Herpes simplex virus mistyping due to inter-species recombination

From the University of Washington, the Vaccine and Infectious Disease Division

Herpes simplex virus (HSV) types 1 and 2 are globally prevalent viruses that establish chronic infections and cause painful ulcers during reactivation periods. Although HSV-1 primarily causes orofacial ulcers and HSV-2 causes mostly genital lesions, both HSV-1 and HSV-2 are capable of infecting either tissue. Clinical guidelines from the CDC and IDSA recommend determining the species of HSV responsible for clinical symptoms as it can guide clinical decision-making and provide prognostic information.

HSV speciation, known as typing, is performed with diagnostic assays that distinguish between HSV types 1 and 2 by species-specific probes that recognize viral DNA or mRNA sequences. Homologous recombination, a natural exchange of DNA between viruses with high sequence homology, can occur between HSV-1 and HSV-2 and this can affect the results of HSV typing. However, the extent of interspecies HSV recombination and its impact on genomic variation and sequence-based HSV typing is unknown.

HSV-1/HSV-2 typing at the University of Washington Clinical Virology Laboratory is performed by PCR. This reaction amplifies a portion of the U27 gene, which encodes an important HSV glycoprotein that varies in sequence between HSV-1 and HSV-2. Dr. Amanda Casto, along with colleagues from the University of Washington and the Fred Hutch Vaccine and Infectious Disease Division, encountered for the first time a case of mistyping due to a recombination event between HSV-1 and HSV-2. This observation, published in Viruses,  “serve[s] as an illustration of how genetic variation within viral species can impact the results of clinical tests,” Dr. Casto said.

The sample, from a participant in an HSV clinical trial, initially was typed as HSV-1. Follow-up testing for antiviral resistance was attempted with HSV-1-specific primers, which then failed to amplify the sequence of interest. However, subsequent use of HSV-2-specific primers successfully amplified the target, suggesting a peculiarity in this clinical sample’s sequence. To clarify the sample’s identity, the authors next performed whole-genome sequencing. This demonstrated that the sample was originally mistyped and was HSV-2. However, this HSV-2 genome carried a small segment of between 114 and 177 basepairs of HSV-1 DNA within the UL27 gene. This segment contained the target sequence for the HSV typing assay used by the UW Clinical Virology Laboratory.

Region in the HSV-2 genome that is targeted by the HSV-1/HSV-2 typing assay and that contains HSV-1 recombinant DNA in the CT_Sample9 HSV-2 genome.
Region in the HSV-2 genome that is targeted by the HSV-1/HSV-2 typing assay and that contains HSV-1 recombinant DNA in the CT_Sample9 HSV-2 genome. Figure from publication, provided by Dr. Casto.

Dr. Casto explained that “it has recently been found that HSV-1 x HSV-2 recombination occurs in human hosts and that most HSV-2 genomes carry recombinant HSV-1 DNA.” This work found, for the first time, a recombination event in HSV gene U27, which encodes surface glycoprotein B. This protein is important for virus entry into the cell and is a target for diagnostics and vaccine candidates. Dr. Casto explained that “this paper raises questions about how frequently viral genomic variation interferes with the results of clinical testing for HSV and for other clinically relevant viruses.” These findings suggest that HSV mistyping may be occurring at an unknown rate. Going forward, Dr. Casto said that she plans to “continue to collect HSV samples from diverse human populations to try to better define the genomic variation present within the HSV-1 and HSV-2 species. This work will provide insights into how robust current testing methodologies are to genomic variation and may inform the development of new methodologies that are less prone to error as a result of this variation.”

Dr. Casto is supported by the Immunocompromised Host T32 training grant to VIDD from the National Institute of Allergy and Infectious Diseases.

Fred Hutch/UW Cancer Consortium members Keith Jerome and Anna Wald contributed to this research.

Casto AM, Huang MW, Xie H, Jerome KR, Wald A, Johnston CM, Greninger AL. 2020. Herpes Simplex Virus Mistyping due to HSV-1 × HSV-2 Interspecies Recombination in Viral Gene Encoding Glycoprotein B. Viruses. 2020 Aug 6;12(8):E860. doi: 10.3390/v12080860.