Corvus bennetti), followed by the detection of immature and intracellular mature virions using electron microscopy. The CRPV genome was 328,768 bp in length and contained 403 predicted open-reading frames. While 356 of the ORFs of CRPV genome had the greatest similarity with other avipoxviruses gene products, a further 47 ORFs were novel. Subsequent phylogenetic analyses showed that the CRPV was most closely related to other avipoxviruses isolated from passerine and marine bird species and demonstrated the highest sequence similarity with an albatrosspox virus (84.4%). Considering the sequence similarity observed between CRPV and other avipoxviruses and phylogenetic position, this study concluded that the CRPV to be a distinct available candidate of avipoxviruses.Avipoxviruses are thought to be restricted to avian hosts and considered significant pathogens that may impact the conservation of many birds. However, reports of avipoxvirus-like viruses from reptiles suggest that cross-species transmission, within birds and other species, may be possible. The vast majority of avipoxviruses in wild birds remain uncharacterised and their genetic variability is unclear. Here, cutaneous pox lesions were used to recover a novel full-length crowpox virus genome from an Australian little crow ( Avipoxvirus  that may cause proliferative, diphtheritic or systemic lesions in birds2. Avipoxviruses represent a diverse virus group, that may infect most avian species. Evidence of poxvirus infection has been found in at least 329 avian species and 20 orders of wild and domestic bird species4, with many more avian hosts considered susceptible. In general, avipoxviruses appear to have been present in bird populations for continuous periods, leading to low levels of infection and relatively mild disease. However, where poxviruses have been introduced to naïve bird populations, they have the potential to cause explosive outbreaks of severe disease with high morbidity and mortality, as occurred in Hawaii, the Galapagos and the Canary Islands6.Avipoxviruses are large, double-stranded DNA (dsDNA) viruses belonging to the genus 7 there are currently 12 species approved under the genus Avipoxvirus: Canarypox virus, Flamingopox virus, Fowlpox virus, Juncopox virus, Mynahpox virus, Penguinpox virus, Pigeonpox virus, Psittacinepox virus, Quailpox virus, Sparrowpox virus, Starlingpox virus and Turkeypox virus. A further two viruses, crowpox virus and peacockpox virus, are putative members of the genus Avipoxvirus, but have not yet been approved as species by the ICTV. There are currently a limited number of complete avipoxvirus genome sequences from ICTV-recognised species available in GenBank. These include a canarypox virus (CNPV)8, a South African strain of pigeonpox virus (FeP2)9, a penguinpox virus (PEPV)9, a Hungarian strain of turkeypox virus (TKPV)10, an American strain of fowlpox virus (FWPV)11, a European strain of fowlpox virus (FP9)12, and an additional eight complete genome sequences of FWPV that have been published since 201815. There are also six further complete avipoxvirus genomes: two shearwaterpox viruses (SWPV1 and SWPV2)16, two magpiepox viruses (MPPV and MPPV2)18, a mudlarkpox virus (MLPV)19, penguinpox virus 2 (PEPV2)20, two albatrosspox virus (ALPV and ALPV2)22, and a poxvirus in house finches (Haemorhous mexicanus)23 available in GenBank that are not yet ICTV recognised species.According to the International Committee on Taxonomy of Viruses (ICTV)Corvus brachyrhynchos) in the United States of America25. Several recent studies have also characterised poxvirus infections in the cutaneous lesions of Australian passerine bird species including the Australian magpie (Gymnorhina tibicen)18, and the mudlark 19. However, there are no sequence data of crowpox virus from the Australian little crow (Corvus bennetti), and consequently, its genetic and phylogenetic relationships with other avipoxviruses are not well known. The aim of this study was to identify and characterise the genome sequence of crowpox virus (CRPV) from an Australian little crow sourced from Victoria in 2021.Avipoxvirus infection in the family Corvidae has been identified in an adult American crow  with the pathogenic avipoxvirus ALPV, isolated from an endangered northern royal albatross (Diomedea sanfordi) in 1997 (GenBank accession no. MW365933)22 . The CRPV genome contained a large central coding region surrounded by two matching inverted terminal repeat (ITR) regions, constituting 4052 bp each  like other characterised avipoxviruses−5)  showed highest similarity to MLPV, five  to ALPV, five  to ChePV1, two (ORF-128 and -129) to FWPV, one (ORF-329) to Hawaiian goose poxvirus (HGPV196), one (ORF-130) to SWPV1, and one (ORF-023) to FGPV  encoding proteins ranging from 30 to 1945 amino acids in length, that were numbered from left to right Fig. . Among t−5) Fig. . Among tGPV Fig. .Figure 127. These unique ORFs encoded proteins of 30 to 100 amino acids in length  were truncated mostly with a single residue compared to CNPV, which may warrant further studies to determine whether they are expressed and functional. Based on a recent study by Carulei et al.3, we also searched for a further 47 genes that are conserved in avipoxviruses (Table Similar to other chordopoxviruses (ChPVs) the CRPV genome contained 89 conserved core genes, which are involved in essential functions such as replication, transcription and virion assembly  tree , northern royal albatross (Diomedea sanfordi), and yellow-eyed penguin (Megadyptes antipodes) with 100% bootstrap support. In the sub-clade B1, FIPV is basal to known avipoxviruses, suggesting that all the avipoxviruses under this subclade evolved from the ancestral house finches (Haemorhous mexicanus) from where MLPV followed by CRPV viruses evolved, and later to the other avian hosts. Using the same set of concatenated protein sequences, we found that the maximum inter-lineage sequence identity values ranged from 98.7 to 97.9% among avipoxviruses under subclade B1, which reflected the phylogenetic position of this novel avipoxvirus sequenced from an Australian little crow, and further inferred that these viruses likely originated from a common ancestor. Furthermore, it was also evidenced that there were many avipoxviruses evolutionarily linked with crowpox virus sequenced in this study when we compared using partial nucleotide sequences of the DNA polymerase gene  and a canary (Serinus canaria) in Chile25, Australian magpies18, a gray-crowned rosy finch (Leucosticte tephrocotis) and a Swainson's thrush (Catharus ustulatus) in the USA25 were the closest evolutionary link with the CRPV isolated in this study.Phylogenetic reconstruction using concatenated amino acid sequences of selected conserved ChPV genes provides clear evidence for the inclusion of CRPV in the genus ree Fig. , CRPV waUsing transmission electron microscopy (TEM) analysis, crowpox virus particles were identified in the sample sourced from the Australian little crow. It showed the presence of intracellular mature virion (IMV) that was brick-ovoid shaped with regular spaced thread-like ridges comprising the exposed surface and was approximately 250 × 230 nm Fig. A, and imAvipoxvirus.This paper documents a novel avipoxvirus, crowpox virus (CRPV), with the complete genome sequence obtained directly from naturally occurring pox lesions in an Australian little crow. Applying various approaches for genomic comparison and transmission electron microscopic analysis, the CRPV genome was shown to be architecturally consistent with other avipoxviruses in terms of genome size, AT content and predicted ORFs; however, it was distinct from other avipoxvirus genomes in multiple ways. Overall, the DNA sequence of CRPV was significantly different from other avipoxviruses but had the closest similarity with the pathogenic avipoxvirus, albatrosspox virus (ALPV), followed by penguinpox virus 2 , shearwaterpox virus 2 , canarypox virus , and magpiepox virus 2 . The novel CRPV genome contained 47 predicted genes that are not found in any other poxvirus, as well as several ORFs that were so truncated/fragmented as to probably cause them to be non-functional. Overall, the CRPV was sufficiently genetically different to other previously classified avipoxviruses to be considered as a distinct new virus species under the genus Phylogenetic tree analysis demonstrated that the subclade B1 consisting of avipoxviruses isolated from several passerine bird species including this Australian little crow, as well as seabirds including a wedge-tailed shearwater, a northern royal albatross, and a yellow-eyed penguin, supports evidence that this CRPV most likely originated from avipoxvirus isolated from other birds in the order Passeriformes Fig. . The bas32, their mode of transmission in Australian avifauna is not well understood. Previous studies suggest that avipoxviruses can be transmitted between birds in several ways: (1) via direct contact with infected birds through broken skin; (2) through contact between skin breaches and contaminated objects including perches; (3) by aerosol transmission33, and (4) via haematophagous arthropods including mosquitoes, which are efficient mechanical vectors through contaminated mouthparts35. These factors may indicate a potential scenario for crowpox virus transmission that merits further attention. At an individual level, poxvirus infections in wild birds can cause primary disease that may be severe in some cases, leading to decreased foraging and mobility. In affected birds, avipoxvirus infection can lead to two different forms of disease. The most common disease is characterised by a proliferative ‘wart-like’ lesions that are commonly restricted to the eyes, beak or unfeathered skin of the body , in which secondary bacterial and fungal infections may aggravate the birds' condition. The second form of poxviral infection is the ‘wet’ or ‘diphtheritic’ form, characterised by lesions on the mucous membranes of the upper alimentary and respiratory tracts36. Avipoxvirus infection in bird can also reduce ability to care for young, and affect vision and/or feeding ability, making them prone to predation, and significantly affecting welfare37, but in some cases, birds are likely sub clinically infected. The repeated occurrence of avian family or order-specific grouping within certain avipoxvirus clades indicates a marked role of host adaptation, while the sharing of poxvirus species within prey-predator systems 25 indicates the potential for cross-species infection and limited host adaptation25. At a population level, these may have serious implications, especially for endangered or endemic species, and hence further studies into the evolution of avipoxviruses in non-model hosts warrants further investigation.Much is still unknown about the host spectrum and epidemiology of poxviruses, in particular for Australian avifauna. Although there is some evidence that poxviruses infecting Australian wild birds including magpies are transmissible to other magpies, but not to chickens, turkeys, pigeons, or canaries after experimental inoculationAvipoxvirus genus, contributing to our understanding of the avipoxviruses more generally, as well as tracking poxvirus evolution in a non-model avian species. By assessing the sequence similarity between CRPV and other avipoxviruses, we concluded that the CRPV complete genome described should be considered a separate avipoxvirus species. Additional investigations will be required to better understand relevant host–pathogen dynamics including routes of transmission and factors leading to infection, associated pathology, and disease prevalence.The novel complete genome sequence of CRPV reported here has enhanced the genomic information for the Corvus bennetti) that was euthanised by inhalational general anaesthesia with isoflurane  in oxygen followed by intravenous injection of pentabarbitone sodium  due to untreatable septic arthritis secondary to proliferative pox lesions by a registered veterinarian at The Unusual Pet Vets, Frankston, Victoria. Animal sampling (ID: 122740) was carried out by the attending veterinarian for the investigation of crusty pox lesions affecting the legs and eyes. Collected samples were stored at − 20 °C until further processing. For DNA extraction, the crusty pox lesion material was aseptically dissected and mechanically homogenised in lysis buffer using disposable tissue grinder pestles and transferred into a 1.5 mL microcentrifuge tube (Eppendorf). Total genomic DNA was isolated according to the established methods39 using a ReliaPrep gDNA Tissue Miniprep System .Samples were obtained from a little crow  by the Genomic Platform, La Trobe University. The prepared library was sequenced with the sequencing reads length of 150-bp paired-end on Illumina® NovaSeq platform according to the manufacturer's instructions through the Australian Genome Research Facility, Melbourne.A total of 250 ng of extracted genomic DNA was used to prepare the library using the protocol adapted previously using the Illumina DNA Prep ® , as described previously41. Briefly, the sequences were processed to remove Illumina adapters, low quality reads and ambiguous bases. Trimmed sequence reads were mapped against the chicken genome  to remove potential host DNA contamination. In addition, reads were further mapped to the Escherichia coli bacterial genomic sequence (GenBank accession no. U00096) to remove possible bacterial contamination. A total of 30.2 million cleaned and unmapped reads were used as input data for de novo assembly using CLC Genomics Workbench (version 9.0.1). This resulted in the generation of a 328,768 bp genome with an average coverage of 1182.51x. The genome was annotated according to the previously published protocol using Geneious software (version 2022.1.1). Open reading frames (ORFs) longer than 30 amino acids, with a methionine start codon (ATG) and minimal overlap with other ORFs (not exceeding 50% of one of the genes), were selected and annotated. ORFs shorter than 30 amino acids that had been previously annotated in other poxvirus genomes were also included. Similarity BLAST searches were performed on the predicted ORFs and were annotated as potential genes if predicted ORFs showed significant sequence similarity to known viral or cellular genes 27. The direct tandem repeats were detected using the Tandem Repeats Finders42.The resulting 39.8 million raw sequence reads were used to assemble the complete genome of CRPV, using CLC Genomics Workbench  and Geneious Prime43 and TMpred44. Additionally, searches for conserved secondary structure (HHpred)45 and protein homologs were conducted using Phyre246 and SWISS-MODEL47.To predict the function of putative unique ORFs identified in this study, the derived protein sequence of each ORF was searched using multiple applications to identify conserved domains or motifs. Transmembrane helices were searched using the TMHMM package (version 2.0)® (version 2022.1.1). Sequence similarity percentages between CRPV and representative ChPV complete genome sequences were determined using tools available in Geneious (version 2022.1.1). Dot plots were created based on the EMBOSS dottup program in Geneious software, with word size = 1248.Genomic features of the newly sequenced CRPV were visualised using Geneious Prime® . Nucleotide sequences of the partial DNA polymerase and partial p4b genes, as well as concatenated amino acid sequences of the selected nine poxvirus core proteins, were aligned as described previously19 using the MAFTT L-INS-I algorithm implemented in Geneious Prime® (version 2022.1.1) (version 7.388)49. To determine the best-fit model to construct phylogenetic analyses, a model test was performed using CLC Genomics Workbench (version 9.0.1), which favoured a general-time-reversible model with gamma distribution rate variation and a proportion of invariable sites (GTR + G + I). Phylogenetic analyses for nucleotide sequences were performed under the GTR substitution model, but the WAG substitution model was chosen for concatenated amino acid sequences with 1000 bootstrap replicates in CLC Genomic Workbench (version 9.0.1).Phylogenetic analyses were performed using the CRPV genome sequence determined in this study, together with other selected ChPV genome sequences available in GenBank Table . The ami30.Cutaneous pox lesions were suspended 1:10 in phosphate-buffered saline (PBS), homogenised, clarified and adsorbed onto 400-mesh copper EM grids, before staining and imaging on a JEOL JEM-2100 transmission electron microscope as previously describedCorvus bennetti) that was euthanatised due to untreatable septic arthritis secondary to proliferative pox lesions by a registered veterinarian at The Unusual Pet Vets, Frankston, Victoria. The animalwas necropsied by the same registered veterinarian for routine diagnostic purposes. All other methods were performed in accordance with the standard guidelines and regulations for PC2 laboratory. The Animal Ethics Committee at La Trobe University was informed that findings from the diagnostic material were to be used in a publication, and a formal waiver of ethics approval was granted.The animal handling and procedures were conducted in accordance with ARRIVE guidelines for experimental procedures. Briefly, samples were obtained from a little crow (Supplementary Information.


During outbreaks of virus diseases, many variants may appear, some of which may be of concern. Stability in an aerosol of several Ebola virus and Marburg virus variants was investigated. Studies were performed measuring aerosol survival using the Goldberg drum but no significant difference in biological decay rates between variants was observed. In addition, historic data on virulence in a murine model of different Ebola virus variants were compared to newly presented data for Ebola virus Kikwit in the A129 Interferon alpha/beta receptor-deficient mouse model. Ebola virus Kikwit was less virulent than Ebola virus Ecran in our mouse model. The mouse model may be a useful tool for studying differences in virulence associated with different variants whereas aerosol stability studies may not need to be conducted beyond the species level.  The COVID-19 pandemic has shown how new variants of a virus may be a cause for concern and may impact infection and pathogenesis or lead to immune escape. New variants may affect the spread of the virus and the efficacy of vaccines as well as hospital admissions and severity of disease . ChangesFiloviruses cause severe disease and remain a problem with multiple outbreaks having occurred in Africa since the viruses were first identified ,4,5. TheEach outbreak of EBOV or MARV is typically associated with the emergence of a different variant and multiple isolates with the result that many variants and isolates exist.Different institutes may have different viruses available for research and different criteria for which virus should be used for the standardization of protocols and procedures . Our defAlthough genome sequencing has rapidly improved over the years, even once the causative agent is known it can take time to get a full sequence and understand genetic relatedness. It may not always be possible to acquire and import an isolate from the latest variant available, for conducting studies, and doing so might take some time, particularly if adaptation is required, for example in the development of animal models. When developing and designing medical countermeasures or putting in control measures for a new outbreak, researchers may choose to work with a recent isolate from that particular outbreak, use pre-existing isolates from earlier outbreaks that are readily available and may be more fully characterized, or, researchers may decide to work with a prototype or type strain as a representative virus for that species.This paper describes the results of investigations comparing the aerosol stability of two different variants of EBOV, the Kikwit variant from 1995 recommended by the Filovirus Animal Nonclinical Group (FANG) for standardizing studies  and the In general, MARV is less studied than EBOV. We present aerosol survival comparison data between an original outbreak isolate from 1967 (Popp), and the well-characterized and FANG recommended variant (Angola), from the largest outbreak to date of MARV which occurred in 2005. These MARV isolates have also been tested in mice for virulence, but only MARV-Popp was tested by the aerosol route so no comparison data is shown here.This work will help inform on the relative aero stability and virulence of different variants and isolates. Studying aerosols is important due to the possibility of deliberate or accidental release or generation of aerosolized virus. Survival and stability studies may help in predicting how a future isolate or variant might perform, based on phylogenetic similarities or differences and thus this data might inform risk assessments and control measures. Understanding the virulence of different variants and isolates in a small animal model may help inform which isolates could be used for vaccine or therapy screening and may help in predicting the severity of new variants. Differences in virulence may also point to targets for intervention.Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Ecran, referred to as Ebola virus E718 in some earlier work  and here2. Enumeration was via the 50% tissue culture infectious dose (TCID50) assay. Historically, the TCID50 assay was performed as described previously   . In compMultiple factors can affect the survival and stability of viruses in aerosols. It is well known that environmental factors such as temperature and relative humidity as well as sunlight ,27 and cOur data can be considered a ‘baseline’ of aerosol survival under ambient conditions in the dark. The baseline can then be used to look at the effect of altering various factors  to see the effect of different aerosol or growth conditions on stability. Some but not all of the factors that could be looked at include a range of temperatures, relative humidity values, and light levels (to simulate sunlight) as well as spraying in the presence of pollutants and other compounds that might be found in the air and looking at different particle sizes. On the virus side, the growth or spray media, the day of harvest, or the passage number might affect stability. There are a huge number of factors to consider, but our data showing minimal variability between different variants/isolates indicates that the work may not need to be done with all, or the most recent virus, but one virus as an exemplar virus for the species.In terms of consistency, the filovirus decay data reported in this study provide similar decay rates and half-life values to that which we have observed with the Lassa virus  and NipaThere is minimal data comparing the aerosol stability of different isolates or variants of the same virus, under the same conditions. The stability of different variants and isolates of SARS-CoV-2 within aerosols were reported as being the same irrespective of isolate  and is c−/− mice have been used to look at the virulence of different species or isolates of filoviruses since this mouse strain was first developed for assessing lethality ) ,5) 4,5]) and thenInvestigations using both the murine model and aerosol decay in the Goldberg drum could be extended into other EBOV or MARV isolates or different variants if available. Based on our observations presented here, we would predict that we might continue to see variability in the virulence of EBOV variants in our murine model, but similar survival of different filovirus isolates in aerosols.In conclusion, the immune-deficient mouse model proved to be a useful model for the discrimination of different lethality rates associated with different filoviruses. Therefore, for rapidly responding and characterizing new isolates or variants, the immune-deficient mouse may prove to be the best first step. Evidence presented in this study suggests that aerosol stability is less likely to differ with new isolates or variants.


Chenopodium album is an economically important weed spread worldwide. Here, we present the molecular characterization of a novel recombinant begomovirus identified from C. album in Lahore, Pakistan. The complete DNA- A genome of the virus associated with the leaf distortion occurred in the infected C. album plants was cloned and sequenced. DNA sequence analysis showed that the nucleotide sequence of the virus shared 93% identity with those of the rose leaf curl virus and the duranta leaf curl virus. Interestingly, this newly identified virus is composed of open reading frames (ORFs) from different origins. Phylogenetic networks and complementary recombination detection methods revealed extensive recombination among the sequences. The infectious clone of the newly detected virus was found to be fully infectious in C. album and Nicotiana benthamiana as the viral DNA was successfully reconstituted from systemically infected tissues of inoculated plants, thus fulfilling Koch’s postulates. Our study reveals a new speciation of an emergent ssDNA plant virus associated with C. album through recombination and therefore, proposed the tentative name ‘Chenopodium leaf distortion virus’ (CLDV).Recombination between isolates of different virus species has been known to be one of the sources of speciation. Weeds serve as mixing vessels for begomoviruses, infecting a wide range of economically important plants, thereby facilitating recombination.  Geminiviridae) in the formation of new DNA virus species is well-documented [Interspecific interactions play a primary role in the diversification and organization of life . Numeroucumented ,13,14 ancumented ,16,17.Geminiviridae includes some of the most damaging plant pathogens that affect a wide host range and cause economic losses throughout the world [Becurtovirus, Begomovirus, Capulavirus, Citlodavirus, Curtovirus, Eragrovirus, Grablovirus, Maldovirus, Mastrevirus, Mulcrilevirus, Opunvirus, Topilevirus, Topocuvirus, Turncurtovirus, [Olea europaea geminivirus [Fraxinus symptomless virus [Geminiviridae having single-stranded circular DNA genome, either monopartite or bipartite components, ranging from 2.5 to 3.0 kb in size and are transmitted by whiteflies, i.e., Bemisia tabaci [AVI/VI) and a movement protein  [AC1/C1), a transcriptional activator protein (AC2/C2), a replication enhancer protein (AC3/C3), and the AC4/C4 protein (AC4/C4) [AC1  initiates viral DNA replication by binding to iterons within the intergenic region and creates a nick into the conserved TAATATT↓AC sequence [AC4, the smallest ORF embedded within the coding region of the Rep protein, is required for monopartite geminivirus infection [BV1) on the virion-sense strand and a movement protein (BC1) on the complementary-sense strand [The family he world ,19. Theshe world ,21,22. Ghe world  and are he world ,25,26. Btovirus,  and a feinivirus  and Fraxss virus . Begomova tabaci ,24,29. Dviruses) ,31, wher(AC4/C4) ,33,34. Asequence . AC4, thnfection ,37. The e strand .Chenopodium album (family: Amaranthaceae) is an erect, branched  cosmopolitan weed, which is widely distributed in Canada, Europe, India, Mexico, New Zealand, Pakistan, and South Africa and is ranked among the six most harmful weeds of the world [C. album is found in all inhabited areas of the world, where it thrives on all soil types and over a wide range of pH [C. album is responsible for important economic losses in agriculture around the world. In Pakistan, weed species have been identified to be responsible for causing monetary loss worth 3 billion USD annually [C. album is used as a vegetable and a medicinal plant in some regions of the world. A mixed infection of the tomato yellow leaf curl virus (TYLCV) and the tomato yellow leaf curl Sardinia virus (TYLCSV) has been reported to occur in C. album. Another begomovirus reported infecting the genus Chenopodium is the chenopodium leaf curl virus found in Chenopodium ambrosiodes [C. album is often used as a herbaceous test plant in plant virology.he world . Except ge of pH . It growge of pH . C. albuannually . Despiterosiodes . C. albuC. album that has emerged from the begomovirus interspecies recombination events.In this study, we characterized a new recombinant begomovirus species in C. album weed grown in a residence area, Lahore, Pakistan  [In June 2018, during a routine survey to record begomoviruses other than the cotton-infecting viruses in Pakistan, leaf distortion was observed in Pakistan A,B. Leaf, Japan) ,43. All , Japan) . All the, Japan) . We also, Japan) ,47. Sout, Japan) ,49.SpeI/BamHI and BamHI/XbaI respectively were amplified using primer sets based on the extracted sequence and were ligated into the pGEM-T Easy vector , using the TA cloning technique according to the manufacturer’s instructions. This was followed by sequencing  and restriction digestion using specific enzymes. The two partial genomes were introduced into the pCAMBIA1303 vector and first transformed into competent Escherichia coli strain DH5α using the heat shock method and then into the GV3101 Agrobacterium strains. GV3101 Agrobacterium strains (both transformed and untransformed) were cultured in LB broth in the presence of a pCAMBIA1303 selection antibiotic, such as kanamycin (50 mg/L), and strain-specific selection antibiotics, such as gentamycin and rifampicin (50 mg/L), at 28 °C with agitation for 30 h . The untransformed GV3101 Agrobacterium with no plasmid was used as the mock. Agro-inoculation was performed by the pin-pricking method [N. benthamiana and C. album plants, respectively. Leaf tissue samples were collected from mock and infected plants 28 days post-inoculation (dpi) to check infectivity through PCR processing using the primers: CLDV-IC1/F (5′-ACTAGTTTTGGCAATCGGTGTCTCAC-3′) and CLDV-IC1/R (5′-GGATCCACACTCGTTTACATCC-3′) specifically designed to amplify IC1  of the infectious clone of CLDV with a target size of 1.4 kb. The infection caused by CLDV in N. benthamiana and C. album plants was further confirmed by Southern blot hybridization. An infectious clone (1.1 mer) of the detected virus was constructed to check its infectivity in the host plants . Two paranamycin  mg/L, anTo explore the intriguing genomic composition of the detected virus, i.e., Chenopodium leaf distortion virus (CLDV), primers were designed based on one ORF sequence of the recombinant viruses rather than their identical ORFs in the new virus genomic composition. The ageratum enation virus (AEV) was found to share the highest identity with the Rep and C4 proteins. New primers were designed to target the gene encoding CP of AEV to confirm whether the detected virus was a separate entity. Similarly, primers were designed to target the genes encoding Rep proteins of the rose leaf curl virus (RLCV), duranta leaf curl virus (DLCV), papaya leaf crumple virus (PaLCrV), and catharanthus yellow mosaic virus (CYMV) .www.ncbi.nlm.nih.gov (accessed on 23 November 2021)) and were aligned at the nicking site in the nonanucleotide motif at the origin of replication (5′-TAATATT//AC-3′). All multiple-sequence alignments were constructed using the MUSCLE method as implemented in MEGA X [A total of 56 full-length DNA-A sequences of AEV (30), RLCV (5), CYMV (6), PaLCrV (9), and DLCV (5) were used in this study including the sequence of CLDV . BLASTn n MEGA X  and manun MEGA X . In addin MEGA X ,53,54. Vn MEGA X . The fulp-value cutoff of 0.05. Putative recombinants and major and minor “parents” within the datasets were determined using the RDP, GeneConv, Bootscan, MaxChi, Chimaera, SiScan, and 3Seq recombination detection methods implemented on the RDP4 v4.100 suite . In RDP4The average pairwise number of nucleotide differences per site  was estimated using DnaSP version 6.12.03 . DnaSP version 6.12.03 was also used to calculate the number of haplotypes (H), the number of segregating sites (S), haplotype diversity (Hd), testing Tajima D , and Fu The method of Templeton, Crandall, and Sing (TCS) resulting genealogical networks identifies both the relationship between the different sequences as well as the number of nucleotide substitutions connecting them. All the sequences of the AEV, RLCV, CYMV, moieties identified in the novel CLDV recombinant begomovirus along with highly identical DLCV and PaLCrV were analyzed using statistical parsimony with the program TCS (v.1.21) implemented in the software Population Analysis with Reticulate Trees (POPART) ,61. BamHI confirmed the presence of a new begomovirus species, i.e., chenopodium leaf distortion virus (CLDV) in all samples  and AC4 showed 92% identity with AC4 of AEV (AGO59954). Similarly, AC2 (TrAP) and AC3 (REn) were 99% identical to the AC2 and AC3 of the RLCV (QAY29069) and (QAY29070), respectively. The ORF AVI (CP) was 100% similar to AV1 of CYMV (YP_009112873), and AV2 showed 100% identity with AV2 of RLCV (ADU33215)  of each ORF using BLASTX showed that DU33215) A,B. (DetDU33215) . In the DU33215) . The simN. benthamiana and C. album. Leaf tissues were harvested and analyzed by PCR to investigate viral replication ability; the virus was detected in the samples, which confirmed its presence  for the aforementioned datasets. The average pairwise nucleotide differences were higher for PaLCrV (π = 0.0651), followed by AEV (π = 0.0494), DLCV (π = 0.0442), CYMV (π = 0.0424), and RLCV (π = 0.0257), respectively (θw) was remarkably higher in the case of PaLCrV (θw = 0.0819), while these values were lower for AEV (wθ =0.0635), DLCV (θw = 0.0480), CYMV (θw = 0.0344), and RLCV (θw = 0.0255) respectively , CYMV (6 sequences), DLCV (5 sequences), PaLCrV (9 sequences) and RLCV (5 sequences) to compare the standing molecular diversity. Despite the difference in sample numbers, we were able to calculate average pairwise nucleotide diversities  these viruses might have been intermixed in any other host and then transmitted to C. album by whiteflies, (iii) the interspecies recombination could have been carried out inside the insect vector, which in this case is the whitefly. To investigate the first scenario, we designed the target-specific primers based on one of the ORF sequences of the recombinant viruses rather than their identical ORFs in CLDV . There might have been a possibility of missing any other host of CLDV during sample collection. Attempt to detect CLDV from the insect vector, i.e., whiteflies did not succeed either and needs further investigation but the presence of whitefly on C. album plants substantiated CLDV as a whitefly vectored begomovirus.Geminiviruses have the ability to adapt and evolve quickly as a result of genome-associated changes and recombination events ,63. Thesirulence ,65,66,67us hosts ,68,69,70 in CLDV . PCR amp in CLDV . In the Since genetic variation influences viral emergence, evolution, and vector transmission , we inveCP of CYMV and Rep and C4 of AEV through recombination events respectively as shown in The possibility of virus existence in the current composition cannot be considered naturally original as clearly recombination events were identified during recombination analysis . PhylogeBased on general ICTV demarcation criteria the newly detected virus (CLDV) should be categorized as a new isolate of RLCV (93% sequence identity between them), but the ICTV report clearly demonstrates exceptions in the case of recombinant viruses  such as


Peach trees can be infected with viruses and viroids. As we do not have efficient plant protection methods against these pathogens, the prevention of infection is crucial. Fruit trees are maintained by vegetative propagation. Planting material such as certified mother trees and rootstocks should be free from viruses and viroids, and this status has to be regularly checked to prevent infections. We surveyed certified peach trees for the presence of viruses and viroids using small RNA high-throughput sequencing (HTS), an unbiased virus diagnostic method. The results of the bioinformatic analysis of HTS were validated by other molecular methods including RT-PCR, Northern blot hybridization and loop-mediated isothermal amplification (LAMP). We found the presence of plum pox virus and peach latent mosaic viroid (PLMVd) in the vector-free isolator houses, whose presence should be regularly tested. Moreover, we detected frequent infection with recently described viruses such as nectarine stem pitting-associated virus and peach-associated luteovirus (PaLV). During the survey, PLMVd and PaLV were detected for the first time in Hungary. The analysis of the presenting virus variants and possible sources of infection suggests that the source of the viral infection could be the infected propagating material. Our study emphasizes the importance of using sensitive and trustworthy diagnostic techniques to be able to detect viral infections and successfully prevent their spread by propagation material. Prunus genus. Cultivars are propagated vegetatively by grafting, and are cultivated all over the world. Several viruses and viroids can infect peach, and they cannot directly be controlled by chemical applications [Peach is a globally widespread and important temperate stone fruit tree belonging to the ications . To prevications . These cications ,4,5. HTSications . The useications ,8,9.Peach can be infected by several viruses and viroids; among them, apple chlorotic leaf spot virus (ACLSV), plum pox virus (PPV), Prunus necrotic ringspot virus (PNRSV) and nectarine stem pitting-associated virus (NSPaV)  have beePrunus species, is one of the ten most devastating plant viruses [Plum pox virus (PPV), a potyvirus that infects  viruses . Its inf viruses .Avsunviroideae, PLMVd has a very definite secondary structure, with a long hammerhead and eight more stem-loop arms. Frequent mutations within its genome are usually maintained only if this specific secondary structure is not, or is only slightly, altered [Aside from PPV, peach is heavily endangered by the infection of a viroid: peach latent mosaic viroid (PLMVd). Although the infection of PLMVd is frequently latent, it is the causative agent of peach latent mosaic (PLM) disease, first described in France and later in Japan and in the USA . As a me altered . PLMVd c altered . In addi altered ,17.In recent years, the use of HTS revealed the presence of new viruses, such as nectarine stem pitting-associated virus (NSPaV) and peach-associated luteovirus (PaLV), which infect peach.Prunus mume trees [Prunus trees [NSPaV was first described in nectarine cultivars imported from France to California  and in nme trees . It has me trees . NSPaV wus trees . Its namus trees , which cus trees .PaLV has been described in the USA, in a quarantine station, from two imported peach varieties  by RNAseCertified peach trees in our research station are kept in an isolator house  or at a In 2017 when we started our sampling, 31 places (24%) in the isolator house were empty, indicating tree loss. To find out the reason for this decline, we sampled peach trees and carried out sRNA HTS .The surveyed trees were not showing any virus specific symptoms. As a result of Illumina sequencing, we obtained 9.7–18.1 million raw reads .. Their Small RNA HTS in the cultivar Springcrest indicated the presence of PPV. In 1_Sc_ih, we found 94 PPV-specific contigs, and the read per million reads (RPM) and the coverage of the genome were 4908 and 68.3%, respectively. On the other hand, in 2_Sc_sn, one PPV-specific contig was detected with 122 RPM and 52% coverage of the PPV genome, respectively .The size distribution of the PPV-derived sRNAs were slightly different in the two libraries. In 1_Sc_ih, most of the PPV-specific contigs were 21–22 nt long, while in 2_Sc_sn, beside 21–22 nt long reads, a peak at 24 nt appeared b. The inNSPaV-specific contigs were detected in 1_Sc_ih and in 5_Peach_ih, but the coverage of the viral genome by sRNA reads was also higher than 80% in the case of 4_Ch_sn .The NSPaV-derived sRNAs were mostly 21–22 nt long, suggesting their DICER (DCL) 2/4 origin. The presence of NSPaV was validated by RT-PCR .The investigation of each individual tree sampled for sRNA HTS for the presence of NSPaV using RT-PCR revealed that all of the Springcrest trees in the isolator, one out of two Cresthaven trees at the stock nursery and two trees, Champion and Aranycsillag, in the isolator were infected a,b.In vitro Springcrest plantlets were also infected with NSPaV, which could explain the source of infection in the case of this cultivar c. HoweveWe found PaLV-specific contigs in 1_Sc_ih, 2_Sc_sn and 5_peach_ih . The covPrunus-infecting luteovirus described from the Czech Republic [In the 5_Peach_ih library, Cresthaven, Suncrest, Aranycsillag and Champion were infected with PaLV, which was further proven by Northern blot hybridization in the first three varieties a. We susRepublic , which hPLMVd-specific contigs were present in three of the sRNA libraries: 3_Ch_ih, 4_Ch_sn and 5_Peach_ih. The RPM was very high  and the coverage of the viroid genome by the mostly 21 nt long viroid-specific reads was 100% a,b.The presence of PLMVd was validated by both RT-PCR and Northern blot hybridization .All of the sampled Cresthaven trees (both in the isolator house and in the open field) and six trees out of ten in 5_Peach_ih  were infected b,c. UnfoThe cultivars of the 5_Peach_ih were tested for the presence of PLMVd by a biological assay using a GF305 indicator. The survival rate of the grafts was very high and enabled a trustworthy evaluation  in the tTo test an alternative assay for PLMVd, we tried colorimetric RT-LAMP in the case of cultivars of 5_Peach_ih. The result of the assay coincided with the RT-PCR either purified RNA or crude extract was used as a template , indicatTo find out if the decline in the isolator can be connected to the infection of the viruses or PLMVd detected by HTS, we surveyed all of the surviving individuals. For the survey we used RT-PCR and Northern blot to detect viruses and PLMVd, respectively . A totalAlthough we amplified only a part of the viral coat protein coding region of PPV, we conducted a phylogenetical analysis of the two different isolates. Our isolates clustered together with a PPV_SK68 isolate sequenced in Hungary  and withIsolates were 95.8–96.1% identical to the reference genome at the nucleotide level, generating only one amino acid change in the encoded coat protein (CP) .To find out the phylogenetic relationship within the different strains of other viruses, we cloned and sequenced several variants of NSPaV, PaLV and PLMVd .NSPaV variants clustered into two distinct clusters, apart from any other NSPaV found in the NCBI GenBank . It suggNSPaV variants were 93–94.8% identical to the reference genome on the nucleotide and 93–94.1% identical on the amino-acid level .Only in recent years has it been described why there are not too many different variants for the reliable phylogenetic analysis of PaLV. However, the tree showing PaLV’s phylogenetic relationships shows that PaLVs from nectarine and peach cluster independently and do not cluster according to their geographical origin .PaLV variants were very conserved, being 98.1–99% identical to the reference genome on the nucleotide and 95.1–98.6% identical on the amino-acid level .We sequenced variants from different individuals from the same cultivars . They did not cluster together, suggesting their independent origin.The result of our virus diagnostic survey showed that 73% of the individuals were infected with PLMVd. The viroid possesses high genetic variability and we found several point mutations , but theThe conserved regions in the hammerhead arm of the viroid responsible for its replication are all maintained, together with the “kissing-loop” side chains .Furthermore, in one case (Ob_166 tree), we identified a PLMVd variant with a 12 nt long insertion located at the same position where the pathogenic determinant of the peach calico (PC) phenotype was found  Figure c. This iCertified mother trees used as a source of propagation material have to be free from any damaging pathogens. Traditionally, the virus-free status of these trees is tested by biological assays using virus-sensitive indicators to detect any possible infection. These tests are land and labor-consuming, requiring up to three years to obtain a result. Serological methods (ELISA) and molecular PCR-based methods can only test the presence of pathogens whose presence is anticipated in the sample. They are valid alternatives to biological assays to test regulated viruses routinely. The concentration of the viral titer fluctuates during the season, which can directly affect the result of these tests, whose sensitivity is also variable. New variants, bearing mutations within the primer sequence or in the antibody-recognized part of the genome, can escape from the detection. While lateral strip-based ELISA can be used in the field, PCR-based methods need a laboratory and experienced technicians to proceed. In recent years, isothermal amplification methods (LAMP and recombinase polymerase amplification (RPA))  emerged.The bioinformatics analysis of the sequenced reads was applied according to our in-house pipeline based on CLC Genomic Workbench. The reliability of bioinformatics pipelines during small RNA HTS analysis is very different  and we tThe size distribution and coverage of the viral genome by virus-specific reads are good indicators of the virus’ presence. Viral specific reads are usually 21–22 nt long, as they are products of the host DCL4 and DCL2 enzymes, respectively, and they usually cover most of the viral genomes by sRNAs that have both sense and antisense orientations . HoweversRNA HTS proved its usefulness, and its cost is constantly decreasing. Moreover, we can prepare mixtures, i.e., pools of different individuals before library preparation, and validate the presence of viruses in all of these individuals later on. In the case of 5_Peach_ih, we followed this strategy. We were able to validate the presence of a virus, NSPaV, even if it was present in two out of ten samples, whose RNA was used in the mixture.The validation of sRNA HTS was performed by different, unrelated methods: RT-PCR, Northern blotting, RT-LAMP and a biological assay. For the first three methods, sequence information about the investigated pathogen was essential to be able to design primers for their amplification and for probe synthesis. Primers used for the validation of the strains present in this research were checked before application by comparison with the consensus sequence of the specific virus prepared from the sequenced small RNA reads. We used previously described primers in the case of PLMVd, but we kept one published primer for PPV diagnostics, fulfilling this requirement. As viruses and viroids evolve fast, this strategy is absolutely essential for correct diagnosis.In contrast to these sequence-specific validation methods, a biological assay can detect any pathogen in the tested cultivar but only if it induces symptoms on a sensitive indicator. Although GF305 is believed to be sensitive to PLMVd infection, we failed to detect it in any of the investigated samples, even if the viroid was present in the grafts. The biological assay lasted for years and because of the latency of the viroid, it failed to diagnose it.Our results on this small pilot test show that there is an urgent need to revise the ability of the biological assay as a current official virus diagnostics method in order to prevent the unwanted dissemination of viruses by not precisely diagnosing the propagation material.RT-PCR and Northern blot are molecular methods which require practice and special hardware, which is why applying them in fast routine assays is difficult. In contrast, RT-LAMP offers a fast, reliable and sensitive alternative. Our results show that RT-LAMP could detect all of the PLMVd-infected cultivars, even if crude extract was used as a template. We used the colorimetric version of RT-LAMP, the result of which can be easily evaluated. This isothermal test works at 65 °C and does not need any special equipment, which is why it could even be carried out in the field or orchard.SRNA HTS made it possible for us to diagnose the frequent infection of NSPaV and PLMVd and also the presence of PaLV, a virus which had not been previously described in Hungary. Surveying every single individual in an isolator house showing decline revealed a high rate of infection even in these controlled circumstances. The investigation of the location of the trees and the phylogenetic analysis of the presenting variants did not give us any clues for finding the original sources of infection. However, as we found PPV, NSPaV and PaLV in the in vitro culture of Springcrest, it is highly possible that the original sanitized plants were the source of infection.Shoot tip grafting offers a very promising possibility for virus elimination from peach , but thiWe found mixed infections on several occasions. During our surveys, the decline in additional trees occurred. Although in some cases it coincided with the presence of mixed viral infections, there were cases where no virus or PLMVd were detected in the declined tree while their causative agent could not be identified. The distribution of virus-infected trees did not follow any pattern, and due to their high frequency, it is very difficult to identify the origin of their infection. We showed that the PPV infection of the Springcrest cultivar could originate from its in vitro source.We could not correlate the presence of viruses to any specific symptoms and not even to the decline in trees. This does not mean that infection did not play any role in the decline, but it is also possible that other factors, i.e., non-viral pathogens or abiotic effects alter or expedite their synergistic effect.Connecting the presence of the pathogen to any specific symptoms requires the molecular characterization of the viruses described by HTS, which needs special attention in the future ,9.During our survey in 2017, we sampled individuals of two peach cultivars (Springcrest and Cresthaven) both in the isolator and in open field stock nursery together with representative members of ten cultivars kept in the isolator . The isoPrunus-infecting viruses. The annotation of these contigs was performed using the BLASTN algorithm with the default options  and the NCBI plant-hosted viral reference genomes (downloaded at 30 June 2021). For the viruses detected by this method , the reads were directly mapped to the reference genome and were counted with and without redundancy . The number of normalized reads (read/1 million reads: RPM) was then calculated from the mapped redundant reads and the number of total sequenced reads. The coverage (%) of the viral genome was calculated based on a consensus sequence generated from this mapping. The presence of the virus was indicated if at least two parameters of the three investigated ones were present: (i) the presence of any virus specific contigs, (ii) a number of normalized redundant virus specific reads >200, or (iii) the coverage of the virus genome was >60% or the coverage of the viroid genome was >80% fulfilled. The result of this analysis is summarized in https://www.geneious.com).For bioinformatics analysis, we used CLC Genomic Workbench. After trimming and quality control, longer contigs were built de novo from the non-redundant reads by employing an assembler of CLC (de novo assembly) using the default options: word size 20, bubble size 50, and simple contig sequences with a minimum 35 nt length , according to the manufacturer’s instructions. The generated cDNA was used as templates for PCR reactions using Phire Hot Start II DNA Polymerase  and published diagnostic primers or new ones , which wFor Northern blot analyses, 1–3 µg of total RNA was separated on formaldehyde-1.5% agarose gel and blotted to the Amersham Hybond-NX membrane , by the capillary method using 20x SSC ). Hybridization was carried out at 65 °C in Church buffer  overnight with the appropriate radioactively labelled probe, washed for 5 min in 2× SSC, 0.1% SDS and for 15 min in 0.5× SSC, 0.1% SDS at the temperature of the hybridization and exposed to an X-ray film. Virus-specific, P32-labelled DNA probes were prepared by using the DecaLabel DNA Labeling Kit . As a template for radioactive probe preparation, the PCR-amplified and purified PCR product of the cloned region of the viruses were used.The RT-LAMP test was performed using the WarmStart Colorimetric LAMP kit from New England Biolabs . Previously published primers were diluted by 10 mM Tris pH 8  (see TabPrunus avium, Prunus persica GF 305, Prunus hibrid GF 31 and Prunus serrulata Kwanzan in August 2016. The sprouting rate was determined after a year (Buds of the certified trees were graft transmitted to r a year . The symFor phylogenetic analysis we used MEGA11 . AlignmeWe proved the incidence of PLMVd and PaLV for the first time in Hungary. We suspect that the source of the viral infection might be the propagation material, which was used as a base for the variety collection in this isolator house.Global markets and climate change affected the habitat and geographical distribution of fruit trees together with their vectors of pathogen diseases, and also insect pollinators . To prev


This cross-sectional study examines out-of-pocket costs for the treatment of invasive breast cancer in employer-insured women younger than 65 years. This population may face significant financial burdens related to long-term hormonal-based prevention4 and enrollment in high-deductible health plans (HDHPs) and consumer-driven health plans (CDHPs). This study examines OOP costs of treatment of invasive breast cancer in employer-insured women younger than 65 years.Oral anticancer prescription drugs are central to breast cancer treatment,3 to identify women 18 to 64 years of age continuously enrolled in a health plan in 2018 with a diagnosis of invasive breast cancer and a claim for at least 1 anticancer drug approved by the US Food and Drug Administration (FDA). Data on race and ethnicity are not collected in the database. Total OOP costs of treatment per patient were calculated as the sum of copayments, coinsurance, and deductibles for pharmaceutical, inpatient, and outpatient claims. Mean oral drug OOP costs were calculated with the same cost variables and standardized to represent a 30-day supply. Costs were inflated to 2022 dollars using the US Bureau of Labor Statistics’ Consumer Price Index for All Urban Consumers. The data were collected from insurers in a Health Insurance Portability and Accountability Act of 1996–compliant manner and are fully deidentified. This study followed the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) reporting guideline and was designated exempt by the Boston University Institutional Review Board for the use of deidentified data.This cross-sectional study used the national Marative MarketScan databaseP < .05 was considered statistically significant.Statistical analysis used Stata software, version 16 (StataCorp LLC) to estimate the mean annual total OOP costs for treatment per patient per year and mean 30-day OOP cost per drug using standard 2-part logit models. A γ log-link regression model estimated potential correlates in adjusted 30-day drug OOP costs, including year of FDA approval and insurance type. A 1-sided P = .001).A total of 25 224 women  with invasive breast cancer diagnosis and claims for 1 or more of 14 oral anticancer drugs were included. A total of 23.1% were HDHP or CDHP insured, and 51.0% had no OOP costs for drugs. Sixteen women had no OOP costs for all care modalities. The total mean estimated annual OOP cost was $1502.23 per patient, with $112.41  in inpatient costs, $1186.27  in outpatient costs, and $203.55  in pharmaceutical costs. The OOP costs for outpatient claims were 79.0% of total costs . Of drug5 Women insured by HDHPs and CDHPs had higher OOP costs compared with those with more generous coverage.Among a prevalent sample of younger women with invasive breast cancer and employer-sponsored insurance who filled prescriptions for oral anticancer drugs in 2018, most used nonproprietary drugs and had limited OOP costs. Outpatient-related OOP costs were larger than drug costs, and OOP costs were higher among those using branded or more recently launched drugs.6 and complications related to treatment. We were also unable to estimate costs by cancer treatment phase6 or clinical history and among those 65 years or older. Recently passed federal policies seeking to reduce prescription drug prices among Medicare-insured patients could be complemented by voluntary employer or insurer efforts and state legislation to reduce OOP costs for commercially insured patients with cancer across care modalities.This study has some limitations. The OOP costs are an underestimate because many patients will also be treated for other comorbidities


The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spreads rapidly and harbors many mutations in the spike protein, but the origin of this virus variant remains unclear. We address the role of unusual virus evolution mechanisms such as hypermutation, out-of-frame reading, and recombination. Rather, regular Darwinian evolution, that is, the repeated selection of beneficial spike mutations, seems to have led to the appearance of the grossly altered spike protein of the Omicron variant. This virus unleashed a true pandemic in 2019 that continues until today, although a fair level of protection can be achieved by several newly developed vaccines and, hopefully, by herd immunity. Multiple SARS-CoV-2 variants of concern (VOCs) have been designated by the World Health Organization (WHO). Omicron (B.1.1.529) is the newest virus variant first reported from South Africa in November 2021 –6. The O–The origin of the Omicron variant remains unclear , 11. A cThe acquisition of many clustered mutations in the spike gene could in theory also be explained by more exotic evolutionary events that we will introduce and critically test. First, viral genomes can be subject to “hypermutation,” a process in which multiple changes occur in a clustered manner in parts of the viral genome. These mutations are introduced either by the viral polymerase, possibly by an error-prone mutant thereof, or by cellular restriction systems as exemplified by the HIV apolipoprotein B mRNA-editing catalytic polypeptide-like protein (APOBEC) case , 14. A cA second evolutionary scenario that could instantaneously generate many mutations is a double-frameshift event that causes the ribosome to temporarily read in a different reading frame . This ouTo broaden the test of the first two hypotheses, we also inspected the hypothetical evolutionary paths from any of the VOCs  to Omicron. This analysis also did not reveal any special mutational profile or frameshift events that could support these scenarios (results not shown).A third scenario that can trigger a gross yet sudden mutational event is recombination, the mixing of Omicron genome sequences with sequences derived from other viruses or the human host. Such swapping of genetic information is a well-known phenomenon in virology and is well documented for coronaviruses . For insHaving considered three unusual scenarios for the evolution of the Omicron variant, we cautiously conclude that regular virus evolution, that is, by the repeated selection of beneficial mutations in the spike gene or Darwinian evolution, underlies the appearance of this variant. Because Omicron first appeared in South Africa, it has been suggested that SARS-CoV-2 could evolve rapidly in this setting because of the weakened immune system of more than 20% of the local human population that is HIV infected . In lighThe most compelling evidence for this scenario of regular Darwinian evolution actually comes from inspection of the genetic changes, which reveals a profound preference for mutations that change the amino acid composition of the spike protein: 30 nonsilent changes versus 1 silent mutation . This evSome or all of these amino acid substitutions may affect the basic viral functions of receptor recognition and cell entry, and other substitutions may represent compensatory changes in the spike protein. We noticed a remarkable adjustment in the usage of charged amino acids: 2 positively charged residues were removed, but some 11 were gained, whereas a more balanced picture was seen for negatively charged residues (1 gained and 3 removed). Such changes in net or local charge could affect the interactions of the spike protein with other molecules, among which are the ACE2 receptor and antibodies raised by natural infection or immunization. Experimental studies are needed to test these ideas. Mutations in key domains of the spike protein may have an impact on the virulence and pathogenicity of this virus, possibly correlating with early epidemiological and clinical data showing that Omicron is less pathogenic than other SARS-CoV-2 variants .In conclusion, it seems likely that strong evolutionary pressure has led to the selection of the grossly altered spike protein of the Omicron variant. However, much remains currently unknown. First of all, extensive sampling may reveal virus isolates that are intermediate between the Wuhan and Omicron strains, which could more firmly prove or deny the exact path of virus evolution. Experiments should then address the biological effects imposed by the many spike protein substitutions, both direct effects on virus replication and also indirect effects through escape from host-induced immune pressure. In the meantime, we should rapidly update the RNA-based vaccines against this rapidly spreading SARS-CoV-2 variant.


Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) allows sensitive detection of viral particles and viruses in epidemic samples but it cannot discriminate noninfectious viruses from infectious ones. Propidium monoazide (PMA) coupled with quantitative polymerase chain reaction (qPCR) was assessed to detect infectious viruses. Currently, there is no established test method to detect the infection of the porcine epidemic diarrhea virus (PEDV). In this study, propidium monoazide coupled with qPCR detects infectivity of PEDV. We optimized the method from the selection of primers, the working concentration of PMA, and the inactivation method using heat or ultraviolet (UV). The viruses which were treated with PMA before qPCR were inactivated using heat or UV. However, the addition of PMA alone did not affect the detection of live viruses, which indicates that a viral capsid break may be essential for PMA to bind to the genome. A repetition of the method on naked PEDV RNA suggests that it can be used to detect potentially infectious PEDV. The results indicated that an optimal plan of PMA could be extremely useful for evaluating infectious and noninfectious viruses. Porcine epidemic diarrhea virus, a single-stranded RNA virus, is one of the significant pathogens causing porcine epidemic diarrhea, with high mortality in neonatal pigs . There wThe detection methods for PEDV include immunological tests, molecular biological detection, and clinical virus isolation. However, none of the above methods can distinguish between infectious and noninfectious viruses. PMA is a dye with an identification function that can bind to DNA molecules. The dye can enter the small grooves of DNA molecules such as damaged bacteria or viruses. The decomposition of PMA produces substances that covalently cross-link with DNA molecules, thereby inhibiting the PCR amplification of DNA in damaged microorganisms. It has been shown that PMA can penetrate the membrane of compromised cells, leading to a covalent bond formation with the DNA strands, which inhibits subsequent PCR amplification , 5. HoweGiven the existing problems in PEDV virus detection, particularly with scant knowledge of the combined application of PMA to detect live bacteria and RNA viruses, this study attempted to use a method based on PMA qPCR technology for the detection of infectious PEDV.2 incubator, blindly passed, and observed for cytopathic effect (CPE). After stable passage for four generations, the cell culture was frozen and thawed three times and centrifuged at 1,700 g at 4°C for 15 min. The supernatant was collected as the PEDV seed and stored at 4°C. The PEDV strain was then selected, inoculated into Vero cells, cultured at 37°C, 5% CO2, and cultivated in the box for 3 days. It was observed for cell CPE, after which the virus liquid was filtered, collected, and diluted 10-fold.PEDV was extracted from clinical specimens in our laboratory. The clinical specimen was ground and centrifuged. The supernatant was collected, filtered, added to Vero cells, cultured in a 37°C, 5% COThe virus was inactivated in a water bath at 100°C for 30 min. For UV inactivation, the virus was subject to ultraviolet irradiation for 4 h. The infectivity of PEDV after inactivation by heat or UV was determined using CPE assays. The virus was inactivated, planted into cells, and cultured in an incubator for 3 days. Then, the cells were examined under a microscope.PEDV-S and N primers were used. In order to test the specificity of the primers and the inactivation methods, the recombinant plasmid pUC19-PEDV-N standard was prepared, and the amplification curve was established . Each experiment was repeated at least three times and fluorescence signals were collected. The criteria for determining the detection of viruses were as follows:(1) When the sample Ct value is ≤ 35, the sample was considered to be positive.(2) If the Ct value ranges between 35 and 40, and if the logarithmic amplification curve is curved, then the sample was judged to be suspicious positive. Alternatively, the sample was judged to be negative and the test was repeated. If the Ct value of the retested sample is ≤ 35, the suspicious sample was judged as positive.(3) If the sample has no Ct value or Ct value > 40, the sample is judged to be negative.The CPE were not observed in this blank control group . Figure The Ct values of the two groups with the PEDV live virus (①②) were 20.56 and 20.36. The Ct values of the two groups with PMA added to the PEDV live virus (③④) were both 22.07, which was <35, indicating that the sample was positive . The conP < 0.05).As shown in These results indicate that the PMA treatment to differentiate between infectious and UV-inactivated viruses had no effect .PEDV-S and N were tested. When using the PEDV-S primers in the final concentration of PMA, the sample Ct value was ≤ 35 , the amplification efficiency was 110.73%, indicating that the primers provided had sufficient sensitivity , when the virus stock was amplified, the Ct value was the same (28.15) and the copy number was 4002.62 copy/μL. When virus stock was diluted 10-fold, the Ct value was 30.05, and the copy number was 971.13 copy/μL. The highest copy number of PEDV detected by PMA qPCR was 971.13 copy/μL. Therefore, in the case of infectious PEDV detected by PMA qPCR , the primers designed by the N gene were used for qPCR amplification to distinguish whether PEDV was infectious or not.The virus stock had a Ct value of 28.15 when was used for amplification. It changed to 30.05 when it was diluted 10-fold . AccordiThe outbreak of PEDV causes serious economic losses throughout the world. Currently, the detection methods for PEDV include immunological tests, molecular biological tests, and clinical virus isolation. However, immunological tests that cannot detect the infectivity status of the microorganisms are problematic. Therefore, the need to develop a rapid, accurate, and simple method that detects infectious PEDV and conducts real-time monitoring of PEDV is rather urgent.via an amplification assay  has received a high level of attention due to the threat posed to pig farming worldwide . There iPMA combined with RT-qPCR could be very effective for determining infectious and noninfectious viruses.2731327322@qq.com.The datasets presented in this article are not readily available because requests to access the datasets should be directed to GL, CH and XT conceived the project, analyzed the data, and revised the manuscript. GL designed and performed the experiments, analyzed the data, and wrote the manuscript. YL, LY, WS, YH, DY, MZ, and GX wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.This study received funding from Wuhan Keqian Biology Co., Ltd. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.All authors were employed by Wuhan Keqian Biology Co., Ltd. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Oryza sativa L.) is one of the major staple foods for global consumption. A major roadblock to global rice production is persistent loss of crops caused by plant diseases, including rice blast, sheath blight, bacterial blight, and particularly various vector-borne rice viral diseases. Since the late 19th century, 19 species of rice viruses have been recorded in rice-producing areas worldwide and cause varying degrees of damage on the rice production. Among them, southern rice black-streaked dwarf virus (SRBSDV) and rice black-streaked dwarf virus (RBSDV) in Asia, rice yellow mottle virus (RYMV) in Africa, and rice stripe necrosis virus (RSNV) in America currently pose serious threats to rice yields. This review systematizes the emergence and damage of rice viral diseases, the symptomatology and transmission biology of rice viruses, the arm races between viruses and rice plants as well as their insect vectors, and the strategies for the prevention and control of rice viral diseases.Rice ( Oryza sativa L.) is a vital food crop for the world’s human populations [Rice , rice stripe virus (RSV), and rice black-streaked dwarf virus (RBSDV) were noticed and described sequentially in Japan. RDV was the first virus documented in rice plants in 1883 ,11. It c8 and directly affecting millions of rice farmers [9 in the 1980s [Alphanucleo-rhabdovirus in the family Rhabdoviridae, was first described in Taiwan and Guangdong provinces of China [Phytoreovirus in the family Sedoreoviridae, was found in Thailand [Bymovirus in the family Potyviridae, was first reported in Japan and later in India [Ludwigia perennis and Corchorus olitorius, leading to an increase in both shoot growth and leaf size, along with characteristic chlorotic lesions on leaves [In 1963, rice grassy stunt virus (RGSV)-causing grassy stunt disease was first observed in the Philippines . In the  farmers . In 1963 farmers , and ric farmers  appearedhe 1980s . In 1965of China ,19. Majoof China . Averageof China . It has Thailand . As of lin India ,24. It an leaves ,47,48.From then to the year 2000, no novel virus was discovered in Asian rice-planting regions. However, the present rice viruses RBSDV, RSV, RGSV, and RRSV frequently caused outbreaks of viral disease in distinct rice-growing areas.Fijivirus species in the family Spinareoviridae, is the agent of this disease. By 2009, rice fields in 19 provinces in northern Vietnam and 9 provinces in southern China had suffered from this disease. In 2010, more than 60,000 ha of paddy fields in 29 provinces of Vietnam and more than 1,300,000 ha in 13 provinces of China were infected, causing crop failure in many regions [Rhabdoviridae family [In 2001, a novel rice viral disease emerged in East and Southeast Asia ,26. It h regions ,50. As oe family ,28. In te family . In 2017e family . These tChaetocnema pulla [Oryza glaberrima and local upland types of O. sativa are very tolerant to RYMV, Fomba considered that RYMV is an indigenous one to which local rice plants have adapted, whereas the apparent equilibrium established between host and virus seems to have been disrupted by the introduction of exotic varieties and an intensification of cropping practices [Rice-growing countries in Africa form another center for viral disease epidemics. In 1966, yellow mottle disease was first reported in rice plants growing around Lake Victoria, Kenya. This disease is caused by RYMV, a sobemovirus transmitted by species of Chrysomelid beetles including ma pulla . The disma pulla . For insma pulla ,54. Asidractices .Polymyxa graminis, is an important causal agent that causes severe viral disease in rice-growing areas in both Africa and America. RSNV-causing disease was first described in 1977 as a new virus infecting rice in Côte d’Ivoire [RSNV, a soil-borne benyvirus transmitted by the fungus d’Ivoire  and was d’Ivoire . RSNV wad’Ivoire . Since td’Ivoire ,58,59,60d’Ivoire , Benin [d’Ivoire , Mali [6d’Ivoire , and Sied’Ivoire , suggestPhenviviridae, is the causal agent for this disease. It was first noticed and described in Colombia in 1935 [In America, hoja blanca disease is a dominant viral disease in rice-growing regions. Rice hoja blanca virus (RHBV), a tenuivirus in the family  in 1935 ,11. Sinc in 1935 . Until 1 in 1935 , of whic in 1935 ; (2) cha in 1935 ; (3) theSusceptible rice plants to virus infections are primarily characterized by a set of disease symptoms such as dwarfism, increased or reduced tillering numbers; leaves being dark green, yellow, or mosaic; inferior heading; and mostly unfilled grains. Most rice viral diseases can be readily distinguished by these symptoms. Here, major disease symptoms including plant height, tillering number, and abnormal leaf morphology exhibited on rice plants infected by distinct rice viruses are listed in Polymyxa graminis [Sesselia pussilla, Chaetocnema pulla, and Trichispa sericea [Conocephalus merumontanus, as well as by mammals  and by birds [www.dpvweb.net (accessed on 10 September 2022)) and CAB International (www.cabi.org/isc/ (accessed on 10 September 2022)).Rice viruses are transmitted by distinct insects or fungi, and most of them cannot be transmitted mechanically or by seeds . RNMV angraminis . RYMV is sericea ,70. It iby birds ,72. Otheby birds ,75,76,77by birds . TherebyDuring the last two decades, the devastating damage on rice production by rice viral diseases has driven a sustained quest for rice viral diseases. Two central points involving rice–virus interactions and vector–virus interactions are explored to comprehensively understanding the complex biological processes during virus infection, from which vital insights into disease prevention and control are providing ,80,81.To resist virus infection, plants have evolved several defense pathways such as RNA silencing, phytohormone, and plant innate immune system ,83,84,85Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), double-stranded RNA binding (DRB), and Argonaute (AGO) genes [RNA silencing is one of the most important antiviral pathways for plants to resist virus infection ,84,85. IO) genes . ThereinO) genes . RDRs arO) genes ,127,128.O) genes ,130. AGOO) genes .AGO1a/1b expression increases the susceptibility to RSV. In contrast, AGO2 enhances rice susceptibility to fijivirus infection through the DNA methylation-controlling transcription inhibition of OsHXK1, by which the ROS-mediated resistance is inactivated [RDR1 transcription, thus activating the RDR1-dependent antiviral RNAi against RSV infection [RDR6 transcripts are more susceptible to both RSV and RDV, indicating its function in host defense [To date, several RNA silencing-associated components have been verified in rice plants by genetic analysis or protein-protein interactions. AGO18, a specific Argonaute protein in monocotyledons, mediates broad-spectrum resistance to virus infections as a molecular lock to binding miR168, thus leading to the alleviated repression of rice AGO1 . They fuctivated . Up-regunfection . Rice pl defense ,90.RDR6 [To counter-defend antiviral RNA silencing, rice viruses versatilely impede antiviral RNA silencing signals by encoding VSRs or other functional proteins. For instance, Pns10, a VSR encoded by RDV, can inhibit both local and systemic RNA silencing, possibly by inhibiting the expression of RDR6 . Pns11, RDR6 . NS3, a RDR6 . Both P2RDR6  and RSMVRDR6  can inteRDR6 . The preRDR6 . RSV infRDR6 . RSV infRDR6 . RGSV p3RDR6 .Either antiviral RNA silencing and the counter-defense performed by VSRs or other viral proteins indicated that the host RNA silencing pathway is indeed a primary way for rice plants to resist virus infection. While rice genome consists of multiple copies of AGOs, DCLs, RDRs, and other RNAi-associated proteins, thereby more antiviral RNAi components in rice need to be determined in the future.Plant hormones exist within plants, contributing to growth and development, but their biosynthesis and signaling can be disrupted or modulated after virus invasion ,135. DurThus far, the well-known plant hormones, including gibberellic acid (GA), salicylic acid (SA) , jasmoniTCP21 was down-regulated for suppression of JA biosynthesis and signaling [Viruses or viral proteins also directly or indirectly target distinct phytohormone-associated components to promote virus infection . RDV Pnsignaling . Recentlignaling . Sometimignaling . For insignaling . Howeverignaling . During ignaling . These fLOC_Os06g03150 is mainly associated with the differentiation of resistance within the Xian subgroup and LOC_Os06g31190 mainly explained the difference in the resistance between Xian and Geng. Using genome-wide association study (GWAS), linkage disequilibrium (LD) decay analyses, RNA-sequencing, and genome editing, a highly RBSDV-resistant variety and its first functional gene, Hap1, was identified [O. glaberrima [CPR5 gene, a regulator of active defense mechanisms [O. glaberrima was identified through fine mapping method by the same group [By forward genetic approaches, a sulfotransferase, STV11, with function in catalyzing the conversion of SA to sulphonated SA, is identified as an available anti-RSV locus . To screentified . Besidesentified ,142. Forentified . Furtheraberrima . Furtherchanisms . Later, me group .OsHAK5 potassium transporter improves virus resistance against RGSV infection [2+ signaling network, has been proven to interact with RGSV p5 [Although the primary plant defense is based in this case mainly on RNA silencing and plant hormones, other cellular pathways are also involved in the rice–virus interactions. Recently, two proteins associated with plant innate immunity, OsRLP1 and OsSOBIR1, are proved to positively regulate rice immunity against RBSDV infection, suggesting pathogen-triggered immunity (PTI)-based innate responses could contribute to antiviral plant defense . Our recnfection . Besides RGSV p5 . PsbP, a RGSV p5 . To over RGSV p5 . These fNicotiana benthamiana, thereby a research model of RSV in N. benthamiana has been developed and applied. To date, several host factors of N. benthamiana with function in regulating RSV infection have been uncovered. Viral proteins can manipulate organelle function to benefit virus infection. For instance, RSV p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement [NbeIF4A activates autophagy and inhibits RSV infection by facilitating autophagic degradation of p3. Further study indicated that overexpression of vsiRNA-4A, a viral siRNA derived from RSV RNA4, targets eIF4A mRNA for cleavage, thus leading to induced autophagy [RSV can be mechanically transmitted to movement . NSvc4 tmovement . Besidesutophagy . Recentlutophagy . These fL. striatellus tissues and thereby lower transmission efficiency [Unlike the huge adverse on rice growth and development by rice viruses, persistent-propagative infection by rice viruses causes a limited pathogenicity in their vectors. For example, RDV , RGDV 1, RSV 14, and SRBficiency . Then, hficiency ,149,150.Persistent transmission by insects firstly needs a successful initiation of viral replication cycle comprising of virus entry, multiplication, and spread in their insect vector cells . Many viApart from pass through the physical barriers in the insects, rice viruses also manipulate vector immune systems for efficient infection of insect cells and persistent transmission by insect vectors. Autophagy pathway is always modulated by rice viruses and often positively regulates persistent viral propagation in insect vectors . For exa6 methylation of adenosine (m6A) regulates RBSDV replication in its vector via modifying viral and insect RNA, and it is used by the virus to maintain a specific titer threshold required for viral transmission [Antiviral RNAi pathway, a primary antiviral response in plants, can also be triggered by the propagation of plant viruses in insect vectors, evidenced by the characterization of vsiRNAs in virus-infected insect vector ,177. Antsmission . This reSuch transovirial viruses that RDV ,181, RGDEmerging evidence has proven that rice viruses can manipulate their vectors either directly or by inducing changes in host plants to promote the spread of viral pathogens. For example, during the late infection of SRBSDV in rice plants, SRBSDV P6 enters the nucleus and interacts with OsEIL2 to suppress ethylene signaling by blocking the dimerization of OsEIL2, thus promoting virus spread via attracting the insect vector . It implWith the long-term efforts of studying on rice viral diseases, many strategies for disease prevention and control have been developed as a system. By collecting the present knowledge, four main points are summarized as follows.MP are highly resistant to the virus [Generation of resistant rice varieties through different breeding strategies is always a more safe and effective way to prevent and control rice viral diseases. While owing to the limitation of antiviral gene resources, antiviral breeding for disease resistance processes slowly. But many other strategies for resistance to viral diseases have been employed, including encapsulation of viral genome, silencing of viral genes by antisense RNA or by small RNA molecules, expression of ribozyme, or modification of host factors. For example, as early as in 1999, transformed lines with expressing the RNA-dependent RNA polymerase of RYMV were found to be resistant to RYMV strains from different African locations, sometimes there were complete suppression of virus multiplication . In addihe virus . Recentlhe virus . These mhe virus ,111. By he virus . Additiohe virus ,194.eIF(iso)4G1, the well-established resistance locus in rice, strongly diminished the interaction with avirulent VPg [Although many strategies are exercisable and effective to generate resistant rice varieties, rare varieties are generated yet. Even worse, some identified resistance loci have potential ability to be overcome by the evolution of virus genome. For example, insertion of an E309K mutation in lent VPg . These sRice plants are more susceptible to viruses at the seedling stage. For example, rice is susceptible to SRBSDV infection during all growth periods, but the symptoms depend on the growth stage at the time of infection, and the earlier the infection occurs, the more severe the symptoms are ,196. ThuR. dorsalis were controlled by applying pesticides, the RSMV transmission was largely suppressed. For RYMV, a soil-borne rice virus, seed pelleting, or coating seedling is recommended to control the rice infection in seedling nurseries.Pesticides should first be applied in rice fields to control overwintering or the first-generation of vector, and in rice seedling nurseries to reduce the population of insect vectors, thereby the primary source of infection largely decreased . For exaIt is of great importance to develop safe and effective chemical agents for controlling plant viral diseases. To date, several immune stimulants such as Dufulin, Amino oligosaccharide, and moroxydine hydrochloride have been developed and applied into viral disease control . HoweverThis review primarily summarizes the occurrence and damage of rice viral diseases, disease symptoms, transmission biology, arms race between rice viruses and rice plants as well as insect vectors, and the strategies in viral disease management. This knowledge tells us about a major challenge during the coming decade that how to balance rice production and consumption.Repeated outbreaks of known and newly emerged rice viruses remind us that rice viruses are tricky. We know little about their law of existence: how they come like SRBSDV emerging since 2001 in China, and how they leave or stash in the vast nature such as RBSV. Most important, under natural conditions, life system of crops, biotic factors, abiotic factors, and human activities should be balanced in the agroecosystem. Once such balance is stricken by external or internal elements such as environment, plant nutrients, transmission ability of vectors, weak link will be vulnerable to cause the outbreak and epidemic disease ,202. The