Human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) genotyping

Purpose of the test

Clinical use

Detect antiviral resistance in patients with Human herpesvirus 6A (HHV-6A) and 6B (HHV-6B) infection.

Clinical background

Human herpesviruses 6A and 6B (HHV-6A and HHV-6B) are genetically related herpesviruses belonging the Roseolovirus genus of the beta-herpesvirinae subfamily. Despite a high genetic sequence identity (~90%), HHV-6A and HHV-6B are recognized as two distinct species. Sequence divergence (e.g., >30%) in key coding regions and important differences in physiological and biochemical properties (such as use of different receptors for viral entry) underscore the conclusion that HHV-6A and HHV-6B are distinct viruses of the β-herpesvirinae. These viruses are ubiquitously distributed worldwide and are responsible for lifelong latent infections, usually asymptomatic in the adult population. Symptomatic disease is often seen in infants or immunocompromised patients who reactivate the virus from latency. HHV-6B primary infection is responsible for a benign and common childhood disease, exanthema subitum, also known as roseola infantum or sixth disease.

Unique to HHV-6 is the covalent integration of its genomic DNA into the telomers of the cell chromosomes [i.e. chromosomally integrated HHV-6, ciHHV-6) in every cell in the body and transmission occurs through Mendelian inheritance. Although infrequent (in about 1% of the population), this is a confounding factor for diagnosis of active viral infection and response to antiviral therapy. 

HHV-6A and HHV-6B are opportunistic pathogens in the immunocompromised host and reactivation or reinfection can lead to serious disease, including encephalitis, colitis, pneumonitis, hepatitis, and bone marrow suppression, interstitial pneumonitis, myelitis, and rash.

Ganciclovir, foscarnet, and cidofovir are known as inhibitors of HHV-6 replication in vitro but to date there is no drug formally approved to manage serious disease in the immunocompromised host. Prophylactic or pre-emptive therapy is not recommended to prevent HHV-6 reactivation or encephalitis post-transplantation. Ganciclovir, foscarnet, and eventually cidofovir can be used to manage HHV-6 severe disease in immunocompromised individuals, in particular those suffering from HHV-6 encephalitis. These three antiviral agents exhibit the same potency against both HHV-6A and HHV-6B in vitro, being the mechanisms of antiviral activity similar for both HCMV and HHV-6.

Criteria for performing this test in the context of reference activities

In vitro studies support the potential for development of HHV-6A or HHV-6B resistance to the above mentioned antiviral agents, though very few studies found drug-resistant isolates following lengthy exposure in the clinical setting. Although (val)ganciclovir treatment for CMV disease did not lead to emergence of drug-resistant HHV-6 mutants in solid organ transplant patients following a large prospective study, it should be considered that drug-resistance HHV-6 can emerge following prolonged prophylaxis or treatment of CMV disease with ganciclovir. Patients presenting HHV-6 encephalitis not responding to antiviral therapy should be rapidly tested for emergence of drug-resistance.

Test details

Includes:

1. (Val)ganciclovir resistance: mutations in the HHV-6A or HHV-6B U69 protein kinase and U38 DNA polymerase genes.
2. Cidofovir resistance: mutations in the HHV-6A or HHV-6B U38 DNA polymerase gene.
3. Foscarnet resistance: mutations in the HHV-6A or HHV-6B U38 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.

HHV-6A/6B gene	HHV-6A/6B encoded protein	Function / drug target	Codons sequenced (partial or complete gene sequence)	Drug(s) for which resistance is predicted
U69	U69 protein kinase	Involved in ganciclovir activation	1-563 (complete sequence)	(Val)ganciclovir
U38	DNA polymerase	Target of ganciclovir, foscarnet, cidofovir	250-800 ( partial 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 HHV-6A strain GS or HHV-6B strain Z29 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 HHV-6A/6B 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 HHV-6A/6B 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.