Experimental Cross-Species Infection of Common Marmosets by Titi Monkey Adenovirus

Adenoviruses are DNA viruses that infect a number of vertebrate hosts and are associated with both sporadic and epidemic disease in humans. We previously identified a novel adenovirus, titi monkey adenovirus (TMAdV), as the cause of a fulminant pneumonia outbreak in a colony of titi monkeys (Callicebus cupreus) at a national primate center in 2009. Serological evidence of infection by TMAdV was also found in a human researcher at the facility and household family member, raising concerns for potential cross-species transmission of the virus. Here we present experimental evidence of cross-species TMAdV infection in common marmosets (Callithrix jacchus). Nasal inoculation of a cell cultured-adapted TMAdV strain into three marmosets produced an acute, mild respiratory illness characterized by low-grade fever, reduced activity, anorexia, and sneezing. An increase in virus-specific neutralization antibody titers accompanied the development of clinical signs. Although serially collected nasal swabs were positive for TMAdV for at least 8 days, all 3 infected marmosets spontaneously recovered by day 12 post-inoculation, and persistence of the virus in tissues could not be established. Thus, the pathogenesis of experimental inoculation of TMAdV in common marmosets resembled the mild, self-limiting respiratory infection typically seen in immunocompetent human hosts rather than the rapidly progressive, fatal pneumonia observed in 19 of 23 titi monkeys during the prior 2009 outbreak. These findings further establish the potential for adenovirus cross-species transmission and provide the basis for development of a monkey model useful for assessing the zoonotic potential of adenoviruses.     

Canine Distemper Virus Associated with a Lethal Outbreak in Monkeys Can Readily Adapt To Use Human Receptors

A canine distemper virus (CDV) strain, CYN07-dV, associated with a lethal outbreak in monkeys, used human signaling lymphocyte activation molecule as a receptor only poorly but readily adapted to use it following a P541S substitution in the hemagglutinin protein. Since CYN07-dV had an intrinsic ability to use human nectin-4, the adapted virus became able to use both human immune and epithelial cell receptors, as well as monkey and canine ones, suggesting that CDV can potentially infect humans.     

New adenovirus species found in a patient presenting with gastroenteritis

An unidentified agent was cultured in primary monkey cells at the Los Angeles County Public Health Department from each of five stool specimens submitted from an outbreak of gastroenteritis. Electron microscopy and an adenovirus-specific monoclonal antibody confirmed this agent to be an adenovirus. Since viral titers were too low, complete serotyping was not possible. Using the DNase-sequence-independent viral nucleic acid amplification method, we identified several nucleotide sequences with a high homology to human adenovirus 41 (HAdV-41) and simian adenovirus 1 (SAdV-1). However, using anti-SAdV-1 sera, it was determined that this virus was serologically different than SAdV-1. Genomic sequencing and phylogenetic analysis confirmed that this new adenovirus was so divergent from the known human adenoviruses that it was not only a new type but also represented a new species (human adenovirus G). In a retrospective clinical study, this new virus was detected by PCR in one additional patient from a separate gastroenteritis outbreak. This study suggests that HAdV-52 may be one of many agents causing gastroenteritis of unknown etiology.     

Host Immune Responses to Chronic Adenovirus Infections in Human and Nonhuman Primates

Recent studies indicate that great apes and macaques chronically shed adenoviruses in the stool. Shedding of adenovirus in the stool of humans is less prevalent, although virus genomes persist in gut-associated lymphoid tissue in the majority of individual samples. Chimpanzees have high levels of broadly reactive neutralizing antibodies to adenoviruses in serum, with very low frequencies of adenovirus-specific T cells in peripheral blood. A similar situation exists in macaques; sampling of guts from macaques demonstrated adenovirus-specific T cells in lamina propria. Humans show intermediate levels of serum neutralizing antibodies, with adenovirus-specific T cells in peripheral blood of all individuals sampled and about 20% of samples from the gut, suggesting a potential role of T cells in better controlling virus replication in the gut. The overall structure of the E3 locus, which is involved in modulating the host''s response to infection, is degenerate in humans compared to that in apes, which may contribute to diminished evasion of host immunity. The impact of adenovirus persistence and immune responses should be considered when using adenoviral vectors in gene therapy and genetic vaccines.Viruses of the Adenoviridae family infect a wide range of vertebrate hosts. Adenoviruses that infect mammals belong to the genus Mastadenovirus and encompass at least seven viral species (formerly called subtypes) that infect primates (5). The molecular biology of human-derived adenoviruses has been characterized extensively for the species C group, for which human adenovirus 2 (HAdV-2) and HAdV-5 serve as prototypes (6). Adenoviruses cause a variety of nonlethal infectious diseases in humans, and lethal disseminated adenovirus infection occurs in immunosuppressed patients (6). Attempts to evaluate the pathogenesis of human adenoviruses in animal models have been difficult since most models demonstrate a very narrow permissiveness for virus replication.The natural history of adenovirus infections in humans was initially evaluated in prospective surveillance studies of family cohorts (7, 8). Self-limited respiratory illnesses in children were the most common syndromes associated with adenovirus infection, although virus was most easily detected in the stool. Excretion of virus in stool usually wanes rapidly following resolution of the infectious syndrome but is prolonged for a subset of individuals. Species C adenoviruses, which are the most prevalent in humans, can establish latency in adenoid and tonsil-derived lymphocytes (9).Adenovirus vectors have been used successfully as vaccine carriers due to their ability to elicit potent cellular and humoral immune response to the encoded transgene product. Recombinant vectors based on HAdV-5 have emerged as a potent platform for activating cytotoxic T lymphocytes against tumor and viral antigens. A problematic response of the host to HAdV-5-based vectors that was not predicted based on animal testing was observed in a clinical trial, namely, there was a paradoxical increased susceptibility to infection with human immunodeficiency virus (HIV) following vaccination with an HIV-based HAdV-5 vector in subjects with evidence of prior exposure to a natural infection with HAdV-5 (2).     

Replication-Defective Vector Based on a Chimpanzee Adenovirus

An adenovirus previously isolated from a mesenteric lymph node from a chimpanzee was fully sequenced and found to be similar in overall structure to human adenoviruses. The genome of this virus, called C68, is 36,521 bp in length and is most similar to subgroup E of human adenovirus, with 90% identity in most adenovirus type 4 open reading frames that have been sequenced. Substantial differences in the hexon hypervariable regions were noted between C68 and other known adenoviruses, including adenovirus type 4. Neutralizing antibodies to C68 were highly prevalent in sera from a population of chimpanzees, while sera from humans and rhesus monkeys failed to neutralize C68. Furthermore, infection with C68 was not neutralized from sera of mice immunized with human adenovirus serotypes 2, 4, 5, 7, and 12. A replication-defective version of C68 was created by replacing the E1a and E1b genes with a minigene cassette; this vector was efficiently transcomplemented by the E1 region of human adenovirus type 5. C68 vector transduced a number of human and murine cell lines. This nonhuman adenoviral vector is sufficiently similar to human serotypes to allow growth in 293 cells and transduction of cells expressing the coxsackievirus and adenovirus receptor. As it is dissimilar in regions such as the hexon hypervariable domains, C68 vector avoids significant cross-neutralization by sera directed against human serotypes.     

Simian homologues of human herpesvirus 8

Gamma-herpesviruses can be found in most primates including Old World an New World monkeys. The gamma-herpesvirinae are grouped into two classes: lymphocryptoviruses (gamma1) and rhadinoviruses (gamma2). The lymphocryptoviruses include Epstein-Barr virus, lymphocryptovirus of rhesus monkeys, and Herpesvirus papio of baboons. Rhadinoviruses that infect New World monkeys include Herpesvirus saimiri, whose natural host is the squirrel monkey, and Herpesvirus ateles, which infects spider monkeys. Rhadinoviruses that infect hominoids and Old World monkeys include Kaposi's sarcoma-associated herpesvirus, also known as HHV-8, and rhesus monkey rhadinovirus.     

Serum leptin in nonpregnant and pregnant women and in old and new world nonhuman primates

Leptin is a hormone that is produced during mammalian pregnancy in the placental trophoblast and other tissues, including! fetal and maternal adipocytes. Synthesis of the polypeptide and the presence of its specific receptors throughout the human maternal fetoplacental unit suggest direct effects on conceptus growth and development. However, both the physiologic roles of leptin and the mechanisms regulating leptin synthesis in human pregnancy differ from those in laboratory and domestic species, necessitating the development of non-human primate research models. Therefore, we compared serum leptin concentrations in nonpregnant and pregnant women with those in both old world nonhuman primates (i.e., baboon, rhesus monkey, cynomolgus monkey) and new world nonhuman primates (i.e., squirrel monkey, titi monkey). As expected, maternal leptin levels were elevated in human and baboon pregnancies (P < 0.05 and P < 0.001, respectively). Levels in both species of old world monkeys were also greatly enhanced (P < 0.001). Although maternal serum concentrations were slightly elevated compared to nonpregnant levels in both species of new world monkeys, overall concentrations were dramatically lower than for either old world primates or humans. Results provide comparisons of serum leptin concentrations in pregnant and nonpregnant humans and baboons with those in both old and new world monkeys and further characterize these nonhuman primates as models for the investigation of leptin dynamics in pregnancy.     

Extension of the Geographical Range of White-browed Titi Monkeys (<Emphasis Type="BoldItalic">Callicebus discolor</Emphasis>) and Evidence for Sympatry with San Martin Titi Monkeys (<Emphasis Type="BoldItalic">Callicebus oenanthe</Emphasis>)

White-browed titi monkeys (Callicebus discolor) have one of the largest distribution ranges of all titi monkey species, occurring from central Peru to southern Colombia. During a long-term study on the distribution of titi monkeys and other primates in Peru, we conducted extensive surveys in the San Martin Department of northeastern Peru. We encountered Callicebus discolor at the left bank of the Huallaga River, where the species most probably lives in sympatry with endemic San Martin titi monkeys (Callicebus oenanthe). Our study reveals an important extension of its formerly known distribution range. Massive deforestation activities in the region make studies on the habitat preferences of both species difficult, as most titi monkeys are confined to the remaining small remnants of the original forest. Urgent conservation measures are necessary to preserve the last lowland forests of San Martin.     

Behavioural Thermoregulation in a Small Neotropical Primate

The maintenance of body temperature in endothermic animals imposes considerable metabolic costs that vary with air temperature fluctuations. To minimise these costs, endotherms can adopt certain behaviours to adjust the pattern of heat transfer between their bodies and the environment. In this study, we evaluated whether a small Neotropical primate, the black‐fronted titi monkey (Callicebus nigrifrons), living in a seasonal environment can use behavioural mechanisms to cope with fluctuations in the air temperature. We monitored the air temperature and the titi monkeys’ behaviour over 1 yr. When the animals were inactive, we recorded the microhabitat used, the huddling between individuals and the body postures adopted. The monkeys primarily responded to air temperature fluctuations through microhabitat selection: they spent more time in sunny places and used higher strata of forest under lower temperatures. Moreover, they used sunny microhabitats during the first hour of their active period after colder nights. The monkeys did not huddle or change body postures in response to air temperature fluctuations. Huddling behaviour seemed to be primarily influenced by social interactions, and body postures were more energy conserving, regardless of temperature. Titi monkeys, however, used more energy‐conserving postures and huddling behaviour under cloudy conditions than sunny conditions, suggesting that these behaviours may be important when they are unable to thermoregulate by microhabitat selection. We concluded that fluctuations in air temperature can promote significant changes in the behaviour of titi monkeys and can impose important restrictions on mammals’ activities, even in tropical regions.     

CD46 is a cellular receptor for group B adenoviruses

Group B adenoviruses, a subgenus of human Adenoviridae, are associated with a variety of often-fatal illnesses in immunocompromised individuals, including bone marrow transplant recipients and cancer and AIDS patients. Recently, group B adenovirus derivatives have gained interest as attractive gene therapy vectors because they can transduce target tissues, such as hematopoietic stem cells, dendritic cells and malignant tumor cells, that are refractory to infection by commonly used adenoviral vectors. Whereas many adenoviruses infect cells through the coxsackievirus and adenovirus receptor (CAR), group B adenoviruses use an alternate, as-yet-unidentified cellular attachment receptor. Using mass spectrometric analysis of proteins interacting with a group B fiber, we identified human CD46 as a cellular attachment receptor for most group B adenoviruses. We show that ectopic expression of human CD46 rendered nonhuman cells susceptible to infection with group B viruses in vitro and in vivo. In addition, both siRNA-mediated knockdown of CD46 and a soluble form of CD46 blocked infection of human cell lines and primary human cells. The discovery that group B adenoviruses use CD46, a ubiquitously expressed complement regulatory protein, as a cellular attachment receptor elucidates the diverse clinical manifestation of group B virus infections, and bears directly on the application of these vectors for gene therapy.     

Genomic and bioinformatics analysis of HAdV-4, a human adenovirus causing acute respiratory disease: implications for gene therapy and vaccine vector development

Human adenovirus serotype 4 (HAdV-4) is a reemerging viral pathogenic agent implicated in epidemic outbreaks of acute respiratory disease (ARD). This report presents a genomic and bioinformatics analysis of the prototype 35,990-nucleotide genome (GenBank accession no. AY594253). Intriguingly, the genome analysis suggests a closer phylogenetic relationship with the chimpanzee adenoviruses (simian adenoviruses) rather than with other human adenoviruses, suggesting a recent origin of HAdV-4, and therefore species E, through a zoonotic event from chimpanzees to humans. Bioinformatics analysis also suggests a pre-zoonotic recombination event, as well, between species B-like and species C-like simian adenoviruses. These observations may have implications for the current interest in using chimpanzee adenoviruses in the development of vectors for human gene therapy and for DNA-based vaccines. Also, the reemergence, surveillance, and treatment of HAdV-4 as an ARD pathogen is an opportunity to demonstrate the use of genome determination as a tool for viral infectious disease characterization and epidemic outbreak surveillance: for example, rapid and accurate low-pass sequencing and analysis of the genome. In particular, this approach allows the rapid identification and development of unique probes for the differentiation of family, species, serotype, and strain (e.g., pathogen genome signatures) for monitoring epidemic outbreaks of ARD.     

Yellow fever outbreak affecting Alouatta populations in southern Brazil (Rio Grande do Sul State), 2008-2009

The natural transmission cycle of Yellow Fever (YF) involves tree hole breeding mosquitoes and a wide array of nonhuman primates (NHP), including monkeys and apes. Some Neotropical monkeys (howler monkeys, genus Alouatta) develop fatal YF virus (YFV) infections similar to those reported in humans, even with minimum exposure to the infection. Epizootics in wild primates may be indicating YFV circulation, and the surveillance of such outbreaks in wildlife is an important tool to help prevent human infection. In 2001, surveillance activities successfully identified YF-related death in a black-and-gold howler monkey (Alouatta caraya), Rio Grande do Sul State (RGS) in southern Brazil, and the YFV was isolated from a species of forest-dwelling mosquito (Haemagogus leucocelaenus). These findings led the State Secretariat of Health to initiate a monitoring program for YF and other 18 arboviral infections in Alouatta monkeys. The monitoring program included monkey captures, reporting of monkey casualties by municipalities, and subsequent investigations. If monkey carcasses were found in forests, samples were collected in a standardized manner and this practice resulted in increased reporting of outbreaks. In October 2008, a single howler monkey in a northwestern RGS municipality was confirmed to have died from YF. From October 2008 to June 2009, 2,013 monkey deaths were reported (830 A. caraya and 1,183 A. guariba clamitans). Viruses isolation in blood, viscera, and/or immunohistochemistry led to the detection of YF in 204 of 297 (69%) (154 A. g. clamitans and 50 A. caraya) dead Alouatta monkeys tested. The number of municipalities with confirmed YFV circulation in howlers increased from 2 to 67 and 21 confirmed human cases occurred. This surveillance system was successful in identifying the largest YF outbreak affecting wild NHP ever recorded.     

The level of CD4 expression limits infection of primary rhesus monkey macrophages by a T-tropic simian immunodeficiency virus and macrophagetropic human immunodeficiency viruses

The entry of primate immunodeficiency viruses into cells is dependent on the interaction of the viral envelope glycoproteins with receptors, CD4, and specific members of the chemokine receptor family. Although in many cases the tropism of these viruses is explained by the qualitative pattern of coreceptor expression, several instances have been observed where the expression of a coreceptor on the cell surface is not sufficient to allow infection by a virus that successfully utilizes the coreceptor in a different context. For example, both the T-tropic simian immunodeficiency virus (SIV) SIVmac239 and the macrophagetropic (M-tropic) SIVmac316 can utilize CD4 and CCR5 as coreceptors, and both viruses can infect primary T lymphocytes, yet only SIVmac316 can efficiently infect CCR5-expressing primary macrophages from rhesus monkeys. Likewise, M-tropic strains of human immunodeficiency virus type 1 (HIV-1) do not infect primary rhesus monkey macrophages efficiently. Here we show that the basis of this restriction is the low level of CD4 on the surface of these cells. Overexpression of human or rhesus monkey CD4 in primary rhesus monkey macrophages allowed infection by both T-tropic and M-tropic SIV and by primary M-tropic HIV-1. By contrast, CCR5 overexpression did not specifically compensate for the inefficient infection of primary monkey macrophages by T-tropic SIV or M-tropic HIV-1. Apparently, the limited ability of these viruses to utilize a low density of CD4 for target cell entry accounts for the restriction of these viruses in primary rhesus monkey macrophages.     

Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes

The common species C adenoviruses (serotypes Ad1, Ad2, Ad5, and Ad6) infect more than 80% of the human population early in life. Following primary infection, the virus can establish an asymptomatic persistent infection in which infectious virions are shed in feces for several years. The probable source of persistent virus is mucosa-associated lymphoid tissue, although the molecular details of persistence or latency of adenovirus are currently unknown. In this study, a sensitive real-time PCR assay was developed to quantitate species C adenovirus DNA in human tissues removed for routine tonsillectomy or adenoidectomy. Using this assay, species C DNA was detected in Ficoll-purified lymphocytes from 33 of 42 tissue specimens tested (79%). The levels varied from fewer than 10 to greater than 2 x 10(6) copies of the adenovirus genome/10(7) cells, depending on the donor. DNA from serotypes Ad1, Ad2, and Ad5 was detected, while the rarer serotype Ad6 was not. When analyzed as a function of donor age, the highest levels of adenovirus genomes were found among the youngest donors. Antibody-coated magnetic beads were used to purify lymphocytes into subpopulations and determine whether viral DNA could be enriched within any purified subpopulations. Separation of T cells (CD4/8- expressing and/or CD3-expressing cells) enriched viral DNA in each of nine donors tested. In contrast, B-cell purification (CD19-expressing cells) invariably depleted or eliminated viral DNA. Despite the frequent finding of significant quantities of adenovirus DNA in tonsil and adenoid tissues, infectious virus was rarely present, as measured by coculture with permissive cells. These findings suggest that human mucosal T lymphocytes may harbor species C adenoviruses in a quiescent, perhaps latent form.     

Detection of a unique genotype of monkey B virus (Cercopithecine herpesvirus 1) indigenous to native Japanese macaques (Macaca fuscata)

The Japanese macaque or snow monkey (Macaca fuscata) is an autochthonous monkey in Japan. It has long been assumed that the monkey population was not infected with Cercopithecine herpesvirus 1 (monkey B virus [BV]) since cases of human BV infection have never been reported in Japan. Although serologic testing of captive snow monkeys in Japan revealed antibodies to BV, it was thought that native Japanese macaques had either been infected with herpes simplex virus from humans or with BV from other imported macaque species. To clarify this issue, we performed polymerase chain reaction (PCR) analysis to amplify BV sequences from trigeminal ganglia of 30 Japanese macaque monkeys that were seropositive for BV. Sequences from two BV genes, UL27 (360 bp) and UL19 (1.0 Kbp), from 3 of 30 monkeys were amplified. Results of restriction fragment length polymorphism analysis and DNA sequencing of the fragments provided evidence that native Japanese macaques are infected with BV. Phylogenetic analysis indicated that these monkeys harbor their own genotype of BV that is different from other known BV genotypes, and provided additional evidence supporting the co-evolution of BV and macaques.     

Hybrids of Human and Monkey Adenoviruses (Adeno-Adeno Hybrids) That Can Reproduce in Monkey Cells: Biological and Molecular Genetic Peculiarities

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.     

Evaluation of adenoviral vectors by flow cytometry

Biological assays for adenoviral gene therapy vectors have included conventional procedures initially developed to detect wild-type adenoviruses. Standard virological assays to quantitate adenoviruses rely on the virus to infect and replicate in the host cell until a cytopathic effect is observed. The appearance of plaques, colonies of rounded, enlarged cells containing infectious virions, usually takes 2 to 3 weeks to reach an endpoint. We describe a flow cytometric bioassay for adenovirus which shortens the time from when the infection takes place to the time that biological titer is determined. A fluorescent focus-forming assay was one of the first rapid adenoviral bioassays developed. Virus titer was determined using fluorescence immunocytochemistry to detect adenovirus proteins and microscopy to count fluorescent foci in cultures of adenovirus-infected cells. In this study, we describe a flow cytometric assay performed on cells stained for adenovirus hexon capsid protein, where virus titer is determined based on the dose-dependent appearance of hexon-positive cells. Adenovirus hexon detection in infected cells can provide data to determine virus titer, inducible promoter function in vector-complementing cells, and vector replication in complementation-deficient cells.