Diagnosis and Prevention of Viral Infection

Section 1 Diagnosis of Viral Infection

Ⅰ.Specimen Collection and Shipment

A. Samples for Virus Isolation

1. Timeliness of Specimen Collection

Specimens for the detection of virus should be collected as early as possible following the onset of disease. Virus may no longer be present as early as 2 days after the appearance of symptoms.

2. Process and Stock Samples

Specimens for viral isolation should not be allowed to sit at room or higher temperature. Specimens should be placed in ice and transported to the laboratory at once. If there is a delay in bringing it to the laboratory, samples were stored at -70 °C or colder. The tissue or organ should be kept with medium containing 50% glycine.

3. Contamination must be avoided by using the disinfected material. antimicrobials may be needed to prevent overgrowth of bacteria and fungi.

B. Samples for Serology Detection

Acute and convalescent serum samples are needed for virus specific antibodies detection, Acute specimens should be collected as soon as possible after the appearance of symptoms. The convalescent specimen is collected a minimum of 2 to 3 weeks after the acute specimen.

Ⅱ.Virus Isolation

A. Methods of viral isolation

1. Animal Inoculation

Animal inoculation is one of the earliest ways of isolating a virus. The animals often used are mice, guinea pigs, rabbits, monkeys, and ferrets. Different virus can be inoculated into different animals, as well as different organs of the same animal.

2. Chick Embryo Inoculation

The chick embryo is very frequently employed in virology experiments. The egg has several cavities into which viruses may be inoculated and cultivated. The site and method of inoculation chosen depend on the purpose of the procedure. Not all viruses can grow in eggs.

3. Cell Culture

(1)Cell culture category

1) Primary cell culture

Primary cultures are derived directly from human or animal fetal tissue. primary cell culture is highly sensitive to virus infection, But, cannot divide indefinitely

2) Diploid cell culture

Diploid cell culture can be passaged for 50 times, and their chromosomes are kept diploid. Diploid cell culture is used for vaccine production and laboratory diagnosis.

3) Continuous Cell Culture

Continuous cultures generally are obtained from malignant tissues which can be propagated indefinitely These cells have lost the contact inhibition property.

(2) Characterization of Viral Intracellular Propagation

1) Cytopathogenic Effect (CPE)

Some viruses produce a characteristic cytopathic effect in cell culture.

For example, respiratory syncytial virus characteristically produces multinucleated giant cells, whereas adenoviruses produce grape-like clusters of large round cells.

2) Hemadsorption phenomenon

Influenza viruses and some paramyxoviruses may be detected within 24–48 hours if erythrocytes are added to infected cultures. Viruses maturing at the cell membrane produce a hemagglutinin that enables the erythrocytes to adsorb at the cell surface (hemadsorption).

3) Viral Interference

Some viruses (e.g., rubella virus) do not produce CPE but can be detected by their interference with the CPE of a second challenge virus.

4) Metabolize of cell

B. Virus Identification

(1) Morphology identification: high concentrated individual virus particles can be observed by electron microscopy.

(2) Serological identification

(3) Molecular techniques

C. Quantification of Viral Infection

1) 50% Tissue Culture Infectious Dose

TCID50 is defined as that dilution of virus which will cause CPE in 50% of a given batch of cell culture. This assay is statistical way of measuring virus populations.

2) Red cell agglutination test

Some viruses with hemagglutinin can bind to surface structures on red blood cells from different species.

3) Plaque-forming Unit (PFU)

Monolayers are inoculated with suitable dilutions of virus and overlaid with medium containing agar or carboxymethylcellulose. The infected cells produce a small area of infection, or plaque. Under controlled conditions, a single plaque can arise from a single infectious virus particle, termed a plaque-forming unit (PFU).

Ⅲ. Serological diagnosis of Viral infection

A. Neutralization Assays

Antibody that inhibits the infectivity of a virus by blocking its host cell receptor site is called a neutralizing antibody. virus can be inhibited by its neutralizing antibody and loss the infectivity.

B. Hemagglutination Inhibition Assays

Viruses with hemagglutinin can agglutinate red blood cells, this phenomenon is called hemagglutination. If antibodies against the hemagglutinins are present, hemagglutination will be prevented. During the hemagglutination inhibition test, serial dilutions of serum are mixed with a known amount of virus. After incubation, RBCs are added, hemagglutination is inhibited, a pellet of RBCs forms at the bottom of the tube.

C. Complement Fixation Assays

Complement fixation test (CF)is one of the classic methods for demonstrating the presence of antibody in a patient's serum. This assay can be used to look for the presence of specific antibody or antigen in a patient's serum. This test is still probably the most common method for diagnosing infection caused respiratory viruses, and arboviruses.

D. Gel immunodiffusion test

Ⅳ. Rapid diagnosis of viral infection

1. Virus Morphology Observation

(1) Electron Microscopy

EM is now mainly used for the diagnosis of viral gastroenteritis by detecting viruses in faeces e.g. rotavirus, adenovirus, astrovirus, calicivirus and Norwalk-like viruses. The sensitivity and specificity of EM may be enhanced by immune electron microscopy, whereby virus specific antibody is used to agglutinate virus particles together and thus making them easier to recognize, or to capture virus particles onto the EM grid.

(2) Optical Microscopy

Optical microscopy is a kind of cytologic or histologic examination through observing viral inclusions and morphologic changes of cells or tissue, respectively. Replicating virus often produce histological changes in infected cells. Viral inclusion bodies are basically collections of replicating virus particles either in the nucleus or cytoplasm. Although not sensitive or specific, histology nevertheless serves as a useful adjunct in the diagnosis of certain viral infections.

2. Detection of viral components

(1) Detection of viral proteins

The detection of viral antigens is widely used in diagnostic virology. The most common of antigen detections are fluoroimmunoassay (FIA) and enzyme immunoassay (EIA) in which antigen is detected on the surface of the organism or in cells present in the infected secretion

(2) Virus Nucleic acid detection

1) Nucleic acid amplification techniques

Nucleic acid amplification techniques are rapidly becoming the standards for diagnostic virology, supplanting the traditional virus culture and antigen detection techniques. The methods include the polymerase chain reaction(PCR), reverse transcriptase-polymerase chain reaction(RT-PCR), the real time quantitative PCR, etc.

2) Nucleic acid hybridization technique

Nucleic acid hybridization methods includes: dot blot, in situ hybridization, DNA blotting and RNA blotting.

(3) DNA chip technique

The advantage of DNA chip technology is the simultaneous detection of a huge quantity of DNA sequences. DNA chip technology solved the problems of traditional nucleic acid hybridization methods and has very broad applications in viral diagnosis and epidemiological study.

3. Detection of virus early antibody

Detection of virus specific IgM antibody is used in early diagnosis of some virual infection, for example rubella virus, encephalitis B virus,hepatitis A virus , etc.

Section 2 Prevention of Viral Infection

Ⅰ.Artificial active immunization

The approach by inoculating antigen substances is known as artificial active immunization. Active immunity is generally more long lasting because the immunized host's own immune response is activated. Artificial active immunization are mainly divided into the following categories.

A. Inactivated vaccine

Inactive vaccine contains the whole virus particles often treated with formaldehyde so that the vaccines are not infectious and are therefore relatively stability and safety. But the inactive vaccine can not induce mucosal and cellular immunity. In order to maintain the immunity, the host have to be immunized the inactive vaccine several times to stimulate the immune system and get better immune response. In addition, they are usually expensive to prepare.

B. Live attenuated vaccines

Live attenuated vaccines are the mutant virus vaccine strains whose virulence has been artificially reduced by in vitro culture under adverse conditions. Attenuated virus vaccines have the advantage of acting like the natural infection with regard to their effect on immunity. They multiply in the host and tend to stimulate longer-lasting antibody production, to induce a good cell-mediated response, and to induce antibody production and resistance at the portal of entry. Potential drawbacks to these vaccines include: the danger of reversion to virulence and the possibility of causing extensive disease in immunocompromised individuals. Therefore those with immune deficiencies or immunosuppression are prohibited to immunize with the live attenuated vaccine.

C. Recombinant carrier vaccine

Use of recombinant DNA techniques to insert the gene coding for the protein of interest into the genome of an avirulent virus that can be administered as the vaccine (such as vaccinia virus).

D. Subunit vaccine

1. Synthetic Peptides: inoculation synthetic peptides that correspond to antigenic determinants on a viral protein, can avoid any possibility of reversion to virulence since no viral nucleic acid would be present, but although the immune response induced by synthetic peptides is considerably weaker than that induced by intact protein.

2. Genetic engineering vaccines: Use purified proteins produced by using cloned genes inoculate into host body, the subunit proteins can elicit immune responses to the target virus. Nowadays, HBsAg produced from yeast cells is widely vaccinated.

Ⅱ.Artificial passive immunization

Passive immunity is provided by the administration of artificial biological agents including the immune serum containing, interleukin-2, the transfer factor, interferon etc. Artificial passive immunization is usually used during communicable disease outbreak and epidemic.

A. immune globulin

"Normal" Immune globulin is a pooled product, prepared from the serum of normal blood donors. For the infectious illness such as hepatitis, measles, chickenpox, mumps and so on, at the acute infection phase, inoculate the immune globulins or gamma-globulins can help to control infection. Hyper-immune globulin may be prepared from the serum of selected individuals who have high titers of antibody to particular viruses.

B. Immunomodulating agents

Immunomodulating agents can enhance immune function. Commonly used Immunomodulating agents include the transfer factor, interleukin, CSF, interferon, etc.

Section 3 Treatment of Viral Infection

Compared with the number of drugs available to treat bacterial infections, the number of antiviral drugs is very small. The major reason for this difference is the difficulty in obtaining selective toxicity against viruses; their replication is intimately involved with the normal synthetic processes of the cell. Currently, antiviral therapy includes two combined strategies: agents are used to inhibit viral replication directly; meanwhile, using drugs to stimulate host immune responds so that the infected cells could be killed. .

Ⅰ. Antiviral Drugs

A. Nucleoside Analogues

The majority of available antiviral agents are nucleoside analogs. They inhibit nucleic acid replication by inhibition of polymerases for nucleic acid replication. In addition, some analogs can be incorporated into the nucleic acid and block further synthesis or alter its function.

1. Acyclovir

Acyclovir is a specific inhibitor of HSV-1, HSV-2 and VZV replication. Acyclovir is unique in that it must be phosphorylated by thymidine kinase to be active, and this phosphorylation occurs only in cells infected by certain herpesviruses. Acyclovir triphosphate inhibits viral replication by competing with guanosine triphosphate and inhibiting the function of the virally encoded DNA polymerase. The selectivity and minimal toxicity of acyclovir is aided by its 100-fold or greater affinity for viral DNA polymerase than for cellular DNA polymerase. A second mechanism of viral inhibition results from incorporation of acyclovir triphosphate into the growing viral DNA chain.

2.Adenine arabinaside

Adenine arabinaside（Ara-a）is used to cure herpetic keratitis and herpes encephalitis.

3. Azidothymidine

Azidothymidine (AZT), a nucleoside analog of thymidine, inhibits the reverse transcriptase of HIV. As with other nucleosides, AZT must be phosphorylated; host cell enzymes carry out the process. The basis for the relatively selective therapeutic effect of AZT is that HIV reverse transcriptase is more than 100 times more sensitive to AZT than is host cell DNA polymerase. Nonetheless, toxicity frequently occurs.

4. Dideoxyinnosine,DDC,3TC,dTC

They can inhibit the reverse transcriptase of HIV.

5. Lamivudine

Lamivudine (3TC), another reverse transcriptase inhibitor, have activity against HIV-1 and have been approved for clinical use. 3TC is also useful for treating hepatitis B.

6. Ribavirin

Ribavirin is another analog of the nucleoside guanosine. It reduces the glycoside needed by gene replication of host cell and virus, so it has the ability both to suppress the proliferation of viruses and inhibit cellular nucleus replication. With its serious adverse effect, ribavarin is used very rarely. However, it can be given to children diagnosed with a respiratory syncytial virus (RSV) infection.

B. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs)

NNRTIs do not need to be metabolized intracellularly to exert antiviral activity. They are active against HIV-1 but not against HIV-2 or other retroviruses. Famous NNRTIs include: nevirapine, delaviradine and pyridine.

C. Protease Inhibitors

Viral protease is essential for viral replication and maturation. So proteases inhibitors are designed based on the three-dimensional crystal structure of the protease enzyme, to block its activity so that to interrupt the viral life cycle. The major protease includes saquinavir, indinavir and ritonavir. Saquinavir was the first protease inhibitor to be approved for treatment of HIV infection. It is a peptidomimetic agent designed by computer modeling as a molecule that fits into the active site of the HIV protease enzyme. Such drugs inhibit the viral protease that is required at the late stage of the replicative cycle to cleave the viral gag and gag-pol polypeptide precursors to form the mature virion core and activate the reverse transcriptase that will be used in the next round of infection. Inhibition of the protease yields noninfectious virus particles.

D. Other type of antiviral agents

Targeting viral release can prevent the viral spread in the individual. The first drug to be useful in preventing symptoms of influenza A infection was amantadine (金刚烷胺), which exhibit two concentration-dependent mechanisms of action against influenza A viruses.

PFA inhibits viral DNA polymerase by blocking the pyrophosphate-binding site of the viral DNA polymerase and preventing cleavage of pyrophosphate from deoxyadenosine triphosphate. It can usually be used to treat patients with CMV,HSV,VZA,EBV infection.

Ⅱ. Interferon and Interferon inducer

Interferons (IFN) can inhibit viral replication. They are produced very quickly in response to viral infection or other inducers and are one of the body’s first responders in the defense against viral infection. Recombinant DNA techniques now allow relatively inexpensive large-scale production of interferons by bacteria and yeasts. Genetically engineered forms of Interferon-α have been approved for human use. The synthetic agent Interferon-α is active against many viral infections, including hepatitis A, B and C, HSV, papillomavirus, and rhinovirus.

Ⅲ. Chinese Herbs

Ⅳ.Gene therapy

Gene therapy is the introduction of genetic material into cells for therapeutic purposes. Genes with broader clinical application are also being utilized to make cells express immune activating agents locally at the disease site or to become susceptible to further drug treatment or to immune response recognition. Gene therapy includes antisense oligonucleotide,short interfering RNA, ribozyne.

Ⅴ. Therapeutic vaccine

A therapeutic vaccine is meant to treat existing infections with vaccine. The first therapeutic vaccine is the rabies vaccine which is developed by Pasteur.Nowadays, the hepatitis B vaccine is given to new born babies whose mothers infected with hepatitis B in order to avoid the transmission of virus from mother to baby. Recently, great effort is being devoted to developing therapeutic vaccines against tumors, AIDS, hepatitis B, tuberculosis etc.