Adenovirus infections: new insights for the clinical laboratory.

Since their discovery in 1953, research on human adenoviruses (HAdVs) has had diverse foci, resulted in groundbreaking discoveries, such as gene splicing, and generated powerful oncolytic constructs and expression vectors for vaccine development and gene therapy. In contrast, virologists working in this field have made relatively little progress toward the prevention and treatment of the wide spectrum of HAdV-associated diseases. The understanding of species-specific features of viral pathogenesis, or of the mechanisms underlying the establishment of latency and reactivation, is still limited. This group of viruses currently comprises 7 species, 51 serotypes, and 116 unique genotypes. This complexity manifests with a challenging pathophenotypic diversity. Some types are highly virulent, and others do not seem to cause disease in immunocompetent hosts. The assessment of viral load in blood and respiratory specimens has well-acknowledged clinical utility, but the lack of virus typing capabilities easily implementable in clinical laboratories represents a lingering major limitation to the interpretation of positive tests. Some HAdV infections do have severe consequences for both immunocompetent and immunocompromised patients, and the understanding of why this is the case will require more research. Clinical isolates and collections of positive specimens can provide unique resources to investigate the molecular bases of viral virulence and fitness and also help gather information of spatial-temporal patterns of viral circulation in susceptible communities, but they are extremely scarce. Clinical laboratories are underutilized interfaces between patients and academic scientists and have, therefore, a high potential to become valuable collaborators in research moving forward.

Surface modification of PVDF ultrafiltration membranes using spacer arms and synthetic receptors for virus capturing and separation.

Although membrane technology has demonstrated outstanding pathogen removal capabilities, current commercial membranes are insufficient for removing small viruses at trace levels due to certain limitations. The theoretical and practical significance of developing a new form of hydrophilic, anti-fouling, and virus-specific ultra-purification membrane with high capturing and separation efficiency, stability, and throughput for water treatment is of the utmost importance. In this study, molecularly imprinted membranes (MIMs) were fabricated from polyvinylidene fluoride (PVDF) membranes utilizing novel surface hydrophilic modification techniques, followed by the immobilization of virus-specific molecularly imprinted nanoparticles (nanoMIPs) as synthetic receptors. Three distinct membrane functionalization strategies were established and optimized for the first time: membrane functionalization with (i) polyethyleneimine (PEI) and dopamine (DOP), (ii) PEI and 3-(chloropropyl)-trimethoxysilane (CTS), and (iii) chitosan (CS). Hydrophilicity was enhanced significantly as a result of these modification strategies. Additionally, the modifications enabled spacer arms between the membrane surface and the nanoMIPs to decrease steric hindrance. The surface chemistry, morphology, and membrane performance results from the characterization analysis of the MIMs demonstrated excellent hydrophilicity (e.g., the functionalized membrane presented 37.84° while the unmodified bare membrane exhibited 128.94° of water contact angle), higher permeation flux (145.96 L m-2 h-1 for the functionalized membrane), excellent uptake capacity (up to 99.99 % for PEI-DOP-MIM and CS-MIM), and recovery (more than 80 % for PEI-DOP-MIM). As proof of concept, the cutting-edge MIMs were able to eliminate the model adenoviruses up to 99.99 % from water. The findings indicate that the novel functionalized PVDF membranes hold promise for implementation in practical applications for virus capture and separation.

Epidemiological impact of human adenovirus as causative agent of respiratory infections: An Italian multicentre retrospective study, 2022-2023.

Human adenoviruses are the causative agents of 5-7% of viral respiratory infections, mainly caused by species B and C. They can infect all age groups, but children are usually at high risk of infections. Adenovirus epidemiology is well documented in East-Asian countries but little is known about adenovirus circulation in Europe in recent years. This multicentre retrospective study aimed to investigate the circulation and molecular epidemiology of hAdVs. This surveillance collected a total of 54463 respiratory specimens between January 1, 2022 and June 20, 2023 were tested for the presence of respiratory viruses. Our results showed that adenovirus was detected in 6.6 % of all cases of acute respiratory infection included in the study and the median age of positive patients was 3 years, with male children in 1-2 years age group being the most affected. 43.5 % of adenovirus cases were co-infected with at least one other respiratory virus, and rhinovirus was co-detected in 54 % of cases. Genotyping of adenovirus allowed the identification of 6 different genotypes circulating in Italy, among which type B3 was the most frequently detected.

High frequency of co-infection between SARS-CoV-2 and respiratory Adenoviruses in the Pediatric population in Hamadan, Iran.

Introduction: The presence of other respiratory pathogens in patients with SARS-CoV-2 infection has been described as a striking feature. However, data on adenovirus co-infection rates and clinical impacts in COVID-19 patients is limited. The purpose of this study is to compare the prevalence of respiratory adenoviruses in children under 15 years of age in Positive and Negative SARS-CoV-2 patients. Methodology: From September 2020 to January 2021, nasopharyngeal swabs were obtained from 280 patients below 15 years old with influenza-like infection symptoms suspected to be COVID-19 and referred to hospitals in Hamadan province. Nucleic acid was extracted using a High Pure Viral Nucleic acid extraction kit for both viral RNA and DNA. Reverse transcription real-time PCR for detecting SARS-CoV-2 and Real-time PCR for Human Adenoviruses were used. Results: Out of 280 examined samples, 11.7% tested positive for AdV, of which 18 samples originated from the SARS-CoV-2 positive group and 15 samples were from the SARS-CoV-2 negative group. Of 18 co-infected samples, which were categorized in three different ranges of age including, 0-5, 6-10, and 11-15 years old were 11, 4, and 3 patients respectively. Also, 14 patients were hospitalized. Compared with AdV-positive patients, children with Co-infection with SARS CoV-2 had lower levels of white blood cell (WBC) count while erythrocyte sedimentation rate (ESR) and C-reaction protein (CRP) had increased levels. Conclusion: We report a substantial burden of AdV co-infection in pediatric COVID-19 patients. This study revealed most AdV infections lead to hospitalization and change in paraclinical parameters.

A Novel Multiplex LAMP Assay for the Detection of Respiratory Human Adenoviruses.

Human adenoviruses (HAdVs) are common pathogens that are associated with a variety of diseases, including respiratory tract infections (RTIs). Without reliable, fast, and cost-effective detection methods for HAdVs, patients may be misdiagnosed and inappropriately treated. To address this problem, we have developed a multiplex loop-mediated isothermal amplification (LAMP) assay for the detection of the species Human adenovirus B (HAdV-B), Human adenovirus C (HAdV-C) and Human adenovirus E (HAdV-E) that cause RTIs. This multiplexing approach is based on the melting curve analysis of the amplicons with a specific melting temperature for each HAdV species. Without the need for typing of HAdVs, the LAMP results can be visually detected using colorimetric analysis. The assay reliably detects at least 375 copies of HAdV-B and -C and 750 copies of HAdV-E DNA per reaction in less than 35 min at 60 °C. The designed primers have no in silico cross-reactivity with other human respiratory pathogens. Validation on 331 nasal swab samples taken from patients with RTIs showed a 90-94% agreement rate with our in-house multiplex quantitative polymerase chain reaction (qPCR) method. Concordance between the quantitative and visual LAMP was 99%. The novel multiplexed LAMP could be an alternative to PCR for diagnostic purposes, saving personnel and equipment time, or could be used for point-of-care testing.

Expanding the Scope of Adenoviral Vectors by Utilizing Novel Tools for Recombination and Vector Rescue.

Recombinant adenoviruses are widely used in clinical and laboratory applications. Despite the wide variety of available sero- and genotypes, only a fraction is utilized in vivo. As adenoviruses are a large group of viruses, displaying many different tropisms, immune epitopes, and replication characteristics, the merits of translating these natural benefits into vector applications are apparent. This translation, however, proves difficult, since while research has investigated the application of these viruses, there are no universally applicable rules in vector design for non-classical adenovirus types. In this paper, we describe a generalized workflow that allows vectorization, rescue, and cloning of all adenoviral species to enable the rapid development of new vector variants. We show this using human and simian adenoviruses, further modifying a selection of them to investigate their gene transfer potential and build potential vector candidates for future applications.

Tat-dependent conditionally replicating adenoviruses expressing diphtheria toxin A for specifically killing HIV-1-infected cells.

HIV-1 infection remains a public health problem with no cure. Although antiretroviral therapy (ART) is effective for suppressing HIV-1 replication, it requires lifelong drug administration due to a stable reservoir of latent proviruses and may cause serious side effects and drive the emergence of drug-resistant HIV-1 variants. Gene therapy represents an alternative approach to overcome the limitations of conventional treatments against HIV-1 infection. In this study, we constructed and investigated the antiviral effects of an HIV-1 Tat-dependent conditionally replicating adenovirus, which selectively replicates and expresses the diphtheria toxin A chain (Tat-CRAds-DTA) in HIV-1-infected cells both in vitro and in vivo. We found that Tat-CRAds-DTA could specifically induce cell death and inhibit virus replication in HIV-1-infected cells mediated by adenovirus proliferation and DTA expression. A low titer of progeny Tat-CRAds-DTA was also detected in HIV-1-infected cells. In addition, Tat-CRAds-DTA showed no apparent cytotoxicity to HIV-1-negative cells and demonstrated significant therapeutic efficacy against HIV-1 infection in a humanized mouse model. The findings in this study highlight the potential of Tat-CRAds-DTA as a new gene therapy for the treatment of HIV-1 infection.

Human adenoviruses in children with gastroenteritis: a systematic review and meta-analysis.

PURPOSE: Human adenoviruses (HAdVs) have always been suggested as one of the main causes of gastroenteritis in children. However, no comprehensive report on the global epidemiology of these viruses in pediatric gastroenteritis is available. METHODS: A systematic search was conducted to obtain published papers from 2003 to 2023 in three main databases PubMed, Scopus, and Web of Science. RESULTS: The estimated global pooled prevalence of HAdV infection in children with gastroenteritis was 10% (95% CI: 9-11%), with a growing trend after 2010. The highest prevalence was observed in Africa (20%, 95% CI: 14-26%). The prevalence was higher in inpatients (11%; 95% CI: 8-13%) and patients aged 5 years old and younger (9%; 95% CI: 7-10%). However, no significant difference was observed between male and female patients (P = 0.63). The most prevalent species was found to be the species F (57%; 95% CI: 41-72%). The most common HAdVs observed in children with gastroenteritis were types 40/41, 38, and 2. Analysis of case-control studies showed an association between HAdV and gastroenteritis in children (OR: 2.28, 95% CI; 1.51-3.44). CONCLUSION: This study provided valuable insights into the importance of HAdVs in children with gastroenteritis, especially in hospitalized and younger children. The results can be used in future preventive measurements and the development of effective vaccines.

Molecular characterization of human adenoviruses associated with pediatric respiratory infections in Karachi, Pakistan.

Human adenoviruses (HAdVs) are a diverse group of viruses associated with respiratory infections in humans worldwide. However, there is a lack of research on the genetic diversity and epidemiology of HAdVs in Pakistan. This study characterized HAdVs in pediatric patients with respiratory tract infections in Karachi, Pakistan, between 2022 and 2023. We analyzed 762 nasopharyngeal samples of children ≤ 5 years. DNA extraction, followed by PCR targeting E2B and hexon genes, was carried out. Data analysis was performed on SPSS 25.0, and phylogenetic analysis of hexon gene was performed on MEGA 11. HAdV was detected in 7.34% (56/762) of patients round the year, but at a significantly higher rate during the winter season. Age was insignificantly associated with HAdV incidence (p = 0.662), but more than 62.5% (35/56) of positive cases were younger than 10 months. The circulating HAdVs were identified as six different types from species B (78.57%) and C (21.42%), with the majority of isolates found to be like B3. HAdV was found to be co-infected with bocavirus (5.4%) and measles (7.14%). These findings revealed a high frequency and genetic diversity of respiratory HAdVs in Karachi, Pakistan. We conclude that periodic and continuous surveillance of adenoviruses and other respiratory pathogens is necessary to improve the prognosis and management of respiratory diseases, thereby reducing the child mortality rate in Pakistan.

Indirubin-3'-monoxime exhibits potent antiviral and anti-inflammatory effects against human adenoviruses in vitro and in vivo.

Human adenovirus (HAdV) infection is a major cause of respiratory disease, yet no antiviral drugs have been approved for its treatment. Herein, we evaluated the antiviral and anti-inflammatory effects of cyclin-dependent protein kinase (CDK) inhibitor indirubin-3'-monoxime (IM) against HAdV infection in cells and a transgenic mouse model. After evaluating its cytotoxicity, cytopathic effect reduction, antiviral replication kinetics, and viral yield reduction assays were performed to assess the anti-HAdV activity of IM. Quantitative real-time polymerase chain reaction (qPCR), quantitative reverse transcription PCR (qRT-PCR), and western blotting were used to assess the effects of IM on HAdV DNA replication, transcription, and protein expression, respectively. IM significantly inhibited HAdV DNA replication as well as E1A and Hexon transcription, in addition to significantly suppressing the phosphorylation of the RNA polymerase II C-terminal domain (CTD). IM mitigated body weight loss, reduced viral burden, and lung injury, decreasing cytokine and chemokine secretion to a greater extent than cidofovir. Altogether, IM inhibits HAdV replication by downregulating CTD phosphorylation to suppress viral infection and corresponding innate immune reactions as a promising therapeutic agent.

m6A Methylation in Regulation of Antiviral Innate Immunity.

The epitranscriptomic modification m6A is a prevalent RNA modification that plays a crucial role in the regulation of various aspects of RNA metabolism. It has been found to be involved in a wide range of physiological processes and disease states. Of particular interest is the role of m6A machinery and modifications in viral infections, serving as an evolutionary marker for distinguishing between self and non-self entities. In this review article, we present a comprehensive overview of the epitranscriptomic modification m6A and its implications for the interplay between viruses and their host, focusing on immune responses and viral replication. We outline future research directions that highlight the role of m6A in viral nucleic acid recognition, initiation of antiviral immune responses, and modulation of antiviral signaling pathways. Additionally, we discuss the potential of m6A as a prognostic biomarker and a target for therapeutic interventions in viral infections.

Radiographic findings of adenoviral pneumonia in children.

OBJECTIVE: Adenovirus pneumonia is a common cause of community-acquired pneumonia in children and can mimic bacterial pneumonia, but there are few publications on its radiographic features. This study has evaluated the chest radiography findings of community-acquired adenovirus pneumonia in children. The frequency of radiological findings mimicking bacterial pneumonia was investigated. The clinical features of patients with adenovirus pneumonia possessing radiological findings mimicking bacterial pneumonia were also evaluated. MATERIALS AND METHODS: The chest radiographs of patients diagnosed with adenovirus pneumonia were retrospectively reviewed. The chest radiographs were interpreted independently by a pediatric infectious disease specialist and a pediatric radiologist. Chest radiography findings mimicking bacterial pneumonia (bacterial-like) were specified as consolidation +/- pleural effusion. Other findings on chest radiography or a completely normal chest X-ray were specified as findings that were compatible with "typical viral pneumonia". RESULTS: A total of 1407 patients were positive for adenovirus with respiratory multiplex PCR. The 219 patients who met the study criteria were included in the study. Chest radiographs were normal in 58 (26.5 %) patients. The chest radiograph findings mimicked bacterial pneumonia in 41 (18.7 %) patients. CONCLUSION: Adenovirus pneumonia occurs predominantly in children aged five years and younger, as with other viral pneumonias. The radiographic findings in adenovirus pneumonia are predominantly those seen in viral pneumonia. Increasing age and positivity for only adenovirus without other viruses on respiratory multiplex PCR were associated with the chest radiograph being more likely to be "bacterial-like". Adenovirus may lead to lobar/segmental consolidation at a rate that is not very rare.

Non-Polio Enteroviruses Isolated by Acute Flaccid Paralysis Surveillance Laboratories in the Russian Federation in 1998-2021: Distinct Epidemiological Features of Types.

More than 100 types of non-polio enteroviruses (NPEVs) are ubiquitous in the human population and cause a variety of symptoms ranging from very mild to meningitis and acute flaccid paralysis (AFP). Much of the information regarding diverse pathogenic properties of NPEVs comes from the surveillance of poliovirus, which also yields NPEV. The analysis of 265 NPEV isolations from 10,433 AFP cases over 24 years of surveillance and more than 2500 NPEV findings in patients without severe neurological lesions suggests that types EV-A71, E13, and E25 were significantly associated with AFP. EV-A71 was also significantly more common among AFP patients who had fever at the onset and residual paralysis compared to all AFP cases. In addition, a significant disparity was noticed between types that were common in humans (CV-A2, CVA9, EV-A71, E9, and E30) or in sewage (CVA7, E3, E7, E11, E12, and E19). Therefore, there is significant evidence of non-polio viruses being implicated in severe neurological lesions, but further multicenter studies using uniform methodology are needed for a definitive conclusion.

Identification of Large Adenovirus Infection Outbreak at University by Multipathogen Testing, South Carolina, USA, 2022.

Using multipathogen PCR testing, we identified 195 students with adenovirus type 4 infections on a university campus in South Carolina, USA, during January-May 2022. We co-detected other respiratory viruses in 43 (22%) students. Continued surveillance of circulating viruses is needed to prevent virus infection outbreaks in congregate communities.

Development of mitochondrial gene-editing strategies and their potential applications in mitochondrial hereditary diseases: a review.

Mitochondrial DNA (mtDNA) is a critical genome contained within the mitochondria of eukaryotic cells, with many copies present in each mitochondrion. Mutations in mtDNA often are inherited and can lead to severe health problems, including various inherited diseases and premature aging. The lack of efficient repair mechanisms and the susceptibility of mtDNA to damage exacerbate the threat to human health. Heteroplasmy, the presence of different mtDNA genotypes within a single cell, increases the complexity of these diseases and requires an effective editing method for correction. Recently, gene-editing techniques, including programmable nucleases such as restriction endonuclease, zinc finger nuclease, transcription activator-like effector nuclease, clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated 9 and base editors, have provided new tools for editing mtDNA in mammalian cells. Base editors are particularly promising because of their high efficiency and precision in correcting mtDNA mutations. In this review, we discuss the application of these techniques in mitochondrial gene editing and their limitations. We also explore the potential of base editors for mtDNA modification and discuss the opportunities and challenges associated with their application in mitochondrial gene editing. In conclusion, this review highlights the advancements, limitations and opportunities in current mitochondrial gene-editing technologies and approaches. Our insights aim to stimulate the development of new editing strategies that can ultimately alleviate the adverse effects of mitochondrial hereditary diseases.

Higher affinities of fibers with cell receptors increase the infection capacity and virulence of human adenovirus type 7 and type 55 compared to type 3.

IMPORTANCE: HAdV-3, -7, and -55 are the predominant types causing acute respiratory disease outbreaks and can lead to severe and fatal pneumonia in children and adults. In recent years, emerging or re-emerging strains of HAdV-7 and HAdV-55 have caused multiple outbreaks globally in both civilian and military populations, drawing increased attention. Clinical studies have reported that HAdV-7 and HAdV-55 cause more severe pneumonia than HAdV-3. This study aimed to investigate the mechanisms explaining the higher severity of HAdV-7 and HAdV-55 infection compared to HAdV-3 infection. Our findings provided evidence linking the receptor-binding protein fiber to stronger infectivity of the strains mentioned above by comparing several fiber-chimeric or fiber-replaced adenoviruses. Our study improves our understanding of adenovirus infection and highlights potential implications, including in novel vector and vaccine development.

Metagenomic and Molecular Detection of Novel Fecal Viruses in Free-Ranging Agile Wallabies.

The agile wallaby (Notamacropus agilis) is one of the most abundant marsupial species in northern Queensland and a competent host for the zoonotic Ross River virus. Despite their increased proximity and interactions with humans, little is known about the viruses carried by these animals, and whether any are of conservation or zoonotic importance. Metagenomics and molecular techniques were used in a complementary manner to identify and characterize novel viruses in the fecal samples of free-ranging agile wallabies. We detected a variety of novel marsupial-related viral species including agile wallaby atadenovirus 1, agile wallaby chaphamaparvovirus 1-2, agile wallaby polyomavirus 1-2, agile wallaby associated picobirnavirus 1-9, and a known macropod gammaherpesvirus 3. Phylogenetic analyses indicate that most of these novel viruses would have co-evolved with their hosts (agile wallabies). Additionally, non-marsupial viruses that infect bacteria (phages), plants, insects, and other eukaryotes were identified. This study highlighted the utility of non-invasive sampling as well as the integration of broad-based molecular assays (consensus PCR and next generation sequencing) for monitoring the emergence of potential pathogenic viruses in wildlife species. Furthermore, the novel marsupial viruses identified in this study will enrich the diversity of knowledge about marsupial viruses, and may be useful for developing diagnostics and vaccines.

Immunogenicity in Mice Immunized with Recombinant Adenoviruses Expressing Varicella-Zoster Virus Envelope Glycoprotein E.

Herpes zoster (HZ) is a disease caused by the reactivation of latent varicella-zoster virus (VZV). The subunit vaccine, Shingrix®, and live attenuated vaccine, Zostavax®, could be used as an HZ vaccine that prevents HZ from being developed due to the reactivation of latent VZV in the sensory ganglia due to aging, stress or immunosuppression. In this study, the recombinant adenoviruses rChAd63/gE expressing glycoprotein E (gE) of VZV based on chimpanzee adenovirus serotype 63 (ChAd63) were constructed and investigated for the immunogenicity of different immune pathways in C57BL/6 mice. The results showed similar CD4+ T and CD8+ T cell responses to Shingrix® were induced in mice vaccinated using rChAd63/gE via different immune pathways. This study elucidates that recombinant adenoviruses expressing VZV gE could be appropriate for further development as a new HZ vaccine candidate via different immune pathways.

Toward the understanding of DSG2 and CD46 interaction with HAdV-11 fiber, a super-complex analysis.

IMPORTANCE: The main limitation of oncolytic vectors is neutralization by blood components, which prevents intratumoral administration to patients. Enadenotucirev, a chimeric HAdV-11p/HAdV-3 adenovirus identified by bio-selection, is a low seroprevalence vector active against a broad range of human carcinoma cell lines. At this stage, there's still some uncertainty about tropism and primary receptor utilization by HAdV-11. However, this information is very important, as it has a direct influence on the effectiveness of HAdV-11-based vectors. The aim of this work is to determine which of the two receptors, DSG2 and CD46, is involved in the attachment of the virus to the host, and what role they play in the early stages of infection.

Postinfectious bronchiolitis obliterans in children: case series at a pediatric hospital in Peru.

BACKGROUND: Postinfectious bronchiolitis obliterans is a rare lung disease; there are limited reports in South America. CASE REPORT: We report 10 patients with this disease diagnosed at the Instituto Nacional de Salud del Niño-Breña (Lima-Peru). The median age at diagnosis was 19 months and all patients had a history of severe acute respiratory infection. The most frequent symptoms were cough, respiratory distress, wheezing, and hypoxemia. The mosaic attenuation pattern was the most frequent on the tomography. All the patients had positive serology for adenovirus. The treatment received was methylprednisolone pulses, azithromycin, hydroxychloroquine, and inhaled corticosteroids. No patient died during the follow-up. CONCLUSIONS: In previously healthy children with a history of severe acute respiratory infection and persistent bronchial obstructive symptoms, the diagnosis of postinfectious bronchiolitis obliterans should be considered. This is the first report in Peru with a therapeutic regimen adapted to our institution.

INTRODUCCIÓN: La bronquiolitis obliterante postinfecciosa es una enfermedad pulmonar poco frecuente; existen limitados reportes en Sudamérica. CASO CLÍNICO: En esta serie se reportan 10 pacientes con esta enfermedad diagnosticados en el Instituto Nacional de Salud del Niño-Breña (Lima-Perú). La mediana de edad al diagnóstico fue de 19 meses. Todos los pacientes presentaron el antecedente de infección respiratoria aguda grave. Los síntomas más frecuentes fueron tos, dificultad respiratoria, sibilancias e hipoxemia; el patrón de atenuación en mosaico fue la característica más frecuente en la tomografía. Todos tenían serología positiva para adenovirus. Se administró tratamiento con pulsos de metilprednisolona, azitromicina, hidroxicloroquina y corticoides inhalados. Ningún paciente falleció durante el seguimiento. CONCLUSIONES: En los niños previamente sanos con antecedente de infección respiratoria aguda grave y sintomatología obstructivo bronquial persistente se debe considerar el diagnóstico de bronquiolitis obliterante postinfecciosa. Este es el primer reporte en Perú con un régimen terapéutico adaptado a nuestra institución.