Clinical Virology Manual, Fourth Edition

Today, medical practitioners rely on the ability of the clinical laboratory to provide key scientific data used for the diagnosis, treatment, and monitoring of persons with viral diseases. Therefore, the accuracy of test results is critical, and ongoing quality assurance (QA) and quality improvement programs are key factors in maintaining service excellence. QA programs must be comprehensive so that all aspects of the laboratory testing are monitored, including the preanalytical, analytical, and postanalytical phases. Another key component of QA is maintaining staff proficiency and competency. Monitoring of equipment and environmental conditions is required at least each day of use for all sections of clinical virology. The main quality standards of the regulatory and accrediting organizations can be categorized as (i) personnel qualifications, responsibilities, and competency assessment; (ii) proficiency testing for all analytes and staff; (iii) written and approved procedures; (iv) method verification and validation; (v) test reagent and equipment quality control (QC) and preventive maintenance; and (vi) patient test management. Written policies defining the process for proficiency testing (PT) must be clearly defined and understood by all personnel. The policies should include (i) requirements for assessment of the preanalytical, analytical, and postanalytical phases of testing; (ii) ongoing verification requirements for proficiency testing; (iii) safety; (iv) technical training; and (v) ongoing competency assessment. QC is an integral part of daily laboratory testing and is intended to ensure the performance of test systems and the accuracy of patient results.

New technology has expanded the selection, collection, and interpretation of test results compared to past years. Basically, the specimen requirements for cell culture-and molecular-based diagnostic tests are similar. The authors aim at defining unique and optimal specimen processing needs for each technology in this chapter. The selection of an appropriate specimen is vital to a correct test result; this includes not only the source of the specimen but also the timing and volume of collections. Information pertaining to all of these issues should be collated into comprehensive tables listing general disease categories and associated viruses, including optimal specimen types, methods of collection, methods of detection, volumes required, and containers for transport. The significance of detection of a virus may have a different level of importance depending on the time of collection. Specimens from the respiratory tract can represent almost one-half of the source material and one-third of the total viruses diagnosed in the clinical laboratory. Detection of viruses from blood specimens provides evidence of disseminated, invasive infections which may lead to systemic disease. Major diagnostic methods for detection of viral infections have been cell culture (conventional tube and shell vial), fluorescent-antibody direct staining of specimen smears on slides, antigen detection by EIA, and amplification and detection of target nucleic acids by polymerase chain reaction (PCR).

Despite the increasing availability of viral antigen and nucleic acid tests, culture methods remain important, especially in the hospital setting. This chapter focuses on the isolation methods currently used in routine clinical laboratories. Recent innovations in monolayer cell culture include the use of mixtures of two or three different cell lines and genetically altered cells to enhance sensitivity or facilitate detection of virus infection. The materials needed for the isolation of viruses in cell culture are those necessary for the safe handling and inoculation of cell cultures, maintenance and observation of cell cultures, and preservation and storage of clinical specimens and virus isolates. Virus-induced cytopathic effect (CPE) must be distinguished from nonspecific CPE caused by toxicity of specimens, contamination with bacteria or fungi, or old cells. The rapid diagnosis of viral infections is important in patient management. One of the most significant contributions to rapid diagnosis in the clinical laboratory has been the application of centrifugation cultures to viral diagnosis. To get the best results from primary isolation in cell culture, healthy cell cultures susceptible to a spectrum of viruses are essential. In addition, conventional culture should be used for lower respiratory tract and tissue biopsy samples to detect additional or unsuspected viruses. While molecular methods are essential for optimal detection of viruses in spinal fluid and to monitor viral load in blood, virus isolation continues to play an important role in viral diagnosis and patient management, especially when performed locally.

This chapter talks about cytopathology of viral infections, and describes the methods used to obtain and prepare cells for cytologic examination. It illustrates characteristic changes encountered in common virus infections. The preparatory methods utilized for the microscopic examination of cytologic specimens can be divided into five categories: direct smears, preparation by cytocentrifugation, membrane filter preparation, monolayer preparation, and cell block preparation. The eye and respiratory, genital, and urinary tracts are locations that readily yield cytologic material for rapid viral diagnosis. Characteristic cytologic changes depend on the cytopathic effect of a virus in infected cells, which need not include all cells of the involved organ. A nonspecific change referred to as ciliocytophthoria is found in various inflammatory diseases of the respiratory tract and, in particular, virus infections. Ciliocytophthoria occurs most frequently with influenza virus, parainfluenza virus, and adenovirus infection but may also occur in bronchiectasis and other nonviral inflammatory conditions. Although many viruses that cause systemic infections have been isolated from urine, those most readily diagnosed by urine cytology are cytomegalovirus (CMV), herpes simplex virus (HSV), and a member of the papovavirus group, designated the BK virus (BKV). Detection of HSV genital tract infection is important in abating the spread of sexually transmitted HSV as well as protecting the neonate from life-threatening infection transmitted to the infant during a vaginal birth. The morphologic changes in conjunctival and corneal cells due to virus infection must be differentiated from the cytologic changes due to chlamydial infections causing trachoma and inclusion conjunctivitis.

In clinical virology, electron microscopy (EM) has achieved a role equivalent to that of conventional light microscopy in clinical microbiology. EM allows for the rapid detection of the virus in a clinical specimen, at least at the level of the family into which it is classified, with a very high degree of specificity. Immunoelectron microscopy (IEM) arose from the combination of EM with the immunospecific interaction of viruses with their respective antibodies. Several methods such as negative staining methods, direct-application method and water drop method will concern only the direct visualization of viruses after negative staining. EM was instrumental in the identification of hendraviruses in cell cultures when this virus first emerged. It has a well-established potential for the rapid differentiation of varicella-zoster virus from poxviruses in skin lesions. With developments of enzyme immunoassays and molecular approaches such as polymerase chain reaction (PCR) and reverse transcription-PCR, EM is now mainly used in reference laboratories or laboratories in tertiary health care centers.

The early immunofluorescence assays (FA) used noncommercial preparations of polyclonal antisera directed against the target virus and a secondary reagent coupled with either rhodamine or fluorescein. Now, more than 60 years after the first report, immunofluorescence remains one of the primary technologies used by diagnostic virology laboratories. A wide variety of fluorochromes have been chemically modified so that they can be directly coupled to proteins. The excitation-emission spectra of one of the most commonly used fluorochromes, fluorescein isothiocyanate (FITC), are illustrated. Direct detection of viruses in clinical specimens has an advantage over culture isolation due to more timely reporting of results. Immunofluorescence has an advantage over enzyme immunoassays, as specimen adequacy can be determined and more than one virus can be detected. Specimens that are considered most appropriate for analysis by immunofluorescence include nasopharyngeal swabs, aspirates, or washes, bronchoalveolar lavage samples, swabs (including the recently introduced flocked swab specimen processing technology) or scrapings from vesicular lesions, tissue biopsy specimens (e.g., lung, liver, and brain), blood leukocytes, conjunctival cells, corneal scrapings, and urine sediment. Quality assurance establishes standard operating procedures for all aspects of testing, including specimen collection and processing, assay protocols, validation requirements, and quality control (QC) for all tests performed in a laboratory. Critical components, such as swabs, buffers, and transport media, etc., should also be included, along with package inserts from the commercial kits used in the laboratory.

This chapter deals with principles of enzyme immunoassay (EIA) and immunochromatography (ICR) and contemporary applications of both methods in viral antibody and antigen detection in the diagnostic virology laboratory. Immunoperoxidase staining, also called histochemical EIA or immunohistologic staining, is a type of EIA. “Immunoperoxidase staining” is the term used to describe the assays because most involve the enzyme horseradish peroxidase. Traditional EIA-type steps involving solid-phase reactants and enzyme-labeled detection complexes are performed. The most prominent application of membrane EIAs in diagnostic virology is detection of viral antigens, most commonly either influenza A and B or respiratory syncytial virus (RSV), in patients’ samples collected from the respiratory tract. Like the membrane-based rapid EIAs, optical immunoassays (OIAs) have several steps involved in testing and are classified as moderately complex according to Clinical Laboratory Improvement Act (CLIA). EIA and ICR methods for detection of rotavirus antigen in fecal and other types of samples are especially popular because this virus does not proliferate in standard cell cultures, and immunofluorescence techniques are not useful in detecting rotavirus antigen. EIAs are sometimes named by their detection system. Chemiluminescence and biotin-avidin EIAs are two of these. Membrane-based EIAs, OIAs, and ICRs, are predictably qualitative in nature, with results reported as positive or negative. The application of membrane EIAs, OIAs, and ICRs to viral antigen and antibody detection have opened an avenue for rapid viral diagnosis that allows testing to be conducted in more venues such as point-of-care and physician's office laboratories.

Since their introduction, immunoenzymatic techniques for the detection of viral antigens have served as important tools to detect, confirm, and identify viruses from direct specimen, cell culture, and tissue. In this chapter, the author reviews the developments in immunohistochemistry (IHC) techniques, with particular emphasis on antibody preparations, pretreatment antigen retrieval (AR), and state-of-the-art enzymatic signal amplification methods. The use of labeled antibodies for the detection of infectious agents in tissue was first demonstrated in 1942, with identification of pneumococcal antigens by direct fluorescent antibody in the livers and spleens of experimentally infected mice. Proper fixation of tissue prior to examination is necessary to maintain tissue architecture, preserve antigenicity, and prevent degradation. Immunohistochemistry method is independent of production of cytopathic effect (CPE) or typical viral inclusions, generates permanent preparations that can be viewed with an ordinary light microscope, and allows simultaneous evaluation of stained and unstained cells in tissue sections. Nonspecific staining due to antibody trapping around defects or edges of culture or tissue sections and endogenous peroxidase activity (e.g., neutrophils and plasma cells) in inflamed tissues or specimens can be sources of false reactivity. Detection and confirmation of cytomegalovirus (CMV) infection in tissue remains one of the most common viral indications for performance of IHC stain. The performance of IHC for detection of herpes simplex virus (HSV) from tissue is similar to that of IHC for CMV.

The neutralization test has been used in virology longer than any other serologic procedure. To test for virus neutralization, virus and serum are mixed together, incubated under appropriate conditions, and then inoculated into a susceptible living host for detection of nonneutralized virus. Nonneutralized virus is detected by looking for viral growth, using indicators such as cytopathic effect (CPE), plaque formation, or metabolic inhibition. The use of standardized components is critical to performing neutralization assays. For viral identification, well-characterized pretitered antiserum or well-standardized immune serum pools are used. Similarly, to measure antibody response to a virus, a well-characterized, pretitered virus is required. Finally, to monitor for viral inactivation (neutralization), a living host system is required. The antiserum must be standardized for use in the neutralization test by titration against both its homologous virus and heterologous virus(es). Standardization of immune serum requires that the serum be tested in the neutralization assay against the virus used to immunize the animal and closely related viruses. The neutralization capacity of antiviral monoclonal antibodies (MAb) is determined by titration against the virus of interest. Neutralization is used for typing viruses and for diagnosing infection based on the host’s immune response. It is also a research tool for dissecting how antibodies protect the host from infection and probing viral function.

The insertion of viral proteins necessary for budding of virus from the cell membrane facilitates the binding of red blood cells (RBCs) to the infected cells. This process is referred to as hemadsorption, and the RBCs from several species can be used in this process. A wide variety of viruses have the ability to bind with and then agglutinate RBCs (hemagglutination). The most common use of the hemagglutination inhibition (HAI) test in laboratories today is for subtyping of influenza virus isolates by state health department or World Health Organization-collaborating influenza surveillance laboratories.

Since the introduction of the first applications of immunoglobulin M (IgM) determinations in diagnostic virology, a variety of methods have been developed and applied. These methods can generally be separated into three groups: (i) those based on comparing IgM titers before and after chemical inactivation of serum IgM, (ii) those based on the physicochemical separation of IgM from other serum Ig classes, and (iii) those based on solid-phase immunologic detection of IgM antibodies. This chapter discusses the relative merits of each of these approaches. Physicochemical separation methods were originally developed to separate IgM antibodies from other serum Igs to facilitate assay by conventional serological tests, e.g., complement fixation (CF) and hemagglutination inhibition (HI) assays. The major distinguishing features of solid-phase immunoassays are the choice of indicator label and solid phase. Solid-phase immunoassays can be further differentiated into indirect and reverse, or “capture,” forms, based on the orientation of the immunoreactants on the solid phase. The indirect and capture formats have advantages and disadvantages that are described in detail. Compared to the whole virus antigen-based IgM immunoassay, the recombinant protein-based assay offers several distinct advantages. First, the use of infectious virus and special safety precautions used for antigen production are not required. Second, recombinant proteins can be easily standardized and quality controlled. Third, recombinant antigen production is efficient and relatively economical, thus eliminating the generally high production costs associated with virus cultivation.

This chapter discusses the clinical situations in which antiviral resistance has emerged, thus necessitating in vitro susceptibility testing, and provides an overview of the phenotypic and genotypic susceptibility testing methods that have been employed to detect resistance. Phenotypic assays are better suited to assess the combined effect of multiple resistance mutations on drug susceptibility. This is especially important for viruses such as hepatitis B virus (HBV), human cytomegalovirus (HCMV), and human immunodeficiency virus type 1 (HIV-1), which acquire resistance-associated mutations in multiple genes that may manifest as new patterns of resistance, cross-resistance, multidrug resistance, or even reversal of resistance. As clinical strains of HCMV and varicella-zoster virus (VZV) are cell-associated and because low-titer cell-free stocks are less stable during storage, infected cell suspensions (obtained by trypsin treatment of the infected monolayer) can be conveniently used for these viruses. Advantages of antiviral susceptibility testing by flow cytometry include the potential for automation, the objectivity of the assay, and a shorter turnaround time relative to the plaque-reduction assay (PRA). PCR amplification followed by restriction digestion could recognize mutant virus when present at 10% of the total virus population. The major advantage of this PCR-restriction endonuclease method is the speed with which UL97 mutations can be identified, since HCMV sequences can be directly amplified from many clinical samples. Genotypic assays do allow more rapid and efficient detection of resistance than phenotypic assays and may allow earlier detection of emerging resistance than phenotypic assays.

Molecular biological techniques have an increasing role in the laboratory diagnosis of viral infections as a consequence of their being more sensitive and specific than methods developed in the past. This chapter discusses the principle, describes the methodology, and provides some practical clinical applications of Western blotting. Enzyme immunoassays (EIA) often are used for diagnosis of viral diseases as well as for screening of blood and blood products for viruses. The basic approach to the use of Western blotting for the diagnosis of viral infections begins with purified virions that are disrupted by ionic detergent treatment, releasing viral proteins. Chemiluminescence has two main advantages compared to chromogenic techniques. First, it enables a >10-fold increase in sensitivity without the use of isotopes, and second, exposure times can be varied to increase or decrease sensitivity. A potential competitor for the Western blot assay has been reported for the diagnosis of HIV infection. This new technique, called “recombinant-antigen immunoblot assay” (RIBA-HIV216), utilizes a set of purified antigens produced by recombinant technology. It remains to be determined whether this recombinant-protein assay is more specific and/or more sensitive than the standard Western blot assay. Nitrocellulose strips with blotted viral proteins or complete kits with all necessary reagents are available from commercial sources for human immunodeficiency virus (HIV-1 ), human T-cell leukemia virus type 1 (HTLV-l), and hepatitis C virus Western blot assays.

Nucleic acid detection methods play an increasingly important role in the detection of viral infection. This chapter describes the major nucleic acid testing methods and assists in test selection. Nucleic acid amplification methods are classified as target or probe amplification methods based upon the source of the nucleic acid that is amplified in the procedure. Target amplification methods are among the oldest and best characterized nucleic acid amplification methodologies. Nucleic acid amplification detection methods include polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA) and transcription-mediated amplification (TMA). Nucleic acid detection methods include SYBR green, fluorescence resonance energy transfer (FRET) system and hydrolysis (TaqMan) probes. Other amplification methods described in the chapter are part of closed assay systems whose manufacturers discourage or prohibit in-house development. Nucleic acid detection methods have also made significant improvements in one's ability to detect fastidious and slow-growing viruses (e.g., human parvovirus, Epstein-Barr virus, and certain enteroviruses), viruses that are dangerous to amplify in culture (e.g., human immunodeficiency virus and certain hemorrhagic fever viruses), and viruses that are present in low concentrations. While nucleic acid detection methods will never completely replace culture and direct fluorescent antibody methods, nucleic acid detection methods will continue to play an important role in the detection and monitoring of viral diseases.

This chapter describes the quantitative molecular techniques that serve as the basis of viral load assays, and discusses key issues and important variables that affect assay performance. Real-time methods offer the advantages of increased dynamic range, simplicity, reduced analysis time, and diminished risk of contamination. Although polymerase chain reaction (PCR) is the best developed and most widely used nucleic acid amplification strategy, other strategies have been developed and several serve as the basis of quantitative assays for viral nucleic acids. Quantitative assays based on nucleic acid sequence-based amplification (NASBA), branched DNA (bDNA), and hybrid capture are commercially available. The specificity of real-time PCR can also be increased by including hybridization probes in the reaction mixture. NASBA is one of several transcription-based amplification methods that amplify RNA targets. Hybrid capture assays (HCAs) employ a signal amplification technology that can be applied to the detection and quantitation of DNA or RNA target molecules without amplification. Prior to the development of quantitative molecular techniques, laboratories were limited to cumbersome culture methods or insensitive antigen detection methods to determine viral load. Each of the quantitative molecular techniques has particular strengths and limitations that are inherent in the underlying nucleic acid amplification strategies. A thorough understanding of the key performance issues and features of the available quantitative molecular techniques is essential to the practice of modern clinical virology.

This chapter covers the following subjects: (i) a brief history of flow cytometry; (ii) a definition of flow cytometry; (iii) a description of the mechanics of a flow cytometer; (iv) the use of flow cytometry in detecting and quantifying human cytomegalovirus (hCMV) infected tissue culture cells; (v) the use of flow cytometry for studying apoptosis of virus-infected cells; (vi) the use of flow cytometry for measuring the effect of virus infection on the cell cycle; and (vii) the use of flow cytometry in drug susceptibility testing. Some flow cytometers are cell sorters that have the capacity to physically separate cells out of a population and collect that specific cell population. However, most of the experiments described in the chapter only require an instrument with a single argon ion laser that has the capacity to simultaneously analyze the light scatter properties of cells and two or three cell-associated fluorochromes. The chapter concentrates on a few studies that have used flow cytometry to measure in vitro drug susceptibility testing of antiviral compounds for cells infected with hCMV, human immunodeficiency virus (HIV), human herpesvirus 6 (HHV-6) and HHV8, Epstein-Barr virus (EBV), and influenza A and B viruses. In summary, the flow cytometry drug susceptibility assay for hCMV clinical isolates is accurate, rapid, and quantitative and can be automated. Expanded use of fluorochrome-labeled monoclonal antibodies to viral antigens and flow cytometry for detection and quantification of virus-infected tissue culture cells will save time and effort and make the diagnostic laboratory more efficient and productive.

Acute respiratory tract illnesses are the most common health conditions affecting humans. Six "classic" respiratory viruses have been known for decades: respiratory syncytial virus (RSV), influenza virus, human parainfluenza virus, rhinovirus, adenovirus, and the coronaviruses (CoVs) OC43 and 229E. The seasonality of most newly described respiratory viruses remains mostly investigational. Classification of viruses that primarily infect the respiratory tract are provided in this chapter. Relative importance of major respiratory viruses in upper and lower respiratory tract diseases are provided in this chapter. Vaccination and antiviral therapy are highly effective at limiting the spread of influenza virus, but these approaches are not available for other respiratory viruses. The major differences between human metapneumovirus (HMPV) and RSV are gene order and the absence of NS1 and NS2 genes in HMPV. The potential for coinfections involving HMPV and other respiratory viruses, especially RSV, is high given their overlap in seasonality. It begins with a profuse watery nasal discharge which may become mucopurulent and viscous. The number of real and potential respiratory viruses has increased considerably, and improved laboratory methods to detect them now abound.

Human enteroviruses (EV) are members of the Enterovirus genus of the family Picornaviridae and are among the most common human viral infections. These investigations had implications for all of virology because they indicated, first, that poliovirus (PV) grew in various tissue culture cells that did not correspond to the tissues infected during the human disease and, second, that PV destroyed cells with a specific cytopathic effect (CPE). The infectious virus is relatively resistant to many common laboratory disinfectants, including 70% ethanol, isopropanol, dilute Lysol, and quaternary ammonium compounds. Poliomyelitis should be considered in all cases of pure motor paralysis and is usually associated with a normal or slightly elevated value for protein, normal sugar value, and moderate mononuclear pleocytosis in cerebrospinal fluid (CSF). The innate immune system is especially important because it is the earliest response and, in addition, it regulates the adaptive immune response. All important information about a virus could potentially be obtained directly by PCR in conjunction with nucleic acid sequencing if all the molecular correlates of viral phenotypic determinants were understood. The most common molecular typing system is based on reverse transcription (RT)-PCR and nucleotide sequencing of a portion of the genomic region encoding VP1. The major advantage of the pan-EV PCR is that rapid detection of an EV is possible, even with very small amounts of clinical specimens such as CSF. Like the EV, human parechoviruses were traditionally detected and identified by virus isolation and antigenic typing.

Acute gastroenteritis is an important cause of morbidity and mortality worldwide, particularly among young children living in developing countries. Among the several causes of gastroenteritis (chemical, bacterial, and parasitic), viral pathogens play an important role. The role of four viral agents, rotaviruses, caliciviruses, astroviruses, and enteric adenoviruses, in causing gastroenteritis in humans is well established. Rotaviruses are members of the Rotavirus genus within the Reoviridae family, which also contains 10 other genera. Cell culture is not used for the diagnosis of rotavirus disease, since human rotaviruses are difficult to cultivate from clinical specimens. Caliciviruses are single-stranded, positive-sense RNA viruses with a broad host range and disease manifestation, such as respiratory disease in cats, vesicular disease in swine, and hemorrhagic disease in rabbits. The first astroviruses were identified in diarrhea stool specimens from children in 1975 by electron microscopy (EM). Later astroviruses have been isolated from numerous animal species and recently grouped into two genera, Mamastrovirus and Avastrovirus, within the Astroviridae family. Human astrovirus infections occur worldwide and are primarily associated with pediatric disease. Human adenoviruses have been linked with a number of diseases, including respiratory illness, conjunctivitis, and diarrhea. They are classified into the Adenoviridae family, Mastadenovirus genus. The recent introduction of rotavirus vaccines to the international markets is a promising start and hopefully will be followed by similar strategies for other viral causes of acute gastroenteritis. This chapter also talks about other enteropathogenic viruses such as coronaviruses and toroviruses, picobirnaviruses, and aichi virus.

This chapter addresses the biology and epidemiology of the waterborne viruses, hepatitis A virus (HAV) and hepatitis E virus (HEV), the value of diagnostic assays for each virus in different settings, and the current and future prospects for prevention and control of HAV and HEV infections. The HEV genome contains three open reading frames (ORFs), organized as 5’-ORF1-ORF3-ORF2-3’, with ORF3 and ORF2 largely overlapping. Acute infections with any of the hepatitis viruses, including HAV and HEV, cannot be distinguished on clinical characteristics or pathological examinations. Although the cyclical nature of HAV incidence suggests that caution should be exercised when interpreting the absolute disease incidence based on short-term trends, there can be no doubt that HAV vaccination in children is an effective public health measure, and it is hoped that other developed countries will implement similar policies in the near future. Although HAV (and potentially HEV) may be partly controlled with vaccines, there is evidence that some cases of waterborne hepatitis are due to as yet unidentified viral agents. A better understanding of HEV disease burden and appropriate use of the candidate vaccine in development will require enhanced efforts in surveillance and diagnosis of HEV, taking advantage of improved serological tests with appropriate use of confirmatory nucleic acid testing in countries where HEV is not endemic.

Blood-borne hepatitis viruses include hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis D virus (HDV). As their names imply, blood-borne hepatitis viruses impact mainly on the liver. Investigation of patients with these cryptogenetic forms of liver disease using a variety of molecular methods aimed at identifying possible causing agents has led to the discovery of a number of novel viruses. Viruses thus unveiled include GB type C virus now known to be the prototype of a vast array of viruses classified within the novel genus Anellovirus, and others. Although the hepatopathogenic potential of these viruses is much less pronounced than originally proposed or possibly nonexistent, these candidate agents of hepatitis can be borne by blood and because, for some of them, the ability to injure the liver, alone or in association with other agents, is still under scrutiny. Importantly, patients with symptoms of hepatitis can alternate clinical manifestations with variably long periods of remission, and the proportion of individuals who progress to cirrhosis and hepatocellular carcinoma (HCC) is roughly proportional to disease activity. The majority of acutely infected individuals progress to chronic infection with almost no propensity to resolve the infection spontaneously. There are also no indications that HCV or other blood-borne hepatitis viruses can be spread by insect vectors. The current standard of care for acute and chronic infection is pegIFN-α in combination with ribavirin, which leads to permanent clearance of the virus in approximately 60% of patients.

The Rhabdoviridae are classified as a group based on a similar conical or bullet-shaped appearance by electron microscopy. The laboratory diagnosis of rabies is most often performed for the postmortem examination of animals that have potentially exposed a human to the disease by a bite or other transdermal contact with saliva or neural tissue. A review of 32 human rabies deaths in the United States from 1980 to 1996 identified agitation and confusion, hypersalivation, hydrophobia or aerophobia, limb pain, and weakness as the most commonly observed signs of clinical rabies. Rabies virus maintained in domestic dog populations accounts for 95% of all animal rabies cases reported globally and still accounts for most of the zoonotic impact of the disease, with 90% of the human exposures to rabies and 99% of the human rabies deaths worldwide attributed to this cycle. The recognition of each human rabies case is very important in identification of highest risk exposure routes, vectors, and rabies virus variants-information essential to the development of effective rabies control and exposure management protocols. The prompt management of potential human exposure to rabies is a critical component of human rabies prevention.

This chapter focuses on the medically important arboviruses. As these viruses are transmitted by arthropods, arboviral disease usually manifests itself during the warmer months in the temperate climates of the world. Laboratory diagnosis of arboviral infections has traditionally been based upon serological identification of antiviral antibodies and/or isolation of virus. The hemagglutination inhibition (HI) test successfully differentiated togaviruses (group A arboviruses, primarily alphaviruses) from flaviviruses (group B arboviruses) long before modern biochemical techniques confirmed this observation. The MAC-enzyme-linked immunosorbent assay (ELISA) is capable of distinguishing among infections caused by the medically important alphaviruses (Eastern equine encephalitis (EEE), Western equine encephalitis (WEE), and Venezuelan equine encephalitis (VEE)). There are murine monoclonal antibodies (MAbs) capable of identifying virus complexes and even larger virus groups (e.g., all alphaviruses or all flaviviruses). In general, the alphaviruses demonstrate replication kinetics similar to those of the flaviviruses and are not commonly detected in acute-phase serum and/or cerebrospinal fluid (CSF) specimens, although detection is generally greater than with the flaviviruses. Consensus reverse transcriptase (RT)-PCR assays have been described for alphaviruses, flaviviruses, and the California and Bunyamwera serogroup bunyaviruses. The bunyaviruses are larger than either alphaviruses or flaviviruses, about 80 to 120 nm in diameter. Dengue (DEN) is currently the most important arboviral disease, with hundreds of thousands of cases occurring each year and millions of people at risk.

Human papillomaviruses (HPVs) infect surface epithelia and produce warts or other pathology at the site of multiplication on the skin or the mucous membrane. All of the open reading frames (ORFs) in papillomavirus DNA are located on only one of the two strands, indicating that only one strand carries the genetic information. The importance of the location of the lesion is best exemplified by laryngeal papilloma. The HPVs naturally fall into two groups, cutaneous HPVs and mucosal HPVs. The HPV-associated illnesses and the most common types of virus responsible for recurrent respiratory papillomatosis conditions are listed in this chapter. Specific HPV types are associated with different morphological types of lesions. It is unlikely that the cutaneous HPVs are associated with skin cancers in the same way as genital HPVs are associated with cervical cancer. The incidence of cervical cancer is high in developing countries, where it is the most common female malignancy and accounts for about 24% of all female cancers. A progressive spectrum of abnormalities, classified as low-grade and high-grade squamous intraepithelial neoplasia, precedes invasive cancer. For detection of virus, an immunologic test for viral capsid antigen is considerably more sensitive than demonstration of virus particles by electron microscopy. HPVs can be specifically identified only by nucleic acid based assays because the viruses cannot be grown in culture and type-specific immunologic reagents are not available. Recently, type-specific serological assays for HPV that use recombinant capsid proteins as antigens have been developed.

The first human polyomaviruses were isolated in 1971 from immunocompromised patients. Very recently, two new human polyomaviruses, KI virus and WU virus, were independently detected by molecular methods in respiratory tract secretions. The major capsid protein, VP1, accounts for more than 70% of the virion mass and has a molecular mass of 39 to 44 kDa. BKV nephropathy (BKVN) has recently been recognized as an important cause of progressive graft dysfunction and graft loss in patients with renal allografts. It is the most common viral infection affecting renal allografts, with an incidence of ~8% and graft loss ranging from 10 to >80%. BK virus (BKV) and JC virus (JCV) are reactivated in some women during normal pregnancy. In a prospective study, cytopathology in cells obtained from urine sediment suggested JCV and BKV infections in 3.2% of pregnant women. This was most frequently observed in the last trimester of pregnancy. In another study, 16% of the women showed an antibody rise to one or the other virus during pregnancy. An etiological role of BKV in hemorrhagic cystitis has been proposed for late-onset hemorrhagic cystitis. The majority of patients with BKV and JCV infections are asymptomatic and do not require treatment. There are no antiviral drugs with proven efficacy against human polyomaviruses. The mainstay of treatment for BKV nephropathy is the judicious reduction, change in drugs, or discontinuation of immunosuppressive therapy.

Herpes simplex virus (HSV) types 1 (HSV-1) and 2 (HSV-2) are members of the family Herpesviridae. They belong to the subfamily Alphaherpesvirinae and the genus Simplexvirus. The majority of the replicative intermediates are long concatemers apparently generated through sequence replacement or insertion. The rate of neonatal herpes projected by this study was 33 of 100,000 live births, and it was highest in the seronegative women. Using this process, trigeminal and sacral dorsal root ganglia were respectively identified as the most common sites for latent HSV-1 and HSV-2 infections, but latency can also be established in the central nervous system (CNS), primarily in the brain stem. The current routine procedure increases the sensitivity and specificity of the antigen detection assays by amplification through short-term (16 to 24 h) growth in tissue culture. The original goals of vaccination were to induce mucosal and systemic immunity to prevent HSV-2 infection and transmission. Oncolytic viruses are promising tools for cancer gene therapy. Cancer gene therapy approach (known as suicide) is based on the ability of HSV thymidine kinase (TK) to preferentially activate ganciclovir, thereby killing tumor cells that contain the TK gene as well as surrounding tumor cells.

The major immediate-early (IE) promoter (MIEP) of cytomegalovirus (CMV) controls the transcription of IE genes, IE1 and IE2, to encode proteins p72 and p86, respectively. Laboratory-based diagnosis is usually required to identify congenital and perinatal CMV disease and to diagnose and monitor viral levels in immunosuppressed hosts. The laboratory techniques commonly employed are the conventional tube and shell vial viral culture techniques, immunological techniques for histopathology, immunohistochemical techniques, antigen and antibody detection, and nucleic acid detection. Acyclovir, which is successfully used against other herpesviruses, such as herpes simplex virus (HSV) and varicella-zoster virus (VZV), is less potent against CMV. As one's understanding of the CMV genome and protein functions further advances, novel vaccine candidates and strategies will evolve that give equivalent or better humoral and cellular immune responses than natural immunity. The availability and versatility of VZV cosmids, which are large DNA fragments of the viral genome that can be recombined to form replication-competent VZV, have allowed targeting of different ORFs with point deletion mutants. A live attenuated vaccine was most effective in persons 60 to 69 years of age, but it also decreased the severity of incident zoster in people 70 years or older. Epstein-Barr virus (EBV) infection in the immunocompromised host is accompanied by the risk of developing lymphoproliferative disease. Using cytotoxic T lymphocyte (CTL) therapy in combination with other treatment modalities for aggressive PT-LPDs that are unresponsive to reduced immunosuppression has successfully induced remission without significant side effects.

Human herpesvirus 6 (HHV-6), HHV-7, and HHV-8 (also known as Kaposi’s sarcoma [KS]-associated herpesvirus) were discovered between 1986 and 1994. HHV-8 is the etiologic agent of KS and has been closely associated with multicentric Castleman’s disease (MCD) and a form of body cavity lymphoma known as primary effusion lymphoma (PEL). The major applications of HHV-6 serology are for (i) discriminating primary HHV-6 infections from adverse vaccine reactions in young children and (ii) detecting temporal changes in serostatus as part of diagnosing possible HHV-6 involvement in an acute clinical event. In addition to the extensive antigenic cross-reactivity between the HHV-6 variants, the variants have sufficient cross-reactivity with HHV-7 to lead to occasional false-positive results unless antigen adsorptions are done or an immunoblot assay is used that is based on non-cross-reactive antigens. During the AIDS epidemic, the incidence of KS dramatically increased, and it is now the most common malignancy in all segments of the population. The major application of HHV-8 diagnostic tests is in research studies of epidemiology, transmission, and new disease associations. Reports of HHV-8 associations with sarcoidosis and angiosarcoma were followed by several reports of no involvement of HHV-8 with these diseases. Other disease associations for HHV-8 have been reported for various lymphomas, cutaneous neoplasms, encephalitis, and Bowen’s disease, but investigations have been too few or the diseases too rare to rigorously confirm or refute any involvement of HHV-8.

Smallpox vaccine, Orthopoxvirus vaccinia, was used extensively for routine vaccination against variola virus. Historically poxvirus infections were laboratory confirmed by a combination of approaches including pock morphology on chicken embryo chorioallantoic membranes (CAMs), serologic reactivity, and electron microscopy (EM). Although EM can distinguish parapoxviruses from other poxviruses, the diagnostic method is constrained by the inability to differentiate between species within genera of poxviruses. Real-time PCR assays currently allow rapid and definitive diagnosis of the species of poxvirus causing an infection but still rely upon time-consuming processing of samples. The poxvirus family is divided into two subfamilies: Entomopoxvirinae (poxviruses of insects) and Chordopoxvirinae (poxviruses of vertebrates). The vertebrate poxviruses were further subclassified into genera by comparing cross-protection in animal studies, cross-neutralization of virion infectivity in cell culture, and through the analysis of genetic polymorphisms in genomic viral DNA. Molluscum contagiosum virus has one of the most limited host cell tropisms of any virus, replicating only in the human keratinocyte of the epidermis. The parapoxviruses, including orf, bovine papular stomatitis, pseudocowpox (milker’s nodule), and sealpox viruses cause occupational infections of humans, with orf infections being the most common. The genus Yatapoxvirus has two members, tanapoxvirus and Yaba monkey tumor virus, which are serologically related. Tanapox virus infection may occur via scratches or possibly via arthropod vectors. Current strategies for preventing human poxvirus infections are ones that stress awareness of the potential for infection and possible behavioral modifications to reduce risk of infection.

This chapter focuses on the two known human parvoviruses: B19 and human bocavirus (HBoV). While most autonomous parvoviruses possess unique sequences at either terminus, B19 differs in that its termini are inverted terminal repeats. B19 replication follows a modified rolling hairpin model of replication characteristic of the autonomous parvoviruses. The viral nonstructural protein NS1 serves as the “nickase,” which reduces the replicative forms to progeny virus. Nickase reduction results in two distinct configurations of the distal 375-nucleotide palindromes, which are inverted complements of each other. Parvovirus B19, like other parvoviruses, replicates and assembles in the cell nucleus. Upon histological analysis, a parvovirus infection may be suspected on the basis of intranuclear inclusions characterized by a peripheral nuclear presence. The polymerase chain reaction (PCR) offers exquisite sensitivity and the ability to detect B19 DNA in an array of clinical specimens.

The nucleoprotein, phosphoprotein, and large polymerase protein, in conjunction with the virion negative-strand RNA, comprise the ribonucleoprotein complex, the replicating and transcriptional unit of measles virus. Traditional antibody tests such as hemagglutination inhibition (HI), plaque reduction neutralization test (PRNT), and enzyme immunoassay (EIA) have been used extensively in the serologic diagnosis of measles. However, because of the availability of sensitive and specific commercial kits, EIAs have become the most widely used test format. The mumps genome is encapsidated by nucleoprotein, and as in the case of measles virus, the phosphoprotein and polymerase are associated with the encapsidated RNA to comprise the ribonucleoprotein complex. Enzyme-linked immunosorbent assay vary greatly in sensitivity (range, 24 to 51%), and the best specificity measured was 82%. Of considerable interest is the use of oral fluid rather than serum in the determination of Immunoglobulin M (IgM) for acute mumps infections and IgG for immune status for measles, mumps, and rubella. Rubella would be of little medical importance were it not for the profound defects rubella virus (RV) infection can cause in the unborn child. Mumps vaccine is given along with measles and rubella vaccines at 12 to 15 months of age and again at school entry. Laboratory diagnosis of both postnatal and congenital RV infections is by serologic and/or virus detection techniques.

This chapter provides an overview of the currently known exogenous human retroviruses and the diseases associated with them. For handling of clinical specimens, all retroviruses, including human immunodeficiency virus (HIV) and human T-lymphotropic retrovirus (HTLV), are classified as biological agents of moderate risk. The currently used Centers for Disease Control and Prevention (CDC) 1993 classification is based on a combination of clinical and CD4 T-cell count categories that defines nine mutually exclusive stages. Western blot (WB) is preferable to in-house methods of confirmation like radioimmunoprecipitation assay, since the commercial WB kits are better standardized and much easier to run. HTLV-1 is associated with a number of inflammatory disorders in addition to adult T-cell leukemia or lymphoma (ATLL). Virus isolation should not only be evaluated with HTLV-specific tests (antigen assay or immunofluorescence) but, where available, also by assays for particle-associated RT, preferentially product-enhanced RT (PERT) assay. Finally, both allogeneic and autologous bone marrow transplantation are evaluated as possible treatments of ATLL.

Chlamydiae are obligate intracellular bacteria which cause many diseases in animals and humans. Originally they were classified as Chlamydia trachomatis, C. pneumoniae, C. psittaci, and C. pecorum. During their growth, chlamydiae produce characteristic intracytoplasmic inclusions that can be visualized by Giemsa stains or direct fluorescent antibody (DFA) stains of infected patient material, such as conjunctival scrapings, as well as cervical or urethral epithelial cells. Chlamydiae are phagocytized by susceptible host cells. The chlamydiae possess group (genus)-specific, species-specific, and type-specific antigens. The major outer membrane protein contains species-, subspecies-, and serovar-specific antigens. Chlamydiae cause the recruitment of lymphocytes to the site of infection by inducing the release of local host factors, which influence the adhesion cascade of cytokines and adhesion molecules. By adult life, active infection is infrequent, but the sequelae of the disease result in blindness. Worldwide, trachoma is considered the most common cause of preventable blindness. C. trachomatis is the most common sexually transmitted bacterial pathogen. Sensitivity and specificity of diagnostic tests for the detection of C. trachomatis have been provided in this chapter. The most widely used serological test for diagnosing chlamydial infections is the genus-specific complement fixation (CF) test. It is useful in diagnosing psittacosis, in which paired sera often show fourfold or greater increases in titer. There are some commercially available serologic tests based on measurement of antibodies reactant to chlamydial inclusions in cell culture or enzyme immunoassay (EIA) using chlamydial antigens.

Small mammals, including rodents, may participate in the sylvatic cycles of some arboviruses, but rodents serve as the primary reservoirs of two major groups of medically important viruses: those of the family Arenaviridae and those of the genus Hantavirus family Bunyaviridae. For both groups of viruses, there is evidence for coevolution with their rodent hosts. Several of the animal models develop diseases that have strong similarities to the human diseases caused by arenaviruses. For both hantaviruses and arenaviruses, reservoir rodents develop chronic infections that result in transient or periodic virus shedding in urine, feces, and saliva. Diagnosis of acute infection with arenaviruses is often problematic because specific antibody responses to arenavirus infections are often delayed until days to weeks after the illness has run its course. For LAS fever, Bolivian hemorrhagic fever (BHF), Venezuelan hemorrhagic fever (VHF), hemorrhagic fever with renal syndrome (HFRS), and hantavirus cardiopulmonary syndrome (HCPS), vaccines represent the most probable route toward control of morbidity and mortality. Modern tools for predicting disease outbreaks have been increasingly developed, which should be in more fluent knowledge for the local health services.