Isolation of a novel intertypic recombinant human mastadenovirus B2 from two unrelated bone marrow transplant recipients.

Human adenoviruses (HAdV) are well-known opportunistic pathogens of immunocompromised adult and pediatric patients but specific associations between HAdV species or individual HAdV types and disease are poorly understood. In this study we report the isolation of a novel HAdV-B2 genotype from two unrelated immunocompromised patients, both recipients of a hematopoietic cell transplant. In both patients, the course of HAdV infection is consistent with a scenario of reactivation of a latent virus rather than a primary opportunistic infection. Archived HAdV PCR-positive plasma, urine, and stool specimens were processed for virus isolation and detailed molecular characterization. Virus isolates were recovered from patient 1 from PCR-positive urine specimens obtained at days 103 and 116 after transplant in association with gross hematuria, and from a stool specimen obtained 138 days after transplant in association with diarrhea. An isolate was recovered from patient 2 from a PCR-positive urine specimen. Hexon and fiber gene amplification and sequencing were carried out for initial molecular typing, identifying the isolates as an intertypic recombinant with a HAdV-11-like hexon gene and a HAdV-77-like fiber gene. Comprehensive restriction fragment length polymorphism (RFLP) analysis was performed on viral DNA purified from urine and stool isolates, and next generation whole genome sequencing was carried out on purified viral genomic DNA. The genomes of the two isolated strains are 99.5% identical and represent the same RFLP genomic variant. The identified virus is a novel HAdV-B2 genotype designated HAdV-78 exhibiting a HAdV-11-like penton base, a HAdV-11-like hexon and a HAdV-77-like fiber (P11H11F77).

A Case of Adenovirus Viremia in a Pediatric Liver Transplant Recipient With Neutropenia and Lymphopenia: Who and When Should We Treat?

Human adenovirus (HAdV) is one of the most feared infections among immunocompromised patients. In particular, in liver transplant patients, HAdV has been implicated in acute liver failure with resultant mortality. The development of current molecular techniques and surveillance testing protocols have provided tools for early detection of HAdV infection, prior to or at the early onset of HAdV disease. Although reduction in immune suppression is the mainstay of therapy, many researchers have also advocated for early administration of antiviral therapy. In multiple reports, cidofovir treatment has been associated with declines in HAdV viral loads or clinical improvement in solid organ and bone marrow transplant recipients. However, there have also been case reports that raise questions about the effectiveness of antiviral therapy in controlling systemic HAdV disease. We report a case of a 26-month-old male recipient of a liver transplantation for hepatoblastoma who developed adenoviremia with an associated hepatitis and gastroenteritis. He recovered with reduced immune suppression but without antiviral therapy, thus avoiding potential toxicities associated with cidofovir therapy. This case a contrast to previous reports, and it highlights the ambiguity regarding which patients should receive HAdV-specific antiviral therapy. Additional knowledge regarding specific pediatric host factors and HAdV factors that predict poor outcomes are needed. Such information would allow clinicians to better stratify patients by risk at the time of adenoviremia detection so that low-risk patients are not unnecessarily exposed to medications with potential toxicities.

Sequence polymorphism in the E3 7.7K ORF of subspecies B1 human adenoviruses.

Sequences corresponding to the 7.7K open-reading frame (ORF) of the E3 region of subspecies B1 adenoviruses (Ads) were compared with prototype strains of Ad3, Ad7, Ad16, Ad21, and Ad50 and field isolates representing a variety of genome restriction types of Ad3 and Ad7 to better assess the extent of genetic variation in this intriguing region of the viral genome encoding a product whose function is still unknown. Alignment of 55 species B1 Ad sequences revealed a marked polymorphism in the 7.7K ORF and allowed the identification of eight distinct sequence profiles (SPs) characterized by (1) deletions that retain or change the reading frame, (2) single-base mutations (SBMs) that change the start codon (ATG to ATT or ATC), and (3) other SBMs. mRNAs of expected size for the observed sequence polymorphisms were identified by RT-PCR from DNAse I-treated total RNA extracts of infected cells. Predicted proteins ranged from 0 to 94 amino acids corresponding to molecular masses of 0-11 K. Together with the hypervariable regions of the hexon gene, the E3 7.7K ORF appears to be another area of the Ad genome in which genetic diversity may be generated by illegitimate recombination.

Comprehensive sequence analysis of the E1A proteins of human and simian adenoviruses.

Despite extensive study of human adenovirus type 5 E1A, surprisingly little is known about the E1A proteins of other adenoviruses. We report here a comprehensive analysis of the sequences of 34 E1A proteins. These represent all six primate adenovirus subgroups and include all human representatives of subgroups A, C, E, and F, eight from subgroup B, nine from subgroup D, and seven simian adenovirus E1A sequences. We observed that many, but not all, functional domains identified in human adenovirus type 5 E1A are recognizably present in the other E1A proteins. Importantly, we identified highly conserved sequences without known activities or binding partners, suggesting that previously unrecognized determinants of E1A function remain to be uncovered. Overall, our analysis forms a solid foundation for future study of the activities and features of the E1A proteins of different serotypes and identifies new avenues for investigating E1A function.

Outbreak of adenovirus genome type 7d2 infection in a pediatric chronic-care facility and tertiary-care hospital.

An outbreak of adenovirus infection that involved residents of a pediatric chronic-care facility, staff of a tertiary-care hospital, and a nosocomial hospital case was studied. In the pediatric facility, 31 (33%) of 93 residents had adenovirus infection, and 8 died. Risk factors for illness were an age of < 7 years (P = .004), presence of a tracheostomy (P = .015), and residence on a particular floor (P < .001). In the tertiary-care hospital, 36 health care workers had adenovirus infection; 26 (72%) had failed to follow strict contact and droplet precautions, and 30 (83%) continued to care for patients while they had symptoms. A 5-month-old patient with underlying lung disease acquired severe adenovirus infection in this hospital. All isolates were adenovirus type 7 (Ad7). DNA restriction analysis revealed the band patterns of all isolates to be identical and characteristic of the genome type d2. Thus, Ad7d2 caused significant morbidity and mortality in persons in the pediatric chronic-care facility and tertiary-care hospital. This is the first published description of Ad7d2 strains in the United States.

Mouse adenovirus type 1 replication in vitro is resistant to interferon.

The effects of mouse interferon (IFN)-alpha/beta and recombinant IFN-gamma on mouse adenovirus type 1 (MAV-1) replication were investigated in single-cycle infectious virus yield reduction assays on mouse L929 cells. Viral yields at 3 days postinfection indicated that wt MAV-1 and pmE314, an early region 3 null mutant, were relatively insensitive to both IFN-alpha/beta and IFN-gamma, whereas early region 1A (E1A) mutants pmE109 (null), dlE105 (conserved region 1 deletion, CR1 Delta), dlE102 (CR2 Delta), and dlE106 (CR3 Delta) were sensitive. MAV-1 E1A that was inducibly expressed in mouse fibroblast 37.1 cells rescued vesicular stomatitis virus from the antiviral effect of IFN-alpha/beta but not from the antiviral effect of IFN-gamma. Interferon-inducible gene expression was reduced in 37.1 cells as compared to the parental 3T6 cell line. Steady-state levels of IFN-inducible gene mRNAs were also reduced in 3T6 cells infected with the wild-type virus and pmE314 but not in cells infected with pmE109. These results suggest that the MAV-1 E1A gene product is capable of interfering with the signaling pathways of both types of IFN, although modulation of IFN-alpha/beta antiviral activity was more pronounced.

Genome type analysis of Brazilian adenovirus strains of serotypes 1,2,3,5, and 7 collected between 1976 and 1995.

A collection of 92 epidemiologically unrelated isolates of Ad1 (n = 14), Ad2 (n = 29), Ad3 (n = 19), Ad5 (n = 16), and Ad7 (n = 14) collected in the cities of Belem do Pará (1 degree S 48 degrees W) and Rio de Janeiro (23 degrees S 43 degrees W) between 1976 and 1995 from patients with respiratory disease and conjunctivitis were characterized by restriction enzyme analysis of genomic DNA. Among the strains of subgenus B, two different genome types of serotype 7, 7b and 7e, were identified. The analysis of their temporal distribution throughout the study period suggested an alternating appearance of these two DNA variants. Only one genome type of Ad3, 3p, was detected during the sampling period. Further analysis with Xba I, Bcl I, and Hpa I indicated that it is a p1-like genome type. Both previously described and new genomic variants were identified among subgenus C strains. Genome types D1, D7, D10, and one not previously described were identified among the 14 Ad1 strains analyzed. Genome types D2, D5, D25, and 13 new DNA variants were identified among the 29 Ad2 isolates. Genome type D38 and 5 new variants were found among the 16 strains of Ad5. In spite of the relatively small size of the sample analyzed, the results of this study confirm the important genetic variability previously observed for members of subgenus C by other authors.

Involvement of epitope mimicry in potentiation but not initiation of autoimmune disease.

We have examined whether the peptide (368-381) from the murine adenovirus type 1 E1B sequence, exhibiting a high degree of homology with the known pathogenic thyroglobulin (Tg) T cell epitope (2695-2706), can induce experimental autoimmune thyroiditis (EAT) in SJL/J mice. The viral peptide was a poor immunogen at the T or B cell level and did not elicit EAT either directly or by adoptive transfer assays. Surprisingly, however, the viral peptide was highly antigenic in vitro, activating a Tg2695-2706-specific T cell clone and reacting with serum IgG from mice primed with the Tg homologue. The viral peptide also induced strong recall responses in Tg2695-2706-primed lymph node cells, and subsequent adoptive transfer of these cells into naive mice led to development of highly significant EAT. These data demonstrate that nonimmunogenic viral peptides can act as agonists for preactivated autoreactive T cells and suggest that epitope mimicry may at times play a potentiating rather than a precipitating role in the pathogenesis of autoimmune disease.

Lymphocyte subsets and cytokines in adenoviral infection in children.

To determine the distribution of major blood lymphocyte subsets we evaluated blood lymphocytes by flow cytometry in adenovirus-infected infants aged 30-730 d. In addition, interleukin-1-receptor antagonist, interleukin-10 and transforming growth factor-beta1 were measured in serum by enzyme-linked immunosorbent assay. According to clinical parameters, mechanical ventilation and outcome, infections were classified as moderate (n = 15), severe (n = 11) and fatal (n = 12). Controls were 13 healthy children. In severe and fatal infection, T cells (CD5+/CD19-), NK effectors (CD16+), CD4+ T subset and B1 subset of B lymphocytes (CD5+/CD19+) were all significantly decreased. CD8+ cells were decreased in severe but not fatal cases. There was no difference in serum values of interleukin-10; however, fatal cases had high interleukin 1-receptor antagonist values. Interestingly, patients with moderate infection showed significantly increased values of transforming growth factor-beta1. These results demonstrate that life-threatening adenoviral infection is associated with marked abnormalities in blood lymphocyte and cytokine profile.

Fatal adenovirus infection associated with new genome type.

The first fatal case caused by the new genome type 7i is described in an 8-month-old boy requiring long-term respiratory support who developed Reye's syndrome, acute respiratory distress, and bronchiolitis obliterans with fatal evolution. Adenovirus was detected in nasopharyngeal secretions and was persistently positive during hospitalization. IgM and IgG adenovirus antibody titers measured in serum by enzyme-linked immunoassay (EIA) were 1:32 and 1:800, respectively. Serum interleukins (IL) and interferons (IFN) measured by EIA were as follows: IL-2, 110 pg/ml; IL-6, 300 pg/ml; IL-8, 7,000 pg/ml; TNF-alpha, 35 pg/ml, IL-1 and IL-4 undetectable, IFN-alpha 2,200 pg/ml, and IFN-gamma 700 pg/ml. Virologic studies showed that adenovirus isolated belonged to subgenus B, and digestion of viral DNA with Bam HI, Sma I, Bgl II, and Hind III identified the isolate as belonging to genome type 7i. Autopsy showed bronchiolitis obliterans with diffuse alveolar damage and perivenular fatty degeneration with polymorphonuclear infiltrates in the periportal spaces. The difficulty in obtaining adequate oxygenation with minimization of iatrogenic oxygen injury is discussed.

Distribution of mouse adenovirus type 1 in intraperitoneally and intranasally infected adult outbred mice.

In situ nucleic acid hybridization and immunohistochemistry were used to determine the histological localization of mouse adenovirus type 1 (MAV-1) during acute infection of adult mice infected either intraperitoneally or intranasally with 1,000 PFU of wild-type virus. Organ samples were collected from days 1 to 17 postinfection for the intraperitoneally infected mice and from days 1 to 13 for the intranasally infected mice. Endothelial cells of the brain and spinal cord showed extensive evidence of MAV-1 infection. Endothelial cells in lungs, kidneys, and other organs were also positive for MAV-1, indicating a widespread involvement of the systemic circulation. The presence of viral nucleic acid and/or antigen was also demonstrated in lymphoid tissue. The spleens, Peyer's patches, and peripheral lymph nodes showed positive staining at various times postinfection in mice infected by either route. Virus-infected cells in the spleen exhibited a stellate shape and were localized to the red pulp and germinal centers, suggesting that they are cells of the mononuclear phagocytic system.

Rapid subgenus identification of human adenovirus isolates by a general PCR.

In most clinical situations involving adenovirus infection, subgenus (subgroup) identification of an adenovirus isolate is as informative as a finer identification by serotype. A PCR method which allows the identification of human adenovirus isolates as members of subgenera A, B:1, B:2, C, D, E, or F is described. It is based on a simple (nonnested) PCR using primers which bind to regions immediately flanking the VA RNA-encoding regions of human adenovirus genomes. The PCR allows amplification of DNA from all 49 human adenovirus prototype strains so far described. Since there are differences in the lengths of the VA RNA-encoding regions in adenoviruses of different subgenera, it is possible to differentiate some subgenera according to the size of the PCR product determined by electrophoresis. This forms the basis of an initial broad categorization of isolates as belonging to either (i) subgenus B:1, C, D, or E or (ii) subgenus A, B:2, or F. Subgenus identification is completed by a one-step restriction enzyme digestion and gel electrophoresis. The method was assessed by blind subgenus identification of 200 miscellaneous primate adenovirus isolates prepared by the reference laboratory at Bilthoven, The Netherlands. Identification at the subgenus level by PCR correlated 91.5% with the results of serotyping. A further 5.5% of isolates were correctly identified as belonging to one of two specified subgenera. Six of the 200 identifications (3%) were unsuccessful for various reasons, including weak PCR products, intermediate strains, and mistaken primate host. The method should serve as a rapid means of confirming adenovirus cytopathic effects in laboratories performing virus culture, with simultaneous subgenus identification of the isolate. It will also have relevance as an aid to conventional serotyping for epidemiological purposes, since for all adenoviruses except those belonging to subgenus D, neutralization tests need only involve a maximum of four type-specific antisera.

Molecular epidemiology of adenovirus acute lower respiratory infections of children in the south cone of South America (1991-1994).

A collection of 165 adenovirus strains isolated from nasopharyngeal aspirates of children hospitalized for acute lower respiratory infection in Argentina, Chile, and Uruguay between 1991 and 1994 was studied by restriction enzyme analysis (work performed in the Department of Virology, University of Umeå). Of the isolates, 71% (n = 117) were identified as members of subgenus B. Of these, 101 (61.2%) corresponded to genome type 7h, four (2.4%) to genome type 3p2, four (2.4%) to genome type 11a, one (0.6%) to genome type 7b, and one (0.6%) to genome type 7c. Two isolates that were neutralized as serotype 3 and four isolates that were neutralized as serotype 7 exhibited novel BamHI cleavage profiles corresponding to three new genome types denominated 3x, 7i, and 7j. Subgenus C members represented 28.5% of all typed isolates. Five different genome types of Ad1, seven genome types of Ad2, and three genome types of Ad5 were identified of, which two, two, and one, respectively, were found to correspond to new DNA variants. Only one isolate (0.6%) corresponded to Ad4 of subgenus E. Ad7h was isolated from 17 of the 18 fatal cases recorded among the patients included in the study.

Sequence analysis of the E3 region and fiber gene of human adenovirus genome type 7h.

Adenovirus type 7h is currently the predominant virulent genome type of serotype 7 isolated in Argentina, Chile, and Uruguay in association with severe infantile pneumonia. In order to characterize possible molecular determinants of pathogenicity, the nucleotide sequence of a 5904-bp fragment (76 to 93 mu) containing the entire E3 region and the fiber gene of Ad7h was established. The organization of the ORFs within the E3 region was similar to that reported for the prototype strains of Ad7 and Ad3. A comparison of the nucleotide and amino acid sequences of all ORFs revealed a higher homology between Ad7h and Ad7p than between Ad7h and Ad3 for 12.0K and 16.1K, whereas the 15.3K ORF and the adjacent fiber gene were strikingly more homologous to those of Ad3 (99.5 vs 81.1% and 98.2 vs 66.6%, respectively). The equivalent to ORF 7.7K in Ad7p was missing in Ad7h due to a deletion and a mutation affecting the start codon (ATG-->ATT). Although the hemagglutinin of the Ad7h fiber could not be characterized due to its lack of activity on monkey erythrocytes, our results indicate that Ad7h is an intermediate strain 7-3.

Genome type analysis of Chilean adenovirus strains isolated in a children's hospital between 1988 and 1990.

In a study designed to evaluate the genetic variability of adenovirus strains associated with infantile cases of respiratory disease requiring hospitalization, a collection of 136 adenovirus isolates obtained in the Roberto del Rio Children's Hospital of Santiago, Chile between June 1988 and November 1990 was studied by restriction enzyme analysis. Nasopharyngeal aspirates were obtained on admission from children under 2 years. During the study period a total of 227 adenovirus respiratory infections (ARI) were diagnosed at the ward for ARI by immunofluorescence, representing 23% of all admissions. Fifty percent of the 136 typed strains were found to belong to subgenus B, and the other 50% corresponded to subgenus C. Digestion with a set of seven enzymes allowed the identification of nine different genome types of subgenus C, three of which had not been previously described, exhibiting novel restriction patterns with either BgI II or BstEII. Ad7h, identified in 66 isolates, was the predominant genome type and was associated with the nine cases requiring mechanical respiratory assistance and with the two fatalities recorded during the 29 months. No differences were found between the age and sex distribution of subgenus B and C genomic variants, but the mean length of hospital stay (X +/- 2 SE) recorded among patients infected with subgenus B types was significantly higher (17.72 + 4.52 days (n = 55) vs. 7.54 + 1.70 days (n = 53); F = 17.22; P < 0.0001).

Genome type analysis of South American adenoviruses of subgenus C collected over a 7-year period.

A collection of 148 adenovirus strains of subgenus C collected in Argentina, Chile, and Uruguay were studied by restriction enzyme analysis of genomic DNA with endonucleases BamHI, BglII, BstEII, EcoRI, HindIII, KpnI, and SmaI. Only strains corresponding to serotypes 1, 2, and 5 were identified representing both already described and new genome types. The most frequently detected type was Ad 2 followed by Ad 1 and Ad 5. Three different genomic variants of both Ad 1 and Ad 5 were discriminated. Ad 2 with 9 genome types exhibited the greatest variability. Novel profiles emerged only in strains corresponding to this serotype by restriction with BglII and BstEII. The circulation of prototype-like strains of Ad 1 throughout the study period is note-worthy. Thirty-six of 43 strains corresponded to genome type D 1. Clearly, one genome type of each serotype predominated over the 7-year period (Ad 1 D 1, n = 36; Ad 2 D 5, n = 32, Ad 5 D 36, n = 17). Some genomic variants were detected sporadically and only in Argentina, Chile, or Uruguay whereas others were widely distributed and circulated for years. A high proportion of the analysed strains was isolated from children under one year and males were more frequently infected than females.

Molecular epidemiology of adenoviruses associated with acute lower respiratory disease of children in Buenos Aires, Argentina (1984-1988).

DNA restriction analysis was carried out on a sample of 73 adenovirus strains isolated in Buenos Aires from nasopharyngeal aspirates of children with lower acute respiratory infection between 1984 and 1988. Thirty-five isolates (47.9%) were classified as members of subgenus B. Of these, three were identified as a new genome type of Ad3p denominated Ad3p3; five strains corresponded to genome type 7b and two to genome type 7c. The other 25 isolates were identified as the recently recognized genome type 7h. All 6 fatalities recorded within this group of 73 children were associated with infection by Adenovirus genome type 7h. Thirty-seven isolates (50.7%) were classified within subgenus C that corresponded to 9 different genome types denominated 1p (n = 5); 1# (n = 2); 2p (n = 4); 2b (n = 6); 2# (n = 5); 5# (n = 4); 5* (n = 7) and 5+ (n = 2). All genome types of subgenus C were compared with the data reported by Adrian et al. (Archives of Virology 112:235-238, 1990). The Ad1p and Ad1# genome types could be allocated to AV1 genome types D1 and D10, respectively. Ad2b genome type could be allocated to AV2 genome type D25. No counterparts were found for the remaining 6 genomic variants. Only one isolate was identified as Ad4a of subgenus E. The comparison of the results of the present study with those of the molecular characterization of Chilean strains isolated between 1984 and 1987 suggests that the adenovirus strains associated with respiratory disease of children may be common in both countries.

Characterization of adenovirus genome type 7h: analysis of its relationship to other members of serotype 7.

Ad7h is a newly identified genome type associated with severe lower acute respiratory infections and has so far been isolated only in South America. To obtain a clue to its possible origin, the degree of restriction enzyme site homology between adenovirus genome type 7h and those representative of the three described genomic clusters (GC) for serotype 7 was studied by analysis of pairwise comigrating DNA restriction fragments (PCRF) after digestion with BamHI, BglI, BglII, BstEII, EcoRI, HindIII, HpaI, SalI, SmaI, XbaI, and XhoI. The most closely related genome types, Ad7b, Ad7d, and Ad7g, displayed 85.7, 83.8, and 82.8% comigrating fragments, respectively. Ad7p (GC1) was clearly distant with only 68.6% PCRF. No remarkable close genetic relationships (%PCRF greater than 90) were observed with any of the genome types grouped within GC3 or with Ad7g (the only member of GC2), so since Ad7h seems to be related to both members of GC2 and GC3, it could equally be considered to represent a new cluster or to end up grouped in either GC2 or GC3, depending on the results of further analysis.

Molecular epidemiology of adenoviruses isolated from hospitalized children with severe lower acute respiratory infection in Santiago, Chile.

Genome analysis was performed on 69 adenovirus isolates from pharyngeal secretions of young children hospitalized with severe lower acute respiratory disease in Santiago, Chile, between 1984 and 1986. As expected, most isolated strains belonged to subgenus B (68.1%). Among the isolates of 1984, Ad7c was the dominant genotype (12 out of 23). The majority of isolates of 1986 were of the recently described genomic variant 3f.