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Laboratory Diagnosis of Viral Infections
Tests to support or establish a specific diagnosis of a viral infection are of five general types: (1) those that demon- strate the presence of infectious virus; (2) those that detect viral antigens; (3) those that detect viral nucleic acids; (4) those that demonstrate the presence of an agent-specific antibody response; (5) those that directly visualize (“see”) the virus. Most available routine tests are agent dependent— that is, they are designed to detect a specific virus and will give a negative test result even if other viruses are present in the sample. For this reason, agent-independent tests such as virus isolation and electron microscopy are still used to identify the unexpected or unknown agent in a clinical sam- ple. Traditional methods such as virus isolation are still widely used; however, many are too slow to have any direct influence on clinical management of an index case. A major thrust of the developments in diagnostic sciences continues to be toward rapid methods that provide a definitive answer in less than 24 hours or, optimally, even during the course of the initial examination of the animal. A second major area of interest and focused effort is the development of multiplexed tests that can screen simultaneously for several pathogens from a single sample. The best of these methods fulfill five prerequisites: speed, simplicity, diagnostic sensitivity, diag- nostic specificity, and low cost. For some economically important viruses: (1) standardized diagnostic tests and rea- gents of good quality are available commercially; (2) assays have been miniaturized to conserve reagents and decrease costs; (3) instruments have been developed to automate tests, again often decreasing costs; (4) computerized analy- ses aid in making the interpretation of results as objective as possible in addition to facilitating reporting, record keeping, and billing. Although less impressive in veterinary medicine in com- parison with human medicine (for reasons of economic return on investment and range of tests required across each spe- cies), there has been recent expansion in the number of com- mercially available rapid diagnostic kits. These tests detect viral antigens, allowing a diagnosis from a single specimen taken directly from the animal during the acute phase of the illness, or they test for the presence of virus-specific antibody. Solid-phase enzyme immunoassays (EIAs) or enzyme-linked immunosorbent assays (ELISAs), in particular, have revolu- tionized diagnostic virology for both antigen and antibody detection, and are now methods of choice in many situations. For laboratory-based diagnosis, polymerase chain reaction (PCR) technology is now widely used to detect viral nucleic acids in clinical specimens, offering a very rapid alternative to other methods of virus detection. Quantitative PCR assays, in particular, facilitate the very rapid, sensitive, and specific identification of many known pathogenic viruses, and auto- mation of these assays allows the processing of large numbers of samples in short periods of time (high sample-throughput). Another major advantage of quantitative PCR assays is that they provide an objective estimate of viral load in a clinical sample. Research efforts in PCR continue, to move testing from the laboratory to the field, particularly for high- consequence agents with which rapidity of diagnosis is critically important. The provision, by a single laboratory, of a comprehen- sive service for the diagnosis of viral infections of domesti- cated animals is a formidable undertaking. Viruses in more than 130 different genera and belonging to 35 families cause infections of veterinary significance. Add to these numbers the rapidly expanding array of viruses that occur in wildlife and fish, and it is not surprising that no single laboratory can have the necessary specific reagents available or the skills and experience for the detection and identification of all viruses of all animal species. For this reason, veterinary diagnostic laboratories tend to specialize [e.g., in diseases of food animals, companion animals, poultry, fish, or labora- tory species, or in diseases caused by exotic viruses (foreign animal diseases)]. Contacting the laboratory to determine its specific capabilities should be a first step in submitting specimens for testing. Table 5. provides a general guide to diagnostic tests currently used in veterinary medicine. These will be defined in more detail later in this chapter.
RATIonAle foR SPeCIfIC
DIAgnoSIS
Why bother to establish a definitive laboratory diagnosis of a viral infection? In earlier times when laboratory diagnos- tic testing was in its infancy, diagnosis of diseases related to viral infections was achieved mainly on the basis of clinical history and signs, and/or gross pathology and his- topathology; laboratory test results were viewed as confirm- atory data. This is no longer the case, for several reasons: (1) the recent development of rapid test formats for spe- cific and sensitive identification of
individual viral infec- tions; (2) many clinical cases occur as disease complexes that cannot be diagnosed on the basis of clinical signs or pathology alone—for example, the canine and bovine res- piratory disease complexes; (3) diagnostic medicine, espe- cially that pertaining to companion animals, increasingly demands reliable and specific antemortem diagnoses; (4) legal/regulatory actions for diseases of production ani- mals and zoonoses can require identification of the specific agents involved, avian influenza being a relevant contempo- rary example. Other areas in which laboratory testing data is essential are considered below.
At the Individual Animal or
Individual Herd level
Diseases in which the management of the animal or its prognosis is influenced by the diagnosis. Respiratory dis- eases (e.g., in a broiler facility, acute respiratory disease in a boarding kennel, shipping fever in a cattle feedlot), diarrheal diseases of neonates, and some mucocutaneous diseases may be caused by a variety of different infectious agents, including viruses. Rapid and accurate identifica- tion of the causative agent can be the basis for establishing a management plan (biosecurity, vaccination, antimicrobial treatment) that prevents additional losses in the stable, ken- nel, flock, or herd. Certification of freedom from specific infections. For dis- eases in which there is life-long infection —such as bovine and feline leukemia virus infection, persistent bovine viral diarrhea virus infection, equine infectious anemia, and certain herpesvirus infections—a negative test certificate or history of appropriate vaccination is often required as a condition of sale, for exhibition at a state fair or show, or for competitions and/or international movement. Artificial insemination, embryo transfer, and blood trans- fusion. Males used for semen collection and females used in embryo transfer programs, especially in cattle, and blood donors of all species are usually screened for a range of viruses to minimize the risk of viral transmission to recipi- ent animals. Zoonoses. Viruses such as rabies, Rift Valley fever, Hendra, influenza, eastern, western, and Venezuelan equine encephalitis are all zoonotic, and are of sufficient public health significance as to require relevant veterinary diag- nostic laboratories to establish the capability for accurate detection of these agents. Early warning of a potential influ- enza virus epidemic through diagnosis of infection and/or disease in an individual poultry flock or in affected swine allows the implementation of control programs to eradicate the infection and/or restrict movement of exposed animals. As an example, laboratory identification of rabies virus in a dog, skunk, or bat that has bitten a child provides the basis for treatment decisions.
At the State, Country, and
International Level
Epidemiologic and economic awareness. Provision of a sound veterinary service in any state or country depends on knowl- edge of prevailing diseases, hence epidemiologic studies to determine the prevalence and distribution of particular viral infections are frequently undertaken. Such programs are also directed against specific zoonotic, food-borne, water-borne, rodent-borne, and arthropod-borne viruses. Internationally, the presence of specific livestock diseases in a country or region requires notification to the Office Internationale des Epizooties (the OIE, syn. the World Organization for Animal Health), which records the occurrence of these notifiable diseases in the approximately 175 member countries of the organization. Test and removal programs. For infections caused by viruses such as equine infectious anemia virus, Marek’s disease virus, bovine herpesvirus 1, pseudorabies virus, and bovine viral diarrhea virus, it is possible to reduce substan- tially the incidence of disease or eliminate the causative virus from herds or flocks by test and removal programs. The elimination of pseudorabies virus from commercial swine facilities in the United States is an example of where differential laboratory tests [the so-called differentiation/ discrimination of infected from vaccinated animals (DIVA) test, which discriminates between naturally infected and vaccinated animals] were essential to the eradication effort. Surveillance programs in support of enzootic disease research and control activities. Surveillance of viral infec- tions based on laboratory diagnostics is central to all epi- demiologic research, whether to determine the
ColleCTIon, PACkAgIng, AnD
TRAnSPoRT of SPeCImenS
The chance of detecting a virus depends critically on the attention given by the attending veterinarian to the collec- tion of specimens. Clearly, such specimens must be taken from the right site, from the most appropriate animal, and at the right time. The right time for virus detection is as soon as possible after the animal first develops clinical signs, because maximal amounts (titers) of virus are usually present at the onset of signs and often then decrease rapidly during the ensuing days. Specimens for virus detection taken as a last resort when days or weeks of empirical therapy have failed are almost invariably a useless endeavor and a waste of consumer and laboratory resources. Similarly, the incorrect collection and storage of specimens, and the sub- mission of inappropriate specimens, will diminish the like- lihood of accurate diagnostic laboratory success.
The site from which the specimen is collected will be influenced by the clinical signs and knowledge of the patho- genesis of the suspected agent(s) (Table 5.2). In viral respi- ratory infection in cattle, for example, the most important diagnostic specimens that should be collected include nasal or throat swabs or transtracheal wash fluid from live ani- mals, and lung tissue and lymph nodes from dead animals; whole-blood samples from this type of case are often use- less because the causative viral agents (bovine respiratory syncytial virus, bovine herpesvirus 1, bovine coronavirus, etc.) may not produce detectable concentrations of virus in blood samples (viremia). Likewise, for routine enteric cases (diarrhea), feces would be the primary sample in calves with rotavirus, coronavirus, or torovirus infections, with whole- blood being useful only if bovine virus diarrhea virus was a likely cause. Timing of sample collection is also critical, particularly with enteric cases, as detection of rotavirus may not be possible more than 48 hours after the onset of clini- cal signs. PCR tests do extend the sampling period because of their high analytical sensitivity and their ability to detect viral nucleic acids even if the causative virus is already com- plexed with neutralizing antibodies, but this longer detection period does not eliminate the need to be attentive to timing. Furthermore, the extended detection of viral nucleic acid by PCR assays increases the likelihood of false-positive results, wherein a virus detected by PCR is not the actual cause of the affected animal’s disease. Tissue specimens should always be taken from any part of the body where lesions are observed, either by sur- gical biopsy or at necropsy of dead animals, as it is criti- cal that laboratory findings be reconciled with lesions that are manifest in the affected animal. Thus separate samples should be split between material that will be fixed (forma- lin or other fixative) and material that will remain unfixed for virus detection assays such as immunohistochemical staining, PCR testing, or virus isolation. Because of the lability of many viruses, specimens intended for virus isolation must always be kept cold and moist, which requires preparation ahead of time. In collec- tion of specimens such as swabs, the discussion immedi- ately turns to viral transport media. The various transport media consist of a buffered salt solution to which has been added protein (e.g., gelatin, albumin, or fetal bovine serum) to protect the virus against inactivation and antimicrobials to prevent the multiplication of bacteria and fungi. A trans- port medium designed for bacteria or mycoplasma should not be used for virus sampling unless it has been proven not to be inhibitory for the intended test. Separate samples should be collected for bacterial testing. In general, speci- mens correctly collected and maintained for virus isolation will be acceptable for antigen and nucleic acid detection testing. An example of a kit containing materials suitable for the collection and transportation of specimens is shown in Figure 5.1. Specimens should be forwarded to the testing labora- tory as soon as possible. With courier services increasingly available throughout the world, overnight delivery services have greatly decreased the time interval required for agent detection, and also greatly increased the rate of diagnostic success (pathogen detection rate). Specimens should not be frozen but should be kept cold (refrigeration temperature), if delivery to the laboratory will be within several days. While viability is not necessary for PCR assays and direct antigen detection, maintaining the specimens under
The morphology of most viruses is sufficiently characteris- tic to identify the image as a virus and to assign an unknown virus to the correct family. In the context of the particular case (e.g., detection of parapoxvirus in a scraping from a pock-like lesion on a cow’s teat), the method may provide an immediate definitive diagnosis. Non-cultivable viruses may also be detectable by electron microscopy. Beginning in the late 1960s, electron microscopy was the means to the dis- covery of several new families of previously non-cultivable viruses, notably rotaviruses, noroviruses, astroviruses, and toroviruses, and unknown members of recognized families such as adenoviruses and coronaviruses. Even today, non- cultivable viruses such as those in the genus Anellovirus (torque teno viruses) have been identified by electron micro- scopy in samples from humans and a variety of animals. Two general procedures can be applied to virus detec- tion by electron microscopy: negative-stain electron micro- scopy and thin-section electron microscopy. For the negative stain procedure, virus particles in a fluid matrix are applied directly to a solid support designed for the procedure. fIguRe 5. Diagnostic electron microscopy. The morphology of most viruses is sufficiently characteristic to assign an unknown virus to the cor- rect family. In this case, direct negative staining of vesicular fluid revealed large numbers of herpesvirus particles, allowing a presumptive diagnosis of infectious bovine rhinotracheitis. Magnification: 10,000. Contrast stains are applied and the virus particles are directly visualized by electron microscopy. Thin-section electron microscopy can be used directly on fixed tissue samples, usually containing “viral” inclusions from the affected ani- mal or on cell cultures growing an unidentified virus. Low sensitivity is the biggest limitation of electron microscopy as a diagnostic tool, followed by the need for expensive equip- ment and a highly skilled microscopist. To detect virus par- ticles by negative-stain electron microscopy, the fluid matrix must contain approximately 10^6 virions per ml. Such con- centrations are often surpassed in clinical material such as feces and vesicle fluid, or in virus-infected cell cultures, but not in respiratory mucus, for instance. Aggregation of virus particles by specific antiserum (immunoelectron micros- copy) can enhance sensitivity and provide provisional iden- tity of the agent. For thin-section electron microscopy, most of the cells in the tissue sample must contain virus if viri- ons are likely to be visualized. Routine electron microscopy procedures have been largely replaced with more sensitive and less expensive procedures such as antigen-capture tests or immunostaining techniques, but because electron micro- scopy is an agent-independent test, it still has use in special- ized cases and in facilities with the necessary equipment and expertise.
Detection of Viruses by
Isolation
Despite the explosion of new techniques for “same- day diagnosis” of viral disease by demonstration of viral anti- gen or viral nucleic acid in specimens, virus isolation in cell culture remains an important procedure. Theoretically at least, a single viable virion present in a specimen can be grown in cultured cells, thus expanding it to produce enough material to permit further detailed characterization. Virus isolation remains the “gold standard” against which newer methods must be compared, but nucleic acid detec- tion tests, particularly quantitative PCR assays, are chal- lenging that paradigm. There are several reasons why virus isolation remains as a standard technique in many non- commercial laboratories. Until recently it was the only technique that could detect the unexpected— that is, identify a totally unanticipated virus, or even discover an entirely new agent. Accordingly, even those laboratories well equipped for rapid
diagnosis may also inoculate cell cultures in an attempt to isolate a virus. Metagenomic and “deep sequencing” techniques can detect unknown agents (so-called pathogen mining), but few lab- oratories outside subsidized research programs have the resources to routinely apply this technology. Culture is the easiest method of producing a supply of live virus for fur- ther examination by molecular methods (genome sequenc- ing, antigenic variation, etc.). Research and reference laboratories, in particular, are always on the lookout for new viruses within the context of emerging diseases; such viruses require comprehensive characterization, as recently shown by the quickly evolving strains of influenza virus. Moreover, large quantities of virus must be grown in cul- tured cells to produce diagnostic antigens and reagents such as monoclonal antibodies. Until recently, vaccine devel- opment has also been reliant on the availability of viruses grown in culture, although this may quickly change in the future with the increasing sophistication of recombinant DNA technology. The choice of cell culture strategy for the primary isola- tion of an unknown virus from clinical specimens is largely empirical. Primary cells derived from fetal tissues of the same species usually provide the most sensitive cell culture substrates for virus isolation. Continuous cell lines derived from the homologous species are, in many cases, an accept- able alternative. As interest in wildlife diseases increases, most laboratories are challenged to have the necessary cell cultures to “match” with the affected species. Testing strate- gies for challenging cases tend to reflect the creativity and bias of the diagnostic virologist and the particular labora- tory, although the clinical signs exhibited by the affected animals will often suggest which virus might be present. Most laboratories also select a cell line that is known to grow many types of viruses, in case an unanticipated agent is present. Arthropod cell cultures are used frequently as a parallel system for isolating “arboviruses.” Even with the best cell culture systems available, some viruses such as papillomaviruses will not grow in traditional cell culture conditions. Special culture systems such as organ cultures and tissue explants can be of value, but contact should be made with the testing laboratory to determine their capa- bilities before requesting such specialized and sophisticated diagnostic expertise. Historically, when standard methods had failed to diag- nose what appeared to be an infectious disease, inoculation of the putative natural host animal was used to define the infectious nature of the problem and to aid in the eventual isolation of the agent. This practice has largely been aban- doned, as a result of costs and animal welfare concerns. Some specialized laboratories still have the capability to inoculate suckling mice, a system that has been valuable for isolating arboviruses that resist cultivation in cell cultures. Embryonated hens’ eggs are still used for the isolation of influenza A viruses, even though cell cultures [Madin– Darby canine kidney (MDCK) cells] are now more com- monly used. Many avian viruses also replicate much better in eggs than in cell cultures derived from chick embryo tis- sues, and there is a lack of widely available avian cell lines for routine virus isolation procedures. According to the virus of interest, the diagnostic specimen is inoculated into the amniotic cavity, or the allantoic cavity, the yolk sac, onto the chorioallantoic membrane or, in rare instances, intravenously into the vessels of the shell membrane and embryo. Evidence of viral growth may be seen on the horioallantoic membrane (e.g., characteristic pocks caused by poxviruses), but otherwise other means are used to detect viral growth (e.g., death of the embryo, hemagglutination, immunofluorescence or immunohistochemical staining of viral antigens, or antigen-capture ELISA). Attempts to isolate viruses require stringent attention by the clinician to the details of sample collection and transport, because success depends on the laboratory receiving a speci- men containing viable virus. Contact with the testing labora- tory before specimen collection is strongly advised in order to clarify the sampling strategy, assess shipping require- ments, and alert the laboratory to the number and type of specimens being shipped. Having cell cultures available on the day of arrival of a specimen can enhance the success of isolation. There is no such thing as an emergency (“stat”) virus isolation; each virus has its own biological clock and no amount of concern will speed up the replication cycle. For viruses such as the alphaherpesviruses, a successful iso- lation can be evident as cytopathic effect in the inoculated cell cultures within 2–3 days, whereas others are consider- ably slower and require repeated serial passage. In gen- eral, the time for detection will depend on the laboratory’s procedures for identifying virus in the culture system. For
Immunohistochemical
(Immunoperoxidase) Staining
In principle, immunohistochemical staining is very simi- lar to immunofluorescence staining of viral antigens, but with several key differences (Figure 5.3B). The “tag” used in immunohistochemical staining is an enzyme, generally horseradish peroxidase. The enzyme reacts with a substrate to produce a colored product that can be visualized in the infected cells with a standard light microscope. The tissue sample will often be formalin fixed, which permits testing of the specimen days to weeks after sampling, without the need for low- temperature storage. Another major advantage for the immunohistochemical staining technique is that it involves an amplification process wherein the product of the reaction increases with increasing incubation, whereas immunofluorescence staining generates a real-time signal that does not get stronger with a longer incubation period. Furthermore, immunohistochemically stained slides can be kept for extended periods of time for several observations, whereas the immunofluorescence slides deteriorate more rapidly. Immunofluorescence does have the advantage of speed; immunohistochemical staining on formalin- fixed tissues requires more than 24 hours to give results. Perhaps the greatest benefit of immunohistochemical staining is that it readily facilitates comparison of virus distribution and cellular localization in tissue sections to determine whether or not viral antigen distribution coincides with that of any lesions that are present (Figure 5.5).
Enzyme Immunoassay—Enzyme-
Linked Immunosorbent Assay
Enzyme immunoassays (EIAs)—often referred to as enzyme-linked immunosorbent assays (ELISAs) —have revolutionized diagnostic testing procedures. Assays can be designed to detect antigens or antibodies. Although EIAs have high relative sensitivity, samples may still require more than 10^5 virus particles/ml for positive reactions with many tests. This level of sensitivity still makes these tests highly valuable, particularly in group settings, where any positive animal defines the herd status. Assays may be con- ducted on a single sample in the veterinarian’s clinic or on many hundred samples at the same time, using automated systems in centralized laboratories. Some commonly used antigen detection test kits include those specific for feline leukemia virus, canine parvovirus, bovine viral diarrhea virus, rotavirus, and influenza virus. There are many dif- ferent types of EIA tests that differ in their geometric properties, detector systems, amplification systems and sensitivity. Not all possible tests will be discussed, as the basic test principles apply to all. Most EIAs are solid-phase enzyme immunoassays; the “capture” antibody is attached to a solid substrate, typically the wells of polystyrene or polyvinyl microtiter plates. The simplest format is a direct EIA (Figure 5.6). Virus and/or soluble viral antigens from the specimen are allowed to bind to the capture antibody. After unbound components are washed away, an enzyme-labeled antiviral antibody (the “detector” antibody) is added; various enzymes can be linked to the antibody, but horseradish peroxidase and alkaline phosphatase are the most commonly used. After a washing step, an appropriate organic substrate for the par- ticular enzyme is added and readout is based on the color change that follows. The colored product of the reaction of the enzyme on the substrate can be detected visually or read by a spectrophotometer to measure the amount of enzyme- conjugated antibody bound to the captured antigen. The product of the enzyme reactions can be modified to pro- duce a fluorescent or chemiluminescent signal to enhance sensitivity. With all such assays, extensive validation testing must be carried out to determine the cut-off values of the test, which define the diagnostic sensitivity and diagnostic specificity of the test. Indirect EIAs are widely used because of their greater analytical sensitivity, but the increase in sensitivity is usu- ally accompanied by a loss of specificity. In this test for- mat, the detector antibody is unlabeled and a second labeled (species-specific) anti-immunoglobulin is added as the “indicator” antibody (Figure 5.6). Alternatively, labeled staphylococcal protein A, which binds to the Fc moiety of IgG of many mammalian species, can be used as the indi- cator in indirect immunoassays. Monoclonal antibodies have especially facilitated the development of EIA tests, because they provide a consistent supply of highly sensitive and specific reagents for commercial tests. However, any variation (antigenic variation of the virus target) in the spe-
cific epitopes recognized by specific monoclonal antibod- ies can lead to loss of binding and loss of test sensitivity because of false-negative results. EIAs have been adapted to formats for use in veterinary clinics on single animal specimens (Figure 5.7).
Immunochromatography
Immunochromatography simply refers to the migration of antigen or antigen–antibody complexes through a filter matrix or in a lateral flow format—for example, using nitro- cellulose strips. In most formats, a labeled antibody binds to the antigen of interest. The antigen–antibody complexes are then immobilized in the support matrix by an unlabeled antibody bound to the matrix. All controls are included in the membrane as well, and results are seen as colored spots or bands, as one of the test reagents is conjugated to col- loidal gold or a chromogenic substance. This test format is especially convenient for point-of- care testing, as the test process is simple and each test unit contains both positive and negative controls to assess test validity.
Detection of Viral nucleic
Acids
Developments in the area of nucleic acid technology in the past few years have relegated some (earlier) techniques to the annals of history with respect to their use in diagnostic testing. For example, classic hybridization techniques are not typically amenable to use for routine testing, especially with the requirement for rigorous quality-control stand- ards. The most dramatic changes in nucleic acid detection technology have been in the evolution of polymerase chain reaction (PCR) testing, and the equally important standard- ization of nucleic acid extraction procedures. In addition, the rapid advances in nucleotide sequencing technology, oligonucleotide synthesis, and development of genetic databases permit inexpensive sequence analysis that has replaced less rigorous procedures for comparing genetic changes in virus strains and isolates. Current technology permits PCR amplification of virus “populations” with direct sequencing of the amplified products from the clini- cal specimen without the potential introduction of cell cul- ture selection bias. More recent developments permit the detection and characterization of unknown agents (viral metagenomics). With the developments in nanotechnol- ogy, one could anticipate the future advent of inexpensive nucleic acid detection units that could reliably detect infec- tious agents when used in the clinician’s office or in the field, without the need for highly trained personnel. Nucleic acid detection methods are invaluable when dealing with: (1) viruses that cannot be cultured readily; (2) specimens that contain inactivated virus as a result of prolonged storage, fixation of tissue, or transport; (3) latent infections in which the viral genome lies dormant and infectious virus is absent; (4) virus complexed with anti- body as would be found in the later stages of an acute infec- tion or during some persistent viral infections. However, the added sensitivity provided by amplification of viral nucleic acid can actually create new problems. Unlike the situation with bacterial pathogens, it has usually been the case that merely detecting a pathogenic virus in a lesion, or from a clinically ill animal, has been considered evidence of its etiologic role (causal relationship). As detection methods have become increasingly sensitive and testing includes more agents, questions of viral “passengers” become more pertinent. Indeed, with viruses such as bluetongue virus, viral nucleic acid can be detected in the blood of previously infected ruminants several months after infectious virus has been cleared. Furthermore, with bovine herpesvirus 1 as an example, detection of viral nucleic acid does not address whether it is present as a consequence of acute infection, reactivation of a latent infection, or vaccination.
Polymerase Chain Reaction
The PCR assay is an in-vitro method for the enzymatic syn- thesis of specific DNA sequences using two oligonucleotide primers, usually of about 20 residues (20-mers), that hybrid- ize to opposite strands and flank the region of interest in the target DNA; the primer pairs are sometimes referred to as forward and reverse primers (Figure 5.8). Primers are neces- sary to provide the DNA polymerase with a substrate upon which to add new nucleotides, and to direct the reaction to the specific region of the DNA for amplification. Primers can also be designed to provide “tags” on the amplified products for purposes of detection. Computer programs are used for the design of optimum
TaqMan® probes (Figure 5.9A), and molecular beacons as exam- ples. Once reactants are added to the reaction tubes, the tubes need never to be opened again, thus preventing any opportunity for laboratory contamination. The real-time detection systems are also more sensitive than standard gel systems, and added assay specificity is achieved through the use of reaction detection probes, because signal is gen- erated only if the probe sequence is also able to bind to the target sequence. Another advantage of the real-time system is that the process can be quantitative. Under optimum conditions, the amount of the amplicon increases by a factor of 10 with each 3.3 amplification cycle (Figure 5.9B). With real-time systems, the generation of product is recorded at each cycle. The amount of product generated in a test reaction can be compared with a copy number control and, with proper extraction controls in the system, a direct measure of the amount of starting sequence can be determined. In humans, for example, this feature has particular value in monitoring responses over time to drug treatments for infections with hepatitis C and human immunodeficiency viruses. A further variation in PCR testing that is becoming more commonly used is multiplex PCR. In this method, two or more primer pairs specific for different target sequences are included in the same amplification reac- tion. In this manner, testing can be done for several agents at the same time and in the same assay tube, thereby sav- ing time and costs. With real-time, multiplex PCR assays, several probes with different fluorescent molecules can be detected simultaneously. This type of application is useful in evaluation of samples from disease complexes, such as acute respiratory disease in dogs. Issues of test sensitivity must be addressed in this format, because several reactions must compete for common reagents in the reaction, thus an agent in high copy number might mask the presence of one at low copy number. Advantages and Limitations of the Polymerase Chain Reaction Technology Given the explosion in use and availability of PCR assays in virological testing, consideration should be given to the potential benefits and limitations of these assays. The PCR assay is especially useful in the detection of viruses that are difficult to grow in culture, such as certain enteric ade- noviruses, papillomaviruses, astroviruses, coronaviruses, noroviruses, and rotaviruses. PCR can be used on any sam- ple that is appropriate for virus isolation; the decision to do PCR as opposed to other virus detection tests is based on speed, cost, and laboratory capability. PCR tests also may be preferred for the initial identification of zoonotic viruses such as rabies virus, certain poxviruses, or influ- enza viruses, to minimize the risk of exposure for labora- tory personnel as amplification of infectious virus is not necessary for detection A limitation of PCR or any nucleic acid amplification technique can be the matrix in which the target sample is embedded. Material in the sample matrix can inhibit the enzymes on which the assay is based, which has been a constant source of concern when dealing with fecal sam- ples and, to some extent, milk samples. Extraction controls need to be included in these types of sample in order to detect problems with the amplification process itself (rather than lack of specific template). Furthermore, PCR and sim- ple nucleic acid amplification tests are agent specific, thus no signal will be generated if the primers do not match the sequence of any virus contained in the sample. With earlier direct PCR assays, and especially with nested PCR assays, false-positive test results were a very significant concern as a result of the ease of laboratory contamination with ampli- fied product. With the availability of single- tube real-time PCR testing formats and real-time PCR tests, this problem has largely been eliminated, although correct performance of PCR assays remains a technically challenging process. Performance of real-time PCR assays is being continually improved with standardized reagent kits, robust instrumen- tation, standardized extraction protocols, and defined labo- ratory operating procedures, and this nucleic acid detection test format has become the mainstay of testing laborato- ries. However, test interpretation still requires evaluation of whether or not a particular test result (either positive or negative) is biologically relevant, which in turn requires a global assessment of history, clinical signs, and lesions in the particular animal from which the sample was obtained.
Microarray (Microchip) Techniques
Another technological advance that is impacting the field of diagnostics is the advent of
microarrays or microchips. The microchip for nucleic acid detection is a solid support matrix onto which have been “printed” spots, each contain- ing one of several hundred to several thousand oligonucle- otides. Increasingly, these oligonucleotides can represent conserved sequences from virtually all viruses represented in the various genetic databases, or can be customized to represent only viruses from a given species involved in a specific disease syndrome, such as acute respiratory dis- ease in cattle. The basis of the test is the capture by these oligonucleotides of randomly amplified labeled nucleic acid sequences from clinical specimens. The binding of a labeled sequence is detected by laser scanning of the chip and software programs assess the strength of the binding. From the map position of the reacting oligonucleotides, the software identifies the species of virus in the clinical sam- ple. This type of test was used to determine that the virus responsible for severe acute respiratory syndrome (SARS) was a coronavirus. With knowledge of the oligonucleotide sequences that bound the unknown agent, primers can be made to eventually determine the entire nucleotide sequence of a new species of virus. The low cost of oligonucleotides synthesis, development of laser scanning devices, nucleic acid amplification techniques, and software development have made this technology available in specialized labo- ratories. A variation of this technique is the re- sequencing microarray. These arrays consist of a set of overlapping probes, which may differ from each other at a single base. The strength of binding to the individual probes in the fam- ily provides information on the genetic sequence of the amplified product. With current technology, the microarray approach is only available in specialized reference laborato- ries and it would not be used for routine diagnostic testing. In the standard format, this technique would probably not detect a new virus family not represented in a current data- base, because oligonucleotides for the new agent would not be included on the microchip.
Gene Amplification by Isothermal
Amplification
For nucleic acid amplification, it is necessary to continu- ally displace the newly synthesized product so that another copy of the sequence can be made. With PCR, the strand displacement is achieved with temperature: the 95°C tem- perature maximum melts (separates) the DNA strands, per- mitting binding of new primers. Isothermal amplification is a technique that does not require the temperature cycling and accompanying equipment used in PCR. Two techniques using different polymerases to achieve sequence amplifi- cation have been developed for isothermal amplification: nucleic acid sequence-based amplification (NASBA) and loop-mediated isothermal amplification (LAMP). If these techniques show significant advantages over PCR, one would expect the availability of an expanding array of avail- able tests.
nuCleIC ACID (VIRAl genomIC)
SeQuenCIng
Perhaps no area in molecular biology has advanced so rap- idly as nucleic acid sequencing. With speed and capacity has come low cost, so that direct sequencing of complete viral genomes is now commonplace. Older techniques such as restriction mapping and oligonucleotide fingerprinting that were used to detect genetic differences among virus isolates have been displaced by sequencing methodology. In the area of diagnostics, new viruses are being discov- ered by techniques that take advantage of random nucleic acid amplification and low-cost sequencing. There are sev- eral basic techniques with numerous modifications that are too detailed to discuss individually. In general, the process involves random amplification of enriched nucleic acid sam- ples or total nucleic acid samples, followed by sequencing of all the amplified products. The process works best if host-cell nucleic acids can be eliminated from the samples by nuclease treatment or subtraction of host sequences by hybridization to normal cell sequences immobilized on solid supports. No prior knowledge of the viral sequence is needed, and there is no need for any virus-specific reagents. Computer analy- sis of the sequenced material can identify sequences that are closely or distantly related to those of specific virus families. The method used to construct the entire genome, if this is desired, is somewhat dependent upon the number and size of sequences identified, but methods are available to “walk” down the entire genome from a single viral sequence. With these types of nucleic acid detection protocols, unknown viruses can be discovered and characterized without the requirement that they first be propagated in cell culture.
domestic species cannot be used. All serological test types will not be discussed in detail (below), but readers should be aware that other test formats may become available and continu- ing communication with their testing laboratory is the most efficient way to learn about the tests available for each spe- cies and for each virus.
enzyme Immunoassay—
enzyme-linked
Immunosorbent Assay
Enzyme immunoassays (EIAs, ELISA) are the serologic assays of choice for the qualitative (positive or negative) or quantitative determination of viral antibodies because they are rapid, relatively cost effective, and may not require the production of infectious virus for antigen if recombinant antigens are used. In the EIA test format for antibody detection, viral antigen is bound to a solid matrix. Serum is added and, if antibodies to the antigen are present in the sample, they bind to it. In direct EIA tests, the bound antibody is detected by an anti-species antibody tagged with an enzyme. With addition of the enzyme substrate, a color reaction develops that can be assessed either visually or with a spectrophotometer. Controls run with the sam- ple define whether the test is acceptable and which sam- ples in the test are positive. Kinetics-based EIAs offer the advantage that quantitative assays can be based on a single dilution of serum. The product of the enzyme reaction is determined several times over a short interval. Software programs convert the rate of product development to the amount of antibody bound to the antigen. A disadvantage of direct EIA tests is that they are species specific. A test developed for canine distemper virus anti- bodies in a dog cannot be used to determine the presence or absence of antibodies to the same virus in a lion. To obviate this problem, competitive or blocking EIA tests have been developed. In this test format, an antibody that binds to the antigen of interest (usually a monoclonal antibody) is tagged with the enzyme. Unlabeled antibody that can bind to the same site as the monoclonal antibody will compete with the labeled monoclonal antibody for that site. A reduction in the binding of the labeled monoclonal antibody indicates that the sample did contain antibody (Figure 5.10). In this test format, the species of the unlabeled antibody is not a factor. The diagnostic sensitivity and specificity of EIA tests, whether direct or indirect, have been greatly enhanced by the development of monoclonal antibodies and the produc- tion of recombinant antigens. In a widely used format for test kits that can be run in a practitioner’s office, the test serum flows through a mem- brane filter that has three circular areas impregnated with antigen, two of which have already interacted with a posi- tive and a negative serum, respectively (Figure 5.7). After the test serum flows through the membrane and a washing step is completed, a second anti-species antibody with an enzyme linked to it is added and the membrane is again rinsed before the addition of the enzyme substrate. The result is read as a color change in the test sample circle, which is compared against the color change in the posi- tive control and no change in the negative control. Such single- patient tests are relatively expensive compared with the economies of testing hundreds of sera in a single run fIguRe 5.10 Competitive enzyme-linked immunosorbent assay (cELISA) for caprine arthritis encephalitis viral (CAEV) antibodies. Undiluted serum samples in duplicate are added to antigen-coated wells of a commercial cELISA test for CAEV antibodies. After removal of the test sera, an antibody specific for CAEV antigen and coupled to horse- radish peroxidase is added. The detector antibody is removed after incu- bation, and a substrate is added to detect the presence of bound detector antibody. If there are antibodies specific for CAEV in the test sera, these antibodies bound to antigen will prevent the detector antibody from bind- ing. A positive sample will therefore show less enzyme product (color) than the negative controls. Cut-off values are determined by reading the intensity of the reaction with a spectrophotometer, although visual inspec- tion can usually detect positive samples. Wells A1–2 and G11–12: posi- tive controls; wells B1–2 and H11–12: negative controls; wells D1–2, H1–2, B3–4, F3–4, C5–6, D5–6, A7–8, E7–8, G7–8, and B9–10: samples positive for antibodies to CAEV. in a fully automated laboratory. The great savings in time and effort to send samples to the laboratory, in addition to the fact that decisions can be made while both client and patient are still in the consulting room, make single tests attractive and useful in the immediate clinical management of critically ill animals.
Serum (Virus) neutralization
Assay
As virus isolation is considered the gold standard for the detection of virus against which other assays must be com- pared, the serum (virus) neutralization test has historically been the gold standard, when available, for the detection and
quantitation of virus-specific antibodies. Neutralizing antibody also attracts great interest because it is considered a direct correlate of protective antibody in vivo. For the assay of neutralizing antibody, two general procedures are available: the constant-serum–variable-virus method and the constant-virus–variable-serum method. Although the constant-serum–variable- virus method may be a more sen- sitive assay, it is rarely used because it utilizes relatively large amounts of serum, which may not be readily avail- able. The basis of the neutralization assay is the binding of antibody to infectious virus, thus preventing the virus from nitiating an infection in a susceptible cell. The growth of the virus is detected by its ability to kill the cell (cytopathic effect) or by its ability to produce antigen in the infected cells that is detected by immunofluorescence or immu- nohistochemistry. The amount of antibody in a sample is determined by serial dilution of the sample and “chal- lenging” each of these dilutions with a standard amount of virus (constant-virus– variable-serum method). The last dilution that shows neutralization of the virus is defined as the endpoint and the titer of the serum is the reciprocal of the endpoint dilution; for example, an endpoint of 1:160 equates to a titer of 160. The disadvantages of serum neu- tralization tests are that they are relatively slow to generate a result, require production of infectious virus for the test, and have a constant high overhead cost in maintaining cell culture facilities for the test. These assays have the benefit of being species independent and, as such, are very useful in wildlife studies. With new agents, a serum neutralization test can be operational with several weeks of isolating the virus, whereas EIA test development may take months or even years to validate.
Immunoblotting (Western
blotting)
Western blotting tests simultaneously but independently measure antibodies against several proteins of the agent of interest. There are four key steps to western blotting. First, concentrated virus is solubilized and the constituent proteins are separated into discrete bands according to their molecu- lar mass ( M r), by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS-PAGE). Secondly, the separated proteins are transferred electrophoretically (“blotted”) onto nitrocellulose to immobilize them. Thirdly, the test serum is allowed to bind to the viral proteins on the membrane. Fourthly, their presence is demonstrated using a radio- labeled or, most commonly, an enzyme-labeled anti-species antibody. Thus immunoblotting permits demonstration of antibodies to some or all of the proteins of any given virus, and can be used to monitor the presence of antibodies to different antigens at different stages of infection. Although this procedure is not routinely used in a diagnostic setting with viruses, western blots were central to the identification of immunogenic proteins in a variety of viruses. Similarly, the assay is used in the analysis of samples for the pres- ence of prion proteins in ruminant tissues. Western blots are more of a qualitative test than a quantitative one, and are not easily standardized from laboratory to laboratory. For this reason, ELISAs and bead-based assays are preferred test formats.
Indirect Immunofluorescence
Assay
Indirect immunofluorescence assays are used for the detec- tion and quantitation of antibody; specifically, these are tests that use virus-infected cells (usually on glass micro- scope slides) as a matrix to capture antibodies specific for that virus. Serial dilutions of test serum are applied to indi- vidual wells of the cell substrate and usually an anti-species antibody with a fluorescent tag is then added as the detec- tor of antibody binding. Slides are read with a fluorescent microscope and scored as positive if the infected cell shows a fluorescent pattern consistent with the antigen distribu- tion of the virus used. This test is rapid (less than 2 hours) and can be used to determine the isotype of the reacting antibody if one uses an anti-isotype-specific serum such as an anti-canine IgM. Non-specific fluorescence can be an issue, particularly with animals that have been heavily vac- cinated as they may contain anti-cell antibodies that will bind to uninfected cells and mask specific anti-virus fluo- rescence. Test slides for some agents can be purchased, so that laboratories offering this test need not have infectious virus or a cell culture facility.
Hemagglutination-Inhibition
Assay
disappear altogether within 3 months, they are usually indicative of recent (or chronic) infection. The most common method used is the IgM antibody capture assay, in which the viral antigen is bound on a solid-phase substrate such as a microtiter well. The test serum is allowed to react with this substrate and the IgM antibodies “captured” by the antigen are then detected with labeled anti-IgM antibody matched to the species from which the specimen was obtained.
new generation Technologies
As with nucleic acid technologies, technological devel- opments for analyte detection are rapidly evolving, and a substantial number of potentially novel platforms for sero- logical assays have been developed that have not yet been fully validated for routine diagnostic use. It is beyond the scope of this text to provide an exhaustive catalog of these technologies, many of which will never find their way into routine diagnostic use. However, one technology that has demonstrated particular promise in both the clinical and research arena is xMAP®, developed by Luminex. The suc- cess of this testing platform probably reflects the maturity of existing technologies that were combined to provide a versatile analyte detection system. xMAP® combines a flow cytometry platform, uniquely labeled microspheres, digital signal processing, and standard chemical coupling reactions to provide a system that can be used to detect either proteins or nucleic acids (Figure 5.13). The micro- spheres carry unique dyes (up to 100 different ones) that emit fluorescent signals that identify the individual beads coupled with a specific ligand. For antibody detection tests, the antigen of interest is coupled to a specific bead. The beads are exposed to the test serum and the bound anti- body is detected with an anti-species antibody tagged with a reported dye. The microspheres are analyzed in a flow cytometer in which lasers excite both the bead dyes and the reporter dyes. Multiple beads for each antigen are analyzed in each test, providing independent readings of the reaction. One distinct advantage of this system is its multiplex capability. Theoretically, 100 or more different antigens can be assessed for antibody reactivity in a single assay. For maximum sensitivity and specificity, recombinant antigens are needed to eliminate extraneous proteins that would reduce specific antigen density on the beads and increase non-specific background reactivity that can confuse test interpretation. Advantages of this bead-based system are: (1) it utilizes small sample volumes; (2) it can be mul- tiplexed; (3) it has been reported to be more sensitive than standard ELISA tests; (4) it can be less expensive than many serology tests; (5) it can be more rapid than ELISA tests, particularly when testing for antibodies to several antigens. As an example, this test platform is ideal for the antibody screening tests that are necessary for maintain- ing research rodent colonies in which antibody responses to several agents are monitored and for which sample vol- umes are often limiting. This platform can also provide DIVA testing as would be applied for control of important regulatory disease such as foot-and-mouth disease. As an example, recombinant antigens representing the capsid proteins present in inactivated foot-and- mouth disease virus vaccines along with non- structural viral protein can be coupled to different beads to analyze the antibody pro- file of a suspect animal. In a single assay, the test can pro- vide evidence of vaccination—response to capsid antigen only—or of a natural infection—response to both types of proteins. One could envision this type of bead-based assay as a quantitative western blot, in that reactivity to several antigens can be assessed. As eradication programs progress for viral diseases of production animals, it is very likely that the requirement for this type of DIVA testing will only increase. The disadvantage for antibody detection is the need for recombinant antigens to achieve acceptable sen- sitivity, and high validation costs associated with multiplex reactions.
InTeRPReTATIon of
lAboRAToRy fInDIngS
As with any laboratory data, the significance of specific results obtained from the virology laboratory must be inter- preted in light of the clinical history of the animal from which the sample was collected. To some extent, the signifi- cance of any result is also influenced by the type of virus that was detected. A fluorescent-antibody positive test for rabies virus on a bat found in a child’s bedroom will elicit a public health response in the absence of clinical data, whereas a positive serological test for bovine leukemia virus from the dam of an aborted fetus is likely to be an irrele-
vant finding if the animal is from an enzootic region. With multiplex PCR testing, it may be possible to detect several different viruses, bacteria, and mycoplasma species in a single dog with acute respiratory disease, raising the obvious question, “what is significant?” Are the virus signals due to a recent vaccination, reactivation of a herpesvirus, or “foot- prints” of the etiological agent? Clearly, several sources of data must be integrated by the clinician to arrive at a coher- ent treatment strategy. However, it is also clear that the speed, the number, and the reliability of virus detection tests have changed the way in which clinicians use laboratory test results, and these results are having greater impact on treatment and management decisions. When attempting to interpret the significance of the detection of a specific virus in a clinical specimen, one may be guided by the following considerations. The site from which the virus was isolated. For exam- ple, one would be quite confident about the etiological significance of equine herpesvirus 1 detected in the tissues of a 9-month-old aborted equine fetus with typical gross and microscopic lesions. However, recovery of an entero- virus from the feces of a young pig may not necessarily be significant, because such viruses are often associated with inapparent infections. The epidemiologic circumstances under which the virus was isolated. Interpretation of the significance of a virus isolation result is much more meaningful if the same virus is isolated from several cases of the same illness in the same place and time. The pathogenetic character of the virus detected. Knowledge that the virus detected is nearly always etiologi- cally associated with frank disease—that is, rarely is found as a “passenger”—engenders confidence that the finding is significant. The identity of the specific virus. The detection of foot- and-mouth disease virus in any ruminant in a virus-free country would, in and of itself, be the cause for great alarm. Similarly, the identification of mouse hepatitis virus in a free colony, or koi herpesvirus amongst highly valuable ornamental fish, would trigger a substantial response.
Interpretation of Serologic
laboratory findings
A significant (conventionally, fourfold or greater) increase in antibody titer between acute and convalescent sam- ples is the basis, albeit in retrospect, for linking a specific virus with a clinical case of a particular disease. However, one must always be aware of the vaccination status of the animal, as sero-responses to vaccines, especially live- attenuated virus vaccines, may be indistinguishable from those that occur after natural infections. The demonstration of antibody in a single serum sample can be diagnostic of current infection in an unvaccinated animal (e.g., with ret- roviruses and herpesviruses), because these viruses estab- lish life-long infections. However, in such circumstances there is no assurance that the persistent virus was responsi- ble for the disease under consideration. Assays designed to detect IgM antibody provide evidence of recent or current infection. A summary of the major strengths and limita- tions of the several alternative approaches to the serological diagnosis of viral infections is given in Table 5.1. Detection of antiviral antibody in pre-suckle newborn cord or venous blood provides a basis for specific diagnosis of in-utero infections. This approach was used, for example, to show that Akabane virus was the cause of arthrogryposis- hydranencephaly in calves. Because transplacental transfer of immunoglobulins does not occur in most domestic ani- mals, the presence of either IgG or IgM antibodies in pre- suckle blood is indicative of infection of the fetus.
Sensitivity and Specificity
The interpretation and value of a particular serologic test is critically dependent on an understanding of two key param- eters: diagnostic sensitivity and diagnostic specificity. The diagnostic sensitivity of a given test is expressed as a per- centage and is the number of animals with the disease (or infection) in question that are identified as positive by that test, divided by the total number of the animals that have the disease (or infection) (Table 5.3). For example, a partic- ular EIA used to screen a population of cattle for antibody to bovine leukemia virus may have a diagnostic sensitiv- ity of 98%—that is, of every 100 infected cattle tested, 98 will be diagnosed correctly and 2 will be missed (the false- negative rate 2%). In contrast, the diagnostic specificity of a test is a measure of the percentage of those
