MicrobiologyBytes

By convention, the human adenovirus replication cycle is divided into two phases, an early and a late phase, which are separated by the onset of viral DNA replication. Based on temporal changes of the gene expression pattern as revealed by DNA microarray analysis, adenovirus type 2 (Ad2) infection in human primary lung fibroblasts can be divided into four periods. The first period is from 0 to 12 h after infection before or shortly after adenoviral gene expression has commenced. During this time, changes in cellular gene expression are likely to be triggered by the virus entry process, such as attachment of virus to cell surface receptors, and its intracellular transport along microtubules.

The second period covers the time from 12 to 24 h after infection and follows activation of the immediate early E1A gene. During this period, there is an increase in the number of differentially expressed cellular genes. About 50% of these genes are involved in cell cycle regulation, cell proliferation and antiviral response. The third period extends from 24 to 42 h after infection. By this time, the virus has gained control of the cellular metabolic machinery, resulting in an efficient replication of the viral genome. Additional changes in cellular gene expression are modest during this phase. During the fourth and last period, when the cytopathic effect becomes apparent, the number of down-regulated genes increases dramatically including many genes involved in intra- and extracellular structure.

The most intensive battle between the adenovirus and its host takes place during the second period after adenovirus genes expression has started. The major functions of the early gene products are to force the host cell to enter the S phase in order to provide optimal conditions for viral DNA replication and to suppress the host antiviral response. Adenoviruses encode several regulatory proteins within the early regions E1A, E1B, E3, and E4. The immediate-early E1A gene encodes two regulators of viral and cellular gene expression, the E1A-243R and E1A-289R proteins. The E1A proteins act as promiscuous transcriptional activators or repressors of cellular genes. E1A proteins are essential for promoting the host cell to enter the S phase. This is achieved by the binding of the E1A proteins to members of the retinoblastoma tumor suppressor (pRB) family, thereby releasing the E2F transcription factors, which are activators of genes required in the S-phase.

The transcriptome of the adenovirus infected cell. Virology. 9 Jan 2012
Alternations of cellular gene expression following an adenovirus type 2 infection of human primary cells were studied by using superior sensitive cDNA sequencing. In total, 3791 cellular genes were identified as differentially expressed more than 2-fold. Genes involved in DNA replication, RNA transcription and cell cycle regulation were very abundant among the up-regulated genes. On the other hand, genes involved in various signaling pathways including TGF-β, Rho, G-protein, Map kinase, STAT and NF-κB stood out among the down-regulated genes. Binding sites for E2F, ATF/CREB and AP2 were prevalent in the up-regulated genes, whereas binding sites for SRF and NF-κB were dominant among the down-regulated genes. It is evident that the adenovirus has gained a control of the host cell cycle, growth, immune response and apoptosis at 24h after infection. However, efforts from host cell to block the cell cycle progression and activate an antiviral response were also observed.

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A novel virus that spread through a California New World titi monkey colony in late 2009 has been shown to have also infected a human researcher and a household family member, in a documented example of an adenovirus “jumping” from one species to another and remaining contagious after the jump. Adenoviruses are known to cause a wide range of clinical illnesses in humans, from cold-like symptoms to diarrhea and pneumonia. Unlike influenza or coronaviruses, adenoviruses had previously not been known to spread from one species to another. Now adenoviruses can be added to the list of pathogens that have the ability to cross species.

The virus, which researchers have named titi monkey adenovirus (TMAdV), infected titi monkeys in the California National Primate Research Center (CNPRC) in 2009. At the time of the outbreak, a researcher caring for the sick monkeys also developed an upper respiratory infection with fever, as did two members of the researchers’ family who had no contact with the monkey colony. But which direction the virus spread – from monkeys to humans or vice versa – remains a mystery. The viral center is now conducting further studies in both humans and monkeys in Brazil and Africa to determine whether TMAdV is common in wild populations of monkeys, as well as whether it has crossed species in those settings to humans who live nearby.

Cross-Species Transmission of a Novel Adenovirus Associated with a Fulminant Pneumonia Outbreak in a New World Monkey Colony. (2011) PLoS Pathog 7(7): e1002155. doi:10.1371/journal.ppat.1002155
Adenoviruses are DNA viruses that naturally infect many vertebrates, including humans and monkeys, and cause a wide range of clinical illnesses in humans. Infection from individual strains has conventionally been thought to be species- specific. Here we applied the Virochip, a pan-viral microarray, to identify a novel adenovirus (TMAdV, titi monkey adenovirus) as the cause of a deadly outbreak in a closed colony of New World monkeys (titi monkeys; Callicebus cupreus) at
the California National Primate Research Center (CNPRC). Among 65 titi monkeys housed in a building, 23 (34%) developed upper respiratory symptoms that progressed to fulminant pneumonia and hepatitis, and 19 of 23 monkeys, or 83% of those infected, died or were humanely euthanized. Whole-genome sequencing of TMAdV revealed that this adenovirus is a new species and highly divergent, sharing ,57% pairwise nucleotide identity with other adenoviruses. Cultivation of TMAdV was successful in a human A549 lung adenocarcinoma cell line, but not in primary or established monkey kidney cells. At the onset of the outbreak, the researcher in closest contact with the monkeys developed an acute respiratory illness, with symptoms persisting for 4 weeks, and had a convalescent serum sample seropositive for TMAdV. A clinically ill family member, despite having no contact with the CNPRC, also tested positive, and screening of a set of 81 random adult blood donors from the Western United States detected TMAdV-specific neutralizing antibodies in 2 individuals (2/81, or 2.5%). These findings raise the possibility of zoonotic infection by TMAdV and human-to-human transmission of the virus in the population. Given the unusually high case fatality rate from the outbreak (83%), it is unlikely that titi monkeys are the native host species for TMAdV, and the natural reservoir of the virus is still unknown. The discovery of TMAdV, a novel adenovirus with the capacity to infect both monkeys and humans, suggests that adenoviruses should be monitored closely as potential causes of cross-species outbreaks.

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During the past three decades, research using the transforming proteins of small DNA tumor viruses such as human adenoviruses (HAdvs), SV40 and human papillomaviruses (HPVs) has illuminated crucial cellular pathways that control proliferation and oncogenic transformation of mammalian cells. Owing to their small genome size, these viruses are heavily dependent on the host cell machineries to express their genes and replicate their DNA. Because these viruses generally replicate in terminally differentiated quiescent target cells, the viral genes expressed during the early phase of the virus life cycle subvert the host cell cycle, inducing transient proliferation of the infected cells to generate a permissive S-phase state to facilitate viral replication. In non-permissive cells, these viruses express only their early genes, which induce cell proliferation, resulting in abortive infection. A fraction of the infected cells recover, and assume oncogenic properties as a result of continued expression of a subset of viral early genes, referred to as ‘transforming genes’. Transduction of subgenomic DNA fragments containing the transforming genes also achieves transformation of the target cells in significant numbers. Defined viral mutants and transduction of isolated genes have been widely used for molecular genetic analysis of viral transforming genes.

Researchers have used biochemical approaches to identify cellular proteins associated with viral proteins to elucidate the mechanisms through which the transforming proteins of small DNA tumor viruses subvert the cell cycle and elicit oncogenic cell transformation. This approach revealed the interaction of viral transforming proteins with tumor-suppressor proteins such as p53 and the retinoblastoma (pRb) protein. The transforming proteins of HAdvs, SV40 and HPVs share common mechanisms of cell transformation, because they target the same cell-cycle regulatory proteins, such as p53 and the pRb family proteins. This paper discusses the role of adenovirus E1A protein in transformation.

Opposing oncogenic activities of small DNA tumor virus transforming proteins. Trends Microbiol. Feb 15 2011 doi: 10.1016/j.tim.2011.01.003
The E1A gene of species C human adenovirus is an intensely investigated model viral oncogene that immortalizes primary cells and mediates oncogenic cell transformation in cooperation with other viral or cellular oncogenes. Investigations using E1A proteins have illuminated important paradigms in cell proliferation and about the functions of cellular proteins such as the retinoblastoma protein. Studies with E1A have led to the unexpected discovery that E1A also suppresses cell transformation and oncogenesis. Here, I review our current understanding of the transforming and tumor-suppressive functions of E1A, and how E1A studies led to the discovery of a related tumor-suppressive function in benign human papillomaviruses. The potential role of these opposing functions in viral replication in epithelial cells is also discussed.

Related:

• MicroRNA regulation of tumor-killing viruses
• Hepatitis B and C Viruses and Hepatocellular Carcinoma
• Vaccines To Prevent Infections by Oncoviruses

Viruses are obligate intracellular parasites which have evolved as natural, biological delivery vehicles. This makes them an attractive choice of vector for various clinical gene therapy applications. Human adenoviruses (Ad) are currently the most widely used viral vectors for gene therapy for several reasons; their basic biology has been studied extensively, the viral genome can accommodate large heterologous transgene insertions, they readily infect quiescent and dividing cells, they can be amplified to high titers and they have previously been shown to be relatively safe for use in humans. The family Adenoviridae consists of five genera, including genus Mastadenovirus and genus Aviadenovirus, which infect mammals and birds respectively. The Adenoviridae are non-enveloped, icosahedral virions which contain a linear, monopartite, double-stranded DNA genome approximately 36 kb in size. As of now, there are at least 55 different human adenoviruses which can be distinguished on the basis of their serological cross-reactivity, hemagglutinating properties or according to their phylogenetic sequence similarity. The adenovirus vector most commonly used for clinical trials and experimental gene therapy applications is species C adenovirus, HAdV-C5 (referred to as Ad5 in this review).

Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors. Viruses 2010, 2(10), 2290-2355 doi:10.3390/v2102290
Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated “stealth” vectors which avoid these interactions. Simultaneous retargeting and detargeting can be achieved by combining multiple genetic and/or chemical modifications.

Related:

• Adenovirus vectors – new genes, new vaccines

Human adenoviruses (HAdV) are non-enveloped double-stranded DNA (dsDNA) viruses associated with acute infections. Although these infections are generally self-limiting, the re-emergence of certain HAdV types has also been linked to potentially fatal respiratory infections in both civilian and military populations. In addition to their disease associations, replication-defective or conditionally replicating HAdVs continue to be evaluated in ~25% of approved phase I to III clinical trials for vaccine and therapeutic gene transfer. However, the lack of accurate details of the virus structure limits the reengineering of HAdV vectors and prevents a better understanding of the virus life cycle. High-resolution HAdV structure determination presents a challenge because of the large size (910 Å average diameter, 150 megadalton) and complexity (pseudo-T = 25) of the virus.

After more than a decade of research, scientists have pieced together the structure of a human adenovirus – the largest complex ever determined at atomic resolution. The new findings about the virus, which causes respiratory, eye, and gastrointestinal infections, may lead to more effective gene therapy and to new anti-viral drugs.

Crystal Structure of Human Adenovirus at 3.5 Å Resolution. (2010) Science 329(5995): 107 -1075 doi: 10.1126/science.1187292
Rational development of adenovirus vectors for therapeutic gene transfer is hampered by the lack of accurate structural information. Here, we report the x-ray structure at 3.5 angstrom resolution of the 150-megadalton adenovirus capsid containing nearly 1 million amino acids. We describe interactions between the major capsid protein (hexon) and several accessory molecules that stabilize the capsid. The virus structure also reveals an altered association between the penton base and the trimeric fiber protein, perhaps reflecting an early event in cell entry. The high-resolution structure provides a substantial advance toward understanding the assembly and cell entry mechanisms of a large double-stranded DNA virus and provides new opportunities for improving adenovirus-mediated gene transfer.

Related:

• Adenovirus vectors – new genes, new vaccines
• MicroRNA regulation of tumor-killing viruses

Acute respiratory viruses cause substantial morbidity and mortality worldwide. Lower respiratory-tract infections are the leading cause of communicable disease death and among the top five contributors to disability-adjusted life years. Viruses are the primary cause of lower respiratory-tract infections in children and a substantial cause of such infections in all age-groups. The incubation period of an infectious disease is the time between infection and symptom onset. This period is widely reported because it is useful in infectious disease surveillance and control, in which the time of symptom onset may be the only indication of the time of infection. The incubation period plays an essential part in surveillance for healthcare-associated infections, and may aid in diagnosis if laboratory facilities are unavailable. The incubation period is clinically relevant in the administration of antiviral medications, many of which are most effective when given before or immediately after symptom onset. Epidemiological studies depend on the incubation period to identify potential sources of infection. Predictive models designed to inform policy decisions use the incubation period to evaluate the potential of surveillance programmes and interventions to confront emerging epidemics.

The length of the incubation period by comparison with the latent period (the time between infection and becoming infectious) determines the potential effectiveness of control measures that target symptomatic individuals. “4–5 days” may refer to the most common range, the highest and lowest incubation periods in a study, or some other interval. Without knowing which summary measure is being stated, it is hard to use this information to make clinical or infection control decisions. Estimates given without attribution or based on few observations do not meet the standards of evidence we demand for modern medical information. A recent paper reviews the literature on nine respiratory viruses selected for their clinical or public-health importance: adenovirus, human coronavirus, SARS-associated coronavirus, influenza, measles, human metapneumovirus, parainfluenza, respiratory syncytial virus (RSV), and rhinovirus. By systematic review and analysis of published estimates and data, the authors aimed to capture the consensus in the medical literature on these incubation periods, characterise the evidence underlying this consensus, and provide improved estimates of incubation periods for these infections.

Incubation periods of acute respiratory viral infections: a systematic review. Lancet Infect Dis 2009;
9: 291–300
Knowledge of the incubation period is essential in the investigation and control of infectious disease, but statements of incubation period are often poorly referenced, inconsistent, or based on limited data. In a systematic review of the literature on nine respiratory viral infections of public-health importance, we identified 436 articles with statements of incubation period and 38 with data for pooled analysis. We fitted a log-normal distribution to pooled data and found the median incubation period to be 5·6 days (95% CI 4·8–6·3) for adenovirus, 3·2 days (95% CI 2·8–3·7) for human coronavirus, 4·0 days (95% CI 3·6–4·4) for severe acute respiratory syndrome coronavirus, 1·4 days (95% CI 1·3–1·5) for influenza A, 0·6 days (95% CI 0·5–0·6) for influenza B, 12·5 days (95% CI 11·8–13·3) for measles, 2·6 days (95% CI 2·1–3·1) for parainfluenza, 4·4 days (95% CI 3·9–4·9) for respiratory syncytial virus, and 1·9 days (95% CI 1·4–2·4) for rhinovirus. When using the incubation period, it is important to consider its full distribution: the right tail for quarantine policy, the central regions for likely times and sources of infection, and the full distribution for models used in pandemic planning. Our estimates combine published data to give the detail necessary for these and other applications.

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• One virus particle can be enough to cause infection
• Adenoviruses
• SARS Virus May Be Tough To Beat

Attenuated viruses have found important applications in medicine, including their use as vaccines (notably for measles, mumps, polio, influenza, and chicken pox) and their experimental development as selective cancer-killing agents, so-called “virotherapy”. Wild-type versions are often most effective in both of these settings; however, attenuated viruses have usually been developed to decrease the risk of significant viral pathology. Recent advances in understanding regulation of gene expression by microRNA now afford the possibility to design viruses that are “selectively attenuated” in sites of potential pathology, by engineering them for inhibition by micro-RNA molecules that are expressed there. A new paper describes how researchers have engineered wild-type adenovirus for recognition by a microRNA expressed in hepatocytes, producing a virus that retains wild-type infection and replication at sites of therapeutic activity (such as cancer cells) but is severely attenuated in hepatocytes, both in vitro and in vivo. This virus caused no significant liver toxicity to mice even when applied at ten times the lethal dose of wild-type virus. The ability to produce replication-competent viruses with key toxicities removed should provide a new platform for development of improved cancer treatments and better vaccines for a broad range of virus diseases.

Cellular microRNA molecules regulate the stability of mRNA in different cell types, and this newly-understood mechanism provides the possibility to engineer viruses for cell-specific inactivation. Adenovirus is a DNA virus widely used in cancer therapy but which causes hepatic disease in mice. Researchers found that introducing sites into the virus genome that are recognized by microRNA 122 leads to hepatic degradation of important viral mRNA, thereby diminishing the virus’ ability to adversely affect the liver, while maintaining its ability to replicate in and kill tumor cells. Tumor-killing replicating viruses are a hot topic in the biotherapeutics arena, with many clinical trials ongoing worldwide. They have now defined a mechanism whereby wild type virus potency could be maintained in tumor cells but the virus could be “turned off” in tissues vulnerable to pathology adds important information to the current base of knowledge.

This approach is surprisingly effective and quite versatile. It could find a range of applications in controlling the activity of therapeutic viruses, both for cancer research and also to engineer a new generation of conditionally-replicating vaccines, where the vaccine pathogen is disabled in its primary sites of toxicity. The present study was intended mainly to explore and demonstrate the potential of this new mechanism to regulate virus activity. Although the current tumor-killing virus is useful in mice, transfer of the technology into the clinical setting will require re-engineering of the virus to overcome virus pathologies seen in humans, and it will be at least two years before this can be tested in the clinics.

Use of Tissue-Specific MicroRNA to Control Pathology of Wild-Type Adenovirus without Attenuation of Its Ability to Kill Cancer Cells. PLoS Pathog 5(5): e1000440
Replicating viruses have broad applications in biomedicine, notably in cancer virotherapy and in the design of attenuated vaccines; however, uncontrolled virus replication in vulnerable tissues can give pathology and often restricts the use of potent strains. Increased knowledge of tissue-selective microRNA expression now affords the possibility of engineering replicating viruses that are attenuated at the RNA level in sites of potential pathology, but retain wild-type replication activity at sites not expressing the relevant microRNA. To assess the usefulness of this approach for the DNA virus adenovirus, we have engineered a hepatocyte-safe wild-type adenovirus 5 (Ad5), which normally mediates significant toxicity and is potentially lethal in mice. To do this, we have included binding sites for hepatocyte-selective microRNA mir-122 within the 39 UTR of the E1A transcription cassette. Imaging versions of these viruses, produced by fusing E1A with luciferase, showed that inclusion of mir-122 binding sites caused up to 80-fold decreased hepatic expression of E1A following intravenous delivery to mice. Animals administered a ten-times lethal dose of wild-type Ad5 (561010 viral particles/mouse) showed substantial hepatic genome replication and extensive liver pathology, while inclusion of 4 microRNA binding sites decreased replication 50-fold and virtually abrogated liver toxicity. This modified wild-type virus retained full activity within cancer cells and provided a potent, liver-safe oncolytic virus. In addition to providing many potent new viruses for cancer virotherapy, microRNA control of virus replication should provide a new strategy for designing safe attenuated vaccines applied across a broad range of viral diseases.

Related:

• Measles Virotherapy
• Adenoviruses
• Adenovirus vectors – new genes, new vaccines

It’s been a bad week for HIV vaccines, with the announcement that the Merck phase II STEP vaccine trial involving 3,000 volunteers was halted because the vaccine did not block HIV infection. This international trial was regarded as “test of concept” of the vaccine – which included three recombinant HIV genes delivered by a weakened adenovirus vector – in uninfected volunteers at high risk for acquiring HIV infection, mainly homosexual men, but including some women in North and South America, the Caribbean, and Australia. Among the 741 volunteers who got at least one of the three planned doses of vaccine, there were 24 cases of HIV infection, compared with 21 infections among the 762 participants who received a placebo.

Related: Adenovirus vectors – new genes, new vaccines

Clearly, there’s still a long road to tread until we have an effective HIV vaccine, and the only way we’re going to get there is not by trial and error but through a much better understanding of what is involved in immune protection against HIV infection. For that reason, a recent paper looking at immunity to HIV-2 infection is of interest:

HIV-2 infection in the majority of infected subjects follows an attenuated disease course that distinguishes it from infection with HIV-1. Antigen-specific T cells are pivotal in the management of chronic viral infections but are not sufficient to control viral replication in HIV-1–positive subjects, and their function in HIV-2 infection is not fully established. In a community-based cohort of HIV-2 long-term non-progressors in rural Guinea-Bissau, the authors performed what they believe is the first comprehensive analysis of HIV-2–specific immune responses. This demonstrated that Gag is the most immunogenic protein in HIV-2. The magnitude of the IFN-gamma immune response to the HIV-2 proteome was inversely correlated with HIV-2 viraemia, and this relationship was specifically due to the targeting of Gag. Furthermore, patients with undetectable viraemia had greater Gag-specific responses compared with patients with high viral replication. The most frequently recognized peptides clustered within a defined region of Gag, and responses to a single peptide in this region were associated with low viral burden. The consistent relationship between Gag-specific immune responses and viraemia control suggests that T cell responses are vital in determining the superior outcome of HIV-2 infection. A better understanding of how HIV-2 infection is controlled may identify correlates of effective protective immunity essential for the design of HIV vaccines.
Robust Gag-specific T cell responses characterize viremia control in HIV-2 infection.
J Clin Invest. 2007 Sep 6