Adenovirus DNA Research Articles

Linearized oncolytic adenoviral plasmid DNA delivered by bioreducible polymers

As an effort to overcome limits of adenovirus (Ad) as a systemic delivery vector for cancer therapy, we developed a novel system using oncolytic Ad plasmid DNA with two bioreducible polymers: arginine-grafted bioreducible poly(disulfide amine)polymer (ABP) and PEG5k-conjugated ABP (ABP5k) in expectation of oncolytic effect caused by progeny viral production followed by replication. The linearized Ad DNAs for active viral replication polyplexed with each polymer were able to replicate only in human cancer cells and produce progeny viruses. The non-immunogenic polymers delivering the DNAs markedly elicited to evade the innate and adaptive immune response. The biodistribution ratio of the polyplexes administered systemically was approximately 99% decreased in liver when compared with naked Ad. Moreover, tumor-to-liver ratio of the Ad DNA delivered by ABP or ABP5k was significantly elevated at 229- or 419-fold greater than that of naked Ad, respectively. The ABP5k improved the chance of the DNA to localize within tumor versus liver with 1.8-fold increased ratio. In conclusion, the innovative and simple system for delivering oncolytic Ad plasmid DNA with the bioreducible polymers, skipping time-consuming steps such as generation and characterization of oncolytic Ad vectors, can be utilized as an alternative approach for cancer therapy.

Chromatin structure of adenovirus DNA throughout infection

For more than half a century, researchers have studied the basic biology of Adenovirus (Ad), unraveling the subtle, yet profound, interactions between the virus and the host. These studies have uncovered previously unknown proteins and pathways crucial for normal cell function that the virus manipulates to achieve optimal virus replication and gene expression. In the infecting virion, the viral DNA is tightly condensed in a virally encoded protamine-like protein which must be remodeled within the first few hours of infection to allow for efficient expression of virus-encoded genes and subsequent viral DNA replication. This review discusses our current knowledge of Ad DNA–protein complex within the infected cell nucleus, the cellular proteins the virus utilizes to achieve chromatinization, and how this event contributes to efficient gene expression and progression of the virus life cycle.

Diagnostic value of quantitative PCR for adenovirus detection in stool samples as compared with antigen detection and cell culture in haematopoietic stem cell transplant recipients

Adenovirus (AdV) infections constitute a significant cause of morbidity and mortality during haematopoietic stem cell transplantation. Recent guidelines recommend repeated screening for AdV in whole blood (WB), with quantitative PCR (qPCR) as the reference standard. Despite pre-emptive antiviral treatment based on qPCR in WB, the mortality rate after disseminated AdV infection remains very high. The aim of our study was to advance early screening for AdV, using a standardized method, so as to enable the earlier initiation of antiviral treatment or adoptive immunotherapy. The diagnostic value of AdV DNA quantification in stool samples was investigated retrospectively and compared with antigen detection and cell culture in 21 patients with AdV infection, from 182 patients followed in the Transplant Unit of Nancy University Hospital Centre, including 18 patients with systemic infection. In 16/18 patients with positive AdV viraemia, AdV DNA was present in stool samples earlier than in WB (median, 42 days; range, 3–199 days), whereas both antigen detection and cell culture were still negative for 11/18 patients with systemic AdV infection. The course of AdV viral loads in stool samples was predictive of adenoviraemia (sensitivity, 89%). Very late and lethal AdV infections were observed in cord blood transplant recipients, and would have been detected much earlier with the use of qPCR on stool samples. This study confirmed that quantification of AdV in stool samples by qPCR is beneficial for the management of transplant recipients, with or without antigen detection.

A 17‐year‐old girl with severe respiratory failure and circulatory shock

A 17-year-old girl presented to her general practitioner with a 1-week history of fever, arthralgia, general malaise and dry cough. She had a history of systemic onset juvenile idiopathic arthritis (SOJIA), diagnosed at age 2 years and treated with aspirin, and had been in remission for 13 years. She was initially treated by her GP with oral roxithromycin, and admitted to hospital 3 days later with worsening of her symptoms. Her admission chest x-ray (Box 1) revealed bilateral perihilar infiltrates. A diagnosis of severe community-acquired pneumonia was made and broad spectrum antibiotics were commenced, including vancomycin, moxifloxacin, and oseltamivir. Despite this treatment, her condition deteriorated. On Day 3 of admission she required endotracheal intubation and circulatory support with noradrenaline 18n50 mg/kg/min, and was admitted to the intensive care unit (ICU). She remained hypotensive, with a mean arterial pressure of 50mmHg, and with sinus tachycardia of 132 beats/min, and subsequently required renal replacement therapy. She remained febrile for the first 3 days after admission (temperature range 37.5dCn39dC), and her temperature settled to normal after appropriate therapy was initiated. Investigations included a computed tomography scan of her abdomen, which showed hepatosplenomegaly, and liver function tests, which showed elevated conjugated bilirubin (36 mmol/L; reference range [RR], l 4mmol/L), g -glutamyl transferase (84U/L; RR,l 24 U/L), lactate dehydrogenase (3540U/L; RR, 150n280U/L), alanine aminotransferase (108U/L; RR, 10n30U/L) and aspartate transaminase (388U/L; RR, l 30U/L). Other abnormal parameters were her haemoglobin level (93g/L; RR, 120n160 g/L), platelet count (65 x 109/L; RR, 150n400 109/L), white cell count (2.9 x 109/L; RR, 4.5n13 x 109/L), international normalised ratio (2.1; RR, 0.9n1.2) and fibrinogen level (0.9 g/L; RR, g 2.5 g/L). Elevated inflammatory markers included C-reactive protein (301mg/L; RR, l 5mg/L) and serum ferritin (50 500mg/L; RR, 7n140 mg/L). A transthoracic echocardiogram showed a left ventricular ejection fraction of 60%, a mild reduction in right ventricular contractility, and a right ventricular systolic pressure of 48mmHg. A full screen for sepsis was performed, including a nasopharyngeal aspirate, bronchoalveolar lavage, blood cultures and serological testing; all were unremarkable. Other immunological tests performed were for Mycoplasma pneumoniae antibodies, Streptococcus pneumoniae urinary antigen, Legionella pneumophila urinary antigen, herpes simplex virus, cytomegalovirus, EpsteinnBarr virus (EBV) IgM and IgG, influenza A and B, H1N1 influenza RNA, respiratory syncytial virus, parainfluenza and adenovirus DNA, Q fever (Coxiella burnetti) IgM and IgG, and serological tests for hepatitis, dengue fever IgM and Leptospira IgM, all of which were non-reactive. Urinalysis revealed a white blood cell count of 140 x 106/L (RR,l 10x 106) and an erythrocyte count of g 500x 106/L (RR,l 10x 106/L) with no microbial growth on culture. A bone marrow aspirate with trephine was performed on Day 4 (Day 2 in the ICU), and EBV DNA was detected in the resulting sample using a qualitative DNA test. The patientrs failure to improve, in combination with her past history of SOJIA, hepatosplenomegaly and a very high ferritin level led to a preliminary diagnosis of macrophage activation syndrome (MAS). Immunosuppressive treatment in the form of high-dose methylprednisolone (10mg/kg daily) and intravenous immunoglobulin (1 g/kg daily) were commenced. Inotrope and ventilatory requirements improved within 24 hours of this treatment. On Day 9 of admission she was extubated, and was discharged home on high-dose steroids 8 days later with no complications. Her bone marrow aspirate histological examination showed haemophagocytosis which confirmed the diagnosis of MAS (Box 2).

Disseminated Adenovirus Disease in Immunocompromised Patient Successfully Treated with Oral Ribavirin: A Case Report

In patients with immunological disorders, adenovirus infections are associated with significant rates of morbidity and mortality. Only few hematological units use molecular virological methods, such as polymerase chain reaction, for surveillance of adenovirus infection, and treatment strategies have never been evaluated in multicenter clinical trials. This report describes the detection and treatment of human adenovirus (HAdVs) disseminated disease in the case of a 46-year-old immunocompromised female having myelodysplastic syndrome with refractory cytopenia with multilineage dysplasia: International Prognostic Scoring System 1. Serum and urine samples were tested for the presence of adenoviral DNA using the quantitative real-time polymerase chain reaction (PCR) assay. For additional confirmation, sequencing of PCR products was also performed. With real-time PCR, we detected HAdV DNA in both serum and urine samples. The viral level constantly decreased with applied oral ribavirin therapy. As the result of sequencing, HAdVs type 11 was determined. Surveillance of adenovirus by real-time PCR is useful in detecting and monitoring disseminated HAdV infection; it is a potential standard diagnostic approach that could assist clinicians to decide whether antiviral therapy ought to be administered.

Adenoviruses in Lymphocytes of the Human Gastro-Intestinal Tract

ObjectivePersistent adenoviral shedding in stools is known to occur past convalescence following acute adenoviral infections. We wished to establish the frequency with which adenoviruses may colonize the gut in normal human subjects.MethodsThe presence of adenoviral DNA in intestinal specimens obtained at surgery or autopsy was tested using a nested PCR method. The amplified adenoviral DNA sequences were compared to each other and to known adenoviral species. Lamina propria lymphocytes (LPLs) were isolated from the specimens and the adenoviral copy numbers in the CD4+ and CD8+ fractions were determined by quantitative PCR. Adenoviral gene expression was tested by amplification of adenoviral mRNA.ResultsIntestinal tissue from 21 of 58 donors and LPLs from 21 of 24 donors were positive for the presence of adenoviral DNA. The majority of the sequences could be assigned to adenoviral species E, although species B and C sequences were also common. Multiple sequences were often present in the same sample. Forty-one non-identical sequences were identified from 39 different tissue donors. Quantitative PCR for adenoviral DNA in CD4+ and CD8+ fractions of LPLs showed adenoviral DNA to be present in both cell types and ranged from a few hundred to several million copies per million cells on average. Active adenoviral gene expression as evidenced by the presence of adenoviral messenger RNA in intestinal lymphocytes was demonstrated in 9 of the 11 donors tested.ConclusionAdenoviral DNA is highly prevalent in lymphocytes from the gastro-intestinal tract indicating that adenoviruses may be part of the normal gut flora.

Safety and Efficacy of CMX001 as Salvage Therapy for Severe Adenovirus Infections in Immunocompromised Patients

No therapeutic agent has yet been established as the definitive therapy for adenovirus infections. We describe the clinical experience of 13 immunocompromised patients who received CMX001 (hexadecyloxypropyl cidofovir), an orally bioavailable lipid conjugate of cidofovir, for adenovirus disease. We retrospectively analyzed 13 patients with adenovirus disease and viremia treated with CMX001; data were available for ≥ 4 weeks after initiation of CMX001 therapy. Virologic response (VR) was defined as a 99% drop from baseline or undetectable adenovirus DNA in serum. The median age of the group was 6 years (range, 0.92-66 years). One patient had severe combined immunodeficiency, 1 patient was a small bowel transplant recipient, and 11 were allogeneic stem cell transplant recipients. Adenovirus disease was diagnosed at a median of 75 days (range, 15-720 days) after transplantation. All patients received i.v. cidofovir for a median of 21 days (range, 5-90 days) before CMX001 therapy. The median absolute lymphocyte count at CMX001 initiation was 300 cells/μL (range, 7-1500 cells/μL). Eight patients (61.5%) had a ≥ 1 log10 drop in viral load after the first week of therapy. By week 8, 9 patients (69.2%) demonstrated a VR, with a median time to achieve VR of 7 days (range, 3-35 days). The change in absolute lymphocyte count was inversely correlated with the change in log10 viral load only at week 6 (r = -0.74; P = .03). Patients with VR had longer survival than those without VR (median 196 days versus 54.5 days; P = .04). No serious adverse events were attributed to CMX001 during therapy. CMX001 may be a promising therapeutic option for the treatment of severe adenovirus disease in immunocompromised patients.

Novel Adenoviruses in Wild Primates: a High Level of Genetic Diversity and Evidence of Zoonotic Transmissions

Adenoviruses (AdVs) broadly infect vertebrate hosts, including a variety of nonhuman primates (NHPs). In the present study, we identified AdVs in NHPs living in their natural habitats, and through the combination of phylogenetic analyses and information on the habitats and epidemiological settings, we detected possible horizontal transmission events between NHPs and humans. Wild NHPs were analyzed with a pan-primate AdV-specific PCR using a degenerate nested primer set that targets the highly conserved adenovirus DNA polymerase gene. A plethora of novel AdV sequences were identified, representing at least 45 distinct AdVs. From the AdV-positive individuals, 29 nearly complete hexon genes were amplified and, based on phylogenetic analysis, tentatively allocated to all known human AdV species (Human adenovirus A to Human adenovirus G [HAdV-A to -G]) as well as to the only simian AdV species (Simian adenovirus A [SAdV-A]). Interestingly, five of the AdVs detected in great apes grouped into the HAdV-A, HAdV-D, HAdV-F, or SAdV-A clade. Furthermore, we report the first detection of AdVs in New World monkeys, clustering at the base of the primate AdV evolutionary tree. Most notably, six chimpanzee AdVs of species HAdV-A to HAdV-F revealed a remarkably close relationship to human AdVs, possibly indicating recent interspecies transmission events.

Checkpoints in Adenoviral Production: Cross-Contamination and E1A

Adenoviruses are widely used for overexpressing proteins in primary mammalian cells. Incorporation of the early viral gene, E1A, or viral cross-contamination can occur during amplification, and identification of these products is crucial as the transcription of unwanted genetic material can impact cell function and compromise data interpretation. Here we report methods for evaluation of contaminating adenovirus and E1 viral DNA.

Severe community-acquired adenovirus pneumonia in an immunocompetent 44-year-old woman: a case report and review of the literature

IntroductionThis case report describes a rare condition: community-acquired adenovirus pneumonia in an immunocompetent adult. The diagnosis was achieved by using a multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and highlights the usefulness of these novel molecular diagnostic techniques in patients hospitalized with acute respiratory illness. We also performed a literature search for previously published cases and present a summary of the clinical, laboratory and radiological features of this condition.Case presentationA 44-year-old immunocompetent Caucasian woman was admitted to our hospital with an acute febrile respiratory illness associated with a rash. Her blood tests were non-specifically abnormal, and tests for bacterial pathogens were negative. Her condition rapidly deteriorated while she was in our hospital and required mechanical ventilation and inotropic support. A multiplex real-time RT-PCR assay performed on respiratory specimens to detect respiratory viruses was negative for influenza but positive for adenovirus DNA. The patient recovered on supportive treatment, and antibiotics were stopped after 5 days.ConclusionsCommunity-acquired adenovirus pneumonia in immunocompetent adult civilians presents as a non-specific acute febrile respiratory illness followed by the abrupt onset of respiratory failure, often requiring mechanical ventilation. Its laboratory and radiological features are typical of viral infections but also are non-specific. Novel multiplex real-time RT-PCR testing for respiratory viruses enabled us to rapidly make the diagnosis in this case. The new technology could be used more widely in patients with acute respiratory illness and has potential utility for rationalization of the use of antibiotics and improving infection control measures.

676. The dNTP Binding Efficiency of Adenovirus DNA Polymerase Contributes to Virus Replication Efficiency and Host Cell Tropism

676. The dNTP Binding Efficiency of Adenovirus DNA Polymerase Contributes to Virus Replication Efficiency and Host Cell Tropism

Molecular Impact and Inducible Factors Associated With Adenovirus Infection in Hematopoietic Skin Cell Transplant Patients

Background and Purpose of Study Latent and active adenovirus infections are detected in 5% to 20% of hematopoietic stem cell transplant (HSCT) patients. In addition to the significant role of adenoviral infection in the pathogenesis of late-onset hemorrhagic cystitis in HSCT patients, adenovirus infections may have possible roles in undefined posttransplant clinical complications. Therefore, pre- and posttransplantation we studied the prevalence and role of adenoviral infections among HSCT clinical syndromes using molecular methods. Materials and Methods In this cross-sectional study between 2005 and 2008. We collected 470 EDTA-treated blood samples from 125 HSCT recipients, including 70 (56%) men and 55 (44%) women. The 52 (41.6%) HSCT patients underwent autologous grafts and the other 73 (58.4%), from related donors. One EDTA-treated blood sample was collected from all recipients pretransplantation. Also once per week for 3 months we were collected blood samples from HSCT patients to evaluate the prevalence of adenovirus DNA infection by a qualitative in house polymerase chain reaction method. Results The adenovirus genome was diagnosed in 2/75 (2.7%) HSCT patient samples pretransplantation. There were 28/395 (7.1%) plasma samples of transplant patients infected with adenovirus DNA. Graft-versus-host disease (GVHD) clinical complications were observed in six adenovirus-infected transplant recipients; there was a significant correlation between these viral infections and GVHD clinical presentation. Conclusion The high prevalence of adenovirus infection in HSCT recipients pre- and posttransplantation, was significantly related to GVHD symptoms, enforcing the important pathogenic role of these viral infections in clinical complications post-HSCT.

Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery

BackgroundAdenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes.MethodsIn vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector.ResultsThe results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo.ConclusionThe results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin.

Anti‐adenoviral effect of interferon‐β and interferon‐γ in serotypes that cause acute keratoconjunctivitis

Interferons are currently used for hepatitis B and C virus infection; the effect of interferons against adenovirus has not been elucidated. It has been reported that interferon-β and interferon-γ were effective against adenovirus serotype 3. We investigated the anti-adenoviral effect of interferon-β and interferon-γ in serotypes that cause acute keratoconjunctivitis, using quantitative polymerase chain reaction in vitro. Experimental study carried out in a university. Five strains of different serotypes of adenovirus, types 3 (AdV3; species B), 4 (species E), 8, 19a and 37 (species D) and interferon-β and interferon-γ for in vitro assay. After pretreatment of A549 with serial dilution of interferons for 24 h, adenovirus was cultivated for 7 days, and adenoviral DNA was quantitatively measured by real-time polymerase chain reaction. Number of virus copies after treatment and the 50% effective concentration (EC(50) ). Interferon-β showed a dose-dependent inhibitory effect on all serotypes. EC(50) of interferon-β ranged between 211 and 843 IU/mL. A similar tendency was observed with interferon-γ. EC(50) of interferon-γ ranged between 133 and 9130 IU/mL. Among the serotypes, interferon-γ exhibited the greatest inhibitory effect on AdV37. In contrast, AdV4 showed the lowest sensitivity to interferon-γ. Statistically significant dose dependency for both interferon-β and interferon-γ was observed in several serotypes. These results suggest that both interferon-β and interferon-γ have anti-adenoviral activity in vitro. Interferons have the potential to be used for local treatment of adenoviral keratoconjunctivitis, although further evaluation in animal models and clinical trials is necessary.

Treatment of adenovirus hepatitis with cidofovir in a pediatric liver transplant recipient

AdV hepatitis is a rarely reported complication after pediatric liver transplantation that is associated with high rates of morbidity, mortality and graft failure. Successful treatment of AdV relies on early diagnosis of disease by quantitative PCR measurement of adenoviral DNA in blood and histological evidence in tissue biopsy. Pharmacologic treatment largely consists of antiviral therapy with CDV, an acyclic nucleoside phosphonate analog and reduction in immunosuppression. This report describes a case of AdV hepatitis in a pediatric liver transplant recipient successfully treated with a modified, renal sparing dosing of CDV.

Lack of Evidence for the Role of Human Adenovirus‐36 in Obesity in a European Cohort

Adenovirus infection has been shown to increase adiposity in chickens, mice, and nonhuman primates. Adenovirus type 36 (Ad-36) DNA was detected in adipose tissues in these animal trials. In the United States, Ad-36 significantly correlates with obesity as illustrated by an Ad-36 seroprevalence of 30% in obese individuals and 11% in nonobese individuals. We investigated the possibility of a similar correlation of Ad-36 in Dutch and Belgian persons. In total, 509 serum samples were analyzed for Ad-36 antibodies using a serum neutralization assay. In addition, PCR was used to detect adenoviral DNA in visceral adipose tissue of 31 severely obese surgical patients. Our results indicated an overall Ad-36 seroprevalence of 5.5% increasing with age. BMI of Ad-36 seropositive humans was not significantly different from seronegative humans. No adenoviral DNA could be found using PCR on visceral adipose tissue. In conclusion, this first Ad-36 study in the Netherlands and in Belgium indicates that Ad-36 does not play a role as a direct cause of BMI increase and obesity in humans in Western Europe.

Clinical features of adenoviral conjunctivitis at the early stage of infection

Because adenoviral conjunctivitis is a contagious disease, prompt and accurate diagnosis in the early stage of infection is necessary to prevent epidemics. We evaluated and compared the clinical features of adenoviral conjunctivitis at the first medical examination with those of nonadenoviral follicular conjunctivitis. The clinical features of 102 patients with suspected adenoviral conjunctivitis at the first medical examination were retrospectively reviewed. Human adenovirus (HAdV) DNA in samples from the patients was detected by polymerase chain reaction, and HAdV DNA-positive and HAdV DNA-negative patients were respectively assigned to adenoviral and nonadenoviral follicular conjunctivitis groups. The two groups were compared for bilaterality, intrafamilial infection, multiple subepithelial corneal infiltrates (MSI), preauricular lymphadenopathy, and severity of conjunctivitis. Adenoviral conjunctivitis and nonadenoviral conjunctivitis were diagnosed in 68 and 34 patients, respectively. Bilaterality, intrafamilial infection, and MSI showed significant intergroup differences. Remarkably, MSI was observed in 42.6% of the patients in the early stage of infection. There were no significant intergroup differences in preauricular lymphadenopathy or severity of conjunctivitis at any stage. To accurately diagnose adenoviral conjunctivitis in the early stage, bilateral conjunctival conditions, history of intrafamilial infection, and MSI should be checked, even in cases of mild or moderate follicular conjunctivitis.

ZFP260 Is an Inducer of Cardiac Hypertrophy and a Nuclear Mediator of Endothelin-1 Signaling

Pressure and volume overload induce hypertrophic growth of postnatal cardiomyocytes and genetic reprogramming characterized by reactivation of a subset of fetal genes. Despite intense efforts, the nuclear effectors of cardiomyocyte hypertrophy remain incompletely defined. Endothelin-1 (ET-1) plays an important role in cardiomyocyte growth and is involved in mediating the neurohormonal effects of mechanical stress. Here, we show that the phenylephrine-induced complex-1 (PEX1), also known as zinc finger transcription factor ZFP260, is essential for cardiomyocyte response to ET-1 as evidenced in cardiomyocytes with PEX1 knockdown. We found that ET-1 enhances PEX1 transcriptional activity via a PKC-dependent pathway which phosphorylates the protein and further potentiates its synergy with GATA4. Consistent with a role for PEX1 in cardiomyocyte hypertrophy, overexpression of PEX1 is sufficient to induce cardiomyocyte hypertrophy in vitro and in vivo. Importantly, transgenic mice with inducible PEX1 expression in the adult heart develop cardiac hypertrophy with preserved heart function. Together, the results identify a novel nuclear effector of ET-1 signaling and suggest that PEX1 may be a regulator of the early stages of cardiac hypertrophy.

Molecular characterization of adenoviruses in fecal samples of captively bred rhesus macaques in China

In an attempt to study the molecular diversity of simian adenoviruses in nonhuman primate (NHP) populations, we screened a colony of captively bred rhesus macaques (Macaca mulatta) in China for the presence of adenoviral DNA in stool samples. This was done by using the nested PCR method that targeted the adenovirus polymerase gene. Among the 57 animals analyzed, fecal samples from 12 animals were positive for the presence of adenoviral DNA and the PCR fragments were cloned for sequencing and phylogenetic analyses. The results suggested that the viral DNA clones were primarily segregated into two large groups: SAdV-6 (2 non-redundant sequences) and SAdV-7 (9 non-redundant sequences). In addition, there were three clones with more similarity to SAdV-1, SAdV-3 and HAdV-52 respectively. Our data confirmed the prevalence of adenoviral DNA in the feces of NHPs and revealed the heterogeneity and phylogenetics of the adenoviruses in the gastrointestinal tract of the study animals.

Complete Genome Analysis of a Novel Intertypic Recombinant Human Adenovirus Causing Epidemic Keratoconjunctivitis in Japan

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.