key: cord-017959-g0nf1iwm
authors: Lipkin, W. Ian; Briese, Thomas
title: Diagnosis, Discovery and Dissection of Viral Diseases
date: 2014-02-27
journal: Viral Infections of Humans
DOI: 10.1007/978-1-4899-7448-8_2
sha: 
doc_id: 17959
cord_uid: g0nf1iwm

Only a few years ago, viral diagnosis was largely an exercise for academic researchers and public health practitioners with focus on epidemiologic analyses and outbreak prevention, detection, and control. Opportunities for therapeutic intervention were limited to only a few applications such as herpesvirus infections, influenza, and HIV/AIDS; hence, once a bacterial or fungal infection was excluded, clinicians were limited to providing supportive care for what was presumed to be a viral syndrome. Public health organizations tracked the incidence of viral infections and the development of resistance to the few antiviral drugs in use and provided input to governments and the pharmaceutical industry regarding selection of vaccine targets. More recently, interest in viral diagnostics has burgeoned with the advent of new tools for detection and discovery, global recognition of pandemic risk, high-throughput drug screening, rational drug design, and immunotherapeutics. An additional impetus has been the implication of viruses in chronic illnesses not previously attributed to infection. The objective of this chapter is to review the factors responsible for the rise in awareness of viral infections, methods for diagnosis and monitoring viral infections, and future prospects for improvements in discovery, detection, and response to the challenges of clinical virology.

In an era when travel and trade are increasingly global, patients with what were once considered exotic infectious diseases restricted to the developing world, like dengue fever, Ebola, or chikungunya, now present in clinics and emergency rooms in North America and Europe. Nonstop fl ights of less than 24 h connect the world's major airports; hence, physicians must be prepared to expect the unexpected. In New York City, for example, more than 12 million passengers annually pass through John F. Kennedy (JFK) airport from more than 100 international destinations. With this traffi c volume in one metropolitan airport alone, it is not surprising that human and stowaway passengers like mosquitoes have been implicated in the transmission of West Nile virus, HIV, infl uenza virus, Mycobacterium tuberculosis , SARS coronavirus, and chikungunya virus. Exotic agents can also transit internationally in legal and illegal (bushmeat) food products and companion animals. The annual traffi c in bushmeat through Charles de Gaulle airport in Paris is estimated at 273 tonnes [ 1 ] . In work with nonhuman primate, rodent, and bat bushmeat seized at JFK by the Wildlife Conservation Society, EcoHealth Alliance, and the Centers for Disease Control and Prevention, we have found evidence of infection with retroviruses, herpesviruses, and pathogenic bacteria [ 2 ] . Illegal importation of companion animals such as birds, primates, and rodents has been linked to outbreaks of poxviruses and Salmonella [ 3 , 4 ] . Approximately 70 % of emerging infectious diseases are zoonoses-infections that are transmitted to humans from wildlife or domestic animals [ 5 , 6 ] . The majority of zoonotic diseases can be attributed to anthropogenic change. Loss of wildlife habitat to development and consumption of bushmeat necessitated by poverty or due to cultural preference increases opportunities for cross-species jumps. Global warming may also increase the geographic range of phlebotomus insects like mosquitoes and ticks that serve as reservoirs and vectors for infectious agents [ 7 ] . Given that there are more than 50,000 vertebrate species, if we assume an average of 20 endemic viruses per vertebrate species, the potential reservoir of vertebrate viruses can be estimated at one million. Although it is unlikely that all of them can be transmitted to humans and cause disease, it is sobering to consider the challenge of detecting and responding even to 1 % of them (10,000 novel viruses).

In the aftermath of the fall of the Twin Towers on September 11, 2001 , in New York City, and the anthrax attacks that followed, many western governments became concerned about bioterrorism. Early investments in surveillance for biological weapons gave way to surveillance for emerging infections when sober refl ection led to recognition that the latter were more likely threats to public health. However, advances in synthetic biology over the past decade have been so dramatic that clinicians and public health practitioners must again consider the possibility that high-threat known and novel pathogens may arise through deliberate genomic manipulation either in the form of bioweaponeering, inadvertent release of high-threat human pathogens, or legitimate gain-of-function research, whereby low-risk agents become high risk. The scientifi c and larger communities are currently grappling with the implications of gain-of-function research in the context of experiments designed to understand virulence and transmission of H5N1 (avian) infl uenza viruses [ 8 -13 ] .

Establishing a causative link between a virus and disease can be straightforward or complex. In some instances, the virus responsible for the induction of disease is present at a site of organ pathology, and there is precedent for the same or a related virus causing similar disease. A classic example is herpes encephalitis where the detection of herpesvirus sequences by polymerase chain reaction (PCR) in the cerebrospinal fl uid (CSF) of a patient with encephalitis provides a clear diagnosis and suggests a specifi c therapeutic intervention [ 14 ] . PCR alone can be inadequate. In West Nile encephalitis, PCR of CSF is less reliable than assays of CSF for IgM antibodies to the virus [ 15 , 16 ] . In some instances, the footprints of an agent cannot be found in or adjacent to the affected organ but can be detected in other compartments. PCR detection of enterovirus, for example, in the feces of a patient with aseptic meningitis, provides strong evidence of enteroviral meningitis [ 17 ] . Despite these examples of success, an etiological agent is not identifi ed by any test in up to 70 % of what is presumed to be viral encephalitis. Similar fi gures pertain in viral pneumonias. There are several explanations for surveillance and diagnostic failure. In some instances, the problem is simply lack of access to the appropriate sample. Infectious agents that do not shed into saliva, nasopharyngeal secretions, blood, urine, CSF, or feces may be detected in tissue biopsies. Alternatively, pathogenetic mechanisms may be indirect, or consequences of infection may be delayed obscuring the relationship between the causative agent and the disease.

The most straightforward mechanisms for viral pathogenesis are cellular damage due to replication and lysis, apoptosis, autophagy, or immune responses to proteins expressed on infected cells. However, viruses can also induce systemic damage through cytokine storm resulting in shock, acute respiratory distress, and/or organ failure, cause immunosuppression resulting in opportunistic infection, or break tolerance for self, resulting in autoimmune disease. Infection can be cryptic, impairing differentiated cell functions like hormone secretion, inducing neoplasia, or impairing developmental programs that may not become apparent for months or years. In summary, the challenge in viral diagnostics is to develop strategies for not only detecting footprints of the agent itself in target tissues but also enduring shadows of infection in accessible compartments.

Once the mainstay of viral diagnostics, culture now receives less emphasis in clinical microbiology, chiefl y because assays require days rather than hours; thus, information obtained is unlikely to directly impact patient management. Culture nonetheless continues to play an important role in public health as well as basic and clinical research because it enables insights into pathogenesis and the effi cacy of drugs, antibodies, and vaccines. The presence of virus can be detected by changes in cell morphology at the level of light microscopy including lysis, rounding, and syncytia formation-fusion of cells as revealed by an increase in size and the presence of more than one nucleus-visualization of pathognomonic structures by electron microscopy; or viral proteins that bind antibodies as revealed through immunohistochemistry or immunofl uorescent microscopy. A wide range of cell lines has been established for culturing viruses. Some are immortalized; others are primary cultures that can only be propagated for a few generations. Although some viruses can grow in many cell types, others have fastidious requirements. Some viruses have never been cultured despite implication in disease. In some instances, propagation failure may be overcome by adaptation with serial passaging in the presence of a second, permissive type of cell (cocultivation), the use of antibodies or RNAi to suppress innate immune responses, or cells obtained from genetically modifi ed animals. However, serial passaging can lead to adaptation, including changes in virulence (the capacity of the virus to cause disease) or tropism (the cells and organs the virus can infect). Indeed, serial passage may be utilized to develop less virulent strains that can be used as vaccines. A potential confound in characterizing samples that may contain more than one virus is that the culture environment can select for the agent that is more fi t to replicate-that may or may not be the agent of interest. In an attempt to address this potential confound as well as to propagate viruses that fail to grow in simple cultures, investigators have developed cultures that include more than one cell type. In some instances these complex cultures are designed to replicate the architecture of an organ like the respiratory tract. An alternative to culture in cells is animal inoculation. An advantage of animal inoculation is that the presence of a wide range of cell types is associated with expression of a wide range of receptors that may allow virus entry. Most investigators use suckling mice because their innate immune responses are immature. Others use mice genetically modifi ed to abrogate immune responses.

Nucleic acid tests (NATs) have largely replaced culture in viral diagnostics due to advantages in cost, speed, and ease of use. Common NAT platforms include polymerase chain reaction (PCR), in situ hybridization, microarray, and highthroughput sequencing.

These assays, designed to detect individual viruses, are the most common NATs employed in clinical microbiology. They take several forms, but quantitative real-time PCR, wherein nucleic acid replication results in either cleavage or release of a fl uorescence-labeled probe oligonucleotide that binds to a sequence region between the regular forward and reverse primers, is the most popular. The continuous ("real time") reading of the reporter fl uorescence signal affords these systems with unprecedented dynamic range and low false-negative rate. The required equipment, thermal cycler with fl uorescence detector and (laptop) computer for data analysis, is cost competitive, and rugged battery-powered instruments are available for fi eld use. Loop-mediated isothermal amplifi cation (LAMP) tests do not require programmable thermal cyclers [ 18 -20 ] . In the laboratory, LAMP products are detected in conventional dye-stained agarose gels, but in fi eld applications the estimation of product accumulation through turbidity or dye reading by the naked eye is also possible [ 21 ] . The sensitivity of all such assays is highest when primers and/or probe sequences perfectly match the selected single genetic target. Fluorescence-based TaqMan or molecular beacon assays, for example, typically have detection limits of <10 molecules per assay. Although ideal for detecting and quantitating a specifi c known agent [ 22 , 23 ] , these assays may nonetheless fail with templates of variable sequence composition, especially if this affects the region of reporter molecule binding. This can be particularly challenging in the diagnosis of RNA virus infections as RNA viruses are characterized by high mutation rates and include species with high genetic strain variability. In comparison, consensus PCR assays are less likely to be confounded by sequence divergence but are also less sensitive than the specifi c PCR assays. Nested PCR tests that can employ consensus or specifi c primers in two sequential amplifi cation reactions with either one (hemi-nested) or two (fully nested) primers located 3′ with respect to the fi rst primer set may both accommodate sequence variation and be more sensitive than fl uorescent or beacon-based singleplex assays. However, whereas in quantitative fl uorescence-or beacon-based realtime assays reporter readings are taken indirectly without opening the reaction vessels ("closed system"), nested PCR systems bear a high risk of contamination because of the transfer of (amplifi ed) material from the fi rst to the second, nested reaction [ 24 , 25 ] , even if scrupulous experimental hygiene is observed. Recently, automated (closed) systems have been developed that allow contamination-free transfer between separate reaction compartments of single-use cartridges that may present new opportunities for nested assay design.

As signs and symptoms of disease are rarely pathognomonic of a single agent, particularly early in the course of an illness, many microbial candidates must be entertained simultaneously. Multiplex NATs provide such an opportunity. The number of candidates considered may range from 10 to 50 with multiplex PCR systems to thousands with microarray platforms to the entire tree of life with unbiased highthroughput sequencing approaches. However, genetic targets compete for assay components in multiplex assays, and thus they may be less sensitive than a singleplex assay. In compensation, multiplex assays provide the advantage of consistently interrogating each sample for a wide range of agents without the selection bias introduced by singleplex testing. This comprehensive coverage is particularly important for surveillance and applications.

Multiplex PCR assays are more diffi cult to establish than singleplex assays because primer sets may differ in optimal reaction conditions (e.g., annealing temperature or magnesium concentration). Furthermore, complex primer mixtures are more likely to result in primer-primer interactions that reduce assay sensitivity and/or specifi city. To advance multiplex primer design, we developed Greene SCPrimer, a software program that automates consensus primer design over a multiple sequence alignment with customizable primer length, melting temperature, and degree of degeneracy [ 26 ] . Gel-based multiplex PCR assays are limited by size differentiation of the amplifi cation products in agarose gels and the concomitant requirement for short product sizes (approx. 90-250 base pairs) to ensure high sensitivity and fi delity [ 24 , 25 ] . Multiplexing can be achieved in fl uorescence-or beacon-based real-time assays to the degree by which different fl uorescent reporter emission peaks can be unequivocally separated. At present up to fi ve fl uorescent reporter dyes are detected simultaneously, although multiplexing may be increased to some extent by double-labeling strategies and/or melting curve analyses. "Sloppy Molecular Beacons" address this limitation in part by binding to related targets at different melting temperatures [ 27 ] ; however, they are not suited to detect targets that differ by more than a few nucleotides.

The Bio-Plex (or Luminex) platform employs fl ow cytometry to detect multiple PCR amplifi cation products bound to matching oligonucleotides that are attached to differently colored fl uorescent beads [ 28 , 29 ] . By combining multiplex PCR amplifi cation systems with various protocols for direct or indirect (tag-mediated) bead hybridization of the products, assay panels have been developed that permit detection of up to approx. 20 genetic targets simultaneously [ 30 -32 ] ; the most commonly used respiratory panels range from 9 to 20 plex [ 33 -37 ] . Like real-time PCR, these assays rely for assay specifi city on a three-oligonucleotide interaction with the target sequence. They are thereby limited in their tolerance for mutated or variant templates when compared to mass spectroscopy (MS)-coupled platforms that require only two oligonucleotide-binding sites, such as MassTag PCR or the Ibis T5000 system.

Two platforms are established that combine PCR with MS for sensitive, simultaneous detection of large numbers of targets. The Ibis T5000 system uses matrix-assisted laser desorption/ionization (MALDI) MS to directly determine the molecular weights of the generated PCR products and to compare them for identifi cation with a database of known or predicted product weights [ 38 -40 ] . MassTag PCR uses atmospheric pressure chemical ionization (APCI) MS to detect molecular weight reporter tags attached via a photo-cleavable linkage to PCR primers [ 41 ] . Whereas the Ibis system or the subsequent electrospray ionization (ESI)-based Plex-ID system requires analytical MS to determine the exact weight of the PCR products and thus depends on advanced mass spectroscopic data analysis, MassTag PCR can be performed using smaller instruments and does not require sophisticated analyses because it only records the known masses of the 40-80 reporter tags used in a given multiplex test. The Ibis system may be able to alert of variants of known organisms via a divergent PCR product weight, but like MassTag PCR, it too requires subsequent sequencing of the product for detailed characterization. A wide variety of syndrome-specifi c MassTag PCR panels have been developed and applied to the detection of viruses, bacteria, fungi, and parasites associated with acute respiratory diseases, diarrheas, tick-borne diseases, encephalitides/meningitides, and hemorrhagic fevers [ 41 -50 ] .

Although multiplex PCR methods are designed to detect known agents, they can nonetheless facilitate pathogen discovery. MassTag PCR requires only two differently tagged primers per target that may include degenerate positions to address genetic variation of larger taxonomic groups such as a whole species or genus, and its use to investigate infl uenzalike illness in New York State revealed the presence of a novel rhinovirus clade by the employed conserved enterovirus/rhinovirus primer set [ 42 ] . This discovery enabled follow-up studies across the globe wherein this third species of rhinovirus, rhinovirus C, was implicated not only in infl uenza-like illnesses but also in asthma, pediatric pneumonia, and otitis media [ 44 , 51 -63 ] .

Whereas multiplex PCR systems support rapid highthroughput diagnosis with highest sensitivity for a limited number of agents, microarray-based systems provide detection of all known pathogens for which sequence information is available, but at the expense of some degree of sensitivity. Modern printing technologies can generate high-quality arrays with several million features, a printing density that enables not only detection of a wide range of infectious agents but also discrimination of medically important types or subtypes. Examples of the latter application include respiratory virus resequencing arrays that identify the different infl uenza virus HA and NA subtypes [ 64 -68 ] .

The discovery array platforms currently in use are the GreeneChip and the Virochip [ 69 , 70 ] . The panmicrobial version of the GreeneChip, addressing viruses and in addition pathogenic bacteria, fungi, and parasites, led to the recognition of Plasmodium falciparum infection in a case of unexplained fatal hemorrhagic fever during the 2004-2005 Marburg virus outbreak in Angola [ 70 ] . A variant of the GreeneChip facilitated recently the implication of Reston Ebola virus in a respiratory disease outbreak on pig farms in the Philippines [ 71 ] . In 2003, the Virochip supported the characterization of the SARS coronavirus and was also used subsequently to diagnose parainfl uenza virus 4 and infection with a human metapneumovirus variant in cases of acute respiratory disease [ 69 , 72 , 73 ] .

Both platforms rely on random PCR strategies to amplify and label nucleic acids for detection. In comparison to multiplex consensus PCR methods employed with some targeted array applications or resequencing arrays, this limits sensitivity especially with complex sample types. In tissue specimens, for example, the sensitivity may not exceed 10 6 -10 7 copies per assay because host and pathogen nucleic acids compete for PCR reagents. Thus, these platforms have been more successful with samples containing comparatively low levels of competing nucleic acid, such as virus culture supernatant, serum, respiratory specimens, spinal fl uid, or urine. Improvements in sensitivity to a range of 10 3 -10 4 copies per assay have been achieved with methods for host DNA digestion and/or the depletion of host ribosomal RNA (rRNA) prior to amplifi cation through subtraction or use of random primers selected for lack of complementarity to rRNA [ 74 ] .

In current array platforms, virus detection is achieved via fl uorescent reporter systems-either through direct incorporation of fl uorescent nucleotides into the PCR product that is bound to the array or with a "sandwich approach" whereby fl uorescent-branched chains of DNA are added to the product after it is bound to the array [ 75 , 76 ] . However, new arrays are in development that will detect viral sequences through changes in electrical conductance. Such platforms would enhance portability by eliminating the need for fl uorescent scanners. They may also increase sensitivity and reduce costs by eliminating the need for PCR amplifi cation.

High-throughput sequencing has transformed microbiology by enabling discovery as well as diagnostics. Unlike PCR or array methods where investigators must choose the pathogens to be considered or are limited by known sequence information, high-throughput sequencing has the potential to simultaneously detect not only all viruses but also bacteria, fungi, and parasites. Although the technology is presently limited to specialized laboratories, sequencing is becoming increasingly accessible as instruments become smaller, methods become more user friendly, and costs decrease. Over the past 10 years, the cost has decreased 10,000-fold from $5,000 per 1,000 nucleotides in 2001 to $0.5 per 1,000 nucleotides in 2012 [ 77 ] . Even more impressive perhaps is the time required to generate sequence data. Projects that required weeks only a decade ago are now completed in hours [ 78 ] . Current sequencing platforms analyze libraries of amplifi ed nucleic acids. However, some platforms in development will have the capacity to directly sequence nucleic acid. Irrespective of the platform, raw sequence reads are fi ltered for quality and redundance before assembly into contiguous sequence streams. These streams, known as contigs, as well as reads that cannot be assembled, are aligned to databases using bioinformatic algorithms that examine homology at the nucleotide and deduced amino acid levels in all six potential reading frames [ 79 ] . The alignments allow identifi cation of known and novel agents, as well as detection of genetic features that may be associated with drug or vaccine resistance, or provide insight into provenance and evolution.

Finding the nucleic acid footprint of a virus is frequently only the fi rst stage in implicating it in disease. There is no functional equivalent in viruses to the pathogenicity islands found in bacteria, wherein specifi c sequences acquired through horizontal gene transfer confer specifi c pathogenic properties. The best established criteria for proof of causation in infectious disease were developed in the late 1800s by Koch and Loeffl er [ 80 ] . Known as Koch's postulates they stipulate that an agent be present in every case of the disease, be specifi c for the disease, and be suffi cient to reproduce the disease after culture and inoculation into a naive host. In the 1930s, Rivers suggested that the development of specifi c immunity to an agent following the appearance of disease could be used in demonstrating causation [ 81 ] . Adapting the original postulates to the molecular era, Fredricks and Relman later established that microbial sequences may be used as surrogates for culturing the actual organism [ 82 ] . Lipkin and colleagues recently established levels of confi dence in the strength of association between an agent and a disease that considers viral burden and distribution, specifi c immunity, and prevention or amelioration of disease with use of specifi c drugs or vaccines [ 12 ] . Given the sensitivity of molecular methods, it is imperative that physicians and researchers consider the biological plausibility of an assay result and, where feasible, pursue confi rmation with an independent assay, particularly when engaged in pathogen discovery.

NATs are rapidly replacing classical culture methods in clinical microbiology laboratories. Although some NATs, such as microarrays and high-throughput sequencing, still require substantial investment in equipment and personnel, diagnostic platforms are becoming more accessible and less expensive through miniaturization and improvements in methods for bioinformatic analysis. Systems using handheld microarrays, for example, are in development that will ultimately enable diagnosis at the bedside or in the fi eld. Benchtop sequencers are also in production. It is inevitable that as sequencing costs continue to decrease, clinicians will seek information concerning not only the presence of a single candidate organism but also the predisposition of the host to disease based on genetic factors and coinfections with other microfl ora. These improvements will bring dramatic benefi ts to medicine and public health.

The scale of illegal meat importation from Africa to Europe via Paris

Zoonotic viruses associated with illegally imported wildlife products

Monkeypox zoonotic associations: insights from laboratory evaluation of animals associated with the multi-state US outbreak

Recent multistate outbreaks of human salmonella infections acquired from turtles: a continuing public health challenge

Host range and emerging and reemerging pathogens

Overviews of pathogen emergence: which pathogens emerge, when and why?

Global climate change and infectious diseases

The H5N1 moratorium controversy and debate

The lessons of Asilomar and the H5N1 "affair

Research on highly pathogenic H5N1 infl uenza virus: the way forward

The pause on Avian H5N1 infl uenza virus transmission research should be ended

The changing face of pathogen discovery and surveillance

Rethinking biosafety in research on potential pandemic pathogens

Diagnosis of herpes simplex encephalitis with PCR

Evaluation of immunoglobulin M (IgM) and IgG enzyme immunoassays in serologic diagnosis of West Nile Virus infection

NS1 protein secretion during the acute phase of West Nile virus infection

Asymptomatic circulation of HEV71 in Norway

Loop-mediated isothermal amplification of DNA

Loop-mediated isothermal amplifi cation method for detection of human papillomavirus type 6, 11, 16, and 18

Diagnosis of human respiratory syncytial virus infection using reverse transcription loopmediated isothermal amplifi cation

Loop-mediated isothermal amplifi cation for infl uenza A (H5N1) virus. Emerg Infect Dis

Real time quantitative PCR

Molecular beacons: probes that fl uoresce upon hybridization

Detection of enteroviral RNA and specifi c DNA of herpesviruses by multiplex genome amplifi cation

Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by infl uenza A and infl uenza B viruses, respiratory syncytial virus, and parainfl uenza viruses 1, 2, 3, and 4

SCPrimer: a rapid comprehensive tool for designing degenerate primers from multiple sequence alignments

The growing utility of microbial genome sequences

Direct screening of clinical specimens for multiple respiratory pathogens using the Genaco Respiratory Panels 1 and 2

Simultaneous amplifi cation and identifi cation of 25 human papillomavirus types with Templex technology

Development and comparison of two assay formats for parallel detection of four biothreat pathogens by using suspension microarrays

Eight-plex PCR and liquidarray detection of bacterial and viral pathogens in cerebrospinal fl uid from patients with suspected meningitis

Environmental monitoring for biological threat agents using the autonomous pathogen detection system with multiplexed polymerase chain reaction

Simultaneous detection and high-throughput identifi cation of a panel of RNA viruses causing respiratory tract infections

Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fl uid microbead-based assay

MultiCode-PLx system for multiplexed detection of seventeen respiratory viruses

Evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections

Comparison of the Luminex Respiratory Virus Panel fast assay with in-house real-time PCR for respiratory viral infection diagnosis

Mass spectrometry provides accurate characterization of two genetic marker types in Bacillus anthracis

Analysis of nucleic acids by FTICR MS

Rapid identifi cation of emerging infectious agents using PCR and electrospray ionization mass spectrometry

Diagnostic system for rapid and sensitive differential detection of pathogens

MassTag polymerasechain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused infl uenza-like illness in New York State during

MassTag polymerase chain reaction for differential diagnosis of viral hemorrhagic fever

Multiplex MassTag-PCR for respiratory pathogens in pediatric nasopharyngeal washes negative by conventional diagnostic testing shows a high prevalence of viruses belonging to a newly recognized rhinovirus clade

Streptococcus pneumoniae coinfection is correlated with the severity of H1N1 pandemic infl uenza

Detection of tick-borne pathogens by MassTag polymerase chain reaction. Vector Borne Zoonotic Dis

Assessment of polymicrobial infections in ticks in New York state

Longitudinal molecular microbial analysis of infl uenza-like illness

Applications of PCR (realtime and MassTag) and enzyme-linked immunosorbent assay in diagnosis of respiratory infections and diarrheal illness among deployed U.S. military personnel during exercise Balikatan

Serotype analysis of Streptococcus pneumoniae in lung and nasopharyngeal aspirates from children in the Gambia by MassTag polymerase chain reaction

Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections

Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity

Clinical features and complete genome characterization of a distinct human rhinovirus (HRV) genetic cluster, probably representing a previously undetected HRV species, HRV-C, associated with acute respiratory illness in children

A diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infant

Characterisation of a newly identifi ed human rhinovirus, HRV-QPM, discovered in infants with bronchiolitis

A recently identifi ed rhinovirus genotype is associated with severe respiratory-tract infection in children in Germany

Global distribution of novel rhinovirus genotype

Novel human rhinoviruses and exacerbation of asthma in children

Distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus C (HRV C). PLoS One

Human rhinovirus group C infection in children with lower respiratory tract infection

Molecular characterization of human rhinovirus fi eld strains isolated during surveillance of enteroviruses

A novel group of rhinoviruses is associated with asthma hospitalizations

Novel species of human rhinoviruses in acute otitis media

A multidisciplinary approach to the diagnosis of cutaneous T-cell lymphomas

Application of broad-spectrum, sequence-based pathogen identifi cation in an urban population

Large-scale evolutionary surveillance of the 2009 H1N1 infl uenza A virus using resequencing arrays

Use of consensus sequences for the design of high density resequencing microarrays: the infl uenza virus paradigm

Ultrasensitive detection of rare mutations using next-generation targeted resequencing

Microarray-based detection and genotyping of viral pathogens

Panmicrobial oligonucleotide array for diagnosis of infectious diseases

Discovery of swine as a host for the Reston ebolavirus

Microarray detection of human parainfl uenzavirus 4 infection associated with respiratory failure in an immunocompetent adult

Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray

Digital transcriptome profi ling using selective hexamer priming for cDNA synthesis

Fluorescent sample labeling for DNA microarray analyses

A sensitive sandwich DNA array using fl uorescent nanoparticle probes

Performance comparison of benchtop high-throughput sequencing platforms

Genetic detection and characterization of Lujo virus, a new hemorrhagic fever-associated arenavirus from southern Africa

A new arenavirus in a cluster of fatal transplant-associated diseases

Viruses and Koch's postulates

Sequence-based identifi cation of microbial pathogens: a reconsideration of Koch's postulates