Approach for defining human adenovirus infection and disease for central review adjudication in clinical studies.

BACKGROUND: Pediatric allogeneic hematopoietic cell transplant (allo-HCT) recipients are at risk for morbidity and mortality from human adenovirus (HAdV). HAdV can be detected in an asymptomatic state, referred to as infection or with signs or symptoms of illness, referred to as disease. Standardized case definitions are needed to distinguish infection from disease and allow for consistent reporting in both observational cohort studies and therapeutic clinical trials. METHODS: A working group of experts in virology, transplant infectious disease, and HCT was assembled to develop HAdV infection and disease definitions with the degree of certainty (i.e., possible, probable, and proven). Definitions were further refined through an iterative process and independently applied by two central review committees (CRCs) to 20 pediatric allo-HCT recipients with at least one HAdV-positive PCR. RESULTS: Initial HAdV infection and disease definitions were developed and updated through an iterative process after reviewing clinical and virological details for 81 subjects with at least one positive HAdV PCR detected in a clinical specimen. Independent application of final definitions to 20 HAdV positive allo-HCT recipients by two CRCs yielded similar number of HAdV infection or disease events but with variation of degree of certainty for some events. CONCLUSIONS: Application of definitions by a CRC for a study of HAdV infection and disease is feasible and can provide consistency in the assignment of outcomes. Definitions need further refinement to improve reproducibility and to provide guidance on determining clinical improvement or worsening after initial diagnosis of HAdV infection or disease.

A Fulminant Case of Adenovirus Genotype C108 Infection in a Pediatric Stem Cell Transplant Recipient with x-Linked Lymphoproliferative Syndrome Type 1.

A 3-year-old male with X-linked lymphoproliferative syndrome type 1 underwent an unrelated umbilical cord blood transplant (UUCBT). The week prior to transplant the patient tested positive for adenovirus (HAdV) with a viral load of <190 copies/mL and was started on cidofovir. UUCBT proceeded as scheduled, and the patient engrafted on day +19. The patient's HAdV load in serum continued to rise with resulting hepatic dysfunction, despite ongoing therapy with cidofovir and HAdV specific T-cell infusions. The patient died 6 months after transplantation having never cleared the virus. Next generation whole genome sequencing and sequence data analyses identified an intertypic recombinant HAdV-C P1H2F2 closely related (99.6% similarity) to genotype C108 in the isolates from three blood specimens obtained during the last week of life. Incidentally, the de novo assembly strategy enabled the detection of an adeno-associated virus type 2 (AAV2) genome in the DNA purified from the plasma isolates. Proteotyping analysis revealed minor differences in the predicted amino acid sequences for E1A, E1B 19K, E1B 55K, DNA polymerase, penton base, and fiber. None of the mutations previously described for HAdV-C5 variants resistant to cidofovir were identified. In silico restriction enzyme analysis revealed a distinct Sac I profile for the identified virus, supporting its designation as a C108 variant.

Characterization of Human Adenoviruses of Medical Importance: Isolation of Infectious Virus from Clinical Specimens and Molecular Typing.

Human adenoviruses (HAdVs) constitute a group of ubiquitous viruses that currently comprises 51 well-defined serotypes and more than 113 genotypes classified into seven species, HAdV-A through HAdV-G. The members of these species differ considerably in their genomic characteristics and also in their tropism and pathogenicity. Virus isolation in cell culture remains critical for the preservation and comprehensive characterization of viruses of biomedical interest but has been almost completely abandoned by diagnostic laboratories. Currently, the most frequently used approach for the detection of HAdV in clinical specimens is real-time qPCR targeting a region of the hexon gene, conserved among all genotypes described to the present. In the absence of typing, the detection of an HAdV in association with disease provides limited information. Molecular typing is therefore highly desirable and required in the epidemiological investigation of HAdV-associated disease. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Virus isolation from plasma and whole blood Alternate Protocol 1: Virus isolation from stool Alternate Protocol 2: Virus isolation from respiratory specimens and urine Alternate Protocol 3: Virus isolation from tissue specimens Support Protocol: Inoculation of shell vials Basic Protocol 2: Extraction of highly pure viral genomic DNA from infected cells Basic Protocol 3: Molecular detection of human adenovirus by real-time PCR Basic Protocol 4: Molecular typing for basic identification of species and hexon type Basic Protocol 5: Typing human adenoviruses by next-generation whole-genome sequencing and analysis.

Dissemination and genome characterization of a human adenovirus F41 in a patient with B-Cell lymphoma.

Adenovirus (HAdV) F41 is a common cause of gastroenteritis and has rarely been reported associated with disseminated disease. In this report, an adult patient with a history of ulcerative colitis, cryptogenic cirrhosis, stage III adenocarcinoma, high-grade diffuse large B-cell lymphoma on chemotherapy was diagnosed with disseminated adenovirus infection. HAdV DNA was quantified in stool, plasma, and urine with viral loads of 7, 4, and 3 log10 copies/mL, respectively. The patient's course was rapidly progressive and he passed away 2 days after initiation of antiviral therapy. The patient's infecting virus was characterized as HAdV-F41 by whole genome sequencing.

Two novel recombinant human mastadenovirus D genotypes associated with acute respiratory illness.

Two novel genotypes of species human mastadenovirus D designated 109 and 110 were isolated from three epidemiologically unrelated cases of acute respiratory disease detected in January 2018 by surveillance efforts at the California/Mexico border. Both genotypes represent examples of intertypic recombination. Genotype D109 is most closely related to genotype D56 (97.68% genomic similarity) and features a type D22-like penton base, a type D19-like hexon gene, and a type D9-like fiber [P22/H19/F9]. On the other hand, genotype D110 is most closely related to type D22 (96.94% genomic similarity) and features a type D67-like penton base, a novel hexon gene, and a type D9-like fiber [P67/H110/F9]. Importantly, the fibers of both novel genotypes are highly similar to those of genotypes D56 and D59, which have also been isolated from a few cases of respiratory infections. The present report shows data contributing to the understanding of the molecular determinants of the expanded tissue tropism of certain members of species HAdV-D.

ICTV Virus Taxonomy Profile: Adenoviridae 2022.

The family Adenoviridae includes non-enveloped viruses with linear dsDNA genomes of 25-48 kb and medium-sized icosahedral capsids. Adenoviruses have been discovered in vertebrates from fish to humans. The family is divided into six genera, each of which is more common in certain animal groups. The outcome of infection may vary from subclinical to lethal disease. This is a summary of the ICTV Report on the family Adenoviridae, which is available at ictv.global/report/adenoviridae.

Human Adenovirus Type 4 Comprises Two Major Phylogroups with Distinct Replicative Fitness and Virulence Phenotypes.

Human adenovirus type 4 (HAdV-E4) is the only type (and serotype) classified at present within species Human mastadenovirus E that has been isolated from a human host. Recent phylogenetic analysis of whole-genome sequences of strains representing the spectrum of intratypic genetic diversity described to date identified two major evolutionary lineages designated phylogroups (PGs) I and II and validated the early clustering of HAdV-E4 genomic variants into two major groups by low-resolution restriction fragment length polymorphism analysis. In this study, we expanded our original analysis of intra- and inter-PG genetic variability and used a panel of viruses representative of the spectrum of genetic diversity described for HAdV-E4 to examine the magnitude of inter- and intra-PG phenotypic diversity using an array of cell-based assays and a cotton rat model of HAdV respiratory infection. Our proteotyping of HAdV-E strains using concatenated protein sequences in selected coding regions including E1A, E1B-19K and -55K, DNA polymerase, L4-100K, various E3 proteins, and E4-34K confirmed that the two clades encode distinct variants/proteotypes at most of these loci. Our in vitro and in vivo studies demonstrated that PG I and PG II differ in their growth, spread, and cell-killing phenotypes in cell culture and in their pulmonary pathogenic phenotypes. Surprisingly, the differences in replicative fitness documented in vitro between PGs did not correlate with the differences in virulence observed in the cotton rat model. This body of work is the first reporting phenotypic correlates of naturally occurring intratypic genetic variability for HAdV-E4. IMPORTANCE Human adenovirus type 4 (HAdV-E4) is a prevalent causative agent of acute respiratory illness of variable severity and of conjunctivitis and comprises two major phylogroups that carry distinct coding variations in proteins involved in viral replication and modulation of host responses to infection. Our data show that phylogroup (PG) I and PG II are intrinsically different regarding their ability to grow and spread in culture and to cause pulmonary disease in cotton rats. This is the first report of phenotypic divergence among naturally occurring known genetic variants of an HAdV type of medical importance. This research reveals readily detectable phenotypic differences between strains representing phylogroups I and II, and it introduces a unique experimental system for the elucidation of the genetic basis of adenovirus fitness and virulence and thus for increasing our understanding of the implications of intratypic genetic diversity in the presentation and course of HAdV-E4-associated disease.

Oral Vaccination Protects Against Severe Acute Respiratory Syndrome Coronavirus 2 in a Syrian Hamster Challenge Model.

BACKGROUND: Vaccines that are shelf stable and easy to administer are crucial to improve vaccine access and reduce severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission around the world. METHODS: In this study, we demonstrate that an oral, adenovirus-based vaccine candidate protects against SARS-CoV-2 in a Syrian hamster challenge model. RESULTS: Hamsters administered 2 doses of VXA-CoV2-1 showed a reduction in weight loss and lung pathology and had completely eliminated infectious virus 5 days postchallenge. Oral immunization induced antispike immunoglobulin G, and neutralizing antibodies were induced upon oral immunization with the sera, demonstrating neutralizing activity. CONCLUSIONS: Overall, these data demonstrate the ability of oral vaccine candidate VXA-CoV2-1 to provide protection against SARS-CoV-2 disease.

Adenovirus: Epidemiology, Global Spread of Novel Types, and Approach to Treatment.

Adenoviruses (AdVs) are DNA viruses that typically cause mild infections involving the upper or lower respiratory tract, gastrointestinal tract, or conjunctiva. Rare manifestations of AdV infections include hemorrhagic cystitis, hepatitis, hemorrhagic colitis, pancreatitis, nephritis, or meningoencephalitis. AdV infections are more common in young children, due to lack of humoral immunity. Epidemics of AdV infection may occur in healthy children or adults in closed or crowded settings (particularly military recruits). The vast majority of cases are self-limited. However, the clinical spectrum is broad and fatalities may occur. Dissemination is more likely in patients with impaired immunity (e.g., organ transplant recipients, human immunodeficiency virus infection). Fatality rates for untreated severe AdV pneumonia or disseminated disease may exceed 50%. More than 100 genotypes and 52 serotypes of AdV have been identified and classified into seven species designated HAdV-A through -G. Different types display different tissue tropisms that correlate with clinical manifestations of infection. The predominant types circulating at a given time differ among countries or regions, and change over time. Transmission of novel strains between countries or across continents and replacement of dominant viruses by new strains may occur. Treatment of AdV infections is controversial, as prospective, randomized therapeutic trials have not been done. Cidofovir has been the drug of choice for severe AdV infections, but not all patients require treatment. Live oral vaccines are highly efficacious in reducing the risk of respiratory AdV infection and are in routine use in the military in the United States but currently are not available to civilians.

Fatal Neonatal Sepsis Associated with Human Adenovirus Type 56 Infection: Genomic Analysis of Three Recent Cases Detected in the United States.

BACKGROUND: Human adenovirus (HAdV)-D56 was first described in 2011 by genomics analysis of a strain isolated in France in 2008 from a fatal case of neonatal infection. Since then, it has been reported in cases of keratoconjunctivitis and male urethritis. Three epidemiologically unrelated fatal cases of neonatal sepsis associated with infection by HAdV-D strains with a similar genetic makeup were documented in the United States between 2014 and 2020. METHODS: Whole genome sequences were obtained for the isolated strains, and genomics analyses were conducted to compare them to phylogenetically related HAdV-D genomic sequences available in GenBank. RESULTS: The three new US strains were indistinguishable by in silico restriction enzyme analysis. Their genome sequences were 99.9% identical to one another and to the prototype strain isolated in 2008 from a similar context of disease. The phylogenetic reconstruction revealed a highly supported clustering of all HAdV-D56 strains isolated in various countries since 1982. Our comparison to serologically intermediate strains 15/H9 described in the literature indicated that HAdV-D56-like viruses have circulated worldwide since the late 1950s. CONCLUSION: As with other HAdV-D genotypes with the ability to infect ocular and genital mucosae, the risk of severe prenatal or perinatal HAdV-D56 infection must be considered.

Complete Genome Sequences of Two Human Adenovirus Type 55 Isolates from South Korea and the United States.

Here, we report two complete genome sequences of human adenovirus 55 (HAdV-55) isolates, from a patient in Pennsylvania in 2006 and a U.S. military member in South Korea in 2019. The findings demonstrate the continued global transmission of HAdV-55 viruses in both military and civilian populations.

Human Adenovirus 7-Associated Hemophagocytic Lymphohistiocytosis-like Illness: Clinical and Virological Characteristics in a Cluster of Five Pediatric Cases.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of immune dysregulation. Children often suffer from primary genetic forms of HLH, which can be triggered by infection. Others suffer from secondary HLH as a complication of infection, malignancy, or rheumatologic disease. Identifying the exact cause of HLH is crucial, as definitive treatment for primary disease is hematopoietic stem cell transplant. Adenoviruses have been associated with HLH but molecular epidemiology data are lacking. METHODS: We describe the clinical and virologic characteristics of 5 children admitted with adenovirus infection during 2018-2019 who developed HLH or HLH-like illness. Detailed virologic studies, including virus isolation and comprehensive molecular typing were performed. RESULTS: All patients recovered; clinical management varied but included immunomodulating and antiviral therapies. A genetic predisposition for HLH was not identified in any patient. Adenovirus isolates were recovered from 4/5 cases; all were identified as genomic variant 7d. Adenovirus type 7 DNA was detected in the fifth case. Phylogenetic analysis of genome sequences identified 2 clusters-1 related to strains implicated in 2016-2017 outbreaks in Pennsylvania and New Jersey, the other related to a 2009 Chinese strain. CONCLUSIONS: It can be challenging to determine whether HLH is the result of an infectious pathogen alone or genetic predisposition triggered by an infection. We describe 5 children from the same center presenting with an HLH-like illness after onset of adenovirus type 7 infection. None of the patients were found to have a genetic predisposition to HLH. These findings suggest that adenovirus 7 infection alone can result in HLH.

Human Adenovirus Type 55 Distribution, Regional Persistence, and Genetic Variability.

Human adenovirus type 55 (HAdV-55) causes acute respiratory disease of variable severity and has become an emergent threat in both civilian and military populations. HAdV-55 infection is endemic to China and South Korea, but data from other regions and time periods are needed for comprehensive assessment of HAdV-55 prevalence from a global perspective. In this study, we subjected HAdV-55 isolates from various countries collected during 1969-2018 to whole-genome sequencing, genomic and proteomic comparison, and phylogenetic analyses. The results show worldwide distribution of HAdV-55; recent strains share a high degree of genomic homogeneity. Distinct strains circulated regionally for several years, suggesting persistent local transmission. Several cases of sporadic introduction of certain strains to other countries were documented. Among the identified amino acid mutations distinguishing HAdV-55 strains, some have potential impact on essential viral functions and may affect infectivity and transmission.

Human mastadenovirus-B (HAdV-B)-specific E3-CR1ß and E3-CR1γ glycoproteins interact with each other and localize at the plasma membrane of non-polarized airway epithelial cells.

The E3 region of all simian and human types classified within species Human mastadenovirus B (HAdV-B) encodes two unique highly conserved ORFs of unknown function designated E3-CR1ß and E3-CR1γ. We generated a HAdV-3 mutant encoding small epitope tags at the N-termini of both E3-CR1ß and E3-CR1γ (HAdV-3 N-tag wt) and a double knock out (HAdV-3 N-tag DKO) mutant virus that does not express either protein. Our studies show that HAdV-3 E3-CR1ß and E3-CR1γ are type I transmembrane proteins that are produced predominantly at late times post infection, are glycosylated, co-localize at the plasma membrane of non-polarized epithelial cells, and interact with each other. At their extreme C-termini HAdV-B E3-CR1ß and E3-CR1γ possess a conserved di-leucine motif followed by a class II PDZ domain binding motif (PBM). HAdV-3 E3-CR1ß and E3-CR1γ are dispensable for virus growth, progeny release, spread, and plaque formation in A549 cells.

Genomic and phylogenetic analysis of two guinea pig adenovirus strains recovered from archival lung tissue.

Next generation sequencing was used to determine the whole genome sequence for two different strains of guinea pig adenovirus (GPAdV) detected in association with outbreaks of pneumonia in Australia in 1996, and in Germany in 1997 using total DNA extracted from infected archival frozen lung tissue as a template. The length of the determined genomic sequences was 37,031 bp and 37,070 bp, respectively. The nucleotide composition showed a relatively high content of guanine + cytosine (G + C) of 62 %. The 99.6 % nucleotide identity between the two sequenced viruses suggests that they may represent variants of the same genotype. The GPAdV genome exhibits the genomic features of a typical mastadenovirus with at least 32 open reading frames identified. Five novel open reading frames were found at the right end of the genomic sequence. One of them maps to the predicted E3 region and encodes a putative CR1 protein, two map to the E4 region, and two map to the l strand of L1 and L3, respectively. Our phylogenetic analysis of whole genome sequences showed that among the mammalian AdV species described to date, GPAdV is most closely related to MAdV-2 The characterization of this mastadenovirus species offers an opportunity to develop a new small animal model to study mammalian adenovirus pathogenesis.

Genomic characterization of human adenovirus type 4 strains isolated worldwide since 1953 identifies two separable phylogroups evolving at different rates from their most recent common ancestor.

Species Human mastadenovirus E (HAdV-E) comprises several simian types and a single human type: HAdV-E4, a respiratory and ocular pathogen. RFLP analysis for the characterization of intratypic genetic variability has previously distinguished two HAdV-E4 clusters: prototype (p)-like and a-like. Our analysis of whole genome sequences confirmed two distinct lineages, which we refer to as phylogroups (PGs). PGs I and II comprise the p- and a-like genomes, respectively, and differ significantly in their G + C content (57.7% ± 0.013 vs 56.3% ± 0.015). Sequence differences distinguishing the two clades map to several regions of the genome including E3 and ITR. Bayesian analyses showed that the two phylogroups diverged approximately 602 years before the present. A relatively faster evolutionary rate was identified for PG II. Our data provide a rationale for the incorporation of phylogroup identity to HAdV-E4 strain designation to reflect the identified unique genetic characteristics that distinguish PGs I and II.

Outcomes of human adenovirus infection and disease in a retrospective cohort of pediatric solid organ transplant recipients.

Information about HAdV infection in SOT recipients is limited. We aimed to describe HAdV infection epidemiology and outcomes in a single-center retrospective cohort during the era of PCR availability. SOT recipients transplanted at the CHOP 2004-2013 were followed up for 180 days post-transplant. HAdV infection was defined as a positive HAdV PCR from a clinical specimen. HAdV disease was defined by organ-specific radiologic and/or laboratory abnormalities. No HAdV surveillance protocols were employed during the study period; testing was solely per clinician discretion. Progression of HAdV infection was defined as HAdV disease or ≥1-log viral load increase since a corresponding site's first positive specimen. Of the assembled 425 SOT recipients, 227 (52.6%) had ≥1 HAdV PCR. Twenty-four (10.6%) had ≥1 HAdV-positive PCR. HAdV-positive subjects were younger than uninfected subjects (2.0 years vs 6.5, P = 0.001). Infection incidence rates were highest in liver recipients (15.3%), followed by heart (8.6%), kidney (8.3%), and lung (4.2%). Four subjects (16.7%) met HAdV disease criteria at virus detection. Five subjects (20.8%) had progression of HAdV infection. All-cause mortality rates in positive and negative subjects were 0% and 3.9%, respectively. HAdV infection was infrequently detected in SOT recipients. Over one-third of HAdV-positive patients met disease criteria at detection or had infection progression, but none died. This low all-cause mortality raises questions about benefits of HAdV surveillance. Larger multicenter studies are needed to assess incidence variance by center and comparative effectiveness of therapeutic interventions.

Emergence and re-emergence of respiratory adenoviruses in the United States.

Human adenoviruses (HAdVs) are prevalent causes of acute respiratory disease (ARD) in military and civilian communities. Over the last 20 years, collaborative efforts between US public health, military and academic laboratories have gathered comprehensive data documenting the emergence and re-emergence of specific HAdV types in association with outbreaks and unrelated cases of ARD, which have attracted national attention. New or reemerging HAdVs have included genomic variants of HAdV-B14, HAdV-B7, and HAdV-E4. Detailed molecular characterizations of virus strains are essential to understand the etiology and epidemiology of HAdV infections. The continuation of such studies is important for ongoing assessment of the national and global evolution of respiratory HAdVs and to inform decisions regarding antiviral drug and vaccine development and implementation.