable in separate wavelength detection channels. By using probes labeled with different reporters, amplification of two distinct sequences can be detected in a single PCR reaction. This is the basis for multiplex real-time PCRs. Multiplexing above 2-3 different targets can pose great problems due to unexpected primer-primer, primer-probe and probe-probe interactions.

Alternative virus nucleic acid detection methods Molecular methods offer several potential advantages over conventional microbiological techniques. Methods based on polymerase chain reaction (PCR) are rapid and sensitive enough to detect very small quantities of microorganisms. There are however alternative molecular techniques.

Multiplex probe based systems (Padlock probes and ViroChip) Padlock probes are molecular tools that combine highly specific target sequence recognition with the potential for multiplexed analysis of large sets of target DNA or RNA sequences. They are linear oligonucleotides designed so that the two end segments, connected by a linker region, are both complementary to a target sequence. Upon hybridization to a target DNA or RNA sequence, the two probe ends become juxtaposed and can be joined by a DNA ligase. The dual recognition ensures a specificity of detection similar

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to PCR. DNA ligases have very little tolerance for mismatches and therefore permit identification of allelic sequence variants.

Padlock probes typically have target-complementary segments of 15–20 nucleotides at both ends, separated by a linker of around 50 nucleotides that may include sequence elements of value to amplify or identify individual probes. As both ends of these relatively long oligonucleotides must be intact for proper probe ligation, they can be relatively challenging to synthesize chemically (Baner, Nilsson et al. 2001; Landegren, Schallmeiner et al. 2004). They can be used for in situ detection, even of single nucleotide variation, via rolling-circle replication (Nilsson, Baner et al. 2002; Landegren, Schallmeiner et al. 2004). The technique is suitable for single nucleotide mutation analysis and many thousand probes can be analyzed on one microarray slide.

DNA microarray technology provides a means of detecting thousands of specific PCR products simultaneously and is thus a useful method of analysis of amplified genes or DNA sequences pathogen (Kostrzynska and Bachand 2006).

DNA microarray technology (also known as DNA arrays, DNA chips or biochips) represents one of the latest advances among in molecular biology. This technology can also be used for microbiological diagnosis. Such techniques started to appear during the late 1990s (DeRisi and Iyer 1999). The new discipline of microarray studies in combination with real-time PCR hold the promise of effective, current, and comprehensive understanding of complex diseases and may be a good approach for reducing the costs and times associated with discovery of causes of disease, diagnosis and improvement of therapy.

Broadly targeted PCR We describe a set of broadly targeted real time PCRs which use several alternative conserved sequences. The broadly reactive TaqMan® PCRs assays developed in this study will be useful tools for performing infection surveys. It can determine whether viruses are present either in human, animal specimens or drinking water to which humans are exposed. The development of broadly reactive real-time PCRs assays for RNA viruses was challenging due to the extensive genetic diversity among the different viruses. TaqMan® assays generally have low tolerance for mismatched sequences within the probe target regions. Nevertheless, the TaqMan® assays in the present study were able to detect all or most of the intended target viruses in less than 3 hours. To achieve the ideals broad targeting, high sensitivity, high specificity and quantitative results we had to optimize several processes.

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Primer and probe design PCR primers provide the first level of specificity for the PCR assay, and primers that only amplify one product will provide the best assay sensitivity. Since real-time PCR also incorporates highly specific homogeneous probe detection, the annealing temperature for probes can be several degrees above the melting temperature of the primers. PCR primers should have a low potential to form secondary structures, including self and cross hybridization with other oligonucleotides in the PCR mixture. This becomes increasingly more difficult as more oligonucleotides are added to the reaction. Details for design of primers and probes are beyond the scope of this study. They are described extensively in the thesis papers. A few key components should be optimized in order to achieve optimal results. These factors include manganese acetate concentration, which allows the polymerase enzyme to function at an optimal level; primer and probe concentrations, which affect the sensitivity and specificity of the assay respectively; and the use of additives such as Uracil N-glycosylase, which can protect against contamination. This enzyme does not function above a critical maximum temperature, it does not significantly affect the PCR and reduces the generation of non-specific sequence fragments (Loftis, Massung et al. 2003; Boeckh, Huang et al. 2004; Pruvost, Grange et al. 2005).

Locked Nucleic Acid (LNA) Locked nucleic acid (LNA) is a nucleic acid analog containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA-mimicking sugar conformation, they are called “locked” or “bridged” because of the furanose ring conformation. This conformation enhances base stacking and phosphate backbone pre-organization and results in improved affinity for complementary DNA or RNA sequences (higher Tm), the thermal stability of a LNA/DNA or LNA/RNA duplex in increased 3°C to 8°C per modified base in the oloigonucleotode. LNA thus provides superior hybridization characteristics and enhanced biostability compared to conventional DNA oligonucleotides. LNA oligos can be designed to enhance a wide variety of genomic applications and technologies that rely upon the use of oligonucleotides (Petersen, Bondensgaard et al. 2002; Mouritzen, Nielsen et al. 2003; Johnson, Haupt et al. 2004; Ugozzoli, Latorra et al. 2004). LNA analogs have other useful properties. The water solubility of LNAs is similar to the solubility of DNAs or RNAs. Modified oligomers can substitute for native nucleic acids in many biological applications (You, Moreira et al. 2006). The optimal use of LNA in broadly targeted diagnostic methods will require larger, more systematic, studies than were possible in this work. It here suffices to say that LNA allowed the use of shorter probes than would otherwise have been possible (papers IV and V). Thus, even relatively short conserved stretches could be considered for probe design.

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Reverse-transcription polymerase chain reaction (RT-PCR)

The RT step is critical for sensitive and accurate RNA quantification. The amount of complementary DNA (cDNA) produced by the reverse transcriptase must accurately represent RNA input amounts. Therefore, the dynamic range, sensitivity and specificity of the RT enzyme are prime considerations for a successful RT-PCR assay. Protocols using a one tube/one enzyme-based approach are significantly more convenient than those using two tube/two enzyme based protocols but have been reported to be less sensitive (Bustin 2000). RT reactions are usually carried out between 40°C and 50°C and at these low temperatures there can be problems with a somewhat lower specificity of the RT reaction. RT-PCR can be primed using specific primers, random hexamer or oligo dT primers (Wang, Doyle et al. 1989; Freeman, Vrana et al. 1996). If the target RNA contains extensive secondary structure, it is advisable to use a single tube (‘one-enzyme/one-tube’) system utilising Thermus thermophilus (Tth) polymerase, a DNA polymerase with intrinsic RT, but no RNAse H, activity) (Myers and Gelfand 1991). This assay uses bicine buffers containing Mn2+ ions that are compatible with both RT and subsequent PCR (Chiocchia and Smith 1997). This permits the RT reaction to be carried out at increased temperatures using primers with significantly higher Tm, while reducing both hands-on time and the likelihood of introducing contaminants into reaction mixtures. There is also evidence that RT-PCR reactions using Tth polymerase may be more robust and resistant to inhibitors present in biological specimen (Bustin 2000).

Uracil N-glycosylase (UNG)

The UNG enzyme hydrolyzes the N-glycosidic bond between the uracilresidue and the deoxyribose sugar of the DNA backbone, generating an apurinic-apyrimidinic (AP) site. The UNG is active on both single- and double-stranded DNA that contains uracil, but has no activity on RNA or 2'-deoxyuridine-5'-monophosphate (Jiang, Krosky et al. 2005). The thermolabile UNG used in this work is fully active at 37-50°C, but is inactivated by a 10-minute incubation at 65°C or higher. If the thermostable UNG is used, inactivation or removal of residual UNG activity after amplification is necessary or PCR products must either be stored at 20°C or held at 72°C to completely prevent degradation of amplicons by the UNG (Rashtchian, Thornton et al. 1992; Thornton, Hartley et al. 1992).

Three sources of contaminating DNA must be considered: i) cross-contamination between samples, resulting in transfer of target DNA from one sample to another; ii) plasmid or synthetic DNA contamination from the

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laboratory environment, e.g., recombinant plasmids containing cloned target sequences; and iii) carry-over contamination of amplified target DNA, i.e., amplification products and primers from previous PCRs. Of these sources, carry-over contamination with amplimers is the major concern due to its relative abundance and its ideal structure for re-amplification (Kwok and Higuchi 1989; Gibbs 1991). Carryover template amplification is preventable if amplifications are performed such that many dTTPs in the amplified DNA are substituted with dUTP. Under this condition, a carryover contaminant from a previous PCR amplified product containing dUTP, but not the specific native target nucleic acid, becomes susceptible to degradation by UNG. Therefore, if the PCR reaction mixture is treated with UNG prior to amplification, the carryover contaminant will not reamplify (Longo, Berninger et al. 1990). After optimisation, a considerable degree of protection from carryover contamination can be achieved. For example, Kleiboeker,showed that 0.25 U/ per PCR reaction protected against several thousand amplimer copies. However he ran it as a separate reaction at 30oCbefore RT (Kleiboeker 2005). A considerable advantage is if the UNG reaction is performed in the same tube as the RT and PCR reactions (like in the methods described in this thesis).

Multiplex real-time PCR

Multiplex real-time PCR is the amplification of more than one target in a single reaction tube by using as many as four dual labeled probes in the same reaction tube. Fluorescent dyes with different emission spectra are attached to the different probes. Multiplexing reduces the handling of the samples, prevents the opportunities for laboratory contamination and the reagent costs. Multiplex TaqMan© assays can be performed using multiple dyes with distinct emission wavelengths. Available dyes for this purpose are FAM, ROX, TET, Cy3, Cy5 and JOE. TAMRA is generally reserved to be the quencher on the probe. The combination of FAM (reporter) and TAMRA (quencher) is a first choice (Mackay, Arden et al. 2002; Mackay 2004; Espy, Uhl et al. 2006).

Touch down quantitative PCR

Touch-Down (TD) PCR is a method for optimizing PCR by circumventing spurious priming during amplification even if the degree of primer–template complementarity is not fully optimal. In some systems, amplification yields were significantly increased compared to a non-touch-down procedure (Hecker and Roux 1996). TD-QPCR starts with a high annealing temperature for the first primer-annealing step. It can reduce non-specific amplification and improve specificity and product yield for variable

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target sequence. The touch-down procedure was designed to achieve more stringent conditions in the first few cycles, to ensure efficient and specific amplification in low-copy-number but well fitting targets in the sample. The annealing temperature is then gradually lowered to a level that increases amplification yield, and includes also less well-fitting targets (Don, Cox et al. 1991; Schunck, Kraft et al. 1995; Marchand, Hajdari et al. 2003).

The advantages of real-time PCR

Theoretically, there is a quantitative relationship between the amount of starting target sequence and the amount of PCR product at any given cycle. In practice, though, it is hard to obtain a reliable quantification with traditional (non-real-time) PCR. Real-time PCR requires less hands on and assay times. It also has a lesser risk of cross contamination since the complete assay, including the RT-step, can be performed using a single, closed, tube. Assay data are collected automatically through the closed lid. In this work, the possibility of combining contamination protection and quantification in a single PCR tube was explored. Quantification is another strength of real-time PCR. Determining Ct values by following the real-time kinetics of PCR diminishes the need for a competitor to be co-amplified with the target. The use of Ct values also expands the dynamic range of quantitation up to around seven orders of magnitude, because data are collected for every cycle of PCR. Compared to endpoint quantitation methods, real-time PCR offers streamlined assay development, reproducible results, and a wider dynamic range.

Computer program for detection of conserved target sequences

The "ConSort" © program (Blomberg J, unpublished) uses alignments of 2-10000 viral sequences made using BLAST, Clustal, Megalign, Multalin or BioEdit. Areas of sequence conservation are identified, and the degree of representativity for all sequences in the alignment, or selected groups included in the alignment, is computed. The program first shows the most common variant for each position, then less common ones in decreasing order. Insertions and deletions are marked especially. BLAST or CLUSTAL W 1.83 alignments were examined by ConSort© to detect conserved regions suitable for primer and probe construction.

Optimisation

Four processes were optimised more or less systematically: (1) Primer, Probe design and addition of LNA, (2) carryover contamination control

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treatment, (3) reverse-transcription of target RNA to generate a complementary DNA (cDNA) and (4) Touch down polymerase chain reaction (TD-QPCR). The target amplification, hybridisation to probe and detection of the probe-bound amplification product by fluorescence was performed using the Corbett Research RotorGene Real Time Amplification system (RG 2000; Mortlake, NSW Australia). The modifications were made on the basis of a reagent kit (One-Step EZ RT-PCR TaqMan reagent kit Applied Biosystems, Foster City, Calif.).

1. Primer and Probe design

Primers and probes were designed with the aim of i. primers having an approximately equal Tm, ii. the probe having a higher Tm than the primers, iii. minimising primer-primer and primer-probe interactions, while iv. still operating within a conserved stretch. All oligonucleotides were checked for predicted primer-primer, primer-probe and probe-probe annealing as well as for self-annealing loops, using a cutoff of –5 kCal/Mol for the Gibbs free energy [ G] of oligonucleotide interactions. The primer-primer interactions were studied using Oligo Analyser 3.0 http://207.32.43.70/biotools/oligocalc/oligocalc.asp,andhttp://www.idtdna.com/analyzer/Applications/OligoAnalyzer/

1.1. Primer degeneracy

Degenerate primers have a number of options at several positions in the sequence so as to allow annealing to and amplification of a variety of related sequences

Y = pYrimidines = C / T (degeneracy = 2X), R = puRines = A / G (degeneracy = 2X), I = Inosine = C / G / A / T, N = Nucleotide = C / G / A / T (degeneracy = 4X). In this work degenerated primers were used to amplify (“fish out”) conserved sequences of a gene or genes from the genome of a virus.

1.2. LNA in probe design

The four LNA/DNA probes used in these studies had several LNA substitutions.They were labeled with FAM, Rox, Cy5 and TET at the 5' end for influenza A, B, C and corona viruses, respectively, and were purchasedfrom MedProbe (Oslo, Norway).

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2. Carryover contamination containment

Thermolabile UNG, “HKTM UNG” is fully active at 37-50 C and is inactivated by 10 minutes incubation at 65 C or higher. Different amounts of HKTM UNG; 0.01, 0.05, 0.1, 0.2, 0.5 and 1 units per PCR reaction were tested in the temperature schemes presented under “optimised QPCR procedure”. Up to 0.1 units per reaction did not have any adverse effect on the PCR. Higher amounts gave a slight inhibition evident by a higher Ct and lower signal strength (data not shown). We therefore settled for 0.1 units of HKTM UNG (and diethylpyrocarbonate treated nuclease-free H2O) to diminish the risk of PCR carryover contamination.

3. Recombinant Thermus thermophilus (rTth) DNA polymerase

The multiple (RNA and DNA dependent DNA polymerase) functions embodied in the single protein recombinant Thermus thermophilus (rTth) DNA polymerase are useful for RT-PCR amplification. It also possesses RNAse H and 5' 3' exonuclease activities (Myers and Gelfand 1991; Auer, Landre et al. 1995; Auer, Sninsky et al. 1996) which are necessary for synthesis of double stranded cDNA and the TaqMan® principle. The RT activity was most efficient at elevated temperatures, a specific pH provided by the temperature-dependent bicine buffer, and the presence of Mn2+. The elevated temperature optimum allows for increased specificity of primer binding and alleviation of secondary structures present in the RNA template, thus reducing non-specific product formation and decreasing premature termination of the polymerase. The recently characterized RNase H activity, which is capable of digesting the RNA strand of a RNA/DNA hybrid exonucleolytically in the 5' 3' direction, also shows optimal activity at elevated temperatures and when Mn2+ is used as the divalent metal ion activator. A strong preference of rTth pol 5' 3' exonuclease activity for RNA/DNA hybrids over DNA/DNA duplexes was observed in the absence of DNA synthesis. Utilizing both the RT and the RNase H activity of rTthpol during the RT step of a RT/PCR allows for reverse transcription of the target RNA strand and subsequent digestion of the RNA strand of the resulting RNA(Myers and Gelfand 1991; Auer, Landre et al. 1995; Auer, Sninsky et al. 1996).

The QPCR methods described here use the dual functions of the rTththermostable polymerase, under special conditions, to preserve enzymatic activity even late during the amplification process, and thermolabile UNG

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with dUTP to protect against inadvertent contamination of samples with amplimers.

4. Touch down quantitative PCR

TD-PCR starts with a high annealing temperature for the first primer-annealing step, and then reduced the annealing temperature by 2ºC in steps for later cycles (Fig. 6). By using the touch-down approach, more stringent annealing conditions are applied during the first cycles of amplification, ensuring high specificity, still allowing the less well fitting targets to be amplified.

Fig. 6

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Assessment of sensitivity and specificity

Sensitivity is the probability of a positive result given that the disease (virus) is present, but it also refers to the minimum detectable quantity of a target that can be measured. Specificity is the probability of a negative result given that the disease (virus) is not present.

Sensitivity was assessed by: titration of synthetic or recombinant DNA as oligos or plasmids, titration of reference viral preparation of known approximate concentration, and titration of clinical samples into the stochastic zone. To explain the principle of the latter, an assessment of sensitivity by end point titration, with four- six observations per dilution step, into the stochastic zone of 1–10 RNA copies per QPCR reaction was done in the calici (paper III) and influenza (paper IV) projects (Fig. 7). If a stochastic positivity is observed before the result becomes negative, the PCR assay is able to detect 1–10 RNA copies per PCR reaction.

Fig: 7. Relationship between Ct value, DNA copies per PCR reaction and standard deviation.

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Sensitivity was also evaluated by comparison of QPCR results with those of alternative methods in collections of clinical samples.

The specificity of QPCR was evaluated by testing RNA and cDNA from several viruses, and by comparison of results with another method in clinical samples.

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Results and Discussion

Enterovirus Project “as a model”: Paper I Viral infections of the central nervous system may involve primarily the brain parenchyma, the meninges, the anterior horn cells, cranial nerves etc.

Meningitis is an infection of the meninges—the tough layer of tissue that surrounds the brain and the spinal cord. If not treated, bacterial meningitis can lead to brain damage and progress to coma and even death (Leite, Barbosa et al. 2005).Viral meningitis is the most common cause of aseptic meningitis. It is generally self limiting, with a good prognosis. However, sometimes it can be more serious. For the doctor it is a common diagnostic dilemma to distinguish between viral and bacterial meningitis. Enteroviruses are the most common cause of aseptic meningitis (meningeal inflammation in the absence of bacterial pathogen). However, the inflammation can also be caused by more rare conditions, such as cancer, a drug reaction, or a disease of the immune system. Children between birth and 2 years of age have the highest incidence of viral meningitis. They also suffer the greatest risk (Fenner 1994; Howley 2001).

Normally, meningitis causes fever, lethargy, and a decreased mental activity (problems of thinking), but these symptoms are often hard to detect in young children. Enterovirus infection in the neonate is characterized by the sudden onset of fever, irritability and poor feeding. If the infection or resulting inflammation progresses past the membranes of the brain or the spinal cord, then the process is called encephalitis (inflammation of the brain). When the infectious agent primarily attacks the brain the more serious clinical picture of 'encephalitis' with fatigue, seizures and coma is produced (Kumar 2005). The prognosis for viral meningitis is much better than that for bacterial meningitis, with most people recovering completely with simple treatment of the symptoms (Howley 2001).

The most common viruses causing aseptic meningitis are the enteroviruses, which account for more than half the cases. Enteroviruses are non-enveloped enteric RNA viruses belonging to the family Picornaviridae.Enteroviruses (EVs) infect billions of people worldwide and cause clinical manifestations such as poliomyelitis, aseptic meningitis, encephalitis, myocarditis, hand-foot and mouth disease and other acute and chronic illnesses.

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EVs are subgrouped into poliovirus, coxsackievirus A and B, echovirus and enterovirus 68 to 78 (Norder, Bjerregaard et al. 2003), partly based on their replication and pathogenesis in humans and in experimental animals. The number of EVs is presently expanding rapidly.

The enterovirus family includes totally 64 serotypes, which usually cause asymptomatic disease. The most prevalent disease manifestation is upper respiratory infections such as the common cold (Rotbart 2002). More rarely aseptic meningitis and myocarditis may develop. Enterovirus infections are common in humans. A seasonal peak is in autumn. They frequently go undiagnosed.

The genome consists of one s/s (+) sense RNA molecule of between 7.2 to 8.5kb. It contains an Open Reading Frame (ORF) which comprises 4 structural and 7 non-structural genes and is flanked by the 5´- and 3´- untranslated region (5´and 3´- URT) (fig 8)..

A number of features are conserved in all Picornaviruses: Genomic RNA is infectious (~1x106-fold less infectious than intact

particles.There is a long (600-1200 base) untranslated region at the 5' end

(important in translation, virulence and possibly encapsidation and a shorter 3' untranslated region (50-100 bases) - important in (-) strand synthesis.

The 5' UTR contains a 'clover-leaf' secondary structure encompassing an IRES: Internal Ribosome Entry Site (fig: 3).

Fig: 8 Schematic view of the enterovirus genome

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Fig 9. Graphical output from ConSort©. The height of a column is proportional to the percentage of the most common (consensus) nucleotide at that position. The upper diagram represents the distribution of variation in a BLAST alignment of all enteroviral sequences in GenBank. Red columns contain invariant stretches. The magenta boxes are regions predicted to be possible for primer and probe design. The lower diagram shows the variation in the 5ÚTR stretch chosen for primer/probe construction.

In paper I, different concentrations of each primer were tested systematically. Final concentrations of 500 and 400 nM for primers NMF1 and NMR1, respectively, and 100 nM of the fluorogenic probe MP, gave an optimal sensitivity with EV RNA (Mohamed, Elfaitouri et al. 2004).

The current diagnostic tests for EV infections have improved significantly with the advent of broadly amplifying PCR for direct detection of specific EV nucleotide sequences in multiple specimen types (Glimaker, Abebe et al. 1992; Glimaker, Samuelson et al. 1992; Casas, Klapper et al. 1995; Kessler, Santner et al. 1997; Rotbart, Ahmed et al. 1997).

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The new enterovirus PCR

Limit of detection and linearity. To test the sensitivity and linearity of the assay, RNA extracts of viral culture supernatants from enteroviruses cultured as described above were serially diluted tenfold (105 to 10-2 TCID50/mL). The titrated viruses represent one member of each major EV group. Each dilution and a negative control sample containing nuclease-free water were tested in duplicate. The linear range of the assay spanned at least 5 10-logs. The highest dilution, 1*10-7, for Coxsackievirus A16 (0.01 TCID50 /mL) corresponded to a threshold cycle (Ct) of 33.6, while the lowest dilution, (1000 TCID50 /mL), corresponded to a Ct of 12.5 of the same virus.

The whole PCR procedure took place in the same tube. In 62 CSF samples from cases diagnosed as meningitis, QPCR detected

EV RNA in 76% of these cases while the EIA-based PCR technique used for comparison detected 47%. This shows that QPCR is a sensitive technique, and confirms that EVs are major causes of aseptic meningitis. High sensitivity is important in establishing a diagnosis in patients with neurological disorders such as aseptic meningitis where only a few copies of virus may be present in CSF.

Meningitis is a common infection that often requires hospitalisation and antibiotic therapy. However, the majority of the cases are caused by viral rather than bacterial pathogens. Previously, the diagnosis of EV meningitis required the isolation of the virus in cell culture. Viral culture is the methodological standard for diagnosis of EV meningitis. Unfortunately, the sensitivity of culture is not always sufficient, as many serotypes grow relatively poorly in culture. Results take several days to weeks (Rotbart, Ahmed et al. 1997). Several authors have developed reverse transcription-PCR (RT-PCR) assays as a more convenient alternative to viral culture (Verstrepen, Kuhn et al. 2001; Monpoeho, Coste-Burel et al. 2002). The QPCR described here can be carried out in 4 hours, including the RNA preparation step. A large number of samples can therefore be screened rapidly, and its sensitivity, simplicity, and reproducibility make it a suitable tool for the routine laboratory use. This rapid screening tool allows time for adequate clinical management and evaluation of antiviral therapy. The QPCR will also be a suitable tool for the study of chronic diseases associated with enteroviral infections (Howard 2005).

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QPIA, Paper II

Current serological tests for enteroviruses

ELISA (enzyme-linked immunosorbent assay): It detects antibodies (IgM, IgG and IgA). IgM is detectable early after the onset of illness. IgG antibodies, which peak later, indicate past infection and immunity in most instances.

Examples of serological tests for enteroviruses are: (i) A PCR-enhanced immunoassay (PIA) to detect enterovirus (EV) immunoglobulin M (IgM) for diagnosis of recent EV infection (Aspholm, Zuo et al. 1999). (ii) The solid-phase reverse immunosorbent test (SPRIST), for measurement of IgM antibodies to EVs (Magnius, Saleh et al. 1988) and ELISA which uses synthetic peptides (Samuelson, Glimaker et al. 1993).

The new enterovirus IgM test

A QPCR-enhanced immunoassay (QPIA) to detect enterovirus (EV) immunoglobulin M (IgM) for diagnosis of recent EV infections.

EV PCR on CSF may directly detect the cause of AM. However, after clearance of viral RNA, detection of a serological EV antibody response remains a diagnostic alternative.

Immunoglobulin M (IgM) antibodies are more useful than neutralizing antibody tests, which mostly measure IgG, because IgM indicates recent infection with a serotype not previously encountered. Specific IgM can often be detected weeks to months after the disappearance of a virus from CSF. PIA provides a new concept for viral serology. It combines the sensitivity and specificity of PCR with an immunoglobulin capture solid-phase immunoassay (Aspholm, Zuo et al. 1999)

In many laboratories, IgM and IgG serological testing is done by indirect immuno-fluorescence (IFA) or enzyme linked immunosorbent assay (ELISA). Especially the former is often poorly standardized and requires specialized personnel for making slides and reading the results. In its real-time PCR form, the PIA assay takes advantage of both QPCR and PIA to become Quantitative PCR-enhanced ImmunoAssay (QPIA). It thus combines solid phase serological techniques with QPCR for the detection of anti-EV IgM. IgM in patient serum is captured onto anti-human IgM-coated microwell plates.

Serum samples were available for 59 of the 62 patients with suspected meningitis, and for 43 of the 45 patients finally diagnosed with AM not due to bacteria, herpes simplex, or TBE. Sixteen of the remaining 17 patients were diagnosed with non-EV infections. QPIA was positive for 24 of the 43

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(56%) cases (Table 3, a and b). However, samples from four cases finally diagnosed as bacterial or probable bacterial meningitis were also (weakly) positive by QPIA. One sample weakly positive by QPIA was from a case of herpes simplex type 2 meningitis, and one was from a case of TBE. Nineteen out of 30 (63%) serum samples obtained on the day of hospital admission were QPIA positive. Of these, 17 were positive for EV by PCR. Sixteen of 43 samples were positive by both QPCR and QPIA. These included samples from two patients from whom EV (E6 and E30) had been isolated. Sixteen of 43 samples were positive by QPCR alone, and 8 of 43 were positive by QPIA alone. None of the 30 control serum samples in group 3 were positiveby QPIA. For the 24 serum samples in group 4, of which 11 were positive and 13 were negative by RIA, the QPIA results were completely concordant. The sensitivity and specificity of QPIA for diagnosis of EV infection were 70 and 80%, respectively.

Table 3: Meningitis due to non-bacterial, non-herpetic and non-TBE cause

Method QPIA/Serum + QPIA/Serum - TotalQPCR/CSF

+16 16 32 (74%)

QPCR/CSF - 8 3 11

Total 24 (56%) 19 43

b) Meningitis due to bacterial (B), herpes (H), TBE and others diseases (O)

Method QPIA/Serum + QPIA/Serum - TotalQPCR/CSF

+0 0 0

QPCR/CSF - 6 10 16

Total 6 10 16

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Promising developments in antipicornavirus drugs (Aspholm, Zuo et al. 1999; Rogers, Diana et al. 1999; Schiff and Sherwood 2000) highlight the need to diagnose EV infections early in the course of the disease. The QPIA combines solid-phase serological techniques with QPCR. The risk of amplimer carryover contamination in the QPCR is reduced by using HK-UNG. The success of an immunoassay depends on choosing a good antigen. Over 10 different enteroviral strains were included in order to cover as many anti-enteroviral IgMs as possible. The complex is allowed to bind the EV antigen from an antigen mixture. Bound virus is heat-denatured and the released RNA, in RNase free water, is used as a template for QPCR. We therefore think that the QPIA method is of great interest for many clinical and research laboratories. If the quality of the real-time PCR method is ensured, and QPIA is further standardized, the accurate measurement of bound virus particles may make the corresponding QPIA suitable as a reference viral IgM method.

Calicivirus Project, Paper III

Viral gastroenteritis

Gastroenteritis means inflammation of the stomach and small and large intestines. Viral gastroenteritis is an infection caused by a variety of viruses those results in vomiting or diarrhea. It is often called the "stomach flu," although it is not caused by the influenza viruses. The major gastroenteritis viruses are rotaviruses, adenoviruses, caliciviruses and astroviruses. All except adenoviruses are RNA viruses.

Caliciviruses (family Caliciviridae) are non-enveloped viruses, 35 nm in diameter with single-stranded RNA positive-strand genomes of 7 to 8 kb. Phylogenetically, members of the family Caliciviridae are placed in four genera. Enteric caliciviruses, including those infecting humans (HuCV), belong to the genera Sapovirus (SV) and Norovirus (NV), whereas the animal caliciviruses belong to the genera Vesivirus and Lagovirus (Pringle 1999).

Noroviruses are listed as the most common cause of non-bacterial gastroenteritis worldwide. They afflict people of all age groups and are the main cause of outbreaks of gastroenteritis in institutions such as nursing homes and hospitals. A study in the Netherlands showed that human caliciviruses are responsible for 13% of all cases of gastroenteritis in the community (de Wit, Koopmans et al. 2001).

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Norovirus is the recently approved official genus name for the group of viruses provisionally described as “Norwalk-like viruses” (NLV). Norovirusis a genus within the positive stranded RNA Calicivirus family. Infection with norovirus manifests as a mild diarrhea associated with nausea, vomiting, abdominal cramps, malaise, headache and low-grade fever. The disease is usually self limited and lasts 1-3 days. Infection with norovirus is commonly known as “stomach flu” or viral gastroenteritis. Individuals of all ages are susceptible to Norovirus. Norovirus induced diarrhea is transmitted by the fecal-oral route and is transmitted via contaminated food and water and also hand to mouth behaviour in infants of contaminated objects. Shellfish, salads, raw clams and oysters are the foods most commonly implicated in the spread of Norovirus during outbreaks. Highly publicized outbreaks on cruise ships are common. Outbreaks at schools, prisons and health care facilities also occur and when investigated, Norovirus has been identified as the causative organism.

The incubation period usually is between 24-48 hours after consumption of affected food or water; and the disease lasts anywhere between 24-60 hours.

A major obstacle in the laboratory diagnosis of NV infection is the lack of a tissue culture system for propagating the viruses. Therefore, electron microscopy (EM) has been used routinely to detect NV particles in stool specimens (Caul and Appleton 1982). Noroviruses are a broad group of enteric pathogens with high sequence diversity. Molecular analyses such as real time PCR are more sensitive than traditional methods for detecting small amounts of viral templates. It can also be automated to allow the examination of large numbers of samples. The great variability of caliciviruses will be a continuous threat of false negativity in future diagnostic work.

Genus Sapovirus Sapovirus (SV), a member of the genus Sapovirus in the family Caliciiridae,is an etiologic agent of human gastroenteritis. (SV)-associated infections can cause both mild and acute gastroenteritis. Symptoms include watery stool, mild and or acute diarrhea, stomach cramps, nausea, and vomiting. The SLVs are also split into two genogroups. The prototype Sapporo virus belongs to genogroup I and the London virus belongs to genogroup II (Johansson, Bergentoft et al. 2005).

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Current diagnostic tests for caliciviruses

(1) Electron microscopy (EM) The most widely used direct detection assays of HuCVs are EM and sensitive generic reverse transcription assays with the RNA polymerase gene (pol) as a target (Ando, Monroe et al. 1995; Green, Gallimore et al. 1995; Nakayama, Ueda et al. 1996; Vinje and Koopmans 1996). EM is only possible in samples with a high viral load. EM is too insensitive to detect HuCVs in samples with less than 106 particles per ml. This requires a specimen where high concentration of virus can be expected, such as diarrhea stool (Caul and Appleton 1982; Otsu, Ishikawa et al. 2000).

(2) Reverse line blot hybridization (RLB) PCR-based reverse line blot hybridization (mPCR/RLB) is a useful method for simultaneous detection and genotyping of microorganisms such as caliciviruses (Vinje and Koopmans 2000).

The new Calicivirus PCR

Diagnosis of caliciviruses by real-time PCR systems have advantages, because they are less liable to be contaminated, and can provide semiquantitative estimates of viral concentration. NVs are highly variable positive-sense single stranded RNA viruses. A combination of reverse transcription and QPCR, using the few conserved sequence stretches, is thus necessary. A ConSort© analysis of a CLUSTALW alignment of 1472 full-length caliciviral sequences indicated that the ORF1-ORF2 junction region within NLV GI or GII was one of the most conserved portions (Fig. 10). Complete genomic sequences of representative caliciviruses were aligned with Blast and examined with ConSort. The stretch which was most highly conserved proved to be the region of overlap between replicase (ORF1) and capsid (ORF2) genes (figure 10). All calicivirus ORF1 and ORF2 sequences available in GenBank were retrieved to refine the analysis.

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Fig:10 Analysis of a Norovirus BLAST alignment. Symbols are as in fig. 9.

Variations in the temperature cycle protocol in real time PCR profoundly affect the sensitivity and specificity of PCR based tests. Several temperature protocols will be evaluated. In touch-down QPCR (TD-QPCR), described above, the annealing temperature starts several degrees above the calculated optimal annealing temperature and is gradually lowered in subsequent cycles (Hecker and Roux 1996; Han, Qi et al. 2004). In TD-QPCR 56 out of 71 (79%) of the specimens were positive. Most of the seventy one outbreaks occurred in semiclosed communities (family party, day-care centre, nursing homes, and hospital). Our method indicated that genogroup II was the most common cause of outbreaks of acute gastroenteritis in Sweden during 1997-2004 in the general population. It was shown that the touch-down protocol increased both the specificity and sensitivity of QPCR assays. In a later evaluation, however, the frequency of positivity with Swedish gastroenteritis samples went down to 45%. The cause for this change is not known. It is likely that mutations in the primer/probe target stretches rendered some samples falsely negative. It will probably be necessary to continuously modify the Calicivirus PCRs to accommodate viral change.

However, in principle, the TD-QPCR technique seems to be a rational screening tool for Norovirus genogroup I and II. The combination of RT and PCR amplification, in a single step real-time PCR with contamination protection as well as preliminary genotyping, could save time and reagent costs.

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Influenza Project, Paper IV

Current influenza diagnostic methods include virus isolation, antigen detection, and serology. Major limitations of these techniques include prolonged time to completion, subjective evaluation, low sensitivity, and low specificity. Use of nucleic acid amplification techniques has made sensitive diagnosis of influenza virus infection feasible, with the possibility of type determination.

The high variability of influenza viruses creates a diagnostic problem. Until now, a PCR based on the matrix gene (Fouchier, Bestebroer et al. 2000; Widjaja, Krauss et al. 2004) was the most general influenza A detection method. For simultaneous detection of influenza A and B, a nested PCR technique based on the hemagglutinin gene was developed (Herrmann, Larsson et al. 2001).

The new influenza PCR

Broadly targeted triplex real time PCR detection of Influenza A, B and C Viruses based on the Nucleoprotein gene (Manuscript submitted for publication)

Respiratory infections are common. Examples are colds in both adults and children. Most are fairly mild, self- limiting and confined to the upper respiratory tract (URI). A viral respiratory infection is an infection caused by a variety of viruses such as: influenza or corona viruses. Most URIs are probably viral induced - at least initially (Mackie 2003).

The term "influenza" refers to illness caused by influenza virus. This is commonly also called "flu", but many different illnesses cause "flu-like" systemic and respiratory symptoms such as fever, chills, aches and pains, cough, and sore throat. In addition, influenza itself can cause many different illness patterns, ranging from asymptomatic illness, mild common cold symptoms, to typical "influenza", or to life-threatening pneumonia and other complications, including secondary bacterial infections. Influenza occurs most often in the winter season. Illnesses resembling influenza may occur in the summer months but they are usually due to other viruses.

Influenza virus particles are highly pleiomorphic (variable), most are spherical/ovoid, 80-120 nm diameters, but many others forms occur, including long filamentous particles (up to 2000 nm long x 80-120 nm diameter) (Howley 2001; Mackie 2003).

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Symptoms of influenza

Typical influenza symptoms include headache, fever, chills, cough or sore throat and body aches. Intestinal symptoms are uncommon. Although most people are ill for only a few days, some people have a much more serious illness, such as pneumonia, and may need to be hospitalized. Influenza A and B virus infections are major causes of morbidity and mortality worldwide. Approximately 20,000 people die each year in the United States from influenza or related complications. Influenza is highly contagious and is easily transmitted through contact with droplets from the nose and throat of an infected person during coughing and sneezing. The incubation period for influenza is one to five days (Howley 2001).

Genome

The influenza genome consists of s/s (-) sense RNA in 8 segments (7 in Influenza C). The structure of the influenza virus genome is known in great detail because of the tremendous amount of investigation which has been done. The 5' and 3' terminal sequences of all the genome segments are highly conserved (Maeda, Horimoto et al. 2003).

The influenza viruses belong to the Orthomyxoviridae family (Monto, Ohmit et al. 1995), are divided into three types, A, B, and C, on the basis of antigenic differences in nucleoprotein and matrix (M) proteins. Influenza A viruses are further classified into subtypes based on the antigenic differences of the surface proteins hemagglutinin (HA) and neuraminidase (NA). Currently, there are 16 distinct HA (H1 to H16) and 9 NA (N1 to N9) subtypes. Three subtypes of HA (H1 to H3) and two subtypes of NA (N1 and N2) are found among influenza A viruses that have caused epidemics among humans (Gregory, Bennett et al. 2002). Only influenza A and B viruses cause epidemic human disease. Influenza B and C viruses are not categorized into subtypes.

Although it has been nearly 60 years since the influenza C virus was first isolated in 1947 (Zakstel'skaia and Govorkova 1987) there have been few reports describing its clinical features. This lack of information may be attributable to a mild pathogenicity of this virus but more to the difficulty in isolating it. Influenza C viruses are known to infect humans and animals such as pigs. Influenza C virus is widely distributed throughout the world. The majority of humans acquire antibodies to the virus early in life. Thus, infection with this virus is common in childhood. It is a significant cause of respiratory tract disease, sometimes in need of hospitalisation, in children <6 years old. However, most of the influenza C infected children <3 years old showed fever and mild upper respiratory tract (URT) symptoms (Matsuzaki, Katsushima et al. 2006).

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The diagnosis of Influenza

The 3QPCR

Nucleoprotein genes are suitable regions for the design of wide range diagnostic primers and probes for real time PCR (figure 1, paper IV). The specificity of the triplex reverse transcription real-time PCR (“3QPCR”) was evaluated on eighteen influenza A reference strains (H1N1, H2N3, H5N1Sc,H5N1Ty, H5N2, H1N7, H7N7, H9N2, H4N6, H5N3, H8N4, H10N8, H12N5, H16N8, H3N8 and AIV-N3). The isolates of influenza B were B/Russia/69and B/Lee/40. The isolates of influenza C were cultured in embryonated eggs and the harvested allantoic fluids were inactivated with formaldehyde. Using parallel endpoint serial dilutions of influenza A, B and C samples, and of known concentrations of synthetic DNA targets, the sensitivity was determined to be 1-10 viral nucleic acid copies per PCR reaction for influenza A, B and C. In 45 sequential nasopharyngeal aspirates collected during 1999-2002 tested in parallel with a nested RT-PCR and the 3QPCR, the nested method detected 15/45 (33%), while 26/45 ( 58%) were positive by 3QPCR. Additionally, 127 nasopharyngeal aspirates were collected during 2000-2005. Of 203 clinical samples tested with both IF and 3QPCR,51 were positive for influenza A in both tests while 22 were Influenza A positive in 3QPCR only. Six samples were Influenza A IF positive and negative with 3QPCR. For influenza B, 33 samples were positive in both tests, but 6 were positive in 3QPCR only. There were 25 samples which were RSV positive by IF. Only one of them was positive by QPCR, probably due to double infection. Among the total set of 203 samples, 3QPCR detected influenza A in 72, influenza B in 39 and influenza C in 1 sample(s). Our study showed that 3QPCR is a sensitive, and rational method for detecting and identification of influenza viruses. Its generic nature should enable detection not only of most human influenza viruses, but also of avian and other influenza strains. Thus, it would provide a powerful novel tool for diagnostic laboratories worldwide. It may also enable the discovery of new influenza viruses.

In summary, 3QPCR described in this study proved to be a rapid, specific and accurate assay for the rapid detection and identification of influenza viruses. The combination of RT and PCR reactions, in a single step real-time PCR with contamination protection, as well as typing, saves time and reagent costs. In addition, this assay can detect a broad range of influenza A, including low and highly pathogenic H5N1, and therefore is suitable for a rational screening tool for influenza viruses in humans and animals.

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Corona Virus Project,”Paper V”

Current diagnostic methodologies for corona virus infection include molecular testing using reverse transcriptase-polymerase chain reaction (RT-PCR); antibody tests by enzyme linked immunosorbent assay (ELISA) or immunofluoresence assay (IFA); and virus isolation using Vero E6 cells. The methods using RT-PCR for detecting SARS-CoV viral RNA are safer and faster than cell culture-based assays (Huang, Lin et al. 2005).

The new corona virus PCR

Broadly targeted TaqMan® QPCR method for the detection of corona viruses in humans and animals (Manuscript)

Corona Viruses

Coronaviruses comprise a genus in the family Coronaviridae. They are large, enveloped, positive-stranded RNA viruses that are highly prevalent in humans and domestic animals and responsible for diseases of the enteric and respiratory systems.

Coronaviruses have the largest genomes (27 to 32 kb) of all the RNA viruses and replicate by a unique mechanism which results in a high frequency of recombination. The genome includes 7 to 10 open reading frames (ORF) that encode both structural and non-structural proteins. Gene 1 consists of two overlapping regions (ORF1a and ORF1b) that are translated into a polyprotein, the precursor of the viral replicase complex (Brian and Baric 2005). There are four structural proteins, (5’-Rep-S-E-M-N-3’) downstream from the replicase gene, interspersed with several ORFs which encode non-structural proteins and, in some strains, the HE glycoprotein. The most important biological and immunological functions seem to reside in the S and M proteins. The coronavirus family is divided into three groups based on antigenic and genetic characteristics (Siddell, Anderson et al. 1983). Groups 1 and 2 infect a large range of mammalian species whereas group 3 is restricted to birds (Saif 2004; Saif 2004). Classification of the severe acute respiratory syndrome coronavirus (SARS-CoV) in group 2 or as the prototype of a new group 4 is subjected to controversy and is complicated by the putative recombinant origin of its genome (Rest and Mindell 2003; Snijder, Bredenbeek et al. 2003; Gorbalenya, Snijder et al. 2004) (Fig.12).

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Coronaviruses, the cause of SARS

SARS is a type of viral pneumonia, with symptoms including fever, a dry cough, dyspnea (shortness of breath), headache, and hypoxaemia (low blood oxygen concentration). Typical laboratory findings include lymphopenia (reduced lymphocyte numbers) and mildly elevated aminotransferase levels (indicating liver damage). Death may result from progressive respiratory failure due to alveolar damage. The typical clinical course of SARS involves an improvement in symptoms during the first week of infection, followed by a worsening during the second week. Studies indicate that this worsening may be related to patient's immune responses rather than uncontrolled viral replication (Chan, Tang et al. 2006; Liu, Fontanet et al. 2006).

Primer and Probe design

Consensus primers and probes were designed using the program Consort (J. Blomberg, unpublished). A ConSort© analysis of a CLUSTALW alignment of 32 full-length coronaviral sequences indicated that ORF1b was one of the most conserved portions (Fig. 11). Complete genomic sequences of representative coronaviruses were aligned with Blast and examined with ConSort. The replicase gene was identified as the most conserved genomic region (figure 1, Paper VI). All coronavirus ORF1a and ORF1b sequences available in GenBank were retrieved to refine the analysis. The most conserved stretches were found in the ORF1b where one pair of degenerate primers was designed to amplify a fragment of 169 bp. The primer sequences, and their positions in the genome of SARS Tor2 (AY274119) are: 11-FW: ´TGATGATGSNGTTGTNTGYTAYAA 3´ (nt 15647-15670) and 13-RV: 5´GCATWGTRTGYTGNGARCARAATT 3´ (15802-15825) probe_a FAM-5´-TAYTAYCARAATAATGTTT-3´-TAMRA and probe_b FAM-5´ YCTAARTGRTGTBTTTATG-3´-TAMRA. Underlined positions were LNA nucleotides.

Thirtytwo animal coronavirus strains and 77 nasopharyngeal aspirates from human lower respiratory infections with the coronavirus QPCR. The assay enabled the detection of all 32 available strains of human and animal coronaviruses.

RNA from 77 human nasopharyngeal aspirates was analyzed by both the NQPCR and the SYBRgreen QPCRs. Both techniques indicated that 8 samples contained coronavirus. The SYBRgreen QPCR products of the eight corona positive samples were sequenced, and aligned with reference sequences. Six of the seven sequenced samples turned out to harbour OC43-like, and one NL63-like sequences.

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The detection of HCoV-OC43-like and HCoV-NL63 sequences in eight out of 77 nasopharyngeal specimens of human origin provides evidence of the efficacy of the technique with clinical material. The NQPCR results were concordant with those of the pan-corona SYBRgreen QPCR technique used as a control. As expected, the 8 QPCR corona positive samples were negative in the respiratory virus IFA, and the nested influenza A+B PCR, both of which do not detect coronaviruses. The method thus should be suitable for diagnostic purposes.

Figure 11. Analysis of an alignment of full length the human NL63, 229E, OC43 and SARS, the porcine PEDV and PTGEV, the avian IBV, the bovine BCov, the and the murine MHV coronaviral sequences. Columns depict the average number of variants per nucleotide position.

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Fig: 12 Classification of the Coronavirus family

In conclusion, the NQPCR described here seems to be a broadly detecting and specific system for coronaviruses. It should be applicable for routine and research, for both humans and animals and as a research tool for the detection of still uncharacterized coronaviruses. To our knowledge, this is the first report of a coronavirus method adapted to real-time format and tested on a wide range of viral strains.

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Concluding remarks and future prospectives

This study focuses on the development and optimisation of sensitive, quantificative broad and rapid viral RNA detection methods intended for diagnostic purposes. .

A sensitive system of real-time polymerase chain reaction for quantification viral RNA amount extracted from various types of human specimens was developed and established in order to lay the ground work for future studies.

In a few comparisons, the LNA probes gave higher fluorescence signals than non-LNA probes of equal length.

There are major advantages of using QPCR compared to conventional semi-quantitative PCR. Firstly, the progression of the PCR reaction may be monitored after each cycle rather than at the end, thereby providing a much better quantification assay; secondly, very little nucleic acid is needed; thirdly it is a non-radioactive assay; and finally it can be performed in approximately two to three hours. The principle used in this assay can be adapted to a large number of biological systems and could therefore have a significant impact on patient care by providing accurate results in a shorter time. The methods developed are rapid and broadly applicable to all specimens and infected tissues.

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Acknowledgements

I wish to express my sincere gratitude to many people who have in one way or another participated to this thesis, I would like to express special thanks and appreciation to:

Jonas Blomberg- who is not only my supervisor in research but my mentor in such an interesting area of research “virology”; for his unlimited support, inspiration, generosity, a no-nonsense approach, his time, constant guidance and unbelievable patience, His great knowledge in virology, helped me to build up my scientific knowledge and his sense of humour that eased-up the monotony of work. I appreciate your excellent scientific guidance and criticism. I acknowledge that I still have a lot of work to do in my training as a scientist; I look forward to working with him in the future.

I owe special debt of gratitude to my co-supervisor Jan Fohlman for his continuous concern and generosity, having me and my husband in his house many times. I will always remember our interesting scientific discussions and for his endless discussions and advices. Jan, you were always there for me whenever I needed help and guidance. In addition, I would like to thank him for his time and valuable input in the writing of this thesis.

All my co-authors and contributors to the studies and in particular Göran Friman for his fruitful collaboration.

I would like to express my thanks to my colleagues Amal, Shamam, Lijuan, Guma and Farid, for their warm friendship and for making the lab a wonderful workplace. I thank all the present and former members of the Public Health (Vårdhygien) lab Eva Tano, Margareta Edvall, Camilla and Ulla Zettersten for their help, encouragement and understanding. I also credit all the people at clinical bacteriology for their patience.

In addition, I would like to thank Eva Haxton for her unlimited help. I would like to thank Afaf Kamal-Eldin, Neil LeBlanc and Eva Haxton

for careful reading and comments on this thesis Also, to the many other members of the Department, thank you for your

assistance and support during my years at the department in particular Birgitta Sembrant.

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I would like to thank Anjuman Hyder and Alia Yacoub for collaboration in the influenza project.

I want to express my gratitude to Prof. Sandor Belak for his positive attitude.

I would like to thank the staff at the department of virology at SVA Frederik Widén and Mikhayil Hakhverdyan that are always supportive and helpful.

There are many others that deserves to be thanks especially the project workers, too numerous to be mention here, but you have many gratitude and appreciation.

I want to express my gratitude to all my friends and colleagues in Uppsala & Stockholm, whose support and good will kept me going through my study time. I would particularly like to thanks my dearest friends Saadia, Azza and Zafir for their support and encouragement.

In addition, I must thank my boss and friend Prof. Mohi Aldin Almagzoub for his encourages me to visit Sweden for the first time.

My profound gratitude to my big family in Sudan, my grand father “habooba Zaineb” my aunts Amna, Liyla, Alawyia, Awatif, Nasra Nuha,my uncles Omer, Aballah, Abdelatif, Badri Sami, my cousins, my dearest friends brother Rida, my lovely sister Ghada and my sister-in-law Amira, my nieces Noona, Shosho, Hayota and my lovely nephew Hadhoodi for all the help and support. Thanks too, to the rest of the family for their interest, unlimited support and encouragement.

I want to express my gratitude to my new family around me all the time, my father-in-law Mohamed abu yousif, my mother-in-law Fayza, my sisters-in-law Miska & Jehan, and my brothers-in-law: Abuobida, Tarig, Abdelmajid, Malik and Khalid.

Last but not least, mountains of gratitude and thanks to my husband Ahmed Abu yousif for his extreme unlimited support and encouragement.

For funding of this research, I gratefully acknowledge the financial support received from SVA, especially through Prof. Sandor Belak in the form of two FORMAS grants.

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References:

Ando, T., S. S. Monroe, et al. (1995). "Detection and differentiation of antigenically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-PCR and southern hybridization." J Clin Microbiol 33(1): 64-71.

Aspholm, R., S. Zuo, et al. (1999). "A novel serological technique: polymerase chain reaction enhanced immunoassay. Application to enterovirus IgM diagnosis." J Virol Methods 80(2): 187-96.

Auer, T., P. A. Landre, et al. (1995). "Properties of the 5'-->3' exonuclease/ribonuclease H activity of Thermus thermophilus DNA polymerase." Biochemistry 34(15): 4994-5002.

Auer, T., J. J. Sninsky, et al. (1996). "Selective amplification of RNA utilizing the nucleotide analog dITP and Thermus thermophilus DNA polymerase." Nucleic Acids Res 24(24): 5021-5.

Baner, J., M. Nilsson, et al. (2001). "More keys to padlock probes: mechanisms for high-throughput nucleic acid analysis." Curr Opin Biotechnol 12(1): 11-5.

Boeckh, M., M. Huang, et al. (2004). "Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR." J Clin Microbiol 42(3): 1142-8.

Bono, C. M. (2006). "Spectrum of spine infections in patients with HIV: a case report and review of the literature." Clin Orthop Relat Res 444: 83-91.

Brian, D. A. and R. S. Baric (2005). "Coronavirus genome structure and replication." Curr Top Microbiol Immunol 287: 1-30.

Bustin, S. A. (2000). "Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays." J Mol Endocrinol 25(2): 169-93.

Bustin, S. A., V. Benes, et al. (2005). "Quantitative real-time RT-PCR--a perspective." J Mol Endocrinol 34(3): 597-601.

Casas, I., P. E. Klapper, et al. (1995). "Two different PCR assays to detect enteroviral RNA in CSF samples from patients with acute aseptic meningitis." J Med Virol 47(4): 378-85.

Caul, E. O. and H. Appleton (1982). "The electron microscopical and physical characteristics of small round human fecal viruses: an interim scheme for classification." J Med Virol 9(4): 257-65.

Chan, P. K., J. W. Tang, et al. (2006). "SARS: clinical presentation, transmission, pathogenesis and treatment options." Clin Sci (Lond) 110(2): 193-204.

Chapman, N. M., S. Tracy, et al. (1990). "Molecular detection and identification of enteroviruses using enzymatic amplification and nucleic acid hybridization." J Clin Microbiol 28(5): 843-50.

Chen, Y., D. B. Carlini, et al. (1999). "RNA secondary structure and compensatory evolution." Genes Genet Syst 74(6): 271-86.

56

Chiocchia, G. and K. A. Smith (1997). "Highly sensitive method to detect mRNAs in individual cells by direct RT-PCR using Tth DNA polymerase." Biotechniques 22(2): 312-4, 316, 318.

de Wit, M. A., M. P. Koopmans, et al. (2001). "Sensor, a population-based cohort study on gastroenteritis in the Netherlands: incidence and etiology." Am J Epidemiol 154(7): 666-74.

DeRisi, J. L. and V. R. Iyer (1999). "Genomics and array technology." Curr Opin Oncol 11(1): 76-9.

Don, R. H., P. T. Cox, et al. (1991). "'Touchdown' PCR to circumvent spurious priming during gene amplification." Nucleic Acids Res 19(14): 4008.

Drake, J. W., B. Charlesworth, et al. (1998). "Rates of spontaneous mutation." Genetics 148(4): 1667-86.

Espy, M. J., J. R. Uhl, et al. (2006). "Real-time PCR in clinical microbiology: applications for routine laboratory testing." Clin Microbiol Rev 19(1): 165-256.

Fenner, D. O. W. F. J. (1994). Medical Virology. San Diego, CA, Academic Press. Firth, A. E. and C. M. Brown (2006). "Detecting overlapping coding sequences in

virus genomes." BMC Bioinformatics 7: 75. Forterre, P. (2006). "The origin of viruses and their possible roles in major

evolutionary transitions." Virus Res 117(1): 5-16. Fouchier, R. A., T. M. Bestebroer, et al. (2000). "Detection of influenza A viruses

from different species by PCR amplification of conserved sequences in the matrix gene." J Clin Microbiol 38(11): 4096-101.

Freeman, W. M., S. L. Vrana, et al. (1996). "Use of elevated reverse transcription reaction temperatures in RT-PCR." Biotechniques 20(5): 782-3.

Gibbs, R. A. (1991). "Polymerase chain reaction techniques." Curr Opin Biotechnol2(1): 69-75.

Gibson, U. E., C. A. Heid, et al. (1996). "A novel method for real time quantitative RT-PCR." Genome Res 6(10): 995-1001.

Glimaker, M., A. Abebe, et al. (1992). "Detection of enteroviral RNA by polymerase chain reaction in faecal samples from patients with aseptic meningitis." J Med Virol 38(1): 54-61.

Glimaker, M., A. Samuelson, et al. (1992). "Early diagnosis of enteroviral meningitis by detection of specific IgM antibodies with a solid-phase reverse immunosorbent test (SPRIST) and mu-capture EIA." J Med Virol 36(3): 193-201.

Gorbalenya, A. E., E. J. Snijder, et al. (2004). "Severe acute respiratory syndrome coronavirus phylogeny: toward consensus." J Virol 78(15): 7863-6.

Green, J., C. I. Gallimore, et al. (1995). "Broadly reactive reverse transcriptase polymerase chain reaction for the diagnosis of SRSV-associated gastroenteritis." J Med Virol 47(4): 392-8.

Gregory, V., M. Bennett, et al. (2002). "Emergence of influenza A H1N2 reassortant viruses in the human population during 2001." Virology 300(1): 1-7.

Han, X. Q., Z. L. Qi, et al. (2004). "Touch-down PCR and single-strand conformation polymorphism for detecting clonal T cell receptor gamma gene rearrangement in lymphoid leukemia." Di Yi Jun Yi Da Xue Xue Bao 24(2): 188-91.

Hecker, K. H. and K. H. Roux (1996). "High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR." Biotechniques 20(3): 478-85.

Hellen, C. U. and T. V. Pestova (1999). "Translation of hepatitis C virus RNA." J Viral Hepat 6(2): 79-87.

57

Herrmann, B., C. Larsson, et al. (2001). "Simultaneous detection and typing of influenza viruses A and B by a nested reverse transcription-PCR: comparison to virus isolation and antigen detection by immunofluorescence and optical immunoassay (FLU OIA)." J Clin Microbiol 39(1): 134-8.

Hofacker, I. L., M. Fekete, et al. (1998). "Automatic detection of conserved RNA structure elements in complete RNA virus genomes." Nucleic Acids Res 26(16): 3825-36.

Hofacker, I. L. and P. F. Stadler (1999). "Automatic detection of conserved base pairing patterns in RNA virus genomes." Comput Chem 23(3-4): 401-14.

Hofmann-Lehmann, R., R. K. Swenerton, et al. (2000). "Sensitive and robust one-tube real-time reverse transcriptase-polymerase chain reaction to quantify SIV RNA load: comparison of one- versus two-enzyme systems." AIDS Res Hum Retroviruses 16(13): 1247-57.

Holland, P. M., R. D. Abramson, et al. (1991). "Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase." Proc Natl Acad Sci U S A 88(16): 7276-80.

Howard, R. S. (2005). "Poliomyelitis and the postpolio syndrome." Bmj 330(7503): 1314-8.

Howley, D. M. K. P. M. (2001). Fundamental Virology. Philadelphia, PA 19106 USA, LIPPINCOTT WILLIAMS & WILKINS.

Huang, J. L., H. T. Lin, et al. (2005). "Rapid and sensitive detection of multiple genes from the SARS-coronavirus using quantitative RT-PCR with dual systems." J Med Virol 77(2): 151-8.

Jiang, Y. L., D. J. Krosky, et al. (2005). "Uracil-directed ligand tethering: an efficient strategy for uracil DNA glycosylase (UNG) inhibitor development." J Am Chem Soc 127(49): 17412-20.

Johansson, P. J., K. Bergentoft, et al. (2005). "A nosocomial sapovirus-associated outbreak of gastroenteritis in adults." Scand J Infect Dis 37(3): 200-4.

Johnson, M. P., L. M. Haupt, et al. (2004). "Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR." Nucleic Acids Res 32(6): e55.

Johnson, Z. I. and S. W. Chisholm (2004). "Properties of overlapping genes are conserved across microbial genomes." Genome Res 14(11): 2268-72.

Kessler, H. H., B. Santner, et al. (1997). "Rapid diagnosis of enterovirus infection by a new one-step reverse transcription-PCR assay." J Clin Microbiol 35(4): 976-7.

Kim, S. H. (1976). "Three-dimensional structure of transfer RNA." Prog Nucleic Acid Res Mol Biol 17: 181-216.

Kleiboeker, S. B. (2005). "Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR." Virol J 2: 29.

Koblet, H. (1988). "Molecular biology and the diagnosis, epidemiology and pathogenesis of infectious diseases." FEMS Microbiol Immunol 1(2): 63-7.

Kostrzynska, M. and A. Bachand (2006). "Application of DNA microarray technology for detection, identification, and characterization of food-borne pathogens." Can J Microbiol 52(1): 1-8.

Kumar, R. (2005). "Aseptic meningitis: diagnosis and management." Indian J Pediatr 72(1): 57-63.

Kwok, S. and R. Higuchi (1989). "Avoiding false positives with PCR." Nature339(6221): 237-8.

Landegren, U., E. Schallmeiner, et al. (2004). "Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes." J Mol Recognit 17(3): 194-7.

58

Lecellier, C. H. and O. Voinnet (2004). "RNA silencing: no mercy for viruses?" Immunol Rev 198: 285-303.

Lee, L. G., C. R. Connell, et al. (1993). "Allelic discrimination by nick-translation PCR with fluorogenic probes." Nucleic Acids Res 21(16): 3761-6.

Leite, C., A. Barbosa, Jr., et al. (2005). "Viral diseases of the central nervous system." Top Magn Reson Imaging 16(2): 189-212.

Liu, W., A. Fontanet, et al. (2006). "Two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome." J Infect Dis 193(6): 792-5.

Loftis, A. D., R. F. Massung, et al. (2003). "Quantitative real-time PCR assay for detection of Ehrlichia chaffeensis." J Clin Microbiol 41(8): 3870-2.

Longo, M. C., M. S. Berninger, et al. (1990). "Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions." Gene 93(1): 125-8.

Lopez de Quinto, S. and E. Martinez-Salas (1997). "Conserved structural motifs located in distal loops of aphthovirus internal ribosome entry site domain 3 are required for internal initiation of translation." J Virol 71(5): 4171-5.

Mackay, I. M. (2004). "Real-time PCR in the microbiology laboratory." Clin Microbiol Infect 10(3): 190-212.

Mackay, I. M., K. E. Arden, et al. (2002). "Real-time PCR in virology." Nucleic Acids Res 30(6): 1292-305.

Mackie, P. L. (2003). "The classification of viruses infecting the respiratory tract." Paediatr Respir Rev 4(2): 84-90.

Maeda, Y., T. Horimoto, et al. (2003). "[Classification and genome structure of influenza virus]." Nippon Rinsho 61(11): 1886-91.

Magnius, L. O., L. H. Saleh, et al. (1988). "A solid-phase reverse immunosorbent test for the detection of enterovirus IgM." J Virol Methods 20(1): 73-82.

Marchand, S., P. Hajdari, et al. (2003). "Touch-down method for high-performance sequencing of polymerase chain reaction products." Anal Biochem 315(2): 270-2.

Martinez-Salas, E., S. Lopez de Quinto, et al. (2002). "IRES elements: features of the RNA structure contributing to their activity." Biochimie 84(8): 755-63.

Martinez-Salas, E., M. P. Regalado, et al. (1996). "Identification of an essential region for internal initiation of translation in the aphthovirus internal ribosome entry site and implications for viral evolution." J Virol 70(2): 992-8.

Matsuzaki, Y., N. Katsushima, et al. (2006). "Clinical features of influenza C virus infection in children." J Infect Dis 193(9): 1229-35.

Mohamed, N., A. Elfaitouri, et al. (2004). "A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA." J Clin Virol 30(2): 150-6.

Monpoeho, S., M. Coste-Burel, et al. (2002). "Application of a real-time polymerase chain reaction with internal positive control for detection and quantification of enterovirus in cerebrospinal fluid." Eur J Clin Microbiol Infect Dis 21(7): 532-6.

Monto, A. S. (2002). "Epidemiology of viral respiratory infections." Am J Med 112 Suppl 6A: 4S-12S.

Monto, A. S., S. E. Ohmit, et al. (1995). "Medical practice-based influenza surveillance: viral prevalence and assessment of morbidity." Am J Epidemiol141(6): 502-6.

Morse, S. S. (1994). THE EVOLUTIONARY BIOLOGY OF VIRUSES. New York, New York 10036, USA, Raven Press.

59

Mouritzen, P., A. T. Nielsen, et al. (2003). "Single nucleotide polymorphism genotyping using locked nucleic acid (LNA)." Expert Rev Mol Diagn 3(1): 27-38.

Myers, T. W. and D. H. Gelfand (1991). "Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase." Biochemistry30(31): 7661-6.

Nakayama, M., Y. Ueda, et al. (1996). "Detection and sequencing of Norwalk-like viruses from stool samples in Japan using reverse transcription-polymerase chain reaction amplification." Microbiol Immunol 40(4): 317-20.

Nilsson, M., J. Baner, et al. (2002). "Making ends meet in genetic analysis using padlock probes." Hum Mutat 19(4): 410-5.

Norder, H., L. Bjerregaard, et al. (2003). "Sequencing of 'untypable' enteroviruses reveals two new types, EV-77 and EV-78, within human enterovirus type B and substitutions in the BC loop of the VP1 protein for known types." J Gen Virol84(Pt 4): 827-36.

Nwachuku, N. and C. P. Gerba (2006). "Health risks of enteric viral infections in children." Rev Environ Contam Toxicol 186: 1-56.

Otsu, R., A. Ishikawa, et al. (2000). "Detection of small round structured viruses in stool specimens from outbreaks of gastroenteritis by electron microscopy and reverse transcription-polymerase chain reaction." Acta Virol 44(1): 53-5.

Petersen, M., K. Bondensgaard, et al. (2002). "Locked nucleic acid (LNA) recognition of RNA: NMR solution structures of LNA:RNA hybrids." J Am Chem Soc 124(21): 5974-82.

Pickering, B. M. and A. E. Willis (2005). "The implications of structured 5' untranslated regions on translation and disease." Semin Cell Dev Biol 16(1): 39-47.

Pringle, C. R. (1999). "Virus taxonomy--1999. The universal system of virus taxonomy, updated to include the new proposals ratified by the International Committee on Taxonomy of Viruses during 1998." Arch Virol 144(2): 421-9.

Pruvost, M., T. Grange, et al. (2005). "Minimizing DNA contamination by using UNG-coupled quantitative real-time PCR on degraded DNA samples: application to ancient DNA studies." Biotechniques 38(4): 569-75.

Rashtchian, A., C. G. Thornton, et al. (1992). "A novel method for site-directed mutagenesis using PCR and uracil DNA glycosylase." PCR Methods Appl 2(2): 124-30.

Rest, J. S. and D. P. Mindell (2003). "SARS associated coronavirus has a recombinant polymerase and coronaviruses have a history of host-shifting." Infect Genet Evol 3(3): 219-25.

Rogers, J. M., G. D. Diana, et al. (1999). "Pleconaril. A broad spectrum antipicornaviral agent." Adv Exp Med Biol 458: 69-76.

Rotbart, H. A. (2002). "Treatment of picornavirus infections." Antiviral Res 53(2): 83-98.

Rotbart, H. A., A. Ahmed, et al. (1997). "Diagnosis of enterovirus infection by polymerase chain reaction of multiple specimen types." Pediatr Infect Dis J16(4): 409-11.

Saif, L. J. (2004). "Animal coronavirus vaccines: lessons for SARS." Dev Biol (Basel) 119: 129-40.

Saif, L. J. (2004). "Animal coronaviruses: what can they teach us about the severe acute respiratory syndrome?" Rev Sci Tech 23(2): 643-60.

Samuelson, A., M. Glimaker, et al. (1993). "Diagnosis of enteroviral meningitis with IgG-EIA using heat-treated virions and synthetic peptides as antigens." J Med Virol 40(4): 271-7.

60

Schiff, G. M. and J. R. Sherwood (2000). "Clinical activity of pleconaril in an experimentally induced coxsackievirus A21 respiratory infection." J Infect Dis181(1): 20-6.

Schunck, B., W. Kraft, et al. (1995). "A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces." J Virol Methods 55(3): 427-33.

Siddell, S. G., R. Anderson, et al. (1983). "Coronaviridae." Intervirology 20(4): 181-9.

Snijder, E. J., P. J. Bredenbeek, et al. (2003). "Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage." J Mol Biol 331(5): 991-1004.

Spriggs, K. A., M. Bushell, et al. (2005). "Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors." Cell Death Differ 12(6): 585-91.

Thornton, C. G., J. L. Hartley, et al. (1992). "Utilizing uracil DNA glycosylase to control carryover contamination in PCR: characterization of residual UDG activity following thermal cycling." Biotechniques 13(2): 180-4.

Thurner, C., C. Witwer, et al. (2004). "Conserved RNA secondary structures in Flaviviridae genomes." J Gen Virol 85(Pt 5): 1113-24.

Ugozzoli, L. A., D. Latorra, et al. (2004). "Real-time genotyping with oligonucleotide probes containing locked nucleic acids." Anal Biochem 324(1): 143-52.

Wang, A. M., M. V. Doyle, et al. (1989). "Quantitation of mRNA by the polymerase chain reaction." Proc Natl Acad Sci U S A 86(24): 9717-21.

Verstrepen, W. A., S. Kuhn, et al. (2001). "Rapid detection of enterovirus RNA in cerebrospinal fluid specimens with a novel single-tube real-time reverse transcription-PCR assay." J Clin Microbiol 39(11): 4093-6.

Widjaja, L., S. L. Krauss, et al. (2004). "Matrix gene of influenza a viruses isolated from wild aquatic birds: ecology and emergence of influenza a viruses." J Virol78(16): 8771-9.

Vinje, J. and M. P. Koopmans (1996). "Molecular detection and epidemiology of small round-structured viruses in outbreaks of gastroenteritis in the Netherlands." J Infect Dis 174(3): 610-5.

Vinje, J. and M. P. Koopmans (2000). "Simultaneous detection and genotyping of "Norwalk-like viruses" by oligonucleotide array in a reverse line blot hybridization format." J Clin Microbiol 38(7): 2595-601.

Wittwer, C. T., M. G. Herrmann, et al. (1997). "Continuous fluorescence monitoring of rapid cycle DNA amplification." Biotechniques 22(1): 130-1, 134-8.

You, Y., B. G. Moreira, et al. (2006). "Design of LNA probes that improve mismatch discrimination." Nucleic Acids Res 34(8): e60.

Zakstel'skaia, L. and E. A. Govorkova (1987). "[Influenza virus C and its characteristics]." Vopr Virusol 32(2): 133-42.

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