Viral discovery as a tool for pandemic preparedness.

Emerging diseases are frequently caused by novel or previously unrecognised zoonotic viral pathogens, which tend to originate in and emerge from wildlife. When human or animal cases are first recognised, molecular or serological diagnostic assays specific to them do not yet exist, causing a delay in the identification of an outbreak's aetiologic agent as well as its source. Preparing for the next virus to emerge is a major public health challenge, impeded by a poor understanding of the diversity of potential candidates that exist in wildlife reservoirs. Characterising the diversity of viruses in key wildlife species will help to reduce the time between detection and response in an outbreak situation, and inform public health strategies that reduce the risk of spillover from animal reservoirs. Pathogen discovery techniques such as consensus polymerase chain reaction (cPCR) and next-generation sequencing (NGS) have been used to identify known and novel viruses in animals and humans, but have not been widely used in surveillance programmes. Metagenomic studies have identified novel viruses, new strains of known viruses, and have characterised host microbiomes. While NGS represents an unbiased approach to viral sequence detection, it is constrained by lower sensitivity than conventional PCR, requires substantial bioinformatics capabilities, and is cost prohibitive and therefore not widely available in the regions of the world that are most vulnerable to zoonotic disease emergence. In contrast, consensus PCR uses standard and widely available technologies, has greater sensitivity than NGS, and has also been used to identify novel viruses in wildlife, livestock and humans, though it is limited to detecting target genetic sequences conserved across known groups of viruses. The use of cPCR, in combination, if possible, with NGS and serology, can offer a powerful approach to rapidly identifying aetiologic agents in an outbreak and characterising the virome of key wildlife known to carry zoonotic viruses. Here, the authors review pathogen discovery techniques currently being used in human and animal surveillance programmes and the challenges of using viral discovery to identify novel zoonotic pathogens.

Les maladies émergentes sont souvent causées par des virus nouveaux ou précédemment inconnus, de portée zoonotique, qui ont généralement leur source dans la faune sauvage, à partir de laquelle s'effectue leur émergence. Lorsque le premier cas d'infection par un virus de ce type est détecté chez l'homme ou chez les animaux, il n'existe encore aucune épreuve moléculaire ou sérologique de détection de l'agent étiologique, ce qui retarde son identification ainsi que l'élucidation de la source du foyer. La préparation aux futures émergences virales est un véritable défi de santé publique et se voit entravée par les lacunes des connaissances sur la diversité des virus potentiellement candidats. La caractérisation des différents virus qui affectent les principales espèces sauvages permettra de réduire le délai entre le moment où un nouveau foyer est détecté et celui où une réponse lui est apportée, et d'élaborer en connaissance de cause des stratégies de santé publique visant à limiter le risque d'un franchissement d'espèce à partir des réservoirs animaux. Les techniques de découverte d'agents pathogènes, par exemple l'amplification en chaîne par polymérase (PCR) pan-générique ou consensus et le séquençage de nouvelle génération (SNG) sont utilisées pour identifier des virus connus ou nouveaux chez l'homme et l'animal, mais ne sont pas d'une utilisation courante dans les programmes de surveillance. Les études métagénomiques permettent d'identifier des virus nouveaux ainsi que les souches nouvelles de virus connus et servent également à caractériser le microbiome de l'hôte. Le SNG constitue une méthode de détection des séquences virales exempte de biais mais sa sensibilité moindre que celle des PCR classiques, les capacités bio-informatiques considérables qu'il requiert et son coût prohibitif sont des contraintes importantes qui en limitent l'utilisation dans les régions du monde les plus vulnérables à l'émergence des maladies zoonotiques. En revanche, la PCR consensus fait appel à des technologies normalisées et largement disponibles, tout en présentant une meilleure sensibilité que le SNG ; elle permet également d'identifier des virus nouveaux présents dans la faune sauvage, chez les animaux domestiques ou chez l'homme, bien qu'elle ne détecte que des séquences génétiques cibles conservées d'un groupe connu de virus à l'autre. Le recours à la PCR consensus, si possible associé aux techniques de SNG et à la sérologie se révèle une stratégie puissante qui permet d'identifier rapidement les agents responsables d'un foyer et de caractériser le virome d'espèces sauvages jouant un rôle majeur en tant que réservoirs de virus zoonotiques. Après avoir passé en revue les techniques de découverte d'agents pathogènes actuellement utilisées dans les programmes de surveillance des maladies animales et humaines, les auteurs font le point sur les enjeux de ces techniques pour l'identification de nouveaux agents pathogènes zoonotiques.

La causa de las enfermedades emergentes reside muchas veces en virus zoonóticos de aparición reciente o hasta entonces no descritos, que en general se originan y surgen en animales salvajes. Cuando se detectan los primeros casos en personas o animales aún no existen pruebas específicas de diagnóstico, ya sea molecular o serológico, y ello retrasa la identificación del agente etiológico del brote y la determinación de su origen. Prepararse para el próximo virus que vaya a aparecer es un gran objetivo de salud pública, lastrado en la práctica por el escaso conocimiento de la gran diversidad de posibles candidatos que moran en los reservorios de la fauna salvaje. La caracterización de los diversos virus que existen en las principales especies de animales salvajes ayudará a reducir el lapso que media entre la detección y la respuesta en caso de brote y a fundamentar a partir de ahí estrategias de salud pública que reduzcan el riesgo de diseminación desde los reservorios animales. Hasta ahora las técnicas de descubrimiento de patógenos, como la reacción en cadena de la polimerasa (PCR) de consenso (PCRc) o la secuenciación de próxima generación, han servido para identificar virus nuevos o ya conocidos en personas y animales, pero no se han aplicado de forma generalizada a los programas de vigilancia. Gracias a estudios de metagenómica se han podido detectar virus recién aparecidos o nuevas cepas de virus ya conocidos y caracterizar los microbiomas de los organismos anfitriones. Aunque la secuenciación de próxima generación constituye un método exento de sesgos para detectar secuencias víricas, adolece de una menor sensibilidad que la PCR convencional, exige una considerable capacidad de gestión informática de datos biológicos y tiene un costo prohibitivo, por lo que no suele aplicarse en las regiones del mundo que están más expuestas a la aparición de enfermedades zoonóticas. La PCR de consenso, en cambio, reposa en técnicas habituales y muy extendidas, ofrece mayor sensibilidad que la secuenciación de próxima generación y también ha sido utilizada para identificar virus nuevos en personas o animales salvajes y domésticos, si bien solo permite detectar las secuencias genéticas «diana¼ conservadas de entre todos los grupos conocidos de virus. El uso de la PCRc, combinado con la secuenciación de próxima generación y técnicas serológicas cuando sea posible, puede ofrecer un potente método para identificar con rapidez los agentes etiológicos de un brote y caracterizar el viroma de los principales animales salvajes de los que se sabe que son portadores de virus zoonóticos. Los autores pasan revista a las técnicas de descubrimiento de patógenos que se utilizan actualmente en los programas de vigilancia sanitaria y zoosanitaria y exponen las dificultades que presenta el uso del descubrimiento de virus para identificar nuevos patógenos zoonóticos.

Coronaviruses in bats from Mexico.

Bats are reservoirs for a wide range of human pathogens including Nipah, Hendra, rabies, Ebola, Marburg and severe acute respiratory syndrome coronavirus (CoV). The recent implication of a novel beta (ß)-CoV as the cause of fatal respiratory disease in the Middle East emphasizes the importance of surveillance for CoVs that have potential to move from bats into the human population. In a screen of 606 bats from 42 different species in Campeche, Chiapas and Mexico City we identified 13 distinct CoVs. Nine were alpha (α)-CoVs; four were ß-CoVs. Twelve were novel. Analyses of these viruses in the context of their hosts and ecological habitat indicated that host species is a strong selective driver in CoV evolution, even in allopatric populations separated by significant geographical distance; and that a single species/genus of bat can contain multiple CoVs. A ß-CoV with 96.5 % amino acid identity to the ß-CoV associated with human disease in the Middle East was found in a Nyctinomops laticaudatus bat, suggesting that efforts to identify the viral reservoir should include surveillance of the bat families Molossidae/Vespertilionidae, or the closely related Nycteridae/Emballonuridae. While it is important to investigate unknown viral diversity in bats, it is also important to remember that the majority of viruses they carry will not pose any clinical risk, and bats should not be stigmatized ubiquitously as significant threats to public health.

Evidence for Nipah virus recrudescence and serological patterns of captive Pteropus vampyrus.

This study aimed to describe the transmission dynamics, the serological and virus excretion patterns of Nipah virus (NiV) in Pteropus vampyrus bats. Bats in captivity were sampled every 7-21 days over a 1-year period. The data revealed five NiV serological patterns categorized as high and low positives, waning, decreasing and increasing, and negative in these individuals. The findings strongly suggest that NiV circulates in wild bat populations and that antibody could be maintained for long periods. The study also found that pup and juvenile bats from seropositive dams tested seropositive, indicating that maternal antibodies against NiV are transmitted passively, and in this study population may last up to 14 months. NiV was isolated from the urine of one bat, and within a few weeks, two other seronegative bats seroconverted. Based on the temporal cluster of seroconversion, we strongly believe that the NiV isolated was recrudesced and then transmitted horizontally between bats during the study period.

Genetic diversity of Nipah virus in Bangladesh.

BACKGROUND: Nipah virus (NiV) infection, often fatal in humans, is primarily transmitted in Bangladesh through the consumption of date palm sap contaminated by Pteropus bats. Person-to-person transmission is also common and increases the concern of large outbreaks. This study aimed to characterize the molecular epidemiology, phylogenetic relationship, and the evolution of the nucleocapsid gene (N gene) of NiV. METHODS: We conducted molecular detection, genetic characterization, and Bayesian time-scale evolution analyses of NiV using pooled Pteropid bat roost urine samples from an outbreak area in 2012 and archived RNA samples from NiV case patients identified during 2012-2018 in Bangladesh. RESULTS: NiV-RNA was detected in 19% (38/456) of bat roost urine samples and among them; nine N gene sequences were recovered. We also retrieved sequences from 53% (21 out of 39) of archived RNA samples from patients. Phylogenetic analysis revealed that all Bangladeshi strains belonged to NiV-BD genotype and had an evolutionary rate of 4.64 × 10-4 substitutions/site/year. The analyses suggested that the strains of NiV-BD genotype diverged during 1995 and formed two sublineages. CONCLUSION: This analysis provides further evidence that the NiV strains of the Malaysian and Bangladesh genotypes diverged recently and continue to evolve. More extensive surveillance of NiV in bats and human will be helpful to explore strain diversity and virulence potential to infect humans through direct or person-to-person virus transmission.

Nipah virus outbreak with person-to-person transmission in a district of Bangladesh, 2007.

In February 2007 an outbreak of Nipah virus (NiV) encephalitis in Thakurgaon District of northwest Bangladesh affected seven people, three of whom died. All subsequent cases developed illness 7-14 days after close physical contact with the index case while he was ill. Cases were more likely than controls to have been in the same room (100% vs. 9.5%, OR undefined, P<0.001) and to have touched him (83% vs. 0%, OR undefined, P<0.001). Although the source of infection for the index case was not identified, 50% of Pteropus bats sampled from near the outbreak area 1 month after the outbreak had antibodies to NiV confirming the presence of the virus in the area. The outbreak was spread by person-to-person transmission. Risk of NiV infection in family caregivers highlights the need for infection control practices to limit transmission of potentially infectious body secretions.

Defect in DNA synthesis in skin of patients with xeroderma pigmentosum demonstrated in vivo.

Exposure of normal human skin in vivo to ultraviolet irradiation at wavelengths shorter than 320 nanometers stimtulated an unscheduled DNA synthesis in all of the cell layers of the epidermis and in the upper dermnial fibrocytes. The skin of patients with xeroderma pigmentosum did not show this response. correlation of these findings with previous tissue culture studies suggests that the defect in repair of the damaged DNA in xeroderma cells occurs in vivo as well as in vitro.

Collaborative research approaches to the role of wildlife in zoonotic disease emergence.

Emerging infectious diseases are a key threat to public health and the majority are caused by zoonotic pathogens. Here we discuss new collaborative approaches to understanding the process of zoonotic disease emergence that link veterinary medicine, public health, and ecological approaches: conservation medicine and one health. We demonstrate how studies on the underlying drivers of disease emergence (bushmeat hunting, wildlife trade, and deforestation) can provide ways to model, predict, and ultimately prevent zoonotic disease emergence and spread.

Efficiency of the Clinical Veterinary Diagnostic Practices and Drug Choices for Infectious Diseases in Livestock in Bangladesh.

As in most low-income countries, adequate laboratory facilities are not available in Bangladesh to assist veterinarians in diagnosing animal diseases. We aimed to determine the efficiency of veterinary diagnoses for two common ruminant diseases in Bangladesh: Peste des petits ruminants (PPR) and foot-and-mouth disease (FMD). We conducted the study from May 2009 to August 2010 in three government veterinary hospitals where veterinarians collected samples from sick livestock and recorded the presumptive diagnosis on the basis of clinical presentations. Samples were tested for PPR and FMD using real-time RT-PCR. We estimated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the presumptive diagnoses when compared to laboratory tests. We tested 539 goats for PPR and 340 cattle and goats for FMD. Our results indicate that the veterinarians' presumptive diagnoses were different from laboratory findings for both PPR (P < 0.05) and FMD (P < 0.05). The overall sensitivity of the presumptive clinical diagnoses was 54% (95% CI: 47-61%) while specificity was 81% (95% CI: 78-84%) compared to real-time RT-PCR tests. The kappa value obtained in our validation process for PPR (kappa: 0.25) and FMD (kappa 0.36) indicated a poor performance of the presumptive diagnoses. Most of the animals (93%) were treated with antibiotics. Our findings indicate that veterinarians can detect animals not infected with FMD or PPR but miss the true cases. The clinical competency of these veterinarians needs to be improved and access to laboratory diagnostic facilities could help veterinarians to improve the diagnostics and outcomes. The rational use of antibiotics by veterinarians in animals must be ensured.

Bat Hunting and Bat-Human Interactions in Bangladeshi Villages: Implications for Zoonotic Disease Transmission and Bat Conservation.

Bats are an important reservoir for emerging zoonotic pathogens. Close human-bat interactions, including the sharing of living spaces and hunting and butchering of bats for food and medicines, may lead to spillover of zoonotic disease into human populations. We used bat exposure and environmental data gathered from 207 Bangladeshi villages to characterize bat exposures and hunting in Bangladesh. Eleven percent of households reported having a bat roost near their homes, 65% reported seeing bats flying over their households at dusk, and 31% reported seeing bats inside their compounds or courtyard areas. Twenty percent of households reported that members had at least daily exposure to bats. Bat hunting occurred in 49% of the villages surveyed and was more likely to occur in households that reported nearby bat roosts (adjusted prevalence ratio [aPR] 2.3, 95% CI 1.1-4.9) and villages located in north-west (aPR 7.5, 95% CI 2.5-23.0) and south-west (aPR 6.8, 95% CI 2.1-21.6) regions. Our results suggest high exposure to bats and widespread hunting throughout Bangladesh. This has implications for both zoonotic disease spillover and bat conservation.

Effects of mechlorethamine (HN2, nitrogen mustard) on UV-induced carcinogenesis in hairless mouse skin.

The influence of mechlorethamine (HN2, nitrogen mustard) on UV-induced carcinogenesis was examined in the hairless mouse skin in vivo. Noncarcinogenic amounts of topically applied HN2 and carcinogenic levels of UVB energy were used in the study. The HN2 applications significantly accelerated the appearance and growth of cutaneous tumors in this study. Thus HN2 acted as either a promoter or cocarcinogen for UV-induced cancer formation. Although the mechanism of this effect was not established, combinations of UVB radiation and HN2 topical therapy pointed to an increased incidence of cutaneous tumors in human skin.

Chemical phototoxicity in humans.

Phototoxic reactions may be induced by endogenous or exogenous chemicals. Endogenous photosensitizers made by the body include porphyrin molecules which are responsible for the photocutaneous syndromes noted in the porphyrias. Exogenous photosensitizers may arrive on the skin through topical applications or may be distributed through the vasculature. Topical photosensitizers are found in cosmetics, medications, plants, and industrial and air pollutant emissions. Systemic photosensitizers consist primarily of therapeutic agents. Acute and chronic phototoxic reactions may occur. The acute response is generally characterized by erythema and edema followed by hyperpigmentation and desquamation. The end point of chronic damage may be cutaneous cancer formation. The mechanisms of these changes may well relate to nucleic acid injury, protein and membrane damage, and alterations in other nuclear and cytoplasmic molecular structures. However, translation of experimental information into human responses remains to be clarified. A great deal of investigation is still needed in developing assays for detecting phototoxic chemicals and for defining mechanisms involved in phototoxic events in human skin.

Stimulation of ultraviolet-induced carcinogenesis by 1,3-Bis(2-chloroethyl)-1-nitrosourea.

The influence of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on ultraviolet-induced carcinogenesis was examined in hairless mouse skin in vivo. Noncarcinogenic amounts of topically applied BCNU and carcinogenic levels of UVB energy were utilized in the study. The applications of BCNU significantly accelerated the appearance and growth of the cutaneous tumors in this study. Thus, the BCNU acted as either a promoting or a cocarcinogenic agent for ultraviolet-induced cancer formation. Although the mechanism of this effect has not been established, avoidance of extensive sun exposure would probably be prudent when topical BCNU is being utilized therapeutically.

3-Aminobenzamide can act as a cocarcinogen for ultraviolet light-induced carcinogenesis in mouse skin.

Chronic irradiation (three times a week) with ultraviolet B light of the skin of hairless mouse Uscd (Hr) strains resulted in the induction of skin tumors after 25 to 41 weeks. Topical applications of 3-aminobenzamide (3AB; 0.1 or 1 M) after each irradiation significantly shortened the earliest time of onset of tumors to 13 to 25 weeks and increased the number of animals that developed tumors over 41 weeks from 67% without 3AB to 73% and 81% with 0.1 and 1 M 3AB, respectively. 3-Aminobenzamide has previously been shown to inhibit radiation-induced transformation in vitro. In vivo, 3AB has the opposite effect, indicating the need for caution in extrapolating from in vitro systems to carcinogenesis in vivo.

Photocarcinogenesis promotion studies with benzoyl peroxide (BPO) and croton oil.

Previous studies demonstrated that BPO can promote chemically initiated tumor formation in SENCAR mice. In addition, a number of chemicals have been shown to promote and/or enhance UVR induced carcinogenesis. This study examined the effect of BPO on UVR initiated tumor formation. One hundred and forty-eight Uscd mice received 270 mJ/cm2 of UVB radiation to the posterior halves of their backs 3 times a week for 8 weeks. Four weeks later the mice were divided into 4 groups. Group I received croton oil in acetone applications to the back 5 times a week for the duration of the study. Group II received acetone, Group III received the BPO diluent, and Group IV received the BPO in an aqueous diluent applications as in Group I. One mouse in Group II (acetone) and one in Group IV (BPO) developed tumors in unirradiated skin. In the UVR initiated skin 38% of the survivors developed tumors in Group I (croton oil), whereas 5% did in Group II (acetone), 8% in Group III (BPO base), and 8% group IV (BPO). Thus under the circumstances of this study croton oil did promote UV initiated tumor formation but BPO did not. These results are consistent with those recently reported by Iversen.

Nitrogen mustard (mechlorethamine) and UVB photocarcinogenesis: a dose response effect.

The effects of UVB energy and nitrogen mustard (mechlorethamine, HN2) on tumor formation in the hairless mouse skin was examined. Twice weekly applications of HN2 produced tumors in 34% of the mice. In addition, the HN2 treatments plus UVB radiation resulted in a significant acceleration in tumor formation as compared to either carcinogenic stimulus alone. The twice weekly application of HN2 plus UVB exposure was more tumorigenic than UVB plus HN2 applied once a week. Thus a positive carcinogenic dose response was noted. No tumor-suppressing effects were detected in this study.

Effects of retinoids on ultraviolet-induced carcinogenesis.

The evidence for effects of the retinoids on UV-induced carcinogenesis is sparse. Clinical observations indicate that topical RA can cause significant regression of premalignant actinic keratoses. Also there is some evidence that this agent can cause dissolution of some basal cell epitheliomas. However this latter effect does not appear to be of therapeutic value. Systemic retinoids are of little value in the treatment of premalignant and malignant cutaneous lesions though 13-cis-retinoic acid might be of use in the basal cell nevus syndrome. Examination of the influence of the retinoids on photocarcinogenesis essentially has been confined to RA and animal experimentation. RA in nontoxic concentrations can both stimulate and inhibit photocarcinogenesis depending upon the circumstances of the study. The mechanisms of these responses are not clear. Influences on DNA synthesis directly and/or indirectly or on immune responses may be involved in both effects. Preliminary studies with oral 13-cis-retinoic acid have not demonstrated any effects to date on UV-induced skin cancer formation.

Inhibition of ultraviolet-induced carcinogenesis by all-trans retinoic acid.

The effects of all-trans retinoic acid (RA) in 0.05%, 0.025% and 0.005% concentrations on ultraviolet (UV) induced carcinogenesis was investigated in the skin of Uscd strain hairless mice. A carcinogenic amount of UV energy was delivered over the 12-mo period of the study. The 0.025% and 0.005% RA solutions did not alter the development of cutaneous cancers. However, the 0.05% RA concentration significantly inhibited the tumor formation in this study.

Ultrastructural study of the nuclei in premitotic and repair DNA synthesis following UVB injury.

Ultrastructural changes in nuclei synthesizing DNA were studied by cytochemical technique. Guninea pigs ears were UVB irradiated and TdR-H3 was injected intradermally into the irradiated sites 1 hr before biopsy. Areas of the epidermis containing more than 80% of cells in DNA (repair or premitotic) synthesis identified by light microscopic autoradiography were selected and cut at 600 A. The glycolmethacrylate sections were stained with uranyl acetate and lead citrate, and consecutive sections were incubated with 0.01% pronase and 0.5% RNase before staining in order to observe DNA. In cells undergoing DNA repair, the zone of DNA became discontinuous and DNA was scattered throughout the entire karyoplasm as small aggregates and fine filaments. Nuclei in S-phase showed essentially the same change, but quantitatively the disappearance of DNA from the nuclear membrane and distribution in the karyoplasm became much greater. These changes were not seen in specimens treated without cytochemical technique.

Repair of pyrimidine(6-4)pyrimidone photoproducts in mouse skin.

The induction and repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts in the epidermal DNA of ultraviolet-irradiated hairless mice were determined by radioimmunoassay. Few cyclobutane dimers were excised by 48 h after ultraviolet (UV) irradiation, whereas 50% of the (6-4) photoproducts were removed by 6 h, correlating with previously determined rates of unscheduled DNA synthesis in mouse skin. After this initial rapid phase of (6-4) photoproduct excision, a slower phase was observed between 6 and 48 h. These repair kinetics contrast with those for fibroblast cell cultures derived from mouse tissues irradiated with UV light yielding similar levels of damage. Although the initial rate of (6-4) photoproduct repair in cultured fibroblasts and epidermal cells was similar, the extent of repair in cultured cells was significantly greater, with most of the damage removed by 24 h. The kinetics for (6-4) photoproduct repair in mouse epidermal cells suggest that a significant population, such as terminally differentiated keratinocytes, may have a reduced repair capacity and that the culture process may select for more rapidly proliferating, repair-proficient stem cells.

Effects of 4-tertiary butyl catechol on melanocytes of hairless mice.

Depigmentary effects of 4-tertiary butyl catechol (TBC) on UV-stimulated melanocytes on the flanks and naturally active melanocytes of ears were compared in Uscd strain hairless mice. UVB irradiation, twice a week for 1 or 2 mo, induced brown-black pigmentation on the flanks. A 1 M TBC application twice a week for 1 mo not only prevented the UV-stimulated pigmentation, but also promoted fading of the post-UV tanning. Dopa-stained split epidermal sheets showed a decrease in the number of melanocytes to less than one-half during the first month of TBC treatment. Melanocytes were often enlarged or lost their dendrites, and both premelanosomes and melanosomes showed ultrastructural changes. However, TBC application on the ears for 1 mo showed neither color change nor a decrease in the number of melanocytes. There were ultrastructural changes in melanocytes but the degree of abnormality was much less than those seen in UV-stimulated melanocytes. Continuation of TBC application for 2 mo with the UV irradiation on the flanks or the nonirradiated ears caused an increase in the number of melanocytes. These results suggest that the initial effect of TBC may be primarily cytotoxicity to melanocytes, and may correlate with their cellular functions. The stimulatory effects of TBC on melanocytes seen during the longer period of time requires further investigation.