New Kaposi’s sarcoma-associated herpesvirus variant in men who have sex with men associated with severe pathologies

Abstract

Background

Kaposi sarcoma (KS)–associated herpesvirus (KSHV) subtype depends mostly on patient origin. The current study aimed to assess KSHV diversity in a population of men who have sex with men (MSM) living in France.

Methods

The study included 264 patients. In 65 MSM, including 57 human immunodeficiency virus (HIV)–infected men with KS, multicentric Castleman disease, or primary effusion lymphoma and 8 HIV-uninfected men receiving HIV preexposure prophylaxis (PrEP), we performed KSHV typing with K1 open reading frame Sanger and KSHV whole-genome sequencing. In 199 other patients, we performed real-time polymerase chain reaction screening for the new variant.

Results

We found that 51% of KSHV-strains were subtype C (85% C3), and 33% were subtype A. Four patients with severe KSHV disease (2 with visceral KS, 1 with multicentric Castleman disease, and 1 with primary effusion lymphoma) and 1 asymptomatic PrEP user had a new variant resembling the Ugandan subtype F, but with different K1 open reading frame and KSHV whole-genome sequences and a different epidemiological context (MSM vs African population). Its prevalence was 4.5% in Caucasian MSM, and it was absent in other epidemiological groups.

Conclusions

Subtype C predominated among MSM living in France. The new F variant was identified in Caucasian MSM and associated with severe KSHV disease, suggesting that subtype F could be split into F1 and F2 variants. Careful screening for this variant may be required in MSM, given the severe clinical presentation of associated diseases.

(See the Editorial Commentary by Bellocchi et al, on pages 1250–3.)

Kaposi (KS) sarcoma–associated herpesvirus (KSHV) is the recognized etiological agent of all epidemiological forms of (KS), including classic, endemic, posttransplantation, and epidemic (human immunodeficiency virus [HIV]– associated) forms. This virus is also involved in the development of 2 lymphoid malignancies: primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD), mostly in immunocompromised patients.

The K1 open reading frame (ORF-K1) encodes a transmembrane glycoprotein with an amino acid sequence varying by 20% to 44% between KSHV subtypes and by about 10% within subtypes (genotype variant) [1]. A molecular epidemiological analysis of ORF-K1 led to the identification of 7 KSHV subtypes (A, B, C, D, E, F, and Z), the worldwide distribution of which depends on patient origin. Subtypes A and C were found in Europe, North America, the Middle East, the Mediterranean, and Asia [2–4]; subtypes B and A5, predominantly in sub-Saharan Africa [5]; subtype D, on Pacific islands and in Taiwan [6]; subtype E, in Brazilian Indians [7, 8]; subtype F, in individuals from Uganda [9]; and subtype Z, in a small cohort of Zambian children [10].

Among men who have sex with men (MSM), KSHV seroprevalence is higher than in the general population of Western Europe (<5%) [11]. A recent systemic review and meta-analysis reported a pooled KSHV seroprevalence of 33% in both HIV-infected and uninfected MSM [12]. The C and A subtypes of KSHV are the most prevalent in European and Asian MSM with KSHV-associated diseases [13–15], consistent with the prevalence of KSHV subtypes in the general populations of these regions [1–3]. However, although the KSHV subtype distribution depends mostly on the patient’s region of origin, several studies have suggested that it may also be affected by clinical presentation or progression, particularly for different forms of KS. For example, the A5 variant has been associated with extensive disease in epidemic KS form [5], the A subtype has been associated with rapidly evolving classic KS form [16], and the A and B’ subtypes have been shown to contrast with the C subtype by occurring at extracutaneous sites in posttransplantation KS form [17].

We conducted a retrospective study analyzing HIV-infected MSM living in France with KS, MCD, or PEL, to describe KSHV subtype diversity and potential associations with the severity of clinical presentation. Because it was clearly demonstrated that the prevalence of KSHV infection was correlated with the number of MSM partners [18], and considering the fact that this population could be exposed to KSHV transmission by frequenting dense sexual network, we also included HIV-seronegative MSM receiving HIV preexposure prophylaxis (PrEP) to compare KSHV strains.

METHODS

Study Population

We studied 65 MSM, including (1) 57 HIV-infected patients with KSHV-associated diseases diagnosed between 2012 and 2017 in France and (2) 8 HIV-uninfected participants receiving HIV PrEP, with positive KSHV antibody results and KSHV DNA shedding in a buccal swab sample. Demographic and medical data were collected, including age, country of origin, CD4⁺ cell count, HIV RNA viral load, KSHV DNA viral load, and clinical presentation of KSHV-associated diseases. Severe clinical presentation was defined as visceral KS or lymphoid malignancies.

We subsequently included 199 patients testing positive for KSHV DNA since 2013, with diagnosis in our department, to estimate the prevalence of the new variant identified. For each patient, demographic data were collected, including sex, age, sexual orientation, country of origin, and KSHV DNA viral load.

KSHV Typing

For HIV-infected patients, we obtained a whole-blood sample at the time of KSHV disease diagnosis. For PrEP-using MSM, we obtained a buccal swab sample during standard medical follow-up. DNA was extracted from this samples and subjected to real-time polymerase chain reaction (PCR) to amplify both ORF-73 (encoding the latency-associated nuclear antigen) and the albumin gene, as described elsewhere [19].

Sanger Sequencing of ORF-K1 or VR1

A 679–base pair (bp) fragment of ORF-K1 including the 2 hypervariable regions, VR1 (amino acids 54–93) and VR2 (amino acids 191–228), was amplified by nested PCR, as described elsewhere [20]. If ORF-K1 amplification was unsuccessful, a second nested PCR was performed to amplify a 363-bp fragment including only VR1, as described elsewhere [2]. Bidirectional sequencing was performed with BigDye Terminator chemistry (Thermo Fisher Scientific), with analysis of the reaction products on an ABI sequencer.

KSHV Whole-Genome Sequencing and Assembly

Sample library preparations and target enrichment were performed according to the SeqCap EZ HyperCap Workflow (Roche). The DNA sample was fragmented mechanically, and specific adapters were added. DNA libraries were pooled and enriched in KSHV sequences by 2 rounds of hybridization with 100-bp overlapping DNA probes designed in conjunction with Roche on the KSHV GK18 (AF148805.2) sequence (excluding repeat regions) and on 28 ORF-K1 reference sequences available from the National Center for Biotechnology Information database (Supplementary Table 1), to give 5× coverage (each base is covered by 5 different probes). Finally, next-generation sequencing was performed with paired-end reads (Mid Output Kit, version 2; 2 × 75 bp) on the NextSeq 500 Illumina system.

Reads were trimmed with Trimmomatic software, using a quality threshold such that only bases with quality >30 (Q-score) were retained and reads <50 bp were filtered out. Paired-end reads were first mapped onto the KSHV reference sequence (GK18) with BowTie v2.3.4.3 software. They were then assembled de novo with SPAdes v3.12.0 and Mira v1.1.1 software to generate 2 other sequences. Finally, we used Mauve v1.1.1 in Geneious v11.1.4 software to align the 3 sequences and establish the consensus whole-genome KSHV sequence (KSHV-WG).

Phylogenetic Analysis

We performed a phylogenetic analysis of ORF-K1 amino acid sequences and KSHV-WG nucleotide sequences, using the maximum likelihood method. Multiple sequence alignments were generated with MAFFT 7 software [21], and a phylogenetic analysis was then performed with PhyML software (version 3.0) [22] and 1000-bootstrap resampling. Pairwise genetic distances between ORF-K1 amino acid sequences were calculated with Mega v7.0.14 software [23], using the JTT model of substitutions and a gamma distribution with 4 parameters.

Screening for the New F Variant With Real-Time PCR

Whole-blood samples with positive KSHV DNA viral load (n = 199) were screened for the new variant using specific real-time PCR. We designed specific primers and probes with Geneious v11.1.4 software, to amplify a fragment about 120 bp long encompassing the VR1 region and specific for the new KSHV variant (see Supplementary Table 2). The specificity of that PCR methods was assessed by testing 25 subtypes/genotype variants and the sensitivity at 25 copies/10⁶ cells.

Statistical Analysis

Continuous variables were expressed as medians with interquartile ranges [IQRs], and discrete variables as numbers with percentages. GraphPad software was used to perform nonparametric tests, specifically Mann-Whitney U tests for quantitative data and Fisher exact tests for qualitative data.

Ethics Statement

The study was carried out in accordance with the Declaration of Helsinki. It was a retrospective noninterventional study with no addition to standard care procedures. Reclassification of biological remnants into research material after completion of the ordered virological tests was approved by the local interventional review board of Pitié-Salpêtrière Hospital. According to the French public health code (CSP article L.1121–1.1), such protocols are exempted from the requirement for individual informed consent.

RESULTS

Patients’ Characteristics

In total, KS was diagnosed in 41 of the HIV-infected men, 12 with MCD and 4 with PEL. Most were Caucasian (50/57, 88%); 46 of 57(81%) originated from France, 6 of 57 (10%) from the Mediterranean Basin, 4 of 57 (7%) from South America, and 1 of 57 (2%) from Asia (Table 1). KSHV DNA viral loads in whole blood were lower in patients with KS than in those with MCD or PEL (P < .0001). Most PrEP users were also Caucasian (7 of 8 [88%]); 4 (50%) originated from France, 1 from Switzerland, 2 from Eastern Europe, and 1 from South America. None of the PrEP users were infected with HIV or had KSHV-associated diseases.

ORF-K1 Phylogenetic Analysis

ORF-K1 amplification was successful for 34 of the 57 MSM with KSHV diseases (60%) and was related to the KSHV DNA viral load. For patients with all KSHV-related diseases considered together, subtype C was the most prevalent (18 of 34 [53%]), followed by subtype A (11 of 34 [32%]) and finally subtype B (1 of 34 [3%]). For subtype C, 15 of 18 cases (83%) were classified as the C3 variant. Subtype classification was possible for 5 of the 8 PrEP users (63%), with the C3 variant in 2 cases and the A4 variant in 2 cases (Figure 1 and Supplementary Table 3). The genetic distance was 34% between subtypes B and A and 39% between subtypes B and C, and the ORF-K1 amino acid sequences of subtypes A and C differed by 19%. Within subtypes, the genotype variant differed from 9% (subtype A) and from 13% (subtype C) at the amino acid level.

Interestingly, 5 KSHV strains (from P030, P035, P075, P076, and PrEP004) were closely related to the F subtype described in an African population from Uganda (AAX55469_F) [9]. However, their amino acid sequences differed from those of the F subtype by 11% and were separated with a bootstrap confidence of 54% (Figure 1).

Overall, we found 3 clusters, corresponding to the C3 variant (17 of 39), the A4 variant (7 of 39), and the F subtype (5 of 39). In most cases, the geographic origin of the MSM was consistent with the subtype detected. Subtypes A and C were the most prevalent in patients originating from France, the Mediterranean Basin, or Asia (Supplementary Table 3).

KSHV Subtype and Clinical Presentation

The proportions of patients with KS and MCD did not differ significantly between subtypes: 26% of patients with KS and 36% of those with MCD had subtype A viruses (P = .69) and 63% of KS and 55% of patients with patients had subtype C viruses (P = .71), suggesting that the subtypes involved in these 2 diseases may reflect the prevalence of the various subtypes in this population.

Among patients with KS, the KSHV DNA viral load tended to be higher for subtype A than for subtype C (P = .051), regardless of immunovirological status (Figure 2), and the C3 variant was more associated with purely cutaneous and/or oral-mucous involvement than the other subtypes (odds ratio, 11.6; 95% confidence interval, 1.1–214.2; P = .023), regardless of immunovirological status (median [IQR] values for C3 variant vs other subtypes: CD4⁺ T-cell count, 70/ µ L [21–325/ µ L] vs 82.5/ µ L [68–213/ µ L] [P = .78]; HIV RNA viral load, 5.62 [1.60–5.89] vs 5.38 [4.71–5.65] log₁₀ copies/mL; P = .75).

New KSHV F Variant

A new genotype variant closely resembling the F subtype was detected in 5 MSM, 4 of whom had severe clinical presentations: 2 cases of visceral KS (P030 with cutaneous and pulmonary involvement and P035 with cutaneous, pulmonary and ganglionic involvement), 1 case of MCD associated with visceral KS (P075), and 1 case of PEL associated with cutaneous KS (P076). The remaining individual was an asymptomatic PrEP user (PrEP004). All these individuals were Caucasian (4 from France and 1 from Peru) (see Supplementary Table 4).

ORF-K1 Sanger Sequencing

Partial ORF-K1 sequences were generated, 191 amino acids long (P030, P075, and P076) and encompassing VR1 and VR2, or 142 and 141 amino acids long (from P035 and PrEP004, respectively) and encompassing only VR1; all sequences were strictly identical. The longest fragment differed by 18 amino acids from the reference subtype F sequence from Uganda (AAX55469_F), and an insertion of 5 amino acids was detected in VR2 (Figure 3). Otherwise, these sequences were closest (genetic distance,10^–6) to the KSHV sequence described in 2000 from a French HIV-positive MSM patient with PEL (AAG01610_F) [15], from which they differed by only 1 amino acid, in position 202 (Figures 1 and 3).

We also compared these sequences with that for a subtype F KSHV strain from our database that was obtained from a Congolese woman with MCD (P072). The ORF-K1 sequence from this woman differed by 23 amino acids from our virus and by 20 amino acids (15 amino acids and the insertion described above) from AAX55469_F. However, P072 and AAX55469_F clustered together in the phylogenetic tree and this sequence was considered to correspond to an “African” F subtype (Figures 1 and 3).

KSHV-WG Sequencing

For the formal identification of a new F variant, we performed KSHV-WG sequencing on the 4 KSHV strains newly identified from patients with KSHV-related diseases, and compared the sequences obtained with that for the P072 virus identified as belonging to the “African” F subtype, because no KSHV-WG F subtype reference sequence was available in the National Center for Biotechnology Information database. A consensus KSHV-WG sequence was obtained for all patients other than P035 (Supplementary Table 4).

As in the phylogenetic analysis for ORF-K1, the phylogenetic tree generated for KSHV-WG sequences presented a separation of subtype B from subtypes A, C, and F, with a bootstrap confidence level of 100%. However, subtypes A and C were not clearly separated, suggesting the probably involvement of other genes in KSHV variability, as well as recombination events, as reported elsewhere [24, 25]. Phylogenetic analysis also confirmed that the 2 F variants (Caucasian MSM vs African origin) differed and were separated with a bootstrap confidence level of 61% (Figure 4). Although ORF-K1 amino acid sequences were strictly identical between P030, P075, and P076, the KSHV-WG phylogenetic tree showed that the new F variant from patients with KS differed from that in patients with lymphoid malignancies.

Prevalence of the New F Variant

Finally, we screened all available samples testing positive for KSHV DNA since 2013 (n = 199) with a new F variant–specific real-time PCR. The median [IQR] KSHV DNA viral load was 1.92 (1.45–2.69) log₁₀ copies/10⁶ cells. The patients tested had a median [IQR] age of 54 (42–62.5) years and 78% were men (156 of 199); 47% were Caucasian (73 of 156), of whom 77% (56 of 73) were MSM. None of the 199 samples tested was positive for the new F variant. Overall, this new F variant was described only in Caucasian MSM, with a prevalence of 4.5% (5 of 113) in this population.

DISCUSSION

This study aimed to assess KSHV diversity in a population of MSM living in France, with or without KSHV-associated disease, and to evaluate the possible correlation between subtype and clinical presentation. The global distribution of KSHV subtypes and variants in our MSM population was consistent with the findings of previous studies conducted in France [15] and other countries (Germany and Taiwan) [13, 14], with KSHV subtypes C (n = 18) and A (n = 11) the most prevalent.

We also found that the C3 variant was the most prevalent KSHV strain in a population of MSM living in France, and that this variant was associated with a less severe clinical presentation of the epidemic form of KS. In patients with epidemic KS, KSHV DNA viral load tended to be higher for subtype A than for subtype C. These results are consistent with those of previous studies reporting a more aggressive clinical presentation in patients with subtype A than in those with subtype C, for the classic and posttransplantation forms of KS [16, 17].

Although subtype F has been described only a few times in the literature [9, 26, 27], we identified a new KSHV variant closely related to the first subtype F virus described in Uganda. This new F variant was identified by means of Sanger sequencing and confirmed by WG sequencing on various samples from 5 patients. Based on the results of WG sequencing, we were able to classify this KSHV strain as a new F variant. Finally, based on our results, we propose the subdivision of subtype F into 2 variants: F1 variant for the KSHV described in Uganda and F2 variant for the KSHV identified in Caucasian MSM.

All of the patients harboring this new F2 variant were Caucasian MSM living in Paris. The screening of all our available samples since 2013 did not disclose this new variant in other patients, which suggests that it is present in a small, restrictive population. The overall prevalence of the F2 variant was 4.5% (5 of 113) in the Caucasian MSM population, and this variant was absent from other epidemiological groups. The patients harboring the new F2 variant included 1 immunocompetent PrEP user who was found to be merely a carrier, with oral shedding but with no KSHV-related disease. The other 4 patients harboring the new variant were immunocompromised (AIDS) and had severe forms of KSHV disease. Further investigations are required to confirm these results and to determine whether specific subtypes or viral determinants of virulence involved in KSHV tumor-associated processes can lead to severe or persistent KSHV-related diseases, as recently reported for KS in HIV-infected patients receiving effective antiretroviral therapies [28, 29]. In conclusion, careful screening of the MSM population may be required for this new F2 variant, which is circulating in Caucasian MSM living in Paris, given the severe clinical presentation of associated diseases in the context of immunosuppression.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Financial support. This work was supported by the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (to the AC43 working group on medical virology in different field including “sexually transmitted infections”).

Potential conflicts of interest. D. D. reports personal fees from Gilead Sciences, Merck Sharp & Dohme, ViiV Healthcare, and Janssen-Cilag, outside the submitted work. A. G. M. reports grants and personal fees from ViiV Healthcare, Gilead, and Merck Sharp & Dohme, and personal fees from Janssen-Cilag, outside the submitted work. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

References

Author notes