Annika Allard

An exhausted antiviral immune response is observed in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and post-SARS-CoV-2 syndrome, also termed long COVID. In this study, potential mechanisms behind this exhaustion were investigated. First, the viral load of Epstein–Barr virus (EBV), human adenovirus (HAdV), human cytomegalovirus (HCMV), human herpesvirus 6 (HHV6), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was determined in sputum samples (n = 29) derived from ME/CFS patients (n = 13), healthy controls (n = 10), elderly healthy controls (n = 4), and immunosuppressed controls (n = 2). Secondly, autoantibodies (autoAbs) to type I interferon (IFN-I) in sputum were analyzed to possibly explain impaired viral immunity. We found that ME/CFS patients released EBV at a significantly higher level compared to controls (p = 0.0256). HHV6 was present in ~50% of all participants at the same level. HAdV was detected in two cases with immunosuppression and severe ME/CFS, respectively. HCMV and SARS-CoV-2 were found only in immunosuppressed controls. Notably, anti-IFN-I autoAbs in ME/CFS and controls did not differ, except in a severe ME/CFS case showing an increased level. We conclude that ME/CFS patients, compared to controls, have a significantly higher load of EBV. IFN-I autoAbs cannot explain IFN-I dysfunction, with the possible exception of severe cases, also reported in severe SARS-CoV-2. We forward that additional mechanisms, such as the viral evasion of IFN-I effect via the degradation of IFN-receptors, may be present in ME/CFS, which demands further studies.

An exhausted antiviral immune response is observed in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and post-SARS-CoV-2 syndrome also termed long COVID. In this study, potential mechanisms behind this exhaustion were investigated. First, the viral load of Epstein-Barr virus (EBV), human adenovirus (HAdV), human cytomegalovirus (HCMV), human herpesvirus 6 (HHV6), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was determined in sputum samples (n = 29) derived from ME/CFS patients (n = 13), healthy controls (n = 10), elderly healthy controls (n = 4), and immunosuppressed controls (n = 2). Secondly, autoantibodies (autoAbs) to type I interferon (IFN-I) in sputum were analyzed to possibly explain impaired viral immunity. We found that ME/CFS patients released EBV at a significantly higher level compared to controls (p = 0.0256). HHV6 was present in ~50% of all participants at the same level. HAdV was detected in two cases with immunosuppression and severe ME/CFS, respectively. HCMV and SARS-CoV-2 were found only in immunosuppressed controls. Notably, anti-IFN-I autoAbs in ME/CFS and controls did not differ, except in a severe ME/CFS case showing an increased level. We conclude that ME/CFS patients, compared to controls, have a significantly higher load of EBV. IFN-I autoAbs cannot explain IFN-I dysfunction, with the possible exception of severe cases, also reported in severe SARS-CoV-2. We forward that additional mechanisms, such as viral evasion of IFN-I effect via degradation of IFN-receptors; may be present in ME/CFS, which demands further studies.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common disease in which inflammatory responses to exogenic stressors, such as viral infections, has been recognised. The role of viruses in CRSwNP pathogenesis is unclear. We aimed to characterise Epstein-Barr virus (EBV), human papillomavirus (HPV), human cytomegalovirus (HCMV), and human adenovirus (HAdV) in nasal polyps and adjacent paired healthy turbinate mucosa. We used real-time PCR for EBV, HCMV, and HAdV DNA detection, combined PCR/microarrays for HPV detection and genotyping, in samples from 45 patients with CRSwNP. Additionally, we used EBER in situ hybridisation for EBV detection. EBV detection with EBER-ISH was significantly higher in polyps (36%) versus turbinate mucosa (12%). None of the viral comparisons with PCR between polyps and turbinate mucosa for EBV-, HCMV- or HAdV-DNA showed statistically significant differences. All samples were HPV negative. We report higher expression of EBV in nasal polyps (36%) than in adjacent healthy turbinate mucosa (12%), using a valid method; EBER-ISH in 45 patients with CRSwNP. EBV might be a possible stressor that can trigger polypoid inflammation.

Purpose: Apart from tobacco and alcohol, viral infections are proposed as risk factors for laryngeal cancer. The occurrence of oncogenic viruses including human papilloma virus (HPV) and Epstein–Barr virus (EBV), in laryngeal squamous cell carcinoma (LSCC) varies in the world. Carcinogenesis is a multi-step process, and the role of viruses in LSCC progression has not been clarified. We aimed to analyze the presence and co-expression of HPV, EBV, human cytomegalovirus (HCMV) and human adenovirus (HAdV) in LSCC. We also investigated if p16 can act as surrogate marker for HPV in LSCC. Methods: Combined PCR/microarrays (PapilloCheck®) were used for detection and genotyping of HPV DNA, real-time PCR for EBV, HCMV and HAdV DNA detection, and EBER in situ hybridization (EBER-ISH) for EBV detection in tissue from 78 LSCC patients. Additionally, we analyzed p16 expression with immunohistochemistry. Results: Thirty-three percent (26/78) of LSCC tumor samples were EBV positive, 9% (7/78) HCMV positive and 4% (3/78) HAdV positive. Due to DNA fragmentation, 45 samples could not be analyzed with PapilloCheck®; 9% of the remaining (3/33) were high-risk HPV16 positive and also over-expressed p16. A total of 14% (11/78) of the samples over-expressed p16. Conclusion: These findings present a mapping of HPV, EBV, HCMV and HAdV, including the HPV surrogate marker p16, in LSCC in this cohort. Except for EBV, which was detected in a third of the samples, data show viral infection to be uncommon, and that p16 does not appear to be a specific surrogate marker for high-risk HPV infection in LSCC.

Purpose: Apart from tobacco and alcohol, viral infections are proposed as risk factors for laryngeal cancer. The occurrence of oncogenic viruses including human papilloma virus (HPV) and Epstein-Barr virus (EBV), in laryngeal squamous cell carcinoma (LSCC) varies in the world. Carcinogenesis is a multi-step process, and the role of viruses in LSCC progression has not been clarified. We aimed to analyze the presence and co-expression of HPV, EBV, human cytomegalovirus (HCMV) and human adenovirus (HAdV) in LSCC. We also investigated if p16 can act as surrogate marker for HPV in LSCC. Methods: Combined PCR/microarrays (PapilloCheck®) were used for detection and genotyping of HPV DNA, real time-PCR for EBV, HCMV and HAdV DNA detection, and EBER in situ hybridization (EBER-ISH) for EBV detection in tissue from 78 LSCC patients. Additionally, we analyzed p16 expression with immunohistochemistry.Results: Thirty-three percent (26/78) of LSCC tumor samples were EBV positive, 9% (7/78) HCMV positive and 4% (3/78) HAdV positive. Due to DNA fragmentation, 45 samples could not be analyzed with PapilloCheck®; 9% of the remaining (3/33) were high-risk HPV16 positive and also over-expressed p16. A total of 14% (11/78) of the samples over-expressed p16.Conclusion: These findings present a mapping of HPV, EBV, HCMV and HAdV, including the HPV surrogate marker p16, in LSCC in this cohort. Except for EBV, which was detected in a third of the samples, data show viral infection to be uncommon, and that p16 does not appear to be a specific surrogate marker for high-risk HPV infection in LSCC.

Background: Sinonasal inverted papilloma (SIP) is a benign tumour originating from the sinonasal mucosa showing an extensive growth pattern, a high risk of recurrence and a 5–10% risk to malignify. Epstein-Barr virus (EBV) is an oncogenic herpesvirus which infects most individuals via the saliva eliciting a latent infection. Previous studies have been reporting variable data on EBV in SIP, and there is no present appreciation regarding the association between these. Aims/objectives: The aims were to investigate the presence and count of EBV in SIP and map the viral distribution in the epithelium versus the connective tissue. Material and method: Fifty-three SIP patients were identified in the Pathology Department register at the University Hospital of Umeå. The biopsies were analysed with Epstein-Barr Encoded Region (EBER) in situ hybridization. EBER-positive cells were counted in the epithelium and connective tissue. Results: We found EBER-stained cells in 30% of the cases, where 19% of these had an abundance of stained cells, and the rest showed a low count. Conclusions/significance: These findings demonstrate a low EBV count in SIP. EBV is less likely to be a causative agent in the formation of SIP, or its malignant transformation.

Human adenoviruses (HAdVs) are common pathogens that can cause respiratory, gastrointestinal, urogenital, and ocular infections. They are divided into seven species containing 85 genotypes. Straightforward typing systems might help epidemiological investigations. As homologous recombination frequently shapes the evolution of HAdVs, information on a single gene is seldom sufficient to allow accurate and precise typing, and complete genome-based methods are recommended. Even so, complete genome analyses are not always easy to perform for practical reasons, and in such cases a multigene system can provide considerably more information about the strain under investigation than single-gene-based methods. Here we present a rapid, generic, multigene typing system for HAdVs based on three main deterministic regions of these viruses. Three PCR systems were used to amplify the genes encoding the DNA polymerase, the penton base hypervariable Arg-Gly-Asp-containing loop, and the hexon loop 1 (hypervariable region 1–6). Using this system, we typed 281 clinical isolates, detected members of six out of seven HAdV species (Human mastadenovirus A–F), and could also detect not only divergent strains of established types but also a new recombinant strain with a previously unpublished combination of adenovirus genomes. This strain was accepted by the Human Adenovirus Working Group as a novel genotype: HAdV-86. Seven strains that could not be typed with sufficient accuracy were also investigated using a PCR based on part of the fiber gene. By analysis of corresponding sequences of the 86 known HAdV genotypes, we determined that the proposed typing system should be able to distinguish all non-recombinant types, and with additional fiber information, all known HAdV genotypes.

Human adenoviruses (hAdVs) of subgroup F (enteric serotypes 40 and 41) display characteristic gut tropism, in vivo, fastidious growth characteristics in cell culture, and are estimated to be associated with 5–20 % worldwide of acute gastroenteritis cases among infants and young children. Adequate hAdV gastroenteritis case management requires laboratory-based diagnosis. The present study aimed to the development and evaluation of a simple and cost-effective, one-step, single-tube adenovirus type 40/41 specific loop-mediated isothermal amplification (LAMP) assay for the detection of hAdV40/41 DNA in environmental and/or clinical samples, since no LAMP assay has previously been reported for the detection of these virus types. The assay targeted the hexon gene and had the advantages of being rapid, simple, specific, and sensitive. Results could be obtained within 60 min, under isothermal conditions at 69 °C. The detection limits for hAdV genomes were between 50 and 100 copies/reaction for hAdV40 and hAdV41, and no cross-reactions with other selected viruses, were found. The assay was evaluated with clinical as well as environmental samples. The developed assay is expected to provide a potential molecular tool in obtaining greater knowledge of the hAdV40/41 importance in the epidemiology and clinical manifestations of gastroenteritis.

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are responsible for lifelong latent infections in humans, with periods of viral reactivation associated with recurring ulcerations in the orofacial and genital tracts. In immunosuppressed patients and neonates, HSV infections are associated with severe morbidity and, in some cases, even mortality. Today, acyclovir is the standard therapy for the management of HSV infections. However, the need for novel antiviral agents is apparent, since HSV isolates resistant to acyclovir therapy are frequently isolated in immunosuppressed patients. In this study, we assessed the anti-HSV activity of the antiadenoviral compounds 2-[2-(2-benzoylamino)-benzoylamino]benzoic acid (benzavir-1) and 2-[4,5-difluoro-2-(2-fluorobenzoylamino)-benzoylamino]benzoic acid (benzavir-2) on HSV-1 and HSV-2. Both compounds were active against both viruses. Importantly, benzavir-2 had potency similar to that of acyclovir against both HSV types, and it was active against clinical acyclovir-resistant HSV isolates.