Human Adenovirus Serotype Research Articles

Heterologous Prime Boost Vaccination Induces Protective Melanoma-Specific CD8+ T Cell Responses

Cancer vaccination aims at inducing an adaptive immune response against tumor-derived antigens. In this study, we utilize recombinant human adenovirus serotype 5 (rAd5) and recombinant lymphocytic choriomeningitis virus (rLCMV)-based vectors expressing the melanocyte differentiation antigen gp100. In contrast to single or homologous vaccination, a heterologous prime boost vaccination starting with a rAd5-gp100 prime immunization followed by a rLCMV-gp100 boost injection induces a high magnitude of polyfunctional gp100-specific CD8+ T cells. Our data indicate that an optimal T cell induction is dependent on the order and interval of the vaccinations. A prophylactic prime boost vaccination with rAd5- and rLCMV-gp100 protects mice from a B16.F10 melanoma challenge. In the therapeutic setting, combination of the vaccination with low-dose cyclophosphamide showed a synergistic effect and significantly delayed tumor growth. Our findings suggest that heterologous viral vector prime boost immunizations can mediate tumor control in a mouse melanoma model.

Preliminary Data Related to the Effect of Climacostol Produced by the Freshwater Ciliate Climacostomum virens on Human Adenovirus.

The new epidemiological scenario has so far focused on the environmental circulation of human viral pathogens. Owing to the side effects of chemical disinfectants, there is an increasing need for knowledge on the use of virucidal compounds, especially those of a natural origin. Climacostol is a molecule produced by a freshwater ciliate and it exhibits activity against bacterial and fungal pathogens. We thus also speculated that there might be an effect on viral viability, which has never been tested. To evaluate such activity, we chose human adenovirus (HAdV), which is representative of waterborne viruses. We conducted experiments using HAdV serotype 5, whose titer was determined by infecting HeLa cell cultures. HAdV5 was shown to be sensitive to climacostol at a concentration of 0.0002 mg/mL, with an approximate 3 Log10 reduction when the initial titer of HAdV5 was approximately 104 and 103 TCID50/mL. These preliminary results could be an important starting point for further research aimed at improving the characterization of climacostol activity under different experimental conditions and against various viruses, including enveloped ones (i.e., the coronavirus). The production of climacostol by a protist living in fresh water also suggests a possible application in the activated sludge of wastewater treatment plants.

Immunogenicity and Efficacy of a Recombinant Human Adenovirus Type 5 Vaccine against Zika Virus.

Zika virus (ZIKV) is a significant public health concern due to the pathogen’s ability to be transmitted by either mosquito bite or sexual transmission, allowing spread to occur throughout the world. The potential consequences of ZIKV infection to human health, specifically neonates, necessitates the development of a safe and effective Zika virus vaccine. Here, we developed an intranasal Zika vaccine based upon the replication-deficient human adenovirus serotype 5 (hAd5) expressing ZIKV pre-membrane and envelope protein (hAd5-ZKV). The hAd5-ZKV vaccine is able to induce both cell-mediated and humoral immune responses to ZIKV epitopes. Importantly, this vaccine generated CD8+ T cells specific for a dominant ZIKV T cell epitope and is shown to be protective against a ZIKV challenge by using a pre-clinical model of ZIKV disease. We also demonstrate that the vaccine expresses pre-membrane and envelope protein in a confirmation recognized by ZIKV experienced individuals. Our studies demonstrate that this adenovirus-based vaccine expressing ZIKV proteins is immunogenic and protective in mice, and it encodes ZIKV proteins in a conformation recognized by the human antibody repertoire.

The Atoh1 expression levels are correlated with the arrangement, ciliary morphology, and electrophysiological characteristics of ectopic hair cell-like cells

Previous reports have suggested that the level and duration of Atoh1 expression are correlated with the survival, arrangement and stereociliary bundle-related morphology of hair cells during development, but whether Atoh1 expression levels are correlated with the arrangement, bundle formation and electrophysiological characteristics of newly formed hair cells is unknown. To address this question, cultured cochlear explants obtained from neonatal rats were treated with different titers of a human adenovirus serotype 5 (Ad5) vector encoding Atoh1 and/or EGFP (EGFP-Atoh1+/-). The results showed that higher EGFP-Atoh1 concentrations led to higher initial Atoh1 mRNA expression levels and induced greater numbers of ectopic hair cell-like cells (EHCLCs) in the lesser epithelial ridge (LER). Furthermore, gradual increases in the number of EHCLCs were associated with the progressive conversion of the LER region similarly to that of hair cells during development. Some of the cilia on EHCLCs with higher Atoh1 expression were regularly arranged in a manner similar to that of normal hair bundles. As demonstrated through patch clamp recordings, high Atoh1 expression was associated with significantly decreased proportions of cells with Ih currents, significantly reduced proportions of transient potassium channel currents, and potassium channel currents with a greatly increased mean amplitude, which indicated that EHCLCs with high Atoh1 expression were more mature than those with low Atoh1 expression. Overall, the evidence suggests that the Atoh1 expression levels affect not only the arrangement and ciliary morphology of hair cells but also the electrophysiological characteristics of Atoh1-induced EHCLCs, and these findings provide important guidance for future therapies aimed at treating deafness.

A tropism-transformed Oncolytic Adenovirus with Dual Capsid Modifications for enhanced Glioblastoma Therapy.

Glioblastoma, the most common human brain tumor, is highly invasive and difficult to cure using conventional cancer therapies. As an alternative, adenovirus-mediated virotherapies represent a popular and maturing technology. However, the cell surface coxsackievirus and adenovirus receptor (CAR)-dependent infection mechanism limits the infectivity and oncolytic effects of Adenovirus type 5. To address this limitation, in this study we aimed to develop a novel oncolytic adenovirus for enhanced infectivity and therapeutic efficacy toward glioblastoma. We developed a novel genetically modified oncolytic adenovirus vector with dual capsid modifications to facilitate infection and specific cytotoxicity toward glioma cells. Modification of the adenoviral capsid proteins involved the incorporation of a synthetic leucine zipper-like dimerization domain into the capsid protein IX (pIX) of human adenovirus serotype 5 (Ad5) and the exchange of the fiber knob from Ad37. The virus infection mechanism and anti-tumor efficacy of modified vectors were evaluated in both in vitro (cell) and in vivo (mouse) models. Ad37-knob exchange efficiently promoted the virus infection and replication-induced glioma cell lysis by oncolytic Ad5. We also found that gene therapy mediated by the dual-modified oncolytic Ad5 vector coupled with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibited significantly enhanced anti-tumor efficacy in vitro and in vivo. This genetically modified oncolytic adenovirus provides a promising vector for future use in glioblastoma gene-viral-based therapies.

Иммуногенные и защитные свойства рекомбинантного аденовируса человека 5-го серотипа, экспрессирующего ген гликопротеина G вируса бешенства вакцинного штамма РВ-97

Иммуногенные и защитные свойства рекомбинантного аденовируса человека 5-го серотипа, экспрессирующего ген гликопротеина G вируса бешенства вакцинного штамма РВ-97

Preclinical Studies of Immunogenity, Protectivity, and Safety of the Combined Vector Vaccine for Prevention of the Middle East Respiratory Syndrome.

The Middle East Respiratory Syndrome (MERS) is an acute inflammatory disease of the respiratory system caused by the MERS-CoV coronavirus. The mortality rate for MERS is about 34.5%. Due to its high mortality rate, the lack of therapeutic and prophylactic agents, and the continuing threat of the spread of MERS beyond its current confines, developing a vaccine is a pressing task, because vaccination would help limit the spread of MERS and reduce its death toll. We have developed a combined vector vaccine for the prevention of MERS based on recombinant human adenovirus serotypes 26 and 5. Studies of its immunogenicity have shown that vaccination of animals (mice and primates) induces a robust humoral immune response that lasts for at least six months. Studies of the cellular immune response in mice after vaccination showed the emergence of a specific CD4+ and CD8+ T cell response. A study of the vaccine protectivity conducted in a model of transgenic mice carrying the human DPP4 receptor gene showed that our vaccination protected 100% of the animals from the lethal infection caused by the MERS-CoV virus (MERS-CoV EMC/2012, 100LD50 per mouse). Studies of the safety and tolerability of the developed vaccine in rodents, rabbits, and primates showed a good safety profile and tolerance in animals; they revealed no contraindications for clinical testing.

Targeting Antigen to the Surface of EVs Improves the In Vivo Immunogenicity of Human and Non-human Adenoviral Vaccines in Mice

Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by “exosome display,” through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5.

Development of pathological process and activity of etiotropic drugs in cell culture under condition of viral co-infection

The aim of the study: to investigate the peculiarities of the development of the pathological process in cells in conditions of mixed viral infection and to study the effectiveness of antiviral drugs in this model. Materials and methods of research. A model of simultaneous mixed infection of MDBK cells with human adenovirus serotype 5 (HAdV-5) and herpes simplex virus type 1 (HSV-1) was developed. Mitochondrial activity, ultrastructure and the state of the cell population were studied using MTT assay, transmission electron microscopy and flow cytometry with propidium iodide dye. The intensity of virus reproduction in cells and their infectious titer were studied by the cytomorphology method. The level of the synthesis of the major proteins of associate viruses was analyzed using flow cytometry and the corresponding monoclonal antibodies. Results of the research. Co-infected cells demonstrated a lower rate of development of pathomorphological changes compared to mono-infections, related to the inhibition of the reproduction of associate viruses. It was found that the co-infection of cells with HSV-1 and HAdV-5 results in a decrease in the number of cells with virus-induced intranuclear inclusions of both viruses by up to 40 % and viruses titer by 1.6 lg and 2.6 lg, respectively. Inhibition of synthesis of major capsid protein and glycoproteins of the herpes virus by 83 % and 64 %, respectively, and a less pronounced decrease in the amount of adenovirus hexon protein (by 17 %) were also noted. It is shown that the mitochondrial activity of co-infected cells increases to 64 % in comparison with herpetic mono-infection. An analysis of the influence of co-infection on cell cycle revealed that the number of cells in G1 phase remained unchanged compared with both mono-infections, while the number of apoptotic cells compared with herpes infection was reduced by 24 %. An analysis of the officinal drugs Acyclovir and Ribavirin effectiveness in conditions of mixed infection showed a reduction in their antiviral activity against associate viruses by 1 to 2.3 lg compared to mono-infections. Conclusions. The presence of a specific innovative cellular model of mixed infection with known aspects of the course of associated infections allows it to be used for preclinical study of antiviral activity of compounds and to obtain new data of the role of viral-viral interactions in the development of inefficient application of antiviral agents in medical practice. Key words: mixed viral infection, cytomorphological changes, cell cycle, reproduction of associate viruses, and antiviral activity. For citation: Biliavska LO, Povnitsa OY, Pankivska YB, Zagorodnya SD. Development of pathological process and activity of etiotropic drugs in cell culture under condition of viral co-infection. Journal of the National Academy of Medical Sciences of Ukraine. 2019;25(4):476–87

A novel human monoclonal antibody potently neutralizes human adenovirus serotype 7 by primarily targeting the adenovirus hexon protein

Human adenovirus serotype 7 (HAdV-7), belonging to species B, has caused severe lower respiratory tract diseases and even deaths recently. However, no adenovirus vaccine or therapeutic is available thus far. In this study, a HAdV-7-specific human monoclonal antibody (HMAb), 3-3E, isolated from single plasma cells obtained from the peripheral blood mononuclear cells of HAdV-7-infected patients showed potent HAdV-7 neutralization activity. The results showed HMAb 3-3E only binds to the hexon protein of intact HAdV-7 or the recombinant hexon protein and it does not bind to other intact virion particles. This could mean the antibody recognizes a conformational epitope of the hexon protein. Further, HMAb 3-3E potently neutralized HAdV-7 in vitro at low concentrations. In vivo studies showed HMAb 3-3E protected from HAdV-7 infection in a murine model. Therefore, HMAb 3-3E is promising as a safe and effective prophylactic and therapeutic treatment for HAdV-7 infection.

Quantitative Proteomics of Uukuniemi Virus-host Cell Interactions Reveals GBF1 as Proviral Host Factor for Phleboviruses

Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.

2788. Case Report: Severe Community-Acquired Human Adenovirus 7 Infection in a Mother and Son

BackgroundWe present the case of a 42-year-old male (Patient 1) with a history of morbid obesity and cirrhosis of unclear etiology who died of acute hypoxic respiratory failure and devastating encephalitis after community-acquired infection with human adenovirus 7 (HAdV7) in spite of antiviral treatment with Cidofovir. Patient 1’s course is outlined in Figure 1. During his stay, the patient’s mother (Patient 2) was also hospitalized in the same unit following a similar presentation with 10 days of flu-like symptoms that progressed to ARDS requiring intubation. Patient 2 recovered from her infection with no specific antiviral treatment.MethodsN/A.ResultsThese cases illustrate particularly fulminant presentations of HAdV7 infection and highlight the high pathogenicity of HAdV7 compared with other adenovirus subtypes. Severe lower respiratory tract HAdV7 infections have been reported most commonly as outbreaks of respiratory illness among military recruits and infants. A notable recent outbreak of HAdV-7d in New Jersey in 2016–2017 resulted in 12 confirmed cases with 4 deaths; however, all deaths occurred in patients with significant medical comorbidities.1 In a case particularly striking for its similarity to the one presented here, a healthy 44-year-old male along with his 68-year-old father were hospitalized with human adenovirus 7 days infection in Chicago in December 2014, with the son requiring ECMO for ARDS and the father briefly requiring ICU-level care.2ConclusionWhile our patient’s case is by no means unprecedented, it does represent an uncommon and potentially serious infection that points to need for continuing nosocomial testing efforts and deserves special attention in outbreak settings. In the future, perhaps use of the extant vaccine for human adenovirus serotypes 4 and 7 (currently licensed for military personnel3) could be expanded for use in civilian populations could be explored and potentially expanded for use in outbreak settings. Disclosures All authors: No reported disclosures.

An Optimized Electroporation-Free Method for Generation of Recombinant Adenoviral Plasmids

Recombinant adenoviruses are widely used for delivery and expression of transgenes for therapeutic purposes and in fundamental research. AdEasy™ adenoviral vector system allows for rapid and efficient generation of replication-deficient adenoviruses. One important crucial step in producing recombinant adenoviruses is the generation of a recombinant adenoviral plasmid in E. coli BJ5183 cells via homologous recombination between the transgene encoding shuttle vector and the pAdEasy-1 plasmid, which carries a significant part of human adenovirus serotype 5 (Ad5) genome. In many published protocols, the necessity of electroporation for the transformation of the shuttle vector and pAdEasy-1 into E. coli cells is emphasized, thus making necessary the availability of the appropriate instruments and consumables. Here, we have proposed an optimized protocol for the generation of recombinant adenoviruses with the use of chemically competent E. coli cells only, without the use of electroporation. We have demonstrated the efficient transformation of linearized nonpurified shuttle vector into chemically competent E. coli BJ5183-pAdEasy-1 cells allowing identification of clones with correct recombinant adenoviral plasmids. Using the optimized protocol, we have generated four functional replication-deficient adenoviruses expressing target proteins in HEK293 cells. The protocol that has been suggested provides a significantly simplified and cheaper method for the generation of recombinant adenoviruses due to lack of requirement to use instruments and consumables for electroporation.

Duration of protection and humoral immunity induced by an adenovirus-vectored subunit vaccine for foot-and-mouth disease (FMD) in Holstein steers

Foot-and-mouth disease (FMD) is a highly contagious viral infection of cloven hooved animals that continues to cause economic disruption in both endemic countries or when introduced into a formally FMD free country. Vaccines that protect against clinical disease and virus shedding are critical to control FMD. The replication deficient human adenovirus serotype 5 (Ad5) vaccine vector expressing empty FMD virus (FMDV) capsid, AdtFMD, is a promising new vaccine platform. With no shedding or spreading of viral vector detected in field trials, this vaccine is very safe to manufacture, as there is no requirement for high containment faciitites. Here, we describe three studies assessing the proportion of animals protected from clinical vesicular disease (foot lesions) following live-FMDV challenge by intradermolingual inoculation at 6 or 9 months following a single vaccination with the commercial AdtFMD vaccine, provisionally licensed for cattle in the United States. Further, we tested the effect of vaccination route (transdermal, intramuscular, subcutaneous) on clinical outcome and humoral immunity. Results demonstrate that a single dose vaccination in cattle with the commercial vaccine vector expressing capsid proteins of the FMDV strain A24 Cruzeiro (Adt.A24), induced protection against clinical FMD at 6 months (100% transdermal, 80% intramuscular, and 60% subcutaneous) that waned by 9 months post-vaccination (33% transdermal and 20% intramuscular). Post-vaccination serum from immunized cattle (all studies) generally contained FMDV specific neutralizing antibodies by day 14. Anti-FMDV antibody secreting cells are detected in peripheral blood early following vaccination, but are absent after 28 days post-vaccination. Thus, the decay in antibody mediated immunity over time is likely a function of FMDV-specific antibody half-life. These data reveal the short time span of anti-FMDV antibody secreting cells (ASCs) and important performance characteristics of needle-free vaccination with a recombinant vectored subunit vaccine for FMDV.

Seroprevalence of Human Adenovirus Type 5 Neutralizing Antibody in Common Marmosets Determined by a New Set of Two Assays.

Preexisting neutralizing antibody (NAb) against human adenovirus serotype 5 (AdHu5) can reduce the immunogenicity of AdHu5 vector-based vaccine, thus inhibiting the host's immune response and utility of other homologous vectors. Common marmoset (Callithrix jacchus), a small new world primate, has attracted considerable attention for its potential as a preclinical research model of vaccine development. However, the prevalence of anti-AdHu5 NAb activity in common marmosets bred in China remains unknown. A recombinant adenovirus expressing luciferase and Zs Green reporter genes were constructed to detect NAb against rAdHu5 by flow cytometry (FCM) and chemiluminescence (CL) assay. Five of 25 marmosets (20%) presented AdHu5 NAb detectable by FCM. Four animals had low titer (1/16), while the fifth one reached 1/64. While by CL assay, 7 of 25 (28%) marmosets were anti-AdHu5 NAb positive. Four animals, two of whom were negative by FCM, also had low titer NAb (1/16), suggesting assay discrepancy at low levels. Two marmosets, 1/32 titer by CL, were at 1/16 by FCM. A single animal showed a high titer with both assays (1/128 and 1/64 by CL and FCM, respectively). The CL method was simpler, more sensitive, accurate, and stable. The low prevalence of preexisting anti-AdHu5 NAb in marmosets provides important background information on the feasibility and applicability of using marmosets as a preclinical research model for vaccine development.

Norovirus and human adenovirus occurrence and diversity in recreational water in a karst aquifer in the Yucatan Peninsula, Mexico

To determine the seasonal occurrence and diversity of norovirus (NoV) and human adenovirus (HAdV) in groundwater from sinkholes, and brackish water used for recreational activities in the karst aquifer of the Yucatan Peninsula, Mexico. Hollow fibre ultrafiltration was used to concentrate viruses and standard plaque assay methods were used to enumerate somatic and F+ specific coliphages as viral indicators. Real-time quantitative polymerase chain reaction assays were used to estimate the number of genome copies for NoV strains GI, and GII, and HAdVs. The predominant NoV genotypes and HAdV serotypes were identified by comparative sequence analysis. Somatic and male F+ specific coliphages were detected at concentrations up to 94 and 60 plaque-forming units per 100ml respectively. The NoV genogroup I (GI) was associated with 50% of the sampled sites during the rainy season only, at concentrations ranging from 120 to 1600 genome copies per litre (GCl-1 ). The NoV genogroup II (GII) was detected in 30 and 40% of the sampled sites during the rainy and dry seasons, respectively, at concentrations ranging from 10 to 290GCl-1 . During the rainy and dry seasons, HAdVs were detected in 20% of the sites, at concentrations ranging from 24 to 690 GCl-1 . Identification of viral types revealed the presence of NoV GI.2, GII.Pe, GII.P16 and GII.P17, and HAdV F serotypes 40 and 41. These findings demonstrate that NoVs and HAdVs are prevalent as virus contaminants in the karst aquifer, representing potential health risks particularly during the rainy season, in one of the most important areas used for tourism in Mexico. This is one of the few studies conducted in karst aquifers that provide a foundational baseline of the distribution, concentrations and diversity of NoVs and HadVs in these particular environments.

Human Adenovirus Serotype 5 Is Sensitive to IgM-Independent Neutralization In Vitro and In Vivo.

Human adenovirus 5 (HAdV-5) is used as a vector in gene therapy clinical trials, hence its interactions with the host immune system have been widely studied. Previous studies have demonstrated that HAdV-5 binds specifically to murine coagulation factor X (mFX), inhibiting IgM and complement-mediated neutralization. Here, we examined the physical binding of immune components to HAdV-5 by nanoparticle tracking analysis, neutralization assays, mass spectrometry analysis and in vivo experiments. We observed that purified mouse Immunoglobulin M (IgM) antibodies bound to HAdV-5 only in the presence of complement components. Active serum components were demonstrated to bind to HAdV-5 in the presence or absence of mFX, indicating that immune molecules and mFX might bind to different sites. Since binding of mFX to HAdV-5 blocks the neutralization cascade, these findings suggested that not all complement-binding sites may be involved in virion neutralization. Furthermore, the data obtained from serum neutralization experiments suggested that immune molecules other than IgM and IgG may trigger activation of the complement cascade in vitro. In vivo experiments were conducted in immunocompetent C57BL/6 or immuno-deficient Rag2-/- mice. HAdV-5T* (a mutant HAdV-5 unable to bind to human or mFX) was neutralized to some extent in both mouse models, suggesting that murine immunoglobulins were not required for neutralization of HAdV-5 in vivo. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis of HAdV-5 and HAdV-5T* after exposure to murine sera showed stable binding of C3 and C4b in the absence of mFX. In summary, these results suggest that HAdV-5 neutralization can be mediated by both the classical and alternative pathways and that, in the absence of immunoglobulins, the complement cascade can be activated by direct binding of C3 to the virion.

Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7

BackgroundHuman adenoviruses are a common group of viruses that cause acute infectious diseases. Human adenovirus (HAdV) 3 and HAdV 7 cause major outbreaks of severe pneumonia. A reliable and practical method for HAdV typing in clinical laboratories is lacking. A simple, rapid and accurate molecular typing method for HAdV may facilitate clinical diagnosis and epidemiological control.MethodsWe developed and evaluated duplex real-time recombinase-aided amplification (RAA) assays incorporating competitive internal controls for detection of HAdV 3 and HAdV 7, respectively. The assays were performed in a one-step in a single tube reaction at 39° for 20 min.ResultsThe analytical sensitivities of the duplex RAA assays for HAdV 3 and HAdV 7 were 5.0 and 14.8 copies per reaction, respectively (at 95% probability by probit regression analysis). No cross-reaction was observed with other types of HAdV or other common respiratory viruses. The duplex RAA assays were used to detect 152 previously-defined HAdV-positive samples. These results agreed with those obtained using a published triplex quantitative real-time PCR protocol.ConclusionsWe provide the first report of internally-controlled duplex RAA assays for the detection of HAdV 3 and HAdV 7. These assays effectively reduce the rate of false negative results and may be valuable for detection of HAdV 3 and HAdV 7 in clinical laboratories, especially in resource-poor settings.

Frequency of adenovirus serotype 8 in patients with Keratoconjunctivitis, in Ahvaz, Iran

Adenoviral keratoconjunctivitis is an extremely frequent ophthalmological disease caused by various serological subtypes of human adenovirus (HAdV) worldwide. Adenoviruses serotypes 8, 11, 19, 37 frequently cause epidemic keratoconjunctivitis (EKC). This study was conducted to evaluate the frequency of adenovirus serotypes in patients with EKC in Ahvaz, Iran. Eighty-eight ocular swabs were collected from patients with EKC. The specimens were analyzed for detection of adenovirus by standard PCR. The PCR products were further sequenced and analyzed to determine the serotypes. The study population consisted of 49/88 (55.7%) males and 39/88 (44.3%) females. Among them 25 (51.02%) males and 22 (56.41%) females were positive for HAdV serotype 8 (p= 0.488). Overall forty-seven (53.4%) samples were positive for AdV serotype 8 while forty-one patients (46.59%) were negative for the adenovirus serotypes. The results of this study revealed predominanance of HAdV 8 with high prevalence of 53.4% among patients with Keratoconjunctivitis. Forty-one patients (46.59%) were negative for adenovirus. Still, the role for other related viruses such as enteroviruses need to be investigated in patients with EKC.

Immuno- and bio-informatic Analysis of Hexon Protein in Human Adenovirus D8 Isolated From Patients With Keratoconjunctivitis

Aim: In silico analysis of the hexon protein of human adenovirus serotype D-8 isolated from a patients with keratoconjunctivitis in Iran. Materials & methods: The hexon gene of HAdV-D8 was amplified by PCR. HAdV-D8 recovered from EKC outbreak was isolated by growing in A549 cells. Results: The hexon gene isolated from a patient with EKC comprised 2829 nt and 942 aa. The analyses of selected B-cell epitopes prediction (KTFQPEPQIGENNWQD) and T-cell epitopes prediction (TENFDIDLAFFDIPQ), showed high score immunogenicity, which may prove this to be a promising candidate for epitope vaccine development. Conclusion: In silico analysis of selected B-cell epitopes prediction (KTFQPEPQIGENNWQD) and T-cell epitopes prediction (TENFDIDLAFFDIPQ) are immunogenic and provoke B- and T-cell responses.