Detection of Low-Copy Human Virus DNA upon Prolonged Formalin Fixation

Mielonen, Outi I.; Pratas, Diogo; Hedman, Klaus; Sajantila, Antti; Perdomo, Maria F.

doi:10.3390/v14010133

Cited by 7 publications

(6 citation statements)

References 40 publications

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“…This raises the possibility that eGCs could originate from thymic B cells rather than peripheral B cells, suggesting a potential protective mechanism of the thymus against viral infection [52]. Formalin fixation, although effective for morphological and molecular preservation, can induce DNA damage and cross-linking, hindering nucleic acid screening, especially for lowabundance targets like persistent DNA viruses [53]. Hence, our rigorous approach of using fresh-frozen tissue and paired blood samples (to evaluate infection history) allowed for a more…”

Section: Discussionmentioning

confidence: 99%

HHV-6B, HHV-7, and B19V Are Frequently Found DNA Viruses in the Human Thymus but Show No Definitive Link with Myasthenia Gravis

Nowlan,

Hannolainen,

Assimakopoulou

et al. 2024

Preprint

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Myasthenia gravis (MG) is an autoimmune disorder characterised by autoantibodies that target components of the neuromuscular junction, primarily the acetylcholine receptor (AChR), resulting in muscle weakness. The thymus plays a significant role in MG pathogenesis, particularly in patients under the age of 50, who display pathological alterations and possess elements conducive to autoimmune reactions. Although viral infections are suspected drivers of thymic pathogenesis, the exact aetiology of MG remains elusive. This study investigates the potential link between MG and DNA viruses within the thymus. Using targeted next-generation sequencing and quantitative PCR, we analysed the presence of human parvovirus B19 (B19V) and nine human herpesviruses (HSV-1, HSV-2, VZV, EBV, CMV, HHV-6A, HHV-6B, HHV-7, and HHV-8) in fresh tissue samples from 19 non-thymomatous MG patients, 16 thymomas (3 with and 14 without MG), 41 normal thymus tissues, and 20 tonsils from healthy individuals. HHV-6B was the most common virus, found in over 50% of all tissue groups. B19V DNA was detected in 40% of adult control thymic tissue, 72% of MG thymus, 7.7% of non-MG thymoma, and 50% of tonsil samples. HHV-7 was present in 15-30% of thymus tissues and 95% of tonsils, while EBV was detected in less than 25% of all thymus samples but 85% of tonsils. In B19V seropositive individuals, B19V DNA was detected in 100% of thymic tissue from both MG patients and healthy individuals, except in thymomatous tissues, where it was found in only one of thirteen seropositive individuals. Immunohistochemistry for B19V protein expression did not show evident B19V VP1/VP2 protein expression, indicating dormant viral persistence. Laser capture microdissection (LCM) and RNAscope in situ hybridisation pinpointed B19V DNA localisation to the thymus medulla. This study is the first to demonstrate the persistence of various DNA viruses in the human thymus. However, neither B19V nor the nine human herpesviruses showed specific enrichment in MG thymic tissue compared to controls, suggesting that these viral infections are unlikely to be sole environmental triggers for MG.

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Section: Discussionmentioning

confidence: 99%

HHV-6B, HHV-7, and B19V Are Frequently Found DNA Viruses in the Human Thymus but Show No Definitive Link with Myasthenia Gravis

Nowlan,

Hannolainen,

Assimakopoulou

et al. 2024

Preprint

Self Cite

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“…This might be due to low virus load in combination with a second freeze-thaw round needed for NGS with possible degraded DNA. In addition, although FFPE samples have previously been shown to be a useful source for virus screening (Jauhiainen et al 2021), it is possible that with this tumor type, larger samples and fresh tumor material would be beneficial as they may increase the amount of intact DNA and improve its quality (Mielonen et al 2022).…”

Section: Discussionmentioning

confidence: 99%

Herpesviruses, polyomaviruses, parvoviruses, papillomaviruses, and anelloviruses in vestibular schwannoma

et al. 2023

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Etiology of vestibular schwannoma (VS) is unknown. Viruses can infect and reside in neural tissues for decades, and new viruses with unknown tumorigenic potential have been discovered. The presence of herpesvirus, polyomavirus, parvovirus, and anellovirus DNA was analyzed by quantitative PCR in 46 formalin-fixed paraffin-embedded VS samples. Five samples were analyzed by targeted next-generation sequencing. Viral DNA was detected altogether in 24/46 (52%) tumor samples, mostly representing anelloviruses (46%). Our findings show frequent persistence of anelloviruses, considered normal virome, in VS. None of the other viruses showed an extensive presence, thereby suggesting insignificant role in VS.

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“…sensitivity being higher when the product is less than 100 bp [ 14 ]. DNA was extracted from tissues with minimal formalinization and before fixation to preserve DNA integrity [ 19 ]. A significantly higher detection rate was observed in PCR results in tumorous tissues versus ANNT group (64% vs 26%, P < 0.001).…”

Section: Discussionmentioning

confidence: 99%

Detection of Human cytomegalovirus UL55 Gene and IE/E Protein Expression in Colorectal Cancer Patients in Egypt

et al. 2023

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Background A possible relation between Human cytomegalovirus (HCMV) and colorectal cancer (CRC) has been widely explored with an unclear role yet speculated. Aim The study aimed at detecting HCMV UL55 gene, immediate early and early (IE/E) proteins in colorectal tumor tissues and adjacent non neoplastic tissues (ANNT). Also, it aimed to correlate HCMV presence with CRC clinicopathological features. Subjects and methods A prospective study of 50 HCMV seropositive patients with resectable CRC were enrolled in the study. Demographic, clinical, and radiological findings were recorded. Pathological assessment was done. Paired CRC tumorous and ANNT were examined for HCMV UL55 by PCR and for IE/ E proteins by immunohistochemistry (IHC). Results 70% of CRC patients enrolled were females and 36% were elderly (> 60y). Adenocarcinoma was the prevalent histopathological type (92%) with Grade 2, higher stages, and nodal involvement accounting for (64%, 64% and 56%) respectively. HCMV detection was significantly higher in tumoral tissue versus ANNT by PCR and IHC (P < 0.001, P < 0.008) respectively. Moderate agreement was found between the two techniques (κ = 0.572, P < 0.001). Univariate analysis identified HCMV presence to be significantly higher in elderly patients, in tumors with higher stage and with nodal involvement (P = 0.041, P = 0.008, P = 0.018 respectively). In multivariate analysis, the latter two retained significance (P = 0.010, P = 0.008). Conclusion CRC tumor tissues are more infected by HCMV than ANNT. A significant association of HCMV presence with a higher CRC tumor stage and nodal involvement in an age-dependent manner was detected. HCMV oncomodulatory and a disease progression role is suspected.

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Detection of Low-Copy Human Virus DNA upon Prolonged Formalin Fixation

Cited by 7 publications

References 40 publications

HHV-6B, HHV-7, and B19V Are Frequently Found DNA Viruses in the Human Thymus but Show No Definitive Link with Myasthenia Gravis

HHV-6B, HHV-7, and B19V Are Frequently Found DNA Viruses in the Human Thymus but Show No Definitive Link with Myasthenia Gravis

Herpesviruses, polyomaviruses, parvoviruses, papillomaviruses, and anelloviruses in vestibular schwannoma

Detection of Human cytomegalovirus UL55 Gene and IE/E Protein Expression in Colorectal Cancer Patients in Egypt

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