Overview of the Methods and Strategies in Virology

Chapter 65


Overview of the Methods and Strategies in Virology*



Objectives



1. Describe the physical components that make up a virion and list a function for each component.


2. Define the viral infectious cycle, including naming the six steps in this process.


3. Explain viral tropism and provide a specific example.


4. Define the properties used to classify a virus and identify the person responsible for classification of a virus.


5. Explain the steps in viral pathogenesis.


6. List some of the reasons the clinical science industry has seen an increased demand for clinical viral services.


7. Name some of the equipment necessary to set up a clinical virology laboratory and give the function of each piece.


8. List some of the viruses associated with the following clinical specimens: throat or nasopharyngeal swab or aspirate, urine, stool, lesion, blood, bone marrow, and stool or rectal swab.


9. List some of the most efficient laboratory tests for detecting the following viruses: enterovirus, herpes simplex virus, influenza virus, norovirus, and respiratory syncytial virus (RSV).


10. Define the Tzanck test and list the viruses for which the test is used.


11. Define monolayer, primary cells, semicontinuous (low passage) cells, and continuous cells.


12. Explain the types of cell lines used in viral cell culture; describe their similarities and differences.


13. Define and differentiate cell culture growth medium and maintenance medium.


14. Explain the incubation conditions for routine cell cultures.


15. Define CPE and explain how it is rated when reading cell cultures.


16. Describe a shell vial cell culture and explain its advantages over conventional cell culture.


17. Define the hemadsorption procedure and name the viruses for which the test is used.


18. Name the virus family capable of establishing viral latency in the human dorsal nerve root ganglion and explain the possible consequence of the latency.


19. Name the preferred tissue type of cell culture for growth of the following viruses: influenza A virus, varicella-zoster virus, herpes virus, and cytomegalovirus (CMV).


20. Associate an appropriate viral pathogen with the following viral syndromes: infant croup, infant bronchiolitis, adult gastroenteritis, parotitis, infectious mononucleosis, and meningitis.


Evidence of viral disease exists in ancient records, dating back to as far as 23 BC, when the Eschunna Code of ancient Mesopotamia noted “the bite of mad dogs to affect disease on humans.” Homer, author of the Iliad, characterizes Hector as “rabid.” Aristotle’s work, The Natural History of Man, written in the fourth century BC, describes a “madness” in dogs that “causes them to become very irritable and all mammals they bite become diseased.” What remains apparent in all these early writings is that all writers realized the communicable nature of something unseen. These writings clearly refer to the rabies virus, transmitted through the saliva of an infected animal.


The survival of viral infectious agents depends on their ability to infect and reside in a living organism. These tiny organisms are thought to have evolved alongside humans and in conjunction with the domestication of animals. Throughout history, evidence shows that viruses are able to survive when established populations of humans are available to provide a means for continued propagation. Viruses that established a long-term relationship with their host (i.e., did not kill the host immediately upon infection) were the first to become adapted to co-evolution with the human race. Some of these earliest viruses were thought to be retroviruses, such as the herpes viruses, and papillomaviruses.


A virus is a submicroscopic, obligate intracellular parasite, among the smallest of all infectious agents, and capable of infecting any animal, plant, or bacterial cell. Viruses are found in every ecosystem. They are strict obligate intracellular parasites, incapable of replication without a living host cell. Virus types are very specific, and each has a limited number of hosts it can infect; this is referred to as viral tropism.


Much is still unknown about the origins of viral agents, although most speculation indicates that viruses affecting man established themselves in the human population through transmission of an animal virus to a human. Transmission of viruses from animals to humans still occurs, as demonstrated in the more recent viral outbreaks associated with the severe acute respiratory syndrome (SARS), West Nile, and influenza A H5 viruses, as well as the 2009 H1N1 virus, formerly known as the pandemic “swine flu.” The influenza virus has proven to be one of the deadliest viruses to affect humans; its history dates back to the 1700s in Italy. The virus was named to indicate disease resulting from the “influence” of miasma (bad air).


The emergence of a new viral disease across a very large geographical region (worldwide) with prolonged human-to-human transmission is called a pandemic. To date, most of the pandemics recorded have been caused by an influenza virus. Pandemics result when an influenza virus undergoes a genetic shift and the reassortment of genes combines with those of another organism, usually an animal. The resulting virus emerges as a completely new or “novel” virus. The genetic changes in viral genomes may result from antigenic shift (major changes that result in novel viral antigens) and/or antigenic drift (minor changes that occur infrequently), which are discussed in Chapter 66. One of the most deadly influenza outbreaks was the Spanish Flu pandemic of 1918-1919. This pandemic was associated with infection with a novel influenza virus of avian origin. After a period of adaptation in humans, the virus emerged in pandemic form and was responsible for more than 50 million deaths worldwide, including 500,000 in the United States. What was so different about this pandemic was that it affected young and healthy individuals, not just the very young or very old. The more recent influenza pandemic of the twentieth century was associated with a human influenza virus in which genes reassorted in combination with an avian influenza virus.


Protection from viral infection has been successful for some viral pathogens. Vaccination (immunization) has proven to be a valuable tool in the control of viral diseases such as yellow fever and rabies and has been instrumental in the eradication of one of the most lethal viruses, smallpox. However, many viral diseases such as influenza, acquired immunodeficiency syndrome (AIDS), and hepatitis continue to pose challenges in treatment, prevention, and control.


The science of clinical virology has seen a rapid expansion in the past few years as new and emerging pathogenic viruses continue to evolve. Since 1988, 50 new viruses have been identified, making prevention and control more and more difficult. The science of virology will continue to evolve and clinicians will continue to rely on the laboratory scientist for the development and implementation of testing to diagnose, treat, and prevent viral disease.



General Characteristics


Viral Structure


Virus particles, referred to as virions, consist of two or three parts:



Because enveloped viruses are very susceptible to drying out and destruction in the environment, they typically are transmitted by direct contact, such as respiratory, sexual, or parenteral contact. This prevents exposure to the environment and successful propagation of the viral agent to another susceptible host. Viruses that do not have an envelope are often referred to as “naked” viruses. Naked viruses are very resistant to environmental factors. Because of their stability, they typically are transmitted by the fecal-oral route. Many viruses have glycoprotein spikes extending from their surface. The term nucleocapsid is often used to describe the nucleic acid genome surrounded by a symmetric protein coat (Figure 65-1).


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Figure 65-1 Illustration of a viral particle. Enveloped and nonenveloped virions have an icosahedral or irregular (usually helical) shape. (Modified from Murray PR, Drew WL, Kobayashi GS, et al, editors: Medical microbiology, St Louis, 1990, Mosby.)

The function of the nucleic acid genome is to encode the proteins required for viral penetration, transmission, and replication. The viral genome structure determines the mechanism for viral replication. A variety of vial genome structures exist, including (+) sense strand RNA, (–) sense strand RNA, and DNA genomes. In addition, viral genomes may be single- or double-stranded molecules. The structural implications of variation in genome organization are discussed in more detail in the section on viral replication.


The viral capsid protects the viral genome and is responsible for the tropism to specific cell types in naked viruses. Viral capsids typically are composed of repeating structural subunits referred to as capsomeres. The capsomeres associate to form the capsid and a characteristic symmetric structure. The most common capsid structures geometrically form a helical or icosahedral structure (see Figure 65-1). Icosahedral capsids are cubical and have 20 flat sides; irregularly shaped capsids usually assume a helical form and are spiral shaped.


As mentioned, in some viruses the nucleocapsid is enclosed in a lipid envelope. The envelope is responsible for viral entry into the host cell (see Figure 65-1). During the infectious process, enveloped virions bud from a host cell’s cytoplasmic, nuclear, or endoplasmic reticular membrane, and a portion of the membrane remains attached to the virion as the viral envelope. Inserted into this viral envelope are viral proteins, such as hemagglutinin (HA), neuraminidase, or glycoprotein spikes. The glycoprotein spikes assist in stabilization of attachment for the lipid envelope and for attachment to the host cell to facilitate viral entry. Some enveloped viruses also contain a matrix protein that lies between the envelope and the nucleocapsid. The matrix protein may have enzymatic activities and/or biologic functions related to infection, such as inhibition of host-cell transcription.


Viruses that cause disease in humans range from approximately 20 to 300 nm. Even the largest viruses, such as the poxviruses, cannot be detected with a light microscope, because they are less than one fourth the size of a staphylococcal cell (Figure 65-2). Not until the invention of the electron microscope in the 1930s were viruses visualized. The electron microscope’s improved magnification (more than 100,000 times) allowed visualization of virus particles and paved the way for viral classification based on structural components.


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Figure 65-2 Relative sizes of representative viruses, bacteriophages (bacterial viruses), and bacteria, including chlamydia. (From Murray PR, Drew WL, Kobayashi GS, et al, editors: Medical microbiology, St Louis, 1990, Mosby.)


Virus Taxonomy


Viral taxonomy is determined by the International Committee on Taxonomy of Viruses (ICTV) of the Virology Division of the International Union of Microbiological Societies. Viral taxonomy is divided into categories: six orders (-virales), 87 families (name ending in -viridae), 19 subfamilies (-virinae), 348 genera (-virus), and 2290 species. Classification of viral species can be problematic and therefore is often polythetic; that is, the members of a group share common characteristics but may not have a single defining characteristic. In addition, some viral families currently are not assigned to an order, and some species are not assigned to a family.


Complex viral taxonomy incorporates a variety of categories, including information related to host range, transmission, disease pathology, antigenicity, and viral particle properties, such as size, envelope, capsid structure, physical properties, genome type, and configuration. For simplicity purposes, many texts limit viral classification to three basic properties: (1) viral morphology; (2) method of replication, including genome organization (whether the genome is RNA or DNA and single- or double-stranded); and (3) presence or absence of a lipid envelope. The term means of replication refers to the strategy the virus uses to duplicate the viral genome. For example, enteroviruses have single-stranded RNA genomes that synthesize additional strands of RNA, whereas retroviruses make RNA in a two-step process by first synthesizing DNA, which subsequently makes RNA.


Characterization of viral genomes has increasingly improved as a result of advances in molecular techniques. Molecular sequencing of viral genomes is becoming more and more common. However, because of the genetic instability of viral genomes, molecular sequencing is limited to providing evidence for species relationships and epidemiologic comparisons of isolates. As a result, clinical virologists generally categorize viruses as containing DNA or RNA and further organize by family and common names.



Viral Replication


Viruses are strict intracellular parasites, reproducing or replicating only inside a host cell. The six steps of virus replication, called the infectious cycle, proceed as follows (Figure 65-3).


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Figure 65-3 Illustration of the viral infectious cycle. (Modified from Murray PR, Drew WL, Kobayashi GS, et al, editors: Medical microbiology, St Louis, 1990, Mosby.)


1. Attachment, also referred to as adsorption, is the first step of the infectious cycle. It involves recognition of a suitable host cell and specific binding between viral capsid proteins (often the glycoprotein spikes) and the carbohydrate receptor of the host cell. Each type of virus specifically recognizes and attaches to a specific type of host cell, allowing infection of some tissues but not others (viral tropism, as previously described).


2. Penetration is the process by which viruses enter the host cell. One mechanism of penetration involves fusion of the viral envelope with the host cell membrane. This method not only provides a mechanism for internalizing the virus, but also leads to fusion between the infected host cell and additional nearby host cells, forming multinucleated cells called syncytia. Detection of syncytia can be used to determine the presence of virus in cell cultures or stained smears of clinical specimens. Other mechanisms of viral penetration include phagocytosis by host cells (endocytosis) or injection of viral nucleic acid.


3. Uncoating occurs once the virus has been internalized. It is the process by which the capsid is removed; this may be by degradation of viral enzymes or host enzymes or by simple dissociation. Uncoating is necessary to release the viral genome before the viral DNA or RNA is delivered to its intracellular site of replication in the nucleus or cytoplasm.


4. Macromolecular synthesis involves the production of nucleic acid and protein polymers. Viral transcription leads to the synthesis of messenger RNA (mRNA), which encodes early and late viral proteins. Early proteins are nonstructural elements, such as enzymes, and late proteins are structural components. Rapid identification of virus in a cell culture can be accomplished by detecting early viral proteins in infected cells using immunofluorescent staining techniques. Replication of viral nucleic acid is necessary to provide genomes for progeny virus particles or virions. Macromolecular synthesis varies, depending on the organization of the viral genome.


5. Viral assembly is the process by which structural proteins, genomes, and in some cases viral enzymes are assembled into virus particles. Envelopes are acquired during viral “budding” from a host cell membrane. Nuclear endoplasmic reticulum and cytoplasmic membranes are common areas for budding. Acquisition of an envelope is the final step in viral assembly.


6. Release of intact virus particles occurs after cell lysis (lytic virus) or by budding from cytoplasmic membranes. Release by budding may not result in rapid host cell death, as does release by cell lysis. Detection of virus in cell cultures is facilitated by recognition of areas of cell lysis. Detection of virus released by budding is more difficult, because the cell monolayer remains intact. Influenza viruses, which are released by budding with minimal cell destruction, can be detected in cell culture by an alternative technique called hemadsorption. Influenza virus–infected cells contain virally encoded glycoprotein hemagglutinins inserted into the host cell’s cytoplasmic membrane, preparing for inclusion in the viral envelope at the time of release by cytoplasmic budding. Red blood cells (RBCs) added to the culture medium adsorb to the outer membranes of infected cells but not to uninfected cells. Each infected host cell results in as many as 100,000 virions; however, as few as 1% of these may be infectious or “viable” in the practical sense. Noninfectious viral particles may result from errors or mutations that occur during the infectious cycle.




Pathogenesis and Spectrum of Disease


Once introduced into a host, the virus infects susceptible cells, frequently in the upper respiratory tract. Viral infections may produce one of three characteristic clinical presentations: (1) acute viral infection, displaying evident signs and symptoms; (2) latent infection, which has no visible signs and symptoms, but the virus is still present in the host cell in a lysogenic state (inserted into the host genome in a resting state); and (3) chronic or persistent infection, in which low levels of virus are detectable and the degree of visible signs or symptoms varies.


After a local viral infection, a viremia occurs (viruses present in the patient’s blood ), which inoculates secondary target tissue distant from the primary site and releases mediators of human immune cell functions. Secondary viremia may occur in a variety of tissues, such as the skin, salivary glands, kidneys, and brain tissues. Symptomatic disease ensues. Disease resolves when specific antibody and cell-mediated immune mechanisms prevent continued replication of the virus. Tissue is damaged as a result of lysis of virus-infected cells or by immunopathologic mechanisms directed against the virus that are also destructive to neighboring tissue. Most DNA-containing viruses, such as those in the herpes group, remain latent in host tissue with no observable clinical impact. Retroviruses and most DNA viruses establish a latent state after primary infection. During the latent state, viral genome is integrated into the host cell’s chromosome and no viral replication occurs. Latent viruses can reactivate silently, resulting in viral replication and shedding but no clinical symptoms, or they can reactivate and cause symptomatic, even fatal, disease. Reactivation may accompany immune suppression, resulting in the recurrence of clinically apparent disease.


Occasionally, pathogenic viruses stimulate an immune reaction that cross-reacts with related human tissue, resulting in damage to host function; this is called autoimmune pathogenesis. When present, it occurs well after the acute viral infection has resolved. Rare viral infection promotes transformation or immortalization of host cells, resulting in uncontrolled cell growth. Viruses with the ability to stimulate uncontrolled growth of host cells are referred to as oncogenic viruses. Some papillomaviruses (wart viruses) are oncogenic, giving rise to human cervical cancer.


Examples of the variety of pathogenic mechanisms of viral infection are illustrated in disease caused by infection with the measles virus. After replication in the upper respiratory tract and subsequent viremia, the virus infects many susceptible cells throughout the body, including endothelial cells in capillaries of the skin. This is accompanied by local inflammation and results in the characteristic rash of measles. Immunocompetent individuals eradicate the virus, resolving the infection, and have lifelong immunity. In some, antibody produced in response to the measles infection cross reacts with tissue in the central nervous system (CNS), causing a postinfectious encephalitis. In others, slow but continuing replication of damaged virus in the brain gives rise to subacute sclerosing panencephalitis. In severely immunocompromised individuals, ongoing primary infection is not aborted by the usual immune mechanisms, and the result is death (Figure 65-4). Because the measles virus is not an oncogenic virus, no cancers result from prolonged infection.


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Figure 65-4 Viral pathogenesis is illustrated by the mechanisms through which the measles virus spreads in the body. (From Murray PR, Drew WL, Kobayashi GS, et al, editors: Medical microbiology, St Louis, 1990, Mosby.)


Prevention and Therapy


Immunizations are available for some viruses capable of causing disease in humans. However, for viruses for which there are no available vaccines, the most effective means of preventing viral infection involves regular, thorough hand washing and avoiding contact with others during episodes of evident signs and symptoms, such as fever, cough, diarrhea, and respiratory infections.




Viruses That Cause Human Diseases


Hundreds of viruses cause disease in humans. Viruses of human medical importance comprise four orders, 25 families, 13 subfamilies and 66 genera. Individual viruses may cause multiple different diseases, and conversely, many viruses may cause the same disease, all of which complicates the understanding of viral disease in humans. For example, viruses that can cause encephalitis include HSV, many arboviruses, rabies virus, HIV, and measles virus. However, HSV also can cause pharyngitis, genital infection, conjunctivitis, and encephalitis. Viruses that are important pathogens in humans and the viral syndromes they cause are summarized in Table 65-1. Specific viral agents and their role in human disease are discussed in Chapter 66.



TABLE 65-1


Viral Syndromes and Common Viral Pathogens

































































































Viral Syndrome Viral Pathogens
Infants and Children  
Upper respiratory tract infection Rhinovirus, coronavirus, parainfluenza, adenovirus, respiratory syncytial virus, influenza
Pharyngitis Adenovirus, coxsackie A, herpes simplex virus, Epstein-Barr virus, rhinovirus, parainfluenza, influenza
Croup Parainfluenza, respiratory syncytial virus, metapneumovirus
Bronchitis Parainfluenza, respiratory syncytial virus, metapneumovirus
Bronchiolitis Respiratory syncytial virus, parainfluenza, metapneumovirus
Pneumonia Respiratory syncytial virus, adenovirus, influenza, parainfluenza
Gastroenteritis Rotavirus, adenovirus 40-41, calicivirus, astrovirus
Congenital and neonatal disease HSV-2, echovirus, and other enteroviruses, CMV, parvovirus B19, VZV, HIV, hepatitis viruses
Adults  
Upper respiratory tract infection Rhinovirus, coronavirus, adenovirus, influenza, parainfluenza, Epstein-Barr virus
Pneumonia Influenza, adenovirus, sin nombre virus (hantavirus), severe acute respiratory syndrome (SARS) coronavirus
Pleurodynia Coxsackie B
Gastroenteritis Noroviruses
Cervical cancer Human papillomavirus
All Patients  
Parotitis Mumps, parainfluenza
Myocarditis/pericarditis Coxsackie B and echoviruses
Keratitis/conjunctivitis Herpes simplex virus, VZV, adenovirus, enterovirus 70
Pleurodynia Coxsackie B
Herpangina Coxsackie A
Febrile illness with rash Echoviruses and coxsackie viruses
Infectious mononucleosis Epstein-Barr virus and CMV
Meningitis Echoviruses and coxsackie viruses, mumps, lymphocytic choriomeningitis, HSV-2
Encephalitis HSV-1, togaviruses, bunyaviruses, flaviviruses, rabies, enteroviruses, measles, HIV, JCV
Hepatitis Hepatitis A, B, C, D (delta agent), E, and non-A, B, C, D, E
Hemorrhagic cystitis Adenovirus, BK virus
Cutaneous infection with or without rash HSV-1 and HSV-2; VZV; enteroviruses; measles; rubella; parvovirus B-19; human herpes virus 6 and 7; HPV; poxviruses, including smallpox, monkeypox, molluscum contagiosum; and orf
Hemorrhagic fever Ebola, Marburg, Lassa, yellow fever, dengue, and other viruses
Generalized, no specific target organ HIV-1, HIV-2, HTLV-1

CMV, cytomegalovirus; HIV, human immunodeficiency virus; HPV, human papillomavirus; HSV-1, herpes simplex virus type 1; HSV-2, herpes simplex virus type 2; HTLV-1, human T-lymphotropic virus type 1; JCV, JC virus; SARS, severe acute respiratory syndrome; VZV, varicella-zoster virus.



Laboratory Diagnosis


Setting Up a Clinical Virology Laboratory


The demand for clinical virology laboratory services has skyrocketed during the past two decades. This growth has resulted from the introduction of virus-specific antiviral drugs; the commercial availability of reagents; the development of rapid diagnostic techniques using conventional methods such as fluorescence microscopy and enzyme immunoassays; the ready availability of cell lines for cell culture procedures; and the introduction of real-time polymerase chain reaction (PCR) assays for detecting viral genomes. Ironically, improved medical care, in the form of organ transplantation and immune suppression with cancer therapy, has resulted in an increased number of patients with viral disease. When these factors are considered along with the appearance of emerging viral pathogens that are threatening local and world populations (e.g., SARS, avian influenza, monkey pox), laboratory diagnosis of viral infection becomes far more important than in previous years.


When determining which virology tests to offer, each clinical laboratory should decide whether the test is required for the appropriate care of their patient population and whether techniques are available that provide an accurate, cost-effective test result. Viral diseases that require laboratory diagnosis include sexually transmitted diseases, diarrhea, respiratory disease in adults and children, aseptic meningitis, arbovirus encephalitides, congenital diseases, hepatitis, and infections in immunocompromised individuals. Table 65-2 presents a representative sample of viruses identified in a community clinical virology laboratory.



TABLE 65-2


Viruses Detected by Culture, PCR, or Assay for Antigen in a Community Hospital Virology Laboratory*






































Infecting Virus Number of Cases (Adults and Children)
Adenovirus 25
Cytomegalovirus 30
Enterovirus 50
Herpes simplex virus 206
Influenza virus 426
Parainfluenza virus 41
Respiratory syncytial virus 151
Rotavirus 163
Varicella-zoster virus 38
Total 1130


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PCR, Polymerase chain reaction.


*Data from 1 year of testing at Evanston Northwestern Healthcare, Evanston, Illinois.


Laboratory scientists in a clinical virology laboratory must be familiar with cell culture, enzyme immunoassay, immunofluorescence methods, and molecular methods (e.g., PCR), in addition to other common laboratory techniques. Large equipment needed for a full-service virology laboratory includes a laminar flow biologic safety cabinet (BSC), fluorescence microscope, inverted bright field microscope, refrigerated centrifuge, incubator, refrigerator and freezer, roller drum for holding cell culture tubes during incubation, and enzyme or molecular testing instrumentation (Figures 65-5 to 65-7).


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Figure 65-5 Roller drum used to hold cell culture tubes during incubation. Slow rotation continually bathes the cells in the medium.

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Figure 65-6 Inverted microscope used to examine cell monolayers growing attached to the inside surface beneath the liquid medium. Note that the objective is under the glass test tube, facilitating observation of the cell monolayer.

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Figure 65-7 Class II biological safety cabinet used in a clinical virology laboratory.

Standard precautions and Biosafety Level 2 conditions are needed for community and most nonretroviral laboratories. Requirements include standard microbiologic practices, training in biosafety, protective clothing and gloves, limited access, decontamination of all infectious waste, and a class I or II BSC. Some viruses should not be propagated in Biosafety Level 2 laboratories, including influenza H5N1, SARS coronavirus, hemorrhagic fever viruses, and smallpox.



Specimen Selection and Collection


General Principles


Specimen selection depends on the specific disease syndrome, viral etiologies suspected, and time of year. Selecting a specimen based on disease is confusing, because most viruses enter through the upper respiratory tract and infect tissues that may produce symptoms distant from the primary inoculation site. For example, aseptic meningitis, caused by infection with various types of enterovirus, may be identified by detecting virus in throat, rectal swab, or cerebrospinal fluid (CSF) specimens. Pharyngitis and gastrointestinal symptoms may not be included in the patient’s complaints.


Specimen selection based on the suspected viral etiology is complicated by the fact that similar clinical syndromes can be caused by many different viruses. When specimens required for identification of a specific virus are collected without thorough consideration of other possible viral agents, additional important etiologic agents may be missed. For example, testing smears of nasal secretions from an infant using fluorescence staining or enzyme immunoassay to detect RSV does not allow for diagnosis of similar disease resulting from infection with influenza virus, parainfluenza virus, or metapneumovirus.


Selection of the appropriate type of specimen is one of the keys to a correct test result. Selection should include the proper specimen source and the correct sample volume and timing of collection. This information should be reviewed institutionally on an annual basis and made available to clinicians.


Appropriate specimen selection dictates that the specimen type and suspected viruses should be included on the requisition. The laboratory should always be notified if rare agents representing a danger to laboratory workers are suspected (e.g., SARS coronavirus, H5N1 avian influenza virus, hemorrhagic fever viruses). Serum for serologic testing may be necessary, and some viral diseases should be considered during specific months. Table 65-3 presents specimens for the diagnosis of viral diseases, noting trends in seasonality.



TABLE 65-3


Specimens for Diagnosis of Viral Diseases*













































































































































































































































































































































































































































































































Disease Categories and Probable Viral Agent Season of Most Common Occurrence Throat/Nasopharynx Stool CSF Urine Other
Respiratory            
Adenoviruses Y ++++        
Influenza virus W ++++        
Parainfluenza virus Y ++++        
Respiratory syncytial virus (RSV) W ++++        
Metapneumovirus W ++++        
Rhinoviruses Y         Nasal (+++)
SARS coronavirus W ++++        
Sin nombre virus SP, S         Serum for antibody detection
Dermatologic and Mucous Membrane            
VESICULAR            
Enterovirus S, F ++ +++     Vesicle fluid or scraping
Herpes simplex virus† Y         Vesicle fluid or scraping
Varicella-zoster virus† Y ++       Vesicle fluid or scraping
Monkeypox Y         Vesicle fluid or scraping
EXANTHEMATOUS            
Enterovirus S, F +++ ++      
Measles Y ++     ++ Serum for antibody detection
Rubella Y       ++ Serum for antibody detection
Parvovirus Y         Serum for antibody detection, amniotic fluid (PCR)
PUSTULAR/NODULAR            
Molluscum contagiosum, orf Y         Tissue
Warts
Papillomavirus		 Y         Tissue/cells, thin-prep cervical
Meningoencephalitis/Encephalitis            
Arboviruses S, F         CSF/serum for antibody detection
Enteroviruses S, F +++   ++ ++++  
Herpes simplex virus Y     ++++   Brain biopsy (PCR)
Lymphocytic choriomeningitis Y         Serum for antibody detection
Mumps virus Y         Serum for antibody detection
HIV Y         Brain biopsy (culture/PCR)
Polyomavirus (JC virus) Y         Brain biopsy (EM/PCR)
Rabies virus Y         Corneal cells, brain
Gastrointestinal Disease            
Adenoviruses (serotypes 40-41) Y   ++++     Stool (EIA or EM)
Noroviruses S   ++++     Stool (EM)
Rotavirus W, SP   ++++     Stool (EIA, latex)
Dermatologic and Mucous Membrane            
CONGENITAL AND PERINATAL            
Cytomegalovirus Y       +++ Serum for antibody (IgM) detection
Enteroviruses S, F +++   +++ +++  
Herpes simplex virus Y         Vesicle fluid
Parvovirus Y         Amniotic fluid, liver tissue
Rubella Y       ++ Serum for antibody (IgM) detection
EYE (OCULAR DISEASE)            
Adenoviruses Y ++       Conjunctival swab or scraping
Herpes simplex virus Y         Conjunctival swab or scraping
Varicella-zoster virus Y         Conjunctival swab or scraping
POSTTRANSPLANTATION SYNDROME            
Cytomegalovirus Y       ++ Blood (++++) shell vial and/or antigenemia; tissue (++++)
Epstein-Barr virus Y         Serology, tissue (PCR) (EBV)
Human herpesvirus-6 (HH6) Y         Serology, blood (PCR)
Herpes simplex Y         Tissue (+++) virus
BK virus Y       ++++  
Myocarditis, Pericarditis, and Pleurodynia            
Coxsackie B S, F +++   ++   Pericardial fluid (++++)
Hemorrhagic Fevers            
Ebola/Marburg viruses Y         Tissue, respiratory secretions, serum for antibody detection
Lassa fever virus   +++     + Serum/throat washes for viral detection
Serum for antibody detection
Hepatitis            
Hepatitis Y         Serology, blood (PCR)


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CSF, cerebrospinal fluid; EBV, Epstein-Barr virus; EIA, enzyme immunoassay; EM, electron microscopy; F, fall; PCR, polymerase chain reaction; HIV, human immunodeficiency virus; S, summer; SARS, severe acute respiratory syndrome; SP, spring; W, winter; Y, Year-round.


*Specimens indicated beside specific viruses should be obtained if that specific virus is suspected (++++, most appropriate; + least appropriate).


†Direct fluorescent antibody studies are available for herpes simplex virus and varicella-zoster virus.


Specimens for the detection of virus should be collected as early as possible after the onset of symptomatic disease. Virus may no longer be present as early as 2 days after the appearance of symptoms. However, other factors, such as the patient’s immune status or age, the type of virus, and the amount of systemic involvement, may play a role in the length of time viral shedding is evident, allowing effective laboratory detection. Certain viruses, such as West Nile virus, produce a brief, low viremia and undetectable levels at the onset of symptoms. Recommendations for collection of various specimens are summarized in this section.


In addition to the type of specimen and collection method, validated devices or containers can enhance the recovery and detection of the viral agent. Swab specimens should not contain chemicals or other compounds that may be toxic to cultured cells and therefore are unsuitable for viral specimen collection. Calcium alginate swabs interfere with PCR, the recovery of some enveloped viruses, and fluorescent-antibody tests and therefore should not be used.



Throat, Nasopharyngeal Swab, or Aspirate


In general, nasopharyngeal aspirates are superior to throat or nasopharyngeal swabs for recovering viruses; however, swabs are considerably more convenient. Throat swabs are acceptable for the recovery of enteroviruses, adenoviruses, and HSV, whereas nasopharyngeal swab or aspirate specimens are preferred for the detection of RSV and influenza and parainfluenza viruses. Rhinovirus detection requires a nasal specimen. Throat specimens are collected with a dry, sterile swab by passing the swab over the inflamed, vesiculated, or purulent areas on the posterior pharynx. The swab should not be touched to the tongue, buccal mucosa, teeth, or gums. Nasopharyngeal secretion specimens are collected by inserting a swab with a flexible shaft through the nostril into the nasopharynx or by washing and collecting the secretions by rinsing with a bulb syringe and 3 to 7 mL of buffered saline. The saline is squirted into the nose by squeezing the bulb and aspirated with a small tubing inserted into the other nostril when the bulb or suction is released.


All respiratory specimens are acceptable for culture of most viruses. However, respiratory and oral samples often are contaminated with bacteria. Contaminants may be removed by concentrating the sample through centrifugation. However, this process may also result in removal of virus-infected cells and reduce the recovery of viral agents from the sample.




Rectal Swabs and Stool Specimens


Stool and rectal swabs of fecal specimens are used to detect rotavirus, enteric adenoviruses (serotypes 40 and 41), and enteroviruses. Many agents of viral gastroenteritis do not grow in cell culture and require PCR or electron microscopy for detection (these are discussed later in the chapter). In general, stool specimens are preferable to rectal swabs and should be required for rotavirus and enteric adenovirus testing. Rectal swabs are acceptable for detecting enteroviruses in patients suspected of having an enteroviral disease, such as aseptic meningitis. The rectal swab is inserted 3 to 5 cm into the rectum and rotated against the mucosa to obtain feces. The swab should then be placed in appropriate transport media. A stool sample is preferred over a rectal swab because of the potential for decreased viral recovery from a small sample size. Five to 10 mL of freshly passed diarrheal stool or stool collected in a diaper from young infants is sufficient and preferred for rotavirus and enteric adenovirus detection.




Skin and Mucous Membrane Lesions


Enteroviruses, HSV, VZV, and in rare cases CMV or pox viruses can be detected in vesicular lesions of the skin and mucous membranes. Once the vesicle has ulcerated or crusted, detection of the virus is difficult.


Collection of specimens from cutaneous vesicles for detection of HSV or VZV may require a Tzanck smear if PCR testing is not available. Tzanck smears are prepared by carefully unroofing the vesicle. The procedure is as follows: If a tuberculin syringe is used, a small “drop” of vesicle fluid should be aspirated first and held for further use in the event a viral or bacterial culture is needed. The needle is flushed with a viral transport medium, and phosphate buffered saline or viral support media (EMEM) is added to the viral transport tube. With the roof of the vesicle folded back, excess fluid is carefully removed by dabbing with sterile gauze. A clean glass microscope slide is pressed against the base of the ulcer. The slide is lifted, moved slightly, and pressed again. Cells from the base of the ulcer stick to the slide, making an “impression smear” of infected and uninfected cells. Additional smears can be made from other vesicles. The slides are sent to the laboratory for fixation and staining. As an alternative, vesicle fluid and cells scraped from the base of an unroofed vesicle can be added to 2 to 3 mL of viral transport medium. Smears can be prepared in the laboratory with cytocentrifugation of fluid medium, or PCR can be performed from the specimen in the viral transport medium.









Specimen Transport and Storage


Ideally all specimens collected for detection of virus should be processed immediately. Although inoculation of specimens into cell culture at the bedside has been recommended in the past, potential biohazards, sophisticated processing steps, and necessary quality controls make this impractical. Specimens for viral isolation should not be allowed to sit at room or higher temperature. Specimens should be kept cool (4°C) and immediately transported to the laboratory. If a delay in transport is unavoidable, the specimen should be refrigerated, not frozen, until processed. Every attempt should be made to process the specimen within 12 to 24 hours of collection. Under unusual circumstances, specimens may need to be held for several days before processing. For storage up to 5 days, specimens are held at 4°C. Storage for 6 days or longer should be at –20° or preferably–70°C. Specimens for freezing should first be diluted or emulsified in viral transport medium. Significant loss of viral infectivity may occur during prolonged storage, regardless of conditions, especially for the more labile enveloped viruses.


If a commercial kit is used for viral identification (e.g., nucleic acid testing), the specimens should be transported and stored according to the manufacturer’s instructions. Specimens for processing using commercial reagents that are not approved by the U.S. Food and Drug Administration (FDA), such as analyte-specific reagents, or assays that have been created and validated in the user’s laboratory (laboratory-developed tests [LDTs]), are transported and stored at refrigeration temperatures. Freezing at –70°C is recommended if processing is delayed for longer than 2 to 3 days.


Many types of specimens for the detection of virus can be collected with a swab. Most types of synthetic swab material, such as rayon and Dacron, are acceptable. Swabs with cotton tips and wooden shafts are not recommended. Once collected, specimens on swabs should be emulsified in viral transport medium before transport to the laboratory, especially if transport will occur at room temperature and require longer than 1 hour. Calcium alginate is not acceptable for the detection of HSV, because it may inactivate the virus. Also, as previously mentioned, it is not recommended for PCR amplification of any respiratory viruses.


Commercially prepared transport media are useful for maintaining viral stability. They are used to transport small volumes of fluid specimens, small tissues and scrapings, and swab specimens, especially when contamination with microbial flora is expected. Transport media contain protein (e.g., serum, albumin, or gelatin) to stabilize the viral agents and antimicrobials to prevent overgrowth of bacteria and fungi. Penicillin (500 units/mL) and streptomycin (500 to 1000 mcg/mL) have been used traditionally; however, a more potent mixture is composed of vancomycin (20 mcg/mL), gentamicin (50 mcg/mL), and amphotericin (10 mcg/mL). If serum is added as the protein source, fetal calf serum is recommended, because it is less likely to contain inhibitors, such as antibodies. Examples of successful transport media include Stuart’s medium, Amie’s medium, Leibovitz-Emory medium, Hanks balanced salt solution (HBSS), Eagle’s tissue culture medium, and the commercially available M4, M5, and universal transport media. Respiratory and rectal and stool specimens can be maintained in modified Stuart’s medium, modified HBSS, or Leibovitz-Emory medium containing antimicrobials.


Blood for viral culture, transported in a sterile tube containing anticoagulant, must be kept at refrigeration temperature (4°C) until processed. Blood for viral serology testing should be transported to the laboratory in the sterile tube in which it was collected. Serum should be separated from the clot as soon as possible. Serum can be stored for hours or days at 4°C or for weeks or months at –20°C or below before testing. Testing for virus-specific IgM should be completed before freezing whenever possible, because IgM may form insoluble aggregates upon thawing, producing a false-negative result.



Specimen Processing


General Principles


Specimens for viral culture should be processed immediately upon receipt in the laboratory. This may be accomplished by combining bacteriology and virology processing responsibilities. Although the threat of cell culture contamination in the past dictated separation of virology procedures, the addition of broad-spectrum antimicrobials to cell cultures has significantly reduced the possibility of cross-contamination with bacteria and fungi. In most laboratories, processing with other microbiology specimens allows viral cultures to be processed 7 days a week. If delays must occur, specimens should be stored in a viral transport medium at 4°C as described previously. Delay in the processing of fluid specimens requires dilution in a transport medium (1 : 2 to 1 : 5) before storage.


In addition to patient identification and demographics, each specimen for virus isolation should be accompanied by a requisition that provides (1) the source of the specimen; (2) the clinical history or viruses suspected; and (3) the date and time of specimen collection. If this information is not available, a call for additional details should be made to the requesting physician or to the person caring for the patient.


Viral specimens should be processed in a BSC whenever possible (see Figure 65-7). This protects specimens from contamination by the processing technologist and protects those in the laboratory from infectious aerosols created when specimens are manipulated. Latex gloves and a laboratory coat should be worn during manipulation of all patient specimens. Vortexing, pipetting, and centrifugation can create dangerous aerosols. Vortexing should be done in a tightly capped tube behind a shield. After vortexing, the tube should be opened in a BSC. Pipetting should be performed behind a protective shield. Pipettes must be discarded into a disinfectant fluid so that the disinfectant reaches the inside of the pipette or into a leak-proof biosafety bag for autoclaving or incineration. When patient cell cultures are manipulated, such as during inoculation or feeding (exchange of cell culture medium), only one patient sample or series of cell culture tubes should be open at one time. Aerosols and microsplashes contribute to cross-contamination of cultures, especially during viral respiratory season when a high percentage of specimens are positive for influenza virus, RSV, and other viruses.


Processing virology specimens is not complicated (Table 65-4). In general, any primary specimen or swab specimen that may be contaminated with bacteria or fungi should be added to a viral transport medium. Normally sterile fluid specimens can be inoculated directly to cell culture. The viral transport medium or fluid specimens not in a transport medium should be vortexed immediately before inoculation to break up virus-containing cells and resuspend the inoculum. Adding sterile glass beads to the transport medium helps break up cell clumps and release virus from cell aggregates. This may not be necessary, because some commercially available mediums contain beads. Grossly contaminated or potentially toxic specimens, such as minced or ground tissue, can be centrifuged (1000× g for 15 minutes) and the virus-containing supernatant used as the inoculum. Each viral cell culture tube is inoculated with 200 to 400 µL of specimen. If insufficient specimen is available, the specimen obtained is diluted with a viral transport medium to increase the volume. Excess specimen can be stored at –70°C in the event the initial culture is contaminated. A set of uninoculated cultures should be maintained simultaneously for continual monitoring of sterility and contamination throughout the process.



TABLE 65-4


Laboratory Processing of Viral Specimens


















































Source Specimen Processing* Cells for Detection of Common Viruses
Blood Anticoagulated blood Separate leukocytes (see Procedure 65-1) PMK, HDF, HEp-2
Cerebrospinal fluid (CSF) 1 mL CSF Inoculate directly PMK, HDF, HEp-2
Stool or rectal swab Pea-sized aliquot of feces Place in 2 mL of viral transport medium vortex. Centrifuge at 1000× g for 15 min and use supernatant fluid for inoculum PMK, HDF, HEp-2
Genital, skin Vesicle fluid or scraping Emulsify in viral transport medium HDF
Miscellaneous Swab, fluids Emulsify in viral transport medium
Fluid, inoculate directly		 PMK, HDF, HEp-2
Respiratory tract Nasopharyngeal secretions, throat swab, respiratory tract washings, sputum Dilute with viral transport medium PMK, HDF, HEp-2
Tissue Tissue in sterile container Mince with sterile scalpel and scissors and gently grind. Prepare 20% suspension in viral transport medium. Centrifuge at 1000× g for 15 min and use supernatant fluid for inoculum. PMK, HDF, HEp-2
Urine Midstream specimen Clear: Inoculate directly.
Turbid: Centrifuge at 1000× g for 15 min and use supernatant fluid for inocula.		 HDF, HEp-2 (if adenovirus suspected)

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Aug 25, 2016 | Posted by in MICROBIOLOGY | Comments Off on Overview of the Methods and Strategies in Virology

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