Detection of recombinant type of human adenovirus C composed of type 89 and type 5 in Aichi Prefecture, Japan, 2016-2019

Budding and secretion of HIV Gag–Env virus-like particles from recombinant human adenovirus infected cells

Background and Objectives:Human adenovirus type 8 is a highly contagious eye disease and is considered as the most common epidemic keratoconjunctivitis worldwide. The virus may alter the course of detection as mutations and recombination in surface antigens are associated with binding and pathogenesis in human adenovirus. The recognition of new recombinant human adenovirus has been based on sequencing of three genes, penton base, hexon and fiber.Materials and Methods:50 suspected samples of ocular keratoconjunctivitis were selected over 6 months. Following DNA extraction from isolates positive for cytopathic effect in each well, the complete sequences of hexon, fiber, and penton regions were performed on the genome of human adenovirus isolates using PCR. The sequences of capsid genes, including hexon, fiber, and penton were assessed to observe the evidence of recombination at the molecular level using genetic tools.Results:The results of nucleotide and amino acid sequence of 5/50 patients with epidemic keratoconjunctivitis positive for hypervariable region of hexon (132aa -449), hypervariable of knob fiber (183aa -362) and hypervariable penton (106aa -466) isolates showed nucleotide and amino acid identity of 98% and 99.41%, 99% and 100%, 95% and 99.72% with hexon, fiber and penton of human adenovirus 8 subtypes. The results of phylogenetic tree and Simplot of the entire sequences and hypervariable regions of isolated hexon, fiber and penton showed all the isolates of human adenovirus from Ahvaz, Iran, were clustered with human adenovirus 8A, B, E, P and J, subtypes isolated strains from different regions of the world.Conclusion:The results of this study revealed that the human adenovirus isolates from patients with epidemic keratoconjunctivitis were closed to human adenovirus 8A, B, E, P and J subtypes. To determine the emergence of new human adenovirus D8 subtypes strain, analysis of complete genome sequence of human adenovirus was required.

For 4 months from September 2008, 102 conjunctival swab specimens were collected for surveillance purposes from patients across Japan suspected of having epidemic keratoconjunctivitis (EKC). Human adenovirus (HAdV) DNA was detected in 61 samples by PCR, though the HAdV type for 6 of the PCR-positive samples could not be determined by phylogenetic analysis using a partial hexon gene sequence. Moreover, for 2 months from January 2009, HAdV strains with identical sequences were isolated from five conjunctival swab samples obtained from EKC patients in five different regions of Japan. For the analyses of the 11 samples mentioned above, we determined the nucleotide sequences of the entire penton base, hexon, and fiber genes and early 3 (E3) region, which are variable regions among HAdV types, and compared them to those of other HAdV species D strains. The nucleotide sequences of loops 1 and 2 in the hexons of all 11 samples showed high degrees of identity with those of the HAdV type 15 (HAdV-15) and HAdV-29 prototype strains. However, the fiber gene and E3 region sequences showed high degrees of identity with those of HAdV-9, and the penton base gene sequence showed a high degree of identity with the penton base gene sequences of HAdV-9 and -26. Moreover, the complete genome sequence of the 2307-S strain, which was isolated by viral culture from 1 of the 11 samples, was determined. The 2307-S strain was a recombinant HAdV between HAdV-9, -15, -26, -29, and/or another HAdV type; however, the recombination sites in the genome were not obvious. We propose that this virus is a novel intertypic recombinant, HAdV-15/29/H9, and may be an etiological agent of EKC.

Computational analysis and identification of an emergent human adenovirus pathogen implicated in a respiratory fatality

A number of novel recombinant human adenoviruses (HAdVs) have recently been identified through sequencing of the complete genomes. The recombinant HAdV sequences share similarity with other types in the major capsid genes, namely the hexon, penton base, and fiber genes, implying recombination events, which may result in escape from the immune response and the acquisition of different organotropisms. Therefore, a surveillance system of HAdVs that considers the effect of frequent recombination on genetic evolution in these genes must be constructed. In this study, we designed new primer sets that can amplify the partial penton base and fiber genes from species HAdV-A to HAdV-F and proteotype HAdVs on the basis of sequence analyses, including previously reported primers that amplify loop 1 of the hexon. Phylogenetic analysis through sequencing with these primers correctly classified clinical HAdV isolates in loop 1 of the hexon gene, the Arg-Gly-Asp (RGD) loop of the penton base gene, and the knob of the fiber gene, which contain neutralizing, hemagglutination, and receptor binding epitopes associated with immunogenicity and tissue tropisms of HAdVs. This study contributes to the accumulation of correct information regarding genetic diversity and evolution in the worldwide HAdV surveillance.

BackgroundAdenovirus is one of the most common causes of viral acute respiratory infections. To identify the types of human adenoviruses (HAdVs) causing respiratory illness in Beijing, a sentinel surveillance project on the viral aetiology of acute respiratory infection was initiated in 2011.Principal findingsThrough the surveillance project, 4617 cases of respiratory infections were identified during 2011-2013. Throat swabs (pharynx and tonsil secretions) were collected from all the patients, and 15 different respiratory viruses were screened by multiplex one-step PCR method. 45 were identified as adenovirus-positive from sporadic and outbreak cases of respiratory infection by a multiplex one-step RT-PCR method, and a total of 21 adenovirus isolates were obtained. Five HAdV types among three species, including HAdV-3 (species HAdV-B), HAdV-4 (species HAdV-E), HAdV-7 (species HAdV-B), HAdV-55 (species HAdV-B), and an undefined HAdV type (species HAdV-C) were identified. The comparison results of the penton base, hexon, and fiber gene sequences of the Beijing HAdV-3, HAdV-4, HAdV-7, and HAdV-55 strains in this study and those from the GenBank database indicated significant spatial and temporal conservation and stability of sequences within the genome; however, the phylogenetic relationship indicated that both strain BJ04 and strain BJ09 isolated in 2012 and 2013, respectively, may have recombined between HAdV-1 genome and HAdV-2 genome within species HAdV-C, indicating intraspecies recombination.ConclusionsThis study confirmed that at least 5 HAdV types including HAdV-3, HAdV-4, HAdV-7, HAdV-55 and an undefined HAdV type were co-circulating and were the causative agents of respiratory tract infections in recent years in Beijing. HAdV-3, HAdV-4, HAdV-7, and HAdV-55 showed the apparent stability of the genomes, while intraspecies recombination was identified in strain BJ04 and BJ09. The recombinants carrying penton base gene of HAdV-1 as well as hexon and fiber genes of HAdV-2 might be a novel type of HAdV worthy of further study.

Evolution of adenoviruses as gene therapy vectors.

Introduction. Recombinant adenoviruses are widely used in the development of vaccines for a variety of infectious diseases. Despite numerous clinical studies, only a few types of human (types 5 and 26) and simian (isolate Y25) adenoviruses are currently used to produce vaccine formulations. Different types of adenoviruses vary in their cellular tropism, which plays a key role in their ability to elicit an immune response. The aim of this study was to investigate the cellular tropism of the simian adenovirus type 25 in vitro and its biodistribution in vivo in comparison with human adenoviruses types 5 and 26. Materials and methods. The efficiency of in vitro transduction was evaluated on 15 different cell lines using recombinant adenovirus vectors expressing the enhanced green fluorescent protein (EGFP) reporter gene. In vivo biodistribution and bioluminescence imaging were evaluated in BALB/c mice after administration of recombinant adenoviral vectors encoding the luciferase reporter gene. The acute toxicity of a recombinant simian adenovirus type 25 vector was assessed in mice and rats following intramuscular or intravenous administration. Results. Recombinant simian adenovirus effectively transduces a wide range of cells. At the same time, a higher tropism to human glioblastoma cells (GL-6) was found compared to the other two studied adenoviruses. In vivo experiments have shown that recombinant adenoviruses are mainly localized at the injection site, and transgene expression persists for 21 days. Acute toxicity studies demonstrated that simian adenovirus type 25 vector was well-tolerated, with no animal deaths or detectable toxic effects. Conclusion. The new platform based on the recombinant simian adenovirus type 25 is not inferior to the existing and well-established delivery systems based on human adenovirus types 5 and 26. Due to its high level of gene transfer and favorable safety profile, the use of the simian adenovirus type 25 in medicine has the potential to offer many benefits for the development of vaccines against future infectious diseases.

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.

Background Human adenovirus (HAdV) is a common cause of acute respiratory illness (ARI) in children, ranging from asymptomatic to life-threatening disease. While previous studies have examined asymptomatic vs HAdV ARI infections, few have analyzed HAdV species and type. We compared frequencies of common HAdV ARI types among children with ARI vs. healthy controls (HCs). Comparison of demographics and HAdV detection between children with ARI and healthy controls: New Vaccine Surveillance Network 12/01/2016–11/30/2019 (N=1,393). Methods Data were analyzed from the New Vaccine Surveillance Network, a multicenter, prospective surveillance network at 7 U.S. children’s hospitals that enrolled children < 18 years with ARI (fever and/or respiratory symptoms) from emergency departments or inpatient settings during 12/01/16–11/30/19. HCs (ARI symptom free ≥3 days and no vomiting/diarrhea ≥14 days) were matched by age and near date of enrollment, and enrolled from outpatient clinics. Nasal and/or throat swabs were tested using molecular assays for HAdV and other respiratory viruses. HAdV-positive specimens were tested by real-time PCR for 11 common respiratory HAdV types representing species B, C, and E based on unique sequences in the hexon gene. Demographics, HAdV species, and HAdV types from HAdV-positive children with ARI were compared to those from HAdV-positive HCs. Percent of HAdV species detections stratified by ARI cases and healthy controls: New Vaccine Surveillance Network 12/01/2016–11/30/2019 Results 1,330 HAdV detections from ARI cases and 63 from HCs were included. Children with HAdV ARI were older, more frequently attended daycare, had higher viral load (based on cycle threshold value), and had more frequent viral co-detection compared to HCs (Table 1). HAdV-C predominated in both ARI and HC groups (Fig. 1); HAdV-C2 was most common, followed by HAdV-C1 (Fig. 2). Among HAdV-positive patients, those with ARI more frequently had HAdV-B detection than HCs (22.3% vs. 1.6%, p< 0.001). HAdV-positive HCs more frequently had HAdV-C detected than HAdV-positive ARI cases (95.2% vs 73.8%, p< 0.001). HAdV-C1 was more likely to be detected in HCs than those with ARI (28.9% vs. 41.3%, p=0.03), and HAdV-B3 was only detected in those with ARI. Percent of HAdV type detections stratified by ARI cases and healthy controls: NVSN, 12/01/2016–11/30/2019. Conclusion While HAdV-C was most frequent in all children, HAdV-B was more often detected in those with ARI than HCs, suggesting that different HAdV species and types have different symptom phenotypes, including HAdV-B3. Further investigations into the identification of specific HAdV types responsible for ARI are necessary. Disclosures Rangaraj Selvarangan, BVSc, PhD, D(ABMM), FIDSA, FAAM, Abbott: Grant/Research Support|Abbott: Honoraria|BioMerieux: Grant/Research Support|Cepheid: Grant/Research Support|Diasorin: Grant/Research Support|GSK: Advisor/Consultant|Hologic: Grant/Research Support|Luminex: Grant/Research Support|Qiagen: Grant/Research Support Christopher J. Harrison, MD, GSK: Grant/Research Support|Medscape: Honoraria|Merck: Grant/Research Support|Pfizer: Grant/Research Support|UpToDate: Honoraria Pedro A. Piedra, MD, Merck: Grant/Research Support|Novavax: Grant/Research Support|Sanofi-Pasteur: Grant/Research Support|Shionogi: Grant/Research Support Mary A. Staat, MD, MPH, Cepheid: Grant/Research Support|Merck: Grant/Research Support|Pfizer: Grant/Research Support|Up-To-Date: Honoraria Elizabeth P. Schlaudecker, MD, MPH, Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant Geoffrey A. Weinberg, MD, Inhalon: Advisor/Consultant|Merck & Company: Honoraria for textbook chapter preparation Janet A. Englund, MD, Abbvie: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Advisor/Consultant|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Advisor/Consultant|Pfizer: Board Member|Pfizer: Grant/Research Support|Pfizer: Speaker at meeting|SanofiPasteur: Advisor/Consultant|Shinogi: Advisor/Consultant James Chappell, MD, PhD, Merck: Grant/Research Support Natasha B. Halasa, MD, MPH, Merck: Grant/Research Support

Adenoviruses have increasingly been recognized as significant viral pathogens causing high morbidity and mortality especially among immunocompromised individuals such as transplant recipients and AIDS patients. Through the infection process, after the adenovirus fiber and penton are bonded to cell surface receptors through special amino acid moieties, secondary messengers activate protein kinases, pro-inflammatory cytokines and chemokines. Serotype and species specific antibodies also are induced. Recombinant human adenoviruses have been pivotal in the development of gene therapy strategies and have shown a great promise for the treatment of genetic disorders and malignancies. Recent studies have enlightened their harmful immunological effects dependent on fiber and hexon polypeptide structure and receptor binding. Pre-existing antibodies or those elicited by vectors neutralize input recombinant adenovirus particles rendering them ineffective. Mediators induce serious even lethal side effects and cytotoxic reactions which extinguish transgene expression. To overcome these difficulties new strategies are required in the application of recombinant adenoviruses to redirect vector entry from the natural receptors to alternative binding sites or using rare human or animal adenovirus fiber molecules to modify the native fiber structure by altering amino acid structure and creating chimeric fibers. This requires searching for, isolating and characterizing new serotypes, mutants or variants for new generation vectors. Human adenovirus 1 feline isolate (feline adenovirus) might fulfil these criteria.

Adenovirus type 41 lacks an RGD α v-integrin binding motif on the penton base and undergoes delayed uptake in A549 cells

Objective: To compare the clinical characteristics of different types of human adenovirus (HAdV) infection in hospitalized children with acute respiratory infection in Beijing, and to clarify the clinical necessity of adenovirus typing. Methods: In a cross-sectional study, 9 022 respiratory tract specimens collected from hospitalized children with acute respiratory infection from November 2017 to October 2019 in Affiliated Children's Hospital, Capital Institute of Pediatrics were screened for HAdV by direct immunofluorescence (DFA) and (or) nucleic acid detection. Then the Penton base, Hexon and Fiber gene of HAdV were amplified from HAdV positive specimens to confirm their HAdV types by phylogenetic tree construction. Clinical data such as laboratory results and imaging data were analyzed for children with predominate type HAdV infection using t, U, or χ2 test. Results: There were 392 cases (4.34%) positive for HAdV among 9 022 specimens from hospitalized children with acute respiratory infection. Among those 205 cases who were successfully typed, 131 were male and 74 were female, age of 22.6 (6.7, 52.5) months,102 cases (49.76%) were positive for HAdV-3 and 86 cases (41.95%), HAdV-7, respectively, while 17 cases were confirmed as HAdV-1, 2, 4, 6, 14 or 21. In comparison of clinical characteristics between the predominate HAdV type 7 and 3 infection, significant differences were shown in proportions of children with wheezing (10 cases (11.63%) vs. 25 cases (24.51%)), white blood cell count >15 ×109/L (4 cases (4.65%) vs.14 cases (13.73%)), white blood cell count <5×109/L (26 cases (30.23%) vs.11 cases (10.78%)), procalcitonin level>0.5 mg/L (43 cases (50.00%) vs. 29 cases (28.43%)), multilobar infiltration (45 cases (52.33%) vs.38 cases (37.25%)), pleural effusion (23 cases (26.74%) vs. 10 cases (9.80%)), and severe adenovirus pneumonia (7 cases (8.14%) vs. 2 cases (1.96%)) with χ²=5.11, 4.44, 11.16, 9.19, 4.30, 9.25, 3.91 and P=0.024, 0.035, 0.001, 0.002, 0.038, 0.002, 0.048, respectively, and also in length of hospital stay (11 (8, 15) vs. 7 (5, 13) d, Z=3.73, P<0.001). Conclusions: HAdV-3 and 7 were the predominate types of HAdV infection in hospitalized children with acute respiratory tract infection in Beijing. Compared with HAdV-3 infection, HAdV-7 infection caused more obvious inflammatory reaction, more severe pulmonary symptoms, longer length of hospital stay, suggesting the clinical necessity of further typing of HAdVs.

Purpose: Recombinant human adenovirus, AdCA35lacZ, was used to examine expression of a reporter gene and its reactivation following UVC (200–280 nm) and oxidative damage in fish cells.Materials and methods: AdCA35lacZ is a recombinant nonreplicating human adenovirus, which expresses the β-galactosidase (β-gal) reporter gene. UVC light produces DNA damage repaired by nucleotide excision repair (NER). In contrast, methylene blue plus visible light (MB+VL) produces oxidative DNA damage, mainly 8-oxoguanine, that is repaired by base excision repair (BER). We examined expression of the reporter gene and host cell reactivation (HCR) of the UVC-treated and MB+VL-treated reporter gene in fish cells.Results: AdCA35lacZ infection of Chinook salmon cells (CHSE-214), eel cells (PBLE) and four rainbow trout cell lines (RTG-2, RT-Gill, RTS-34st and RTS-pBk), but not zebrafish (ZEB) or carp (EPC) cells resulted in expression of β-gal. HCR of UVC-treated AdCA35lacZ in fish cells varied from that obtained in NER-deficient xeroderma pigmentosum group A fibroblasts to greater than that for NER-proficient normal human fibroblasts. HCR of UVC-treated AdCA35lacZ correlated with β-gal expression levels for untreated AdCA35lacZ. Exposure of cells to fluorescent light (400–700 nm) increased expression of the undamaged reporter gene in normal human fibroblasts and in all fish cells except PBLE and increased HCR of the UVC-damaged reporter gene in fish cells but not in human fibroblasts. HCR of the MB + VL-treated reporter gene was similar to that in human cells for PBLE, CHSE-214, RTG-2 and RTS-pBk, but was reduced in RT-Gill and RTS-34st cells.Conclusions: These results indicate the detection of functional photoreactivation (PR) of UVC-induced DNA damage in fish cells but not in normal human fibroblasts and a link between NER and transcription of the reporter gene in the fish cells in the absence of PR. We show also efficient BER of the reporter gene in several fish cell lines.

A recombinant replication-defective human adenovirus type 3: A vaccine candidate