Monkeypox virus (MPXV) genotyping and phenotyping

Purpose of the test

Clinical use

Detect antiviral resistance in patients with monkeypox virus (MPXV) infections.

Clinical background

Monkeypox virus (MPXV) is a member of the Orthopoxvirus genus, family Poxviridae, which encompasses large, enveloped, double-stranded DNA viruses.

The MPXV clade IIb was associated with the global 2022 outbreak while the 2024 outbreak was linked to the MPXV clade I strains. Mpox is typically a mild disease, however clade I virus strains are responsible for a severe form of mpox in humans. Furthermore, mpox disease can be fatal for people with a weak immune system with disseminated mpox including secondary infections, sepsis, and encephalitis.

Patients with mild mpox typically have a self-limited disease and do not require treatment with antivirals.  However, in patients presenting severe mpox disease, tecovirimat, brincidofovir, and, to a less extent, cidofovir (antivirals developed against variola virus) can be used to treat severe cases of mpox. Tecovirimat is approved by the European Medicines Agency (EMA) to treat smallpox, mpox, and cowpox (three infections caused by viruses belonging to Orthopoxvirus genus), as well as to treat complications that may occur following smallpox vaccination.

Tecovirimat inhibits the viral envelope protein VP37, blocking viral maturation and release of extracellular enveloped viruses from infected cells. Tecovirimat is not widely used to treat mpox because of its low barrier to resistance. Brincidofovir, a lipid conjugate analogue of cidofovir orally bioavailable, was designed to avoid the drawbacks of its parental counterpart, i.e. poor oral bioavailability and nephrotoxicity. Brincidofovir is intracellularly converted to cidofovir, a phosphonate analogue that after two phosphorylations carried out by cellular enzymes inhibits  viral DNA replication by targeting viral DNA polymerases. Cidofovir, initially approved by the FDA for cytomegalovirus retinitis therapy, can be used off-label for the therapy of infections caused by different DNA viruses, including poxviruses.

Emergence of tecovirimat-resistance is a real concern in the clinic and requires careful monitoring. Tecovirimat resistance is linked to the emergence of F13L mutations and has been described in the clinic in severely immunocompromised patients with disseminated and progressive mpox despite prolonged tecovirimat treatment (>14 days). The mechanism of action and of resistance is similar for cidofovir and its oral prodrug brincidofovir, and was shown in vitro to be associated with the acquisition of specific mutations in poxvirus DNA polymerases. Therefore, no cross-resistance between (brin)cidofovir and tecovirimat exists.

Criteria for performing this test in the context of reference activities

Emergence of tecovirimat resistance in the setting of prolonged drug exposure in immunodeficient patients should be monitored to offer alternative treatment options. Because compartmentalization of viral MPXV mutants can occur, specimens from different body sites (such as various mucocutaneous lesions and blood) should be investigated for drug-resistance longitudinally to also allow evaluation of the viral dynamics.

Test details – MPXV genotyping

Includes:

1. Tecovirimat resistance: mutations in the MPXV OPG057 gene (Palmytilated EEV membrane protein) / homologous to vaccinia virus F13L).
2. (Brin)cidofovir resistance: mutations in the MPXV OPG071 DNA polymerase gene.

Test description – MPXV genotyping

1. Isolation of DNA from the sample.

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

MPXV gene	MPXV  encoded protein	Drug target of	Codons sequenced (partial or complete gene sequence)	Drug(s) for which resistance is predicted
OPG057 (homologous to vaccinia virus F13L gene)	Palmytilated EEV (extracellular enveloped virus) membrane protein	Tecovirimat	372 (complete sequence)	Tecovirimat
OPG071	DNA polymerase	Cidofovir	1006 (complete sequence)	Cidofovir and brincidofovir

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 reference MPXV 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 MPXV infection is due to viral drug-resistance.

Interpretation of the MPXV genotypic 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 MPXV 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 genotypic tests for MPXV drug-resistance

• Test results fail to detect mutations that are present in 20-30% of the viral population.
• Because genotypic artifacts may occur, in particular in mixed mutant populations from specimens with low viral load, retesting in a new sample is advised.
• Some variants in the MPXV genes have not yet been phenotypically characterized and the impact of these novel changes cannot be predicted. Therefore, the combination of genotypic and phenotypic testing is necessary to diagnose  whether the novel changes in the viral genes are associated with drug-resistance or with a natural polymorphism.

Test details – MPXV phenotyping

1. Growth of viral sample in human embryonic lung (HEL) fibroblasts until 100% cytopathic effect (CPE) is reached.
2. Preparation of the viral stock.
3. Antiviral assay performed in HEL cells using at least two different viral inoculums and a reference MPXV strain.
4. Determination of the EC₅₀ (50% effective concentration) or drug concentration required to inhibit viral CPE by 50% for reference anti-poxvirus agents, i.e. cidofovir, brincidofovir, and tecovirimat.
5. Comparison of EC₅₀ values between the reference MPXV strain and patient sample(s) and determination of the fold-resistance (Ratio EC₅₀ patient sample/EC₅₀ reference strain).

Interpretation of the MPXV phenotypic results

Results include EC₅₀ values and fold-resistance for different classes of anti-MPXV drugs, DNA polymerase inhibitors (cidofovir and brincidofovir), and inhibitors of viral maturation (tecovirimat).

Limitations of phenotypic tests for MPXV drug-resistance

• Phenotypic tests cannot be carried out for cerebrospinal fluid, ocular fluid, blood, or e-swab specimens because of failure to grow the virus in cell culture from these sample types. 
• For successful viral isolation, the sample should be collected immediately in a virus transport medium, stored refrigerated at 2-8°C, and transported refrigerated within 48 hours of sample collection.
• Antiviral drug-resistance mutations can confer a significant reduction in viral fitness. Therefore, in cases of mixed mutant populations, viruses with reduced fitness will be overgrown in cell culture by the virus with a superior replication capacity. Isolation of viral clones from the original sample is then necessary to evaluate the phenotype of each mutant virus.

Instructions for samples and transport

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

 Unacceptable requests 

• Insufficient sample.
• Incorrect transport or storage of the sample.
• Test request form not 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 for genotypic testing.

                            10-20 working days for phenotypic testing.

Reporting of test results

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