Rigorous wildlife disease surveillance
Evidence suggests that zoonotic (animal origin) coronaviruses have caused three recent emerging infectious disease (EID) outbreaks: severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the current coronavirus disease 2019 (COVID-19) pandemic. In the search for an intermediate host for SARS coronavirus 2 (SARS-CoV-2, which causes COVID-19), studies have identified SARS-CoV-2–like strains in bats (1) and pangolins (2), but these do not contain the same polybasic cleavage site that is present in SARS-CoV-2 (3). It is unknown what the intermediate host for this spillover event was because to date there are no international or national conventions on pathogen screening associated with animals, animal products, or their movements, and capacity for EID diagnostics is limited along much of the human-wildlife interface. EID risks associated with the wildlife trade remain the largest unmet challenge of current disease surveillance efforts.
Although viruses represent a fraction of ∼1400 known human pathogens, they place a disproportionate burden on global health (4). Around 89% of the 180 recognized RNA viruses with the potential to harm humans are zoonotic. Coronaviruses are only the tip of the spillover iceberg: HIV came from nonhuman primates, Ebola came from bats, and H5N1 and H1N1 influenza strains came from birds and pigs, respectively. Indeed, 60% of EIDs are zoonotic in nature, and more than 70% of these have an origin in wildlife (5).
Unchecked exploitation of wildlife—whether for sustenance or profit, legal or illegal—puts humans in direct contact with myriad unfamiliar species. Increased contact occurs in the global practice of bushmeat and game hunting and in wildlife farms, which often unsustainably and illegally supply wildlife for consumption or trade (6). Imported, hunted, and farmed wildlife then reach a common endpoint, wildlife markets. There, animals endure debilitating and immunocompromising conditions that promote disease transmission: packed cages, poor biosecurity, and unhygienic shedding of animal excreta (7). Direct human-wildlife contact, mixing of nonendemic wildlife species, and limited health and safety standards are all criteria for a zoonotic hotspot. Many wildlife markets around the world meet these criteria, yet disease surveillance in them is largely absent. More broadly, although the Convention on the International Trade in Endangered Species (CITES) regulates international wildlife trade on the basis of species' endangered status, only a few countries use strict veterinary import controls, and there are no global regulations on pathogen screening associated with the international trade in wildlife.
Pathogen biosurveillance and how humans interact with wildlife are at the crux of EID risk management and response. After bats were identified as likely reservoirs for a range of zoonotic events (such as Hendra, Nipah, SARS, MERS, and Ebola) (8), surveillance of a single cave in southwest China between 2011 and 2015 revealed 11 novel coronaviruses (9). From 2015 to 2017, of 1497 people tested in the surrounding Yunnan, Guangxi, and Guangdong districts, nine (0.6%) were positive for prior bat coronavirus antibodies, and 265 (17%) reported SARS- or influenzatype symptoms associated with contact with poultry, carnivores, rodents, shrews, or bats (10). These findings, formally reported in September 2019, provided a warning about the risk of zoonotic coronaviruses that was neither heard nor heeded. The COVID-19 pandemic is evidence that bridging the gap between research and response is critical to anticipating and mitigating future spillover events.
PREDICT, the intermittently federally funded offshoot of the 2009 United States Agency for International Development (USAID) Emerging Pandemic Threats program that partially financed the study of bat coronaviruses (10), screened 164,000 animals and humans and detected 949 novel viruses in zoonotic hotspots across 30 countries between 2009 and 2019. The Global Virome Project—a collaboration between experts in global health and pandemic prevention—aims to sequence all animal virus strains over a 10-year period, with a projected cost of $1.2 billion. Both projects share stakeholders, and although their missions are likely to adapt to a post–COVID-19 world, one of their stated goals includes strengthening existing laboratory capacities along the human-wildlife interface. But are there sufficient numbers of animal pathogen reference laboratories? According to the World Organisation for Animal Health (OIE) (11), there are 125 reference laboratories certified to screen for one or more target pathogens (and not for broad pathogen surveillance). Their global distribution does not reflect EID risks. Southeast Asia, Africa, and Central and South America carry the burden of EID risk, yet 78 (62%) of reference laboratories are in Europe and North America; only 33 (26%) are in Asia (14 in China and 8 in Japan), with 12 (34%) spread between 7 countries; 3 (∼2%) are in Africa; 4 (∼3%) are in Australia, and 8 (∼6%) are in South America. Although this does not account for laboratory size or screening methods and capacity, it is evident that many regions with zoonotic hotspots lack testing facilities with the capability of conducting disease surveillance.
Markets selling live animals, including wildlife such as this slow loris at the Borito Market in Jakarta, Indonesia, are hotspots for zoonotic spillover and should be monitored to better manage emerging infectious diseases.
PHOTO: TOM LE LIEVRE/REDUX
What can be done to mitigate future zoonotic EIDs? Centralized biosurveillance efforts produce results but are expensive, maintained by a select few countries, and subject to political whims, as evidenced by the 2019 shift in funding for PREDICT, a recent recall of U.S. National Institutes of Health (NIH) support for the EcoHealth Alliance, and the withdrawal of the United States from the World Health Organization (WHO). As such, they are not immediately scalable, nor do they stimulate widespread capacity. The international wildlife trade is a substantial global industry in need of greater oversight. Because ill-conceived restrictions would affect millions of people and likely drive these activities deeper underground, further impeding regulation (12), the first step is to establish a more cost-effective, decentralized disease surveillance system. It would empower local wildlife and public health professionals to test for diseases year round, at source, without criminalizing public participation in screening programs. Such screening was not technologically feasible after the emergence of the H1N1 influenza virus in 2009, but now, affordable modern technologies enable quick in situ biosample processing, whole-genome sequencing, metagenomics, and metabarcoding of pathogens. This would enable proactive, broad, routine wildlife pathogen screening in remote areas rather than reactive targeted testing.
Decentralized laboratories must be able to extract genomic material and conduct metagenomic sequencing and targeted pathogen testing if necessary. As demand increases, individual technologies have evolved to be smaller, simpler, and more affordable. Multiplex polymerase chain reaction (PCR)–based viral enrichment protocols with portable DNA sequencers (such as MinION) have been deployed for in situ monitoring of Ebola virus, Zika virus, and now SARS-CoV-2 infections (13). Practical training in applying these laboratory solutions (such as miniature centrifuges, thermocyclers, and electrophoresis setups) is well documented, even in remote locations (14). However, most pathogen screening efforts that use such equipment have been in response to human disease outbreaks. These technologies could be used to regularly monitor entire pathogen families of increased global concern in animals and areas with increased risk of zoonotic spillover, including wildlife markets or farms and free-ranging high-risk taxa such as primates and bats (15). Local wildlife scientists and health care workers can be trained on how to safely use facilities with broadly accessible molecular equipment in local facilities with standard biosecurity practices to prevent risk of pathogen spillover into the community. Restricting such training and activities to relatively few specialized centers impedes broad surveillance efforts. Any animal surveillance program should integrate with testing programs for humans to capture early zoonotic pathogen circulation between human and nonhuman populations. Sources of zoonotic pathogens are frequently unclear and often not possible to determine after the early stages of a spillover event. Monitoring could remove much of this uncertainty, allowing molecular epidemiology to inform short- and long-term responses on both a local and global level.
To complement decentralized laboratories, a publicly accessible, centralized, curated system for monitoring pathogens must be established for three main reasons: (i) This would provide instant pathogen classifications based on comparative genomics, further cross-linked to reference data on prevalence by species and region. (ii) A centralized curated system could alert to EID indicators, including gains and losses of strains, pathogen-specific changes in host species numbers, rapid increases in mutation rates that may indicate pathogen spillover into a naive host, and pathogen detection in traded animals that do not occur in wild counterparts. (iii) For virus families that are poised to spillover into human populations, genomic sequence data can reveal diversity of key pathogen proteins in circulating strains (for example, the spike protein that mediates human cell entry of coronaviruses, and the RNA-dependent RNA polymerase that is important for viral replication). Such approaches assist in identifying broad-spectrum antivirals and vaccination targets as well as treatment-resistant pathogen variants that pose a risk of generating future EIDs.
An example of a disease-focused public database that could be expanded is the GISAID (global initiative on sharing all influenza data) EpiFlu repository, a global initiative developed for sharing influenza virus sequence data and currently also documenting SARS-CoV-2 sequences. It facilitates data access for registered users while securing data ownership by requiring that contributors be acknowledged in derivative research. Additionally, the database could include report-generating features such as those in the Zoological Information Management Software (ZIMS), used by more than 1000 Species360–accredited zoological institutions worldwide to upload biomedical data and compute reference ranges across multiple variables and species.
An internationally recognized standard for managing wildlife trade on the basis of known disease risks should be established. Currently, few countries consider disease risk as a factor in regulating wildlife imports and exports, and a disease status equivalent to CITES is lacking. Pathogen screening is also not required nor facilitated before, during, or after translocating wildlife products, leaving pathogen status to be declared by the shipper, who may not have the experience to make such determinations. Because a large number of animals naturally carry pathogens that could spillover to humans if improperly handled, the means to identify the species for which security standards should be enhanced, or for which trade and consumption should potentially be prohibited, is needed. An important caveat is that such classifications can stigmatize animals to their detriment and incite fear-based human behaviors that may threaten species conservation.
A decentralized network could improve feedback between those who screen samples and those who curate data to bolster the safety of wildlife and humans, a fundamentally “One Health” approach. This would increase localized knowledge of EID risks, provide earlier warnings and faster global responses to spillovers, and inform wildlife trade policy. This model is more robust to shifting political landscapes and funding and does not ignore the role of advanced regional research laboratories, which also provide vital targeted pathogen screening. Research laboratories can also provide samples for or generate high-quality host de novo reference genome assemblies and expand regional capacity for biobanking, including cell cultures, which will improve understanding of the co-evolutionary processes that underlie pathogen-host range and susceptibility. By giving more parties a stake in the effort, decentralization is more likely to succeed in garnering geographically representative participation that explicitly includes the most at-risk, under-resourced regions.
Supplementary Material
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References and Notes
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10 July 2020
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Building Biorepository Capacity Internationally Would Stimulate More Effective Pathogen Surveillance and Mitigation: A Response to Watsa et al. 2020
We commend Watsa et al. (2020) for pointing out that decentralized pathogen surveillance networks are the most effective way to improve the prediction, monitoring, and mitigation of emerging infectious diseases (EIDs). Watsa and colleagues underscore the value of One Health approaches to stimulate integration across currently siloed efforts in zoonotic research and mitigation. Yet, to achieve comprehensive decentralized pathogen surveillance, there is an urgent need to develop not only public health facilities, but also environmental and biodiversity infrastructure in biodiverse countries experiencing high rates of habitat conversion, wildlife trafficking, and human-wildlife interactions. Approximately one-third of One Health networks lack an environmental component, less than half are active in wildlife surveillance, and almost none is led by developing countries (Khan et al. 2018). We submit that international support for development of natural history museums with frozen vertebrate tissue collections remains a key component missing from the One Health equation.
A majority of pathogens causing severe outbreaks in humans are zoonotic in origin (Jones et al. 2008); consequently, understanding their wild animal hosts is imperative. As with Covid-19 (Cohen 2020), identifying wild animal reservoirs can be challenging when biorepositories are lacking (Leendertz et al. 2016). Yet, in most countries, natural history biorepositories remain poorly supported and largely disconnected from public health initiatives. For example, most studies of bat coronaviruses (Hu et al. 2017) to date, including the PREDICT animal surveys discussed in Watsa et al. (2020), did not preserve host specimens or tissues, thus limiting potential for molecular host identification or replication and extension of the science (Cook et al. 2020). EID response hinges on sampling depth across space, time, and taxonomy, the very sampling enabled by museum biorepositories. As primary biological infrastructure, in-country development of museum collections, following best practices (Dunnum et al. 2017), and cyber-linked specimen data should be an international imperative (Paknia et al. 2015) for effective global surveillance and mitigation of EIDs.
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M. Watsa and Wildlife Disease Surveillance Focus Group. Rigorous wildlife disease surveillance. Science. 369, 145-147. (2020)
SARS-CoV-2, forth and back from animals to humans: What's next?
Based upon the data reported by Dr Shi and coworkers (1) as well as by other investigators, cats, ferrets, hamsters, tigers, lions, and minks represent, along with macaques and other non-human primates, mammalian species that are susceptible to natural and/or experimental infection by the "Severe Acute Respiratory Syndrome (SARS) Coronavirus (CoV)-2", the seventh officially recognized human coronavirus, which is also the causative agent of "CoronaVirus Disease 2019" (CoViD-19). A less pronounced SARS-CoV-2 sensitivity has been additionally shown in dogs, with resistance to experimental challenge having been reported in chickens and ducks (1). As firmely ascertained in mankind, cats would be also prone to acquire SARS-CoV-2 infection through the respiratory route, with infected (and asymptomatic) felines shedding the virus by means of aerosolization, thereby infecting their conspecifics housed in close proximity to them (1). Noteworthy, SARS-CoV-2 infection has been recently diagnosed in mink farms in The Netherlands, with minks likely acquiring the virus from infected caregivers, in a similar fashion to what previously found in tigers and lions from New York City Bronx Zoo as well as in privately owned cats in Hong Kong, Belgium, and USA.
Since the viral isolates characterized from some of the aforementioned patients in The Netherlands had a genome sequence closer to that of SARS-CoV-2 strains detected in minks, as compared to the isolates identified in the "general population" residing in the same area, it seems plausible that humans (mink caregivers) might have acquired the infection from minks rather than by interhuman transmission.
This is of special concern when dealing with the intricate and complex eco-epidemiological dynamics of a natural (and pandemic) infection as the one caused by SARS-CoV-2 betacoronavirus, a pathogen likely originating from bats (Rinolophus affinis) and which could have subsequently "jumped" into an "intermediate" (and hitherto unidentified) species before making its "definitive" spillover into mankind.
This is not an "unprecedented" finding, given that the SARS and the "Middle East Respiratory Syndrome" (MERS) coronaviruses had done (more or less) the same in 2002-2003 and 2012, respectively, and provided also that, even more important, no less than 70% of "emerging infectious diseases" (EIDs) are caused by pathogens originating, either certainly or suspectedly, from animals (2).
As a concluding remark, SARS-CoV-2 infection and CoViD-19 disease, which have now reached the dramatic figures of 17 million cases and over 650,000 deaths worldwide, are a complex issue, in a similar way to the vast majority, if not to all the other zoonotic infections and diseases. As a consequence, a multidisciplinary approach is absolutely needed in handling zoonotic EIDs, thereby taking into special consideration the "One Health" concept, a crucial "common denominator" mutually and indissolubly linking human, animal, and environmental health into a common and unique "triangle".
References
1) Shi J., et al. (2020) - Science 368: 1016-1020.
2) Casalone C. & Di Guardo G. (2020) - Science (Letter to the Editor, e.Letter).
--
Giovanni DI GUARDO, DVM, Dipl. ECVP,
Scientific Editor,
Research in Veterinary Science,
Professor of General Pathology and
Veterinary Pathophysiology,
University of Teramo,
Faculty of Veterinary Medicine,
64100 - Teramo, Italy
(E-mail address: [email protected];
Telephone: +39-861-266933;
Telefax: +39-861-266865)
RE: Watsa et al.'s Rigorous wildlife disease surveillance
Watsa et al. [1] call for a global wildlife-disease surveillance system. We completely agree, but two major challenges have not been addressed:
1. Novel viruses are likely to be missed by PCR- and Crispr-based kits because only a small fraction of viral diversity has been sequenced. Any surveillance system must include capacity to characterize novel viruses.
2. It is exceedingly difficult to survey wildlife for viruses. Many viruses have low prevalence, requiring enormous amounts of sampling (e.g. 25000 wild birds were sampled in Germany, resulting in an avian-influenza prevalence <1% [2]). Trying to trap thousands of individuals per species will likely be both futile, especially in the tropics, and unethical, since trapping can kill, injure, and infect wildlife. Using the shortcut of sampling in markets potentially introduces two biases. First, wildlife can exchange viruses along the supply chain [3], obscuring the original hosts. Second, wildlife that host the more human-pathogenic virus strains might be more likely to die in the wild or early in the supply chain and thus be less likely to reach markets. Sampling in the wild is the only way to avoid these biases.
Fortunately, methods are now available to at least partially overcome these bottlenecks.
1. Viral enrichment methods such as hybridization capture allow us to sequence the nucleic acids of highly divergent, unknown viruses.
2. Aquatic environmental DNA (eDNA) and invertebrate-derived DNA (iDNA) sampling provides efficient, non-invasive access to low-abundance wildlife species and their viral loads, even in remote areas [4]. For instance, mass-collected leeches, coupled with hybridization capture, have detected a novel coronavirus associated with tropical deer [5].
The tools exist to overcome the challenges related to sampling pathogens in wildlife, and we urge the designers of surveillance systems to integrate metagenomics and e/iDNA.
[1] Watsa, M., Wildlife Disease Surveillance Focus Group. 2020. Rigorous wildlife disease surveillance. Science 369:145–147.
[2] Wilking H, Ziller M, Staubach C, Globig A, Harder TC, Conraths FJ. (2009) Chances and limitations of wild bird monitoring for the avian influenza virus H5N1 - detection of pathogens highly mobile in time and space. PLoS One 4:e6639.
[3] Huong, N. Q., N. T. Thanh Nga, N. Van Long, et al. 2020. Coronavirus testing indicates transmission risk increases along wildlife supply chains for human consumption in Viet Nam, 2013-2014. bioRxiv doi:10.1101/2020.06.05.098590.
[4] Bitome-Essono, P.-Y., B. Ollomo, C. Arnathau, P. Durand, N. D. Mokoudoum, L. Yacka-Mouele, A.-P. Okouga, L. Boundenga, B. Mve-Ondo, J. Obame-Nkoghe, P. Mbehang-Nguema, F. Njiokou, B. Makanga, R. Wattier, D. Ayala, F. J. Ayala, F. Renaud, V. Rougeron, F. Bretagnolle, F. Prugnolle, and C. Paupy. 2017. Tracking zoonotic pathogens using blood-sucking flies as "flying syringes." eLife 6:e22069.
[5] Alfano, N. Dayaram, A., Axtner, J. Tsangaras, K. Kampmann, M.-L., Mohamed, A., Wong, S.T., Gilbert, M.T.P. Wilting, A., Greenwood, A. D. Non-invasive surveys of mammalian viruses using environmental DNA. bioRxiv https://doi.org/10.1101/2020.03.26.009993.