Nicotiana benthamiana with the binary vector pCambia-crTMV-CPPLRV. The significant levels of the chimeric virus enabled direct visualization of crTMV-CP-PLRV in the cell and to investigate the mechanism of the pathogenesis. Localization of the crTMV-CP-PLRV in plant cells was examined by immunoblot techniques, as well as light, and transmission electron microscopy. The chimera can transfer between vascular and nonvascular tissues. The chimeric virus inoculum is capable to infect N. benthamiana mechanically. The distinguishing feature of the chimeric virus, the RNA virus with the positive genome, was found to localize in the nucleolus. We also investigated the role of the N-terminal sequence of the PLRV P3 coat protein in the cellular localization of the virus. We believe that the gene of the PLRV CP can be substituted with genes from other challenging-to-study plant pathogens to produce other useful recombinant viruses.A genetically engineered chimeric virus crTMV-CP-PLRV composed of the crucifer-infecting tobacco mosaic virus (crTMV) RNA and the potato leafroll virus (PLRV) coat protein (CP) was obtained by agroinfiltration of  Polerovirus, family Luteoviridae. It is a highly pathogenic, phloem-limited cytoplasmic virus that accumulates in infected plants in low quantities. The PLRV genome encodes two coat proteins (CP)—the major protein P3 (22.3 kDa) and the minor protein P5 (55 kDa), which is an extended version of the P3 protein produced by translational read-through [Physalis are 400 and 1300 ng/g leaf [Potato leafroll virus (PLRV) is the type species of the genus -through . PLRV, lotein P3 2.3 kDa a-through  and beca-through ,7. In pl-through ,9,10. Thg/g leaf ,12, respPLRV to overcome the problem of the low PLRV yield; its complete nucleotide sequence (14410 bp) is presented [N. benthamiana with this binary vector resulted in the multiplication of the tobamovirus RNA and PLRV CP, and the formation of spherical chimeric virus particles tentatively called a chimeric virus, crTMV-CP-PLRV. The morphology, size, and antigenic specificity of the wild-type PLRV and the purified chimeric virus were found to be similar. The yield of crTMV-CP-PLRV was about a thousand times higher than the WT PLRV [N. benthamiana, the chimeric virus genome is first transiently expressed in the mesophyll tissue through RNA synthesis in all transfected cells. As with the transport of similar viruses in the infected cell [In our previous study , we conssequence 410 bp isresented , in whic WT PLRV ,12. The  WT PLRV . We belited cell , we hypoted cell ,19. We pted cell . The higDr. Yu. Varitzev  kindly provided tube potato plants infected with wild type PLRV (Canadian strain). Antiserum to chimeric PLRV obtained as described . AntibodPLRV, which produced hybrid virus particles, crTMV-CP-PLRV, of chimeric crTMV RNA encapsidated by PLRV CP, was engineered by replacing the crTMV CP gene with the PLRV 22.3 kDa CP (ORF 3) gene [Binary vectors—The crTMV-based vector, pCambia-crTMV-CP 3) gene .PLRV, where the PLRV CP gene was substituted with three different genes coding GFP and its two modifications (will be published elsewhere). Three control vectors were constructed similar to pCambia-crTMV-CPN. benthamiana plants were grown in a chamber with a cycle of 16 h light at 20 °C and 8 h dark at 15 °C.Plant growth—Agrobacterium tumefaciens strain GV-3101 was chemically (20 mM CaCl2) transformed [PLRV. Bacteria were grown in 2YT medium containing antibiotics: rifampicin, gentamycin, and kanamycin at 50, 25, and 100 µg/mL, respectively, at 28 °C overnight. The cells were collected and re-suspended in agroinfiltration buffer  to a density of 0.6 at OD600. Approximately 200 µl of the transformed bacterial suspension was infiltrated into the abaxial side of N. benthamiana leaves, using a syringe without a needle, mostly using 10 plants at 5–6 leaf stage. The injected mixture also contained agrobacterium harboring a plasmid expressing the tomato bushy stunt virus p19, a potent protein-suppressor of RNA silencing [Agroinfiltration—emically  mM CaCl2N. benthamiana were mixed with the three-times the weight of 10 mM Tris-HCl (pH 8.0) or 10 mM phosphate buffer (pH 7.5) in a chilled mortar and were homogenized with a pestle with Celite® R 281 . Ten uninfected young, at the 5–6 leaf stage, plants with very thin and soft cuticles were used for mechanical transfection with the sap obtained above. Celite powder and 20–30 µL of homogenized sap from the infected plant were rubbed into 1–2 leaves of each plant. As rubbed areas on leaves often become dry, we prevented covering the plant with polyethylene bags to allow the recovery of the cuticle. Symptoms of infections appeared 6 days after inoculation. In parallel, plants were inoculated in the same manner with 20 µg of the chimeric virus previously purified and stored at −20 °C. As a control, WT PLRV was rubbed into 10 young Nicotiana plants.Mechanical transmission—First, fresh leaf tissue homogenate of an agroinoculated plant (6–7 days post agroinfiltration) was obtained. For this purpose, leaves of 5–6 leaf stage We used quantitative DAS-ELISA to compare the mechanically infected and uninfected plants. For this purpose, 5 mm diameter disks from three leaves of two sample plants with a total mass of approximately 45 µg were obtained with the hole-punch. These discs were ground in 5 µl of PBS-0.05% Tween20 buffer with 1 mL Potter homogenizer. Then, the homogenate was diluted 20-fold with the same buffer and clarified by centrifugation at 1000 rpm for 5 min. Serial double dilutions of the supernatant and the isolated crTMV-CP-PLRV (1 µg/mL) were transferred into the MaxiSorp immune plates (Nunc) covered with antibodies against the chimeric virus and incubated overnight at 4 °C. After washing, the of antibody-virus complex was analyzed with the secondary antibodies conjugated with the horseradish peroxidase as described .Working with the artificial chimeric virus, we were guided by the general rule that infectious material should be sterilized, and the infection should be killed. We ensured that no virus infection would to go outdoors from the lab.N. benthamiana were stripped with forceps and scraped off partially with a small brush. These leaves were then pressed onto Protran BA 85 0.45 µm nitrocellulose membrane filters  under a 400 g weight for 15–20 s. Then, the leaf blots were placed in Petri dishes and incubated for one hour at room temperature with TBS-T buffer  containing 5% membrane blocking agent . Membranes were subsequently washed three times for 10 min at room temperature in TBS-T and incubated overnight at 4 °C with antiserum to the chimeric virus diluted 1:15000 with TBS buffer (TBS-T without Tween). Then the membranes were washed three times for 10 min with TBS-T and incubated for 1 h at room temperature with goat anti-rabbit secondary antibody conjugated to alkaline phosphatase . Detection performed using a chromogenic substrate of alkaline phosphatase 5-Bromo-4-chloro-3-indolyl phosphate and tetranitroblue tetrazolium (BCIP/NBT) [Leaf tissue blot-immunoassay—The epidermis of leaves of CIP/NBT) . Petiole cross-sections immunoblot analysis— Several prints of N. benthamiana petiole cross-cuts agroinoculated with the chimeric virus or wild type CMV (cucumber mosaic virus), as a control virus that causes a systemic infection, were obtained on 0.45µm nitrate cellulose membranes. Membranes were treated with the blocking agent, incubated with commercial virus-specific antibodies, followed by secondary antibodies, and then analyzed with the chromogenic substrate substantially as described above.N. benthamiana plants were fixed with 2.5% glutaraldehyde solution  in 0.1 M Sorensen’s phosphate buffer (pH 7.2) and 1.5% sucrose. After washing out the residual glutaraldehyde, the plant material was stained with 1% osmium tetroxide , and dehydrated in ethanol through stepwise increasing concentrations of 30, 50, 70, 96, and 100%. Residual ethanol was then removed with propylene oxide , and the leaf tissue was embedded in a mixture of epoxy resin and Epon 812 Araldite  according to standard procedures [Sample preparations for microscopy— Six days after agroinfiltration, small leaves of the fourth and fifth tier of healthy and infected ocedures . For analysis by light-optical microscopy, semi-thin (2.1 µm) tissue sections mounted on glass slides were stained with a 0.1% aqueous solution of methylene blue  and embedded in epoxy resin. Slides were analyzed with the Olympus BX51 microscope  and photographed with a Color View II camera . Ultrathin sections were prepared using a diamond knife on an LKB-V ultramicrotome . They then stained with 5% aqueous uranyl acetate and Reynolds lead citrate , and vis1MSTVVVKGNVNGGAHQPRRRRRQSLRRRANRVQPVVMVTAPGQPRRRRRRRGGNRRSRRTGVPRGRGSSE70 . Regarding the bipartite nucleus/nucleolus PLRV CP signal, one can conclude that the second sequence, 45RRRRRRRGGNRRSRR59, is a stronger signal for nucleolar localization than the first sequence, 18RRRRRQSLRRR28. We deleted 15 nucleotides in the CP gene of pCambia-crTMV-CPPLRV, encoding five arginine residues (aa 18–22), to produce pCambia-crTMV-CPPLRV(Δ5R) using overlapping PCR with two pairs of primers: 1)5‘-ttctcgagatgagtacggtcgtggttaaaggaaacgtcaacgg-3‘ PLRV-CP (+)2)5‘-ttggtaccctatttggggttttgcaaagcca-3‘PLRV-CP (-)3)5‘-ggtggtgcacaccaaccacaatcccttcgcaggcgc-3‘ PLRV (+)4)5‘-gcgcctgcgaagggattgtggttggtgtgcaccacc-3‘ PLRV (-),Genetic engineering of the nucleolar localization sequence (NoLS)—The amino-terminus of PLRV CP has the sequence: Where  represent coding and complementary nucleotide sequences. N. benthamiana, this vector showed limited accumulation in Nicotiana. It did not move systemically, likely because the TMV CP is essential for TMV long-distance movement, and PLRV CP was unable to complement this function as was hypothesized. Additionally, Barker et al. [N. benthamiana transformed with Agrobacterium tumefaciens containing the plasmid pBNUP110 or in the plant infected by rubbing the PLRV transcripts onto carborundum-dusted leaves. Again, the PLRV yield was no more than 1,600 ng/g of the transgenic plant, even when mixed with PVY infection.Earlier , a vector et al.  examinedThere are several differences between these constructions and those made by us, including different strains of TMV, deletion of regulatory sequences, absence of the P5 protein, and different movement proteins. Additionally, we employed a more efficient method of plant infection using agroinfiltration with the powerful P19 silencer , insteadN. benthamiana, we examined the dynamics of the systemic infection spread in the host plant and the chimeric virus localization.To characterize interaction between the chimera obtained by us and N. benthamiana. After two-to-three days following infiltration with pCambia-crTMV-CPPLRV, the inoculated region of N. benthamiana became necrotic. Moreover, six days post-agroinfiltration, petioles of the upper tier of leaves became much thinner in addition to showing necrosis. The leaves associated with these petioles were shrunken and were smaller in size .We also found that the inoculum of the chimeric virus, but not WT PLRV, can infect mechanically, causing a very similar infection for all ten ltration b. This oiana see . Our calSolanum sarrachoides mechanically. Both of these findings were consistent with Peter et al. 2009 , who shoSurprisingly, fresh virus inoculum in plant sap but not purified virus preparations that were kept frozen at −20 °C was infectious mechanically. The likely explanation is the virus RNA degradation before or during its extraction from the frozen chimeric virus . Bald 2 observedN. benthamiana, a representative of the family Solanaceae, has internal and external phloem tissues and large parenchyma cells with large vacuoles, surrounded both xylem and phloem. Companion cells in the leaf stalks are much larger than sieve elements. Their characteristic features are large nuclei and dense cytoplasm because these cells usually have a significantly larger number of ribosomes and mitochondria. In sieve tubes, the cytoplasm is more often seen near the thickened walls as a thin layer. In this study, 5 non-agroinfiltrated plants and 10 agroinfiltrated plants were used in the experiment. Under a low magnification of light microscopy (250×), internal and external phloem cells, companion cells of sieve tubes, xylem, and parenchyma were visible at the base of petioles a. N. benN. benthamiana from uninfected  . BecauseN. benthamiana plants. Infection with crTMV-CP-PLRV dramatically changed phloem cells, sharply reducing the number of cells containing a nucleolus and decreasing the content of chromatin in the nucleus. In the early stages of infection, at 5–7 days post-inoculation, numerous viral particles appeared in the majority of leaf blade cells, i.e., in infected companion cells, cells of phloem, and xylem, and parenchyma cells adjacent to the vascular bundle. Virus particles formed dense clusters, localized predominantly in the nucleolus, nucleus, and at a much lower level in the chloroplasts and vacuoles. Cells of the vascular bundle containing nuclei, i.e., primary phloem cells and companion cells from uninfected and infected plants 6–7 days post-inoculation, were selected for more detailed study. Images of more than 2000 cells of infected plants (341 cells contained nuclei), and more than 500 for control samples of uninfected cells (100 cells contained nuclei), were compared. In this study, three non-agroinfiltrated plants and six agroinfiltrated plants were examined independently, while the earlier-characterized chimeric virus was isolated from the same infected plants. High-resolution transmission electron microscopy (TEM) was used to obtain images of fixed, ultra-thin sections of petioles and veins of fourth-tier leaves of uninfected and infected High-resolution TEM study at the sub-cellular level of crTMV-CP-PLRV infection showed that the virus first appeared in the granular component of the nucleolus a,b. ThisThe chimeric virus continued to accumulate in the nucleolus, displacing the contents of this organelle. The virus then spread out of the nucleolus and filled the entire volume of the nucleus, where it formed multilayered honeycomb-like pseudo-crystal structures a. By thiIn rare instances, we also observed also clusters of the chimera virus particles in chloroplasts a,b.Tobamoviruses of subgroup III, but not in Tobamoviruses of subgroups I and II, including TMV U1 [N. benthamiana, suggesting that the nuclear function of P30 is needed for active systemic infection, but not for the nucleus localization.The TMV U1 MP (P30) lacks nuclear localization signal (NLS) sequences, which are present in the crTMV MP  and whicg TMV U1 ,40. Earlg TMV U1 , this reThe coat protein of PLRV contains in its structure both NLS and NoLS signals . Short sN. benthamiana plants with pCambia-crTMV-CPPLRV(Δ5R) resulted in an active and severe infection very similar to the infection of pCambia-crTMV-CPPLRV, precisely as shown in 45RRRRRRRGGNRRSRR59 targets the coat protein and the virus to the nucleolus. Interestingly, agroinfiltration of five The chimeric virus has features of both viruses. As tobamoviruses, the chimera grows to high concentration. This virus transfers between vascular and nonvascular tissues, and is infectious mechanically. As poleroviruses, it is spherical and is recognized by antibodies to PLRV , its capIt should be emphasized that all other parts of the chimeric virus belong to the cytoplasmic tobamovirus, with the PLRV P3 coat protein being the only polerovirus component of the chimera. It appears the PLRV CP has to be a multifunctional protein as many other virus coat proteins involved in the particle assembly  and longWe propose that the PLRV CP binds to the tobamovirus RNA, thus protecting the transfer form of the virus for moving through phloem tissue and substitutes the tobamoviral CP in systemic transport.Nicotiana benthamiana cells. The fresh chimeric virus inoculum can mechanically transmit to N. benthamiana. The chimeric virus exits phloem tissue.Summarizing the findings from obtained data, we can state that the CP of PLRV can substitute for the tobamoviral CP to produce powerful virus. The major coat protein of the potato leafroll virus targets the chimeric virus with the positive RNA genome to the nucleolus of The chimera is a robust system that gives high CP yields thus overcoming a serious limitation in luteovirid research. We believe that the gene of the PLRV CP can be substituted with genes of other difficult-to-access plant pathogens to produce other useful recombinant viruses.The particles of the obtained chimeric virus may be a fascinating object of study under cryo-electron microscopy  taking i


Over the past decade, pandemics caused by pandemic H1N1 (pH1N1) influenza virus in 2009 and severe acute respiratory syndrome virus type 2 (SARS-CoV-2) in 2019 have emerged. Both are high-impact respiratory pathogens originating from animals. Their wide distribution in the human population subsequently results in an increased risk of human-to-animal transmission: reverse zoonosis. Although there have only been rare reports of reverse zoonosis events associated with the ongoing coronavirus disease 2019 (COVID-19) pandemic from SARS-CoV-2 so far, comparison with the pH1N1 influenza pandemic can provide a better understanding of the possible consequences of such events for public and animal health. The results of our review suggest that similar factors contribute to successful crossing of the host species barriers in both pandemics. Specific risk factors include sufficient interaction between infected humans and recipient animals, suitability of the animal host factors for productive virus infection, and suitability of the animal host population for viral persistence. Of particular concern is virus spread to susceptible animal species, in which group housing and contact network structure could potentially result in an alternative virus reservoir, from which reintroduction into humans can take place. Virus exposure in high-density populations could allow sustained transmission in susceptible animal species. Identification of the risk factors and serological surveillance in SARS-CoV-2-susceptible animal species that are group-housed should help reduce the threat from reverse zoonosis of COVID-19. Emerging viral diseases are an important threat for public health. Manyof these diseases are zoonotic, in the sense that their original source is animals. Sporadically, these emerging viral diseases can lead to pandemics in humans. When such a pandemic occurs, the high number of infected people can in turn form a source of infection for animals: reverse zoonosis. There are 2 main concerns about such reverse zoonosis events. First, the infected animals can become ill and even die; second, the population of animals in question can become a virus reservoir, from which reintroduction into humans can take place.24 There must be sufficient contact between donor species  and recipient species and enough compatibility between the virus and the new host to allow replication and the possibility of transmission to other members of the recipient species. If this transmission can occur, the contact network structure of the recipient species, together with variations in transmission through this network, are critical in determining whether the virus will persist or die out.For any virus spillover between species, including reverse zoonosis, several barriers need to be breached.78 Already there are several reports of reverse zoonosis events, involving domestic cats and dogs, tigers, lions, and American mink  in 2009  is ongoing, and the causative virus, severe acute respiratory syndrome virus type 2 (SARS-CoV-2), is becoming more widely distributed in the human population.can mink .37,38,42 in 2009 . Althoug18 Epidemiologic evidence for human-to-pig transmission was based on pigs testing positive by qPCR (quantitative polymerase chain reaction) for pH1N1 virus RNA after contact with farmers who were infected with pH1N1 virus.17 Sequencing analysis showed that the viruses infecting humans and pigs were highly similar, indicating no virus adaptation was necessary for replication in pigs.63 Since then, pH1N1 virus has repeatedly spread from human to pigs globally, resulting in genetic diversity of pH1N1 viruses in pig populations.35 In pigs infected with pH1N1 virus, mild or asymptomatic respiratory diseases were observed. Histopathologic changes consisted of multifocal broncho-interstitial pneumonia with suppurative alveolitis, suggesting secondary bacterial coinfections. Sustained transmission between pigs has been reported in both natural and experimental studies,67 suggesting that pigs can act as a reservoir for pH1N1 virus.Events of human-to-pig transmission were the most frequently reported reverse zoonosis of pH1N1 influenza. The first cases of human-origin pH1N1 virus infections in a pig farm were reported in Canada just 1 month after pH1N1 influenza had spread worldwide in the human population.61 Evidence of pH1N1 antibody in serum, viral RNA from oropharyngeal and cloacal swab specimens and virus isolation from specific pathogen free fowl embryonated eggs indicated pH1N1 influenza in turkey breeder flocks.61 No to mild clinical signs except for reduced numbers of eggs were observed in affected flocks.61 pH1N1 virus was detected in reproductive tissues as well as the cecal tonsils and bursa of Fabricius, and no lesions were detected in other tissues.61 This corresponds to experimental infections, where turkeys developed clinical signs only after intracloacal but not after intranasal inoculation with pH1N1 virus, even though intranasal inoculation is a successful route of pH1N1 virus inoculation in other species.47 Remarkably, besides domestic turkeys, pH1N1 virus infections have not been reported in other avian species.The domestic turkey is another food animal species in which reverse zoonosis of pH1N1 influenza has occurred. Sporadic infections of pH1N1 virus have been reported mainly in turkey breeder flocks. Human-to-turkey transmission of pH1N1 virus potentially occurred during artificial insemination (AI), based on the evidence that farm workers had been sick with flu-like symptoms during the AI period and subsequently had antibodies against pH1N1 virus. Phylogenetic analysis of pH1N1 virus isolated from infected farm workers and turkeys were grouped in the same cluster, supporting human-to-turkey transmission.Neovison vison) were first reported in a mink farm in Norway during the pandemic. Although the source of transmission was unclear, phylogenetic analysis of sequences derived from affected mink were highly similar to human derived-isolates during the pH1N1 pandemic. The infection might have been transmitted from subclinically infected farm workers or virus-contaminated feed from infected pig offal. The infected mink kits developed severe respiratory disease and had increased mortality rates. Pathological changes included acute to subacute interstitial pneumonia with edema, lymphocyte infiltration, and epithelial hyperplasia. pH1N1 virus antigen was mainly observed in the nuclei of the epithelial lining of bronchi and bronchioles and also of pneumocytes in the alveolar septa.1pH1N1 virus infections in American mink , a Bornean binturong (Arctictis binturong penicillatus), an American badger (Taxidea taxus), a black-footed ferret (Mustela nigripes), and a giant panda (Ailuropoda melanoleuca) were susceptible to pH1N1 virus infection.58 The source of infection in these cases was not determined due to lack of availability of clinical samples from humans with close contact. However, all affected animals were in contact with caretakers or veterinarians, and were housed separately from other wildlife.58 This suggests that animals may have been infected by humans even though the latter did not show clinical symptoms. Although no viral RNA detection or virus isolation from the potential human sources of virus were performed, phylogenetic analysis showed that viruses isolated from infected animals were highly similar to pH1N1 virus circulating among humans during the pandemic and the subsequent seasonal influenza period.11 While the cheetah, the Bornean binturong, the American badger, and the giant panda exhibited severe respiratory signs of infection—hematopnea and dyspnea—no clinical signs were reported in the black-footed ferret.58pH1N1 virus was reported to infect wild animals maintained in captivity, nearly all of which were carnivores held in zoos. Several zoo carnivores including a cheetah (Elephas maximus) based on the presence of pH1N1 antibodies. The source of infection remains unknown, but most likely was infected mahouts, or infected tourists who attended activities such as elephant riding and feeding.49 Similarly to Asian elephants, pH1N1 virus–specific antibodies in nonhuman primates have been reported in several studies. However, there is no evidence that nonhuman primates had clinical signs of disease or mortality from pH1N1 virus infection.28 Whether animal-to-animal transmission can be sustained among captive wild animals, with the risk of becoming a new reservoir, remains unknown since serological and epidemiological studies in captive wild animals are limited.Besides in zoo carnivores, there was evidence of pH1N1 virus infection in Asian elephants (Mephitis mephitis). Sequencing and phylogenetic analysis of virus isolated from affected animals were highly related to pH1N1 virus circulating in humans.5 The source of infection was unclear. In one study, the skunks lived near a mink farm, suggesting that spillover of pH1N1 virus from infected mink farm workers or infected mink may have occurred.4 In the other study, the skunks were found in an urban park where hand feeding by park visitors normally took place.5 Clinical signs were not noted, but purulent nasal exudate was observed in fatally infected skunks. Histopathological changes ranged from moderate, acute, suppurative rhinitis to severe bronchopneumonia.5The only free-living wild animal species in which pH1N1 virus has been reported is the striped skunk  with respiratory signs such as dry cough and wheezing.42 Subsequently, another 3 tigers and 2 lions (Panthera leo) at the same facility were all confirmed with SARS-CoV-2 infection, based on viral RNA detection in fecal samples.37 Sequencing analysis showed that viruses from infected animals were identical to SARS-CoV-2 in humans. These data suggest that the virus could have been transmitted by a zookeeper who might not have developed symptoms of COVID-19 at the time of exposure to these animals.74 Whether subsequent animal-to-animal transmission occurred is not known.SARS-CoV-2 infections in captive wild animals were reported from 2 enclosures at a zoo. Viral RNA was first detected in a nasal swab of a tiger  compared to roaming cats (11%) tested by neutralization assay.77 Similar to pH1N1, neutralization titer in pet cats owned by COVID-19 patients was higher than the titer from pet hospital cats and stray cats.76 These observations from serological surveillances suggest a likely transmission from infected owners to their pets by close contact, thus addressing the role of sufficient interaction between donor hosts and recipient hosts in crossing the species barrier. Although 2 host species share the same geographical area and habitat, host behavior can either limit or enhance pathogen transmission. Certain behaviors of humans, which enhance close contact between infected owners or keepers and their animals, increase the chance of reverse zoonosis. For example, artificial insemination of domestic turkeys likely caused pH1N1 virus spillover from infected humans to domestic turkeys. It could be that particular procedures of caretakers in mink farms, such as weaning pups and vaccination, may have led to human-to-mink transmission. Also, hand-feeding by visitors, surface contamination of bedding or other fomites, or contaminated food could be sources of spillover from humans to animals.45 After the global spread of pH1N1 virus in 2009, the virus continued to circulate in humans, resulting in normal seasonal epidemics of influenza. This contributed to repeated virus introductions from humans to susceptible animals. Multiple events of human-to-pig transmission occurred worldwide during 2009 to 2014.36 The introduction of human pH1N1 viruses into the pig populations and subsequent co-circulation with endemic swine influenza viruses resulted in an increase of genetic diversity by exchange of genome segments.35 For example, a novel reassortment (A/swine/Hong Kong/201/2010 [H1N1]) was found during virological surveillance. This reassortant was composed of a neuraminidase (NA) gene from pH1N1, a hemagglutinin (HA) gene from a European avian-like H1 virus, and the 6 internal genes from triple reassortant H1N1 viruses.72 If the new reassortments result in increased transmission, virulence, or immune escape, they may cause a massive threat to humans and public health by potential generation of a new pandemic influenza virus.75Interaction of several factors are involved in limiting transmission of a virus infection from a donor to a recipient host species; these represent the host species barriers to virus infections. Thus, generally, viruses only sporadically jump from one species to another. Specific interactions are required to accomplish such species jumps and sustain transmission.46 Further surveillance and sequencing analysis are required in order to monitor amino acid substitutions that may be associated with changes in disease severity or transmission. In some cases, both pH1N1 virus and SARS-CoV-2 infections caused relatively no or only mild signs, and virus was transmitted before clinical symptoms became apparent.45 These could facilitate under-detection of human-to-animal transmission.Not much is known about the association between genetic diversity and pathogenicity during SARS-CoV-2 outbreaks in animals. Whole-genome sequencing analysis in SARS-CoV-2-infected mink revealed high genetic diversity in farms which tested negative before, suggesting a fast evolution of viruses in the mink populations. To date, no specific mutations have been observed that are common to all mink isolates. Even though one specific substitution (D614G) associated with increased virulence in vitro was present in some farm clusters, no clear differences in clinical signs, virulence, or transmissibility to humans were found.73 Angiotensin converting enzyme 2 (ACE2), a receptor for SARS-CoV-2, is expressed in tissues of cats and ferrets.70 Once the virus attaches to a new host cell, compatibility between virus proteins and host cell machinery is required for efficient virus replication and potential transmission. Viruses that replicate to a high level are generally more easily transmitted to other hosts. For example, pH1N1 virus replicates efficiently in pigs, cats, and ferrets, allowing subsequent transmission.71 Similarly to pH1N1 virus infections, SARS-CoV-2 replicates efficiently in cats and ferrets resulting in transmission to sentinel animals. In contrast, low replication efficiency limits virus transmissibility. For example, limited SARS-CoV-2 replication in dogs and absence of virus replication in pigs and chickens are associated with limited or no transmission.59In the second place, virus-host interaction is important in determining susceptibility of a new host species to a virus, and virus transmission to other individuals in the new host species. Similarity of biological host factors between humans and animals  can partially determine the potential of a virus to switch species. For example, pH1N1 virus preferentially attaches to α2,6-linked sialic acids, which are abundantly expressed in the upper respiratory tract of animals including pigs, cats, and ferrets.24 Several lines of evidence in both pH1N1 virus and SARS-CoV-2 infections suggest that group-housed animals have a higher chance of spreading the viruses to other individuals of the same species, compared to animals kept individually. High population density, high farm density, and large herd size are the most common risk factors for influenza virus infection in pig farms.68 The same risk factors could also be valid for efficient SARS-CoV-2 virus transmission in mink farms. In human COVID-19 patients, generation interval or time between infection events in a donor and recipient pair is around 4 to 5 days; however, high dose of virus exposure in a high-density population could potentially reduce this interval resulting in broad spread of infections in mink farms.46 Several examples of efficient animal-to-animal transmissions have been reported in a cat colony, a mink farm, and a pig farm.45In the third place, if the transmission can occur, intraspecific contacts in the recipient population are crucial in determining whether the virus will persist or die. The possibility of maintenance of infection in a new host species depends in a complex way on the population sizes and the degrees of mixing of donor and recipient host species as well as the rate of virus transmission in each.15 These approaches are also used to investigate reverse zoonosis in large-scale outbreaks, for example, pH1N1 transmission between pig farms and clustering of SARS-CoV-2 outbreaks in 16 mink farms within the same province.69 Together with chronology of infection, serologic evidence for infection with pH1N1 or COVID-19 in employees and workers in outbreak areas point out the occupational risk of human-to-animal transmission and vice versa.46For the assessment of risk for animals and humans involved with reverse zoonosis outbreaks of pH1N1 or COVID-19, a combination of clinical, epidemiological, sequencing analysis, and laboratory investigations are needed. Particularly, some infected animals show no or only mild clinical signs, which can make it difficult to detect or apply quarantine measures. However, those animals can develop antibodies against these virus infections. Thus, conducting seroepidemiological studies in outbreak areas can assist in identifying susceptible animals and transmission within the population.77 To date, few serosurveillance studies have been performed for SARS-CoV-2 in domestic dogs, cats, and mink, indicating a need for further serosurveillance studies in human-animal interfaces that represent a critical point for cross-species transmission.76Once the reverse zoonosis events occur, it is crucial to know whether infection is maintained in an animal population and has a potential to spillover back to humans. Therefore, continued serosurveillance in susceptible animals and other animal species in the same area are recommended. For example, increased seroprevalence of pH1N1 virus in cats after the 2009 pandemic was detected by serosurveillance studies suggested sustained transmission of this virus in cat populations.Although the number of reported reverse zoonosis events of COVID-19 are limited so far, sufficient contact between humans and animals, compatibility between SARS-CoV-2 and the new animal host, group housing of large numbers of animals, and the structure of the animal host’s contact network can overcome the host species barriers. In order to assess the risk of reverse zoonosis of COVID-19, it is crucial to determine whether factors that allow the host species barriers to be overcome are present for those situations where SARS-CoV-2-infected humans are in contact with animals. Additionally, knowledge of clinical and pathological features of SARS-CoV-2 infection in different animal species will raise awareness of the possibility of this diagnosis during the ongoing pandemic period. Following the pandemic, serological surveillance in animal populations at risk should be conducted, particularly in group-housed animals. Together, this knowledge will improve our understanding of the potential risk for reverse zoonosis of COVID-19.


Rice black‐streaked dwarf virus, genus Fijivirus, family Reoviridae) is the causal agent of rice black‐streaked dwarf and maize rough dwarf diseases, which occur in intermittent epidemics in East Asian countries and are responsible for considerable yield losses. Intermittency of epidemics make accurate forecasting and designing of effective management strategies difficult. However, recent insights into host–virus–vector insect interactions are now informing forecasting and disease control measures. Resistance genes are also being identified and mapped.Rice black‐streaked dwarf virus (RBSDV) , and six proteins. The genome of RBSDV contains 10 segments of dsRNA, named S1 to S10 in decreasing order of molecular weight. Segments 1, 2, 3, 4, 6, 8, and 10 encode the RNA‐dependent RNA polymerase (RdRp), the major core structural protein, a protein with guanylyltransferase activity, an outer‐shell B‐spike protein, viral RNA‐silencing suppressor, the major capsid protein, and the outer capsid protein, respectively. Each of the segments 5, 7, and 9 encodes two proteins: P5‐1, a component of viroplasms; P5‐2 of unknown function; nonstructural protein P7‐1, involved in forming the structural matrix of tubular structures in infected tissues; P7‐2 of unknown function; P9‐1, the main component of viroplasms in infected cells and involved in viral replication; and P9‐2 of unknown function.Laodelphax striatellus in a persistent propagative manner. The vector insect is the only means of virus spread in nature, so its migration and transmission efficiency are obligatory for disease epidemics to develop. Susceptible varieties are widely planted, but efficient transmission by vectors is the primary reason for the epidemics. Cultivation system, pesticide overuse, and climatic conditions also contribute to epidemics by affecting the development of the vector insects and their population dynamics.RBSDV is transmitted by In the absence of resistant varieties, integrated disease management aims at disrupting the cycle of virus transmission by the insect vector. Inheritance studies have indicated that resistance is mostly governed by quantitative trait loci or multiple genes. Genetic engineering through RNA‐interference and gene‐editing strategies are potential approaches for disease control. Rice black‐streaked dwarf virus, which causes rice black‐streaked dwarf and maize rough dwarf diseases.This pathogen profile summarizes the current knowledge on  In addition, Avena fatua, Avena sterilis subsp. ludoviciana, Cynosurus echinatus, Festuca arundinacea, Festuca pratensis, Lolium perenne, and Vulpia myuros var. megalura can also be infected by RBSDV, although the incidence in each species varies  Figure . A recen3Fijivirus . The outer capsid has a T13 icosahedral symmetry and the inner capsid has a T2 icosahedral symmetry. An RNA‐dependent RNA polymerase (RdRp) in virions is used by the genomic dsRNAs to synthesize mRNAs that are extruded from the virus particles  for RBSDV detection in field plants and vector insects have been produced using crude extracts from white waxy enations (tumours) of RBSDV‐infected maize as the immunogen , then acquire the virus by feeding on infected overwintered host plants. They develop into adults, migrate to rice and maize seedlings or other gramineous weeds to feed and lay eggs, and transmit the virus in the newly colonized areas in late May and early June. In most cases, each successfully mated female lays 150–200 eggs on infected seedlings. The newly hatched nymphs become viruliferous then disperse into other fields, resulting in new disease outbreaks. After mid‐October, nymphs migrate to wheat, barley, and some gramineous weed species for overwintering and can transmit the virus to these hosts again, thus finishing the infection cycle , and salicylic acid decrease after RBSDV infection  construct targeting the P7‐2 or P8 genes of RBSDV induce a high‐level resistance in rice against the disease  how does the virus overcome barriers to transmission in insects by interacting with insect components to achieve highly efficient transmission, (b) epigenetic methylation of genes of viruses and insects, which may affect interactions between the virus and vector insect, (c) mutually beneficial or antagonistic mechanisms among viruses transmitted by SBPHs, and (d) impact of gut microbiota in vector insects on the transmission. Some of this research work has been done for other virus–vector insect combinations (Wei and Li, Second, because genetic resistance for disease control is the most sustainable and environmentally friendly solution, plant pathologists and breeders should use currently identified resistant germplasms and search for novel sources of resistance. Research is needed to investigate the genetics of resistance in the identified rice and maize cultivars and breeding lines, and find more DNA markers linked to the resistance genes or QTLs. As soon as possible the known QTLs should be used in breeding programmes to quickly incorporate resistance into local cultivars. Although it could take a long time to transfer the resistance genes from wild or indica rice to japonica cultivars, it is worth doing because these genes could confer high levels of resistance to RBSDV.Third, a better understanding of the interactions between the host, RBSDV, and the vector insect is needed to develop rice or maize cultivars with durable resistance and illuminate the deeper reasons for the intermittent nature of the epidemics. Next‐generation sequencing, comparative genomics, and transformation techniques can serve as powerful tools to identify host genes involved in resistance and susceptibility and host‐range determinants. The susceptibility genes in the host that either directly or indirectly interact with virus genes can be subsequently identified, then altered using gene‐editing technologies.Last and more immediately, real‐time monitoring of viruliferous SBPHs and an accurate forecasting system are the bases for making an effective management strategy. To accomplish this purpose, relevant research institutions should strengthen cooperation with agricultural extension services which can develop a sound, evidence‐based forecasting system based on the data obtained by research institutions. Moreover, effective management strategies must be carried out by farmers. The researchers should give concise guidance for the farmers to prevent and control disease outbreaks in the fields. Click here for additional data file. Click here for additional data file.


Drosophila, encapsidates one of the largest single-stranded RNA virus genomes known. Its taxonomic affinity is uncertain as it has a picornavirus-like cassette of enzymes for virus replication, but the capsid structure was at the time for genome publication unknown. By solving the structure of the virus, and through sequence comparison, we clear up this taxonomic ambiguity in the invertebrate RNA virosphere. Despite the lack of detectable similarity in the amino acid sequences, the 2.7 Å resolution cryoEM map showed Nora virus to have T = 1 symmetry with the characteristic capsid protein β-barrels found in all the viruses in the Picornavirales order. Strikingly, α-helical bundles formed from the extended C-termini of capsid protein VP4B and VP4C protrude from the capsid surface. They are similar to signalling molecule folds and implicated in virus entry. Unlike other viruses of Picornavirales, no intra-pentamer stabilizing annulus was seen, instead the intra-pentamer stability comes from the interaction of VP4C and VP4B N-termini. Finally, intertwining of the N-termini of two-fold symmetry-related VP4A capsid proteins and RNA, provides inter-pentamer stability. Based on its distinct structural elements and the genetic distance to other picorna-like viruses we propose that Nora virus, and a small group of related viruses, should have its own family within the order Picornavirales.Nora virus, a virus of  Drosophila species including Drosophila melanogaster and Drosophila simulans1. The virus infects the midgut cells and the infection is transmitted vertically by the fecal–oral route. Interestingly, the infected insects exhibit no obvious pathological effects5. Nora virus has several characteristic features of the order Picornavirales6, such as an isometric capsid about 30 nm in diameter and a replicative cassette that includes an RNA helicase, a protease and an RNA-dependent RNA polymerase1. Based on the conserved polymerase and helicase sequences, Nora virus is similar to viruses in the Picornavirales families Iflaviridae, Secoviridae, Picornaviridae and Dicistroviridae8. However, uniquely, the Nora virus genome is organised into four open reading frames (ORF), and for a Picornavirales virus it has a relatively large RNA genome of 12,333 nucleotides9  and icosahedral image reconstruction, which helps us to understand the architecture of this virus, to investigate the presence of VP3 in the capsid and to investigate the function of VP4C.Nora virus ORF4, situated at the 3′-terminal end of the genome, encodes a 931 amino acid-long polyprotein. The translated polyprotein is cleaved at two positions (264 aa and 515 aa) post-translationally by a virus-encoded protease to form the virion’s mature structural proteins VP4A (29 kDa), VP4B (28 kDa) and VP4C (48 kDa)T = 1 (pseudo T = 3) triangulation number; a capsid architecture previously described as one of the main characteristics of the order Picornavirales , VP4B is most similar to human Aichi virus VP0  and VP4C is most similar to hepatitis A virus VP3 . The β-barrels account for the alignment. However, the most prominent capsid surface features are the 60 mainly α-helical protrusions that are situated at the interface between VP4C contributing most of the residues and VP4B contributing one α-helix  and the non-structural ORF 12 of the virulent lactococcal phage p2 (PDB: 3D8L). All these three molecules are involved in signalling, suggesting that the Nora virus surface protrusion may have a similar role, binding to a cell surface receptor and causing downstream signalling to bolster virus entry. Additionally, the surface charge distribution of these protrusions shows a positive patch which, similar to the C-terminal extension of S protein in cowpea mosaic virus, may have a role in capsid assembly by stabilizing the formation of pentamers during assembly14.Structural alignment with DALI (13) showed that VP4A is most similar to FMDV VP2 14. However, unlike HPeVs, there is a crossover of the VP4A N-terminus (residues 1–13) with a symmetry-equivalent N-terminus of another VP4A from the neighbouring pentamer at the two-fold axis  of the two VP4A N-termini on the two-fold axis closely interact with the ssRNA genome. This causes a localised condensation of RNA density in this region around the twofold symmetry axis Figs. b, 6. Arg8 have previously defined a “Nora Virus Related Clade” of viruses, but we found that Nora-like capsid protein sequences are only present in a subset of these viruses. This Nora-like subset forms a well-defined monophyletic group with a conserved genome organization  and one from a sea anemone (A. equina). The capsid protein sequences are well conserved among the members of this group, suggesting that the structure described here is well adapted to the genome size and/or the biology of these viruses.Sequences related to the Nora virus capsid proteins can be identified in a number of virus-like sequences in the databases Fig. . Based oion Fig. a, which ion Fig. b. The NoFopius arisanus is a parasitoid wasp, feeding on Bactrocera dorsalis and Ceratitis capitata, and the virus may well derive from the gut contents of this wasp. Similarly, dragon flies (Odonata) are voracious predators on flies and other flying insects, and their viromes may include viruses present in their diet. A similar argument could be made about the spider. It is of course also possible that the viruses have adapted to replicate in these predators. However, the sea anemone remains a mystery.The phylogenetic tree of the Nora-like viruses tends to mirror that of their hosts Fig. , suggestPicornavirales, but with an α-helical domain protrusion from the virion surface and an unusual interaction of two N-termini from symmetry-related VP4A around the two-fold axes. Taken together, both global and detailed analysis of the capsid structure, the genome organization and the genetic distance to other viruses, suggest that Nora virus and the clade of related viruses can be described as representatives of a new virus family within the order Picornavirales.In conclusion, we showed that the Nora virus has a T = 1 arrangement where VP4C is present around the five-fold axes of symmetry, VP4A around the two-fold axes of symmetry and VP4B around the three-fold axes of symmetry. Each protein shows the β-jelly roll fold characteristic of Drosophila24. To avoid cross-contamination with other viruses, dechorionated Drosophila melanogaster eggs were infected with Nora virus as described earlier9. Nora virus was propagated and purified essentially as described earlier9.Persistent viral infections are common in −/Å2 and a sampling of 1.06 Å/pixel. The initial dataset consisted of 3516 frame stacks, and the frames were aligned prior to processing using motioncorr25. We used Ethan26 for automated particle picking, CTFFIND4 for CTF estimation and correction27. An initial model was built with 150 picked particles using random model generation module in AUTO3DEM28 with icosahedral-symmetry imposed. Both 2D and 3D classification in Relion version 1.329, were used to reduce the dataset heterogeneity. A total of 16,131 particles were refined using the 3D autorefine module, followed by particle polishing, an additional 3D autorefine step, and finally B-factor correction in the “post processing” module using a B-factor value of − 20. The final refinement step combining two independent datasets gave a resolution of 2.7 Å as assessed by the 0.143 criterion Fourier shell correlation from the deposited half maps using the EMDB server  of purified virus in 10 mM Tris–HCl pH 7.4. buffer were vitrified in a Leica EM GP device at 22 °C and 70% humidity on glow discharged Quantifoil 2/2 holey carbon grid in liquid ethane. CryoEM data were collection at eBIC at the Diamond Light Source, UK on a FEI Krios 300 kV TEM equipped with a Gatan post-GIF K2 Summit detector. The GIF was set to 20 eV slit width and FEI EPU software was used to automatically the data. Each exposure was written out as a 20 frames stack with an estimated total electron dose of 40 e30 to trace C-α backbones of the constituent proteins from capsid density. Volumes corresponding to the three distinct subunit densities were then segmented with “Zone”-tool in UCSF Chimera at a radius of 8 Å. The models were built de novo into each segmented EM density in COOT31 using the known amino acid sequences of VP4A, VP4B and VP4C. The models were refined in real space using Phenix32 and in Fourier space using Refmac33. This step was iterated with local refinements in COOT until no further improvement in the refinement statistics were observed (Table 34 were used for all visualizations.We used the “Volume tracer”-tool in UCSF Chimera13. The buried surface area for all the viruses were obtained from VIPERdb association energy data analysis35.The structural alignments were performed on the DALI web serverSupplementary Figures.


Jatropha leaf curl Gujarat virus (JLCuGV), which encodes six ORFs with each one having RNA silencing suppressor activity. Therefore, three artificial microRNAs  were designed employing overlapping regions, each targeting two ORFs of JLCuGV genomic DNA and transformed in tobacco. The C1/C4 and C2/C3 amiRNA transgenics were resistant while V1/V2 amiRNA transgenics were tolerant against JLCuGV. The relative level of amiRNA inversely related to viral load indicating a correlation with disease resistance. The assessment of photosynthetic parameters suggests that the transgenics perform significantly better in response to JLCuGV infiltration as compared to wild type (WT). The metabolite contents were not altered remarkably in amiRNA transgenics, but sugar metabolism and tricarboxylic acid (TCA) cycle showed noticeable changes in WT on virus infiltration. The overall higher methylation and demethylation observed in amiRNA transgenics correlated with decreased JLCuGV accumulation. This study demonstrates that amiRNA transgenics showed enhanced resistance to JLCuGV while efficiently maintaining normalcy in their photosynthesis and metabolic pathways as well as homeostasis in the methylation patterns.The leaf curl disease of Jatropha caused by geminiviruses results in heavy economic losses. In the present study, we report the identification of a new strain of a  Geminiviridae is the second largest family of plant viruses2, possessing the potential to exploit a broad range of plants as its host. The geminiviruses are characterized by geminate virus particles that are categorized into nine genera based on their genome organization, insect vector, host range and phylogeny reconstruction, viz., Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus, Turncurtovirus, Capulavirus and Grablovirus. Based on the number of DNA molecules in the viral genome, they can be: monopartite (one DNA molecule) or bipartite (two DNA molecules: DNA-A and DNA-B)3. The viral genome in monopartite viruses and the DNA-A component of bipartite viruses are homologous and have a similar organization that includes the V1/AV1, C1/AC1, C2/AC2, C3/AC3, and C4/AC4 ORFs3.Viruses have emerged as major plant pathogens causing severe losses to agricultural production worldwide4, US$ 5 billion for cotton in Pakistan, US$ 300 million for grain legumes in India5 and the US$ 140 million in Florida for tomato alone6. The DNA viral mutation rates roughly range between 10–8 and 10–6 substitutions per nucleotide per cell infection7. This high rate of recombination and mutation leads to their great diversity worldwide8, thereby increasing their host range and geographical distribution. Thus, geminiviruses are emerging as widely spread and diverse plant DNA viruses posing severe threat to crop production.The geminiviruses infect a wide range of economically important crops like cassava, cotton, grain legumes and tomato, causing significant financial losses across the world. These economic losses have been estimated to about US$ 1300–2300 million for cassava in Africa9. It has also gained importance as a biodiesel producing crop as Jatropha seeds contain 27–40% oil10. The oil yield is affected by attacks of various pathogens such as viruses, bacteria, fungi and nematodes. The leaf curl disease in Jatropha, caused by the whitefly transmitted geminiviruses, is the most devastating. In India, it causes a reduction of up to 80% in seed yield11 and 40% in oil production12. The symptoms of the disease include mosaic and curling of leaves and malformed fruits. To date, both monopartite and bipartite geminiviruses have been detected in Jatropha from India, Kenya, Nigeria, Dominic Republican, Jamaica and the United States of America (USA)13.Jatropha, belonging to family Euphorbiaceae, has various medicinal properties14, pathogen-derived resistance15, expression of double-stranded (ds) RNA homologues16 and CRISPR/Cas9 system17. The RNA silencing mechanism in plants forms the first line of defence by targeting the viral mRNA or the RNA genome18. Interestingly, viruses have evolved to acquire a variety of unrelated RNA silencing suppressors (RSSs) that affect different yet multiple steps in the silencing pathway19 to favour virus replication. Several reports have demonstrated that different geminiviral proteins encode RSS activity22 and may supplement each other to overcome host RNA silencing response. A molecular strategy to target the virus-encoded suppressors is expected to boost plant tolerance to infection. The artificial miRNA (amiRNA) provide a highly specific alternative to silence the viral transcripts for targeting the RSS for developing disease resistance23.For generating resistance to geminiviruses, various molecular strategies based on RNA silencing and gene editing have been reported including antisense RNAsTherefore, in this study, we adopted the strategy of identifying and inactivating the virus-encoded RSSs to boost Jatropha plant resistance against geminiviruses. For this purpose, amiRNAs were designed from genomic regions producing overlapping ORFs to target multiple viral transcripts simultaneously.J. curcas CP9 accession (IC 565735), from CSIR-CSMCRI Jatropha plantation fields, Neswad . The infected plants showed severe to moderate leaf curling with > 70% disease incidence.The geminivirus infected leaves were collected from Jatropha leaf curl Gujarat virus (JLCuGV). Its genomic DNA possessed two ORFs in the sense strand (AV1 and AV2) and four ORFs in complementary strand  and sequenced. Sequence analysis confirmed the presence of only one genomic DNA  in the isolated virus. Based on sequence homology to other begomoviruses, it was identified to be a novel isolate and named as Flock house virus B2 gene), as a positive control24 and empty vector and mock (only buffer) infiltration as a negative control [Fig. For identification of potential RSS in the JLCuGV, the six ORFs of the genome  were individually cloned in pCAMBIA-1301 , hptII (1.2 kb) and amiRNA (259 bp) genes ; so, these regions were selected as targets for the designing of amiRNAs. Three amiRNAs  were designed in silico  using specific primers , 1.15-fold C2/C3) and 0.97-fold (V1/V2) at 14 dpi to 5.4-fold (C1/C4), 5.4-fold (C2/C3), 2.88-fold (V1/V2) at 35 dpi in amiRNA transgenics , C2/C3 , V1/V2 , VA and WT plants were used for MSAP (methylation-sensitive amplified polymorphism) analysis with 20 primers, which were earlier characterized to be best polymorphic MSAP primers in tobacco . Among 20 primers, 15 primers  were infected (in 3 replicates) with JLCuGV and analyzed for various photosynthetic parameters.2 (Ci) and the ratio of intercellular to ambient CO2 (Ci/Ca) increased in virus infiltrated WT and V1/V2 plants, but decreased in C2/C3 and C1/C4 transgenics, as compared to their respective uninfected control plants  decreased in WT and V1/V2 transgenics and increased in C1/C4 and C2/C3 transgenics after virus infiltration. Upon virus infiltration (35 dpi), ETR and ɸCO2 increased significantly in C1/C4 , C2/C3  and V1/V2 transgenics  than WT  and COThe efficiency of photosystem II (Fv/Fm) was similar in both WT and transgenics with and without virus infiltration Fig. a. The quMetabolite analysis was performed in both WT and amiRNA transgenics with and without JLCuGV agroinfiltration. Twenty-one common metabolites were detected in both healthy and virus infiltrated tissue  and virus infiltrated tissues. In WT, C2/C3 and V1/V2 transgenics, fructose and glucose content increased, with a maximum increase of 8.76- and 8.10-fold, respectively, in WT on infiltration. The C1/C4, however, showed a decrease in fructose and glucose content on virus infiltration. The sucrose content increased in transgenics (1.19–1.26-fold), whereas decreased by 1.95-fold in WT with virus infiltration  was detected in the highest amount, followed by myo-inositol (14.30%) and malic acid (13.52%), while in C1/C4 and C2/C3 transgenics, malic acid  was detected in the highest amount, followed by myo-inositol , independently show strong RSS activity. The reversal of GFP expression in GFP silenced tobacco lines on agroinfiltration of AC1, AC2, AC3, AC4, AV1 and AV2 ORFs, independently, confirmed the strong RSS activity in all the six ORFs. The positive amplification of GFP gene from the cDNA generated from infiltrated leaves, gave the molecular confirmation of RSS activity exhibited by JLCuGV ORFs. The RSS activity acquired by individual JLCuGV ORFs seems to have increased its virulence. In the case of geminiviruses, ORFs like AV2, AC1, AC2, and AC4 were reported as RSSsN. benthamiana transgenics independently expressing C1/C4 and C2/C3 amiRNAs showed resistance against JLCuGV while transgenics expressing V1/V2 amiRNAs showed tolerance to JLCuGV. The WT and VA plants showed high viral load with severe disease symptoms, whereas no viral load and no symptoms were detected in C1/C4 and C2/C3 transgenics. This indicated that JLCuGV resistance in C1/C4 and C2/C3 transgenics could be due to the expression of amiRNA. Several other reports are available that have shown that amiRNAs are effective in bringing out the degradation or silencing of the target viral genes29.As a strategy to boost plant resistance to virus infection, we designed amiRNAs targeting overlapping regions between ORFs C1/C4, C2/C3 and V1/V2 to silence their transcripts and hence inhibit their RSS activity. The 29. The lower amiRNA expression level in V1/V2 amiRNA transgenics correlates with the development of mild symptoms and limited tolerance to virus infection. It was observed that the viral load in V1/V2 amiRNA transgenics was much lower than that observed in WT or VA plants. This shows the important role of miRNAs in providing protection from virus infection and the manifestation of pathogen symptoms30. Earlier, Vu et al.29 have shown an inverse correlation between the expression levels of amiRNA and the development of disease symptoms.Interestingly, amiRNA levels were lower in V1/V2 amiRNA transgenics as compared to the C1/C4 and C2/C3 transgenics. The reason needs to be investigated though, in all probability, it may be due to the allelic states of amiRNA precursor, based on the integration of T-DNA into the plant genome, which may have altered the level of amiRNA expression30. The changes in methylation in Nicotiana tabacum genome were observed after the Cucumber mosaic virus (CMV) infection31. The predominance of CHH hypomethylation has emerged as a major defence response against viral infection31. The transgenics C1/C4 and C2/C3 amiRNA exhibited higher methylation across the genome after virus infection as compared to WT, VA and V1/V2 amiRNA transgenics. The higher methylation and demethylation in amiRNA transgenics, corelated with decreased JLCuGV accumulation. This suggested that the homeostasis in methylation pattern in C1/C4 and C2/C3 amiRNA transgenics helped to block the JLCuGV accumulation, leading to resistance against JLCuGV. It has been reported that geminiviruses counteract plant defence strategy by inhibiting DNA methylation. The geminiviral AC2 genes act as RSS by interacting and inhibiting adenosine kinase (ADK), an essential gene of the methylation pathway32. ADK plays a role in the production of the methyl group donor S-adenosyl methionine (SAM) and defects in ADK lead to a loss in methylation in plants33. As C2/C3 amiRNA transgenics target the AC2 gene, the viral counter defence to suppress ADK was not functional, and the plants could successfully follow the methylation pathway to inhibit the virus growth.DNA methylation plays a major role in protecting plants under both biotic and abiotic stresses. Several reports state that plants can methylate the invading virus genome to suppress their growth35, such that decreased photosynthesis is correlated with increased disease severity and increased virus multiplication37. JLCuGV infiltrated WT showed severe symptoms and significantly decreased photosynthesis as compared to amiRNA transgenics (C1/C4 and C2/C3). The V1/V2 transgenics showed mild symptoms and relatively less reduction of photosynthesis parameters as compared to WT. The decrease in photosynthesis rate has been previously reported in geminivirus-infected leaves of Eupatorium makinoi35 and mustard after turnip mosaic virus infection37. Naidu et al.38 suggested that a reduced level of chlorophyll-a may be responsible for the reduced rate of photosynthesis.Virus infection usually affects the process of photosynthesis37. The Ci and Ci/Ca were significantly higher in WT, suggesting that the CO2 taken by the plants was not utilized during photosynthesis and got accumulated in the cells, increasing the Ci and Ci/Ca ratio. It also correlates with the reduced rate of photosynthesis observed in WT. Similar results were reported with tobamovirus infection for reduced stomatal conductance, transpiration rate and Ci in pepper39. The ɸCO2 was significantly higher in transgenics, suggesting efficient CO2 assimilation, which correlated with both increased photosynthesis rate and reduced Ci of transgenics. The reduced ɸCO2 is reported for pea on Pea enation mosaic virus (family Luteoviridae) infection40. The higher ETR seen in amiRNA transgenics suggests better redox homeostasis. The high ETR was found associated with improved reactive oxygen species (ROS) homeostasis during salinity41. Although Guo et al.37 report no change for ETR during geminivirus infection, a significant reduction in ETR was observed in grapevine on virus infection42.The transpiration rate, WUE and stomatal conductance were significantly higher in virus-infected transgenics compared to WT. The reduced transpiration rate and stomatal conductance were also observed in mustard after geminivirus infection31 in both WT and transgenics even after virus infiltration. Similarly, no change in Fv/Fm was reported for geminivirus infected mustard37, and Pepper mild mottle virus (PMMoV) infected pepper39. ɸPSII was significantly higher in transgenics, suggesting proper functioning of PSII as it is a measure of PSII performance. In WT, ɸPSII was significantly reduced, and reduced ɸPSII is reported to cause the accumulation of reduced bound quinone, which causes damage to the primary electron acceptor plastoquinone of PSII43. Therefore, reduced ɸPSII can be correlated to the reduction of ETR observed in WT during infection. The qP indicates the efficient conversion of light into chemical energy37 and significantly improved qP in transgenics can be attributed to the maintenance of photochemical quenching followed by increased photosynthesis rate in transgenics. The overexcitation of the photochemical system after turnip mosaic virus infection caused reduced qP in mustard37.Fv/Fm was maintained at a healthy 0.8 levelTobacco mosaic virus-infected tobacco44, Tobacco rattle virus-infected Arabidopsis45 and CMV infected beet plants46. Similarly, the decrease in carboxylic acids of WT is correlated with virus infection in Papaver somniferum on geminivirus Ageratum enation virus (AEV) infection47. The decrease in sucrose concentration is reported in Potato virus Y (PVY)-inoculated potato leaves, while the increase in sucrose content reported in Jatropha infected by geminivirus48. In WT, a decrease in sucrose and an increase in fructose and glucose content after virus infiltration might be due to activation of invertase enzyme which converts sucrose into fructose and glucose50. An increase in glucose concentration is also reported in tomato infected with Tomato mosaic virus50 and PVY-inoculated potato leaves51.In WT, the sugar content was significantly increased and intermediates of the tricarboxylic acid (TCA) cycle  reduced after virus infiltration. This suggests that sugar metabolism is affected and the normal functioning of the TCA cycle is disturbed in WT after JLCuGV infection. In transgenics, no remarkable changes are observed, indicating better maintenance of metabolic pathways on virus infiltration. The increase in sugar content of WT is correlated with the virus infection in in-planta. Three amiRNAs were designed against C1/C4, C2/C3 and V1/V2 transcripts by targeting the overlapping regions. Transgenics expressing these amiRNAs showed a resistance (C1/C4 and C2/C3) and tolerance (V1/V2) response to viral infection. A direct correlation of the viral load and disease symptoms with relative levels of amiRNA was observed. The photosynthetic parameters were significantly better in amiRNA transgenics as compared to WT on virus infiltration, suggesting that both light and dark centres of photosynthesis are maintained due to reduced viral load. Likewise, the metabolite profiles were not much altered in amiRNA transgenics, while sugar metabolism is disturbed in WT on virus infiltration, again suggesting resistance of the amiRNA transgenics to the virus. MSAP analysis revealed an increase in methylation and demethylation profiling in amiRNA transgenics as compared to VA and WT, suggesting that methylation pathways get activated in amiRNA transgenics, and increased level of methylation profiling is correlated with a degree of virus resistance in amiRNA transgenics. All the results indicated that the amiRNA transgenics were better adapted to resist/tolerate JLCuGV infection. Importantly, as amiRNAs designed in this study share homology to related geminiviruses that infect other crops like tomato, pepper and papaya, so these have the potential to be employed for developing geminiviral resistance in other susceptible economically important crops.In conclusion, the study reports that a new isolate of JLCuGV was responsible for the leaf curl disease in Jatropha CP9 accession. All six ORFs of JLCuGV genomic DNA exhibited RSS activity J. curcas leaves CP9 accession showing mosaic and leaf curl symptoms were collected from Nesvad, Jatropha experimental fields of Plant Omics division, CSIR-CSMCRI, Gujarat. Collections were made during July and September 2014. The collected samples were stored at − 80 °C until use.The 51. The detection and characterization of begomoviral DNA components  from the samples was performed by PCR with degenerate primers , cloned in pBluescriptII KS(+) vector and sequenced.The genomic DNA of the collected samples was isolated by modified CTAB methodPstI  or HindIII  were cloned in pRT101, thereafter, the entire cassette with CaMV35S promoter-ORF- PolyA was cloned in pCAMBIA-1301 vector using Agrobacterium cultures  of individual JLCuGV ORF constructs were injected into leaves of stable N. tabacum cv. Xanthi GFP silenced lines into separate patches in each leaf for identification of RSS activity. Agroinfiltration was performed by a method described by Hamilton et al.52. The Agrobacterium cultures  were injected into leaves of the N. benthamiana (WT and transgenics) at 4–6 leaf stage using a 2 ml syringe. The plants were kept in an insect-free chamber at a constant temperature (25–28 °C) under 14–16 h lighting.The The agroinfiltrated leaves of the GFP silenced lines were observed under UV light for visualization of green fluorescence at 7, 11 and 15 dpi. The ORFs having inherent RSS activity were expected to restore the GFP expression by suppressing the RNA silencing machinery.52 agroinfiltrated independently with single JLCuGV ORF  and FHVB2 construct, a well-known RSS used as control. The cDNA was prepared using 5 μg of total RNA using SuperScript IV First-Strand synthesis system (Invitrogen), as per manufacturer’s instructions. cDNA (1 μl), from each sample was used for amplification of gfp gene (~ 300 bp) with specific primers . While designing of amiRNA, we also tried to take the sequences, which can be used to target the begomovirus infecting tomato, papaya, pepper, ageratum etc.  were used for designing amiRNA Table a. The am.53. The pre-amiRNA fragment was then cloned in pRT101, using XhoI and BamHI sites for directional cloning, under CaMV-35S promoter. Then the entire cassettes of C1/C4, C2/C3 and V1/V2 amiRNA with CaMV-35S promoter were cloned in pCAMBIA-1301 using HindIII site. The schematic representation of the amiRNA gene construct in pCAMBIA-1301 showed in Supplementary Fig. The three designed amiRNAs Table  were ampA. tumefaciens strain LBA4404. The transformed agrobacterium was used for the transformation of N. benthamiana leaf discs, according to Clemente54.Three amiRNA constructs  cloned in pCAMBIA-1301 vector and vector backbone alone (VA) were mobilized into the 1) were checked by GUS assay to confirm the transgene expression. These were later confirmed for gene integration by PCR. The PCR was carried out using the primers specific for amiRNA gene, gus gene and hygromycin (hptII) gene  was cloned in pCAMBIA-1301 to get a recombinant vector pCAM-JLCuGV-Partial. In the second step, the full-length genome of RCA product generated by HindIII was ligated with pCAM-JLCuGV-Partial, to get a complete infectious clone pCAM-JLCuGV-Inf. The orientation of the inserts was confirmed with sequencing. The positive clones that contain tandemly repeated viral genomes having two ori (origin of replication) sites were selected. The infectious clone was transformed in A. tumefaciens LBA4404 strain. For causing infection, the agroinfectious clone of JLCuGV  according to Singh and Jha58. The quality of the purified RNA was examined by 1% agarose gel electrophoresis and the absorbance ratio (A260/A280 and A260/A230) using Epoch Microplate Spectrophotometer .The total RNA was isolated from transgenic lines by Tris-SDS buffer59 and actin gene was used as an internal control.The cDNA was synthesized using small RNA as template and specifically designed stem-loop primers . All other conditions for gas exchange and chlorophyll fluorescence measurement were kept the same as reported in Shukla et al.60. Photosynthetic gas exchange parameters and chlorophyll fluorescence parameters were recorded in WT and amiRNA transgenics  at 21, 28 and 35 dpi.Photosynthetic gas exchange parameters were measured at photosynthetic photon flux density (PPFD) of 1000 μmol m.61. The derivatized samples were then transferred into glass vials suitable for GC–MS analysis. The GC–MS analysis was carried out in a SHIMADZU prominent instrument  equipped with EI mass spectra and autosampler. The injection volume for each sample was 1.0 μl throughout the analysis. Gas chromatographic separation was performed using the silica capillary SH-Rtx-5 column (30 m × 0.25 mm)62. Injection temperature was 230 °C, the interface was set to 250 °C and the ion source was adjusted to 200 °C. Helium flow was 1 mL min−1. After a 5-min solvent delay time at 70 °C, the oven temperature was increased at 5 °C min−1 to 310 °C, 1 min isocratic, cool-down to 70 °C, followed by an additional 5-min delay. The ion source temperature was 200 °C and mass ions in the range of 45–600 m/z were scanned at a rate of 1.0 scans s−1.The sample preparation and derivatization by N-methyl-N-trimethylsilyl trifluoroacetamide (MSFTA) was carried out as given in Lisec et al25. For MSAP analysis, WT, VA and two representative lines of C1/C4 , V1/V2  and C2/C3  amiRNA transgenics were selected. The DNA was isolated from each plant with and without virus agroinfiltration after 35 dpi.MSAP analysis was performed as reported earlierP ≤ 0.05 to determine the significance of the difference in viral load of WT, VA and V1/V2 amiRNA transgenics and relative expression of amiRNA in the C1/C4, C2/C3 and V1/V2 amiRNA transgenics at 14, 21, 28 and 35 dpi. Similarly, the analysis of variance was also calculated in the photosynthesis parameters between the means of control and virus infiltrated plants  at 21, 28 and 35 dpi. Different alphabets indicated mean values of treatments that were significantly different from each other.The mean values and standard deviations were calculated of three independent biological replicates and the bars in figures represent the mean ± SD. Analysis of variance was calculated using the Tukey test by InfoStat software (version 2018) at Supplementary Information.


Range of movement (ROM) assessment is an important strategy to increase physical-technical performance and minimize the risk of sports-related injuries. Currently, there is no consensus regarding which ROM assessment method is the most appropriate. The main objective of this study was to perform a systematic review of the test batteries available for the assessment of lower limb ROM; additionally, we compare the ROM-SPORT I battery with those previously reported in the literature. The systematic review was conducted following the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines. The identification of publications was made by using the databases SciELO, Medline, Scopus, PubMed, and Web of Science. Based on the inclusion criteria, sixteen publications were selected and analyzed. The ROM-SPORT I battery is the most valid of the analyzed methods. This battery evaluates the ROM of eleven lower limb movements. The inclinometer with a telescopic arm and a box is a simpler, more comfortable, and faster procedure than others. The Lumbosant support and use of two examiners are essential to avoid compensatory movements to obtain reliable measurements during ROM assessment. The ROM-SPORT I is a field-based battery of tests that may be used by sports professionals, clinics, and researchers in applied settings to accurately assess and monitor lower extremity ROM. Flexibility, which is defined as the intrinsic ability of tissues to achieve the maximum range of movement without sports injury , is one A limited or restricted ROM has been considered an important intrinsic and modifiable risk factors for the most prevalent sports-related injuries, such as groin pain , and this might predispose such players to high injury risk . LikewisIn addition, the ROM can be decreased by high training loads and repeated movements used in technical sports actions during both training and competition throughout the season, which induces physical stress and fatigue on the muscles ,24. WhenCertain articles in the scientific literature that assess athletes´ flexibility show major differences in results depending on the sport ,35,40,41There are many published assessment tests to measure the ROM of the major joints in the lower extremities  ,54,55,56n = 7], knee [n = 1], and ankle [n = 2]) in athletes and general population ) might not accurately assess ROM, as they may be biased by other factors, such as anthropometry (length of the limbs), and inter-muscular coordination (dynamic stability) which could limit the validity of the results.Furthermore, all the selected passive tests specifically measure a single joint movement. The tests that imply using more than one joint  for measuring flexibility, report a high concordance  with the ROM measurement method using a goniometer or inclinometer ,98. SomeAll the tests represented in According to absolute reliability values, clinicians and sport practitioners can be 95% confident that an observer’s change between two measures larger than 1.3–6.9° for the ROM values obtained from the ROM-SPORT I would likely indicate a real change  The majority of the tests of the ROM-SPORT I battery (9 of the 11) involve two examiners to avoid any possible compensatory movements; it reduces measurement error and may give more accurate ROM values, avoiding false diagnostic of limited ROM; (2) The inclinometer (the instrument used in the ROM-SPORT I procedure) is easy and simple to use, as it does not require the estimation of the joint’s axis nor the initial position. Also, this tool reduces measurement errors since the examiner can systematically and repeatedly locate the same position to place the inclinometer by just following the parallel imaginary bisector of the segment assessed; (3) It is recommended to use a telescopic arm to facilitate the inclinometer’s placement, which may improve the precision and reproducibility of the measurement and reduce the duration of the ROM-SPORT I battery; (4) The lower-back protection support “Lumbosant” , used in the ROM-SPORT I battery, helps to standardize the lumbar curvature (20°) during the assessments, avoiding higher ROM values, due to anterior or posterior pelvis tilt compensatory movements.−To accurately quantify the ROM measures of the major lower extremity joints ;−To identify athletes with limited or restricted joint ROM values. This knowledge may help in the decision-making process regarding the identification of athletes at high risk of sustaining an injury (mainly soft tissue injury);−To detect those athletes  that should improve their ROM values to successfully perform the technical actions that are awarded the highest points by a judge;−To monitor the efficacy of intervention programs  designed to maintain or improve lower extremity joints ROM;−Furthermore, in rehabilitation processes, the ROM-SPORT I battery may be used to determine if the ROM of the injured joint has been fully restored, which may help to achieve a safe return to play (athletes) or activities of daily life .Although this review has been focused mainly on a sports context, the ROM-SPORT I battery can also be applied in research  and clinical fields. This battery has the following practical applications:Although different batteries have been used to assess ROM previously, they all have some limitations. The ROM-SPORT I battery seems to be the most complete procedure that meets the requirements of a battery of tests for the ROM assessment of the lower limbs. The novelty and new contributions of the ROM-SPORT I battery over other procedures described previously by other authors are: (1) The rapidity of the ROM-SPORT I battery. This procedure evaluates 11 tests in one athlete, including both lower limbs in 8–11 min; (2) the importance of the assistant examiner, together with using the lumbar support tool, “Lumbosant” , to reduce compensatory movements; (3) the simplicity of the procedure. The inclinometer with an extensible rod is a simple and cheap tool that minimizes the measured variability and the error of the examiner.; (4) the validity of the ROM-SPORT I battery is based on criterion validity (radiographic) and content validity; and (5) all the ROM tests of the selected publications reported moderate to excellent reliability values; it is excellent for tests of ROM-SPORT I battery.


Clone CC3 is not only able to neutralize a wild-type (WT) strain of chikungunya virus (CHIKV), but also neutralizes WT strains of Mayaro virus (MAYV) and Ross River virus (RRV); both arthralgic, Old World alphaviruses. Interestingly, CC3 also demonstrated a degree of neutralizing activity against the New World alphavirus, Venezuelan equine encephalitis virus (VEEV); albeit both the vaccine strain, TC-83, and the parental, WT Trinidad donkey strain had PRNT50 values ~1,000-fold higher than that of CHIKV. However, no neutralization activity was observed with Western equine encephalitis virus (WEEV). Ten CC3 variants designed to possess a range of isoelectric points, both higher and lower, were constructed. This approach successfully identified several lower pI mutants which possessed improved thermal stabilities by as much as 10°C over the original CC3 (Tm = 62°C), and excellent refolding abilities while maintaining their capacity to bind and neutralize CHIKV.A single domain antibody (clone CC3) previously found to neutralize a vaccine strain of the chikungunya virus (PRNT Chikungunya fever is a reemerging infectious disease caused by the chikungunya virus (CHIKV), a mosquito-borne alphavirus. Old World alphaviruses typically result in persistent, or recurring, arthralgia after acute infection , 2, whilE. coli , offer an alternative format for antiviral therapeutics . ComprisE. coli , 13. SevE. coli , abilityE. coli , 16, andE. coli , 18. FasE. coli –21.Previously, we described five sdAb able to bind CHIKV virus-like particles (VLPs), or recombinant CHIKV envelope protein. Two of the clones (CC3 and CA6) were evaluated for their ability to neutralize CHIKV; whereas both clones showed neutralization, CC3 was ~80 times more effective . In thisExpanded methods are provided in the The CHIKV-specific sdAb CC3 was previously described . All enzE. coli and purified as previously described  with flanking NcoI and NotI sites. Mutants were designed based on the consensus sequence of the CA6 neutralizing sdAb as well as toxin binding sdAb , 27. Theescribed .Thermal denaturation was monitored using circular dichroism (CD) and binding ability was assessed by MagPlex direct binding assays as previously described .Neutralization studies were similar to those previously described . Minor dWe had previously identified five CHIKV binding sdAb. The neutralization capacity of two of the five (clones CC3 and CA6) was determined by IBT Bioservices . Both neutralized CHIKV 181/25 ; however50 values all <0.625 μg/ml; 50: 4.0 μg/ml) as well as the parental WT Trinidad donkey strain (PRNT50: 1.9 μg/ml), no neutralization was observed with WEEV  to provide better pharmacokinetics while possessing superior thermal stability and improved refolding, qualities that may be of value in cases where maintenance of the cold-chain of transport is difficult.The original contributions presented in the study are included in the article/EG, JL, GA, EW, and PG designed the experiments. EG, JL, GA, EW, CB, and CG performed the experiments. EG and GA wrote the manuscript. All authors analyzed data and edited the manuscript.JL, GA, and EG are inventors on a patent application: United States Patent Application 16513881 July 17, 2019 Single Domain Antibodies to Chikungunya Virus. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To find out the location ofcorona virus mutations of one type to another, DNA sequences can be alignedusing the Needleman-Wunsch algorithm. Corona virus data was taken from GenbankNational Center for Biotechnology Information from 1985-1992. TheNeedleman-Wunsch algorithm is a global alignment algorithm in which alignment isperformed to all sequences with the complexity of O (mn) and is capable ofproducing optimal alignment. An important step in this reserach is the sequencealignment of two corona viruses using the Needleman-Wunsch algorithm. Second,identification of the location of mutations from the DNA of the virus. Theresult of this reserach is an alignment and the location of mutations of bothsequences. The results of identification DNA mutation can be used to find outother viruses mutation corona virus as well as can be used in the field ofhealth as a reference for the manufacture of drugs for corona virus mutationoutcome.