Genotyping Epstein-Barr virus (EBV)

Purpose of the test

Clinical use

Detect antiviral resistance in patients with Epstein-Barr virus (EBV) infection.

Clinical background

Epstein-Barr Virus (EBV) or human herpesvirus 4 (HHV-4) is a member of the gamma 1 or lymphocryptovirus genus of the gammaherpesvirinae subfamily. EBV is a highly successful pathogen and infects more than 90% of the world’s population. Primary infection is often asymptomatic and occurs during childhood and the virus is intermittently shed in saliva from healthy individuals. When primary infection occurs during adolescence or early adulthood, it can cause infectious mononucleosis. The virus establishes latency in memory B cells and in healthy people is asymptomatic without causing any disease. However, in heavily immunosuppressed individuals, EBV latency can be associated with the development of various lymphoproliferative disorders and epithelial/endothelial malignancies. 

There are no approved treatments for EBV-related disease, though several antiviral drugs are known to inhibit replication of EBV in vitro, including acyclovir, ganciclovir, foscarnet, and cidofovir, all of which have the EBV DNA polymerase as target. Despite their potency in vitro, these drugs have limited use in vivo either for treatment of acute primary EBV infection or EBV-associated malignancies.

Criteria for performing this test in the context of reference activities

In vitro studies using surrogate models of EBV support the potential for development of gammaherpesvirus drug-resistance to the above mentioned DNA polymerase inhibitors. Therefore, EBV drug-resistance could emerge following prolonged prophylaxis or treatment of other herpesvirus disease, such as those caused by CMV, HSV1/2, VZV, and HHV-6A/B with (val)acyclovir, (val)ganciclovir, foscarnet, or cidofovir.

Test details

Includes:

1. (Val)ganciclovir and (val)acyclovir resistance: mutations in the EBV BGLF4 protein kinase gene.
2. Cidofovir resistance: mutations in the EBV BALF5 DNA polymerase gene.
3. Foscarnet resistance: mutations in the EBV BALF5 DNA polymerase gene.  

Test description

1. Isolation of DNA from the sample.

2. Amplification of the viral genes involved in drug-resistance by PCR according to the therapy administered to the patient.

EBV gene	EBV encoded protein	Function / drug target	Codons sequenced (partial or complete gene sequence)	Drug(s) for which resistance is predicted
BGLF4	protein kinase	Involved in ganciclovir activation	1-429 (complete sequence)	(Val)ganciclovir
BALF5	DNA polymerase	Target of ganciclovir, foscarnet, cidofovir	1-1015 (complete sequence)	(Val)ganciclovir, foscarnet, cidofovir

3. Direct sequencing of the amplicons by Sanger dideoxy sequencing, which has a limit of detection of viral mutant subpopulations of 20%-30%.

4. Sequence alignment (derived sequences of the patient sample are aligned with the EBV strain Mutu reference sequences).

5. Detected mutations are compared to a database of known mutations associated with drug-resistance or natural occurring polymorphisms (inter-strain variability) to determine whether clinical resistance EBV infection is due to viral drug-resistance.

Interpretation of the results

Results include a list of the detected mutations and their association with  resistance to each specified drug, inter-strain variability, or unpredicted significance (novel changes). In function of the viral genotype, alternative therapeutic options are suggested.

A result of unavailable or incomplete genotyping indicates that not all viral amplicons could be amplified and sequenced. This can be due to insufficient viral load, quality of the sample (storage and transportation conditions, age of the sample), and/or presence of polymerase chain reaction inhibitors.

Limitations of the genotypic tests for EBV drug-resistance

• Test results fail to detect mutations that are present in 20-30% of the viral population.
• Tests are most reliable if the viral load in the specimen is at least 3 log₁₀ IU/ml (1,000 IU/ml). Results for specimens with lower viral loads should be interpreted with caution and should be confirmed with in a new sample.
• Because genotypic artifacts may occur, in particular in mixed mutant populations from specimens with low viral load, retesting in a new sample is advised.

Instructions for samples and transport

https://rega.kuleuven.be/regavir/shipping

 Unacceptable requests 

• Inappropriate or insufficient sample.
• Improper transport or storage of the sample.
• Test request form not fully completed without specifying the virus for which drug-resistance needs to be investigated.

Turnaround time (and frequency of analysis)

Frequency of analysis: every working day, during working hours.

Response time: 3-5 working days.

Reporting of test results

Results will be sent via e-mail according to the requesting laboratory or physician’s wishes.