Phenotypic and genetic characterization of MERS coronaviruses from Africa to understand their zoonotic potential.

Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV-infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.

Incorporating heterogeneous sampling probabilities in continuous phylogeographic inference - Application to H5N1 spread in the Mekong region.

MOTIVATION: The potentially low precision associated with the geographic origin of sampled sequences represents an important limitation for spatially explicit (i.e. continuous) phylogeographic inference of fast-evolving pathogens such as RNA viruses. A substantial proportion of publicly available sequences is geo-referenced at broad spatial scale such as the administrative unit of origin, rather than more precise locations (e.g. geographic coordinates). Most frequently, such sequences are either discarded prior to continuous phylogeographic inference or arbitrarily assigned to the geographic coordinates of the centroid of their administrative area of origin for lack of a better alternative. RESULTS: We here implement and describe a new approach that allows to incorporate heterogeneous prior sampling probabilities over a geographic area. External data, such as outbreak locations, are used to specify these prior sampling probabilities over a collection of sub-polygons. We apply this new method to the analysis of highly pathogenic avian influenza H5N1 clade data in the Mekong region. Our method allows to properly include, in continuous phylogeographic analyses, H5N1 sequences that are only associated with large administrative areas of origin and assign them with more accurate locations. Finally, we use continuous phylogeographic reconstructions to analyse the dispersal dynamics of different H5N1 clades and investigate the impact of environmental factors on lineage dispersal velocities. AVAILABILITY AND IMPLEMENTATION: Our new method allowing heterogeneous sampling priors for continuous phylogeographic inference is implemented in the open-source multi-platform software package BEAST 1.10. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A 'what-if' scenario: Nipah virus attacks pig trade chains in Thailand.

BACKGROUND: Nipah virus (NiV) is a fatal zoonotic agent that was first identified amongst pig farmers in Malaysia in 1998, in an outbreak that resulted in 105 fatal human cases. That epidemic arose from a chain of infection, initiating from bats to pigs, and which then spilled over from pigs to humans. In Thailand, bat-pig-human communities can be observed across the country, particularly in the central plain. The present study therefore aimed to identify high-risk areas for potential NiV outbreaks and to model how the virus is likely to spread. Multi-criteria decision analysis (MCDA) and weighted linear combination (WLC) were employed to produce the NiV risk map. The map was then overlaid with the nationwide pig movement network to identify the index subdistricts in which NiV may emerge. Subsequently, susceptible-exposed-infectious-removed (SEIR) modeling was used to simulate NiV spread within each subdistrict, and network modeling was used to illustrate how the virus disperses across subdistricts. RESULTS: Based on the MCDA and pig movement data, 14 index subdistricts with a high-risk of NiV emergence were identified. We found in our infectious network modeling that the infected subdistricts clustered in, or close to the central plain, within a range of 171 km from the source subdistricts. However, the virus may travel as far as 528.5 km (R0 = 5). CONCLUSIONS: In conclusion, the risk of NiV dissemination through pig movement networks in Thailand is low but not negligible. The risk areas identified in our study can help the veterinary authority to allocate financial and human resources to where preventive strategies, such as pig farm regionalization, are required and to contain outbreaks in a timely fashion once they occur.

Emergence of Influenza A(H7N4) Virus, Cambodia.

Active surveillance in high-risk sites in Cambodia has identified multiple low-pathogenicity influenza A(H7) viruses, mainly in ducks. None fall within the A/Anhui/1/2013(H7N9) lineage; however, some A(H7) viruses from 2018 show temporal and phylogenetic similarity to the H7N4 virus that caused a nonfatal infection in Jiangsu Province, China, in December 2017.

The impact of surveillance and control on highly pathogenic avian influenza outbreaks in poultry in Dhaka division, Bangladesh.

In Bangladesh, the poultry industry is an economically and socially important sector, but it is persistently threatened by the effects of H5N1 highly pathogenic avian influenza. Thus, identifying the optimal control policy in response to an emerging disease outbreak is a key challenge for policy-makers. To inform this aim, a common approach is to carry out simulation studies comparing plausible strategies, while accounting for known capacity restrictions. In this study we perform simulations of a previously developed H5N1 influenza transmission model framework, fitted to two separate historical outbreaks, to assess specific control objectives related to the burden or duration of H5N1 outbreaks among poultry farms in the Dhaka division of Bangladesh. In particular, we explore the optimal implementation of ring culling, ring vaccination and active surveillance measures when presuming disease transmission predominately occurs from premises-to-premises, versus a setting requiring the inclusion of external factors. Additionally, we determine the sensitivity of the management actions under consideration to differing levels of capacity constraints and outbreaks with disparate transmission dynamics. While we find that reactive culling and vaccination policies should pay close attention to these factors to ensure intervention targeting is optimised, across multiple settings the top performing control action amongst those under consideration were targeted proactive surveillance schemes. Our findings may advise the type of control measure, plus its intensity, that could potentially be applied in the event of a developing outbreak of H5N1 amongst originally H5N1 virus-free commercially-reared poultry in the Dhaka division of Bangladesh.

A spatial assessment of Nipah virus transmission in Thailand pig farms using multi-criteria decision analysis.

BACKGROUND: Thailand's Central Plain is identified as a contact zone between pigs and flying foxes, representing a potential zoonotic risk. Nipah virus (NiV) has been reported in flying foxes in Thailand, but it has never been found in pigs or humans. An assessment of the suitability of NiV transmission at the spatial and farm level would be useful for disease surveillance and prevention. Multi-criteria decision analysis (MCDA), a knowledge-driven model, was used to map contact zones between local epizootic risk factors as well as to quantify the suitability of NiV transmission at the pixel and farm level. RESULTS: Spatial risk factors of NiV transmission in pigs were identified by experts as being of three types, including i) natural host factors (bat preferred areas and distance to the nearest bat colony), ii) intermediate host factors (pig population density), and iii) environmental factors (distance to the nearest forest, distance to the nearest orchard, distance to the nearest water body, and human population density). The resulting high suitable areas were concentrated around the bat colonies in three provinces in the East of Thailand, including Chacheongsao, Chonburi, and Nakhonnayok. The suitability of NiV transmission in pig farms in the study area was quantified as ranging from very low to medium suitability. CONCLUSIONS: We believe that risk-based surveillance in the identified priority areas may increase the chances of finding out NiV and other bat-borne pathogens and thereby optimize the allocation of financial resources for disease surveillance. In the long run, improvements of biosecurity in those priority areas may also contribute to preventing the spread of potential emergence of NiV and other bat-borne pathogens.

Changing Geographic Patterns and Risk Factors for Avian Influenza A(H7N9) Infections in Humans, China.

The fifth epidemic wave of avian influenza A(H7N9) virus in China during 2016-2017 demonstrated a geographic range expansion and caused more human cases than any previous wave. The factors that may explain the recent range expansion and surge in incidence remain unknown. We investigated the effect of anthropogenic, poultry, and wetland variables on all epidemic waves. Poultry predictor variables became much more important in the last 2 epidemic waves than they were previously, supporting the assumption of much wider H7N9 transmission in the chicken reservoir. We show that the future range expansion of H7N9 to northern China may increase the risk of H7N9 epidemic peaks coinciding in time and space with those of seasonal influenza, leading to a higher risk of reassortments than before, although the risk is still low so far.

Reassortant highly pathogenic influenza A(H5N6) virus in Laos.

In March 2014, avian influenza in poultry in Laos was caused by an emergent influenza A(H5N6) virus. Genetic analysis indicated that the virus had originated from reassortment of influenza A(H5N1) clade 2.3.2.1b, variant clade 2.3.4, and influenza A(H6N6) viruses that circulate broadly in duck populations in southern and eastern China.

Development and application of a vaccination planning tool for avian influenza.

The vaccination planning tool for avian influenza supports evidence-based planning and preparedness for vaccinating poultry at national and regional levels. This study describes the development, testing, and application of a vaccination planning tool for H5N1 highly pathogenic avian influenza (HPAI) used in two South Asian countries. The tool consists of eight planning clusters, 37 planning elements, and 303 referenced planning criteria. Both countries attained a score of 52% among planning clusters as a measure of preparedness. The highest and lowest planning cluster scores included vaccination strategies and financial readiness, respectively. The comprehensive vaccination program was identified as the most-useful planning cluster for assessing preparedness, and 86% of participants indicated that the objectives of the planning tool were achieved. Based on these results, the planning tool provides a structured approach for decision makers to develop their national vaccination program for HPAI as part of an overall strategy for the progressive reduction and control of endemic influenza viruses in poultry.

Highly pathogenic avian influenza (H5N1) in Myanmar, 2006-2010.

Highly pathogenic avian influenza (HPAI) virus subtype H5N1 was first reported in Myanmar in 2006. In this study, we have characterized 6 HPAI (H5N1) viruses recovered from 2007-2010 as well as three additional available nucleotide sequences representing Myanmar AI outbreaks. Phylogenetic analysis showed that the Myanmar viruses belong to HPAI (H5N1) clades 7, 2.3.2 and 2.3.4. The result suggested that the HPAI (H5N1) viruses recovered from Myanmar had been introduced into the country by multiple introductions. Genetic analysis of the viruses confirmed the HPAI characteristics of the viruses.

Mapping H5N1 highly pathogenic avian influenza risk in Southeast Asia.

The highly pathogenic avian influenza (HPAI) H5N1 virus that emerged in southern China in the mid-1990s has in recent years evolved into the first HPAI panzootic. In many countries where the virus was detected, the virus was successfully controlled, whereas other countries face periodic reoccurrence despite significant control efforts. A central question is to understand the factors favoring the continuing reoccurrence of the virus. The abundance of domestic ducks, in particular free-grazing ducks feeding in intensive rice cropping areas, has been identified as one such risk factor based on separate studies carried out in Thailand and Vietnam. In addition, recent extensive progress was made in the spatial prediction of rice cropping intensity obtained through satellite imagery processing. This article analyses the statistical association between the recorded HPAI H5N1 virus presence and a set of five key environmental variables comprising elevation, human population, chicken numbers, duck numbers, and rice cropping intensity for three synchronous epidemic waves in Thailand and Vietnam. A consistent pattern emerges suggesting risk to be associated with duck abundance, human population, and rice cropping intensity in contrast to a relatively low association with chicken numbers. A statistical risk model based on the second epidemic wave data in Thailand is found to maintain its predictive power when extrapolated to Vietnam, which supports its application to other countries with similar agro-ecological conditions such as Laos or Cambodia. The model's potential application to mapping HPAI H5N1 disease risk in Indonesia is discussed.

A literature review of the use of environmental sampling in the surveillance of avian influenza viruses.

This literature review provides an overview of use of environmental samples (ES) such as faeces, water, air, mud and swabs of surfaces in avian influenza (AI) surveillance programs, focussing on effectiveness, advantages and gaps in knowledge. ES have been used effectively for AI surveillance since the 1970s. Results from ES have enhanced understanding of the biology of AI viruses in wild birds and in markets, of links between human and avian influenza, provided early warning of viral incursions, allowed assessment of effectiveness of control and preventive measures, and assisted epidemiological studies in outbreaks, both avian and human. Variation exists in the methods and protocols used, and no internationally recognized guidelines exist on the use of ES and data management. Few studies have performed direct comparisons of ES versus live bird samples (LBS). Results reported so far demonstrate reliance on ES will not be sufficient to detect virus in all cases when it is present, especially when the prevalence of infection/contamination is low. Multiple sample types should be collected. In live bird markets, ES from processing/selling areas are more likely to test positive than samples from bird holding areas. When compared to LBS, ES is considered a cost-effective, simple, rapid, flexible, convenient and acceptable way of achieving surveillance objectives. As a non-invasive technique, it can minimize effects on animal welfare and trade in markets and reduce impacts on wild bird communities. Some limitations of environmental sampling methods have been identified, such as the loss of species-specific or information on the source of virus, and taxonomic-level analyses, unless additional methods are applied. Some studies employing ES have not provided detailed methods. In others, where ES and LBS are collected from the same site, positive results have not been assigned to specific sample types. These gaps should be remedied in future studies.

Informing resilience building: FAO's Surveillance Evaluation Tool (SET) Biothreat Detection Module will help assess national capacities to detect agro-terrorism and agro-crime.

Attacks using animal pathogens can have devastating socioeconomic, public health and national security consequences. The livestock sector has some inherent vulnerabilities which put it at risk to the deliberate or accidental spread of disease. The growing concern of countries about the risks of agro-terrorism and agro-crime has led to efforts to prepare against potential attacks. One recent international effort is the launch of a joint OIE, FAO and INTERPOL project in 2019 to build resilience against agro-terrorism and agro-crime targeting animal health with the financial support of the Weapons Threat Reduction Programme of Global Affairs Canada. Given the importance of strong animal health surveillance systems for the early and effective response to agro-terrorism and agro-crime, the project will use the FAO Surveillance Evaluation Tool (SET) and its new Biothreat Detection Module to evaluate beneficiary countries' capacities to detect criminal or terrorist animal health events. This paper presents the development of the new SET Biothreat Detection Module and how it will be used to evaluate surveillance for agro-terrorism and agro-crime animal disease threats. The module will be piloted in early 2021 and, once finalized, will be used by beneficiary countries of the joint OIE-FAO-INTERPOL project. Results from evaluations using SET and its Biothreat Detection Module are expected to provide a baseline from which countries can build targeted capacity for animal disease surveillance including early detection and investigation of potential terrorist or criminal events involving zoonotic and non-zoonotic animal pathogens.

Two decades of one health surveillance of Nipah virus in Thailand.

BACKGROUND: Nipah virus (NiV) infection causes encephalitis and has > 75% mortality rate, making it a WHO priority pathogen due to its pandemic potential. There have been NiV outbreak(s) in Malaysia, India, Bangladesh, and southern Philippines. NiV naturally circulates among fruit bats of the genus Pteropus and has been detected widely across Southeast and South Asia. Both Malaysian and Bangladeshi NiV strains have been found in fruit bats in Thailand. This study summarizes 20 years of pre-emptive One Health surveillance of NiV in Thailand, including triangulated surveillance of bats, and humans and pigs in the vicinity of roosts inhabited by NiV-infected bats. METHODS: Samples were collected periodically and tested for NiV from bats, pigs and healthy human volunteers from Wat Luang village, Chonburi province, home to the biggest P. lylei roosts in Thailand, and other provinces since 2001. Archived cerebrospinal fluid specimens from encephalitis patients between 2001 and 2012 were also tested for NiV. NiV RNA was detected using nested reverse transcription polymerase chain reaction (RT-PCR). NiV antibodies were detected using enzyme-linked immunosorbent assay or multiplex microsphere immunoassay. RESULTS: NiV RNA (mainly Bangladesh strain) was detected every year in fruit bats by RT-PCR from 2002 to 2020. The whole genome sequence of NiV directly sequenced from bat urine in 2017 shared 99.17% identity to NiV from a Bangladeshi patient in 2004. No NiV-specific IgG antibodies or RNA have been found in healthy volunteers, encephalitis patients, or pigs to date. During the sample collection trips, 100 community members were trained on how to live safely with bats. CONCLUSIONS: High identity shared between the NiV genome from Thai bats and the Bangladeshi patient highlights the outbreak potential of NiV in Thailand. Results from NiV cross-sectoral surveillance were conveyed to national authorities and villagers which led to preventive control measures, increased surveillance of pigs and humans in vicinity of known NiV-infected roosts, and increased vigilance and reduced risk behaviors at the community level. This proactive One Health approach to NiV surveillance is a success story; that increased collaboration between the human, animal, and wildlife sectors is imperative to staying ahead of a zoonotic disease outbreak.

The Costs, Benefits and Human Behaviours for Antimicrobial Use in Small Commercial Broiler Chicken Systems in Indonesia.

There are growing concerns over the threat to human health from the unregulated use of antimicrobials in livestock. Broiler production is of great economic and social importance in Indonesia. This study used a structured questionnaire approach to explore the human behaviours and economic drivers associated with antimicrobial use in small commercial broiler systems in Indonesia (n = 509). The study showed that antimicrobial use was high with farmers easily able to access antimicrobials through local animal medicine, however, it was difficult for farmers to access veterinary advice on responsible antimicrobial use. The most significant finding was that the relative cost of antimicrobials was low, and farmers observed improvements in productivity rates from routine antimicrobial administration. However, farmers seldom kept detailed records on farm productivity or economic costs; this is a hurdle to undertaking a more detailed economic analysis of antimicrobial use. There is a need for further research on the cost-effectiveness of alternative methods of preventing disease and ensuring that feasible alternatives are easily available. Farm-level economics and securing the food supply chain need to be central to any future policy interventions to reduce antimicrobial use in broiler systems in Indonesia and this observation is relevant at a regional and global level.

New frontiers in applied veterinary point-of-capture diagnostics: Toward early detection and control of zoonotic influenza.

Among the chief limitations in achieving early detection and control of animal-origin influenza of pandemic potential in high-risk livestock populations is the existing lag time between sample collection and diagnostic result. Advances in molecular diagnostics are permitting deployment of affordable, rapid, highly sensitive, and specific point-of-capture assays, providing opportunities for targeted surveillance driving containment strategies with potentially compelling returns on investment. Interrupting disease transmission at source holds promise of disrupting cycles of animal-origin influenza incursion to endemicity and limiting impact on animal production, food security, and public health. Adoption of new point-of-capture diagnostics should be undertaken in the context of promoting robust veterinary services systems and parallel support for operationalizing pre-authorized plans and communication strategies that will ensure that the full potential of these new platforms is realized.

Social network analysis for the assessment of pig, cattle and buffalo movement in Xayabouli, Lao PDR.

The aim of this study is to understand the role that the movement patterns of pigs, cattle and buffalo play in the spread of foot-and-mouth disease (FMD). A cross-sectional survey consisting of a questionnaire was used in a hotspot area for FMD: Xayabouli Province, Lao People's Democratic Republic. A total of 189 respondents were interviewed. We found that the key players in this network were people who were involved with more than one species of animal or occupation (multipurpose occupational node), which represents the highest number of activities of animals moved off the holding (shown with the highest out-degree centrality) and a high likelihood of being an intermediary between others (shown with the highest betweenness centrality). Moreover, the results show that the animals moved to and away from each node had few connections. Some nodes (such as traders) always received animals from the same group of cattle owners at different times. The subgroup connection within this network has many weak components, which means a connection in this network shows that some people can be reached by others, but most people were not. In this way, the number of connections present in the network was low when we defined the proportion of observed connections with all possible connections (density). These findings indicate that the network might not be busy; only one type of node is dominant which enables increased control of disease spread. We recommend that the relevant authorities implement control measures regarding the key players, which is the best way to effectively control the spread of infectious diseases.

A field-deployable insulated isothermal RT-PCR assay for identification of influenza A (H7N9) shows good performance in the laboratory.

BACKGROUND: Avian influenza A (H7N9) remains circulating in China. For countries at risk of introduction of H7N9, such as Vietnam, early detection of H7N9 virus is essential for the early containment of the virus. Insulated isothermal reverse transcriptase PCR (iiRT-PCR) is a portable PCR system that can be deployed under field conditions to identify pathogens at the sampling site. Applying PCR at the sampling site will greatly reduce the time to obtain a diagnostic result which allows the veterinary authority to take immediate action to contain disease spreading. OBJECTIVE: To determine analytical and diagnostic sensitivity and specificity of the portable iiRT-PCR for H7N9 virus detection. METHODS: A panel of 59 virus isolates, including H7N9, avian influenza viruses of subtype H1 to H13, swine and human influenza viruses, Newcastle disease virus, and infectious bursal disease virus, were tested by H7 and N9 iiRT-PCR reagents, using probes and primers specific to H7 or N9, in comparison with laboratory-based real-time RT-PCR assays to determine analytical sensitivity and specificity. Fifty oropharyngeal samples from experimentally infected chicken and ducks with H7N9 and 50 non-infected control swabs were tested by the H7 iiRT-PCR to determine diagnostic sensitivity and specificity. RESULTS: The H7 and N9 iiRT-PCR reagents yielded comparable levels of analytical sensitivity and specificity with real-time RT-PCR for the detection of H7N9 virus. Diagnostic sensitivity and specificity of H7 iiRT-PCR were 98% and 100%, respectively. CONCLUSION: The observed high sensitivity and specificity of iiRT-PCR for H7N9 detection show its potential for early detection of H7N9 in risk-based surveillance.

Characterizing Antimicrobial Use in the Livestock Sector in Three South East Asian Countries (Indonesia, Thailand, and Vietnam).

A framework was developed to characterize the antimicrobial use/antimicrobial resistance complex in livestock systems in Indonesia, Vietnam, and Thailand. Farm profitability, disease prevention, and mortality rate reduction were identified as drivers toward antimicrobial use in livestock systems. It revealed that antimicrobial use was high in all sectors studied, and that routine preventative use was of particular importance to broiler production systems. Misleading feed labeling was identified as a hurdle to the collection of accurate antimicrobial use data, with farmers being unaware of the antimicrobials contained in some commercial feed. Economic analysis found that the cost of antimicrobials was low relative to other farm inputs, and that farm profitability was precariously balanced. High disease and poor prices were identified as potential drivers toward economic loss. The research indicates that antimicrobial use in small-scale poultry production systems improves feed conversion ratios and overall productivity. However, data were limited to quantify adequately these potential gains and their impacts on the food supply. During the study, all countries embraced and implemented policies on better management of antimicrobial use in livestock and surveillance of antimicrobial resistance. Future policies need to consider farm-level economics and livestock food supply issues when developing further antimicrobial use interventions in the region.

A Tool for Assessment of Animal Health Laboratory Safety and Biosecurity: The Safety Module of the Food and Agriculture Organization's Laboratory Mapping Tool.

The Laboratory Management Tool (LMT) is a standardized spreadsheet-based assessment tool developed to help support national, regional, and global efforts to maintain an effective network of animal health and veterinary public health laboratories. The safety and biosecurity module of the LMT (LMT-S) includes 98 measures covering administrative, operational, engineering, and personal protective equipment practices used to provide laboratory safety and biosecurity. Performance aspects of laboratory infrastructure and technical compliance considered fundamental for ensuring that a laboratory is able to appropriately function in a safe and biosecure manner are systematically queried and scored for compliance on a four-point scale providing for a semi-quantitative assessment. Data collected is used to generate graphs and tables mapping levels of compliance with international standards and good practices, as well as for documenting progress over time. The LMT-S was employed by trained auditors in 34 laboratories located in 19 countries between 2015 and 2017. The tool is intended to help standardize animal health laboratory assessments, document compliance with recognized laboratory safety and biosecurity measures, serve as a self-help and training tool, and assist global laboratory development efforts by providing an accurate measurement of laboratory safety and biosecurity at local, national, and regional levels.