Laboratory diagnosis of viral infections
Indications for Laboratory Diagnosis of Viral Infections
- Diagnosis of diseases caused by viruses for which antiviral chemotherapy is already available (herpesviruses, human immunodeficiency virus (HIV).
- For proper management of the patient Example:
- Rubella, rabies diagnosis.
- Rabies in the animal and postexposure immunization of the patient prevents the development of rabies.
- Primary genital herpes.
- Baby born to an HBsAg positive mother.
- Screening of blood donors: For the prevention of some diseases (such as screening for HBV and HIV in blood donors).
- Early detection of dangerous epidemics: Such as yellow fever, encephalitis, influenza, poliomyelitis, etc.
- To discover new viruses: HIV was discovered in 1983.
The appropriate specimens should be collected from patients, preserved and transported to the laboratory in the proper manner along with pertinent clinical and epidemiological information.
Methods for Laboratory Diagnosis of Viral Infections
- Direct detection of virus
- Virus isolation and growth
- Detection of viral proteins
- Detection of viral genetic material
Direct Detection of Virus
- Electron Microscopy (EM): Clinical applications of electron microscopy include detection of rotavirus and hepatitis A virus in fecal specimens, poxviruses in vesicle fluid and herpes virus in brain biopsy tissue.
- Immunoelectron Microscopy: The addition of virus-specific antibody to a sample can cause viral particles to clump, thereby facilitating the detection and simultaneous identification of the virus (immunoelectron microscopy). This method is useful for the detection of enteric viruses, such as rotavirus.
- Fluorescence Microscopy: Direct or indirect fluorescent antibody technique can be used to detect virions or viral antigens in frozen tissue sections, acetone-fixed cell smears, cells from virus infected cultures or vesicles fluid. Fluorescence microscopy of brain biopsy can be used for the verification of rabies in the brain of animals suspected to be rabid. This method is also useful for the rapid diagnosis of respiratory infections caused by paramyxoviruses, orthomyxoviruses, adenoviruses and herpes viruses.
- Light Microscopy: Demonstration of the inclusion body is a routine diagnostic method. Rabies may be detected through the finding of Negri bodies (rabies virus inclusions) in brain cells of animals.
For virus isolation it is imperative that the specimen be collected properly and transported with least delay to the laboratory. The reasons are that many viruses are labile and that the samples are susceptible to bacterial and fungal overgrowth. Viruses are best transported and stored on ice and in special media.
In general, the methods used for isolation consist of inoculation into animals, eggs or tissue culture, after the specimen is processed to remove bacterial contaminants. The isolates are identified by neutralization or other suitable serological procedures. Many viruses (for example adenoviruses, enteroviruses) are frequently found in normal individuals. The results of isolation should always be interpreted in the light of the clinical data.
Detection of Viral Proteins
The viral proteins can be assayed by the following methods:
- Protein patterns (by electrophoresis)—HSV.
- Enzyme activities (e.g. reverse transcriptase)—Retrovirus.
- Hemagglutination and hemadsorption—Influenza virus.
- Antigen detection: Viral antigens on the cell surface or within the cell can be detected by immunofluorescence and enzyme immunoassay (EIA).
- Virus or antigen released from infected cells can be detected by enzymelinked immunosorbent assay (ELISA), radioimmunoassay (RIA), and latex agglutination (LA).
Detection of Viral Genetic Material
- The electrophoretic patterns of RNA (influenza, reovirus) or restriction endonuclease fragment lengths from DNA viral genomes are like genetic fingerprints for these viruses.
- Different strains of HSV-l and HSV-2 can be distinguished in this way by restriction fragment length polymorphism.
- DNA probes:
- DNA probes with sequences complementary to specific regions of a viral genome can be used, like antibodies, as sensitive and specific tools for detecting a virus.
- Enzyme-labelled or radiolabeled nucleic acid (DNA or RNA) sequences complementary to unique regions in nucleic acid sequences of most viruses are now manufactured commercially. These labeled complementary sequences are known as nucleic acid probes.
- Two strands of the target DNA molecule in the clinical specimens are first separated, then, allowed to hybridize with a labeled single-stranded DNA or RNA probe present in excess.
- Depending on the type of label attached to the probe, hybridized labeled probe can be detected by radiography, gamma-counting or a simple colorimetric evaluation (dot-blot hybridization).
- The DNA probes are detected with autoradiography or with fluorescent or EIA-like methods. By use of nucleic acid probes cytomegalovirus, papillomavirus and Epstein-Barr virus have been identified.
- Southern, Northern andd Dot blot analysis:
- Viral genomes can also be detected in clinical samples with the use of dot blot or Southern blot analysis.
- Electrophoretically separated viral RNA (Northern blot-RNA: DNA probe hybridization) blotted onto a nitrocellulose filter can be detected in a similar manner.
- Polymerase chain reaction:
- The polymerase chain reaction (PCR) for DNA, reverse transcriptase polymerase chain reaction(RTPCR) for RNA, and branched-chain DNA (DNA, RNA) assays are becoming very important for viral detection.
- Use of the appropriate primers for PCR can promote a million-fold amplification of a target sequence in a few hours.
- This technique is especially useful for detecting latent and integrated sequences of viruses, such as retroviruses, herpes viruses, papillomaviruses, and other papovaviruses as well as the sequences of viruses present in low concentrations.
- Reverse transcriptase polymerase chain reaction (RT-PCR) for RNA: This approach was very useful for identifying and distinguishing the Hantaviruses.
- Branched-chain DNA assays: Quantitate viral DNA or RNA much like ELISA.
- The demonstration of a rise in titer of antibodies to a virus during the course of a disease is strong evidence that it is the etiological agent.
- For this, it is essential to examine paired sera, the ‘acute’ sample collected early in the course of the disease and the ‘convalescent’ sample collected 10–14 days later.
- Examination of a single sample of serum for antibodies may not be meaningful except when IgM specific tests are done which is present during the first 2 or 3 weeks of a primary infection, generally indicates a recent primary infection.
- The serological techniques employed would depend on the virus but those in general uses are neutralization, complement fixation, ELISA, hemagglutination inhibition tests, indirect fluorescent antibody test, latex agglutination test.
Reference and Sources
- Southern Blotting
- Water Purification and Sanitary Analysis of Water Sample
- Virology: Introduction, Virus classification and Viral diseases
- Fundamental Principle of Clinical Specimen Collection
- Biosafety Cabinet: Introduction, Development and Safety guidance
- Classification of viruses on the basis of genome