Virus-virus interactions in plants can modify host symptoms. As a result, disease management strategies may be unsuccessful if they are based solely on visual assessment and diagnostic assays for known individual viruses. Papaya ringspot virus is an important limiting factor for papaya production and likely has interactions with other viruses that are not yet known. Using high-throughput sequencing, we recovered known and novel RNA and DNA viruses from papaya orchards in Chiapas, Mexico, and categorized them by host and, in the case of papaya, symptom type: asymptomatic papaya, papaya with ringspot virus symptoms, papaya with nonringspot symptoms, weeds, and insects. Using network analysis, we demonstrated virus associations within and among host types and described the ecological community patterns. Recovery of viruses from weeds and asymptomatic papaya suggests the need for additional management attention. These analyses contribute to the understanding of the community structure of viruses in the agroecological landscape. Potyvirus, Comovirus, and Tombusvirus (RNA viruses) and Begomovirus and Mastrevirus (DNA viruses). We analyzed the network of associations between viruses and host-location combinations, revealing ecological properties of the network, such as an asymmetric nested pattern, and compared the observed network to null models of network association. Understanding the network structure informs management strategies, for example, revealing the potential role of PRSV in asymptomatic papaya and that weeds may be an important pathogen reservoir. We identify three key management implications: (i) each region may need a customized management strategy; (ii) visual assessment of papaya may be insufficient for PRSV, requiring diagnostic assays; and (iii) weed control within orchards may reduce the risk of virus spread to papaya. Network analysis advances understanding of host-pathogen interactions in the agroecological landscape.The study of complex ecological interactions, such as those among host, pathogen, and vector communities, can help to explain host ranges and the emergence of novel pathogens. We evaluated the viromes of papaya orchards, including weed and insect viromes, to identify common viruses in intensive production of papaya in the Pacific Coastal Plain and the Central Depression of Chiapas, Mexico. Samples of papaya cultivar Maradol, susceptible to papaya ringspot virus (PRSV), were categorized by symptoms by local farmers . These analyses revealed the presence of 61 viruses, where only 4 species were shared among both regions, 16 showed homology to known viruses, and 36 were homologous with genera including IMPORTANCE Virus-virus interactions in plants can modify host symptoms. As a result, disease management strategies may be unsuccessful if they are based solely on visual assessment and diagnostic assays for known individual viruses. Papaya ringspot virus is an important limiting factor for papaya production and likely has interactions with other viruses that are not yet known. Using high-throughput sequencing, we recovered known and novel RNA and DNA viruses from papaya orchards in Chiapas, Mexico, and categorized them by host and, in the case of papaya, symptom type: asymptomatic papaya, papaya with ringspot virus symptoms, papaya with nonringspot symptoms, weeds, and insects. Using network analysis, we demonstrated virus associations within and among host types and described the ecological community patterns. Recovery of viruses from weeds and asymptomatic papaya suggests the need for additional management attention. These analyses contribute to the understanding of the community structure of viruses in the agroecological landscape. Pathogen emergence results from interactions between susceptible hosts and pathogenic viruses in conducive environments, causing disease outbreaks in new geographic regions or hosts , 2. EmerCarica papaya L.) have been observed in recent years. A disease outbreak of a mixed infection of RNA and DNA viruses, the interaction of potyvirus, crinivirus, and begomovirus, caused severe symptoms in papaya orchards in Texas  report 52 near-full-length genome sequences identified as plant viruses, 29 being novel viruses, associated with papaya, weeds, and insects, (ii) report the diversity and distribution of viruses in two contrasting physiographic regions, the Central Depression and the Pacific Coastal Plain of Chiapas, and (iii) evaluate the bipartite network structure of viruses and host-location combinations in the papaya orchard agroecosystem. The papaya orchard virome network has structures beyond stochastic associations. Bipartite network analysis of these complex viromes, generated through viral metagenomics, revealed virus associations and identified the roles of particular hosts in connecting the network.To evaluate the papaya orchard virome, papaya samples were collected based on symptoms evaluated by local growers. Growers are generally familiar with PRSV symptoms, because management in orchards commonly includes roguing papaya plants with typical PRSV symptoms or other virus symptoms. To evaluate where PRSV and other viruses were present in papaya orchards, we used a viral metagenomics approach to assess the diversity of viruses in the region. We identified 82 sequences corresponding to at least 57 viruses, of which 52 sequences were nearly full viral genomes within 10 genera, and 10 were unassigned genera in 10 viral families. The bipartite network of viruses, based on metagenomic results and host-location combinations for the papaya orchards, was evaluated in terms of modularity, nestedness, and centrality measures .Bipartite networks have frequently been described for cases such as pollinator networks, where the two types of nodes are pollinators and plants . We concThe diversity of viruses was first evaluated by observing the purified virus-like particles (VLPs) with an electron microscope. Several viral particle morphotypes were confirmed, including filamentous, icosahedral, and pleomorphic. We estimated the diameters for icosahedral particles as ranging from 15 to 65 nm and lengths of up to 750 nm for filamentous particles. Viral morphologies resembling flexuous filamentous or rigid helical rod particles were observed in papaya plants from both the Pacific Coastal Plain and Central Depression. Large numbers of icosahedral particles were observed in all samples. The presence of pleomorphic particles suggested the presence of rhabdoviruses in papaya plants from the Pacific Coastal Plain . Negative staining transmission electron microscopy (TEM) micrographs of VLPs obtained from the papaya orchard samples of the Central Depression (A) and the Pacific Coastal Plain (B). The numbers on images refer to VLPs extracted from papaya plants (1 to 6), weeds (7 and 8), and insects (9 and 10). Papaya with PRSV symptoms (PS) contained icosahedral, filamentous, and pleiomorphic particles . Papaya with non-PRSV symptoms (OS) showed rigid helical rods, flexuous filamentous, and pleomorphic particles . Papayas that were visually asymptomatic (VA) contained icosahedral, filamentous, and pleomorphic particles . Weeds (W) contained icosahedral and filamentous particles . Insects (I) contained icosahedral and filamentous particles . Download Copyright © 2020 Alcalá-Briseño et al.2020Alcalá-Briseño et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Euphorbia sp., Ipomea sp., Sida sp., Portulaca sp., wild grasses, and maize)  were recovered from both physiographic regions: PRSV, PapMV, and Euphorbia mosaic virus (EuMV). Thirty-three viral sequences  were obtained from samples from the Pacific Coastal Plain, and 32 sequences (47%) were obtained from the Central Depression . The per10.1128/mSystems.00423-19.2TABLE S1Table S1, XLSX file, 0.01 MB.Weed accessions. Download Copyright © 2020 Alcalá-Briseño et al.2020Alcalá-Briseño et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00423-19.3TABLE S2Table S2, XLSX file, 0.01 MB.Insect accessions. Download Copyright © 2020 Alcalá-Briseño et al.2020Alcalá-Briseño et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Comovirus, Crinivirus, Potexvirus, Potyvirus, Nucleorhabdovirus, and Begomovirus) and an unclassified double-stranded RNA (dsRNA) virus, PMeV-2, a toti-like virus, were identified from papaya. In the Pacific Coastal Plain, potexviruses, comoviruses, potyviruses, nucleorhabdoviruses, and begomoviruses were present, and potexviruses, criniviruses, potyviruses, unclassified dsRNA viruses, and begomoviruses were present in the Central Depression  and two DNA genera (Begomovirus and Mastrevirus) as two novel unclassified potyviruses, one unclassified caulimovirus, a DNA pararetrovirus, and a satellite virus , indicating limited differences between the ranks for unweighted and weighted versions of betweenness centrality.Betweenness centrality indicates the importance of a species as a connector forming bridges between hosts. We calculated unweighted and weighted versions of betweenness centrality. The highest betweenness centrality (unweighted and weighted) was observed for papaya with PRSV symptoms , visually asymptomatic papaya , and weeds  in the Pacific Coastal Plain and for papaya with PRSV symptoms  and weeds  in the Central Depression. The rest of the nodes had no role in connecting the network . To compP = 0.07) for differences between the unweighted and weighted versions of closeness centrality  to all other nodes (host-locations) in the network. Surprisingly, asymptomatic papaya in the Pacific Coastal Plain had the highest closeness centrality (0.122) in the network, followed by papaya with PRSV symptoms (0.112 and 0.115) for the Pacific Coastal Plain and Central Depression, respectively; however, the weighted closeness was significantly lower (0.001) . For thentrality .We represented the host-location interactions by generating a one-mode projection of the network  showing Begomovirus) was identified in weeds from both regions, but only in the Central Depression was it associated with PRSV and lettuce chlorosis virus  in papaya showing symptoms of PRSV. Interestingly, PRSV and LCV were present in asymptomatic papaya in the Central Depression. A novel bipartite comovirus was recovered from both weeds and insects in the Central Depression and was different from PCV from the Pacific Coastal Plain and two comoviruses tentatively named unknown host comovirus 1 (UHCV-1) and 2 (UHCV-2).Viral metagenomics revealed previously unknown viruses in the papaya orchard and their associations with other known viruses such as PRSV. Network metrics indicate host associations with viruses. We can show more clearly how host and locations are linked to each other by removing virus nodes that are linked to only a single host and location . PRSV waWe used two network metrics to summarize the viral community structure: modularity and nestedness . These mP < 0.001) but little evidence to reject null model 2 (P = 0.216) and null model 3 (P = 0.213)  . The nes= 0.213) . In geneExpanding virome databases offer new opportunities for analysis and understanding of virome complexity. Typical approaches to virome analysis include the use of descriptive statistics, sequence analysis for the identification and discovery of novel viruses, and studies of evolution and viral diversity –49. More–Carica papaya is the only species in the family Caricaceae, a cultivated species originating and domesticated in southern Mexico , Cnidoscolus chayamansa, and Jacaratia mexicana images with pleomorphic virions  and EuMV  that are new reports for viruses infecting papaya in Chiapas. The crinivirus, LCV, and a begomovirus, Tomato yellow leaf curl virus (TYLCV), were recently reported in papaya in Southern Texas, causing severe symptoms , hosts and parasitoids , and bacteria and bacteriophages , 60–62. 60–Bipartite network analysis of the papaya orchard virome also focused on hosts and locations, and a one-mode projection of the network was generated. Information about the role of hosts and viruses in the network can be translated into potential risk management strategies. For example, weeds and visually asymptomatic papayas in the Pacific Coastal Plain had the highest betweenness centrality ; howeverThe phytobiome is conceptualized as the interactions among microorganisms, the environment, and plants . Virome Plant-pollinator networks are asymmetric, relying on generalist species to maintain the nested structure of the network that supports specialized species , 66. HosIn our study, there was strong evidence for the observed modularity of the papaya orchard virome for null model 1 but not for null models 2 and 3. There was strong evidence for nestedness of the papaya orchard virome for all three null models, indicating that the observed nestedness may be due to first-order properties . It is i2, were kept in a plastic bag (see below). Weeds were sampled by collecting asymptomatic and symptomatic leaves in the orchards or their surroundings and insects were actively sampled using insect sweep collecting nets by walking along the perimeter of the orchards and in a cross section inside the orchard and passively collecting insects from sticky traps placed in the orchard by the owner. Samples from these orchards were weed species in the Euphorbiaceae, Poaceae, Solanaceae, Convolvulaceae, Asteraceae, Lamiaceae, Anacardiaceae, Malvaceae, Salicaceae, Moraceae, Plantaginaceae, Cucurbitaceae, Portulacaceae, Caryophyllaceae, and Amaranthaceae families . The regions have different levels of ecosystem fragmentation, with patches of deciduous forest, secondary vegetation, and farmland in the Central Depression, contrasting with former deciduous tropical forest replaced with grasslands and farms in the Pacific Coastal Plain , 68. Thelies see . Within g for 30 min. The supernatant was filtered through a 0.22-μm-pore-size sterile filter  to eliminate particles of higher density and mass, including bacteria, eukaryotic cells, or their fragments. Afterwards, VLPs were precipitated with 10% (wt/vol) polyethylene glycol 8000 (PEG), incubated overnight at 4°C, and further centrifuged at 13,000 × g for 1 h. The pellet was resuspended in PBS and washed with an equal volume of chloroform 2 or 3 times; each time, the mixture was incubated for 15 min at room temperature and centrifuged at 13,000 × g for 10 min at 4°C, and the supernatant was recovered. Insect VLPs were partially purified only with SM buffer  , to which 60 μl of 0.25 mM iodoacetamide and 125 μl of Triton X-100 (33%) were added. The homogenate was stirred for 10 min and centrifuged at 13,000 ×  pH 7.5) . Samples pH 7.5) . Once thg for 4 min at 4°C, the aqueous phases were transferred to clean tubes, and a second step of extraction with chloroform/isoamyl alcohol (24:1) was repeated. One volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of ethanol were added to the samples. After centrifugation at 12,000 × g for 20 min at 4°C, the pellet was resuspended in 35 μl of RNase-free water.Nucleic acids were isolated from the VLP pellets using a procedure described for the extraction of PMeV-RNA in latex . VLPs weg for 15 min at 4°C, and the aqueous phase was recovered and deposited into a new 1.5-ml tube containing 0.02 g of CF-11 cellulose . Ethanol was added to a final concentration of 16%, and the mixture was shaken and then centrifuged at 8,000 × g for 5 min at 4°C. The cellulose was resuspended thoroughly, and the centrifugation process was repeated several times until there were no traces of color. Thereafter, 200 μl of STE was added, and the mixtures were centrifuged at 8,000 × g for 5 min at 4°C. This step was repeated three times, followed by 95% ethanol precipitation of dsRNA at −20°C overnight. After a centrifugation of 10,000 × g at 4°C for 30 min, the dsRNA pellet was dissolved in a volume of 30 μl of RNase-free water and stored at −20°C for further downstream applications.The dsRNA enrichment procedure is a microscale adaptation of a published method . Five gr18 primer or 20 μM of each of three random primers based on the reported 5′-CCTTCGGATCCTCCN6-12-3′ (2), 2 μl dithiothreitol, and 1 μl of Superscript II  were added to each microtube, and the mixture was then incubated first at 42°C for 60 min and then at 70°C for 15 min. This was followed by alcoholic precipitation using 3 M ammonium acetate and absolute ethanol. The second-strand cDNA was synthesized according to the manufacturer’s recommendations; 20 μl of purified single-stranded cDNA was mixed with 20 μl NEB 10× buffer, 6 μl dNTPs (10 mM), 2 μl RNase H (2 U/μl), 3 μl DNA polymerase I , and 80 μl deionized H2O. The sample was incubated for 2.5 h at 16°C and purified with phenol/chloroform/isoamyl alcohol (24:25:1).For the RNA viruses, the first DNA strand was synthesized with murine reverse transcriptase  according to the manufacturer’s recommendations for cDNA synthesis, with 2 μl of 10 μM dTN6-12-3′ : 5′-CCTTC. papaya reference genome (accession number ABIM01) with BOWTIE2 , using BOWTIE2, and SAMtools . Three types of papaya symptom types were sampled  along with bulked samples of weeds and insects. The duplicated cDNA libraries for each of the ten host-location combinations were obtained using the Nextera XT library preparation kit and sequenced by Illumina HiSeq 2500 fast mode with paired-end reads (2 × 100) . The total number of raw reads we obtained was 66,406,113, and an average of 2 million reads per library were obtained after removing low-quality reads. The libraries from papaya were filtered using the draft genome sequence of the  BOWTIE2 , 73. Alles v.3.7  enablinges v.3.7  using a es v.3.7 , where sSAMtools . All seqhttps://talk.ictvonline.org/), either by identities using BLAST or by pairwise similarities. Sequences were submitted to NCBI  or for sequences with high similarity to known virus species. The sequences assembled were aligned among them using Needleman and Wunsch in Geneious . SequencNCBI see .10.1128/mSystems.00423-19.4TABLE S3Table S3, XLSX file, 0.01 MB.Papaya orchard virome accession numbers and classification. Download Copyright © 2020 Alcalá-Briseño et al.2020Alcalá-Briseño et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We constructed a bipartite incidence matrix, where one group comprised viral taxa and the other group comprised the combinations of host type and region. The entries in the incidence matrix indicated the presence or absence of each viral taxon in each host-location combination. We also considered weighted matrices with weights representing the read coverage across the contig length as an indicator of relative sequence abundance. Network metrics such as node degree, nestedness, and others were calculated using the bipartite package in R . HypothePRJNA592837. Ten BioSamples were submitted corresponding to visually asymptomatic papaya (SAMN13440963 and SAMN13440956), papaya with non-PRSV symptoms (SAMN13440964 and SAMN13440957), papaya with PRSV symptoms (SAMN13440965 and SAMN13440960), weeds (SAMN13440966 and SAMN13440961) and insects (SAMN13440967 and SAMN13440962). Virus accession numbers (MN203139 to MN204623) are also listed in The read sequence accessions of the papaya orchard virome from the Central Depression and the Pacific Coastal Plain were deposited in the NCBI database as BioProject


The effect of fluorination on the film structure, composition, and magnetic properties is compared with the case of oxygen-deficient and fully-oxidized LSMO films. Although incorporation of F− anions does not significantly alter the volume of the LSMO unit cell, a strong impact on the magnetic characteristics, including a remarkable suppression of Curie temperature and saturation magnetization accompanied by an increase in magnetic coercivity, was found. The change in magnetic properties can be ascribed to the disruption of the ferromagnetic exchange interactions along Mn-anion-Mn chains driven by F− doping into the LSMO lattice. Our results indicate that F− doping is a powerful means to effectively modify the magnetic functional properties of perovskite manganites.Chemical doping via insertion of ions into the lattice of a host material is a key strategy to flexibly manipulate functionalities of materials. In this work, we present a novel case study on the topotactic insertion of fluoride ions into oxygen-deficient ferromagnetic thin films of La For Lperature . This caxAExMnO3 .1−xSrxMnO3 (LSMO) is that, for a composition around x = 0.25 [One of the significant features of Lax = 0.25 , the parx = 0.25 ,11. Sincx = 0.25 ,12 direc− anions, was found to be [2 gas [In recent years, novel routes based on X-site anion doping, such as intercalation of oxygen  anions, nd to be ,15,16,17nd to be . Althougnd to be  performe2CF2 or HF) are transported to the film surface, where they react further [This polymer-based fluorination method was previously applied to perovskite-based thin films ,20,21,222− anions, one could expect a significant variation of the magnetic properties of LSMO upon partial substitution of O2− with F− ions. In this article, we present a thin film fluorination study of epitaxially-grown films of La1−xSrxMnO3−δ . We show that the reactivity towards fluorine incorporation strongly depends on the degree of anion vacancies within the lattice and can lead to remarkable changes of the magnetic properties.Since magnetism in LSMO and related perovskite-type manganites originates from the indirect exchange interactions , namely 3 (STO) substrates using a large-distance magnetron sputtering (LDMS) chamber featuring an unconventionally-large target-to-substrate separation of 285 mm. STO was chosen as a substrate, because it has a perovskite structure and a low lattice mismatch with LSMO, and both conditions can facilitate the epitaxial growth of LSMO onto STO. Prior to the deposition process, the surface quality of the STO substrates was analyzed via atomic force microscopy , which revealed a flat morphology with a root mean square roughness of about 0.2 nm. For all depositions, an LSMO ceramic disc of 3 inches in diameter was used as the sputtering target. The LSMO samples were prepared at a temperature of about 800 °C and a pressure of 0.018 mbar in Ar/O2 = 3/2 gas mixture. A sputtering power of 200 W led to a deposition rate of 0.045 Å/s, which was monitored with a quartz crystal microbalance. As we were aiming to produce LSMO films with inherent oxygen vacancies, after completing the deposition the samples were cooled to room temperature at a rate of 10 K/min under a relatively-low oxygen pressure of 0.08 mbar. Further details about the LSMO growth conditions can be found elsewhere [The LSMO films were grown on (001)-oriented single-crystalline SrTiOlsewhere .0.74Sr0.26MnO3−δ films with inherent oxygen vacancies were subjected to various heat treatments and fluorination processes in order to be able to study the influence of different anion-incorporating conditions on the film characteristics. Those treatments are summarized in The obtained Laθ–2θ configuration with Cu-Kα1 (λ = 1.540596 Å) radiation in order to analyze the phase evolution of the LSMO films treated with different reaction conditions. Analysis of the out-plane lattice parameter of the various films was performed by using the Pawley method as implemented in the TOPAS software package [The surface roughness of the samples was characterized by tapping-mode AFM under ambient conditions. High resolution X-ray diffraction  measurements were carried out using a Bruker D8 Advance thin film diffractometer in  package , after t package . This es package , which iTC. Magnetic hysteresis loops carried out at the temperature of 5 K allowed to analyze the saturation magnetization and the magnetic coercivity.The magnetic characterization was performed using a Quantum Design MPMS SQUID Magnetometer . Field cooled (FC) curves were measured in the 5 to 400 K temperature range with an external magnetic field of 500 Oe applied parallel to the in-plane film direction to determine the value of Transmission electron microscopy  was performed within an aberration (image) corrected FEI Titan 80–300 operated at an acceleration voltage of 300 kV. Elemental distributions were imaged by energy filtered TEM using the “3-windows approach” with a Gatan Tridium 863 energy filter. The study was performed on cross-sections of the LSMO thin film specimens prepared by focused ion beam milling  roughness as small as 0.24 nm (not shown here). After fluorination (LSMO_F), only a slight increase of the rms roughness (~0.7 nm) was observed, thus confirming that the fluorinating agent (PVDF) does not cause a substantial alteration of the LSMO surface topography due to decomposition and/or polymer deposition.2). The out-of-plane lattice parameter of the different films is summarized in HRXRD patterns revealed that the reflections of as-grown and topochemically-treated LSMO films follow the same (001)-orientation of the STO substrate peaks see  with the4+ ion as compared to Mn3+ [Oxidation of the as-grown samples (LSMO_O) results in a significant shift of the LSMO peaks towards higher angles, which is related to the shrinkage of the LSMO unit cell volume owing to the smaller size of the Mn to Mn3+ . The con to Mn3+ , but als to Mn3+ ,33.(a)Reductive fluorination: One fluoride ion replaces one oxygen ion, accompanied by a reduction of the transition metal oxidation state(b)Substitutive fluorination: Two Fluoride ions replace one oxygen ion and fill a vacancy under maintenance of the transition metal oxidation state(c)Oxidative fluorination: One Fluoride ion fills a vacancy, accompanied by an increase of the transition metal oxidation stateIn contrast, the fluorination of the as-grown film (LSMO_F) does not strongly alter the out-of-plane lattice parameter of LSMO. In this respect, the reader should be aware that fluorination reactions of perovskite materials  can give a strong indication for which fluorination mechanism has taken place. This is explained by the empirical finding that the volume per formula unit mainly depends on the transition metal oxidation state and not on the detailed composition of oxygen and fluoride ions as well as vacancies of the anion sublattice [3+O2.5 and BaFe3+O2.5 under formation SrFe3+O2F [3+O2F [3 to SrFeO2F (mechanism (a) goes with a strong increase of the unit cell (ΔV~+6%) [2.5 to AMO2.5F0.5 (mechanism (c)) is accompanied by a strong shrinkage of the unit cell [Usually, the cell volume per formula unit  , whereas− anions predominantly at the film surface , a cross-section of sample LSMO_F was analyzed by means of elemental mapping via energy filtered TEM (EFTEM), see In order to confirm fluorination of the film and in order to verify if the small influence of fluorination on the LSMO unit cell volume may be caused by incorporation of FThe importance of the presence of anion vacancies, which are required for a substitutive fluorination according to mechanism (b), was further examined by reacting differently treated films. Application of fluorination treatment to an oxidized film (LSMO_O+F) resulted in a mainly unreactive response with the absence of significant changes of the LSMO lattice parameter (and in agreement with the absence of changes of magnetic properties as discussed in 1−xSrxMnO3−y with HF (from PVDF decomposition) and O2 by the following equations, again highlighting the importance of the presence of anion vacancies VO•• for the fluorine incorporation:1−xSrx3+1−x+2yMn4+x−2yMnO3−y + 2y HF ➔ La1−xSrx3+1−x+2yMn4+x−2yMnO3−2yF2y + y H2O (LSMO_F): La1−xSrx3+1−x+2yMn4+x−2yMnO3−y + y/2 O2 ➔ La1−xSrx3+1−xMn4+xMnO3(LSMO_O): La1−xSrx3+1−x+2yMn4+x−2yMnO3−2yF2y + O2 ➔ no reaction(LSMO_F+O): La1−xSrx3+1−xMn4+xMnO3 + HF ➔ no reaction (LSMO_O+F): LaWe, therefore, summarize the reaction of as-grown oxygen deficient LaTc~135 K compared to bulk LSMO and a rather smeared out para/ferromagnetic transition is found, in agreement with the behavior encountered in oxygen-deficient LSMO thin films [Tc up to about 320 K. Differently, in case of incorporation of fluoride ions (LSMO_F), the magnetic transition temperature decreases down to 100 K. Processing of LSMO_O and LSMO_F with a sequential fluorination/oxidation treatment, i.e., LSMO_O+F and LSMO_F+O respectively, did not bring any substantial modification of the measured FC curves.in films . As expeMs~1.5 µB/Mn., Hc~270 Oe), oxidation treatment led to an increase in saturation magnetization (Ms~3 µB/Mn.) and decrease in coercive field (Hc~8 Oe), whereas fluorination produced a much lower magnetic moment (Ms~0.9 µB/Mn) and a broader hysteresis loop (Hc~760 Oe). It is worth to notice that fluorinated LSMO is magnetically harder than oxidized LSMO with a difference in magnetic coercivity of almost two orders of magnitude. Additionally, the measurements confirmed that an a-posteriori change of the nearly-completely filled anion lattices of the LSMO_O/LSMO_F films is not possible at the chosen reaction conditions, since no prominent changes of magnetization curves could be found for the LSMO_O+F and LSMO_F+O films. However, it is acknowledged that such changes might be induced in different reaction conditions .Further insights into the influence of different conditions of anion incorporation on the magnetic properties were gained from the analysis of low-temperature magnetic hysteresis loops see b. StartiTC, saturation magnetization MS, and coercive field HC are listed in TC, MS, and HC indicate that anion intercalation affects a large portion of the LSMO volume, thus corroborating the results of the microstructural analysis.The obtained values of Curie temperature 3+/4+ ions originates from double-exchange mechanism, whereas antiferromagnetic interactions are promoted between Mn3+/3+ or Mn4+/4+ via superexchange mechanism.In a conventional scenario, the magnetic properties of manganites are determined by the exchange interactions along Mn-O-Mn bonds due to the overlap of the manganese d-orbitals with the p-orbitals of the bridging anions . Ferroma3+ and missing oxygen bonds. With the insertion of oxygen anions, Mn-O-Mn bonds are restored and the Mn3+/4+ balance increases owing to the covalency of O2−, which amounts to doping of the LSMO unit cell with two holes. As a consequence, oxidized films present enhanced Tc and Ms, in agreement with the values expected from the LSMO magnetic phase diagram [The fact that as-grown oxygen-deficient films reveal a suppressed magnetization and Curie temperature is compatible with an excess of Mn diagram  for a coTo our knowledge, the effect of the substitutive fluorination on the magnetic properties of manganites has not been theoretically investigated yet. Experimental studies only exist for the oxidative fluorination of Ruddlesden-Popper type manganites ,39, whic3+/4+ ratio, driven by the fact that F− insertion amounts to doping the LSMO unit cell with only one hole. In this respect, fluorinated LSMO should give a similar magnetic response comparing to as-grown LSMO films, since fluorination follows a substitutive mechanism and the Mn3+/4+ ratio is maintained  and oxidation (LSMO_O) may be primarily attributed to the different balance in Mn2−, and more ionic for X = F−. In this regard, F− doping may trigger antiferromagnetic coupling or extinguish any exchange interaction between adjacent Mn ions. This observation is in principle agreement with the recently reported weakening of Fe-F-Fe superexchange interactions as compared to Fe-O-Fe for the series LaFe3+O3F0 (TN~750 K), (Sr/Ba/Pb) Fe3+O2F1 (TN = 650–700 K) and AgFe3+O1F2 (TN~500 K) [2Ni2+O3F2 (TN = 49 K) as compared to Ni-O-O-Ni in La2NiO4 (TN = 330 K) [One point that, in accordance with the Goodenough–Kanamori rules , could jN~500 K) , as well= 330 K) .Γ [Another factor to keep in mind, related to a figure of merit known as tolerance factor Γ , is the Γ , vacancyΓ  at the L2− with monovalent F− anions on the structural and magnetic properties of oxygen-deficient thin films of LSMO epitaxially grown on STO substrate. Both oxidation and fluorination processes involve anion incorporation into the bulk of the LSMO films, although no remarkable expansion or shrinkage of the LSMO unit cell volume occurs upon F− doping. Concerning the magnetic properties, the processes of oxidation and fluorination have completely different effects on the as-grown LSMO films. The former makes LSMO a stronger and softer ferromagnet, whereas the latter leads to an increase of the magnetic coercivity at the expense of the Curie temperature and magnetization saturation. As to the physical origins of the strong suppression of the ferromagnetic order, further theoretical studies are necessary to unveil the complex mechanisms controlling the magnetic exchange interactions in LSMO upon F− doping.In this work, we have compared the influence of topochemical oxidation as well as substitution of divalent O3−y → AMnO3−yFy), or reductive fluorine deintercalation (AMnO3−yFy → AMnO3−y), shall open new opportunities for tuning material properties.This work suggests that topochemical fluorination can offer an alternative route to conventional doping approaches based on divalent cation or oxygen anion incorporation to manipulate the functional properties of strongly correlated perovskite manganites. Since fluorine ions are more mobile than oxygen ions , their r


Chickpea chlorotic dwarf virus (CpCDV) is a threat to chickpea production leading to yield losses of 75–95%. Chickpea chlorotic dwarf virus is a monopartite, single-stranded circular DNA virus in the genus Mastrevirus and family Geminiviridae. It is transmitted by Orosius albicinctus in a circulative (persistent) and nonpropagative manner. Symptoms of CSD include very small leaves, intense discoloration (yellowing (kabuli type) and reddening (desi type)), and bushy stunted appearance of the plant. Presently, CpCDVs occurs in Africa, Asia, Australia, and the Middle East, causing extensive losses on economically important crops in in the families Fabaceae, Asteraceae, Amaranthaceae, Brassicaceae, Cucurbitaceae, Caricaceae, Chenopodiaceae, Leguminosae, Malvaceae, Pedaliaceae, and Solanaceae. High frequency of recombinations has played a significant role in the wide host range, diversification, and rapid evolution of CpCDVs. This review highlights the extensive research on the CpCDV genome diversity, host range, plant–virus–insect interactions, and RNA interference-based resistance of CpCDV, providing new insights into the host adaptation and virus evolution.Chickpea stunt disease (CSD), caused by  Cicer arietinum L.) is an important pulse crop grown and consumed all over the world. As the world’s population rises, the demand for grain legumes is also rising, and it is a permanent challenge to meet increasing demands. However, abiotic and biotic factors affect plant growth and pose a threat to sustainable agriculture and food production. Pathogens include fungi, bacteria, viruses, nematodes, and mycoplasma . T. T26]. TThe Rep protein, which is the only protein responsible for the initiation of replication, is a ~39 kDa protein comprising ~334 amino acids. In silico analysis of the Rep protein  revealed the presence of motif I (FLTYP), motif II (HY/CHALI/VQ or HYHASYS) and motif III (VLD/EYIS) ,29. The Mastrevirus, like other geminiviruses, is a multifunctional protein. It is a structural protein and is required for encapsidation, accumulation of ssDNA, vector-mediated transmission, nuclear import of genomic DNA, and cell-to-cell movement of the viral genome. The movement protein encoded by the virion sense strand ORF V2 is positioned upstream of the capsid protein gene . H. (1994)  reported for MSV  and BCTV for MSV ,63,64. M for MSV  demonstrmastrevirus-infected plant from plants affected by other pathogens. In these circumstances, it is necessary to have virus-specific diagnostic reagents to detect the virus present in naturally infected plants. The diagnostics are also required to detect the virus in the insects visiting the plants in order to identity the vectors. Serological diagnostic methods such as DAS-ELISA, dot-blot ELISA, and tissue-blot immunoassay (TBIA) have been developed to detect the presence of CpCDV from infected field plant samples and viruliferous vector [The symptoms caused by mastreviruses in graminaceous hosts are often similar to symptoms caused by abiotic agents like nutritional deficiencies. In the case of dicot-infecting mastreviruses, it is difficult to distinguish a s vector ,46,65,66Over the past decade, rolling circle amplification (RCA) and restriction fragment length polymorphism (RFLP) has been extensively used to identify geminiviruses in most virus-infected plants. RCA was developed using the bacteriophage varphi 29 DNA polymerase, and was central in revolutionizing the detection and diagnosis of geminiviruses . This teKanakala et al. 2013)  detected  detecteMastrevirus diagnosis and control vector population to transmit disease. The relationships between mastreviruses infecting different host species need to be understood to develop management strategies that will prevent the further emergence of new viruses. An extensive global sampling and metagenomics analysis using next-generation sequencing of these viruses will identify the global diversity of dicot-infecting mastreviruses and inform better strategies for diagnostics and disease management.All these molecular methods corroborate the importance of the extensive In the past decade, an upsurge of chickpea viral diseases has been experienced, resulting in economic losses of chickpea production across the growing regions. Successful plant breeding programs for disease resistance depend on the successful identification of sources of resistance and the incorporation of resistance genes into commercial varieties ,69. ChicAgrobacterium tumefaciens. Over 70 genotypes screened genotype SCGP-WR-29, which showed resistance in the field condition but exhibited 80% incidence under agroinoculation. Three agroinoculated genotypes , and IPC 09-07) showed virus resistance and did not express any symptoms, and plants remained alive compared to susceptible genotypes. More interestingly, resistant plants were shown to be virus-free under PCR tests. These kinds of resistance screening tests have yet to be adopted to generate CpCDV-resistant cultivars on a wide scale. In the same study, an objective scoring to assess the response of chickpea genotypes to CSD by agroinoculation of CpCDV construct was also developed.Among 10,000 germplasm lines screened for resistance to stunt disease, two lines (GG669 and ICCC10) were found to be field-resistant to CpCDV . More reN. benthamiana to provide immunity to CpCDV inoculation [N. benthamiana plants expressing CRISPR-Cas reagents and challenged with BeYDV had reduced virus load and symptoms [Mastrevirus in chickpea. This will require significant progress in tissue culture and transformation technologies to CpCDV-resistant chickpea genotypes.RNA interference (RNAi) is a very promising strategy that has been employed to control both plant viruses and insect vectors ,71. Hairculation . Baltes culation  demonstrsymptoms . Howeverlaccase-2/peptidoglycan recognition protein (PGRP-LC) resulted in significant mortality in leafhoppers [Mastrevirus insect vector. The identification of such candidate genes and the development of transgenic plants expressing dsRNA/SiRNA that target insect genes are necessary to control insect virus transmission under field conditions. Recent genome editing tools like CRISPR/Cas9 are highly suggested to modify virus/vector genes in order to develop effective resistance against CpCDV/O. albicinctus.Similarly, RNAi has been successfully demonstrated in other leafhopper insect vectors. Silencing/knockdown of insect genes fhoppers ,75. UntiCpCDV continues to be a threat to chickpea production worldwide. Although the virus was first reported in India in the year 1993, today CpCDV has been reported in Africa, the Middle East, and Australia, because of the polyphagous and widespread insect vector. At present, mixed infections, the emergence of new strains, and inter/intra recombinations among CpCDV strains/species might have increased its host range and caused new epidemics. Some major questions remain to be answered concerning (1) Mastrevirus–satellite interactions, (2) virus–host–insect interactions, (3) insect vector and its endosymbiont’s efficacy in virus transmission, and (4) the discovery of CSD-resistant chickpea varieties. Considering the continuing new reports of CpCDV strains from new hosts and regions of the world, and given the importance of the fourth most widely grown pulse, continued research is needed to understand the biology, ecology, and epidemiology of CpCDV and its insect vector.Over the past few years, a very low number of resistant chickpea varieties were screened through virus inoculations ,56. One


DNA hybridization-based assays are well known for their ability to detect and quantify specific bacteria. Assays that employ DNA hybridization include a NanoGene assay, fluorescence in situ hybridization, and microarrays. Involved in DNA hybridization, fragmentation of genomic DNA (gDNA) is necessary to increase the accessibility of the probe DNA to the target gDNA. However, there has been no thorough and systematic characterization of different fragmented gDNA sizes and their effects on hybridization efficiency. An optimum fragmented size range of gDNA for the NanoGene assay is hypothesized in this study. Bacterial gDNA is fragmented via sonication into different size ranges prior to the NanoGene assay. The optimum size range of gDNA is determined via the comparison of respective hybridization efficiencies (in the form of quantification capabilities). Different incubation durations are also investigated. Finally, the quantification capability of the fragmented (at optimum size range) and unfragmented gDNA is compared. DNA hybridization is a well-known and critical process in assays that targets specific genomic DNA (gDNA). Essentially, it is the formation of a hydrogen bond between complementary sequences of probe single-stranded DNA (ssDNA) and the target ssDNA. It is employed widely in numerous bioassays, such as membrane hybridization ,2,3, fluThe target gDNA can be from a variety of sources, such as microorganisms (like pathogenic bacteria) to human body analytes (such as blood). Since the gDNA is double-stranded and has a large supramolecular structure, it needs to be fragmented (as a pretreatment) prior to hybridization with the probe ssDNA. The fragmentation of long double-stranded gDNA into shorter gDNA will increase its accessibility to the probe ssDNA and, hence, allow for the achievement of higher hybridization efficiency.Characterization of pretreatment conditions to determine optimum environments for a particular assay is a mundane but, unfortunately, a very important exercise. It enables the particular assay to subsequently perform at its optimum conditions in its respective applications, such as identification of pathogenic bacteria. This study is no exception. Specifically, the authors are interested in the existence of an optimum fragmented gDNA size range for the NanoGene assay. The NanoGene assay has previously demonstrated its ability to detect and quantify specific bacteria (both airborne and waterborne) based on their gDNA. Furthermore, it could be implemented via portable apparatus, thus enabling the possibility for mobile monitoring as well as point-of-investigation diagnostics ,18,19,20The NanoGene assay uses particle complexes consisting of magnetic beads, a set of quantum dots, and probe and signaling DNAs. Both probe and signaling DNA complexes can be tethered together via DNA hybridization with the target gDNA. Magnetic beads are used as carriers that are suitable for magnetic manipulation. The normalized fluorescence intensity from a set of quantum dots will be indicative of the amount of target gDNA. The normalized fluorescence also depends strongly on the hybridization efficiency . Therefore, in the absence of photobleaching and quenching while all else being equal, the hybridization efficiency is also representative of the NanoGene assay’s quantification capability and vice versa.Pseudomonas putida) is first extracted, followed by sonication for DNA fragmentation. The authors have shown previously that sonication is the most efficient method for gDNA fragmentation with sizes ranging from ~200–1000 base pairs or bp [During this study, the gDNA of the target bacterium  was selected as a model bacterium for this study. The dry cells were revived in 1 mL of the tryptic soy broth  in a Gallenkamp orbital shaker at 300 rpm at ambient temperature for 5 days. Afterward, the cells were streaked on a tryptic soy agar (Difco) plate and a single colony was chosen. The single colony inoculated in the tryptic soy broth (Difco) was incubated in a Gallenkamp orbital shaker at 300 rpm at ambient temperature for 5 days to achieve a stationary phase of growth (OD ≅ 1.2). The growth of the bacteria was monitored by measuring the optical density at 600 nm using a SpecraMax® M2 Multi-Mode Microplate Reader . Later, the bacteria were collected from 1 L medium via centrifugation at 5000× g for 30 min . The pellet was subjected to gDNA extraction using a FastDNA® SPIN Kit for Soil , followed by additional purification using Zymo Genomic DNA Clean and ConcentratorTM . The quantity and the purity of the extracted gDNA were determined via the NanoDropTM 1000 spectrophotometer .® tissue. Subsequently, the gDNA fragments were subjected to agarose gel electrophoresis for sizing. The fragmented gDNA was loaded on a 2% agarose gel, and 110 V was applied for 1 h , followed by visualization with a UV Transilluminator . The size of the fragmented gDNA was measured against a 1 kb ladder . Subsequently, the electrophoresis gel was excised manually for each size range: 200–500 bp, 500–1000 bp, 1000–1500 bp, 1500–2000 bp, 2000–2500 bp, and 2500–3000 bp. The respective fragmented gDNA for each size range was obtained from the excised gel using the ZymocleanTM Gel Recovery kit (Zymo Research). To validate, the fragmented gDNA was visualized with 2% agarose gel electrophoresis.Sonication was performed via XL-2000 Ultrasonic Dismembrator with a P-3 microprobe  for 10 s. The output power of the sonicator was set to 10 W. The samples were placed on ice to minimize heat generation during sonication. The microprobe was washed three times with 70% ethanol and a KimwipesBriefly, as shown in α  gene was selected as a target gene for the quantification of P. putida. The PAH-RHDα gene is involved in the initial step of the PAHs metabolism by the P. putida [P. putida by the NanoGene assay was performed based on the protocol previously developed by Kim and Son [(i)® 565 ITK™ carboxyl quantum dot nanoparticles  were immobilized on the surface of 100 µL of 107 beads/mL aminated Dynabeads® M-270 magnetic beads . Dynabeads® M-270 MB with 2.8 µm in diameter are superparamagnetic, and have a monolayer of amine functional group on their surface. This was followed by conjugating 5 µL of 100 nmol/L aminated probe DNA  on the surface of QD565 by forming an amide bond via the reaction of ethylcarbodiimide hydrochloride  and N-hydroxysuccinimide .Preparation of probe complex: 8 µL of 2 µmol/L Qdot(ii)2-3′) were attached covalently to the surface of 8 µL of 2 µmol/L carboxyl quantum dots  via EDC-NHS reaction. Qdot® ITK™ carboxyl quantum dots are made from nanometer-scale crystals of a semiconductor material (CdSe), which are shelled with an additional semiconductor layer (ZnS). The size of QDs that were used in this study were within 15–20 nm.Preparation of signaling complex: Aliquots (1.6 µL) of 100 nmol/L aminated signaling probe DNA (5′-CAA CCM ACG TGG TAT GCA TCT CAT C-NH(iii)P. putida, ~4 Mbp) were mixed with the probe and signaling complexes. Subsequently, a slow vertical rotation in a hybridization oven  was used to enable DNA hybridization overnight at 37 °C.gDNA hybridization: Five µL of the target gDNA in six size ranges  with a negative control (water) and a positive control (unfragmented gDNA of (iv)565 and QD655 were measured using a SpectraMax M2 spectrofluorometer  at 570 and 660 nm, respectively, under excitation at 360 nm. All experiments were performed in triplicate, unless otherwise stated. The output of quantification was expressed by the ratio of the fluorescence intensity between QD655 and QD565 (QD655/QD565), as the signal (QD655) was normalized by the internal standard (QD565) to comprehend the different numbers of nanoparticles in each reaction.Fluorescence measurement: Following DNA hybridization, the hybridized complexes were washed three times using a phosphate buffer (pH = 7.4) to remove the untethered complexes. The endpoint fluorescence of QDThe PAH-RHD. putida . The qua and Son .(i)PrepaDifferent hybridization durations  were investigated using the optimum fragmented gDNA size range. The fragmented gDNA size range used was 1000–1500 bp. A total of 18 samples (triplicate for 6 time points) were prepared and used in the experiment. Three triplicate samples were taken out of the hybridization oven, washed with phosphate buffer three times, and transferred to a 96-well plate  at each time point. The fluorescence of the samples was measured using a SpectraMax M2 spectrofluorometer as described above.Using a gDNA size range of 1000–1500 bp and 4 h of hybridization time, the NanoGene assay was performed with different amounts of target gDNA . To compare, similar amounts of unfragmented gDNA were also used with a NanoGene assay.p-value = 9.5 × 10−12, ANOVA). Pairwise t test was performed as a post-ANOVA, to show the statistical difference between the 1000–1500 bp gDNA and other sizes. As a result, all P-values of the t test (95% confidence level) between 1000–1500 bp, and all other fragmented DNAs  were 0.005, 0.009, 0.004, 0.010, 0.008, and 0.004, respectively. This suggested all other fragmented DNA were statistically different from the 1000–1500 bp gDNA. Additionally, the normalized fluorescence of the unfragmented gDNA was similar to that of 2500–3000 bp gDNA. The p-value of t test comparing between unfragmented gDNA and 2500–3000 bp gDNA was 0.858 (95% confidence level). This suggested that gDNA with a size range equal to or larger than 2500 bp would behave like unfragmented gDNA during hybridization. As described in α gene of P. putida [The negative control yielded a normalized fluorescence of 12.6 ± 0.263. As shown in . putida ,23. GiveAs shown in Considering an incubation duration of 4 h, the gDNA with an optimum size range (1000–1500 bp) yielded a higher normalized fluorescence than unfragmented gDNA . Using 4The presented work describing the optimum size of a gDNA template and the accompanying incubation time is a part of the continuing efforts to complete the development of the NanoGene assay by optimizing conditions of DNA hybridization in particle suspension. DNA hybridization in the NanoGene assay was previously evaluated and optimized for buffer stringency, effects of additives, washing and passivation, and probe lengths and configuration ,25,26. EThe authors have shown that the optimum size range of gDNA for the NanoGene assay was 1000–1500 bp. It yielded the highest normalized fluorescence at 35 ± 2.895, as compared to that of 200–500 bp at 20 ± 0.789, and 2500–3000 bp at 16 ± 1.053. Additionally, its quantification capability was almost twice that of unfragmented gDNA for a gDNA amount of 400 ng and incubation of 4 h. This study will allow for the improvement of the performance of NanoGene assay-based diagnostics in terms of kinetics and analytical sensitivity. Finally, an important implication of the result presented in this study is that it will become part of the custom optimized pretreatment process prior to DNA hybridization-based assays. Therefore, the improvement of DNA hybridization can be achieved through the use of size-specific fragmentation, rather than the existing procedure that involves whole gDNA and other supporting agents.


Background: Endemic and seasonally recurring respiratory viruses are a major cause of disease and death globally. The burden is particularly severe in developing countries. Improved understanding of the source of infection, pathways of spread and persistence in communities would be of benefit in devising intervention strategies.Methods: We report epidemiological data obtained through surveillance of respiratory viruses at nine outpatient health facilities within the Kilifi Health and Demographic Surveillance System, Kilifi County, coastal Kenya, between January and December 2016. Nasopharyngeal swabs were collected from individuals of all ages presenting with acute respiratory infection (ARI) symptoms (up to 15 swabs per week per facility) and screened for 15 respiratory viruses using real-time PCR. Paediatric inpatient surveillance at Kilifi County Hospital for respiratory viruses provided comparative data.Results: Over the year, 5,647 participants were sampled, of which 3,029 (53.7%) were aged <5 years. At least one target respiratory virus was detected in 2,380 (42.2%) of the samples; the most common being rhinovirus 18.6% , influenza virus 6.9% (390), coronavirus 6.8% (387), parainfluenza virus 6.6% (371), respiratory syncytial virus (RSV) 3.9% (219) and adenovirus 2.7% (155). Virus detections were higher among <5-year-olds compared to older children and adults  =177.3, P=0.0001). Frequency of viruses did not differ significantly by facility (χ2(8) =13.38, P=0.072). However, prevalence was significantly higher among inpatients than outpatients in <5-year-olds for RSV  = 159.4, P=0.0001), and adenovirus  =56.6, P=0.0001).Conclusions: Respiratory virus infections are common amongst ARI outpatients in this coastal Kenya setting, particularly in young children. Rhinovirus predominance warrants further studies on the health and socio-economic implications. RSV and adenovirus were more commonly associated with severe disease. Further analysis will explore epidemiological transmission patterns with the addition of virus sequence data. Viruses are recognized as major cause of both mild ARI and of severe acute lower respiratory tract infection (LRTI)3. Respiratory syncytial virus (RSV), rhinovirus and influenza A are often identified as the most common viruses associated with ARI5, but a wide range of viruses are to be found in ARI presentations to the hospital and outpatient settings5. The changing landscape of ARI due to the widespread use of conjugated bacterial vaccines could lead to an increased prominence of viral causes of these illnesses7.Acute respiratory infection (ARI) is a major cause of morbidity and mortality globally, principally affecting young children and the elderly, and with majority of the burden occurring in low-resource settings9, high serotype diversity (e.g. rhinovirus)10 or target populations not appropriate for current vaccines (e.g. RSV)11. Consequently, innovation in vaccination strategies for these pathogens (e.g. targeting of schools and households), mass use of antivirals or non-pharmacological methods such as social distancing (e.g. school closures), are options to consider. Designing strategies to control respiratory viruses would be assisted by detailed knowledge of the patterns of spread, i.e. of distinct pathways of transmission in communities, at various social organizational levels from the household, school, local community, nationally and beyond country boundaries.Given this context, greater emphasis on the control of virus-associated ARI is likely. Vaccination as an intervention for the control of viral respiratory infections faces considerable hurdles. These include continuous or rapid pathogen evolution (e.g. influenza)SPReD (Studies of the Pathways of transmission of Respiratory virus Disease), which aims to map patterns of spread of a range of respiratory viruses using epidemiological and nucleotide sequence data across different settings in Kenya. Here we present the baseline results of 1 year of respiratory virus epidemiological surveillance at health facilities within a well-defined coastal Kenyan population and a comparison with contemporaneous data on inpatient admissions to Kilifi County Hospital (KCH).The present work forms part of a larger project under the title14. The KHDSS area was defined and mapped for demographic surveillance, clinical and epidemiological research by the KEMRI Wellcome Trust Research Programme (KWTRP) in the year 2000. It is located in Kilifi County along the coastal fringe and covers an area of 891 km2, 50 km north and south, and 30 km west, of the KCH. The KHDSS monitors a population of around 296,000 residents (2016 census) through household enumeration visits conducted every 4 months. The major economic activity of most residents is subsistence farming of maize, cassava, cashew nuts and coconuts, as well as goats and dairy cattle14.This study was conducted on the coast of Kenya, within the Kilifi Health and Demographic Surveillance System (KHDSS)The KHDSS area has 21 public health facilities (including the KCH) receiving out-patients, which operate under the Kenya Ministry of Health (MoH). In total, nine of these facilities were selected for this study: Matsangoni, Ngerenya, Mtondia, Sokoke, Mavueni, Jaribuni, Chasimba, Pingilikani and Junju . The fac15) were eligible. New-borns aged less than 7 days and patients with ARI for more than 30 days were excluded. Written individual informed consent was sought from adult patients and parents/guardians of patients below 18 years. The study commenced in December 2015 with piloting before enhanced surveillance of 15 samples per site per week from January to December 2016.Participant recruitment and specimen collection was integrated within the routine patient care at the nine selected outpatient facilities led by a resident clinician or nurse. Each facility had one or two sampling days per week, usually scheduled from Monday to Friday, between 9.00 am and 1.00 pm. On each sampling day, a study fieldworker stationed at the health facility, assisted by the resident clinician or nurse, would describe the study to the attending patients. Any person present with listed signs and symptoms of ARI would be asked to see the fieldworker for further screening and obtainment of consent as they await review by the nurse or clinician. Patients of any age presenting with one or more ARI symptoms of cough, sneezing, nasal congestion, difficulty breathing, or increased respiratory rate for age , 700 (10%), 490 (7%) and 490 (7%) specimens to be positive for rhinoviruses, RSV, coronaviruses, and influenza viruses, respectively.16. The NPS sample was stored in universal virus transport media  and kept at approximately 8°C in an ice-packed cool box for return to KWTRP within 4 h of collection.The selection of participants each week was on a ‘first-come first-served’ basis as they presented to the health facility on the set sampling days. A standardised questionnaire  was usedAll individuals, parents and guardians gave written informed consent for themselves or their children to participate in this study. The study was approved by the KEMRI-Scientific and Ethical Review Unit (SERU# 3103) and the University of Warwick Biomedical and Scientific Research Ethics Committee (BSREC# REGO-2015-6102).19, RNA was extracted from the respiratory specimens by Qiacube HT using an RNeasy extraction kit  and screened for RSV (A and B), rhinovirus (HRV), human coronaviruses , influenza viruses , parainfluenza viruses (PIV 1-4), adenovirus (ADV) and human metapneumovirus (HMPV), using a multiplex real-time PCR assay system. Samples with cycle threshold (Ct) of <35.0 were defined as positive for the target virus. Residual NPS samples were stored at −80°C.NPS collections received at KWTRP virology laboratory were stored in 2-ml vials at −80°C until use. Using previously described methods23. Chi-squared and Fisher’s exact tests were used to test associations of virus occurrence with age, calendar month, facility, setting (outpatient or inpatient) and other demographic characteristics. Frequency distribution graphs were generated for all virus targets. Graphs for temporal patterns for each virus were generated.Statistical analysis was conducted using STATA version 13.1 . Summary statistics were produced for the data to give the proportions of virus positives by age and by location. Comparative data was obtained from the paediatric ward of KCH for patients aged <5 years admitted with acute LRTI from a contemporaneous respiratory virus surveillance2(11) =78.26, P=0.001). There were interruptions during the 5th to 13th of December 2016 due to a health workers’ industrial dispute and 1 week during the Christmas break that led to lower recruitment for that month. Only 56 participants were recruited in December from all the nine facilities out of the 585 expected per month.A total of 5647 participants were recruited from January to December 2016. The median age was 4 years (interquartile range (IQR): 1–15 years), 53.6% (n=3029) were children under 5 years of age, and 42.3% (2389) were male. The frequency of distribution of symptoms among participants by site, age and virus target are shown in2 (1) =177.3, P=0.0001). Infants aged below 6 months with ARI constituted 9.3% (n=523) of the participants, and 58% (302) of these had one or more respiratory virus targets detected. Of all virus-positive individuals, 70% (1665/2380) were not in any form of education, with the vast majority of these pre-school, i.e. 83.2% (1385/1665). During study piloting (month of December 2015), respiratory samples from 153 participants were collected; 24.2% (n=37) were children under 5 years of age and 37.9% (n=58) were positive for one or more virus.The prevalence of NPS collections positive for one or more respiratory virus target was 42.1% (n=2380). The median age of the virus-positive patients was 2 years . The proportion of virus-positive individuals differed significantly by age, sex and education status, but not by sampling site . The pre2(8) = 13.38, P=0.072) (The most common respiratory viruses detected in this study were rhinovirus 18.6% (n=1050), influenza virus 6.9% (n=390), coronavirus 6.8% (n=387), parainfluenza virus 6.6% (n=371), RSV 3.9% (n=219) and adenovirus 2.7% (n=155). The frequency of viruses did not differ significantly by facility of recruitment (χ=0.072) . The dis=0.072) . Further=0.072) . Note foThe age distribution of detections showed variation between viruses . For exaMultiple viruses were detected in 4.4% of the participants (n=246), with 2, 3 and 4 co-infecting viruses detected in n=221, n=19 and n=6 of the participants, respectively. Rhinovirus occurred in 153 participants with multiple viruses; coronaviruses in 131 participants; adenovirus in 55 participants; parainfluenza virus type 3 in 36 participants and RSV B in 28 participants. Rhinoviruses and adenoviruses co-infected with all the other 14 target viruses while RSV group B was found not to co-infect with all, except HMPV.Only 1.4% of the participants (n=33) with a virus-positive NPS sample were referred to the KCH for specialized management. Treatment given to participants with ARI symptoms who tested positive for any of the virus targets were: antibiotics 81.4% (n=1947), antihistamines  60.4% (n=1437) and paracetamol 86% (n=2045). The most common antibiotic drugs prescribed were amoxicillin  and trimethoprim/sulfamethoxazole . Amoxicillin/clavulanic acid  and ciprofloxacin  were less commonly prescribed.We observed a seasonal pattern in occurrence for some of the viruses . Rhinovi2(1)=159.4, P=0.0001). Adenovirus was also of high prevalence and significantly more common 12.4% (n=71) than in outpatients 4.4 % (134) (χ2(1) =56.6, P=0.0001). Rhinovirus was at high prevalence in both settings, but more so in the outpatients. Coronavirus OC43 and parainfluenza virus type 4 were more prevalent in outpatient than inpatients. All remaining viruses, though proportionally less common, showed higher prevalence in the outpatient setting. The distribution of Ct values for each virus target was similar for both inpatient and outpatient samples.A comparison of the distribution of viruses between the outpatient and the inpatient setting among children under 5 years of age is shown inIn this study on the coast of Kenya, we describe the frequency of occurrence, and the spatial and temporal distribution of 15 viruses associated with respiratory illness in the outpatient setting. We also compare the distribution of viruses associated with respiratory infection among children under 5 years of age in the outpatient and inpatient settings.2 in rural coastal Kenya. We observed a prevalence of 42% virus infection among the selected patients presenting with symptoms of ARI in outpatient departments of nine health facilities. These findings indicate that viruses are prominently associated with respiratory tract infections of sufficient severity for individuals to seek medical attention within this community.To provide baseline data for investigating the transmission patterns and pathways of spread for common respiratory viruses, we used molecular techniques to detect viruses circulating throughout a well-defined population of nearly 300,000 inhabitants in an area of ~900 km24, most of the ARI outpatients in this study were young children under 5 years; 50.3% of the participants in this age group had a virus positive NPS sample. This demonstrates that the under 5’s are a highly vulnerable age group for medically important ARI. The results are comparable to those from the outpatient health facilities in refugee camps and at a national referral hospital, in Kenya, where the proportions of virus-positive NPS samples in those under 5 years old with cases of ARI was 49.8%25 and 54%26, respectively. This age group appears to experience the highest burden of acute illnesses linked to the studied respiratory viruses and should therefore be a focus for future intervention strategies. However, additional data should be collected on the social and economic burden of these viruses, such as days of school missed, medical costs and time off work for caregivers.Consistent with observations elsewhere28. There is need for empirical evidence to define the relationship between contact rates and respiratory virus transmission.Of all virus-positive patients presenting to the outpatient facilities, only a small proportion (30%) were in education (mostly kindergarten or primary.) This is of interest as the school age groups might be expected to be important agents in the community spread of infectious disease, certainly given their high rates of contact through which transmission is presumed to be effected29. Serological and molecular epidemiological studies show rhinoviruses to exist as many types (currently over 160)21, with little cross-type protection , which may explain the high prevalence and absence of seasonality in respiratory illness caused by rhinovirus infection in the community. Persistence of rhinovirus might be ascribed to the frequent introduction of new virus strains into the community unconstrained by prior circulation of other types.The most frequently detected virus in this study was rhinovirus. This is in accordance with previous community-based studies, for example the Tecumseh project in Michigan2). This allows for the possibility of population mixing and frequent interactions, especially during social events, leading to the rapid spread of viruses across the KHDSS area. However, definitive understanding of the temporal and spatial patterns of spread requires the addition of sequence data to infer relatedness of circulating viruses.In this study, we did not see any major difference in distribution of viruses across the health facilities. This could have been attributed to the relatively small size of the demographic surveillance system area , and to explore the possible effect of eliminating a virus such as RSV through vaccination. Such information will be useful to guide policy on priority respiratory viruses to focus for intervention.We also find the wide use of antibiotics to treat majority of patients presenting with symptoms of ARI most likely caused by viruses. This raises concern over antimicrobial stewardship, with increased risk of antimicrobial resistance to first-line antibiotic agents and unnecessary use of expensive second-line antibiotics in treating mild acute respiratory disease.In contrast to the outpatient setting, where rhinovirus is the most common virus associated with ARI among children under 5 years; in the inpatient setting, RSV and adenovirus are the leading cause of severe respiratory illness. From the long-term in-patient surveillance at KCH the observation for RSV is not unusual, but 2016 had an unusually high occurrence of adenovirus cases (data not shown for other years). The pattern in distribution of virus load  suggests that the cut off of 35.0 for the MPX real time PCR diagnostic method is generally suitable for all viruses, both in outpatient and inpatients , excepting for HMPV, PIV-1 and CoV 22E in outpatients and PIV-2 in inpatients and outpatients, where sensitivity may be an issue and a contributor to low prevalence in this study.The major limitation of this study is that data are for 1 year only and caution should be applied in inferring seasonal patterns. This is exacerbated by the low numbers of participants recruited in December due to countrywide industrial action by nurses. In addition, there is competition for viruses due to the design of sampling we used, of selected 15 samples per facility per week. An epidemic for one target virus might influence the proportions of other viruses observed but this might not necessarily imply seasonality. The detection of multiple viruses in one individual makes it difficult to determine the viral pathogen responsible for the respiratory illness at the time of recruitment. Only a sub-sample of all ARI presentations were recruited and the underlying denominator not recorded, which prevents an estimation of community incidence of presentations that would be useful for comparative purposes.In a sample of 5647 participants, about 40% of the ARI outpatient visits to the Kenyan Coast were associated with respiratory virus infection. Virus ARI is predominant among children <5 years, and relatively uncommon amongst school-going children. Rhinoviruses, influenza viruses, parainfluenza viruses, coronaviruses and RSV are most commonly associated with ARI over one year of community surveillance, whereas RSV and adenoviruses are the predominant respiratory virus among hospitalized patients with ARI. Virus occurrence  is similar across all facilities within the KHDSS area. Studies of the socio-economic implications of this burden are warranted, especially for rhinovirus infections that predominated. Further analysis of virus sequence data will delineate patterns of spread of viruses causing ARI illness in this setting.https://doi.org/10.7910/DVN/ZX7NS430. As the dataset contains potentially identifying information on participants, it is stored under restricted access. Details on eligibility for access and a request form are available fromhttp://kemri-wellcome.org/about-us/#ChildVerticalTab_15 for consideration by our Data Governance Committee (dgc@kemri-wellcome.org).The replication data and analysis scripts for this manuscript are available from the Harvard Dataverse: This study on the molecular epidemiology of respiratory viral illness in community and hospital settings in costal rural Kenya is an important addition to the growing literature on respiratory viral epidemiology in sub-Saharan African children. Future publications from this group of researchers is eagerly anticipated, particularly with regards tracking temporality and seasonality of viral prevalence over multiple years, and detailing the frequently-occurring viral-viral co-detections in this cohort. Kenya is generally known to be a low human immunodeficiency virus type 1 (HIV) prevalence setting. It would, however, be useful to factor in HIV status of study participants in future studies from this well-delineated catchment area. Point for clarification in the analysis:Results section, 5647 participants were enrolled – considerably fewer than the 7000 participants sought in the sample size calculation: how this ‘under-count’ impacted on the ability to establish certainty of statistical inference needs to be mentioned in the Discussion section; 1. In the first sentence of theFigures 6 B and6 C seem superfluous, as there is no text which describes the statistical comparison between pathogen density and inpatient or outpatient status. Suggest add in one or two sentences regarding this analysis in the last paragraph of yourResults section. 2. Introduction, rather state ‘and with the majority of the burden’;In the first sentence of theIntroduction, rather state ‘are recognized as the major cause’;In the second sentence of theIntroduction, rather state ‘Given this context, it is likely that greater emphasis will be placed on the control of virus-associated ARI.’;In the first sentence of the second paragraph of the 3. In order to strengthen the important assertions made in sentence four of the second paragraph of theIntroduction, especially with regards ‘social distancing’, I would suggest that the authors reference this sentence; Recommended minor stylistic adjustments:Introduction, suggest reword as ‘Here we present the baseline results of 1 year of respiratory virus epidemiological surveillance at health facilities within a well-defined coastal Kenyan population, and compare them with contemporaneous data from inpatient admissions to Kilifi County Hospital (KCH).’; 4. In the last sentence of paragraph three of thePatient recruitment and specimen collection, reword as ‘they awaited review’; 5. In the fourth sentence of the first paragraph ofPatient recruitment and specimen collection, I would suggest that the authors reference their proportional split between respiratory viruses; 6. In the last sentence of the second paragraph ofStatistical analysis, suggest reword as ‘proportion of virus positives’; 7. In the second sentence of the paragraph onStatistical analysis, insert a space between the preceding full stop and the words ‘Chi-squared’; 8. In the third sentence of the paragraph onTable 1, it makes logical sense to reorder the columns according to order in which a clinician would examine a patient, starting with history and moving to physical examination: I propose that an optimised order would be:  ‘Cough’, ‘Difficulty breathing’, ‘Fever’, ‘Nasal discharge’, ‘Nasal flare’, ‘Chest indrawing’, ‘Crackles’, ‘Wheeze’; 9. Inpage 6, in the second last sentence of column one (starting ‘The number of participants in each facility…’ gives aΧ2 degree of freedom of 11: is this in fact correct? My reading is that this should be 8 (in view of 9 included outpatient facilities having been included in the sampling strategy). Please correct if appropriate; 10. OnΧ2(8/11)’ and ‘ =78.26’; 11. Also, close the gap between ‘Virus detection, reword as ‘respiratory virus targets’; 12. In the first sentence underTable 2, add a space between ‘positive’ and ‘(n)’ in the second cell in the top row, and between ‘Total’ and ‘(n)’ in the sixth cell. Widen the Table slightly to accommodate a slightly wider columns; 13. Inpage 7, suggest close the gaps between ‘Χ2’, ‘ (1)’, and ‘ =177.3’; 14. In the first line onpage 7, the last sentence of the first paragraph (which describes the pilot phase of the study in December 2015) does not seem to add to the ‘meat’ of the results: suggest delete this sentence; 15. Onpage 7, suggest reword to ‘except with HMPV.’; 16. In the last sentence of the fourth paragraph onpage 7, suggest reword influenza A activity to ‘while influenza A had biphasic activity in January/February and November/December’; 17. In the second sentence of the second column onComparison of inpatient vs outpatient virus detections, suggest reword to ‘inpatient settings’; 18. In the first sentence under the headingComparison of inpatient vs outpatient virus detections, suggest close the gap between ‘Χ2(1)’ and ‘ =56.6’; 19. In the fourth sentence under the headingpage 7, suggest qualify as ‘, both in Kenya,’; 20. In the last line onFigure 2, replace the full stop after ‘RSVB’ with a comma and change ‘E229’ to ‘229E’; 21. In the legend toFigure 3, replace the lower case ‘e’ in ‘229e’ with an upper case ‘E’; 22. In the legend topage 8, suggest reword as ‘under 5 years old with ARI were 49.8% and 54%, respectively.’; 23. In the first line onpage 8, place the full stop outside the closing parentheses: ‘kindergarten or primary).’; 24. In the first sentence of the second paragraph onpage 8, suggest change wording from ‘in accordance’ to ‘in keeping’; 25. In the first sentence of the third paragraph onFigure 4, replace the full stop after ‘RSVB’ with a comma and change ‘E229’ to ‘229E’; 26. In the legend toFigure 5, the same seasonal (grey) line plot is included in each of the graphs and represents the total number of samples collected per month in the study: this should be mentioned in the Figure legend; 27. InFigure 5, replace the full stop after ‘RSVB’ with a comma and change ‘E229’ to ‘229E’; 28. In legend toFigure 6 A, suggest quote the percentage ‘49%’ to one decimal place, and add a space between ‘Inpatients:’ and ‘Virus’ in the text superimposed on the plot area; 29. InFigure 6, replace the full stop after ‘RSVB’ with a comma and change ‘E229’ to ‘229E’; 30. In the legend topage 13, suggest reword as ‘We observed a high prevalence of antibiotic prescribing to outpatients presenting with ARI most likely cause by viruses.’; 31. In the first sentence of the second paragraph onpage 13, suggest reword to ‘Based on long-term in-patient surveillance’; 32. In the second sentence of the third paragraph inpage 13, suggest replace ‘MPX’ with ‘multiplex’, as the abbreviation ‘MPX’ has not been defined before in the text; 33. In the third sentence of the third paragraph inpage 13, suggest reword to ‘both in outpatients and inpatients’; 34. In the third sentence of the third paragraph inpage 13, correct ‘CoV 22E’ to ‘CoV 229E’; 35. In the third sentence of the third paragraph in 36. In the second sentence of the second column on page 13, suggest reword as ‘respiratory virus among children hospitalized with LRTI’.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Would be nice to know the severity of participants with multiple viruses and see whether there is an association. What about the multiple viruses infection in inpatients?Newborns aged less than 7 days were excluded. Would this affect the detection rate of respiratory viruses in infants? Would this underestimate the burden?This is a well described manuscript reporting the epidemiological data of respiratory viruses at nine outpatient health facilities in rural coastal Kenya. More than half (53.7%) of participants with ARI symptoms were from children younger than 5 years. The most common respiratory viruses detected were rhinovirus, influenza virus, coronavirus, parainfluenza virus, respiratory syncytial virus (RSV) and adenovirus. In comparison group with young children admitted to hospitals, the frequency of RSV and adenovirus was significantly higher. However, a few points need to be clarified. The detection of viruses in participants with ARI symptoms does not necessarily indicate a causal attribution of these viruses . Would be nice to add a few sentences discussing this point.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Henipavirus of the family Paramyxoviridae. It is a biosafety level-4 pathogen, which is transmitted by specific types of fruit bats, mainly Pteropus spp. which are natural reservoir host. The disease was reported for the first time from the Kampung Sungai Nipah village of Malaysia in 1998. Human-to-human transmission also occurs. Outbreaks have been reported also from other countries in South and Southeast Asia. Phylogenetic analysis affirmed the circulation of two major clades of NiV as based on currently available complete N and G gene sequences. NiV isolates from Malaysia and Cambodia clustered together in NiV-MY clade, whereas isolates from Bangladesh and India clusterered within NiV-BD clade. NiV isolates from Thailand harboured mixed population of sequences. In humans, the virus is responsible for causing rapidly progressing severe illness which might be characterized by severe respiratory illness and/or deadly encephalitis. In pigs below six months of age, respiratory illness along with nervous symptoms may develop. Different types of enzyme-linked immunosorbent assays along with molecular methods based on polymerase chain reaction have been developed for diagnostic purposes. Due to the expensive nature of the antibody drugs, identification of broad-spectrum antivirals is essential along with focusing on small interfering RNAs (siRNAs). High pathogenicity of NiV in humans, and lack of vaccines or therapeutics to counter this disease have attracted attention of researchers worldwide for developing effective NiV vaccine and treatment regimens.Nipah (Nee-pa) viral disease is a zoonotic infection caused by Nipah virus (NiV), a paramyxovirus belonging to the genus  Apart from this there may be development of pulmonary illness and sometimes the infection may be asymptomatic in nature  and Hendra virus (HeV). Molecular studies have significantly improved our understanding of the genetic diversity of Henipaviruses , an emerging virus that can cause severe respiratory illness and deadly encephalitis in humans. It is a negative sense, single-stranded, nonsegmented, enveloped RNA virus possessing helical symmetry. The RNA genome, from the 3´-5´, contains consecutive arrangement of six genes, viz., nucleocapsid (N), phosphoprotein (P), matrix (M), fusion glycoprotein (F), attachment glycoprotein (G) and long polymerase (L). The N, P and L attached to the viral RNA forming the virus ribonucleoprotein (vRNP). F and G proteins are responsible for cellular attachment of the virion and subsequent host cell entry  is a paramyxovirus  protein makes fusion of virus-cell membranes for cellular entry. The G protein of NiV binds to host ephrin B2/3 receptors and induces conformational changes in G protein that trigger the F protein refolding  production  kept at 22 °C. The virus has a half-life of 18 h in the urine of fruit bats. NiV is relatively stable in the environment, and remains viable at 70 °C for 1 h . It can be completely inactivated by heating at 100 °C for more than 15 min .Bats serve as reservoir hosts for several high risk pathogens, including Nipah, rabies and Marbug viruses. Such viruses are not associated with any significant pathological changes in the bat population  might play a role in transmitting NiV during close contact  revealed bat-to-man spillovers every winter with 36% annual variation and the distance to surveillance hospitals showed 45% of spatial heterogeneity  initially revealed the seropositivity of Nipah viral antibodies in them and laterconfirmed the existence of virus also by isolation studies  in Singapore where pig meat was imported from Malaysia, with one dead. Patients showed higher level of IgM in serum and some unusual symptoms of atypical pneumonia and encephalitis with characteristic focal areas of increased signal intensity in the cortical white matter in MRI. Symptoms of hallucination along with abnormal laboratory results including low lymphocyte and platelet counts, high levels of CSF proteins and of aspartate aminostransferase were present. The patients were treated by intravenous acyclovir and eight were cured ]. Various risk factors for NiV infection in pteropid bats were also explored. Among the two bat species, the risk of NiV and seroprevalence were higher for P. vampyrus (33%) than P. hypomelanus (11%). NiV seroprevalence and distribution showed variation (1–20%) in the P. hypomelanus batsand also in between the years 2004–2006 irrespective of seasons  in Negri Sembilan region of Malaysia. Genome of the NiV was sequenced at the CDC, Atlanta, Georgia, USA. The Ministry of Health declared total of 101 human deaths and approximately 900,000 pigs were culled Uppal . ResearcP. lylei (Lyle’s flying foxes). In Indonesia, such antibodies were also detected by ELISA in the sera collected from P. vampyrus . In both outbreaks, circumstantial evidences suggested the human-to-human transmission, as most people who acquired the infection were either care-givers, or family members of infected persons.NiV was detected for the first time in Siliguri, West Bengal, India in the year 2001 during an outbreak characterized by febrile illness in association with altered sensorium (poor thinking capability or poor concentrating capacity). A close resemblance had been found between the isolates of Siliguri outbreak and those obtained during the outbreak in Bangladesh. Such resemblance is justified, as Siliguri is located at the vicinity of Bangladesh . Twenty cases (87%) showed respiratory symptoms and the case fatality rate was 91% with only two survivors. Sequencing and phylogenetic analysisrevealed NiV isolate to be closer to the Bangladesh lineage  on the pathogenesis of Henipaviruses. Herein bioluminescence imaging technique has been used for monitoring of the replication of the virus as well as spread in knockout mice. This reverse genetics system may be a useful tool to investigate Henipa-like viruses  N and G genes was carried out after aligning the sequences by the Clustral V program in MegAlign software of the DNASTAR software package. For the genetic relatedness study, representative 1599- and 1809 bp-length for N gene (27 strains) and G gene (15 strains), respectively, were investigated. NiV strains from different countries including Malaysia, Cambodia, Bangladesh, India and Thailand, submitted during 2001–2018 were retrieved from the NCBI database. Phylogenetic analysis was performed using the maximum likelihood method (1000 bootstrap replicates) in MEGA 6 software (v 6.06) , the immune responses might be host-specific and/or anterogradely via olfactory nerves , 8.2.Highly pathogenic NiV causes symptomatic infections in pigs and humans. Respiratory symptoms are much more severe in pigs as compared to humans.8.2.1.www.medicinenet.com). There may be development of septicemia along with impairment of the renal system and bleeding from the gastrointestinal tract. In severe cases within a period of 24–48 h, there may be development of encephalitis along with seizures that ultimately leads to coma  mainly getting affected , barking pig syndrome (BPS)  or one-mile cough. An acute febrile illness has been reported in pigs below six months of age wherein there is development of respiratory illness that ranges from rapid labored breathing to non-productive cough which is harsh in nature. With the exception of young piglets, the mortality is relatively low  studies in human patients have revealed that the cortex, pons as well as temporal lobes of brain get involved. There may be bilateral abnormalities in the white matter of the brain. In the cerebral cortex, there may be more than one hyperintensities (T1-weighted) which are very much similar to necrosis of the laminar cortex. Lesions may also become evident in corpus callosum, brain stem, as well as cortex of the cerebrum. It is crucial to note in this regard that diffusion-weighted (DW)-MRI is employed to detect such lesions  in the lungs of affected pigs at necropsy. Froth-filled bronchi along with trachea are commonly observed. In certain instances, there may be presence of blood stained fluids in the trachea and bronchi. Congestion along with generalized edema is present in kidneys and brain. Both the cortex as well as suface of kidneys may become congested  serve as natural hosts (wildlife reservoir) and pigs are the intermediate hosts for NiV zoonotic cycle  bear the virus in saliva as well as urine, indicating their potential to act as natural reservoir of the virus .Besides Malaysia, the fruit bats of Consumption of fruits, vegetables or water contaminated with saliva, urine or fecal matter of infected bats could also be a possible mode of transmission to man and animals  laboratory facilities are required for NiV isolation as well as propagation. However, BSL-3 may prove to be sufficient to primarily isolate the virus from suspected clinical materials. Following confirmation of the virus in infected cells (fixed by acetone) by immunofluorescent technique, there should be immediate transfer of the culture fluid in BSL-4 laboratory , Bhopal. By the use of pseudotyped particles, a serum neutralization test for NiV can be performed under BSL-2 conditions. This test uses a recombinant vesicular stomatitis virus that expresses secreted alkaline phosphatase (SEAP). Neutralization titer can be obtained by measurement of SEAP activity . Microsphere assay (luminex based) has been used for detection of antibodies against a glycoprotein of NiV, namely NiV sG, in the sera of pigs and ruminants like goats and cattle  along with real-time RT-PCR (qRT-PCR) and duplex nested RT-PCR (nRT-PCR) have been found useful for detection of NiV infection, with subsequent confirmation by nucleotide sequencing of amplicons. A unique primer set targeting the N gene has been reported. Internal controls may also beincluded in nRT-PCR tests for detection of NiV RNA. Further, such kind of nRT-PCR has helped to detect two different viral strains from 11.Vaccination of humans is an integral part of preventing infection due to NiV. Prevention also includes vaccination of livestock  in endemic areas . It was formed in January 2017 for developing safe, efficacious and affordable vaccines against diseases associated with pandemic potential, like Nipah , and other advanced vaccines have been developed as strategies of immunization against both HeV and NiV  vaccine that expresses envelope glycoprotein of NiV has been found to be promisingfor use in man Yoneda . A repliNipah virus-like particles (NiV-VLPs) composed of three NiV proteins G, F and M derived from mammalian cells have been produced and validated as vaccine in BALB/c mice. The immunogenicity of the NiV-VLP vaccine was high because the VLPs possess the native characteristics of the virus including the size, morphology and surface composition . A more recent in silico analysis using bioinformatics tools indicated that the epitopes from G (VDPLRVQWRNNSVIS) and M (GKLEFRRNNAIAFKG) proteins can be helpful for designing common B- and T-cell epitope-based peptide vaccinesagainst HeV and NiV, and this approach needs to be evaluated  by targeting the envelope glycoprotein of the virus  is presented in 12.viz., drinking of contaminated date palm sap, would be necessary. In Bangladesh, date palm sap is usually harvested overnight. The nocturnal activities of bats such as drinking from, defecating or urinating in the date palm sap collection jars have become evident through infrared cameras. Measures to prevent the access of bat to the sap stream of the date palm tree as well as shaved surface can minimize the risk of human exposure to NiV in such settings .Outbreaks of NiV invoke costly emergency responses around the world. NiV poses a greater threat in regions associated with risk factors and with poor indicators of development . Sporadic nature of NiV outbreaks, lack of information of exact correlates of protective immunity, lack of interest among private pharmaceutical companies, and inadequate availability of BSL4 laboratory facilities to test the vaccines or therapeutics against NiV in preclinical models pose challenges to NiV vaccine development. Development of diagnostics suitable for field conditions, immunotherapeutic approaches, vaccines and antiviral drugs are urgent priorities for long-term measures aimed at prevention and control of NiV disease. Utmost care should be taken in order to avoid direct contact with the persons infected by NiV. Personal protection devices, viz., masks, glasses and gloves, should be used properly. Hydration of the patient is also important . In nations having no past history of Nipah viral outbreak, anticipatory preparedness for rural as well as urban outbreaks of the disease will ultimately help in prevention and control of potential outbreaks  reported that implementation of infection control and precaution at both household and hospital levels helps to limit the NiV disease outbreak. Active surveillance and contact tracing are important along with quarantine of health professionals and peopleat high risk . A recent bibliometric study identified a sudden increase in the number of publications referring to the eight pathogens of global concern identified by WHO, viz., Lassa, Rift Valley, Marburg, Ebola, Middle Eastern Respiratory Syndrome, Severe Acute Respiratory Syndrome, and Crimean-Congo Hemorrhagic Fever viruses . Supportive therapies such as hydration and ventilator support constitute important aspects of clinical management of NiV cases.The essence of treatment modalities along with effective therapeutics is understood, once there is an outbreak of an infectious disease. There is a need for administering therapeutics to manage the patients during NiV outbreaks and to prevent the mortality. No specific drug has been yet approved for the treatment of this important disease. Limited work has been done to develop therapeutics against NiV infection. In preclinical studies, monoclonal antibodies have been used for treatment purposes. Due to the expensive nature of the drugs based on antibodies, identification of broad spectrum antivirals is essential along with focusing on small interfering RNAs (siRNAs) , etc. have been studied extensively to develop therapeutics effective against infection caused by Henipavirus including NiV  especially after the outbreaks in Bangladesh and India and marked the necessity of development of network communicating between veterinary and medical services concerning this disease. By involving the multiple sectors and with multidisciplinary approach, precise and concrete preventive strategies can be planned and implemented.The ‘One Health’ approach is also the utmost importance. There is requirement of coordination between institutes as well as at the international level among virologists from both medical and veterinary fields as well as ecologists for understanding to the fullest the period and mechanism involved in excretion of the virus by the bats. At the same time, the common people should be educated about food hygiene as well as hygiene at personal level. Inspection of all the imported livestock at the time of arrival and also before travel at the point of origin is essential. Proper isolation, quarantine and disinfection protocol including infrastructure facilities and trained personnel with protective clothing should be in place to respond quickly upon identification of any new case. There should be maintenance of proper hygiene at maximum level for slaughtering such livestock. For preventing future NiV outbreaks, a continuous surveillance in the area of human health, animal health, and reservoir hosts should be carried out to determine the prevalence and to predict risk of virus transmission in human and swine populations. Successful accelerated development of preventive vaccines and therapeutic antibodies or antivirals are need of the hour to control the spread and treat the infected patients during an outbreak. Collaborative efforts such as CEPI and biotech companies will accelerate the vaccine or therapeutic development for NiV.