Rhabdovirus - Lab diagnosis, Treatment, Vaccination

Lab diagnosis of Rhabdovirus

In most cases, human rabies is diagnosed in laboratory primarily on the basis of clinical symptoms and signs, and a corroborative history of or evidence of an animal bite, death of the animal, and incomplete or no vaccination following exposure:

Sample

• Saliva, CSF, Blood

• Skin biopsy from the nape of the neck (nuchal skin biopsy)

• brain biopsy (mostly on post-mortem cases)

• CSF (but virus not always detectable in ante-mortem)

Microscopy

Demonstration of Negri bodies in the brain and spinal cord tissue by microscopy is the characteristic histopathological feature of rabies.

Virus isolation

• Rabies tissue culture isolation (RTCIT) is used to isolate the virus

• Cell lines such as WI-38, BHK-21 (Baby Hamster Kidney), and CER (Chicken Embryo Related) are used for virus isolation.

• Since cytopathic effects are minimal, DDFA tests are used.

• Samples are inoculated onto a neuroblastoma mono later followed by 4-day incubation. The plates are fixed and then stained using fluorescein-labeled monoclonal Ab (mAbs) and the presence/absence of the Rhabdovirus was determined

Note: Negri bodies

• Negri bodies are round or oval inclusion bodied seen in the cytoplasm and sometimes in the processes of neurons of rabid animals after death

• Negri bodies are eosinophilic, sharply outlined bodies (2-10µm in diameter) found in the cytoplasm of certain nerves.

• The mouse Inoculation Test (MIT) involving inoculation of a sample into the mouse brain and observation of the animal for a period of 28 days or until clinical signs developed was the gold standard

  The brain tissues are examined for the presence of Negri bodies by microscopy or for viral Ag by the DFA test

  MIT has been superseded and replaced by tissue culture methods

Ag detection techniques

• Fluorescent Antibody test (FAT)

  It detects virus Ag in brain samples using fluorescently labeled anti-rabies antibodies, It is reliable and provides results within 2 hours

  Sensitivity depends upon the quality of the sample received

• ELISA can be used to detect virus nucleocapsids. It has the advantage of being able to detect viruses in autolyzed samples

Serological assays

• The presence of rabies virus-neutralizing Ab can be detected by using

  • Fluorescent Ab virus neutralization assay (FAVN)

  • rapid fluorescent focus inhibition test (RFFIT)

  • ELISA based test

  FAVN assesses Ab titers following pre-exposure vaccination. Due to the use of live viruses in FAVN and RFFIT, high containment facility is required

• Recently, a virus neutralization assay using a rabies virus-pseudotyped lentivirus has been developed.

  It uses the lentiviral expression of viral glycoprotein as a surrogate for live Lyssavirus

  Its advantage over other serological assays is such that;

  • no need for high containment facility although it requires tissue culture facilities

  • lower volumes of serum required

Molecular methods

• widely accepted and increasingly used:

  Not a prescribed diagnostic technique by OIE or WHO

  Used as a screening procedure for research purposes

• RT-PCR

• Taqman PCR (distinguishes between lyssavirus species)

• NASBA (nucleic acid sequence-based amplification)

  More sensitive than RT-PCR while assessing ante-mortem samples

• Microassay

  Utilizes oligonucleotide probes designed to bind to difficult lyssavirus species. It has limited sensitivity but can be used for CSF sample

Treatment of Rhabdovirus

• Rabies, a species of Rhabdovirus, cannot be treated after the appearance of symptoms

• It can be prevented if treatment is initiated within a day or two after biting

• The biting animal should be kept in strict isolation for 10 days. It remains healthy after 10 days, rabies is extremely unlikely

Vaccination of Rhabdovirus

• Currently, the available vaccine includes:

  • Human diploid cell vaccine (HDCV)

  • Purified chick embryo cell vaccine (PCEV)

  • Vero Cell vaccine

• These vaccines are safe and immunogenic but they do not protect from all lyssavirus species and are not available in many developing countries

Pre-exposure prophylaxis

• Normally administered to personnel who handle potentially infected animals, a person traveling to rabies endemic countries

• Three intramuscular doses of HDCV/PCEV were administered on days 0, 7, and 28 followed by an assessment of neutralizing antibodies. A booster dose is required every 1-3 years depending upon the type of vaccine used

• Response to these vaccines can persist for at least 2 years post-vaccination

Post-exposure prophylaxis

• It is started immediately after exposure to infection

• It involves cleaning of wound with soap and water and applying antimicrobials

• The biting animal should be observed and if suspected of rabid, HDVC or PCEVC alone or in combination with human rabies immunoglobulin (HRIG) is administered based on the risk of exposure

• Usually, 2 doses and four doses of vaccines are recommended for previously immunized and non-immunized individuals respectively