Volume 10 - 2023 | https://doi.org/10.3389/fvets.2023.1026296 Newcastle disease virus (NDV) infects a wide range of bird species worldwide and is of importance to the poultry industry Although certain virus genotypes are clearly associated with wild bird species the role of those species in the movement of viruses and the migratory routes they follow is still unclear we performed a phylogenetic analysis of nineteen NDV sequences that were identified among 21,924 samples collected from wild and synanthropic birds from different regions of Ukraine from 2006 to 2015 and compared them with isolates from other continents The fusion gene sequences of these strains were similar to strains detected in birds from different geographical regions of Europe and Asia it is noteworthy to mention the isolation of vaccine viruses from synanthropic birds suggesting the possibility of their role in viral transmission from vaccinated poultry to wild birds which may lead to the further spreading of vaccine viruses into other regions during wild bird migration here we present the first publicly available complete NDV F gene from a crow (genus Corvus) our phylogenetic results indicated a possible connection of Ukrainian NDV isolates with genotype XXI strains circulating in Kazakhstan NDVs of genotype 1 of class I and genotype I of class II were detected The phylogenetic analysis highlighted the possible exchange of these NDV strains between wild waterfowl from the Azov-Black Sea region of Ukraine and waterfowl from different continents studies performed at the major stopping point locations for migratory birds can be of great contribution toward gaining more knowledge on the potential host range of NDV therein Sampled regions are indicated in green color The number of collected samples are indicated in parenthesis under the name of each region Genotypes detected in this study are shown in red (genotype 1 Sampling from wild birds was carried out in cooperation with ornithologists Cloacal swabs were collected from apparently healthy live-trapped and hunted wild birds Fresh feces were collected from places of mass bird accumulation Feces were collected only if the origin and type of bird had been established samples of the biological material from wild birds were placed in tubes with a transport medium (Hank's balanced salt solution containing 0.5% lactalbumin and 50 U nystatin per ml) then stored and transported on ice to the laboratory and further stored in liquid nitrogen and intestines were collected from dead synanthropic birds (pigeons and gray crows) in Ukraine between 2006 and 2015 All dead synanthropic birds were found in locations of their natural habitat within cities in Kharkiv samples were thawed and suspended in transport media (10% w/v) Pathogenicity evaluation was performed on eleven selected NDV isolates (KF851268–KF851270, KJ914671, KJ914672, KU133362–KU133365, KY042127, KY042128, MZ101338) using the intracerebral pathogenicity index (ICPI) assay on 1-day-old SPF chickens following established procedures at the Southeast Poultry Research Laboratory (SEPRL), U.S. Department of Agriculture (USDA), Athens, GA, USA (3) All RT-PCR products were subjected to electrophoresis in a 1% agarose gel (0.5X TBE) The appropriately sized DNA bands were excised from the gel and purified using the QuickClean II Gel Extraction Kit (GenScript Nucleotide sequencing was performed for 19 samples on an ABI Sanger sequencer (Applied Biosystems USA) with fluorescent dideoxy-nucleotide terminators at SEPRL Sequence editing and assembly were performed using the SeqMan software of the LaserGene package (DNASTAR with branch lengths measured in the number of substitutions per site and all positions containing gaps and missing data were eliminated A total of 1,661 positions were included in the pilot analysis of the complete F gene dataset For each genotype detected (1 of class I and I more detailed phylogenetic trees were constructed using the most closely related sequences detected in BLAST The Roman numerals presented in the name of each sequence in the phylogenetic tree represent the respective sub-genotype The complete F gene data set used for the phylogenetic analysis was also used to estimate the average evolutionary distances comparing Ukrainian NDV isolates to other relative strains. Pair-wise analysis was conducted using the maximum composite likelihood model using MEGA7 software (56) The rate variation among sites was modeled with a gamma distribution (shape parameter = 4) The six isolates from wild birds all came from the Kherson region in the south of Ukraine Background information data for NDV isolates recovered in Ukraine between 2006 and 2015 The cleavage site motifs of these Ukrainian isolates were 112KRQKR↓117 (n = 5) and 112RRQKR↓F117 (n = 2) All seven viruses were isolated from synanthropic pigeons in the Dnipro The rest of the cleavage site motifs were represented by sequences 112GRQGR↓L117 (n = 6) and 112ERQGR↓L117 (n = 1) that are typical for viruses of low virulence All positions containing gaps and missing data were eliminated There were a total of 1,661 positions in the final data set The isolates used in this study are shown in colors The Roman numerals presented in the taxa names in the phylogenetic tree represent the respective sub-genotype for each isolate followed by the GenBank identification number The single NDV isolate of class I was collected from a mallard in AR Crimea in 2010 (KF851268) and belongs to sub-genotype 1.2 of class I (Figure 3), previously known as sub-genotype 1c (5, 58) The highest homology of this isolate is with the two strains—pochard in Finland from 2006 (EU493454) and duck in China from 2007 (JF893453) The single NDV genotype VI isolate was derived from a pigeon located in the Kharkiv region of Ukraine (MZ101342) and belongs to sub-genotype VI.2.1.1.2.2, formerly classified as sub-genotype VIk (Figure 5) (5, 58). The highest homology of this isolate is to a strain isolated from a pigeon in Belgium in 2011 (JX901124) (61) The single NDV genotype VII isolate was derived from a pigeon located in Simferopol, AR Crimea (KU710277) and belongs to sub-genotype VII.1.1, formerly classified as sub-genotype VIId (Figure 5) (5, 58). The highest homology of this isolate is to a strain isolated from a chicken in the Kharkiv region of Ukraine in 2003 (KU295454) (44) provide essential information on the epidemiology of the NDV isolates from synanthropic and wild birds in Ukraine we used Sanger sequencing to obtain complete F gene sequences of NDV in Ukraine from 2006 to 2015 Our study reports repeated NDV detection of sub-genotype I.2 in wild birds collected at the stopping points of migratory birds in Ukraine as well as the first occurrence of sub-genotype VI.2.1.1.2 and the continuous presence of sub-genotypes II and XXI.1.1 in Ukrainian synanthropic birds we present the first publicly available complete NDV F gene from a crow (genus Corvus) The Azov-Black Sea region of Ukraine is part of three transcontinental wild bird migration routes: the West Asia-East Africa, East Atlantic, and Black Sea-Mediterranean flyways (30, 31, 45) This region is comprised of areas for transit which makes it one of the highly important regions in Eurasia for monitoring and studying the global circulation of NDV and predicting the emergence of new strains possibly transmitted by wild birds The single class I NDV isolate was detected from wild waterfowl at a major stopping point location in AR Crimea and belonged to the 1.2 sub-genotype This virus clustered together with strains also isolated from wild waterfowl of the order Anseriformes (family Anatidae) in China and Finland The high identity between these isolates from Europe and Asia supports our hypothesis of intercontinental viral transmission by migratory birds The high identity between isolates from wild birds in Europe and Asia and their close phylogenetic relationship with strains from Africa support our hypothesis of virus exchange along the Black Sea-Mediterranean and Asian-East African migratory flyways and highlight the possibility of intercontinental viral transmission special attention must be paid to the distribution of vaccine strains in wild birds in order to understand the consequences of global vaccination This isolate weakly cross-reacted with APMV-1 and APMV-7 antisera in serology but we were unable to amplify NDV's F gene by utilizing the set of primers for routine NDV detection random whole-genome next-generation sequencing allowed us to assemble the complete genome and discover a new serotype of APMV (named APMV-13) which explained the previous inability to amplify the complete F gene using primers specific for NDV This highlights the need for implementing random next-generation sequencing as a routine diagnostic tool in order to better perceive the complete epidemiological situation To the best of our knowledge, only viruses of sub-genotypes II, VII.1.1, and XXI.1.1 have been previously reported to circulate in pigeons in Ukraine (23, 27, 44, 65) we report the first identification of genotype VI NDV in Ukraine was obtained from a deceased pigeon in the Kharkiv region in 2015 To obtain a complete picture of the distribution of NDV along the migratory flyways and to determine all circulating genotypes among wild waterfowl it is necessary to continue the annual monitoring of NDV in the Azov-Black Sea region as one of the major stopping points for migratory birds Additional data will help to assess the degree of involvement of wild birds in the spread of virulent strains that can be especially dangerous for poultry production Further monitoring of NDV in synanthropic bird species will provide useful data for the study of vaccine strains widespread globally The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/ The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the National Scientific Center Institute of Experimental and Clinical Veterinary Medicine Project administration and coordination: CA All authors have read and agreed to the published version of the manuscript The work with Ukrainian isolates was supported by the U.S Defense Threat Reduction Agency and by the USDA Part of the research was funded by USDA Project P444 through the Ukrainian Science and Technology Center Part of the research was done in the frame of the Joint Ukrainian-Austrian R&D Project (contract #M7-2021 We would like to acknowledge Timothy Olivier and Dawn Williams-Coplin for their technical assistance The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2023.1026296/full#supplementary-material Number of samples of biological material collected from wild birds of different species in different regions of Ukraine from 2006 to 2015 Number of samples of biological material collected from synanthropic birds of different species in different regions of Ukraine from 2006 to 2015 ICTV virus taxonomy profile: paramyxoviridae Newcastle Disease in Free-Living and Pet Birds PubMed Abstract | Google Scholar Newcastle Disease (Infection with Newcastle Disease Virus) Manual of Diagnostic Tests and Vaccinesfor Terrestrial Animals Geneva: World Organization for Animal Health (2021) Google Scholar Google Scholar Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus and host distribution of avian paramyxovirus 1 (Newcastle disease virus) Newcastle disease virus: detection and characterization by PCR of recent German isolates differing in pathogenicity Google Scholar Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus The spread of a pathogenic and an apathogenic strain of Newcastle disease virus in the chick embryo as depending on the protease sensitivity of the virus glycoproteins Two mutations in the HR2 region of Newcastle disease virus fusion protein with a cleavage motif “RRQRRL” are critical for fusogenic activity Structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of Newcastle disease virus A host range mutant of Newcastle disease virus with an altered cleavage site for proteolytic activation of the F protein Role of fusion protein cleavage site in the virulence of Newcastle disease virus Evolutionary dynamics of Newcastle disease virus Newcastle disease virus fusion protein is the major contributor to protective immunity of genotype-matched vaccine Effect of probe-site mismatches on detection of virulent Newcastle disease viruses using a fusion-gene real-time reverse transcription polymerase chain reaction test Evolutionary changes affecting rapid identification of 2008 Newcastle disease viruses isolated from double-crested cormorants Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge Development of an improved vaccine evaluation protocol to compare the efficacy of Newcastle disease vaccines Empirical analysis suggests continuous and homogeneous circulation of Newcastle disease virus in a wide range of wild bird species in Africa Avian Paramyxovirus Serotypes Circulating in Wild Bird Populations of the Azov-Black Sea Region of Ukraine in 2006–2011 Google Scholar Presence of vaccine-derived newcastle disease viruses in wild birds International biological engagement programs facilitate newcastle disease epidemiological studies Oncolytic effect of wild-type Newcastle disease virus isolates in cancer cell lines in vitro and in vivo on xenograft model Nearly complete genome sequence of a Newcastle disease virus strain isolated from a wild garganey Phylogenetic assessment reveals continuous evolution and circulation of pigeon-derived virulent avian avulaviruses 1 in Eastern Europe Characteristics of pigeon paramyxovirus serotype-1 isolates (PPMV-1) from the Russian Federation from 2001 to 2009 Wild bird surveillance for avian paramyxoviruses in the Azov-Black Sea regions of Ukraine (2006–2011) reveals epidemiological connections with Europe and Africa 30. 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Available online at: http://polj.uns.ac.rs/wp-content/uploads/arhiva-savremena-poljoprivreda/2009Savremenapoljoprivreda12.pdf Google Scholar Afonso CL and Muzyka D (2023) Genetic diversity of Newcastle disease viruses circulating in wild and synanthropic birds in Ukraine between 2006 and 2015 Received: 23 August 2022; Accepted: 02 January 2023; Published: 19 January 2023 Copyright © 2023 Goraichuk, Gerilovych, Bolotin, Solodiankin, Dimitrov, Rula, Muzyka, Mezinov, Stegniy, Kolesnyk, Pantin-Jackwood, Miller, Afonso and Muzyka. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Denys Muzyka, ZG11enlrYTc3QGdtYWlsLmNvbQ== Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Tesla shared on X that it’s ending its current referral program globally by the end of April but that is still “a few months away” When Musk was asked if the current credits would be lost, Musk responded by saying “No just that new credits would not be earned until we launch a future referral program.” That means there’ll be a gap of a couple of months when owners will not be able to earn referral credits Tesla was always famous for relying on its community to promote its vehicles rather than spending millions (or even billions) in ad campaigns Through Tesla’s referral program — which started back in 2015— customers have been earning referral credits by having friends use their referral link when ordering a Tesla product or taking their first demo drive. At the time it started with a direct $1,000 prize for the referrer but later evolved to a more complex system with different tiers and points Our current referral program benefits will end after April 30 in all markets Tesla’s first referral program was released in July 2015 where customers who referred new buyers would receive $1,000 for every new Tesla Model S delivered For those who were able to accrue 5 or 10 referrals for the US automaker Tesla raised the bar and offered invitations to the opening of its Giga Factories or even a fully loaded Model X Over the first 2 years, Tesla significantly increased the value of the prizes and even offered free or discounted Roadsters with enough referrals Elon Musk decided to stop amid cost concerns The referral program was re-launched in late 2019 offering new buyers free supercharging for a period of time Given the considerable gap of “a few months” between the current program and the next one it is expected that Tesla will bring a new structure with updated awards focused on minimizing Tesla’s cost while increasing the brand’s awareness Subscribe to our newsletter to stay up to date on the latest Tesla news There are a few features to keep in mind when taking your Tesla through a car wash, but Tesla’s Car Wash Mode makes it easy by enabling or disabling several features for you These settings don’t only prevent damage to your vehicle such as locking the charge port door so that it doesn’t automatically open when a cleaning brush touches it but they also improve the experience by recirculating the air in the vehicle to prevent cleaning chemical smells from coming in Car Wash Mode makes it easy by giving you a checklist of items and their real-time status When activated from Controls > Service > Car Wash Mode Locks the charge port door to prevent accidental opening Enables easy access to the Fold Mirrors and Free Roll options (you can also put your vehicle in neutral through the gear stalk) While Car Wash Mode monitors more than a handful of items it doesn’t continuously check the status of your windows the vehicle will automatically roll up any open windows it won’t alert you if a window has been lowered after Car Wash Mode was enabled This could spell disaster for your vehicle's interior demonstrating what happened to his friend and requesting that Tesla add open windows to the list of flags in Car Wash Mode noticed the post and decided to take action offering to pass on the suggestion to the vehicle software team While Troy’s response doesn’t confirm the feature being added in a future update it’ll at least be put in front of the software team to potentially address Tesla could add this window-specific solution in several ways Tesla could add this as another on-screen flag while the vehicle is in Car Wash Mode simply alerting the driver that there’s a window open they could also go one step further and lock the rear windows to prevent accidental opening while Car Wash Mode is enabled Tesla could present an on-screen button that pops up when a user tries to open a window while Car Wash Mode is activated We’re interested to see what Tesla would do here as these little quality-of-life changes really improve the end-user experience What else would you like to see added to Car Wash Mode Tesla offers a range of home energy products — from the widely used Wall Connector to the Powerwall and the innovative Solar Roof a unique residential development in Houston combines all of these technologies — and more — to move toward energy self-sufficiency located in Oaks of Shady Acres and built by Utopia Homes each designed using Tesla technology to be self-sufficient Utopia has equipped the homes with Solar Roofs and Wall Connectors to complete the entire ecosystem Tesla’s Solar Roof replaces traditional roofing materials while doubling as a clean energy source during daylight hours The best part is that it mimics the look of conventional shingles while improving durability and longevity Any excess energy generated is stored in the home’s Powerwall 3 units Tesla’s Universal Wall Connector which can charge any EV equipped with either a NACS or CCS port (through a J1772 adapter) If you added on a Cybertruck with Powershare (more vehicles will support Powershare in the future) you’d have a backup system that would last an extremely long time on batteries alone A Powerwall 3 stores about 13.5 kWh of energy while a Cybertruck has a battery pack of 123 kWh which is roughly equivalent to about nine Powerwalls the Cybertruck could be used as a “mobile battery pack,” which can get additional energy from Superchargers and bring it back to the home if there’s an extended power outage Utopia markets these homes with “100% energy security,” - but they’re still grid-connected they appear to have made quite a point with this - as many people in Texas with its notoriously unstable electricity grid - were excited to get into these homes.  so they’re not entirely green and disconnected This likely comes down to the fact that powering an induction range alongside a heat pump in the winter could draw more energy than Powerwall 3 is capable of outputting instantaneously These 11 homes attracted a lot of attention - according to a broker working on Utopia’s team they had requests to see or buy these homes coming from across the country these homes are about $150,000 higher than Houston’s median list pricing for similarly sized townhomes but the benefits are clear for many buyers who will recoup these additional costs over the home’s life Utopia has acknowledged the demand for Tesla-powered and future-proofed homes like these and is already planning to build more in the future This is an excellent showcase of what an electric-powered future could look like and we’re excited to see more of these types of homes and neighborhoods in the future There are a few features to keep in mind when taking your Tesla through a car wash, but Tesla’s Car Wash Mode makes … Tesla offers a range of home energy products — from the widely used Wall Connector to the Powerwall and the innovative… With most vehicles moving on to Tesla’s 2025.14 Spring Update, we’re now taking a look at all the undocumented featu… Tesla has issued a strong denial in response to a Wall Street Journal report (Paywall) which claimed that Tesla’s Boar… Tesla’s Megapack is rapidly becoming a key component in energy storage and grid modernization efforts worldwide. Thoug… Take a look at features that Elon Musk has said will be coming soon We may all be familiar with the need to reset our PCs or phones. They may freeze or just not operate properly sometimes.… A drop-down menu is added to the "About Your Tesla" menu, which allows one to choose any version of the car that has eve… The 2025 Q1 Earnings Call gave us the opportunity to learn about a lot of things, from Unsupervised FSD, to the Robotaxi… Tesla is heavily leaning into artificial intelligence, and its insurance offering is just another example of how it’s … Sentry Mode is an invaluable tool for owners - capable of keeping the vehicle safe and secure even when you’re not aro… Tesla’s Dan W Priestley attended the Advanced Clean Transportation (ACT) Expo in Anaheim, California, and provided an … Tesla’s Vice President of Vehicle Engineering, Lars Moravy, recently took to X and opened the floor for user input. Th… Sometimes, even with Tesla’s intensive bug-testing regime, bugs manage to make it out into the wild. In this particula… We encourage you to republish Dialogue Earth articles, online or in print, under the Creative Commons license. Please read our republishing guidelines to get started Weighing wood from Zambia’s miombo forests to see if it is dry enough for making charcoal. Demand for charcoal, the main heating and cooking fuel in much of sub-Saharan Africa, is driving deforestation. (Image: Gabriel Mulenga/CIFOR, CC BY-NC-ND 2.0) delegates from nine African countries convened in Maputo Mozambique to sign a transnational blueprint for sustainably managing Africa’s miombo woodlands The Maputo Declaration issued in August now looks to halt and address the threats to the biome “The declaration was drafted to deliver three key objectives,” said Claudio Afonso national director of forestry at Mozambique’s Ministry of Land and Environment to promote miombo forest management for climate resilience and community development; to protect miombo forests as a carbon sink to ensure emission reductions for the regional NDCs [nationally determined contributions to the Paris climate agreement]; and to conserve miombo forests to save the Zambezi Basin.” The nine countries to sign the declaration are Angola which will serve as the Miombo Initiative’s focal point South Africa is not home to miombo woodlands but is included because of its experience in forestry management Across central and southern Africa, miombo woodlands contribute to erosion reduction, climate and microclimate regulation, and mitigation of and adaptation to climate change through carbon sequestration. They are also responsible for maintaining the greater Zambezi Basin – one of the most important transboundary river basins in southern Africa which coincides with much of the miombo woodlands’ range A 2014 study on the significance of miombo woodlands detailed how they support biodiversity and contribute to the global carbon balance sequestering between 0.5 and 0.9 tonnes of carbon per hectare per year with increased sequestration in young miombo trees The study explained how miombo woodlands can increase resilience against the impacts of floods droughts and other extreme weather events exacerbated by climate change as they provide a habitat for popular – and often endangered – animal species Perhaps most importantly, the UN’s Food and Agricultural Organization estimates that miombo forests support the livelihoods of more than 100 million rural people and 50 million urban residents through their provision of various non-timber forest products where around 93% of the country’s total forest cover consists of miombo woodlands deforestation and degradation has reached an average of 400,000 hectares per year chief executive of the Tanzania Forest Services Agency told China Dialogue The critical significance of the miombo woodlands and their spread across national boundaries has moved the nine countries to embark on their joint conservation initiative Speaking at the signing of the Maputo Declaration, Mozambican president Filipe Jacinto Nyusi emphasised that miombo woodlands conservation is of “collective interest for the good of the people and the planet” “Climate change does not respect borders,” President Nyusi added The Southern Africa Development Community’s Protocol on Forestry notes that miombo is of immense value to communities and countries But much as the Miombo Initiative is regional it also aspires to play a part in broader continental and global environmental goals The stated aim of the initiative is “to leverage the capacities and alliances of the African continent to jointly and decisively use and manage miombo woodlands sustainably to achieve the objectives of the 2030 Agenda for Sustainable Development… the African Union Agenda 2063 the nationally determined contributions (NDCs) and those called for in the 2014 New York Declaration on Forests at the Climate Summit.” Afonso said the implementation of the initiative would be a secure and assured way for the southern Africa region to work towards achieving the climate mitigation and adaptation goals proposed in the African Union’s (AU’s) new Climate Change Strategy and Agenda 2063 “The two AU documents place emphasis on a number of issues that have been adequately addressed in the Maputo Declaration,” Afonso said He highlighted its mention of issues such as modern agricultural practices to increase productivity; sustainable natural resource management and biodiversity conservation; the building of environmentally sustainable and climate-resilient economies and communities; and the promotion of renewable energy use Among the targets of the initiative are expanding emissions reduction schemes via the REDD+ programme; diversifying income sources from timber and non-timber forest products; reducing illegal trade in flora and fauna products; and promoting programmes of education and knowledge sharing on forest management There are as yet few numerical or measurable targets laid out under the Maputo Declaration nor much clarity on funding for implementation these will be determined in an action plan set by a new regional commission for sustainable miombo woodland management to be released and approved within a year of the declaration’s adoption On the improvement of technologies to aid and enhance forest management Afonso said this will be achieved through research and outreach from regional universities and research institutions He highlighted the role South Africa can play in this field as the most technologically advanced country in the region Some of the main actions ahead for the initiative include the establishment of the new regional commission and the approval of its strategy for the implementation of the Maputo Declaration Afonso said the main goals stipulated in the declaration will be evaluated in a midterm review in 2027 and again as the initiative’s initial 10-year phase reaches its end in 2032 Afonso told China Dialogue that interim coordination of the initiative is being led by the President of Mozambique and assisted by the ministers of environmental affairs of the member countries until the regional commission is founded and functioning The forestry director was optimistic on the broader backing the initiative will receive: “There will be mobilisation of technical and financial support for the implementation of the initiative as part of Africa’s climate action effort.”  a doctoral candidate in social-ecological systems and development practice at the University of Zimbabwe highlighted funding as a potential major challenge for the initiative He said substantial funds will be needed for member countries to effectively monitor and control the exploitation of resources in miombo woodlands “This is a long-standing problem not only in southern Africa,” Mataruse told China Dialogue, citing the UN Convention on Biological Diversity’s failure to establish a dedicated financing mechanism for biodiversity protection in Montreal last year Afonso, however, is optimistic that in the next three to five years, the Miombo Initiative will raise the necessary funds through investment projects financing (IPF) in cooperation with partners such as the World Bank “The timeframe for fundraising is for the next three years The initiative’s first phase is from 2022 to 2032 with a possible extension for another 10 years from 2032.” Another major challenge will be to ensure communities’ dependency on the woodlands can be sustainable “There is high dependence on miombo forests,” said Dr Robertson Khataza head of the Forestry Department at Lilongwe University of Agriculture and Natural Resources in Malawi “More than 70% of the population in the region rely on wood fuel and charcoal as the primary household energy source for cooking and heating.” A critical path to establishing sustainable use and conservation of the wooldands is therefore by providing diversified means of living and income generation to the woodlands’ most dependent local communities “A range of anti-poverty and economic empowerment programmes such as improved agricultural technologies to enhance productivity and alternative energy options should be promoted among forest-dependent communities,” he said “This would require more partners and stakeholders and stronger partnerships to make conservation financing a reality.” Dr Khataza also proposes that the initiative should enhance environmentally friendly conditional cash transfers to tackle poverty and build household resilience to shocks: “Social cash transfer programmes or safety nets should be conditional on conservation practices such as tree planting and care to stimulate positive attitudes towards sound environmental stewardship.” Professor Silayo of the Tanzanian forestry agency noted that member countries’ commitment to the initiative may vary based on their respective national priorities Mataruse similarly highlighted that differences in terms of interests and priorities between member states could derail the initiative’s progress He emphasised that the processes put in place by the regional committee and the strategy for implementing the Maputo Declaration will be important for keeping member states‘ commitment on track “Above all, I would reiterate the [importance of] proper implementation of co-management arrangements between member states and local communities,” Mataruse said. Temwani Mgunda is East Africa editor at Dialogue Earth He previously worked as an editor for Malawi News and as lecturer in journalism and media studies at University of Malawi He has an MA in international communication from the Communication University of China Temwani’s interests are in how policies and community involvement advance environmental and food security issues Select from our bespoke newsletters for news best suited to you We’ve sent you an email with a confirmation link 我们向您的邮箱发送了一封确认邮件，请点击邮件中的确认链接。如果您未收到该邮件，请查看垃圾邮件。 If you would like more information about the terms of our republication policy or permission to use content, please write to us: [email protected] We use cookies to ensure that we give you the best experience on our website. By continuing to use our site, you are agreeing to our use of cookies. 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highly-repetitive genomic regions and genomic arrangement and to close the sequences of bacterial chromosomes and plasmids we report the use of long-read nanopore sequencing to simultaneously sequence the entire chromosome and plasmid of Salmonella enterica subsp enterica serovar Bareilly and Escherichia coli O157:H7 We developed a rapid and random sequencing approach coupled with de novo genome assembly within a customized data analysis workflow that uses publicly-available tools we obtained full-length genomes with an average identity of 99.87% for Salmonella Bareilly and 99.89% for E coli in comparison to the respective MiSeq references These nanopore-only assemblies provided readily available information on serotype We also demonstrate the potential of nanopore sequencing assemblies for rapid preliminary phylogenetic inference Nanopore sequencing provides additional advantages as very low capital investment and footprint and shorter (10 hours library preparation and sequencing) turnaround time compared to other NGS technologies there is a need for an approach that generates inexpensive Such approach will offer benefits for rapid detection of an organism complete sequencing of bacterial chromosomes and plasmids and complementation to other sequencing technologies used in both outbreak investigations and foodborne pathogen surveillance we aimed to simultaneously sequence and assemble complete genomes of two pathogenic bacterial strains that can cause human illness worldwide we aimed to develop an improved bioinformatics workflow that provides accurate assemblies and to determine whether shorter sequencing time would still provide reliable results we report a reproducible bioinformatics workflow which assembled the circularized bacterial genomes and associated plasmids with the lowest error rate reported to date We also demonstrate that utilizing the proposed sequencing and bioinformatics approach sequencing of the entire chromosome and plasmid can be achieved with significantly shortened run time This study shows that long-read nanopore sequencing can be used as a low-cost method to sequence the whole microbial genomes of foodborne pathogens These closed assemblies provide information on genome organization and can complement existing characterization data from other technologies such as short-read sequencing The Salmonella Bareilly isolate (CFSAN000189) was isolated from raw shrimp in India (Biosample SAMN04364135) coli O157:H7 isolate (FSIS11705876) was isolated from domestic Department of Agriculture Food Safety and Inspection Services (USDA-FSIS) as part of routine sampling of a U.S Both bacterial isolates were grown on sheep blood agar (SBA) for 24 hours at 35 °C Total DNA from each isolate was extracted using the DNeasy Blood and Tissue Kit (Qiagen USA) following manufacturer’s instructions DNA concentrations throughout the experiment were determined by using the Qubit® dsDNA HS Assay Kit on a Qubit® fluorometer 3.0 (Thermo Fisher Scientific final genome sequences were submitted to GenBank to be processed through the NCBI Prokaryotic Genomic Annotation Pipeline (PGAP) before being released The second tree was constructed by replacing the reference Illumina data of the CFSAN000189 strain with the MinION-only data obtained by sequencing the same strain in this study (240 and 1500 mins time points were used) A third tree that contained both the Illumina and the MinION-only data of the CFSAN000189 strain was also built for comparison The full NextFlow workflow, Conda environment configuration, and other associated code used in the analyses are publicly-available on GitHub (https://github.com/jvolkening/minion_bacterial) while the longest read was from Salmonella Bareilly and was 129,119 nt Both sets of MinION data had a mean read quality score above the standard (Q ≥ 10) The minimum read length from both sets of bacterial sequences was 35 nt while the longest was 151 nt for Salmonella Bareilly and 251 nt for E coli; the MiSeq mean read quality was above the Q30 benchmark the following analyses herein refer to the data collected in the first four hours of sequencing Polishing Results of the MinION-only Assemblies Using Multiple Rounds of Nanopolish Due to the errors remaining in the MinION-only assemblies was used to increase the assembly accuracy the Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness and number of SNPs per kb are shown after 0 the overall accuracy and the number of Indels and SNPs per kb did not considerably change after two rounds of polishing with Nanopolish were annotated using Geneious and the most closely related annotated genomes for each bacterial species Since the Salmonella Bareilly genome was already completed and closed by a hybrid Illumina/PacBio approach and published we confirmed that the Geneious genome annotation of the sequence of the same bacterium produced by MinION was accurately reconstructed (loci of protein-coding genes) by using the PGAP annotations tool on the final the two major serotyping antigens located on the chromosome: the flagellin FliC CDS and the O-antigen polymerase Annotation of the MinION assembly of Escherichia coli coli O157:H7 chromosome was sequenced and assembled into a final consensus of 5,482,542 nucleotides The annotation of the genome provided the location of 5,748 coding sequences (CDS) the one repeat region (brown) and the CDS of two virulence factors (yellow) are shown magnified The LEE (locus of enterocyte effacement) is highlighted at position 4,603,699 to 4,636,299 and the Shiga Toxin subunits are shown at position 3,181,004 to 3,180,992 for demonstration purposes coli pO157 plasmid was sequenced and assembled into a final consensus of 94,503 nucleotides The annotation shows all 124 coding sequences (CDS) in yellow The CDS of three well-known virulence factors are highlighted: hemolysin (ehx) at position 16,584 to 19,578 catalase-peroxidase (katP) at position 76,704 to 78,356 and the type II secretion system (T2SS) at position 64,056 to 85,694 for demonstration purposes One of the main objectives of the presented work is to determine if MinION alone can be utilized to obtain fully closed genomes and plasmids from important foodborne pathogens for submission of final sequences to GenBank the most accurate assemblies attainable were used assemblies produced using the full run length were utilized and further error-corrected using Pilon Pilon utilizes the low error rate of Illumina reads mapped to the draft assembly to drastically improve the local accuracy of the final sequence The error rate for both samples after Pilon polishing decreased and BUSCO completeness rates of 99.7% and 99.99% for Salmonella and E.coli There were also a reduction in SNPs per kb to 0.002 and 0.001 and indels per kb to 0.008 and 0.002 for Salmonella and E and short-read error-corrected data from the full 25-hour run were the final assemblies annotated and submitted to GenBank (Accession numbers CP034177- CP034178 and CP035545-CP035546 along with other associated code used in the analyses Based on the results of barcoding and simultaneous sequencing of two whole bacterial genomes and plasmids shown here we estimate that six bacterial samples could be multiplexed together to further decrease cost and sequenced in approximately 16 hours to obtain complete genomic data with high accuracy The effects of increased sequencing run lengths different criteria and weights to subsample data for assembly were examined for their effect on the final assembly completeness and accuracy Filtlong subsampling is not random but keeps the longest and highest quality reads from the input which targets maximum sequencing depth (total bases) It was observed that the nanopore reads were long enough on average that over-aggressive length-based filtering resulted in reduced representation Such extensive subsampling would result in less complete assembly of small plasmids which can contain virulence factors of great interest for diagnostic and food safety purposes It therefore proved critical to evaluate filtering and subsampling criteria to take full advantage of the technology Read quality was weighted more heavily than length as testing showed this was necessary to retain sufficient coverage of small plasmids this value was observed to be greater than 20% of expected coding genes which must be taken into consideration during annotation with only 0.1% error are accurately reconstructed and reveal serotype and important genes responsible for the virulence random-priming nanopore sequencing approach provides sufficient data where complete genomes can be assembled into a single contiguous sequence with 99.89% accuracy (highest reported-to-date) These data allowed accurate gene identification and genomic organization without the need for additional sequencing tools to close gaps that are required by other sequencing methods As the nanopore chemistry and bioinformatics continue to 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Journal of clinical microbiology 55, 3530–3543, https://doi.org/10.1128/JCM.01069-17 (2017) Download references Ruth Timme and James Pettengill with the FDA Center for Food Safety and Applied Nutrition for the Salmonella Bareilly isolate used in this manuscript The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S The USDA is an equal opportunity provider and employer Present address: Office of Public Health Services Present address: Texas A&M Veterinary Medical Diagnostic Lab Exotic and Emerging Avian Viral Diseases Research Unit The authors declare no competing interests Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-019-52424-x Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. 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Volume 9 - 2022 | https://doi.org/10.3389/fvets.2022.931272 New variants of infectious bronchitis viruses (IBVs; Coronaviridae) continuously emerge despite routine vaccinations we report genome sequence variations of IBVs identified by random non-targeted next generation sequencing (NGS) of vaccine and field samples collected on FTA cards from commercial flocks in Mexico in 2019–2021 Paired-ended sequencing libraries prepared from rRNA-depleted RNAs were sequenced using Illumina MiSeq IBV RNA was detected in 60.07% (n = 167) of the analyzed samples from which 33 complete genome sequences were de novo assembled The genomes are organized as 5'UTR-[Rep1a-Rep1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b]-3'UTR except in eight sequences lacking non-structural protein genes (accessory genes) 4b Seventeen sequences have auxiliary S2' cleavage site located 153 residues downstream the canonically conserved primary furin-specific S1/S2 cleavage site The sequences distinctly cluster into lineages GI-1 (Mass-type; n = 8) and GI-17 (California variant; CAV; n = 1) with regional distribution in Mexico; this is the first report of the presence of 793B- and CAV-like strains in the country insertions and deletions are present in the S1 hypervariable regions (HVRs I-III) across all 5 lineages and 69 that are critical in viral attachment to respiratory tract tissues Nine intra-/inter-lineage recombination events are present in the S proteins of three Mass-type sequences two each of Holte/Iowa-97 and Ark-like sequence and one each of 793B-like and CAV-like sequences This study demonstrates the feasibility of FTA cards as an attractive adoptable low-cost sampling option for untargeted discovery of avian viral agents in field-collected clinical samples our data points to co-circulation of multiple distinct IBVs in Mexican commercial flocks underscoring the need for active surveillance and a review of IBV vaccines currently used in Mexico and the larger Latin America region The apparent regionality of IBV diversity underscores the importance of viral characterization which can be used to assess and properly deploy existing vaccines and potentially identify when new vaccines need to be developed we sequenced 30 complete IBV genome sequences from clinical field samples collected from commercial flocks in Northern Central and Southern Mexico in 2019–2021 We performed comparative analysis of sequence variation (vaccine vs field sequences) and phylogenetic relationships with other IBVs and assessed potential recombination events and deletions in the HVRs of the S glycoprotein The data presented here expand current knowledge of the IBVs circulating in Mexico which can inform vaccination strategies to control IB outbreaks in the country and in Latin America The samples used in this study were randomly collected from “apparently healthy” (i.e. no observable clinical signs of disease at the time of sampling) commercial broiler (aged 21–42 days) and layer (aged 7 weeks) chicken flocks in central and southern regions of Mexico between April 2019 and December 2021 The samples were derived from respiratory (choana and lung) immunological (spleen and bursa) and digestive (cloaca) tissues from 100 birds per flock using sterile flocked swabs and pooled (25 samples per pool) in sterile 1.5 mL viral transport media Samples were then spotted on 4-sample-area (125 μl per area) Whatman Flinders Technology Associates (FTA) cards® (Millipore-Sigma) within 24 h of collection and dried for at least 2 h at room temperature (RT; 15–25°C) each sample-spotted FTA card was individually enclosed in double leak-proof zip-lock plastic bags with Whatman desiccant packets (GE healthcare) 4/91 variant and Mass-type/Connecticut recombinant strains) from Boehringer Ingelheim Animal Health (BIAH) The detailed information about what flocks were vaccinated and with what type of vaccine is not publicly available because of propriety and confidentiality between BIAH and their clients All samples were shipped to the Southeast Poultry Research Laboratory (SEPRL) and stored at −80°C in a BSL-3 laboratory until further processing Sequencing libraries were prepared using sequence-independent, single-primer amplification (SISPA) as previously described (39) cDNAs were synthesized from 10 μL of RNA using random K-8N primer with SuperScript TM IV First Strand synthesis Kit (Invitrogen and then purified using Agencourt AMPure XP beads (Beckman Coulter Life Sciences Purified cDNAs were amplified by Phusion® High-Fidelity PCR Kit (NEB Inc. and used to prepare sequencing libraries by the Nextera TM DNA Flex kit (Illumina which were then quantified by Qubit™ dsDNA HS Assay Kit (Thermo-Fisher Scientific) and Agilent 4150 TapeStation HS D5000 System (Agilent Technologies Based on their concentrations and average fragment sizes 8 μL of each library) of the libraries were pooled then digested by incubation with 0.2 N NaOH (5 min at RT) Pooled libraries were further diluted to 10 pM final concentration a control library added (5% PhiX library v 3) and paired-end (2 × 300 bp) sequencing performed using a 600-cycle MiSeq Reagent Kit v3 (Illumina Each NGS run consisted of 48 multiplexed samples BWA-MEM/samtools was used to re-call consensus sequences from the NGS reads aligned to the de novo-generated contigs (minimum coverage depth to call a base set at 3X) at highest p-value of 0.05 with Bonferroni multiple correction and SEQ-GEN parametric data simulations Confirmation of putative recombinant event was accepted only when the recombination breakpoints were detected by at least five of the seven algorithms and with breakpoints of the transferred fragments (recombinant regions) supported by corrected p-values of ≤1 × 10–6 Total trimmed/filtered read counts ranged from 16,541 to 1.3 million reads Proportions of chicken-specific reads ranged from 0.82 to 77.8% with only six samples having over 50% of the reads mapping to the host genome IBV RNA was detected in 60.07% (n = 167) of the analyzed samples. Fifty-five of the samples (20 immunological tissue samples, 32 respiratory tissue samples, and the 3 vaccine samples) had enough IBV-specific reads to allow for assembly of complete or nearly-complete genome or S-gene consensus sequences (Supplementary Table 1) 10 spleen/bursa and nine choanal/lung tissues contained RNA of avian viruses belonging to families Astroviridae (chicken astrovirus Birnaviridae (infectious bursal disease virus Paramyxoviridae (avian paramyxovirus type 1 Pneumoviridae (avian metapneumovirus subtype A) were overrepresented (detected together with IBV in 73.68% of the 19 samples) avian pathogenic bacterial species were detected in 29 out of the 55 samples Sequence assembly coverage of 33 complete genome sequences assembled in this study All 33 complete sequences, with lengths ranging from 27,022 to 27,805 nt excluding poly(A) tails, contain the six IBV genes flanked by 5′- and 3′-UTRs (294–643 nt and 136–446 nt in length, respectively); 14 of the sequences, including the 3 vaccine sequences, have poly(A) tails of variable lengths (Supplementary Table 2) with 25 sequences having a cassette of seven “accessory” genes (3a and 6b) interspersed variably downstream of gene 2 (S) genomic region and 6b are absent in eight sequences as follows: both genes 4b and 4c are absent in field sequence 2360/20 from Southern Mexico 4b is absent in sequences 2359/20 and 2754/21 (from Southern Mexico) and 2723/21 (from Northern Mexico) and 6b is absent in the Mass-type vaccine strain 1616/19 and Mass-type/Conn recombinant vaccine strain 1623/19 sequences and the field sequences 2523/21 and 2598/21 from Central and Northern Mexico cleavage sites and lengths of the accessory genes in Rep1a/1ab are conserved across all the sequences including amino acid residues Q/S required by the 3CL pro for the cleavage of Rep1ab into Rep1a and 1ab which releases products of genes10 (exonuclease; 145 amino acids) Although the cleavage sites of PL pro are conserved in all sequenced their length varies (1,529–1,619 amino acids) compared to the consistent lengths of the main CoV protease Nucleotide overlaps between genes and ORFs in the 33 IBV sequences analyzed in this study respectively) vary among the 33 sequences; HVR I of 27 or 28 residues (in 11 and 22 sequences HVR II of 25 or 27 residues (in 8 and 25 sequences and HVR III of 18 residues (in all sequences) Figure 1. Schematic and amino acid alignment of the overall features of the S glycoprotein. The scheme was constructed in Geneious Prime based on the S protein sequence of the field sequence 2602/21; all 33 S protein sequences in this study have similar general features (see Supplementary Table 4) Sequence IDs are shown on the alignments; the positions indicated at the top of the sequence alignments are in reference to ungapped amino acid residues; dots and dashes indicate identical and deleted amino acid residues Features of subunit S1 include N-terminal signal peptide (SP) which harbor the receptor-binding domains (RBD) Shown is a 20- residue (running from positions P14–P6') motif furin S1/S2 cleavage site consisting of a core region (8-residues; position P6–P2') which harbors the canonical 4-residue motif (Rx[K/R]-R↓S); the core region is flanked by two solvent accessible regions (8-residue; P7 to P14 Note the backward and forward numbering of P1-P14 and P1'-P6' starting at the conserved R immediately upstream of the cleavage S1/S2 site The auxiliary S2′ (PS(G)SPRxR↓S) cleavage site positioned 153-residues downstream the primary S1/S2 cleavage site is also shown Subunit S2 domains include fusion peptide (FP) Purple vertical bars represent predicted N-linked glycosylation sites 4b and 4c overlap by 80 nt; the two 2 accessory genes overlap by 17 and 95 nt in sequences 2731/21 and 2743/21 In all the sequences, gene 4 and gene 5 (5a-5b) overlap by 17 nt; 5a and 5b are 198 and 249 nt in length, respectively, all overlapping by 4-nt (see Table 2 and Supplementary Table 2). The presence and apparent conservation of 5a and 5b across all the 33 sequences in this study agree with reports that all avian CoVs contain gene 5, whose protein products are postulated to contribute to virus/host interactions (64) Genes 5 and 6 (N and 6b) overlap by 58 nt in 29 of the 33 sequences (6b is absent in the Mass-type vaccine 1616/19 and Mass-type/Conn recombinant vaccine 1623/19 sequences, and field sequences 2523/21 and 2598/21) (Table 2). Lengths of VncRNAs between N and 6b varies among the sequences; 8 nt (n = 16 sequences), 23 nt (n = 11 sequences), 17 nt (in sequence 2960/21), and 27 nt (in sequence 2725/21)—Supplementary Table 2 all N gene sequences are of the same length (1,230 nt) the lengths of 6b varies widely from 129 to 321 nt amongst the sequences Supplementary Table 5 presents the results of comparative pairwise nt sequence identities of vaccine vs the highest identity (100%) is between gene 3 (3a and 3b) the Mass-type vaccine 1616/19 and Mass-type/Conn recombinant vaccine 1623/19 sequences and the lowest (77.31%) between subunit S1-gene of sequences of the Mass-type/Conn recombinant vaccine 1623/19 and the 4/91 vaccine variant 1619/19 Comparing the vaccine vs. field sequences, the highest identities (99.43–100%) are between genes 3 and 4, and 5a and 5b of field sequence 2598/21 and their homologs in the vaccine sequences. The lowest identity is between 6b (60%) and the 3'-UTR (64.1%) of the field sequences 2723/21 and 2360/20, respectively, and their homologous genomic regions in the 4/91 vaccine variant 1619/19 sequence. For the S1-gene sequence, which is used for IBV classification (18) the highest identity (98.14%) is between field sequence 2860/21 and the Mass-type vaccine 1616/19 sequence while lowest (75.78%) is between the field sequence 2944/21 and the 4/91 vaccine variant 1619/19 sequence The two field sequences also showed similar identities in their complete S-gene sequences (highest and lowest identities of 98.15 and 80.15% the most conserved genomic regions amongst the 33 sequences are Rep1ab (88.5–93.24% nt identity) and 5b (93.98–100% nt identity) while the least conserved region are 6b (60–94.22% nt identity) and 4c (74.27–100% nt identity) Relationships between the 33 sequences in this study with other IBVs are summarized in Table 3 (complete S-gene sequences) and Supplementary Table 6 (complete genome sequence). Phylogenetic trees based on nt sequences of complete S-gene, S1-gene, and HVRs I-III classified the 33 sequences in this study within five different lineages (Figure 2) BLASTn results of the 33 complete S-gene sequences in this study Maximum likelihood phylogenetic tree of nt sequences of subunit S1-gene hypervariable regions I-III (HVRs I-III) of the S1-gene and complete S-gene using GTR model in MEGA 6 The 33 sequences in this study (3 vaccine sequences highlighted in blue; 30 field sequences; color-coded based on sampling regions in Mexico) clustered with serotypes in 5 lineages of IBVs lineages GI-1 (Mass-type serotypes; n = 8 sequences) GI-3 (Holte/Iowa/97 serotypes; n = 2 sequences) GI-9 (Ark-like serotypes; n = 8 sequences) GI-13 (793B also known as 4/91 serotypes; n = 14 sequences) All positions with <95% site coverage were eliminated and 3,431 positions for the complete S1-gene Recombination events in the complete S-gene sequences in this study We also performed phylogenetic analyses based on complete genome and Rep1ab (Figure 3), structural genes E, M and N (Supplementary Figure 1), and 3ab, 5a/b, and 6b (Supplementary Figure 2). Notably, none of the 33 sequences cluster with lineage GI-11 (SAI serotypes), which have been described as serologically and phylogenetically unique to South America (29–31) the previously described Mexican SAI viruses group in distinct subclades within larger clades containing Mass-type (based on nt sequences of the complete genome Rep1b and 3a/b) and 793B (based on nt sequences of the complete S-gene Maximum likelihood phylogenetic tree nt sequences of complete genome The 3 vaccine sequences are highlighted in blue color; the 30 field sequences are color-coded based on sampling regions in Mexico The final dataset had 26,895 (complete genome) Alignment of translated S1 HVR I (residues 37–88), HVR II (residues 115–146) and HVR III (residues 282–301) sequences revealed considerable variations among the 33 IBVs (Figure 4) HVR I. Six residues in HVR I of the Mass-type prototype AY851295/M41 (i.e., N38, H43, S56, P63, I66, and T69) are critical for viral attachment to respiratory tract tissues (14) field sequence 2598/21 from Northern Mexico and the Mass-type vaccine 1616/19 and Mass-type/Conn recombinant vaccine 1623/19 sequences have an asparagine in position 38 (N38) similar to the M41 sequence while all five Mass-type sequences from Southern Mexico have an aspartic acid at position 38 (D38) similar to the Brazilian Ma5 vaccine The consensus histidine at position 43 (H43) is conserved across all 33 sequences except in the 4/91 vaccine variant 1619/19 sequence (lineage GI-13) which has a substitution of histidine with tyrosine (H43Y) similar to the CR88 strain from UK Serine and threonine residues at positions 56 (S56 and T56) are conserved amongst the 793B-like and Mass-type sequences the critical amino acid at position 63 (S63) is conserved in the sequences across the five IBV lineages used in the alignment except in field sequences 2731/21 and 2960/21 (from Central Mexico) and sequence 2944/21 (from Northern Mexico) and sequence 2598/21 (from Northern Mexico) with an S63R substitution Position 66 is largely conserved across sequences in all 5 lineages while position 69 appear to be lineage-specifically conserved except in the Mass-type vaccine 1616/19 and Mass-type/Conn recombinant vaccine 1623/19 sequences which have T69 compared the six field sequences from Southern (n = 5) and Northern (n = 1) Mexico in the Mass-type group One is in field sequences 2731/21 and 2960/21 from Central Mexico (in lineage GI-3) with a 2-residue deletion (positions 61–62) which is also present in the Mexican BL-56 strain Another instance is a 4-residue deletion (positions 57–60) in the field sequence 2944/21 from Northern Mexico (lineage GI-17) which is also present in the DMV strains MK878536/ck/GA9977/19 and MN512438/ck/Can/18-049707/17 The third is a 3-residue deletion (positions 60 to 62) in field Mass-type sequence 2598/21 from Northern Mexico which is present in strain QKE31017/T&T/18RS1461-8/14) and three Conn vaccine strains from USA HVR II. Most amino acid variations in HVR II are between the positions 116–121 and 138–147 (Figure 4) all the six field sequences from Northern Mexico and the 4/91 vaccine variant 1619/19 sequence have N117 (similar to CR88 strains from UK and a field variant from Ireland) while all six field sequences from Southern Mexico have S117 (similarly present in a pathogenic 4/91 strain from UK and two other Chinese strains) The Mass-type vaccine 1616/19 and Mass-type/Conn recombinant vaccine 1623/19 sequences and all the field sequences in lineage GI-1 (one sequence from northern and five from Southern Mexico) have a 2-residue deletion between positions 117 and 120 some of the field sequences from Northern (n = 5) and Central (n = 2) Mexico have a G145R substitution also present in the Canadian and USA DMV-like strains All eight field Ark-like sequences (four each from Central and Southern Mexico) and the CAV-like sequence 2944/21 from Northern Mexico have a single residue insertion between positions 142 and 143 One of the variations in the HVR III is in the Ark-like sequences where all eight field sequences have E284V substitution compared to the USA pathogenic field strain AYA44731/US/Ark99 This substitution is also present in the CAV viruses including in field sequence 2944/21 from Northern Mexico Another variation is in the 4/91 vaccine variant 1619/19 sequence and the Holte/Iowa-97-like field sequence 2731/21 from Central Mexico which have a single amino acid (alanine) insertion at position 296 which is also present in the CR88 strains AJ618986/UK/FR-88061-88 and MN548285/ck/UK/CR88/11 One of the recombination events (involving the 4/91 vaccine variant 1619/19 sequence) has field sequence 2833/21 as “major parent” and an unknown “minor” parent inferred to be a 1996 Mass-type vaccine strain (FJ904716/US/Conn46 vaccine/96) the nt identities between the recombinant region and the “parental” sequences is low (96–96.8%) which probably imply the recombinant regions could have accumulated further mutations or that the recombination event happened long before the isolation and eventual divergence of the recombinant and parental viruses the phenomenon remains to be convincingly demonstrated Studies have demonstrated the versatility of FTA cards as a low-cost option for collection, storage, transportation and preservation of genetic materials from field samples for the surveillance of various viral agents (71–74) despite the weakness of FTA cards in yielding lower quantities of mostly fragmented RNAs resulting data is of sufficiently high quality to allow the assembly of full-length viral genomes even when the clinical samples are prepared under field conditions This opens up the advantages of FTA cards to NGS-based diagnostics of avian viruses via direct RNA sequencing from field-collected samples without the need of passage in eggs or transportation in liquid media Avoiding egg-passage is advantageous for several reasons including biosafety issues (there is no live virus manipulation and the absence of selective virus amplification (passage may select variants with a minority representation in the host High abundance of host/bacterial RNAs in samples prepared under field conditions, which, from our experiences and those of others, can constitute over 95% of NGS reads (38) results in low sensitivity in detection of viral RNAs our optimized protocols increased NGS sensitivity (high levels of virus-specific reads vs low levels of host-/bacteria-specific reads) which produced complete genome and/or S-gene sequences (with optimal read coverage depth and coverage) Some of the assembled genome sequences were missing nt at the 5′- and/or 3′- termini but all coding regions had sufficient depth coverage (≥10X) within single NGS runs (48 multiplexed samples per run) we obtained complete and/partial genome/gene sequences of various RNA viruses belonging to six taxonomic families it remains unclear why the S2' cleavage site is absent in the sequences belonging to lineages GI-3 (n = 2 sequences) and GI-13 (n = 14 sequences) None of the 33 sequences in this study clustered with indigenous SAI viruses based on the S-/S1 and HVRs I-III trees based on complete genome and other specific genomic regions the SAI viruses clustered in distinct subclades within larger clades containing strains from other lineages coupled with the clustering observed from S-/S1 and HVRs I-III trees could be interpreted to imply decreased prevalence of the SAI viruses Whether these changes in the S1 spike result in subsequent change in host cell binding or evasion of neutralizing antibodies remains to be determined This study has demonstrated FTA cards as adoptable low-cost option for untargeted discovery and full-length sequencing of avian viruses from field-collected clinical samples from various tissues Our data demonstrates that multiple distinct IBV serotypes/strains are co-circulating in Mexican commercial chickens with the high likelihood of intra- and inter-lineage recombination as well as indels and point mutations which are potentially driving the generation of new subpopulations of field variants capable spreading and adapting to chicken populations in the country It should be further investigated whether strains emerging from the commercial enterprises may have spilled over to backyard poultry and whether they have further evolved into strains that are distantly related to the predominantly SAI strains our data reiterates the need for enhanced surveillance of IBVs in Mexico and the Latin America region as well as a review of the vaccines currently used in the control of IBVs and ED: project administration and coordination HK: methodology and writing—original draft and CA: writing—review and editing All authors read and agreed to the published version of the manuscript This research was funded by Agricultural Research Service (ARS) and Oak Ridge Institute for Science and Education (ORISE) postdoctoral appointment The funders had no role in data analyses or interpretation The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest The authors acknowledge Dawn Williams-Coplin and the field sampling teams (BIAH) for technical assistance in sample collection and preparation The authors also acknowledge the contributions made by the BIAH field sample collection team (Alberto Carrillo The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2022.931272/full#supplementary-material Background information and sequencing data of the 55 samples in which IBV RNA was detected in this study The quantification of the dsDNA (HiFi PCR products) and the IBV-specific reads obtained from the NGS data are shown and partial S-gene sequences (n = 11) were assembled from NGS reads in the current study Other identified microbial agents (viral and bacterial) that are of avian interest based on literature and expert knowledge are listed in the table coding regions and nt lengths of the 33 IBV sequences identified in this study The nt lengths (and gene intervals in brackets) of each of the genes and the 5'-/3'-UTRs are shown The 15 accessory proteins (2–15) that are proteolytically processed from Rep1ab by the virally-encoded PL pro and 3CL pro For each of the 33 IBV sequences assembled in this study the size (amino acid) of each cleavage product are indicated The position (amino acid residue) of each product on the Rep1a (2–11) and Rep1b (12–16) are shown in brackets Analysis of the S-gene nt sequences assembled in this study The arrow (↓) indicates the S1/S2 proteolytic cleavage site The conserved cysteine-flanked region in the S2 is indicated (amino acid residues in bold and underlined) The numbers in subscript indicate the position of the amino acid residues in the S protein sequence Supplementary Table 5. Comparative pairwise homologies of IBV gene nt sequences between the field and vaccine sequences assembled in this study. Field sequences with the highest pairwise homologies to the vaccine sequences are in bold font, while sequences with the lowest are underlined. The lineage (serotype) classification is based on S1-gene sequence according to Valastro et al. (18) Supplementary Table 6. BLASTn results of the 33 complete genome nt sequences assembled in this study. The lineage (serotype) classification is based on S1-gene sequence according to Valastro et al. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Henry M. Kariithi, aGVucnkua2FyaWl0aGlAa2Fscm8ub3Jn; David L. Suarez, ZGF2aWQuc3VhcmV6QHVzZGEuZ292 Sign In Register Jamie Garratt is paying his employees to watch Hockey Night in Canada this upcoming NHL season the president of Vancouver-based Idea Rebel has been working with Rogers Communications (TSX:RCI) to develop an interactive app for hockey fans Garratt said it wasn’t tough finding staff at the digital marketing agency to manage the Pick ‘N Play app which responds to action on the ice as it happens each Saturday once the NHL season kicks off October 8 “Hockey is such a huge part of Canadian culture and to be part of a large digital experience that’s tied to the broadcast Instead of just sitting back and watching the Vancouver Canucks play the Edmonton Oilers on Hockey Night in Canada fans pick players from opposing teams to go head-to-head in the week leading up to the game As those Saturday games air on TV screens at sports bars viewers can check their phones to see how many points (and possible prizes) they earned depending on how their players do After Rogers signed a $5.2 billion deal last fall to secure TV and Internet rights for the NHL Garratt said the media giant was eager to get fans engaged with the new network “Most people are using devices as a second screen when watching TV anyway,” said Claudio Afonso “[Rogers] can get them locked in as fans by creating a really simple game.” Efforts to pull hockey fans into the second-screen experience aren’t unique to Idea Rebel Vancouver-based app developer Atimi Software partnered with NHL teams such as the Canucks and Chicago Blackhawks to provide fans with custom features geared toward their respective markets Its sports framework – the basis for its other team apps – has 3.3 million downloads and about 50,000 active monthly users “[NHL teams] found fans in other places that they didn’t know and that allowed their market to go after that,” said Scott Michaels Partnerships with NHL teams ended earlier this year when the league pushed all 30 franchises to start using its own app But Atimi is still in the local sports game and has created an app for the B.C it’s photos published by the team and any information that the team wants [to] let out so that fans can comment in the app and about everything that’s happening with the Lions,” Michaels said He said the apps also function when the season is over: “For example draft information or what the team does in the off-season around their charities.” Michaels said the key has always been securing involvement of team sponsors to make sure fans are don’t have to pay for that second-screen experience And although NHL apps are no longer in the works at Atimi Michaels said the company is now eyeing U.S markets that are big on college and high school teams But these second-screen experiences also aren’t limited to sports or TV Vancouver’s Work At Play specializes in creating services for gamers that stretch beyond when the playing systems are turned off “We keep [gamers] engaged when they’re not on the console “We do that by creating experiences and applications across multiple devices – consoles This could be anything from a mobile app that breaks down player statistics to a game-within-a-game app that furthers a player’s status in the main console game Haman said both social media and the changing video game market have pushed Vancouver tech companies toward developing second-screen experiences “A lot of small startups have surfaced and you don’t know who’s doing what,” she said “Instead of just being large console titles being released on a two-year cycle