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.

A significant outbreak of respiratory human adenovirus infections among children aged 3-6 years in Hokkaido, Japan, in 2023.

Human adenovirus (HAdV) infections present diverse clinical manifestations upon infecting individuals, with respiratory infections predominating in children. We surveyed pediatric hospitalizations due to respiratory HAdV infections across 18 hospitals in Hokkaido Prefecture, Japan, from July 2019 to March 2024, recording 473 admissions. While hospitalizations remained below five cases per week from July 2019 to September 2023, a notable surge occurred in late October 2023, with weekly admissions peaking at 15-20 cases from November to December. There were dramatic shifts in the age distribution of hospitalized patients: during 2019-2021, 1-year-old infants and children aged 3-6 years represented 51.4%-54.8% and 4.1%-13.3%, respectively; however, in 2023-2024, while 1-year-old infants represented 19.0%-20.1%, the proportion of children aged 3-6 years increased to 46.2%-50.0%. Understanding the emergence of significant outbreaks of respiratory HAdV infections and the substantial changes in the age distribution of hospitalized cases necessitates further investigation into the circulating types of HAdV in Hokkaido Prefecture and changes in children's neutralizing antibody titers against HAdV.

An Observational Study Suggests That Natural HAdV-36 Infection Decreases Blood Glucose Levels without Affecting Insulin Levels in Obese Young Subjects.

Human adenovirus-36 (HAdV-36) infection has been linked to obesity, low lipid levels, and improvements in blood glucose levels and insulin sensitivity in animal models and humans, although epidemiological studies remain controversial. Therefore, this study investigated the relationship between HAdV-36 seropositivity and glycemic control in youths. This observational study examined 460 youths (246 with normal weight and 214 obese subjects). All participants underwent assessments for anthropometry, blood pressure, circulating fasting levels of glucose, lipids, insulin, and anti-HAdV-36 antibodies; additionally, the homeostatic model assessment of insulin resistance (HOMA-IR) was calculated. In all, 57.17% of the subjects were HAdV-36 seropositive. Moreover, HAdV-36 seroprevalence was higher in obese subjects compared to their normal weight counterparts (59% vs. 55%). BMI (33.1 vs. 32.3 kg/m2, p = 0.03), and waist circumference (107 vs. 104 cm, p = 0.02), insulin levels (21 vs. 16.3 µU/mL, p = 0.003), and HOMA-IR (4.6 vs. 3.9, p = 0.02) were higher in HAdV-36-positive subjects with obesity compared to seronegative subjects. In the obese group, HAdV-36 seropositivity was associated with a reducing effect in blood glucose levels in a model adjusted for total cholesterol, triglyceride levels, age and sex (ß = -10.44, p = 0.014). Furthermore, a statistically significant positive relationship was observed between HAdV-36 seropositivity and insulin levels in the obesity group. These findings suggest that natural HAdV-36 infection improves glycemic control but does not ameliorate hyperinsulinemia in obese subjects.

Influence of Seropositivity against Adenovirus-36 on the Risk of Obesity and Insulin Resistance in the Child Population of Southern Chile.

Background: Previous infection with Adenovirus-36 (HAdv-D36) has been associated with adipogenesis and glycemic regulation in cell culture and animal models. In humans, HAdv-D36 antibodies correlate with increased obesity risk yet paradoxically enhance glycemic control across various demographics. This study assesses the association of HAdv-D36 seropositivity with obesity, lipid, and glycemic profiles among school-aged children. Methods: We evaluated 208 children aged 9-13, categorized by BMI z-scores into normal weight (-1 to +1), overweight (+1 to +2), and obese (>+3). Assessments included anthropometry, Tanner stage for pubertal development, and biochemical tests (relating to lipids, glucose, and insulin), alongside HAdv-D36 seropositivity checked via ELISA. Insulin resistance was gauged using Chilean pediatric criteria. Results: The cohort displayed a high prevalence of overweight/obesity. HAdv-D36 seropositivity was 5.4%, showing no correlation with nutritional status. Additionally, no link between HAdv-D36 seropositivity and lipid levels was observed. Notably, insulin levels and HOMA-RI were significantly lower in HAdv-D36 positive children (p < 0.001). No cases of insulin resistance were reported in the HAdv-D36 (+) group in our population. Conclusions: HAdv-D36 seropositivity appears to decrease insulin secretion and resistance, aligning with earlier findings. However, no association with obesity development was found in the child population of southern Chile.

Alpha-defensin binding expands human adenovirus tropism.

Mammalian α-defensins are a family of abundant effector peptides of the mucosal innate immune system. Although primarily considered to be antimicrobial, α-defensins can increase rather than block infection by certain prominent bacterial and viral pathogens in cell culture and in vivo. We have shown previously that exposure of mouse and human adenoviruses (HAdVs) to α-defensins is able to overcome competitive inhibitors that block cell binding, leading us to hypothesize a defensin-mediated binding mechanism that is independent of known viral receptors. To test this hypothesis, we used genetic approaches to demonstrate that none of several primary receptors nor integrin co-receptors are needed for human α-defensin-mediated binding of HAdV to cells; however, infection remains integrin dependent. Thus, our studies have revealed a novel pathway for HAdV binding to cells that bypasses viral primary receptors. We speculate that this pathway functions in parallel with receptor-mediated entry and contributes to α-defensin-enhanced infection of susceptible cells. Remarkably, we also found that in the presence of α-defensins, HAdV tropism is expanded to non-susceptible cells, even when viruses are exposed to a mixture of both susceptible and non-susceptible cells. Therefore, we propose that in the presence of sufficient concentrations of α-defensins, such as in the lung or gut, integrin expression rather than primary receptor expression will dictate HAdV tropism in vivo. In summary, α-defensins may contribute to tissue tropism not only through the neutralization of susceptible viruses but also by allowing certain defensin-resistant viruses to bind to cells independently of previously described mechanisms.

High-Frequency Recombination of Human Adenovirus in Children with Acute Respiratory Tract Infections in Beijing, China.

Recombination events in human adenovirus (HAdV) have led to some new highly pathogenic or infectious types. It is vital to monitor recombinant HAdVs, especially in children with acute respiratory tract infections (ARIs). In the retrospective study, HAdV positive specimens were collected from pediatric patients with ARIs during 2015 to 2021, then typed by sequence analysis of the penton base, hexon and fiber gene sequence. For those with inconsistent typing results, a modified method with species-specific primer sets of a fiber gene sequence was developed to distinguish co-infections of different types from recombinant HAdV infections. Then, plaque assays combined with meta-genomic next-generation sequencing (mNGS) were used to reveal the HAdV genomic characteristics. There were 466 cases positive for HAdV DNA (2.89%, 466/16,097) and 350 (75.11%, 350/466) successfully typed with the most prevalent types HAdV-B3 (56.57%, 198/350) and HAdV-B7 (32.00%, 112/350), followed by HAdV-C1 (6.00%, 21/350). Among 35 cases (7.51%, 35/466) with inconsistent typing results, nine cases were confirmed as co-infections by different types of HAdVs, and 26 cases as recombinant HAdVs in six genetic patterns primarily clustered to species C (25 cases) in pattern 1-5, or species D (1 case) in pattern 6. The novel recombinant HAdV of species D was identified with multiple recombinant events among HAdV-D53, HAdV-D64, and HAdV-D8, and officially named as HAdV-D115. High-frequency recombination of HAdVs in six genetic recombination patterns were identified among children with ARIs in Beijing. Specifically, there is a novel Adenovirus D human/CHN/S8130/2023/115[P22H8F8] designed as HAdV D115.

NPP-669, a prodrug of cidofovir, is highly efficacious against human adenovirus infection in the permissive Syrian hamster model.

Human adenoviruses can cause serious, disseminated infections in immunocompromised patients. For pediatric allogeneic stem cell transplant patients, the case fatality rate can reach 80%. Still, there is no available antiviral drug that is specifically approved by the Food and Drug Administration for the treatment of adenovirus infections. To fill this pressing medical need, we have developed NPP-669, a prodrug of cidofovir with broad activity against double-stranded DNA viruses, including adenoviruses. Here, we report on the in vivo anti-adenoviral efficacy of NPP-669. Using the immunosuppressed Syrian hamster as the model, we show that NPP-669 is highly efficacious when dosed orally at 1 mg/kg and 3 mg/kg. In a delayed administration experiment, NPP-669 was more effective than brincidofovir, a similar compound that reached Phase III clinical trials. Furthermore, parenteral administration of NPP-669 increased its efficacy approximately 10-fold compared to oral dosing without apparent toxicity, suggesting that this route may be preferable in a hospital setting. Based on these findings, we believe that NPP-669 is a promising new compound that needs to be further investigated.

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.

Clinical manifestations, prognostic factors, and outcomes of adenovirus pneumonia after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Severe pneumonia is one of the most important causes of mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Adenovirus (ADV) is a significant cause of severe viral pneumonia after allo-HSCT, and we aimed to identify the clinical manifestations, prognostic factors, and outcomes of ADV pneumonia after allo-HSCT. METHODS: Twenty-nine patients who underwent allo-HSCT at the Peking University Institute of Hematology and who experienced ADV pneumonia after allo-HSCT were enrolled in this study. The Kaplan-Meier method was used to estimate the probability of overall survival (OS). Potential prognostic factors for 100-day OS after ADV pneumonia were evaluated through univariate and multivariate Cox regression analyses. RESULTS: The incidence rate of ADV pneumonia after allo-HSCT was approximately 0.71%. The median time from allo-HSCT to the occurrence of ADV pneumonia was 99 days (range 17-609 days). The most common clinical manifestations were fever (86.2%), cough (34.5%) and dyspnea (31.0%). The 100-day probabilities of ADV-related mortality and OS were 40.4% (95% CI 21.1%-59.7%) and 40.5% (95% CI 25.2%-64.9%), respectively. Patients with low-level ADV DNAemia had lower ADV-related mortality and better OS than did those with high-level (≥ 106 copies/ml in plasma) ADV DNAemia. According to the multivariate analysis, high-level ADV DNAemia was the only risk factor for intensive care unit admission, invasive mechanical ventilation, ADV-related mortality, and OS after ADV pneumonia. CONCLUSIONS: We first reported the prognostic factors and confirmed the poor outcomes of patients with ADV pneumonia after allo-HSCT. Patients with high-level ADV DNAemia should receive immediate and intensive therapy.

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.

Activation of the RIG-I/MAVS Signaling Pathway during Human Adenovirus Type 3 Infection Impairs the Pro-Inflammatory Response Induced by Secondary Infection with Staphylococcus aureus.

The exacerbation of pneumonia in children with human adenovirus type 3 (HAdV-3E) is secondary to a Staphylococcus aureus (S. aureus) infection. The influence of host-pathogen interactions on disease progression remains unclear. It is important to note that S. aureus infections following an HAdV-3E infection are frequently observed in clinical settings, yet the underlying susceptibility mechanisms are not fully understood. This study utilized an A549 cell model to investigate secondary infection with S. aureus following an HAdV-3E infection. The findings suggest that HAdV-3E exacerbates the S. aureus infection by intensifying lung epithelial cell damage. The results highlight the role of HAdV-3E in enhancing the interferon signaling pathway through RIG-I (DDX58), resulting in the increased expression of interferon-stimulating factors like MX1, RSAD2, and USP18. The increase in interferon-stimulating factors inhibits the NF-κB and MAPK/P38 pro-inflammatory signaling pathways. These findings reveal new mechanisms of action for HAdV-3E and S. aureus in secondary infections, enhancing our comprehension of pathogenesis.

DMSO and Its Role in Differentiation Impact Efficacy of Human Adenovirus (HAdV) Infection in HepaRG Cells.

Differentiated HepaRG cells are popular in vitro cell models for hepatotoxicity studies. Their differentiation is usually supported by the addition of dimethyl sulfoxide (DMSO), an amphipathic solvent widely used in biomedicine, for example, in potential novel therapeutic drugs and cryopreservation of oocytes. Recent studies have demonstrated drastic effects, especially on epigenetics and extracellular matrix composition, induced by DMSO, making its postulated inert character doubtful. In this work, the influence of DMSO and DMSO-mediated modulation of differentiation on human adenovirus (HAdV) infection of HepaRG cells was investigated. We observed an increase in infectivity of HepaRG cells by HAdVs in the presence of 1% DMSO. However, this effect was dependent on the type of medium used for cell cultivation, as cells in William's E medium showed significantly stronger effects compared with those cultivated in DMEM. Using different DMSO concentrations, we proved that the impact of DMSO on infectability was dose-dependent. Infection of cells with a replication-deficient HAdV type demonstrated that the mode of action of DMSO was based on viral entry rather than on viral replication. Taken together, these results highlight the strong influence of the used cell-culture medium on the performed experiments as well as the impact of DMSO on infectivity of HepaRG cells by HAdVs. As this solvent is widely used in cell culture, those effects must be considered, especially in screening of new antiviral compounds.

Expanding the adenovirus toolbox: reporter viruses for studying the dynamics of human adenovirus replication.

To streamline standard virological assays, we developed a suite of nine fluorescent or bioluminescent replication competent human species C5 adenovirus reporter viruses that mimic their parental wild-type counterpart. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. Moreover, they permit real-time non-invasive measures of viral load, replication dynamics, and infection kinetics over the entire course of infection, allowing measurements that were not previously possible. This suite of replication competent reporter viruses increases the ease, speed, and adaptability of standard assays and has the potential to accelerate multiple areas of human adenovirus research.IMPORTANCEIn this work, we developed a versatile toolbox of nine HAdV-C5 reporter viruses and validated their functions in cell culture. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. The utility of these reporter viruses could also be extended for use in 3D cell culture, organoids, live cell imaging, or animal models, and provides a conceptual framework for the development of new reporter viruses representing other clinically relevant HAdV species.

In Silico Intensive Analysis for the E4 Gene Evolution of Human Adenovirus Species D.

Adenovirus (Ad) is a ubiquitous pathogen capable of infecting a wide range of animals and humans. Human Adenovirus (HAdV) can cause severe infection, particularly in individuals with compromised immune systems. To date, over 110 types of HAdV have been classified into seven species from A to G, with the majority belonging to the human adenovirus species D (HAdV-D). In the HAdV-D, the most significant factor for the creation of new adenovirus types is homologous recombination between viral genes involved in determining the virus tropism or evading immune system of host cells. The E4 gene, consisting of seven Open Reading Frames (ORFs), plays a role in both the regulation of host cell metabolism and the replication of viral genes. Despite long-term studies, the function of each ORF remains unclear. Based on our updated information, ORF2, ORF3, and ORF4 have been identified as regions with relatively high mutations compared to other ORFs in the E4 gene, through the use of in silico comparative analysis. Additionally, we managed to visualize high mutation sections, previously undetectable at the DNA level, through a powerful amino acid sequence analysis tool known as proteotyping. Our research has revealed the involvement of the E4 gene in the evolution of human adenovirus, and has established accurate sequence information of the E4 gene, laying the groundwork for further research.

Liver injury and prolonged hospitalization as indicators of severity in patients with adenovirus infections.

BACKGROUND: Adenovirus (ADV) is a prevalent infective virus in children, accounting for around 5-10% of all cases of acute respiratory illnesses and 4-15% of pneumonia cases in children younger than five years old. Without treatment, severe ADV pneumonia could result in fatality rates of over 50% in cases of emerging strains or disseminated disease. This study aims to uncover the relationship of clinical indicators with primary ADV infection severity, regarding duration of hospitalization and liver injury. METHODS: In this retrospective study, we collected and analyzed the medical records of 1151 in-patients who met the inclusion and exclusion criteria. According to duration of hospitalization, all patients were divided into three groups. Then the difference and correlation of clinical indicators with ADV infection were analyzed, and the relationship among liver injury, immune cells and cytokines was evaluated. RESULTS: The study revealed that patients with a duration of hospitalization exceeding 14 days had the highest percentage of abnormalities across most indicators. This was in contrast to the patients with a hospitalization duration of either less than or equal to 7 days or between 7 and 14 days. Furthermore, correlation analysis indicated that a longer duration of body temperature of ≥ 39°C, bilateral lung lobes infiltration detected by X ray, abnormal levels of AST, PaO2, and SPO2, and a lower age were all predictive of longer hospital stays. Furthermore, an elevated AST level and reduced liver synthesis capacity were related with a longer hospital stay and higher ADV copy number. Additionally, AST/ALT was correlated positively with IFN-γ level and IFN-γ level was only correlated positively with CD4+ T cells. CONCLUSIONS: The study provided a set of predicting indicators for longer duration of hospitalization, which responded for primary severe ADV infection, and elucidated the possible reason for prolonged duration of hospitalization attributing to liver injury via higher ADV copy number, IFN-γ and CD4+ T cells, which suggested the importance of IFN-γ level and liver function monitoring for the patients with primary severe ADV infection.

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.

Activation of the NLRP3 inflammasome by human adenovirus type 7 L4 100-kilodalton protein.

Human adenovirus type 7 (HAdV-7) is a significant viral pathogen that causes respiratory infections in children. Currently, there are no specific antiviral drugs or vaccines for children targeting HAdV-7, and the mechanisms of its pathogenesis remain unclear. The NLRP3 inflammasome-driven inflammatory cascade plays a crucial role in the host's antiviral immunity. Our previous study demonstrated that HAdV-7 infection activates the NLRP3 inflammasome. Building upon this finding, our current study has identified the L4 100 kDa protein encoded by HAdV-7 as the primary viral component responsible for NLRP3 inflammasome activation. By utilizing techniques such as co-immunoprecipitation, we have confirmed that the 100 kDa protein interacts with the NLRP3 protein and facilitates the assembly of the NLRP3 inflammasome by binding specifically to the NACHT and LRR domains of NLRP3. These insights offer a deeper understanding of HAdV-7 pathogenesis and contribute to the development of novel antiviral therapies.

Genomic Evolution and Recombination Dynamics of Human Adenovirus D Species: Insights from Comprehensive Bioinformatic Analysis.

Human adenoviruses (HAdVs) can infect various epithelial mucosal cells, ultimately causing different symptoms in infected organ systems. With more than 110 types classified into seven species (A-G), HAdV-D species possess the highest number of viruses and are the fastest proliferating. The emergence of new adenovirus types and increased diversity are driven by homologous recombination (HR) between viral genes, primarily in structural elements such as the penton base, hexon and fiber proteins, and the E1 and E3 regions. A comprehensive analysis of the HAdV genome provides valuable insights into the evolution of human adenoviruses and identifies genes that display high variation across the entire genome to determine recombination patterns. Hypervariable regions within genetic sequences correlate with functional characteristics, thus allowing for adaptation to new environments and hosts. Proteotyping of newly emerging and already established adenoviruses allows for prediction of the characteristics of novel viruses. HAdV-D species evolved in a direction that increased diversity through gene recombination. Bioinformatics analysis across the genome, particularly in highly variable regions, allows for the verification or re-evaluation of recombination patterns in both newly introduced and pre-existing viruses, ultimately aiding in tracing various biological traits such as virus tropism and pathogenesis. Our research does not only assist in predicting the emergence of new adenoviruses but also offers critical guidance in regard to identifying potential regulatory factors of homologous recombination hotspots.