Developing African countries face health problems that they struggle to solve. The major causes of this situation are high therapeutic and logistical costs. Plant-made therapeutics are easy to produce due to the lack of the safety considerations associated with traditional fermenter-based expression platforms, such as mammalian cells. Plant biosystems are easy to scale up and inexpensive, and they do not require refrigeration or a sophisticated medical infrastructure. These advantages provide an opportunity for plant-made pharmaceuticals to counteract diseases for which medicines were previously inaccessible to people in countries with few resources.The techniques needed for plant-based therapeutic production are currently available. Viral expression vectors based on plant viruses have greatly enhanced plant-made therapeutic production and have been exploited to produce a variety of proteins of industrial, pharmaceutical and agribusiness interest. Some neglected tropical diseases occurring exclusively in the developing world have found solutions through plant bioreactor technology. Plant viral expression vectors have been reported in the production of therapeutics against these diseases occurring exclusively in the third world, and some virus-derived antigens produced in plants exhibit appropriate antigenicity and immunogenicity. However, all advances in the use of plants as bioreactors have been made by companies in Europe and America. The developing world is still far from acquiring this technology, although plant viral expression vectors may provide crucial help to overcome neglected diseases.Today, interest in these tools is rising, and viral amplicons made in and for Africa are in progress. This review describes the biotechnological advances in the field of plant bioreactors, highlights factors restricting access to this technology by those who need it most and proposes a solution to overcome these limitations. Least developed countries (LDCs) encounter major health problems that are difficult to solve mainly for economic reasons, such as medicine acquisition and logistic organization. Among the deadly diseases occurring in Africa, human immunodeficiency virus (HIV) disease and malaria are old diseases that still occur with high mortality among the infected population. In 2016, there were 37 million people living with HIV, 1 million HIV deaths and 1.8 million new infections worldwide. Approximately 70% of HIV-infected people live in Africa [Neglected tropical diseases (NTDs) are mainly curable. However, they still cause damage in tropical areas. NTDs impair the lives of 1 billion people worldwide and threaten the health of millions more . While LOne of the main obstacles to health problem resolution for LDCs is a lack of economic resources. For example, in Burkina Faso, where more than half of the population lives on less than 2 US$ per day, an efficient malaria cure costs approximately 14 US$. Malaria in these areas represents a public health problem with substantial risks for the pregnant woman, her fetus and the newborn child . VaccinaTo manage human diseases such as HIV, malaria or new and emerging diseases, vaccines and therapeutics are crucial. These therapeutics and vaccines need to be inexpensive to be suitable for LDCs. To meet these challenges, plant-produced vaccines and therapeutic agents offer enormous potential for providing relief to LDC healthcare systems. In recent decades, plants have been recognized as a possible solution for global health. Plant-made biopharmaceuticals have long been considered a promising technology for providing inexpensive and efficient medicines for LDCs –10. IndeTobacco mosaic virus (TMV), Potato virus X (PVX) and Cowpea mosaic virus (CPMV) [Plant virus expression vectors have revolutionized the use of plants as bioreactors. Once introduced to host plant cells, viruses engineered to contain a gene of interest replicate, allowing the expression of foreign proteins at high levels in infected plants , 16. Plas (CPMV) . More res (CPMV) , or useds (CPMV) .The driving forces behind the rapid growth of plant bioreactors include low production costs, product safety, posttranslational modifications and easy scale-up compared to other production systems based on bacteria, yeast or mammalian cells  Fig. 1)1). TheseApproximately 15 million deaths per year worldwide (> 25%) are estimated to be related directly to infectious diseases . VaccinaIn the early 1990s, plants were recognized as useful bioreactors for the production of high-value proteins . First, Agrobacterium tumefaciens as a vector to deliver DNA encoding proteins of interest directly into leaf cells by syringe infiltration: agroinfiltration [Tomato bushy stunt virus (TBSV) or Artichoke mottle crinkle virus (AMCV) on the RNA silencing pathway has been exploited with success to enhance the transient or constitutive expression levels of recombinant proteins in plants [The first crucial advance was the use of transient expression systems relying on ltration . These tltration . RNA silltration , 43. Thiltration . Proteinn plants , 46. Finn plants .Table 1Another significant development in the plant bioreactor field was the coupling of agroinfiltration with the delivery of cDNA encoding viral RNA, which is used as an “amplifier” for production of the protein of interest. Once introduced into host plant cells, viruses engineered to contain a gene encoding a candidate protein replicate many times, and the corresponding protein can be produced in higher quantities than in a nonviral context , 47 Fig.Fig. 2STransient expression systems exploiting either RNA silencing suppression or viral-based vectors have revolutionized the use of plants as bioreactors. They shorten production time and increase the yield of the protein of interest while avoiding the concerns of the general public about the use of stably genetically modified organisms. The use of plant virus suppressors of RNA silencing was demonstrated to increase the expression levels of heterologous proteins several-fold in agroinfiltrated leaves . Virus vTobacco mosaic virus), has been established as a viral vector and enabled the production of diverse proteins of interest [The first discovery of a virus in the nineteenth century has led to the emergence of virology and important biotechnological advances. In the field of biotechnology, plant viruses have become the basis of practical tools for plant fundamental genomic studies and protein production. The first exploited virus, TMV . Several scientific works have reported the successful expression of different immunogenic epitopes as fusions with viral coat proteins [in trans to participate in chimeric virus multiplication. This trans complementation could be achieved through coagroinfiltration [trans-complementation approach for the accurate use of CPMV-based vectors for protein expression [Cucumber mosaic virus (CMV) accumulate to the wild-type level only when inoculated in transgenic tobacco expressing the removed open reading frame [Plant viral vectors can be divided into three categories based on the manner they have been Plant viral vectors can be divided into three categories based on the manner in which they were designed. The first strategy uses the unmodified native virus to maintain its full properties. It is called the “full-virus strategy”. Therefore, this first strategy led to the design of fully functional viruses that, despite carrying and expressing heterologous sequences, have retained infectivity, stability and systemic virulence in their hosts. The foreign gene of interest is expressed either as part of a fusion protein with a viral protein or separately from an additional strong subgenomic promoter that is incorporated into the viral genome Fig. .a. Severproteins , 63. In proteins . This kiproteins . Howeverproteins . There aproteins . The decltration  or establtration –73. Thuspression . Open reng frame . Here, iRed clover necrotic mosaic virus (RCNMV) encapsulating the anticancer drug doxorubicin (DOX) worked more efficaciously than pegylated liposomal doxorubicin (PLD) in an ovarian cancer model [Hibiscus chlorotic ringspot virus (HCRSV). The authors demonstrated its ability to transport the drug molecules polystyrene sulfonic acid (PSA) and polyacrylic acid (PAA) acting on cancer cells [More recently, plant viruses have been utilized as nanoparticles to transport drugs and active molecules into cancer cells. Plant viruses play a key role in the fight against cancer by acting as shuttles that are able to reach tumor cells and deliver drugs or elicit a highly localized and powerful immune response . This ther model . Moreoveer model . Anotherer cells . They aler cells .Table 3Tobacco mosaic virus (TMV) was the first virus engineered as a deconstructed vector system because of its high number of capsid protein (CP) copies (2130 copies) and the relatively simple organization of its genome. The TMV expression system is composed of two modules, one containing the replicase and the other containing the site for foreign gene integration. TMV has been used successfully for the production of several proteins of interest. The expression of vectors has been improved by genetic engineering with modifications of transcript splicing sites, codon usage patterns and insertion of introns into coding sequences [These three strategies, the full-virus, deconstructed virus and nanoparticle strategies, have been exploited for the construction of several viral expression vectors based on viruses belonging to some families, including Potexviruses, Comoviruses, Geminiviruses and Tobamoviruses. equences . Human pequences . Influenequences . Others equences  or vacciequences .Potato Virus X (PVX), a flexuous, rod-shaped virus containing a plus-sense RNA molecule, has also been engineered extensively as an expression vector for biopharmaceutical production. The capsid protein gene and a triple gene block whose products are responsible for PVX movement are targeted to engineer deconstructed PVX vectors. PVX has been used to express full-length proteins, fusion proteins and epitopes for medical purposes [purposes , 85. Morpurposes , 86, 87.Cowpea mosaic virus (CPMV) is an icosahedral virus of 30 nm in diameter. The genome of CPMV is bipartite, and its RNA-2 is the principal component used for expression vector development. CPMV has been utilized extensively in antigenic presentation and full-length protein expression as part of a fusion protein that can undergo proteolytic cleavage to release the therapeutic protein [The Comovirus  protein . Medicag protein . The CPM protein  allows t protein , 92.Agrobacterium tumefaciens suspensions has allowed the development of a set of expression vectors that rival the deconstructed RNA virus vectors in their yield and application, with some potential advantages over the latter that still need to be explored [Bean yellow dwarf virus (BeYDV) by the Rybicki group in South Africa. This ssDNA expression vector have been exploited to address African health concerns like HIV or HPV [Besides RNA viruses that have been widely exploited as viral expression vector, there is a rise of single strand DNA (ssDNA) virus exploitation as viral vector for potential expression of a various single or even multiple proteins of interest in a wide range of plant families. The use of vacuum or other infiltration of whole plants by explored . RecentlV or HPV .The technology of viral vectors based expression is more and more mastered; it allows nowadays the production of complex proteins. It has also opened up to new horizons such as the exploitation of viral vectors for the fight against cancers. Yet the developing African countries remains on the sidelines of these technologies, which would be undoubtedly a ready-made solution to the health problems that undermine this part of the world. In the past, vaccines produced through other expression systems have not had a positive impact in developing African countries. Those needing the most these product, populations of remote, impoverished regions, could not access vaccines and other biopharmaceutical proteins. This is mainly due to the high costs estimated for a successful immunization strategy and the absence of accessible infrastructure for vaccine production, distribution and delivery .All these difficulties are reduced in the case of the expression of the proteins of interest in plants through viral expression vectors. Indeed, plant systems have the capacity to produce properly folded proteins at low cost, making them suitable expression systems for poor nations . In addiThe level of knowledge of the exploitation of plants as bioreactors and, more particularly, amplicon technology is such that it could be accessible for developing Africa. Several preliminary works based on plant and viral vector exploitation have already begun to target the diseases found in developing countries, such as NTDs, HBV (Hepatitis B virus), cancers, malaria and HIV.Nicotiana benthamiana and spinach. The recombinant Tat produced in these plant systems allowed the systemic production of anti-HIV antibodies when it was used as a vaccine to immunize mice [Nicotiana tabacum [A safe and effective preventative vaccine is urgently needed to curb the HIV pandemic. Several strategies and candidates have been considered to achieve HIV-efficient vaccines , 96. Parize mice . Nef pro tabacum . Finally tabacum . With thPlasmodium yoelii merozoite surface protein 1 (PyMSP1), P. yoelii merozoite surface protein 4/5 (PyMSP4/5), and P. falciparum merozoite surface protein 1 (PfMSP1), at satisfactory yields [Nicotiana benthamiana system [RTS,S/AS01 (trade name Mosquirix), a recombinant protein-based malaria vaccine has been approved for use by European regulators in July 2015 , 109. Sey yields , 111. Soy yields , 110–113a system , 110.Because the production of pharmaceuticals against NTDs is not economically profitable for international pharmaceutical companies, plants represent an alternative choice. The World Health Organization (WHO), in an effort to reduce rabies burden, has chosen a plant-based expression system to procure and deliver safe and efficacious rabies vaccines in countries with a crucial need .Alfalfa mosaic virus (AMV) capsid protein (CP). This chimeric protein has been successfully expressed in infected plants, yielding viral particles that were successfully purified from infected plant tissue and directly used to immunize mice. This work demonstrated that plant-produced rabies virus antigen was capable of inducing an immune response in mice in an adjuvant-free system [Rabies surface glycoprotein (G-protein) is the most important protein involved in viral pathogenesis. It has been recognized as the major protective antigen . Severale system .Nicotiana benthamiana [Nicotiana species and thus is more suitable for administration [In addition to NTDs, there are new emerging diseases that also threaten poor nations. Ebola virus is a recently identified filovirus that causes hemorrhagic fever in both humans and nonhuman primates . Ebola vthamiana . This Ebstration . The usePlants have also been useful in the production of an antibody against dengue fever virus, another filovirus causing tremendous and widespread damage in developing countries. A TMV-based transient expression system has been used to produce a dengue-virus-derived antigen in plant cells that exhibits appropriate antigenicity and immunogenicity .Plant-based expression systems and viral expression vectors represent a versatile technology allowing the expression of several proteins displaying vaccine properties against diverse diseases, such as viral diseases, that may eventually lead to cancer. Human papilloma viruses (HPVs) represent the main cause of cancer-related death for women in developing countries and the third leading cause of cancer-related death among women worldwide . 120 HPVViral hepatitis is the most common causative agent of inflammation and damage of the liver. Hepatitis A, B or C viruses frequently cause viral hepatitis. While Hepatitis A Virus (HAV) causes the least amount of liver damage, Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) can lead to chronic liver disease and cancer. These viruses are found worldwide, but predominate in developing countries. An efficacious vaccine exists for Hepatitis A and B, but not for hepatitis C . VaccineHepatitis B virus (HBV) is a worldwide disease that represents a significant burden on developing countries. Yeast-derived subunit vaccine was generated . This vaPlant-based pharmaceutics and viral expression vectors represent a boon to resolve the health problems of the population of developing Africa. However, this technology is mainly applied by northern firms that are not economically interested in pharmaceutical production for diseases present in LDCs. In Africa, most countries that are involved in biotech activities are still at the level of tissue culture applications and they are generally limited to genetic engineering , 166. SoThe rest of Africa, especially developing Africa, is on the sidelines of this technological advance for a number of reasons. The expertise and the infrastructure necessary for the implementation of this technology are absent in these countries. In addition, the viral expression vectors that are key tools for the efficient production of therapeutics are patented, limiting their exploitation to large northern industrial firms. Other major obstacles to the implementation of plant bioreactors relate to biosafety concerns in these states. Moreover, the institutional frameworks for legislation on this type of product are poorly defined. The populations concerned must also be addressed, because they are generally resistant to biotech products using genetic engineering.Rice yellow mottle virus (RYMV) and Imperata yellow mottle virus (IYMV), and DNA virus belonging to the group of begomoviruses, such as Pepper yellow vein Mali virus (PepYVMLV). Sobemoviruses present the advantage of having a small genome with just five open reading frames. Research groups from south Saharan Africa, especially those from Burkina Faso and their northern partners, already have a solid background in these three viruses [Nicotiana species and natural virus host plants are ongoing . These viral vectors could be used to treat human diseases such as malaria, HIV, Ebola, dengue, NTDs and animal diseases such as avian influenza (H5N1) but could also contribute to the production of antibodies for medical and research purposes.Plant viral vector technology could be a platform allowing developing nations to grow their own pharmaceutical industries. This effect could be long-lasting only if politicians adopt laws allowing LDCs to freely acquire and use virus constructs. This will necessarily go through the development of virus constructs based on African viral resources by African researchers. Excellent candidate viruses already exist, such as RNA viruses from the sobemovirus group, such as  viruses –176. DevImprovements in manufacturing infrastructure, financial support and the maturation of a regulatory framework surrounding plant-derived pharmaceuticals are all essential prerequisites for the implementation of this technology in the developing world. If these obstacles were overcome, many LDC health issues would find suitable solutions. Then, the plant-made pharmaceutical industry could become a reality for the developing world.Such an implementation project requires solid infrastructure and expertise for both the medical and plant biological domains. This intersectional and multidisciplinary work implies different specialized institutions with deep knowledge of both plant science and medicine. Burkina Faso is one of the West African LDCs meeting these requirements. Indeed, Burkina Faso contains specialized structures for both aspects. The International Research Institute on Health Science (IRSS: Institut de Recherche en Science de la Santé) has strong expertise in parasites and pathogens such as malaria. LABIOGENE (Laboratoire de Biologie Moléculaire et de Génétique) is a molecular biology and genetic laboratory with crucial knowledge of genetic engineering for human health. On the plant side, the National Institute of Environment and Agronomy Research (INERA: Institut de l’Environnement et de Recherches Agricoles) displays a deep expertise on cultivated plants and their pathogens. In accordance with the “one health concept”, these three institutes would provide a complementary context to discover and develop plant viral expression vectors. IRSS and LABIOGENE are institutes with subregional missions recognized as centers of excellence of by the UEMOA (Union Economique et Monétaire Ouest Africaine). Experts on the plant side can benefit from the technical platforms of these two institutes to strengthen their activities in the “one health concept”, as the techniques used for human and animal diagnostics are similar to those used for experiments based on plants. Such collaborations would help reduce the costs of equipment and infrastructure. Furthermore, Africa has diverse biological resources that are underexploited. Indeed, some plants and pathogens endemic to Africa may be excellent candidates for viral amplicon/bioreactor technologies. African resources could then be exploited by and for Africans, as the Nagoya protocol advises . Figure Plant made therapeutics are safe with a reduced cost and thanks to viral vector they can be obtain in days rather months as at their beginning. By the past, poor resource communities could not afford medicines because of the production cost and logistic cost involved. Plant made therapeutics have been produced for major disease that prevail in these countries through either time-consuming transgenic approach or quick transient expression system using viral vectors. Most of these viral vectors that are patented can be used through win-win collaborations with research laboratories or private companies. To support plant-made therapeutics as a strategic means to overcome poor countries’ diseases, these countries should be equipped with their own viral constructs to provide them with the freedom to operate. Some plant viruses are present in Africa and have already been recognized as excellent candidates for viral amplicons made in and for Africa. This will be possible with the support of resource-poor countries’ governments through infrastructure, financial funding and the establishment of an efficient regulatory framework. Regarding the context, the implementation of plant-made biopharmaceuticals in the developing world is an ineluctable event. This would require new biotechnological tools for protein expression, such as new strong promoters, silencing suppressors or efficient viral expression vectors. Industrial firms should not avoid this field, as they have the opportunity to benefit from the advent of African viral-based biotechnological tool development.


I thank all the teams of authors, the scientists who reviewed submitted manuscripts and made suggestions that improved the reports, and the editorial staff workers who put this special issue together. Thanks for your contributions.Charavirus canadensis, in which genome segments have origins from viruses related to hepacivirusees, benyviruses, tobamoviruses, and hepegiviruses. As the number of canonical genome segments increases, so will the number of viruses with missing genome components that may have to be discovered. A report of the discovery of the missing components of the subterranean clover stunt virus is presented, authenticating its identification as a Nanovirus. Bean-associated cytorhabdovirus is proposed as a virus found in resistant bean cultivars. The articles published in this special issue highlight many of the processes that contribute to the evolution of viral genomes. A major theme is the reassortment of multi-partite genomes. At its extreme, this results in genomes such as that of Ecology has also played a role in virus spread. An investigation of disease in cyclamen revealed the presence of a strain of the fig mosaic virus. A review of chickpea stunt disease underscored the importance of plant–virus–insect interactions. Glasa et al report an analysis of turnip mosaic virus isolates from pepper, which confirmed that recombination played an important role in the evolution of this virus and the shaping of its genome.Thanks to all.


Phylogenetic analysis showed that Formica fusca virus 1 grouped within the family Nyamiviridae, Lasius neglectus virus 2 grouped within the family Rhabdoviridae and Myrmica scabrinodis virus 2 belongs to the family Dicistroviridae. All three viruses are highly divergent from previously sequenced viruses.Three novel RNA viruses, named Formica fusca virus 1 (GenBank accession no. MH477287), Lasius neglectus virus 2 (MH477288) and Myrmica scabrinodis virus 2 (MH477289), were discovered in ants collected in Cambridge, UK. The proposed virus names were given based on the hosts in which they were identified. The genome sequences were obtained using The online version of this article (10.1007/s00705-018-4093-2) contains supplementary material, which is available to authorized users. Insect-infecting viruses comprise a large and diverse group, but their full diversity has as yet been poorly investigated . Furtherhttp://www.ebi.ac.uk/arrayexpress) under the accession number E-MTAB-5781. Trimmed RNA-Seq reads were assembled de novo into contigs using TRINITY v2.3.2 [We generated RNA-Seq libraries from ants sampled from various locations in Cambridge, UK . SequencY v2.3.2 , and assTo determine the taxonomy of the three viruses, related sequences were identified within GenBank , using the amino acid sequence of the entire ORF encoding the RNA-dependent RNA polymerase (RdRp) as the query in TBLASTN. Reference (RefSeq database) and selected non-reference (nr/nt database) sequences with > 25% coverage match were used for phylogenetic analysis. Amino acid sequences of the RdRp-encoding ORF were aligned using MUSCLE v3.8.31) , and phy.31 , andMononegavirales gene order with five ORFs, encoding proteins in the sequence 3′ – N  – P  – M  – G  – L  – 5′ in negative polarity  comprises 9,851 nt. The genome organization of the virus follows a typical Mononegavirales genome organisation, encoding proteins in the same order as FfusV-1, N (470 aa), P (433 aa), M (248 aa), G (506 aa) and L (2124 aa)  comprises 12,041 nt. The 5′ leader and 3′  trailer sequences of the antigenome are of 115 and 174 nt, respectively, and share nine nucleotides of terminal complementarity. LneV-2 follows a typical Triatovirus within the family Dicistroviridae of the order Picornavirales  comprises 10,663 nt. It contains two ORFs of 5553 nt and 4089 nt in length separated by an intergenic sequence of 196 nt and flanked by 5′ and 3′  UTRs of 630 and 199 nt plus the poly(A) tail, respectively Below is the link to the electronic supplementary material.


Chrysothrixchlorina and Lepraria incana lichens. Comparison of encoded genome proteins revealed classification of the viruses to the genus Alphachrysovirus and a relationship to chrysoviruses from filamentous ascomycetous fungi. We propose the names Chrysothrix chrysovirus 1 (CcCV1) and Lepraria chrysovirus 1 (LiCV1) as acronyms for these viruses. Surprisingly, observation of Chrysothrix chlorina hybridization with fluorescent-labelled virus probe by confocal microscope revealed that the CcCV1 virus is not present in the lichen body-forming fungus but in accompanying endolichenic Penicilliumcitreosulfuratum fungus. These are the first descriptions of mycoviruses from a lichen environment.A lichen body is formed most often from green alga cells trapped in a net of ascomycetous fungi and accompanied by endolichenic or parasitic fungi, other algae, and symbiotic or free-living bacteria. The lichen’s microcosmos is inhabited by mites, insects, and other animals for which the lichen is a source of food or a place to live. Novel, four-segmented dsRNA viruses were detected in saxicolous  Fusarium species, and these parasites attack a variety of lichens and are tolerant of many lichen defense compounds [Caloplaca are known to have parasitic phases in their life cycles during which they take over other crustose lichens [Athelia arachnoidea, which has been shown to attack and destroy Lecanora conizaeoides lichen [Cladonia arbuscula lichen, as has been an Apple mosaic virus (a plant ilarvirus) from different Usnea sp., Xanthoria parietina, and Cladonia arbuscula samples [Fungi are ancient organisms whose major lineages originated in the Precambrian . They arompounds . Moreove lichens ,9. A kils lichen . Neithers lichen ,12,13. I samples . Convers samples ,16, and  samples .Chrysothrix Mont. is characterized by the yellow, lemon to bright yellow, or golden yellow thallus containing pulvinic acid derivatives that give the lichens their characteristic color. Chrysothrix chlorina (Ach.) J. R. Laundon  is a morphologically simple lichen-forming ascomycetous fungus in the family Chrysothricaceae, class Lecanoromycetes. The lichen is a widely distributed saxicolous (growing on rocks) and crustose (strongly adhered to the substrate) organism from the lowlands to the mountains in the temperate and arctic zones of both the Northern and Southern hemispheres. It has a thallus without cortex and reproduces asexually. The lichen never has been found with ascomata or conidiomata [Lepraria incana (L.) Ach.  is a morphologically simple, granular, grayish or greenish lichen growing all over the world on bark, acidic rock, wood and soil in shady places [The lichen genus idiomata . There iidiomata . Vulpiniidiomata . Leprariy places . This liBymo-, Beny-, Furo-, Peclu-, and Pomovirus genera [Mycoviruses are widely distributed cohabitants in fungi. Recently, they have been found in Basidiomycota as well as in Ascomycota. Furthermore, Plasmodiophorids and Chytrids (Chytridiomycota) are known vectors of some plant viruses belonging to the s genera . Only a s genera  is widelChrysothrix chlorina sample ZSH was scraped from granite rocks in the Vltava River valley  in the České Budějovice countryside (Czech Republic). The Lepraria incana lichen sample DK10 was from growths in crevices of shadowed, wet rock at a location about 20 km upriver from the first . Material of non-lichen origin was manually removed before further manipulation. Samples were washed in distilled water, surface-sterilized in 5% sodium hypochlorite for 15 min, and washed twice in water. Due to a limited amount of the DK10 sample, only partial sequencing was performed with this lichen. All other experiments were performed with the ZSH sample.Double-stranded RNA was extracted from about 0.1 g of wet lichen thalli by the CF-11 cellulose chromatography method, as described previously . ExtractEscherichia coli cells. The 5′ and 3′ terminal sequences of each viral genome segment were obtained using 5′/3′ rapid amplification of cDNA ends (RACE) protocol with specific primers (see Complementary DNA (cDNA) was prepared with Superscript III reverse transcriptase  and tagged random primer 5′-CGATCGATCATGATGCAATGCNNNNNN-3′. The random cDNA products were then amplified using a single specific primer, 5′-CGATCGATCATGATGCAATGC-3′ . The poomers see . The 18Sg for 5 min. The pellet was resuspended in sorbitol buffer and loaded onto 80% Percoll™ in sorbitol buffer. After centrifugation at 10,000× g for 10 min, a green layer near the top was collected, resuspended in the buffer, and a second round of centrifugation was performed on 80% Percoll™. Collected green layer was diluted in sterile water and centrifuged at 1000× g for 10 min. The pellet was then resuspended in 2 mL of sterile water, sonicated for 30 s, then centrifuged at 500× g for 5 min. This treatment was repeated 5 times.Lichen alga isolation was prepared according to Gasulla et al.  from 20 Total nucleic acid was prepared from the final pellet containing the algal cells and, after the first centrifugation, from the sediment containing both algal and fungal cells using a NucleoSpin RNA Plant Kit (Macherey Nagel) according to the manufacturer’s instructions.Multiple sequence alignments were carried out using the CLUSTALx  and MEGARelative concentration of the virus in lichen thalli was estimated from quantitative real-time PCR (q-RT-PCR) performed in the CFX96 Real-Time System  with primers 1901 and 1902 see  amplifyi®, sectioning to 6 µm, deparaffinization, and rehydration. Penicillium citreosulfuratum samples for hybridization were taken from aerial hyphae of the fungus cultivated 10 days on potato dextrose agar (PDA) plates. The samples were then treated with chitinase for 15 min in 1× phosphate-buffered saline buffer with 1% sodium dodecyl sulfate (SDS), pH 5.5, at 30 °C and then rinsed with distilled water. Prehybridization was performed in the hybridization buffer , 0.03% SDS and 20% formamide) at 46 °C for 3 h. The samples were then hybridized with the probes in concentration 5 ng per µl of hybridization buffer at 46 °C overnight. They were then rinsed with 20 mM Tris-HCl (pH 7.2), 0.01% SDS, and 250 mM, 88 mM, 62 mM, and 31 mM NaCl buffer at 46 °C for 20 min each. Some samples were stained with calcofluor white  for 5 min. Stained sections were rinsed in distilled water, dehydrated, mounted in DPX mounting medium , then viewed and imaged under a Fluoview FV3000 confocal laser scanning microscope .For in situ hybridization, Cy3-labeled probes MY1574 5′-TCCTCGTTGAAGAGC-3′ specific for a wide range of Eumycota  and 859 Chrysothrix chlorina. The sequence of the algal symbiont (MN396489) was 98% identical to the GU017647 sequence of Asterochloris phycobiontica. Partial ribosomal sequence of the lichen fungus was amplified with ITS1 and ITS4 primers, sequenced, then compared in BLAST with the GenBank database. The lichen of the DK10 sample was identified as Lepraria incana. The 18S rDNA sequence (MN393971) was 95% identical with L. incana sequence AF517899 deposited in GenBank. The algal symbiont of this virus (MN396484) was 97% identical to uncultured Trebouxia sp. sequence AY250848.Based on morphological features, the lichen sample ZSH was identified as In ZSH, a high molecular weight dsRNA band around 3.5 kbp was visible after separation on agarose gel . After sWe had expected the lichen-forming fungus to be the host of these extrachromosomal dsRNAs, because no high molecular weight dsRNA was visible after extraction from partially purified algal symbiont. Algal layer after the third round of sonication/centrifugation was used for RNA isolation and q-RT-PCR detection. Virus-specific detection primers were prepared according to the obtained sequences and RT-PCR detection was performed in parallel in algal symbiont as well as fungal symbiont. No signal increase was observed after 40 rounds of amplification.Relative concentration of the virus in lichen thalli was estimated from total RNA isolations from three different samplings. In a single isolation, only the virus-specific signal in q-RT-PCR occurred 10 cycles after the GAPDH signal, while in the other two isolations the virus concentration was below the detection level. This means that the relative concentration of the virus in ZSH samples varies significantly and was as little as one-thousandth the concentration of GAPDH.n repeat that works as the enhancer element in chrysoviruses was present 5 to 9 times in the 5′-untranslated regions (UTR) of distinct segments. Only for the DK10 isolate were 5′-terminal regions of dsRNA1 and dsRNA2 segments obtained. Both revealed significant nucleotide sequence identity in that region, and this supports the assumption that they belong to a single virus.We determined the complete nucleotide sequence of all dsRNA segments in the ZSH sample. The 5′- and 3′-termini of the four dsRNAs share conserved sequences . The (CAThe dsRNA1 segment from the ZSH sample was 3552 nt long and contained a single open reading frame (ORF) of 1089 amino acids (aa) with predicted molecular mass 125.5 kDa. The eight conserved RNA-dependent RNA polymerase motifs (RdRP_4) characteristic of RNA mycoviruses were localized in the central part of the protein . The polPartial sequences of dsRNA1, dsRNA2, and dsRNA4 segments of a chrysovirus were obtained from the DK10 sample. We did not obtain a sequence of the fourth segment visible on the gel. These sequences represent 84%, 78%, and 72% of segments size for the most similar Colletotrichum gloeosporioides chrysovirus 1  and reveChrysoviridae reflect host isolation, less than 70% aa sequence identity in the RdRp, size of segments, length of 5′-UTR, and serological relationships [Alphachrysovirus. We propose for these viruses the names Chrysothrix chrysovirus 1 (CcCV1) and Lepraria chrysovirus 1 (LiCV1), reflecting the names of their host sources.The taxonomic criteria for differentiating species within the family ionships . In the Universal fungus-specific probe and CcCV1 RNA3 segment probe were used to localize the virus inside the lichen components. In the conditions used for the hybridization , the fungus probe penetrated hyphae and gave strong signal in the majority of hyphae A. The acChrysothrix but of some accompanying or endolichenic fungus. To resolve this question, we cultivated fungus-enriched fraction on PDA plates for 10 days. Newly growing fungi were picked and cultivated individually on fresh PDA plates and used for dsRNA extraction, RT-PCR virus detection, and hybridization. Penicillium citreosulfuratum from the P. citreonigrum clade [P. citreosulfuratum. In the hybridization experiments with the virus probe, strong signal is observed from conidiophores and from single spores of P. citreosulfuratum. That is consistent with the fact that the virus is frequently transmitted by conidia  [Chrysoviridae, with 3–5 segments (25 classified species and about 20 chryso-like viruses), make up the second [Quadriviridae and genus Mycoreovirus (one species each), respectively.Chrysoviruses and their related viruses comprise three to five linear monocistronic dsRNA segments encapsidated separately in identical capsids 35–40 nm in diameter . Most ofosquitos . Furtherosquitos . The ovespecies) . Virusese second . MycovirP. citreosulfuratum.The transmission of chrysoviruses and especially CcCV1 is uncertain. Mycoviruses themselves have limited ways of transmission and, with several exceptions, they generally do not have extracellular routes for infection and the viral particles are not infectious. Usually they are transmitted via hyphal anastomosis and heterokaryosis or via sexual and asexual spores. In chrysoviruses, hyphal fusion experiments have demonstrated that MoCV1 is transmissible via anastomosis . TransmiChrysothrix- and Lepraria-lichenized fungi are related to mycoviruses infecting filamentous fungi and could be classified as regular species of the genus Alphachrysovirus. One can only speculate thus far about the significance of these viruses in lichens, but, analogously with viruses of higher plants and fungi, we could expect an influence on lichen viability, stress tolerance, changes in morphology and/or in gene expression, and other effects. This influence could be directly on the lichen or on the accompanying endogenous fungus. In any case, the cohabitation of the Chrysothrix lichen, Penicillium citreosulfuratum endolichenic fungus and CcCV1  was obtained from Lepraria incana lichen. Both these viruses should be classified in the genus Alphachrysovirus.A new virus distantly related to Aspergillus fumigatus chrysovirus and named Chrysothrix lichen chrysovirus 1 (CcCV1) has been found to inhabit


Acanthamoeba polyphaga mimivirus. Since then, giant viruses of amoeba enlightened an unknown part of the viral world, and every discovery and characterization of a new giant virus modifies our perception of the virosphere. This notably includes their exceptional virion sizes from 200 nm to 2 µm and their genomic complexity with length, number of genes, and functions such as translational components never seen before. Even more surprising, Mimivirus possesses a unique mobilome composed of virophages, transpovirons, and a defense system against virophages named Mimivirus virophage resistance element (MIMIVIRE). From the discovery and isolation of new giant viruses to their possible roles in humans, this review shows the active contribution of the University Hospital Institute (IHU) Mediterranee Infection to the growing knowledge of the giant viruses’ field.The history of giant viruses began in 2003 with the identification of  Viruses, members or former members (mostly former PhD) of our team working at the University Hospital Institute (IHU) Mediterranee Infection prepared this review that presents the last 15 years of our work on giant viruses, from the discovery and description of Mimivirus to our latest studies, some under review and others not yet completed.For this 10th-year anniversary special issue of the journal Rickettsia and rickettsial diseases, was traditionally expert in the isolation and study of fastidious and intracellular microorganisms. In the 1990s, we were the first French hospital laboratory to routinely identify rare bacteria using 16S ribosomal RNA (rRNA) gene sequencing .,75.Tupanp ocean” . Tupanviiviruses ,79,80,81 of life ,86,87,88Marseilleviridae family was the second family of giant viruses to be described [Marseillevirus marseillevirus (MSRV), the prototype virus, was discovered in 2007 in our laboratory [Marseillevirus isolates were described including six at the IHU Méditerranée Infection , ite sample . To datelineages A. All isent sera ,112,113.Faustovirus E12 replication cycle in V. vermiformis lasts 18 to 20 h after individual viral particles’ phagocytosis by the amoeba. The eclipse phase occurs from 4 to 6 h p.i. along with a reorganization of the host nucleus. From 8 to 10 h p.i., a donut-shaped virus factory appears with newly formed viral particles, released through cell lysis in the last step of the cycle [Faustovirus isolates showed an open pan-genome of over 1000 genes and a stable core-genome of around 207 genes, 74% of which have best matches with members of the Asfarviridae family [e family .Faustoviruses are most closely related to the African swine fever virus (ASFV), a tick-borne virus responsible of highly epidemic hemorrhagic fever in domestic pigs. This is most clearly demonstrated when DNA polymerases are used for phylogenetic studies [Faustovirus E12, the gene coding for the major capsid protein (MCP) forming the external protein layer of the capsid is 17 kbp long and comprises 13 exons separated by large group I and spliceosomal-like introns defined by non-canonical splice sites [MCP gene in other faustoviruses showed the presence of six different splicing profiles that correlate with the described lineages (Unpublished under characterization).Among giant viruses,  studies . Interes studies . In Fausce sites . HomologKaumoebavirus strain Sc was the second giant virus of amoeba isolated on Vermamoeba vermiformis [Kaumoebavirus shares several characteristics with faustoviruses: (i) a comparable morphology with 250-nm large icosahedral particles protected by a double-layered capsid, (ii) replication cycles of similar lengths in their common amoebal host, and (iii) the spliced structure of the MCP gene, predicted to be 5 kbp long in Kaumoebavirus. Moreover, a recent study showed the presence of an AT-rich motif similar to a known promotor of ASFV in the intergenic regions of Faustoviruses and Kaumoebavirus suggesting a potential shared gene expression regulation mechanism in these viruses [miformis  , harboring a genome of intermediate length (395 kbp). Pacmanvirus shares 31 genes with Faustovirus, Asfarvirus, and Kaumeobavirus, but is the only virus in this group that encodes for a transfer RNA  [In the tellanii C. This vne-tRNA) .Pithovirus sibericum [Pithovirus sibericum with two corks [The first member of this group of giant viruses was isolated from a >30,000-year-old Siberian permafrost sample and was named ibericum . This viibericum  , and are possible exceptions to the allometric law involving virion volume and genome length [Mimivirus particle could be detected in Pithovirus [Pithoviruses and Cedratviruses reveal an extreme variation rate of their particles. Given the genomic variations we observed between Cedratvirus lineages, this particle size could be a powerful adaptation mechanism in light of evolution where the volume of the capsid does not become a constraint for the selection process and for genomic evolution. There are currently two lineages of Cedratviruses, a possible new genus of the expanding “Pithoviridae” family [New members of the putative “tviruses ,120,121.articles   [Solivirus (276 kb) and Solumvirus (316 kb) from soil samples [Pithoviridae familly [More recently, various viruses and virophages were detected from metagenomic studies in this emerging edvirus) ; 15 gianedvirus)  and two  samples . For the familly .Pandoravirus salinus and Pandoravirus dulcis, were described in 2013, isolated from samples collected on the coast of Chile, and from a freshwater pond near Melbourne, Australia [Pandoravirus inopinatum, described few years earlier as an endosymbiont, was recognized as another strain of pandoravirus [Pandoravirus massiliensis BZ81c, Pandoravirus pampulha 8.8, and Pandoravirus braziliensis SL2, were isolated from soil samples collected from Pampulha lagoon and Belo Horizonte city, and from a soda lake (Soda Lake 2), Brazil [Pandoravirus quercus, Pandoravirus neocaledonia, and Pandoravirus macleodensis, collected from ground soil in Marseille, France; from water of a mangrove in New Caledonia; and from a freshwater pond near Melbourne, Australia, respectively [Acanthamoeba castellanii.The first two strains of pandoraviruses, ustralia . Pandoraustralia ,126,127.oravirus ,129. Thr, Brazil . Then, tectively . All theFurthermore, metagenomic studies showed that pandoraviruses are ubiquitous, as sequences related to these viruses were detected in environmental metagenomes collected worldwide, and even from human plasma ,136,137.Pandoravirus virion morphology stands apart from other giant viruses, by their ovoid particles of 1.5–2 µm in length with an apical pore via which the content of particles is emptied in the amoebal cytoplasm. The study of the replication cycle showed that pandoraviruses can recruit mitochondria and membranes, inducing a modification of the amoebal cytoplasm, especially into and around the viral factories. Interestingly, the authors also showed that pandoravirus infection induces a complete degradation of the host nucleus, after which viral factories start forming [ forming .P. massiliensis) to 2693 (P. braziliensis) open reading frames (ORFs), and some tRNAs [P. salinus. Among the other predicted proteins, more than half encode MORN, F Box, and Ankyrin repeats. The proportion of duplicated genes is very high, varying from 16% to more than half of the gene content (55%), according to the parameters used [Pandoraviruses harbor a linear double-stranded DNA genome, with a high GC content ranging from 59 to 64% ,126. Theme tRNAs . A remarers used .P. salinus genome also encompasses a high number of transposable elements, named MITE for miniature inverted repeat transposable elements [P. salinus, P. dulcis, P. inopinatum, P. massiliensis, P. braziliensis, and P. pampulha, and revealed a very small core genome consisting of 4.7% of the total pangenome [P. salinus, P. dulcis, P. inopinatum, P. quercus, P. neocaledonia, and P. macleodensis [Genomes of pandoraviruses do not encompass any gene encoding for a known capsid, another interesting and unique characteristic distinguishing further away giant viruses from canonical viruses . As deteelements . Two panangenome . The seceodensis . It reveP. massiliensis showed that at least 25% of the predicted genes are transcribed in the conditions used in the experiment. Two-thirds of the total reads provided by the sequencing were found 6 h post infection [P. salinus, 357 for P. quercus, 387 for P. dulcis, and 337 for P. neocaledonia [P. massiliensis was detected by transcriptomics, and 11.4% of viral gene products were found by proteomic analysis in virions, more than half of which belong to the core genome [Transcriptomics experiments of nfection . The traaledonia . In addie genome . On the e genome .These findings show that further analyses and investigations will be necessary to improve our knowledge on these very intriguing giant viruses.In many features, giant viruses have similar characteristics to the three major branches of the tree of life. The mobilome is no exception and, in our laboratory, genetic elements were highlighted as independent or integrated genomic sequences.Acanthamoeba castellanii mamavirus, a mimivirus belonging to the lineage A of the Mimiviridae family cultivated from waters of cooling towers in Paris, France [Mamavirus at a specific location and earlier stage of the co-infection cycle. Sputnik reproduction impaired the infectivity of its virus host, leading to a decrease in amoebae lysis. In addition, the morphogenesis of the mimivirus was significantly impacted resulting in a high abnormal particle production [The concept of a virophage as a parasitic agent that depends on and predates the replicative cycle of a host mimivirus dates back to 2008, when La Scola et al. observed the presence of small virions of approximately 50 nm, infiltrating the viral factory of , France . This sm, France ,141. Spuoduction . The repoduction . So far,oduction ,142,143.oduction ,34,35, Zoduction , Guaranioduction , and Sisoduction ,148,149.Mimivirus genome that could have been cannibalized and that would confer a selective advantage in the defense of Mimivirus against virophages [Mimiviridae and, among them, lineage A had the unique ability to resist against infection by a specific virophage, Zamilon [R349) present in the lineage A of Mimivirus. In addition, we localized, in the vicinity of these repeated sequences, genes that we assigned as a putative helicase and putative endonuclease (R350 and R354). Although this system is distinct from the structure described in the well-known prokaryotic CRISPR/Cas system, a functional analogy was proposed, and we defined this system as a defense system in giant viruses, named MIMIVIRE (Mimivirus virophage resistance element) [Mimivirus was successfully achieved and the deletion by knockout of the R349 gene containing the repeated sequences of Zamilon restored the susceptibility of the infection by Zamilon [R349 gene as an essential element of MIMIVIRE. An alternative hypothesis based on protein-based interaction was also proposed to explain the resistance of Mimivirus against Zamilon [According to the Red Queen hypothesis , microorrophages . As desc Zamilon . Accordielement) . Based o Zamilon . These r Zamilon . Finally Zamilon .Acanthamoeba species, and other giant virus genes encoding family B DNA polymerase, DNA-dependent RNA polymerase subunits, or D5 helicase-primase in the genome of Phycomitrella patens and Selaginella moellendorffii. In the case of genomes of Acanthamoeba spp., results suggested that the sequence flow between these amoebae and giant viruses could be in both directions [Hydra magnipapillata or Phytophtora parasitica corresponded to giant viral sequences integrated in these eukaryotes’ DNA, or alternatively were possibly previously overlooked [Genes from giant viruses were identified in the genomes of eukaryotic organisms, including amoebae and plants ,155,156.erlooked .Mimivirus discovery and the extension of giant virus diversity [Mimiviruses and Marseilleviruses, but also more recently discovered representative members such as Faustoviruses, Pithoviruses, and Pandoraviruses, as well as virophage-like sequences. In addition, we detected sequences from putative giant virus relatives in the NCBI GenBank environmental protein sequence database (env nr), using giant virus sequences encoding a DNA-dependent RNA polymerase subunit, as well as reconstructed putative ancestral sequences for this protein as baits [Marseillevirus-like sequences in a metagenome from the stools of a young and healthy Senegalese man led to the isolation, for the first time, a giant virus named Senegalvirus from this human sample [Marseillevirus-like sequences and could be assembled in two 10–13 kbp-large contigs closely related to the genome of the Marseillevirus prototype strain [The onset of metagenomics for the study of microorganims and its expansion coincided with the iversity . The powiversity . We alsoiversity . It homoas baits . Metagenn sample ,159. Thee strain . Thus, ge strain ,161.Mimiviruses and Marseilleviruses, the two families of giant viruses of amoebas first described.The presence of giant viruses in humans was documented especially for Senegalvirus, a close relative of Marseillevirus, from a stool of a healthy young Senegalese man [Marseillevirus were detected in the metagenome generated from the blood of blood donors from France [Marseillevirus was detected by PCR in 10% of the 20 blood samples tested. However, these results were controversial, with other studies showing negative results for all blood samples tested by PCR, with the same primers used [Marseilleviruses was unexpectedly high with rates varying between 1.7 and 15% [Marseilleviruses with humans are frequent.The first giant virus isolated from a human sample was lese man . It was m France . In the ers used . Seropre and 15% ,44,104, Marseilleviruses were also detected in lymphoid organs in three reports. A Marseillevirus-like organism was detected in a lymph node of an 11-month-old boy who presented an adenitis of undetermined etiology [Marseillevirus in humans. These reports may raise questions about the pathogenicity of these Marseilleviruses, particularly in the case of lymphomas. The prolonged carriage, the presence in the blood, the detection in pathologic lymph nodes are arguments for a possible causal link between the virus and the disease. Many other infectious agents, as the Epstein–Barr virus, are linked with lymphomas, and the question on the link between Marseillevirus and lymphomas could be open; however, further investigations are required. Furthermore, a murine model confirmed the prolonged detection of the virus in a mammalian organism, after an experimental inoculation by pulmonary or intravenous routes [etiology , in the etiology ,47. In as routes .Marseillevirus sequences were also detected in metagenomic studies in buccal mucosa, retro-auricular crease, vagina, and stools from healthy individuals [Moreover, ividuals ,160.Mimiviruses were first isolated in an amoebal co-culture of water samples from cooling towers during the investigation of an outbreak of pneumonia [Mimiviruses and pneumonia in humans was explored. Firstly, seroprevalence studies showed that the rates found in community-acquired pneumonia and nosocomial pneumonia were 9.7% and 10%, respectively, versus 2.3% and 3% in control patients [Mimivirus could be the fourth most common etiology of acquired nosocomial and community pneumonia [neumonia . Then, tpatients ,163. Morneumonia .Acanthamoeba polyphaga mimivirus and developed pneumonia, for which all other pathogens were negative, was a strong argument for the association between Mimivirus and pneumonia [Mimivirus was able to inhibit the regulation of IFN-stimulated genes, thus evading the IFN system. These results suggest that Mimivirus and humans have host–pathogen interactions [Mimivirus exposure is a risk factor for the development of auto immunity against collagen [The seroconversion of a laboratory technician who handled huge quantities of neumonia . Indeed,neumonia . Moreoveneumonia ,40,167. neumonia . Howeverneumonia ,168. Theneumonia . Furtherractions . Anothercollagen .Mimivirus sequences in human stools, nasopharyngeal aspirates from patients with respiratory tract infections, buccal mucosa, saliva and retroauricular crease, vagina, blood from healthy people, and even in blood from patients with liver diseases [Mimivirus sequences were the most represented viral sequences together with Poxviridae sequences in the human gut.Recent metagenomic studies also reported the presence of diseases ,160. RamViruses gave us the opportunity to overview 15 years of research and discoveries on giant viruses. The most recent metagenomic works [This 10th-year anniversary special issue of the journal ic works ,52 or thic works  suggest


Nipah virus (NiV) is an emerging bat-borne pathogen. It was first identified 20 years ago in Malaysia and has since caused outbreaks in other parts of South and Southeast Asia. It causes severe neurological and respiratory disease which is highly lethal. It is highly infectious and spreads in the community through infected animals or other infected people. Different strains of the virus show differing clinical and epidemiological features. Rapid diagnosis and implementation of infection control measures are essential to contain outbreaks. A number of serological and molecular diagnostic techniques have been developed for diagnosis and surveillance. Difficulties in diagnosis and management arise when a new area is affected. The high mortality associated with infection and the possibility of spread to new areas has underscored the need for effective management and control. However, no effective treatment or prophylaxis is readily available, though several approaches show promise. Given the common chains of transmission from bats to humans, a One Health approach is necessary for the prevention and control of NiV infection. Paramyxoviridae. It belongs to the genus Henipavirus which also contains Hendra virus (HeV) and the recently described Cedar virus. Bats are the natural reservoir of Henipaviruses [Pteropus bats, have a worldwide distribution. It is likely that new areas where they reside will be the location of spillover events in the future. A recent outbreak in a new geographical area in Kerala, India is just the latest such event [Nipah virus (NiV) is an RNA virus belonging to family aviruses . While Caviruses . NiV is aviruses . It firsaviruses . The resch event . ResearcA One Health approach that takes into account humans, domestic and peri-domestic animals and the environment is required to control the disease effectively.We conducted a literature search using the digital archives Pubmed, Google Scholar and the Cochrane library. The following MeSH terms were used: ‘Nipah Virus Infection’, ‘Epidemiology Nipah virus’, ‘Clinical features Nipah virus’, ‘Diagnosis Nipah virus’, ‘Surveillance Nipah virus’, ‘Vaccine Nipah virus’, ‘Nipah virus Malaysia’, ‘Nipah virus Bangladesh’, Nipah virus infection India’ and ‘Nipah virus Philippines’. All literature reviews, original papers and case reports referring to all aspects of NiV origin, modes of transmission, clinical presentation, diagnosis and management were reviewed. The cross-references from these publications were also included. Additionally, epidemiological reports from the WHO and the National centre for disease control (NCDC), India and other public health organisations were assessed for this paper. The aim of this search strategy was to find literature describing the transmission of the disease, diagnosis of infection and control strategies.Human NiV infection was first identified in Malaysia from 1998 to 1999 . The namIn these outbreaks, close contact with pigs or pig excreta was shown to be a risk factor , 10. ThePteropus bats were shown to be the reservoir of infection in Malaysia [Dogs were also found to be commonly infected  and dogsMalaysia  which inPteropus bats have been identified as the reservoir [Pteropus bats have been found to visit date palm trees and lick the sap streams being used for collection. Bats may also contaminate the sap collection pots with urine or faeces [The epidemiology of NiV is significantly different in Bangladesh. Since 2001, seasonal outbreaks of NiV have occurred in Bangladesh in the winter months, primarily in 20 districts  in centreservoir . Though eservoir . Transmieservoir . Outbrear faeces . Domestir faeces  though tr faeces , 20. Perr faeces . NiV is r faeces  and NiV r faeces . Other pr faeces . Consumpr faeces .In India, there was a large outbreak (66 probable cases and 45 deaths) in Siliguri, West Bengal in 2001 and another smaller outbreak  in 2007 in Nadia district, West Bengal. These outbreaks were across the border from the Nipah belt in Bangladesh. In May 2018, an outbreak of NiV was declared in Kozhikode and Malappuram districts of Kerala, a southern state in the west coast, which is geographically disconnected from previously affected areas. Date palm sap consumption is not a common practice in this area. There were 18 confirmed cases and 17 deaths as of 1 June 2018 . All casAn outbreak of NiV infection occurred in the Philippines in 2014. Seventeen cases were confirmed, the case fatality rate was 82%. Ten patients had a history of close contact with horses or of horse meat consumption. Deaths of 10 horses were reported in the same time period, of which nine showed neurological symptoms. However, samples from horses were not tested for NiV. Five patients, including two healthcare personnel, acquired the disease through person to person transmission. This strain was closely related to the Malaysian strain where definite person to person spread had not been previously identified . This suThe incubation period of NiV varies from 4 to 21 days. NiV primarily causes acute encephalitis and respiratory illness and is highly fatal. A small percentage of infected people are asymptomatic .A short incubation period is followed by prodromal signs and symptoms such as a fever headache and myalgia . FeatureResidual neurological deficits are seen in 20% of survivors and range from fatigue to focal neurological deficits and depression . Some caThere are some differences in clinical features seen in the Malaysian and Indian outbreaks. A higher mortality rate has been seen in India and Bangladesh (70%) as opposed to Malaysia (40%). Respiratory illness is seen in 70% of patients in India and Bangladesh , 33, wheRisk factors for poor prognosis include old age, comorbidities, thrombocytopenia and raised aminotransferases on admission, brainstem involvement and seizures .The Henipaviruses are the only zoonotic paramyxoviruses. They are also exceptional in their broad host range and high case fatality rates. They have a nonsegmented negative-stranded RNA genome consisting of helical nucleocapsids encased in an envelope forming spherical to filamentous, pleomorphic virus particles. Both HeV and NiV have a significantly larger genome than other paramyxoviruses . The genThe virus enters its host through the oro-nasal route and causes infection. Since human tissues have only been studied from the terminal stages of the disease, the site of initial replication is unknown. However, high concentrations of antigen found in lymphoid and respiratory tissues indicate these tissues as probable sites of initial replication . Early vIn Malaysia, two major strains of NiV were found circulating in pigs, one in the northern part of the country and the other in the south and at least two introductions of the virus into pigs are thought to have occurred . The NiVSpecimens for virus detection may be collected from symptomatic patients or at post-mortem examination. Specimens for serological testing should be collected late in the course of infection, 10–14 days after onset. The NCDC, India, recommends throat swabs , urine, blood and/or CSF for diagnosis. Samples must be collected safely and transported in triple container packing at 2–8 °C. Storage at −20 °C is recommended beyond 48 h of collection. Processing of the clinical samples requires a BSL 4 facility. However, virus inactivation by sample irradiation may be an effective technique to make the samples safe to use in a BSL-2 laboratory , 46.The best test for direct detection is polymerase chain reaction (PCR) due to its high sensitivity, specificity and the rapidity with which results can be reported. Specimens that may be used include tissue samples, swabs, CSF and urine. Diagnosis by direct detection in animals may be difficult, as virus detection has low sensitivity.Conventional PCR targeting the N (nucleocapsid protein) gene has been developed by the US Centers for Disease Control and Prevention (CDC) . NiV RNAFormalin-fixed tissue may be used for immunohistochemistry. Since the site of viral replication is vascular endothelium, a wide range of tissues may be used including brain, lung, spleen, kidney and lymph nodes. Uterus, placenta and products of conception are also analysed in pregnant animals. Previously, convalescent human serum was used for immunohistochemistry. This has now been replaced by rabbit serum against NiV .Virus isolation from respiratory secretions, urine, cerebrospinal fluid or other tissue specimens must be done in a BSL-4 laboratory. The cell line of choice for both NiV and HeV is the Vero cell line. Pteroid bat cell lines have also been developed . CytopatOther tests that may be used include sequencing, which is used for virus characterisation and electron microscopy. However, they are infrequently available and are inappropriate for primary diagnosis.IgM antibody in serum or CSF is used for diagnosis. Detection of IgG antibodies is a good test for surveillance in humans and for the identification in reservoir animals during epidemiological investigations. It has also been used for diagnosis in humans during outbreaks.It is the most commonly used test for serological diagnosis due to its high sensitivity, rapidity, ease and safety of use. ELISAs for the detection of IgG and IgM developed by the CDC were used in the confirmation of diagnosis in Malaysia . It has This is considered the gold standard test but requires the use of a BSL-4 laboratory. In this test, test sera are incubated with the virus and then allowed to infect Vero cells. Positive sera block the development of cytopathic effects and tests can be read at 3 days. A modified neutralisation test which can be read at 24 h has been developed . Here, tPseudo typed viruses can be used to perform a surrogate neutralisation test. A pseudo typed virus is an enveloped virus with one or more foreign envelope proteins. These viruses can be handled safely in the BSL-2 laboratory but contain NiV envelope proteins capable of being neutralised by positive sera .In non-endemic areas where NiV outbreaks have not occurred, a high index of suspicion is required for rapid identification and containment of an outbreak. The NCDC, India has issued guidelines on the definitions of a Suspected, Probable and Confirmed case of NiV infection  which haPatients must be isolated and rigorous infection control practices implemented. Treatment of NiV infection is primarily supportive- maintenance of airway, breathing and circulation. Fluid and electrolyte balance is maintained. Patients with severe pneumonia and acute respiratory failure must be supported by mechanical ventilation. Invasive mechanical ventilation is preferred.et al. [et al. [in vitro [Ribavirin, which is effective against other Paramyxoviruses  was used to treat infected patients in Malaysia. Chong et al.  reported [et al.  found no [et al. . However [et al. . Acyclov [et al. . Chloroq [et al. . The natin vitro . Favipirin vitro . Neutralin vitro . Use of in vitro .Disease surveillance is carried out regularly in the Nipah belt in Bangladesh. Surveillance activities consist of event-based and sentinel surveillance. Print and electronic media surveillance is carried out in 10 national newspapers and eight national news channels and hotlines have been set up for healthcare personnel to report outbreaks. Suspected outbreaks and deaths due to unknown causes are rapidly identified through these methods. Under sentinel surveillance clusters of encephalitis are investigated. Clusters are defined as two or more cases within 21 days and half an hour's walk from each other . A team Efforts towards prevention have primarily focussed on the prevention of contamination of date palm sap, increasing awareness about the dangers of consuming date palm sap and prevention of person-to-person spread. The use of skirts to cover the sap producing areas of date palm trees has been found to effectively prevent contact with bats . In 2015Prevention of person-to-person transmission includes the implementation of infection control practices such as isolation of patients, use of personal protective equipment and good hand hygiene practices. Contacts identified through contact tracing are tested and kept under observation until they test negative. Hospital surfaces have been found to be contaminated by NiV around patients . HealthcA number of vaccine strategies have been developed for NiV, several of which have been tested in animal models. The most studied approach has been a subunit vaccine based on G glycoprotein (sG) of NiV and HeV. HeV-sG elicits a cross-protective immune response against both HeV and NiV . It has NiV has emerged as a deadly zoonotic disease. Bats, the natural reservoir of the virus, are effective at virus dissemination and human outbreaks continue to be reported regularly. Due to the worldwide distribution of bats, outbreaks in new areas are likely to occur. The high case fatality rate and acute course of disease make the infection difficult to diagnose. This is further compounded by the lack of easily available low-cost diagnostic tests and facilities equipped to handle viral samples. Effective treatment and prophylaxis are unavailable due to a lack of studies in human subjects because the overall case burden is small and the course of infection is acute. The recent outbreak in India highlights the possibility of potential spillover events in areas where currently known risk factors do not exist. Establishment of surveillance systems for NiV is necessary, particularly in South and Southeast Asia. There is an urgent need for countries in South and Southeast Asia to work together to strengthen surveillance systems in order to monitor spillover events and prevent transmission. A better understanding of bat ecology and the causes of spill-over events, the development of effective treatment and prophylaxis for humans and animals and strengthening of surveillance systems to prevent outbreaks is required to curb the threat posed by NiV.


But the hopping conducting mechanism of boron doping a-C (a-C:B) is still mysterious. To exploring the intrinsic reasons behind the semiconductor properties of a-C:B, in this work, the boron doping a-C (a-C:B) nano-film was prepared, and the growth rate and Eopt changing were analyzed by controlling the different experimental conditions of magnetron sputtering. A rapid deposition rate of 10.55 nm/min was obtained. The Eopt is reduced from 3.19 eV to 2.78 eV by improving the substrate temperature and sputtering power. The proportion of sp2/sp3 increasing was uncovered with narrowing the Eopt. The shrinking Eopt can be attributed to the fact that boron atoms act as a fluxing agent to promote carbon atoms to form sp2 hybridization at low energy. Furthermore, boron atoms can impede the formation of σ bonds in carbon atom sp3 hybridization by forming B–C bonds with high energy, and induce the sp3 hybridization transfer to sp2 hybridization. This work is significant to further study of amorphous semiconductor films.Amorphous carbon (a-C) films are attracting considerable attention to due their large optical band gap (E Carey et al. described a-C as a conductive sp2 phase embedded in electrically insulating sp3 matrix [2 hybridization is preferred to narrow the Eopt [2 and sp3 hybridization are still unclear, and further study to uncover the basis of the semiconductor properties of a-C is required.Semiconductor films are the most common form of amorphous carbon (a-C) films, owing to the superior mechanical, optical, and opto-electronic properties ,2,3,4,5. to 4 eV ,7. Thus,le light ,9. This osphorus , which cosphorus ,12. For  content . The readization ,14. A hiC (ta-C) , and the3 matrix . Furtherthe Eopt . However3 content in boron-incorporated ta-C (ta-C:B) [2 hybridization. Schenk et al. among others found that sp3 hybridization becomes dominated with increasing boron concentration in boron doping hydrogenated a-C film [3 hybridization and C–H bonds in hydrogenated a-C [3 hybridization decreases with increasing boron content. For example, Tan et al. found that sp3 content decreased from 73.8% to 42.5% with boron concentrate increasing from 0.59% to 6.04% in boron-incorporated a-C (a-C:B) [opt of a-C:B was almost unchanged with a boron content below 10%, but that Eopt decreased with higher boron content, i.e., more than 10%, and they considered that the decreased Eopt was not caused by an increased sp2 content [opt of a-C, and to understand the inherent reasons behind the interconverted mechanism of sp2 and sp3 hybridization in a-C:B.Boron has one fewer electrons than carbon, which allows it to generate p-type doping in a-C ,18. Boro(ta-C:B) ,19, and (ta-C:B) . In genea-C film . Nastasiated a-C . However (a-C:B) . Meanwhi content . Until nopt was analyzed at a fast growth rate. The interconvert between sp2 and sp3 in a-C:B films were analyzed under different experimental conditions of increasing boron target power, carbon target power, substrate temperature, and working pressure. Finally, a reason for the doping boron atoms inducing to form sp2 hybridization is put forward. This work is significant in the development of new functional amorphous semiconductor films.In this work, a-C:B nano-films were prepared by magnetron sputtering. The most superior growth rate was determined to obtain a maximum thickness of 5 μm, and the Eopt for a-C:B films was obtained via the Tauc relationship, (αhν)2 = c(hν−Eopt), where α is the absorption coefficient, c is a constant, and hν is photon energy [Magnetron Sputtering  was used to prepare the a-C:B films. The substrate material was glass, and both the boron and carbon targets were pure to 99.999%. The distance between the substrate and target was 55 mm in all cases. The working gas was Ar. Synthesized films were tested by X-ray photoelectron spectroscopy  with a focused Al Kα radiation (1486.6 eV), scanning electron microscopy . High resolution transmission electron microscopy ) was performed to determine the morphology. Raman spectra were studied on a Renishaw  in Via micro-Raman spectrometer at room temperature with a 532 nm laser. The thickness of films was analyzed by atomic-Profiler  to determine the deposition rate. Optical transmittance spectra were measured with a UV-3150 double-beam spectrophotometer  in the range from 300 nm to 900 nm. The En energy .The deposition rate related to the experimental conditions is shown in The deposition rate relates to the sputtering power of boron and carbon targets, indicating an undulant curve b. The sp2O3 on the surface, which is due to surface oxidation. The morphology of a-C:B films is granulated with a nano size of about 30–80 nm in SEM results  with increasing the temperatures from 200 °C to 400 °C. Thus, temperature may not directly influence the electronic structure, in contrast to changing the content of defect or sp2 cluster size [opt narrowed from 3.19 eV to 2.90 eV  appears at a high temperature, i.e., 300 °C   b,d,f. Annductors . The naropt is mainly attributed to the increase in sp2 content. This is consistent with previous reports [opt, and the supplied energy is the crucial to the generation of sp2 hybridization. We propose the following reasons for this narrowing Eopt: (a) with lower energy, the boron atoms only act on weakly π bonds of sp2 hybridization in carbon atoms; hence, boron atoms can’t react with carbon atoms, and boron atoms act as mixture in a-C:B films. However, the mixture of boron atoms can also serve as a fluxing agent for carbon to form sp2 clusters due to the fact that boron has a lower melting point (about 2075 °C) compared to carbon (3550 °C). Thus, boron atoms can still promote the formation of sp2 clusters with carbon; (b) with higher energy, the boron atoms are able to act on σ bonds in sp3 hybridization to form B–C bonds with terminate edges, and can also induce sp3 hybridization transfer to sp2 hybridization. The intrinsic reason for this is that the π bonds are the weakest, and σ bonds in sp2 hybridization are stronger than σ bonds in sp3 hybridization; thus, boron atoms can influence different carbon bonds with different energies.Thus, the narrowing of the E reports . Howeveropt were obtained in a-C:B films. Increasing boron and carbon target powers can increase the sp2 content to narrow the Eopt from 3.19 eV to 2.78 eV. The content of boron may not the most important factor in the modulation of the Eopt, but the supplied energy is crucial in generating sp2 hybridization. So, these results show that boron atoms can act as a fluxing agent to promote the formation of sp2 clusters at lower energy with carbon. Also, boron atoms prefer to act on carbon σ bonds in sp3 hybridization to induce a sp3 hybridization transfer to sp2 hybridization under higher energy. This work is significant to advances the present understanding of a-C and the preparation of new amorphous films.In this work, variations of E


Three Komondor dogs in a small family and 3 sporadic cases exhibited a constellation of signs that included juvenile-onset of failure-to-thrive, inappetence, vomiting and/or diarrhea, and weakness. In each we documented dyshematopoiesis, increased anion gap, methylmalonic acidemia/-uria, and serum cobalamin deficiency. Urine protein electrophoresis demonstrated excretion of cubam ligands. All clinical signs and metabolic abnormalities, except proteinuria, were reversed by regular parenteral cobalamin administration. The pattern of occurrence and findings in the disorder suggested an autosomal recessive inheritance of cobalamin malabsorption with proteinuria, a condition in humans called Imerslund-Gräsbeck syndrome. The purpose of this study was to determine the molecular cause of this disorder in Komondors.CUBN intron 55 splice donor consensus sequence (NM_001003148.1: c.8746 + 1G > A) that was homozygous in affected dogs and heterozygous in the unaffected parents. Alleles of the variant co-segregated with alleles of the disease locus in the entire family and all more distantly-related sporadic cases. A population study using a simple allele-specific DNA test indicated mutant allele frequencies of 8.3 and 4.5% among North American and Hungarian Komondors, respectively.Whole genome sequencing of two affected Komondor dogs of unknown relatedness and one parent and a clinically-normal littermate of an affected dog revealed a pathogenic single-base change in the DNA testing can be used diagnostically in Komondors when clinical signs are suggestive of cobalamin deficiency or to inform Komondor breeders prospectively and prevent occurrence of future affected dogs. This represents the third cubilin variant causing inherited selective cobalamin malabsorption in a large animal ortholog of human Imerslund-Gräsbeck syndrome.The online version of this article (10.1186/s12917-018-1752-1) contains supplementary material, which is available to authorized users. SuccessIn addition to the distal small intestine, cubam is expressed in renal proximal tubular epithelial cells. In the kidney, cubam mediates reabsorption of a variety of protein ligands, such as albumin, apo AI (involved in lipid metabolism), haptoglobin, and vitamin D-binding protein from the glomerular filtrate –9. ThereCUBN and AMN are found causing hereditary selective intestinal cobalamin malabsorption with mid- to low-molecular weight proteinuria, an autosomal recessive trait also known as Imerslund-Gräsbeck syndrome (I-GS) [AMN or CUBN variants are breed-specific. Description of cobalamin malabsorption in dogs caused by AMN variants (OMIA 000565–9615) and CUBN variants (OMIA 001786–9615) is curated in the Online Mendelian Inheritance in Animals data base (https://omia.org).The functional cubam complex is composed of cubilin (CUBN) and amnionless (AMN) subunits . The rece (I-GS) , 22. ThoWe describe here autosomal recessive selective intestinal cobalamin malabsorption with proteinuria in Komondor dogs characterized by failure to thrive, dyshematopoiesis, and metabolic disturbances during the juvenile period, as occur in I-GS of other dog breeds and human patients. The purpose of this study was to determine the molecular defect underlying I-GS in Komondors and to determine the disease-allele frequency in Komondor populations of Hungary and North America.Owners of affected Komondors first noted signs of failure-to-thrive between 2.5 and 5 months of age that were progressive until institution of parenteral cobalamin administration. Typical clinical signs included inappetence, vomiting, diarrhea, failure to thrive, and weakness. One dog exhibited a fine head tremor, and another had intermittent seizures. On physical examinations the puppies were lethargic and underweight but of near-normal linear size.Routine complete blood counts revealed a non-regenerative, normocytic, normochromic anemia or low-normal hematocrit. Neutrophil counts were between 900 and 3700/μL  and some appeared toxic and/or hypersegmented. The dogs also had a mild thrombocytopenia. Serum chemistry indicated mildly increased blood urea nitrogen, normal serum creatinine concentrations, and increased anion gaps, suggesting a metabolic acidosis. Serum bile acid concentrations were normal. Serum cobalamin concentrations were undetectable (<150 ng/L) or below the lower limited of normal (250 ng/L). Urinalysis showed mild to moderate proteinuria by dipstick (2+ to 4+ in concentrated urine). Urine metabolic screens showed strongly positive methylmalonic acid (MMA) spot test results, and quantitative organic acid analyses revealed extremely high MMA concentrations of 20,000–37,000 mmol/mol creatinine . The mid- to low-molecular weight proteins found in urine from affected dogs were identified as cubam ligands, including transferrin, albumin, vitamin D binding-protein, haptoglobin, and apo A1 .Detailed clinicopathological information was available from 5 affected dogs, 3 males and 2 females that presented over a period of 12 years from both coasts of the United States and the Midwest  administered subcutaneously or intramuscularly led to rapid clinical responses. One affected dog was treated with 800 μg of cyanocobalamin spray administered to oral mucous membranes and responded well and rapidly. Cyanocobalamin administration continued with subcutaneous injections every other week to monthly or with sublingual spray daily, and the dogs remained clinically asymptomatic. Each affected puppy rapidly gained weight and became active, and all other clinical signs resolved, including tremors and seizures. The seizing dog’s phenobarbital and potassium bromide treatments were discontinued without seizure recurrence after initiating cobalamin treatment. All laboratory abnormalities resolved except for the ligand-specific mild to moderate proteinuria, recurring low serum cobalamin concentrations, and moderately elevated serum MMA concentrations.CUBN or AMN [CBLIF locus (previously designated GIF) and thus far have only been reported in human patients [Clinical, hematological, and biochemical signs compatible with cobalamin deficiency and mild proteinuria suggested dysfunction of cubam in both ileal brush border and renal proximal tubular epithelia, but there was also the possibility of IF dysfunction accompanied by proteinuria of unrelated cause , 23. CubN or AMN –20 that patients , 23. To AMN and a second informative SNV ~ 2.1 kb telomeric of the 5′ end of AMN  they created segregated with the disease locus  of the affected dogs: c.8C > G (p.Ala3Gly) and c.313C > T (p.Leu105Phe). Both variants were also homozygous in the clinically normal sire of 1 of the affected dogs, and both variants were considered tolerant by SIFT [CBLIF (NM_001005759.1) that we considered nonpathogenic because, while homozygous in an affected dog, they were also homozygous in his clinically normal sire: c.41C > T ; c.913G > A ; and c.1074-10_1074-2delAATCTTGCA (does not change the splice acceptor site of intron 7). A variant c.244A > G (p. Ser82Gly) was heterozygous in both an affected dog and his sire. All the CBLIF missense variants were considered tolerant by SIFT. There were no disruptive or missense variants of TCN2, encoding transcobalamin, in any affected dog sequenced.Whole genome sequences of 3 I-GS affected, the sire of 1 of them, and a clinically normal Komondor (20–25 coverage each) were compared to the CanFam 3.1 canine reference genome sequence. This revealed 2 homozygous missense variants in  by SIFT . SimilarCUBN (NM_001003148.1) we observed 2 homozygous SIFT-tolerant variants in the affected dogs that were also homozygous in the clinically normal sire and, therefore, which we did not consider pathogenic: c.6201C > A in exon 41  and c.7175 T > C in exon 46 . The clinically normal Komondor’s sequence had an additional homozygous variant c.9362G > C , which was tolerant by SIFT analysis.In CUBN variant  at genomic position chr2:g.19,981,457 (CanFam 3.1), which was homozygous (A) in the affected dogs, heterozygous (A/G) in the clinically normal sire, and wildtype sequence (G/G) in the normal Komondor. This variant obliterates a splice donor consensus sequence in intron 55, predicting an abnormal RNA splicing pattern, and was thereby considered pathogenic. We confirmed the variant in all 6 affected Komondors by Sanger sequencing , including WGS of 282,822 healthy humans, revealed 38 variants in the + 1 position of the 66 intronic splice donor sites (frequency of 2.0 × 10− 6). None were homozygous.However, and of particular note, there was a Database   and 5 of 56 Komondors from Hungary  were heterozygous . Pedigree analyses revealed distant relatedness among some carriers in Hungary and to some Komondors in North America, including 1 of the 3 sporadic cases  of cyanocobalamin corrected the metabolic disturbances of cobalamin deficiency in affected Komondors within 2–3 days and began correction of the hemogram over the ensuing few weeks. Affected dogs recovered normal weight-for-age within 2 months. Following treatment, however, the initial supernormal serum cobalamin concentration, which may be 2–5-fold greater than the high end of the normal reference range, fell rapidly to below the normal range in about 2 weeks. This is because serum cobalamin is bound entirely to transcobalamin in dogs  and is rAlso demonstrated here in 1 of the affected Komondors (case 2), a daily dose of cobalamin (800 μg/day) administered by sublingual spray can be a successful treatment, but such a regimen does not produce a superior effect and costs more. The mechanism by which a few micrograms of an oral cobalamin megadose is absorbed in the absence of functional IF or cubam is unknown, but the phenomenon of IF-independent cobalamin absorption from the proximal small intestine exposed to large doses was observed in dogs long ago , 33. TheAMN and CUBN each present difficulty. AMN is GC rich (65–70%) throughout the gene (GenBank® accession no. KF445236), so much so that the canine reference genome has large gaps in the AMN locus and is missing several exons. The CUBN gene is large, with 67 exons extending over 259 Kb, and again, the CanFam 3.1 reference genome is missing exon 32 (compare GenBank® accession no. AF137068 to reference genome). Of greatest concern was that without genetic positional information, the inevitably numerous sequence variants observed by WGS would be difficult to interpret. We excluded CBLIF and AMN as disease loci because alleles of internal or flanking markers did not segregate in our study kindred with alleles of an autosomal recessive disease locus.We opted for genetic exclusion analyses prior to whole genome sequencing in this instance because, while the clinical disorder suggested few candidate genes, sequencing of The all-important assumption of autosomal recessive inheritance depends on the definition of the trait under study, which in this case is juvenile-onset cobalamin deficiency with compatible clinical signs and ligand-specific proteinuria. This definition does not rule out the possibility that heterozygous Komondors may have subclinically reduced cobalamin absorption. However, heterozygotes in this study were healthy, had serum cobalamin concentrations equally within the normal range as normal control dogs (data not shown), and had no detectable urinary cubam ligands lane 2, Fig. , Fig. 2.CUBN by WGS, we dismissed them as causative, because they were tolerated by SIFT and were also homozygous in clinically healthy Komondor dogs. However, the intronic CUBN c.NM_001003148.1; c.8746 + 1G > A variant was only homozygous in affected dogs and is likely pathogenic because it disrupts the splice donor consensus sequence of intron 55. The G nucleotide in that position is 100% conserved in mammalian introns [CUBN mRNA expression [CUBN c.NM_001003148.1; c.8746 + 1G > A variant is a highly likely pathogenic null allele. The small amount of uncertainly that remains due to lack of direct experimental evidence of an effect on CUBN mRNA or protein expression will be further reduced as more Komondor I-GS cases come to light and are genotyped.While we found several missense variants in  introns . Loss ofpression , most lipression , and faipression , we concAMN or CUBN create embryonic lethality [AMN and CUBN, including early (5′) frameshift variants and large deletions of the entire CUBN locus, have been described without evidence of embryonic or fetal developmental abnormalities [CUBN variants that cause I-GS in humans are clustered in the first 28 exons, which encode regions important for IF-cobalamin binding and AMN-mediated membrane localization and internalization , [CUBN variants of dogs are 2 single-base deletions (c.786delC in Beagles and c.8392delC in Border Collies), and the splice-site variant reported here, each predicting a frameshift [In mice, targeted genetic disruptions of either ethality , 39. In ng early ′ frameshmalities –20. Repong early ′ frameshameshift –20. Two 3 concentrations in urine, due to failed tubular reabsorption of vitamin D binding protein, and ~ 55% decreases of serum 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 concentrations in serum [The ascertainment of human I-GS patients suggests that cobalamin malabsorption is the single aspect of cubam dysfunction that alerts medical clinicians to the need for treatment intervention and molecular investigation. Urinary loss of the cubam ligands, albumin and vitamin D-binding protein, may cause phenomena such as low-grade tubular albuminuria , 40 or ain serum . Despitein serum , 41–43.CUBN c.NM_001003148.1; c.8746 + 1G > A variant permitted design of simple RFLP and Taqman® assays to differentiate between homozygous affected, asymptomatic heterozygous, and wildtype healthy Komondors. While we did not pursue a randomized survey, screening of Komondors in North America revealed an 8.3% prevalence of the disease-allele in this small population. Moreover, surveying Komondors in Hungary, the breed’s country of origin, revealed a disease-allele prevalence of 4.5%. Common ancestors trace back to dogs born in Hungary in the early 1970s. Thus, the I-GS allele has been segregating in Komondors for decades and is widespread in the breed worldwide. The higher allele-frequency in North America could be due to a founder effect subsequent to importation of a popular sire or dam.The CUBN splice-site variant  segregating among Komondors. The disease-allele appears to be widespread among Komondors in North America and Hungary. Simple and reliable allele-specific DNA tests are available so that Komondors with clinical signs suggestive of I-GS may be diagnosed early and treated effectively. We also recommend that Komondors intended for breeding be tested to identify carriers and prevent production of additional affected dogs.Breed-specific cubam mutations that cause life-threatening cobalamin deficiency in dogs now include a These investigations included clinical case studies and/or laboratory analyses of samples from 4 I-GS affected Komondors, their parents, and 6 healthy littermates in 1 nuclear family plus 3 affected Komondors that occurred as sporadic cases and 1 of their sires. Owners submitted samples for DNA isolation and genotyping from 98 additional clinically-normal Komondors in North America and Hungary to assist with their breeding programs. Additionally, we studied DNA samples from 100 dogs of other breeds that were in laboratory archives. Blood and urine sampling from Komondors were according to principles and protocols approved by the respective Institutional Animal Use and Care Committees at Michigan State University, University of Pennsylvania, and University of Tennessee. Owners and local veterinarians submitted case histories and samples for routine diagnostic tests such as blood tests, DNA isolation, metabolic analysis, and urinalysis from dogs of Komondor kindreds or sporadic cases. Pedigrees were gathered to determine shared ancestry. Characterization of cobalamin-deficient dogs included clinical signs, medical history, routine complete blood counts, serum chemistry panels, serum cobalamin and folate concentrations, examination of bone marrow aspirates, and routine and special urinalyses.In affected dogs, excess methylmalonic acid excretion in urine was documented either qualitatively by a specific reaction with diazotized p-nitroaniline or quantitatively by gas chromatography-mass spectroscopy. Urine proteins were concentrated by centrifugation through a 10 kDa molecular weight cut-off membrane and separated by one-dimensional SDS-polyacrylamide gel electrophoresis, and protein identities were confirmed by antibody reactivity on immunoblots, as previously described , 17, 18.CBLIF, CUBN and AMN were chosen from a canine reference genome sequence  as viewed on the University of California Santa Cruz genome browser [2, and 2 U Taq polymerase  in 50 μL reactions. For higher GC templates 1 M betaine was added to the reaction, as noted in Table 1; Additional file Genomic DNA was prepared from buccal brushes or EDTA-anticoagulated whole blood by standard methods . Single  browser  with Rep browser  and Sing browser . Howeverhttp://www.ncbi.nlm.nih.gov/bioproject/486818 and BioSample accessions: SAMN09865705, SAMN09865706, SAMN09865707, SAMN09865708, SAMN09865709; Release date: 2019-01-01.DNA samples of 5 Komondor dogs, including 3 affected dogs of widely separated ancestry, 1 clinically normal sire, and 1 clinically normal littermate of an affected dog were prepared for WGS. Genome libraries were prepared using Illumina® TruSeq Nano LT Kit . Briefly, genomic DNA was fragmented, end-repaired and adenylated before ligation with the corresponding unique index adapters and amplified for WGS. All samples were sequenced on the Illumina HiSeq 2500 platform by paired-end sequencing. Average genome sequence coverage for each dog was 20-25X  analysis [Genomic data were searched for variants using the CanFam 3.1 reference sequence with a focus on the potential candidate genes analysis  and reseCUBN c.NM_001003148.1; c.8746 + 1G > A  was genotyped in all members of the I-GS Komondor kindred and sporadic cases using PCR amplification primers as listed in Table 1; Additional file The sequence variant Additional file 1Format is Adobe Portable Document Format (.pdf), titled Detailed clinical descriptions and course of disease before and following specific treatment. The file summarizes 4 clinical cases of I-GS in young Komondors as they were presented to veterinary clinicians across the USA. (DOCX 22 kb)Additional file 2Format is Adobe Portable Document Format (.pdf), titled Genetic exclusion markers. The file contains 2 tables: Table 1 describes genetic exclusion markers used to query biological candidate genes for segregation of alleles with the disease locus, and Table 2 shows alleles of the markers described in Table 1 in each member of a family of Komondors segregating I-GS. (DOCX 33 kb)