Measurement of Adenovirus-Based Vector Heterogeneity.

Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ∼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities.

The changed endemic pattern of human adenovirus from species B to C among pediatric patients under the pressure of non-pharmaceutical interventions against COVID-19 in Beijing, China.

BACKGROUND: Under the pressure of non-pharmaceutical interventions (NPIs) targeting severe acute respiratory syndrome coronavirus 2, the prevalence of human adenovirus (HAdV) was monitored before and after NPIs launched on Jan 24, 2020 in pediatric patients in Beijing, China. METHODS: Respiratory samples collected from children hospitalized with acute respiratory infections from Jan 2015 to Dec 2021 were screened by direct immunofluorescence test or capillary electrophoresis-based multiplex PCR assay. The hexon, penton base, and fiber genes were amplified from HAdV positive specimens, then sequenced. For HAdV typing, phylogenetic trees were built by MEGA X. Then clinical data of HAdV positive cases were collected. All data were evaluated using SPSS Statistics 22.0 software. RESULTS: A total of 16,097 children were enrolled and 466 (2.89%, 466/16,097) were HAdV-positive. The positive rates of HAdV varied, ranging from 4.39% (151/3,438) in 2018 to1.25% (26/2,081) in 2021, dropped from 3.19% (428/13,408) to 1.41% (38/2,689) from before to after NPIs launched (P < 0.001). There were 350 cases typed into nine types of species B, C, or E and 34 recorded as undetermined. Among them, HAdV-B3 (51.56%, 198/384) was the most prevalent types from 2015 to 2017, and HAdV-B7 (29.17%, 112/384) co-circulated with HAdV-B3 from 2018 to 2019. After NPIs launched, HAdV-B3 and B7 decreased sharply with HAdV-B7 undetected in 2021, while HAdV-C1 became the dominant one and the undetermined were more. CONCLUSIONS: The endemic pattern of HAdV changed in Beijing because of the NPIs launched for COVID-19. Especially, the dominant types changed from HAdV-B to HAdV-C.

Analysis of the Prevalence of Binding and Neutralizing Antibodies against 39 Human Adenovirus Types in Student Cohorts Reveals Low-Prevalence Types and a Decline in Binding Antibody Levels during the SARS-CoV-2 Pandemic.

Human adenoviruses (HAdVs) are important tools for vector development for applications such as immunization, oncolytic therapy, or gene therapy. However, their potential is limited by preexisting immunity against HAdV; therefore, it is important for future vector design to identify HAdV types of low seroprevalence. To provide such data, we performed an analysis of both binding and neutralizing antibodies in sera from three student cohorts. Among these young adults, we found the highest levels of binding antibodies against HAdV-C1, -D33, -A31, -B35, -C5, -D26, -E4, and -B7. The highest levels of neutralizing antibodies were detected against HAdV-C2, -B3, -C1, -F41, -G52, -C5, -A31, -E4, and -C6. While binding and neutralizing antibody levels were not different in males and females or in samples collected before and after the cold season, we found significantly lower levels of binding antibodies in sera collected 20 months after the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, indicating a waning of HAdV-specific antibody responses on that time scale. Our data indicate that mainly HAdV types of species A, B, and D show low seroprevalence with regard to both binding and neutralizing antibodies and may represent good candidates for further characterization and future development as novel vector systems. IMPORTANCE Vectors based on human adenoviruses (HAdVs) are important for the development of novel immunizations, oncolytic therapies, and gene therapies. The use of HAdV-based vaccines against Ebola virus, the rapid adaptation of the vector technology for vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and their very good efficacy have shown the great potential of HAdV-based vaccines. Preexisting immunity against HAdV-based vectors can limit their efficacy significantly; therefore, it is highly desirable to identify HAdV types with low seroprevalence. The identification of new suitable HAdV types for vector development will broaden the repertoire and contribute to future epidemic preparedness.

Human Adenovirus Type 26 Infection Mediated by αvß3 Integrin Is Caveolin-1-Dependent.

Human adenovirus type 26 (HAdV26) has been recognized as a promising platform for vaccine vector development, and very recently vaccine against COVID-19 based on HAdV26 was authorized for emergency use. Nevertheless, basic biology of this virus, namely, pathway which HAdV26 uses to enter the cell, is still insufficiently known. We have shown here that HAdV26 infection of human epithelial cells expressing low amount of αvß3 integrin involves clathrin and is caveolin-1-independent, while HAdV26 infection of cells with high amount of αvß3 integrin does not involve clathrin but is caveolin-1-dependent. Thus, this study demonstrates that caveolin-1 is limiting factor in αvß3 integrin-mediated HAdV26 infection. Regardless of αvß3 integrin expression, HAdV26 infection involves dynamin-2. Our data provide for the first-time description of HAdV26 cell entry pathway, hence increase our knowledge of HAdV26 infection. Knowing that functionality of adenovirus vector is influenced by its cell entry pathway and intracellular trafficking, our results will contribute to better understanding of HAdV26 immunogenicity and antigen presentation when used as vaccine vector. IMPORTANCE In order to fulfill its role as a vector, adenovirus needs to successfully deliver its DNA genome to the host nucleus, a process highly influenced by adenovirus intracellular translocation. Thus, cell entry pathway and intracellular trafficking determine functionality of human adenovirus-based vectors. Endocytosis of HAdV26, currently extensively studied as a vaccine vector, has not been described so far. We present here that HAdV26 infection of human epithelial cells with high expression of αvß3 integrin, one of the putative HAdV26 receptors, is caveolin-1- and partially dynamin-2-dependent. Since caveolin containing domains provide a unique environment for specific signaling events and participate in inflammatory signaling one can imagine that directing HAdV26 cell entry toward caveolin-1-mediate pathway might play role in immunogenicity of this virus. Therefore, our results contribute to better understanding of HAdV26 infection pathway, hence, can be helpful in explaining induction of immune response and antigen presentation by HAdV26-based vaccine vector.

Human Adenovirus Type 26 Infection Mediated by αvß3 Integrin Is Caveolin-1-Dependent.

Human adenovirus type 26 (HAdV26) has been recognized as a promising platform for vaccine vector development, and very recently vaccine against COVID-19 based on HAdV26 was authorized for emergency use. Nevertheless, basic biology of this virus, namely, pathway which HAdV26 uses to enter the cell, is still insufficiently known. We have shown here that HAdV26 infection of human epithelial cells expressing low amount of αvß3 integrin involves clathrin and is caveolin-1-independent, while HAdV26 infection of cells with high amount of αvß3 integrin does not involve clathrin but is caveolin-1-dependent. Thus, this study demonstrates that caveolin-1 is limiting factor in αvß3 integrin-mediated HAdV26 infection. Regardless of αvß3 integrin expression, HAdV26 infection involves dynamin-2. Our data provide for the first-time description of HAdV26 cell entry pathway, hence increase our knowledge of HAdV26 infection. Knowing that functionality of adenovirus vector is influenced by its cell entry pathway and intracellular trafficking, our results will contribute to better understanding of HAdV26 immunogenicity and antigen presentation when used as vaccine vector. IMPORTANCE In order to fulfill its role as a vector, adenovirus needs to successfully deliver its DNA genome to the host nucleus, a process highly influenced by adenovirus intracellular translocation. Thus, cell entry pathway and intracellular trafficking determine functionality of human adenovirus-based vectors. Endocytosis of HAdV26, currently extensively studied as a vaccine vector, has not been described so far. We present here that HAdV26 infection of human epithelial cells with high expression of αvß3 integrin, one of the putative HAdV26 receptors, is caveolin-1- and partially dynamin-2-dependent. Since caveolin containing domains provide a unique environment for specific signaling events and participate in inflammatory signaling one can imagine that directing HAdV26 cell entry toward caveolin-1-mediate pathway might play role in immunogenicity of this virus. Therefore, our results contribute to better understanding of HAdV26 infection pathway, hence, can be helpful in explaining induction of immune response and antigen presentation by HAdV26-based vaccine vector.

Human adenovirus (HAdV) infection in children with acute respiratory tract infections in Guangzhou, China, 2010-2021: a molecular epidemiology study.

BACKGROUND: Human adenovirus (HAdV) infection can cause a variety of diseases. It is a major pathogen of pediatric acute respiratory tract infections (ARIs) and can be life-threatening in younger children. We described the epidemiology and subtypes shifting of HAdV among children with ARI in Guangzhou, China. METHODS: We conducted a retrospective study of 161,079 children diagnosed with acute respiratory illness at the Guangzhou Women and Children's Medical Center between 2010 and 2021. HAdV specimens were detected by real-time PCR and the hexon gene was used for phylogenetic analysis. RESULTS: Before the COVID-19 outbreak in Guangzhou, the annual frequency of adenovirus infection detected during this period ranged from 3.92% to 13.58%, with an epidemic peak every four to five years. HAdV demonstrated a clear seasonal distribution, with the lowest positivity in March and peaking during summer (July or August) every year. A significant increase in HAdV cases was recorded for 2018 and 2019, which coincided with a shift in the dominant HAdV subtype from HAdV-3 to HAdV-7. The latter was associated with a more severe disease compared to HAdV-3. The average mortality proportion for children infected with HAdV from 2016 to 2019 was 0.38% but increased to 20% in severe cases. After COVID-19 emerged, HAdV cases dropped to 2.68%, suggesting that non-pharmaceutical interventions probably reduced the transmission of HAdV in the community. CONCLUSION: Our study provides the foundation for the understanding of the epidemiology of HAdV and its associated risks in children in Southern China.

NIRVANA for Simultaneous Detection and Mutation Surveillance of SARS-CoV-2 and Co-infections of Multiple Respiratory Viruses.

Detection and mutation surveillance of SARS-CoV-2 are crucial for combating the COVID-19 pandemic. Here we describe a lab-based method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes. It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus and monitor mutations for up to 96 samples in real time. The method proved to be rapid and sensitive (limit of detection: 29 viral RNA copies/µL of extracted nucleic acid) in detecting SARS-CoV-2 in clinical samples. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses.

RANBP2 and USP9x regulate nuclear import of adenovirus minor coat protein IIIa.

As intracellular parasites, viruses exploit cellular proteins at every stage of infection. Adenovirus outbreaks are associated with severe acute respiratory illnesses and conjunctivitis, with no specific antiviral therapy available. An adenoviral vaccine based on human adenovirus species D (HAdV-D) is currently in use for COVID-19. Herein, we investigate host interactions of HAdV-D type 37 (HAdV-D37) protein IIIa (pIIIa), identified by affinity purification and mass spectrometry (AP-MS) screens. We demonstrate that viral pIIIa interacts with ubiquitin-specific protease 9x (USP9x) and Ran-binding protein 2 (RANBP2). USP9x binding did not invoke its signature deubiquitination function but rather deregulated pIIIa-RANBP2 interactions. In USP9x-knockout cells, viral genome replication and viral protein expression increased compared to wild type cells, supporting a host-favored mechanism for USP9x. Conversely, RANBP2-knock down reduced pIIIa transport to the nucleus, viral genome replication, and viral protein expression. Also, RANBP2-siRNA pretreated cells appeared to contain fewer mature viral particles. Transmission electron microscopy of USP9x-siRNA pretreated, virus-infected cells revealed larger than typical paracrystalline viral arrays. RANBP2-siRNA pretreatment led to the accumulation of defective assembly products at an early maturation stage. CRM1 nuclear export blockade by leptomycin B led to the retention of pIIIa within cell nuclei and hindered pIIIa-RANBP2 interactions. In-vitro binding analyses indicated that USP9x and RANBP2 bind to C-terminus of pIIIa amino acids 386-563 and 386-510, respectively. Surface plasmon resonance testing showed direct pIIIa interaction with recombinant USP9x and RANBP2 proteins, without competition. Using an alternative and genetically disparate adenovirus type (HAdV-C5), we show that the demonstrated pIIIa interaction is also important for a severe respiratory pathogen. Together, our results suggest that pIIIa hijacks RANBP2 for nuclear import and subsequent virion assembly. USP9x counteracts this interaction and negatively regulates virion synthesis. This analysis extends the scope of known adenovirus-host interactions and has potential implications in designing new antiviral therapeutics.

Comparison of Insertion, Deletion, and Point Mutations in the Genomes of Human Adenovirus HAdvC-2 and SARS-CoV-2.

Virus genome mutation profiles with insertion, deletion, and point mutations have recently been revealed to differ remarkably between viruses. In RNA viruses like human coronaviruses or influenza viruses, genome samples collected over two to three decades usually show point mutations in 10-20% of the bases, while the rate of insertion and/or deletion mutations (indels) largely depends on the virus. This study evaluates the mutation profiles of DNA viruses by comparing a recently sampled genome of human adenovirus species C type 2 (isolate SG06/HAdvC2/2016) with a genome of the same species sampled in the 1970s. It was found insertions of 23 bases at seven sites and deletions of 22 bases at nine sites. The longest indels were 6-base insertions in E2B and L4. All indels in the coding regions were in-frame mutations with base lengths in multiples of three. In the non-coding regions, the lengths of the indels ranged from 1-4 consecutive bases. Long indels with more than 10 consecutive bases, which comprise nearly half of indels in the SARS-CoV-2 genome, were absent. In other sites, the point mutation rate was approximately 0.3%, which was significantly lower than in RNA viruses. In summary, the estimated point mutation rate in human adenovirus species C type 2 (HAdvC-2) was over 10 times lower than in RNA viruses. Unlike the relatively long indels in the SARS-CoV-2 genome, the indels in HAdvC-2 were short, with 6 or fewer consecutive bases.

Viral nucleic acid analysis with PCR in lacrimal tissue and nasal swab samples of primary acquired nasolacrimal duct obstruction cases.

PURPOSE: To evaluate the role of viral infections in the pathogenesis of primary acquired nasolacrimal duct obstruction. METHODS: The study included 48 patients diagnosed with primary acquired nasolacrimal duct obstruction undergoing dacryocystorhinostomy surgery. Prior to dacryocystorhinostomy surgery, nasal swab sample was taken from the inferior meatus at the same side. During dacryocystorhinostomy, tissue biopsy sample (2 × 2 mm) was taken from the junction area of the lacrimal sac and nasolacrimal duct. Following nucleic acid extraction, polymerase chain reaction was performed. RESULTS: The patients consisted of 9 (18.8%) men and 39 (81.2%) women with a mean age of 51.0 ± 14.3 years. Qualitative polymerase chain reaction showed viral genome in the nasal swabs of 10 (20.8%) patients, including coronavirus 229E (three cases), coronavirus HKU1 (two cases), respiratory syncytial virus (two cases), coronavirus OC43 (one case), coronavirus NL63 (one case), and adenovirus (one case). In the dacryocystorhinostomy samples, viral genomes were detected in four (8.3%) cases, including respiratory syncytial virus (two cases), coronavirus HKU1 (one case), and adenovirus (one case). There was a statistically significant agreement between nasal mucosal swab and dacryocystorhinostomy biopsy samples in terms of respiratory syncytial virus positivity (kappa = 1.000, p = 0.001). CONCLUSION: Although the viral genome was detected in the samples, a direct relationship between viruses and pathogenesis of primary acquired nasolacrimal duct obstruction could not be revealed because of the low number of positive results. However, considering the profibrotic characteristics of specific viruses such as respiratory syncytial virus and adenovirus, viral infections may be one of the many predisposing factors of primary acquired nasolacrimal duct obstruction.

Human species D adenovirus hexon capsid protein mediates cell entry through a direct interaction with CD46.

Human adenovirus species D (HAdV-D) types are currently being explored as vaccine vectors for coronavirus disease 2019 (COVID-19) and other severe infectious diseases. The efficacy of such vector-based vaccines depends on functional interactions with receptors on host cells. Adenoviruses of different species are assumed to enter host cells mainly by interactions between the knob domain of the protruding fiber capsid protein and cellular receptors. Using a cell-based receptor-screening assay, we identified CD46 as a receptor for HAdV-D56. The function of CD46 was validated in infection experiments using cells lacking and overexpressing CD46, and by competition infection experiments using soluble CD46. Remarkably, unlike HAdV-B types that engage CD46 through interactions with the knob domain of the fiber protein, HAdV-D types infect host cells through a direct interaction between CD46 and the hexon protein. Soluble hexon proteins (but not fiber knob) inhibited HAdV-D56 infection, and surface plasmon analyses demonstrated that CD46 binds to HAdV-D hexon (but not fiber knob) proteins. Cryoelectron microscopy analysis of the HAdV-D56 virion-CD46 complex confirmed the interaction and showed that CD46 binds to the central cavity of hexon trimers. Finally, soluble CD46 inhibited infection by 16 out of 17 investigated HAdV-D types, suggesting that CD46 is an important receptor for a large group of adenoviruses. In conclusion, this study identifies a noncanonical entry mechanism used by human adenoviruses, which adds to the knowledge of adenovirus biology and can also be useful for development of adenovirus-based vaccine vectors.

Cross-infection of adenovirus among medical staff: A warning from the intensive care unit in a tertiary care teaching hospital in China.

RATIONALE: In 2019, a small HAdV55-associated outbreak of adenovirus infection occurred among the intensive care unit (ICU) staff in Xiangya Hospital of Central South University in Hunan Province, China, during the treatment of a patient. OBJECTIVE: To investigate the characteristics of a nosocomial adenovirus outbreak in an ICU. METHODS: We evaluated all the patients treated and the medical staff working in the ICU from August 1 to September 4, 2019. We further performed an epidemiological and molecular analysis for this outbreak from patient to healthcare workers and between healthcare workers. After the outbreak, we adopted exposure prevention and droplet prevention measures based on standard precautions. MEASUREMENTS AND MAIN RESULTS: Between August 1 and August 27, 2019, 27 cases of human adenovirus cross-infection were reported in our institution. Among the cases, eleven were doctors (41%), eleven were nurses (41%), three were respiratory therapists (11%), and two were caregivers (7%). The attack rate was 28.4%, and the fatality rate was 0. The results showed that contact with the index case, lack of hand hygiene or gloving adherence were risk factors for infection after adenovirus exposure. After taking specific precautions, no new cases of infection have appeared since August 27. CONCLUSIONS: Our results show that HAdV55 in a single patient had strong transmission potential in an intensive care unit with adequate facilities and standardized operation. We provide convincing evidence indicating that attention could be highlighted on the role of standard and specific precautions for controlling the spread of adenovirus in ICUs.