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Virol Sin
Virol Sin
Virologica Sinica
1674-0769
1995-820X
Wuhan Institute of Virology, Chinese Academy of Sciences

S1995-820X(24)00102-0
10.1016/j.virs.2024.06.006
Letter
Coexistence of adeno-associated virus 2 with adenovirus 18 or herpesvirus may be associated with severe lingual papillomatosis in an immunocompromised individual
Yu Jie-Mei a1
Liang Ze-Yin b1
Fu Yuan-Hui a
Peng Xiang-Lei a
Zheng Yan-Peng a
Dong Yu-Jun dongy@bjmu.edu.cn
b⁎
He Jin-Sheng jshhe@bjtu.edu.cn
a⁎
a College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing, 100044, China
b Department of Hematology, Peking University First Hospital, Beijing, 100034, China
⁎ Corresponding authors. dongy@bjmu.edu.cnjshhe@bjtu.edu.cn
1 Jie-Mei Yu, Ze-Yin Liang contributed equally to this article.

22 6 2024
8 2024
22 6 2024
39 4 705707
16 10 2023
19 6 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• Viral metagenomics analysis was conducted on samples from an HSCT recipient experiencing severe lingual papillomatosis.

• Coexistence of AAV2 with AdV18 in fecal and HSV-1 in tissue samples was detected.

• A second complete genome of AdV18 was obtained in this study and is available in public databases.
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pmcDear Editor,

Patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) often experience a significant reduction in immune function due to factors such as severe lymphopenia, immunosuppressive treatment, and the development of graft-versus-host disease, rendering them more susceptible to severe infections (Sahin et al., 2016). Traditional diagnostic approaches are limited in sensitivity, speed, and assay targets, thus failing short of meeting the diagnostic demands (Qu et al., 2022). However, next-generation sequencing (NGS) has greatly improved the accuracy and efficiency of diagnosing various diseases (Schuler et al., 2022), and has been widely utilized for pathogen detection in samples from HSCT patients (Armstrong et al., 2019; Peng et al., 2021). In this study, we employed viral metagenomics analysis using high-throughput sequencing on fecal and tissue samples from a patient with HSCT experiencing severe lingual papillomatosis. Our findings revealed the co-detection of adeno-associated virus 2 (AAV2) and its potential helper viruses, human adenovirus serotype 18 (AdV18) and herpesvirus type 1 (HSV-1) in the fecal and tissue samples, respectively.

In June 2018, a fecal sample (coded MI-F), along with a tissue sample (coded MI-T) of excised lingual papilloma from a 13-year-old allo-HSCT recipient at Peking University First Hospital were collected. This patient was diagnosed with acute lymphoblastic leukemia in 2017 and underwent allo-HSCT. During the transplant period, the patient experienced prolonged severe oral mucositis. Four months post-transplant, the patient presented with multiple proliferative lesions on the tongue (Supplementary Fig. S1A). Blood tests indicated the presence of cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Following treatment with ganciclovir, both CMV and EBV became undetectable. Subsequently, the proliferative lesion on the tongue was surgically removed, and tissue samples from the excised lingual papilloma were collected. Total nucleic acids were extracted from the two samples, MI-F and MI-T, which then respectively underwent random RT-PCR amplifications using primers with unique barcodes before undergoing NGS. Results showed that in the MI-F sample, the Parvoviridae family was the predominant viral family, accounting for up to 85.6% of the viral reads. Additionally, Caudovirales and Adenoviridae constituted 10.2% and 2.1% of the viral reads, respectively. Other virus families, including Anelloviridae and phages, contributed to the remaining 2.1% of viral reads (Fig. 1A). Similarly, in the MI-T sample, Anelloviridae, Parvoviridae, Herpesvirales, and Caudovirales collectively represented over 93% of the viral reads, with proportions of 36.6%, 34.6%, 14.5%, and 6.7% respectively. The remaining 7.7% of viral reads encompassed nine virus families (Fig. 1B). Notably, there was less than 0.01% of Herpesvirales in the fecal samples and 0.3% of Adenoviridae in the tissue samples, which may be attributed to potential low-level cross-contamination between samples during NGS. Alternatively, it is plausible that viral genomes with low abundance are genuinely present in the samples. The viral metagenomic data were provided in Supplementary Table S1.Fig. 1 Viral metagenomic analysis of the patient with lingual papillomatosis. A Viral metagenomics of fecal sample MI-F. B Viral metagenomics of tissue sample MI-T. High reads of Parvoviridae and Adenoviridae were detected in feces, while Parvoviridae and Herpesvirales were detected in tissue. C, D Phylogenetic trees for polymerase and hexon genes by neighbor-joining method with 1000 bootstrap replicates using MEGA 7.0.26. Our AdV18 differed from the previous ones in one amino acid in the hexon (G349C) and three in the polymerase (E303D, S359T and Q1079H). A close relationship was observed between both regions of AdV18 in our study and previously reported AdV18 sequences. Sequences acquired in this study were indicated by “•” in red.

Fig. 1

Remarkably, our analysis revealed that 85.6% of the reads in the fecal sample and 34.6% of the reads in the tissue sample were identified as belonging to the Parvoviridae family. Subsequent investigation revealed that these Parvoridae-related reads corresponded to AAV2. As the AAV2 reads were not detected in other samples from the same run of the NGS (data not shown), it was speculated that the presence of the AAV2 reads in the fecal and tissue samples of the patient was not due to contamination. The nearly full-length genome of the AAV2 detected in this study, designated as MI-AAV2, was assembled from the NGS data (4518 bp) and has been deposited in public databases (GenBank under accession number: PP621512, Science Data Bank: https://doi.org/10.57760/sciencedb.j00001.00828). Sequence analysis showed that MI-AAV2 exhibited the highest nucleotide homology of 97.3% with an AAV2 sequence obtained from a liver biopsy conducted in the UK in 2022, followed by 96.9% homology with an AAV2 sequence derived from a liver tumor in France in 2015. Furthermore, comparison of the amino acid sequences of each protein encoded by MI-AAV2 demonstrated homologies exceeding 99% with previously reported AAV2 sequences.

Typically, AAV is inherently deficient in replication and relies on co-infection with a helper virus such as adenovirus and herpesvirus for productive propagation (Meier et al., 2020). Initially considered non-pathogenic and safe for human use, AAV has been extensively utilized as a gene therapy vector (Wang et al., 2019). However, a recent study has highlighted that recurrent clonal AAV2 insertions were associated with the development of hepatocellular carcinoma (La Bella et al., 2020). Furthermore, AAV2 infection has been implicated in acute pediatric hepatitis outbreaks in both the United States and the United Kingdom (Gates et al., 2023; Ho et al., 2023). In this study, we identified the sequences from Adenoviridae in the fecal sample specifically as AdV18, while the sequences from Herpesvirales in the tissue sample were identified as HSV-1. This observation suggested that these two viruses could potentially serve as helper viruses facilitating AAV2 replication within host cells. The freshly excised tissue was promptly frozen in a cryostat, sectioned, and then placed in a fixative solution. Rapid hematoxylin and eosin (HE) staining was conducted, revealing the inflammatory hyperplasia of the lingual papilloma (Supplementary Figs. S1B and C). Unfortunately, we were unable to conduct the in situ hybridization experiment to ascertain the presence of the virus within the tissue due to insufficient specimen quantity. Thus, we cannot exclude the possibility that the lingual papilloma may be linked to AAV2 with AdV18 and/or HSV-1 acting as helper viruses. Further validation from clinical cases is imperative to elucidate this potential association.

Significantly, AdV18 is a rarely detected serotype of adenovirus that has garnered considerable interest due to its oncogenic potential in neonatal rodents (Huebner et al., 1962). Initially isolated from an anal swab of a child with Niemann-Pick syndrome in the 1950s, AdV18 has been sporadically detected in children's fecal samples, as evidenced by a study from Tanzania in 2014 (Moyo et al., 2014). Currently, only one complete genome sequence of AdV18 (GU191019.1) is available in public databases, originating from a sample collected in 1954 in the United States. Additionally, there are 22 AdV18-related fragments of varying lengths (ranging from 235 bp to 3164 bp) in public databases, located at different positions within the viral genome (Supplementary Fig. S1D). Among the three relatively long sequences, one represents a near full-length sequence of the polymerase, the other two are full-length sequences of the hexon (Liu et al., 2000; Hoeben and Uil, 2013). Our sequence differed from the previous sequences by one amino acid in the hexon (G349C) and three in the polymerase: E303D, S359T and Q1079H. The first two alterations in the polymerase are located in the exonuclease domain, while the latter resides in the thumb domain. Further investigation is warranted to determine whether these changes impact polymerase activity. Evolutionary trees were constructed using nucleotide sequences from the polymerase and hexon regions of related AdVs. Our results demonstrated a close association between both regions of AdV18 in our study and previously reported AdV18 sequences, forming a coherent evolutionary cluster (Fig. 1C and D).

The role of AdV18 in human health remains poorly understood, including its potential association with the lingual papillomatosis observed in the patient of this study. Although AdV18 has been recognized for its oncogenic potential since its initial discovery in 1954, it has been rarely reported in the literature. Currently, only one complete genome sequence of AdV18 is available in public databases. In our investigation, we successfully obtained a complete genome sequence of AdV18 through high-throughput sequencing (GenBank under accession number: OR609364, Science Data Bank: https://doi.org/10.57760/sciencedb.j00001.00828), which exhibited 99.3% nucleotide identity with the previously reported sequence. This represents the second complete genome sequence accessible in the public domain. The newly acquired data serve as a valuable resource for future studies focusing on the molecular evolution, origins, and pathogenicity of adenoviruses.

In conclusion, our analysis identified a notable abundance of reads for AAV2, AdV18 in the feces and HSV-1 in the tissue of an individual exhibiting severe lingual papillomatosis following HSTC. Despite failing to establish a direct causal relationship between these viral infections and the development of lingual papillomatosis, this study provides the first documentation of the concurrent presence of non-replicative AAV2 alongside potential helper viruses AdV18 or HSV-1 in fecal or tissue samples of a patient.

Footnotes

This work was supported by the 10.13039/501100004826 Natural Science Foundation of Beijing Municipality (L222074) and Key R&D Program of Shandong Province, China (2019JZZY011011). We acknowledge the support and resources from the Center for High Performance Computing at 10.13039/501100005022 Beijing Jiaotong University (https://hpc.bjtu.edu.cn). None of the authors declare a conflict of interest. The study protocol was approved by the Ethics Committee of the Peking University First Hospital. Due to the retrospective nature of the study, informed written consent was waived.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.virs.2024.06.006.

The following are the Supplementary data to this article.Supplementary Material

Multimedia component 1

Supplementary Table S1

Viral metagenomic data of the sample MI-F and MI-T.

Supplementary Table S1

Supplementary Fig. S1 Lingual papillomatosis information and AdV18-related sequences distribution. A The image of severe lingual papillomatosis. B A significant presence of plasma cells in the circle. Arrow bars indicated typical plasma cells. C The left side of the purple line is necrotic tissue of an ulcer, and the right side of it is granulation tissue. Fibroblasts, newly formed capillaries and a mixed inflammatory cell such as neutrophils and eosinophils can be seen in the granulation tissue. D Genome distribution of AdV18-related sequences in public databases. Except for one full-length genome sequence, two full hexon gene sequences, and one near full-length polymerase gene sequence, the other sequences are very short.

Supplementary Fig. S1
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