Rhabdoviridae contain important human and mammalian pathogens that are vectored by different arthropod species. The ground supernatants of mosquitoes were used to inoculate in BHK-21 and C6/36 cells for virus isolation. Then, the viral complete genome sequence was obtained and used for phylogenetic analysis. In this study, we observed a cytopathic effect (CPE) in mosquito cells (C6/36) and rod-like virion after inoculating a pool of Armigeres subalbatus samples collected in Shanxi Province, China, in 2019 (SX1916). Meta-transcriptomics sequencing revealed the presence of two distinctive rhabdoviruses with similar abundance levels, namely, Shanxi Armigeres subalbatus rhabdovirus (SXARV) and Shanxi Arboretum virus (SXABTV). Despite the fact that the SXARV genome (9590 nt) was much shorter than that of SXABTV , both belonged to the Almendravirus group within Rhabdoviridae whose genomes encoded five proteins  and a small hydrophobin (U1) and the difference in lengths is mainly caused by a substantially shorter N protein encoded by SXARV. On the phylogenetic tree, SXABTV was closely related (90.7% amino acid identity at L protein) with the Arboretum virus isolated from Psorophora albigenu mosquitoes in Peru in 2014, whereas SXARV was distantly related to Rio Chico virus (63.3% amino acid identity), a genetic distance large enough to be defined as a new species within Rhabdoviridae. Collectively, we report a simultaneous isolation of two related rhabdoviruses from Armigeres subalbatus that marked the circulation of almendraviruses in Shanxi, China.The family  Rhabdoviridae comprises 45 genera and 275 species [Rhabdoviridae viruses have a wide host range, including arthropods, such as mosquitoes, ticks, sandflies, birds, fish, and mammals [Rhabdovirus is a single-stranded negative-sense RNA virus that is rod or bullet shaped, with a particle length of 100–430 nm and diameter of 45–100 nm. According to the latest virological classification report from the International Committee on Taxonomy of Viruses (ICTV), the family Almendravirus (Rhabdoviridae) virus had a full-length genome of approximately 11,000 nt, which mainly encode five proteins, namely, nucleoprotein (N), phosphoprotein (P), and matrix protein (M), glycoprotein (G), RNA-dependent RNA polymerase protein (L), as well as a small hydrophobin (U1) between the G and L genes, following the order 3′N-P-M-G-U1-L5′ [Almendravirus viruses [G-U1-L5′ . The U1  viruses ,7.Almendravirus: Arboretum virus and Puerto Almendras viruse isolated from Peru [Almendravirus has not been isolated from bloodsucking insects collected in China.To date, the ICTV has established seven viral species within the genus rom Peru , Balsa vrom Peru , Coot Barom Peru , Menghairom Peru , and Japrom Peru  and Rio rom Peru . Among trom Peru  and fiveArmigeres subalbatus, both belonging to the Almendravirus genus (Rhabdoviridae).From June to August 2019, we conducted comprehensive sampling of different species of vectors, including sandfly and mosquito in Wuxiang County, Shanxi Province, Central China. A number of viruses have been identified from these vectors, including Wuxiang virus isolated from sandfly samples [Anopheles sinensis, 360 Armigeres subalbatus, 37 Aedimorphus vexans, and 45 Culex pipiens pallens. Anopheles sinensis and Armigeres subalbatus accounted for 43.48% (340/782) and 46.04% (360/782), respectively, of total mosquito specimens collected and are, therefore, considered the dominant species in Wuxiang County.A total of 782 mosquitoes from four genera and four species was collected in this study , includiAnopheles sinensis, seven for Armigeres subalbatus, one for Aedimorphus vexans, and one for Culex pipiens pallens. The resulting supernatant was inoculated onto BHK-21 or C6/36 cells and the cells were cultured continuously and observed daily for CPE. For the pooled Armigeres subalbatus sample SX1916, the first passage in C6/36 cells showed no CPE. CPE appeared in the second generation on day 4, characterized by the aggregation of large numbers of cells and cell space enlargement . Importantly, both rhabdoviruses had very high mean coverage, namely, 33,038-fold for SXABTV and 76,101-fold for SXARV . The sequencing resulted in 9,144,290 reads, which were assembled into 4969 contigs. Taxonomy annotation of all contigs revealed two rhabdovirus genome sequences in the SX1916 isolate, with lengths of 11,480 and 9590 nt, respectively. These viruses were tentatively named as Shanxi Arboretum virus  and Shanxi or SXARV , which aor SXARV . Howevern = 12); they were thawed and diluted with cell culture medium to extract RNA and subjected to gene detection. The results of parallel detection of SXARV and SXABTV genes showed that all 12 plaques were SXARV gene positive and SXABTV gene negative. Plaques that tested positive for SXARV were diluted with cell culture medium and subjected to additional plaque purification. After three rounds of plaque purification, an SXARV isolate (SXARV1916-2) was obtained. All subsequent studies involving SXARV came from the SXARV1916-2 strain.Because the NGS results identified two rhabdoviruses with large genomic differences in the SX1916 isolate, we applied virus plaque purification methods to purify each rhabdovirus. The SX1916 isolate was used for four consecutive passages of the fourth-passage C6/36 cells for virus plaque purification. In the first and second plaque purification, the virus-infected C6/36 cells were cultured for 7 days (a short-diameter plaque was observed on day 6) to obtain a single virus plaque. We obtained nine and three single virus plaques in the first and second purification tests, respectively . All 12 To confirm that sample used before the plaque purification test contained both viruses, we performed parallel genetic testing of both viruses in fourth-generation C6/36 cells inoculated with the SX1916 isolate to verify whether both viruses were present in the cell supernatant used in the plaque purification test. The results showed simultaneous amplification of the viral gene amplification products of both SXARV and SXABTV in the fourth-generation supernatant of the C6/36 cells . The plaque-purified SXARV1916-2 isolate was inoculated into C6/36 cells to observe its cytopathic characteristics. In second-generation C6/36 cells inoculated with plaque-purified SXARV1916-2, CPE was clearly observed after 4 days of culture, manifesting as large numbers of fused cells and cell shedding C. HowevePlaque-purified SXARV was inoculated into C6/36 cells and ultra-thin section detection was performed when the cells showed clear CPE. Rod-shaped virus particles  were observed via electron microscopy .https://www.ncbi.nlm.nih.gov/tools/primer-blast (accessed on 5 April 2021)), and the primers used are listed in According to the virus genome sequence information obtained via NGS sequencing, we designed amplification primers to cover the full-length genome nucleotide sequence of SXARV and SXABTV, respectively, using Primer-BLAST  between the G and L proteins of the small hydrophobin . The difAlmendravirus viruses, including 13 isolate strains in GenBank, and found that SXABTV and Arboretum virus (ABTV) had the highest identity. We identified nucleotide (amino acid) identity of the coding region of the N gene at 83.9% (97.5%), the P gene at 70.8% (68.5%), the M gene at 79.3% (88.5%), the G gene at 75.6% (84.1%), the U1 gene at 69.4% (52.9%), and the L gene at 78.9% (90.7%).We examined the identity of nucleotides and amino acids in the coding regions of SXABTV and SXARV and other Nucleotide and amino acid identity of the coding regions of the SXARV isolate had the highest identity with the Rio Chico virus. We identified nucleotide (amino acid) identity of the coding region of the N gene at 59% (52.8%), the P gene at 55% (54.8%), the M gene at 59.5% (48.1%), the G gene at 53.4% (41.7%), the U1 gene at 57.5% (49.4%), and the L gene at 64.6% (63.3%) .Almendravirus contains seven virus species: ABTV, Balsa virus, Menghai rhabdovirus virus, Rio Chico virus, SXARV, Coot Bay virus, and Puerto Almendras virus. Recent studies have reported that amino acid identity of the coding region of the L protein of <90% is sufficient for identifying new rhabdovirus species. The amino acid identity of the L gene coding region varies among the seven viruses from 40%  to 64.1% (ABTV and Puerto Almendras virus).The virus nucleotide and amino acid identity data  indicateAlmendravirus viruses, we prepared an analysis dataset of the L gene sequence of Rhabdoviridae viruses, including all Almendravirus viruses and representative strains of 29 other genera of Rhabdoviridae  [Viruses in the bin (U1) . The conbin (U1) ; RdRp inbin (U1) . Recent bin (U1) .Almendravirus species  and 11,876 nt (Puerto Almendras virus) .SXARV isolated in this study had a full-length genome of 9590 nt, whereas that of SXABTV was 11,480 nt, and the nucleotides and amino acids of the coding regions of the six protein genes differed significantly between the two virus isolates , with ons virus) , whereas species . In summhttps://guangzhou.11467.com/info/8049209.htm (accessed on 5 June 2019)) comprising a 12-V, 12-Ah battery, 0.1-A bulb, and 0.14-A fan. All mosquito specimens were placed in a −40 °C low-temperature refrigerator for 30 min and then removed for processing in an ice bath. Each specimen was labeled according to morphology, collection time, collection environment, and then stored in liquid nitrogen until laboratory testing.From June to August in 2019, we collected blood-sucking insects overnight (18:00 p.m. to 6:00 a.m.) at animal pens including chicken pens, dog houses, and mule pens in Wuxiang County, Shanxi Province, China  using a blood-sucking insect collection tool  cells were cultured in 89% RPMI 1640 (Invitrogen), 10% FBS (Invitrogen), and 1% penicillin and streptomycin (100 U/mL) in a 28 °C incubator [Golden hamster kidney (BHK-21) cells were cultured in 90% Eagle medium (laboratory preparation) containing 7% fetal bovine serum , 1% penicillin and streptomycin (100 U/mL), 1% glutamine (30 g/L), and 1% NaHCOncubator ,16,17.3. We then added 1.5 mL grinding solution and ground the pooled sample in an ice bath. After centrifugation at 4 °C and 12,000 rpm for 30 min, 70 μL of ground supernatant was inoculated into 80% monolayer BHK-21 and C6/36 cells in a 24-well plate . BHK-21 and C6/36 cells were continuously cultured in an incubator at 37 °C with 5% CO2 and at 28 °C, respectively. Cytopathic effect (CPE) was monitored under a microscope every 12 h. When CPE appeared, the virus solution was collected and stored in the refrigerator at −80 °C until identification. First-generation cell culture supernatant that did not show CPE was blind passaged for three generations and discarded if CPE did not appear after that [To isolate viruses from mosquitoes, we pooled groups of 30–50 individuals according to collection site and species, put them into a glass grinder, and washed them twice with grinding solution consisting of 93% Eagle medium, 5% penicillin and streptomycin (100 U/mL), 1% glutamine (30 g/L), and 1% NaHCOter that ,16,17.–1–10–6) were sequentially added to the 6-well culture plate (0.1 mL/well). The wells were placed in a 28 °C incubator for 1 h, and 1% agarose-MEM medium containing 2% FBS was added to each well to cover the cells (3 mL/well). After culturing for 5 days, a second layer of 1% agarose-MEM medium containing 7% neutral red and 2% FBS was spread over the cells (3 mL/well). The time and number of instances of plaque appearance were observed, and the plaque diameter was measured as previously described [C6/36 cells were transferred into 6-well culture plates  to grow into monolayer cells with a coverage rate of 80%. Virus dilutions  .Total RNA was extracted from the supernatant of virus-infected cells using the QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions. The extracted RNA was placed in a 65 °C water bath for 10 min and then immediately placed in an ice bath for 2 min. Next, 32 μL RNA was added to the first-strand reaction tube of the Ready-To-Go kit , which was maintained at room temperature for 1 min before the addition of 1 μL pd(N)6 random primer . This mixture was centrifuged immediately and incubated in a water bath at 37 °C for 1 h. The total volume of the cDNA library was 33 μL; it was stored at −40 °C until later use ,20.To examine the viral genome of the SX1916 isolate, we extracted the total RNA of the supernatant of C6/36 cells infected with the SX1916 isolate for next-generation sequencing (NGS) using the QIAamp Viral RNA Mini Kit . We used the TruSeq total RNA library kit to construct the cDNA library, and sequencing was performed on the HiSeq4000 platform . After low-quality reads were removed from the sequencing data, we performed de-novo assembly. We extracted virus-related contigs based on the taxonomic information of the blast hits. To avoid mis-assembly, reads were mapped back to the virus genome with Bowtie2  and inspected with Geneious Program  . The virWe applied polymerase chain reaction (PCR) for viral gene amplification in a 25 μL system comprising 2 μL cDNA as template, 12.5 μL 2× GoTaq Green Master Mix, 8.5 μL nuclease-free water, and 1 μL each of 10 pmol/μL upstream and downstream primers. After the PCR was complete, 5 μL of gene amplification products was detected by 1% agarose gel electrophoresis ,20. The https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 5 May 2021)) using blastn program. We used the Seqman software  for nucleotide sequence splicing and quality analysis and the BioEdit software  for multiple sequence alignment. We used the MEGA software  to complete the systematic evolution analysis based on the maximum likelihood method, with a bootstrap value of 1000; the General Time Reversible model is used in analysis. We used the MegAlign  tool for identity analysis of nucleotide and amino acid sequences [The nucleotide sequence was compared to non-redundant protein (nr) database available at the National Center for Biotechnology Information (NCBI) website . Despite the isolation of a variety of Almendravirus viruses in Asia, North America, and South America, it remains unclear whether these viruses could infect mammalian hosts. Therefore, further investigations of the infection status of Almendravirus in domestic and wild animals will be of great importance.In the study, two rhabdoviruses, SXARV and SXABTV, were found in a pool of


The epidemiology of yam viruses remains largely unexplored. We present a large-scale epidemiological study of yam viruses in Guadeloupe based on the analysis of 1124 leaf samples collected from yams and weeds. We addressed the prevalence of cucumber mosaic virus (CMV), Cordyline virus 1 (CoV1), Dioscorea mosaic associated virus (DMaV), yam asymptomatic virus 1 (YaV1), yam mosaic virus (YMV), yam mild mosaic virus (YMMV), badnaviruses, macluraviruses and potexviruses, and the key epidemiological drivers of these viruses. We provide evidence that several weeds are reservoirs of YMMV and that YMMV isolates infecting weeds cluster together with those infecting yams, pointing to the role of weeds in the epidemiology of YMMV. We report the occurrence of yam chlorotic necrosis virus (YCNV) in Guadeloupe, the introduction of YMMV isolates through the importation of yam tubers, and the absence of vertical transmission of YaV1. We identified specific effects on some cropping practices, such as weed management and the use of chemical pesticides, on the occurrence of a few viruses, but no crop-related factor had a strong or general effect on the overall epidemiology of the targeted viruses. Overall, our work provides insights into the epidemiology of yam viruses that will help design more efficient control strategies. Dioscorea spp.) is an important staple crop for hundreds of millions of people in the tropics and subtropics, particularly in West Africa, which accounts for 97.8% of the world yam production [Yam , the cultivated yam species of diseases .Yams are planted on mounds or ridges, grown on stakes, or crawling on the ground, and weed control is achieved through mulching or by manual or mechanical weeding since the use of herbicides in yam plots has been prohibited in Guadeloupe since 2005. Other crops, such as banana, sugarcane, or pineapple, are often grown in association with yams in the same plots or in nearby plots [D. trifida from the Caribbean and the Amazon region, from which this species originates, likely results from its high sensitivity to yam mosaic virus (YMV) [Yam remains the most cultivated food crop in Guadeloupe, although its production has decreased sharply from 22,500 tons/year in 1968 to 3000 tons/year in 2006 [us (YMV) , a potyvAmpelovirus, Badnavirus, Macluravirus, Potexvirus, Potyvirus, Sadwavirus and Velarivirus have been reported in the germplasm collection of the Biological Resource Center for Tropical Plants (BRC-TP) and from small-scale surveys of yam plots in farms [Twenty-five virus species infecting yam are currently recognised by the International Committee on Taxonomy of Viruses (ICTV) . This fiin farms ,12,13,14in farms ,11,13,14D. trifida. Our results set a key milestone toward understanding the epidemiology of yam viruses, which is a prerequisite for the design of more efficient control strategies.Here, we report a large-scale epidemiological survey of cucumber mosaic virus (CMV), Cordilyne virus 1 (CoV1), Dioscorea mosaic-associated virus (DMaV), yam asymptomatic virus 1 (YaV1), YMV, yam mild mosaic virus (YMMV), badnaviruses, macluraviruses and potexviruses infecting yams in Guadeloupe. We studied the key factors that could potentially affect the occurrence, prevalence and epidemiology of these viruses in the main yam production areas of Guadeloupe, such as cropping practices, the potential of weeds as viral reservoirs, and the role of tuber imports and trade in the introduction and spread of exotic yam virus strains or species. We investigated the vertical (mother-to-daughter plant) transmission of YMMV and YaV1 in D. alata, D. bulbifera, D. cayenensis, D. esculenta, D. rotundata and D. trifida).Two surveys were carried out, serving two distinct purposes, respectively. From October 2019 to December 2019, 16 farm plots and 2 experimental plots scattered along the main yam production areas of Guadeloupe A were suD. alata, which is the main cultivated yam species in Guadeloupe. A total of 116 leaf samples were collected from the same two experimental plots that were sampled in 2019  was collected per plant, regardless of the presence of symptoms, to avoid bias towards viruses causing more severe symptoms at the expense of viruses causing symptomless infections or mild symptoms.The samples from the most abundant annual and perennial weed species were collected during both surveys  within or at the immediate vicinity (0–5 m away from the edges) of the sampled yam plots, with the aim of searching for potential reservoirs of yam viruses. All samples were stored at −80 °C until processing. A complete and detailed list of the samples collected, including the location and characteristics of the surveyed plots, is provided in Two pieces of 1 × 2 cm were cut from each leaf sample and placed in two separate grinding tubes . One set of tubes was used for total nucleic acid (TNA) extractions, according to Foissac et al. . TNAs we® cloning vector  using the supplier’s protocol. Cloned amplification products  . Double D. alata and 2 D. rotundata samples collected in 2019, and 13 D. alata samples collected in 2020, respectively, were indexed positive -occurrence of viruses across the different field plots after integrating the effects of the crop-related variables. The first observation from the ordination  showed tComparing the residual covariances from the model fit without and with the crop-related variables revealed that 70% of the covariance in virus abundances is explained by the covariance of the crop-related factors. However, the strong effects of these variables were observed only for CoV1, DMaV, potexviruses and YMV . The facCleome viscosa (family: Capparidaceae) originating from a D. alata plot (Le Petit Portland), while the remaining seven samples  originated from another D. alata plot (Roujol). No other virus was detected in any of the 126 analysed weed samples  generated from the YMMV-infected weed samples, corresponding to nucleotide positions 9236–9495 of the YMMV-Brazil reference genome (JX470965), were cloned and sequenced. Phylogenetic analyses were performed on the coding part of these sequences, which was 139 nt long and corresponded to the 3′ end of the CP domain. The analyses showed that the sequences amplified from the weeds were 97.1–100% similar to each other and 79.9–100% similar to the sequences of the YMMV isolates sampled in the yams collected in Africa, the Caribbean, India and Brazil . The seqD. trifida plants. For this, fifty-five tubers from ten D. trifida mother plants co-infected by YMMV and YaV1, the most frequent co-infection situation encountered during the surveys, were planted in individual pots in an insect-proof greenhouse to avoid possible external vector transmission. One and three months after sprouting, the 55 daughter plants were indexed for CMV, CoV1, DMaV, YaV1, YMMV, YMV, badnaviruses, macluraviruses and potexviruses. Only YMMV was detected in 96.4% (53/55) of the daughter plants at one and three months after sprouting, whereas neither YaV1 nor any other virus was ever detected in any of the daughter plants at any time point  and D. trifida variety ‘Cousse-couche’ (two tubers) found on local markets and that had been imported from Costa Rica. The plants originating from these tubers were indexed for CMV, CoV1, DMaV, YaV1, YMMV, YMV, macluraviruses and potexviruses, and only YMMV was detected in two of the D. alata plants and both D. trifida plants , as described above. The sequences obtained from the two infected D. trifida plants shared 97.8–99.2% identity with a sequence  obtained from an infected D. trifida plant originating from Costa Rica [D. trifida and D. alata. Interestingly, the only sequence generated from D. alata (CR3YMMVDa) was 100% identical to that from a D. alata plant of the same variety, ‘Kabusah’, collected in 2019 in Guadeloupe for this study  thanks to constantly improving yields. Meanwhile, yam production was plummeting in some smaller producing countries such as Guadeloupe, which is no longer self-sufficient and relies heavily on imports to meet local demand. Reversing this trend has become of utmost importance to restore food self-sufficiency, as yam remains one of the pillars of food security in Guadeloupe ,30. The CMV is one of the most ubiquitous plant viruses, with an estimated host range exceeding 1000 species in 85 families . It is tD. rotundata, which is known to be mainly infected by YMV [D. alata (66.8%), considering that 80.3% of all the collected samples originated from this species. Some associations between yam species and viruses were not found. For instance, macluraviruses were not found in D. trifida, D. esculenta or D. bulbifera, although this result may be biased by a lack of representativeness due to the very low frequency at which these species were encountered during the surveys, coincidentally with the overall low prevalence of macluraviruses in yams in Guadeloupe. Nevertheless, Umber et al. [D. trifida plants from the BRC-TP germplasm collection. On the contrary, CoV1 was not detected in D. cayenensis, while Diouf et al. [D. cayenensis accessions of the BRC-PT, although at a low prevalence .All the other virus species and genera targeted by this work had been previously described on yam in Guadeloupe ,12,13,14d by YMV ,16 and Dd by YMV . YaV1 waD. alata samples collected in 2019 is consistent with that reported by Umber et al. [D. alata accessions of the BRC-TP germplasm collection. The absence of YMV from the D. alata samples collected in 2020 might result from a statistical artefact resulting from a smaller sample size . A similar situation may apply to potexviruses, which were detected at a very low prevalence (0.3%) in the D. alata samples collected in 2019 and not at all in those collected in 2020.The low prevalence of YMV (9.8%) in the r et al.  (8.6%) iD. alata and D. nummularia from Africa, China, India, and Vanuatu [D. rotundata is also a host species for YCNV and that there is a very high degree of relatedness (94.6–100% identity in a 131 nt sequence) among YCNV isolates from Guadeloupe, regardless of their host yam species and location, suggesting either a more recent introduction into Guadeloupe through a founder effect or a slower rate of evolution as compared to YMMV.Although macluraviruses were reported previously in the BRC-TP yam germplasm collection , their d Vanuatu ,27,28 buBadnavirus) so far [Ampelovirus, Badnavirus, Macluravirus, Potexvirus, Potyvirus, Sadwavirus and Velarivirus. It is, therefore, likely that more yam viruses in these genera are also transmitted by vectors, although research effort is needed to provide experimental evidence supporting this hypothesis. In yam, viral infections are primarily attributed to vertical transmission through vegetative propagation, although this mechanism has never been quantitatively assessed. We addressed this issue for the two most prevalent viruses in our sampling, YaV1 and YMMV. Firstly, we showed that seed-tuber transmission of YMMV occurred in plants originating from non-certified imported tubers. This finding raises concerns about the risk of introduction in Guadeloupe of exotic YMMV isolates and possibly other yam viruses through imported tubers. It highlights the need for tighter controls on the sanitary status of imported yam tubers and for enforcing the ban on the use of tubers imported for consumption purposes as planting material. Our data confirmed that mixed infections by distinct virus species are the rule rather than the exception in yams, similar to other vegetatively propagated crops ,44,45,46) so far ,48,49. HD. trifida whereas YMMV is transmitted to almost 100% of daughter plants originating from D. trifida tubers coinfected by YMMV and YaV1. Our results relate to those of Bertschinger et al. [Phenacoccus and Planococcus, have been reported on yam in Guadeloupe [Secondly, we showed that YaV1 is not vertically transmitted in r et al. , who shor et al. ,51,52, ar et al. , of whicadeloupe . Whethere.g., aphids), which is common in plants [Cotransmission of several viruses by the same vector (n plants , or thron plants  through n plants ,57,58. Rn plants ,61,62,63n plants ,66,67,68n plants ,70,71. In plants .Aphis craccivora, an aphid vector of YMMV [Cropping practices such as monoculture, intensive production, or the introduction of new resistant cultivars have been shown to affect the incidence of viral diseases in crops ,72,73. I of YMMV , is poly of YMMV ,75. ConvIn this study, our focus was on identifying factors that could potentially affect the occurrence, prevalence, and epidemiology of yam viruses in Guadeloupe. The knowledge gained from our survey allows us to identify some prophylactic strategies for better protection of yam fields, such as the more careful management of weeds and proscribing the use of imported tubers as planting material. Combining these measures with the use of certified virus-free plant material by farmers should benefit yam production in Guadeloupe. However, by limiting our research on virus introductions to viruses already known to Guadeloupe, it is likely that we overlooked other yam viruses not yet reported in Guadeloupe. Future analyses of introduced tubers using HTS technologies will help draw a more detailed picture of the nature of the yam virome introduced into Guadeloupe through imported tubers and compare it to the indigenous yam virome. An important body of work remains to be undertaken, which concerns the role of vector transmission in the epidemiology of yam viruses, but it will first require identifying which vectors may be involved, for such essential information is still lacking at the present time.


While there is absolutely no evidence to ensure recovered patients are either likely or unlikely to get reinfected. But studies in non-human primates indicate that reinfection of recovered patients is highly unlikely. It is also clear that primary immune responses or induced immunity to severe acute respiratory syndrome coronavirus 2 remain in circulation for several months and at least temporarily confer immunity to protect from reinfection. In addition, negative virus culture analysis of re-positive suggests that positive reverse transcriptase-polymerase chain reactions in recovered patients are more likely to be false-positive, or detection of genetic remnants of virus discharged from lesions of lungs or better sampling at the time of repeat analysis. However, emerging severe acute respiratory syndrome coronavirus 2 variants are likely to be causing the infections observed in some of the recovered patients. Yet, the immunity of this novel virus is not defined, and there is rising global speculation about the risk of reinfection. Few recent reports from china have confirmed the positive reverse transcriptase-polymerase chain reaction (RT-PCR) in numerous patients after complete recovery of clinical symptoms or with previous negative test results.6 Conversely, another study indicated that repeat analysis of RT-PCR 48 hours apart is necessary to ensure virus clearance.3 Besides, laboratory tests, including D-dimer, absolute lymphocyte count, and antibody test in combination with RT-PCR negative testing, are essential to ensure that the virus is no longer transmitted by discharged or recovered patients.3 Few recent reports have shown that asymptomatic or moderately symptomatic or in close contact with patients recovered is also a means of transmission or infection in the population.8 Furthermore, persistent viral shedding has been recorded for an extended period of 60 days from the onset of symptoms, and even for 36 days after the recovery of symptoms.9 This suggests that patients with COVID-19 or those recovered may require prolonged isolation. Also, more precautions are obviously needed to be certain that a virus has not been reactivated after recovery, but the patient has fully recovered. Although reactivation may be rare or very unlikely, the possible risks are too high to ignore, warranting an urgent call for more in-depth research.Despite the fact that there is limited evidence, KCDC thus postulates the new hypothesis about the probability of COVID-1 9 viral reactivation instead of reinfection. Taking this into consideration, they issued testing guidelines that patients are only considered fully recovered if RT-PCR is tested negative twice within 24 hours.10 A similar study from US groups demonstrated that 5log10 reductions in median viral load and neither led to respiratory failure or mortality.11 Another group developed six candidates deoxyribonucleic acid (DNA) vaccines formulated using variants of the critical viral protein. These investigational vaccines were immunized in adult rhesus macaques to investigate the immunological response against the virus. The study revealed that most of the vaccinated animals showed no detectable virus, while few showed dramatically lower viral loads compared to that of control group.12 Those studies on non-human primates (rhesus macaques) suggest that immunity (antibody- and cell-mediated immune responses) acquired following primary infection provided immunity against SARS-CoV-2 relapse , which protects against infection . Such immunity remains in the body, which provides protective efficacy against secondary challenges (large peak-orange). During the infection phase, RT-PCR may be used to detect viral nucleic acid in the early infection phase (blue line) followed by detection of IgM antibodies (red line), while IgG antibodies may be detected in the later infection phase which serves as a long-term immunity (green line).It is therefore clear that higher antibody levels are directly proportional to lower levels of the virus that indicates neutralizing antibodies are a reliable marker of protection. Such findings raise the probability that immunological interventions will ultimately be effective for prevention and treatment. But it is critical to stress that notable variations exist between viral infection in macaques and humans. This suggests that rigorous clinical studies are required to evaluate immunity against SARS-CoV-2 reinfection in humans.2 RT-PCR will once again test absolutely negative upon full recovery of lesions in the lungs and clinical symptoms.2 Interestingly, KCDC has demonstrated negative results of virus isolation in cell culture on further analysis of respiratory samples from re-positive RT-PCR. Also, they showed that 96% positive for viral neutralizing antibodies in a serum sample of re-positive RT-PCR cases.4 In a row, experts in South Korea have confirmed that reactivation or reinfection is possibly due to the results of a false-positive test that is detected from the remains of dead viruses.13 According to new research, immunity to SARS-CoV-2 induced by infection or vaccination appears to be long-lasting. During an infection, cells that recognize the virus survive in the bone marrow and produce antibodies as needed.14 As a result, COVID-19 recovered individuals who were later immunized will not require boosters. While vaccinated people who have never been infected, or who have been infected but have not produced a strong immune response, will certainly need boosters. Vaccine breakthrough infection reported in some Nepalese might result from emerging variants circulating in Nepal. It is also possible that SARS-CoV-2 variants are less susceptible to vaccines than the wild-type virus.Besides, re-positive RT-PCR in recovered patients are unlikely to be caused by the reinfection from the residual virus, as described in Figure 1 . Actually, it emerges from the residual virus transferred to the upper respiratory tract (throat or nose) with a cough from the lower respiratory tract.Infection observed in some of the recovered patients is more likely as a result of SARS-CoV-2 emerging variants than reinfection with wild-type SARS-CoV-2. More research is required to assess if recovering patients are at risk of reinfection, and further precautions tend to be required to ensure that a patient has fully recovered from COVID-19 or is unable to reactivate or patients are fully vaccinated. Furthermore, genomic surveillance and vaccinations are only a key to drive evidence-based policies and implement effective control strategies in Nepal.


Aspergillus lentulus). We separately utilized rolling circle amplification and next-generation sequencing to identify ssDNA viruses specifically. We identified 13 new cressdnaviruses across all libraries, but unlike the RNA viruses, they could not be confirmed by PCR in either the original unamplified samples or freshly amplified nucleic acids. Their distributions among sequencing libraries and inconsistent detection suggest low-level contamination of reagents. This highlights both the importance of validation assays and the risks of viral host prediction on the basis of highly amplified sequencing libraries. Meanwhile, the detected RNA viruses provide a basis for experimentation to characterize possible hypovirulent effects, and hint at a wealth of uncharted viral diversity currently frozen in biobanks.Fungi host viruses from many families, and next-generation sequencing can be used to discover previously unknown genomes. Some fungus-infecting viruses (mycoviruses) confer hypovirulence on their pathogenic hosts, raising the possibility of therapeutic application in the treatment of fungal diseases. Though all fungi probably host mycoviruses, many human pathogens have none documented, implying the mycoviral catalogue remains at an early stage. Here, we carried out virus discovery on 61 cultures of pathogenic fungi covering 27 genera and at least 56 species. Using next-generation sequencing of total nucleic acids, we found no DNA viruses but did find a surprising RNA virus diversity of 11 genomes from six classified families and two unclassified lineages, including eight genomes likely representing new species. Among these was the first jivivirus detected in a fungal host (IMPORTANCE Fungal pathogens of humans are a growing global health burden. Viruses of fungi may represent future therapeutic tools, but for many fungal pathogens there are no known viruses. Our study examined the viral content of diverse human-pathogenic fungi in a clinical biobank, identifying numerous viral genomes, including one lineage previously not known to infect fungi. Candida yeasts and Aspergillus moulds have caused the majority of IFIs in cancer patients and hematopoietic stem cell transplant recipients, recently Rhizopus, Mucor, Fusarium, and others have emerged as threats  has been growing for decades , 2, part threats –5. Next demicity . Meningopatients , while tpatients , 9. In Ipatients , 11, andpatients .Aspergillus fumigatus), while emerging species may be unaffected by standard empirical treatments (Genomoviridae were identified (–Cryphonectria parasitica. Infected trees can be treated with the RNA virus Cryphonectria hypovirus 1 (CHV1), resulting in a significantly reduced virulence of the fungus (Sclerotinia sclerotiorum infection of plants using the virus Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1)  . InfectisHADV-1) . AnothersHADV-1) .Not all mycoviruses are viable as biological control agents, especially for fungal pathogens of humans. It may be important that the virus is not recognized by the innate immune system  or the Rhizopus microsporus, R. oryzae, Syncephalastrum racemosum, Aspergillus niger, A. lentulus, Cladosporium sphaerospermum, and Penicillium vanoranjei. Surprisingly, upon analysis we found that all resulting assemblies, including the control, contained at least one RNA virus, and two had mixed infections with three viral species each , but a different species. The last was made in a different genus (Trichophyton) to the index (Aspergillus), both of which are in the class Eurotiomycetes.The 61 initial fungal samples belonged to 27 genera and at least 56 species . VIDISCA sequencing produced an average of 7,600 reads per sample. Bioinformatic analysis identified six samples containing between 1 and 1,306 RNA virus reads belonging to distinct viral lineages. To recover and characterize full genomes of these viruses, Illumina reads were generated  and assembled from the six positive samples plus one without detected viruses to serve as a control of screening sensitivity. Sequenced isolates belonged to ies each . A totalionships . PCR scrhttps://figshare.com/projects/Viruses_infecting_clinical_mycology_cultures/128186). The other 13 sequences  for circular ssDNA. These had an average of 1.7 million reads per library after quality control. Removal of poor quality contigs left 14 that appeared to be of cressdnaviral origin, since they possessed at least partial Rep and Cap proteins with BLASTp identity to known viruses. We found that 12 of the 14 sequences were complete circular genomes and the last two were truncated. Each was derived from a different sample. Since RCA indiscriminately amplifies any primed circular ssDNA, we explored the possibility that they represented viral contaminants amplified from reagents rather than mycoviruses , 27. We equences  did not Endornaviridae were found in a single Rhizopus microsporus culture. They were distantly related to each other, sharing 13% amino acid (aa) identity across the RdRp, and interestingly both were distant to all other public endornavirus sequences. The Committee on Taxonomy of Viruses (ICTV) species demarcation for endornaviruses is <75% nucleotide (nt) identity across the genome . The closest relative of RMEV1 was Rhizoctonia solani endornavirus 7 (QDW65434.1) at 15.6% RdRp aa identity, while for RMEV2 it was Phytophthora endornavirus 2 (BCL84886.1) at 17.14% RdRp aa identity. Phylogenetic analysis showed both RMEV1 and RMEV2 cluster within the genus Alphaendornavirus  as the closest relative (37.4% RdRp aa identity). Narnaviruses were found in cultures of Rhizopus oryzae and Aspergillus lentulus. They shared only 8% RdRp aa with each other and clustered in different parts of the family tree (QJT93774.1). The virus found in A. lentulus belongs to the previously described species Aspergillus fumigatus narnavirus 2 (AFNV2), sharing 98% RdRp aa identity with accession AXE72934.1. Although no data on biological impact are currently available for these viruses, some mitoviruses and narnaviruses do have the potential to impact fungal biology either by conferring hypovirulence or affecting reproductive capabilities . The closest relative of the virus identified here was Trichoderma koningiopsis totivirus 1 (QGA70771.1) at 60.9% RdRp aa identity. Despite this, we suggest the genome be given the provisional name Syncephalastrum racemosum totivirus 1 (SRTV1). Our rationale is the large phylogenetic distance between the host genera Syncephalastrum and Trichoderma and the current lack of biological data to support assignment to the same species. Members of the genus Totivirus have been previously associated with hypovirulence  with 77.1% RdRp aa identity and 54.9% capsid aa identity. We suggest the name Aspergillus niger partitivirus 1 (ANPV1). The finding is in line with the known ascomycete host range of the genus Gammapartitivirus.A virus belonging to the family itivirus . Criteriitivirus , and theBotourmiaviridae were found, one in the A. lentulus culture also containing AFNV2 and another in a Cladosporium sphaerospermum culture. The former belonged to the genus Magoulivirus, while the latter belonged to the genus Penoulivirus (BCH36640.1) with 97.7% RdRp aa identity, while the penoulivirus belongs to Erysiphe necator-associated ourmia-like virus 69 (QKI79899.1) with 90.7% RdRp aa identity. At least one member of the family has previously been shown to be associated with the hypovirulence of its host  and Grapevine-associated jivivirus 1 (QIJ25698.1), each with approximately 40% RdRp aa identity across >96% query coverage. Though no ICTV guidelines on species demarcation currently exist for this lineage, we propose this genome be named Aspergillus lentulus jivivirus 1 (ALJV1) on the basis of low sequence identity to relatives  in combination with a novel host record; notably, this is the first jivivirus identification in an axenic fungal culture. As the first record, no data are currently available regarding the biological impact of jivivirus infection on their fungal hosts. The closest relative of the virus infecting P. vanoranjei was an unclassified virus recorded as Riboviria sp. (QDH88072.1), at 49% RdRp aa identity. We gave it the temporary name Penicillium vanoranjei-associated RNA virus 1 (PVRV1) until proper taxonomic classification. Notably, BLASTp results suggested ALJV1 was related to the Virgaviridae and Bromoviridae, while PVRV1 hit one sequence labeled as virga-like (BBB86779.1). We therefore analyzed their relationships together, alongside representatives of both families. This confirmed a close relationship between ALJV1 and members of the Virgaviridae and Bromoviridae , but all remaining sequences were resolved as distant relatives of lineages, including Kirkoviridae, Smacoviridae, Naryaviridae, Redondoviridae, CRESSV1, and Vilyaviridae. These lineages in particular are conspicuous since all are found associated with the gastrointestinal tracts of humans and other animals, and also in the human respiratory environment in the case of Redondoviridae (LC671626) did cluster alongside MZ824233.1 and MZ824234.1.As described above, analysis of RCA libraries returned 14 cressdnavirus sequences suspected of being contaminants due to PCR validation failure in the original samples. Phylogenetic analysis of the Rep proteins showed they clustered in distinct locations across the es class . The onloviridae . Gastroioviridae , though Decades of research have uncovered numerous mycoviruses, with the bulk of sampling effort directed toward industrially relevant hosts, such as plant-pathogenic fungi or edible mushrooms , 40. LarAspergillus fumigatus). In five cases, the closest mycovirus relatives were identified in phytopathogenic fungi, one was found in an endophytic species, one in an entomopathogenic fungus, and in two cases the relatives were from uncertain hosts. This is likely partly due to low sampling effort toward human pathogens as mentioned above; however, it probably also reflects the fact that human-pathogenic fungi are phylogenetically nested within nonpathogenic lineages across the fungal radiation  has been observed to naturally infect the phytopathogenic fungus Rhizoctonia solani, which can in turn transmit CMV to uninfected plants under laboratory conditions , and we were thus confident they represented true mycoviruses and not contamination. The notable exception was ALJV1, which represents the first unambiguous detection of a jivivirus in a fungus. Previous identifications of the recently named jiviviruses  have beeapevines . Besidest plants , 45. Intt plants , and cucnditions . Such crnditions , hintingt plants . Cross-kt plants .We also detected ssDNA viruses in RCA libraries, 12 with complete genomes. We universally failed to validate these by PCR, both in the original unamplified samples and after repeating RCA. Recently, more attention has been given to the detection of viral contaminants in sequencing libraries, since they can easily result in incorrect assessments of virus–host relationships , 50. HerMucorales species) were included later and used only in PCR screening.Bronchial aspirates, bronchoalveolar lavage fluid, bone marrow, and biopsy specimens sent in for culture of (dimorphic) fungi were inoculated on two containers of brain heart infusion agar with penicillin and gentamicin. One container was incubated for 3 weeks at 20 to 25°C, and the other at 35 to 37°C. Nails, hair, and skin scrapings were inoculated on dermatophyte test medium agar and Sabouraud agar with gentamicin and chloramphenicol (SabGC). Incubation was for 3 weeks at 25 to 28°C, with one container of SabGC incubated for 3 weeks at 35 to 37°C. All other materials sent in for fungal culture were inoculated on two SabGC containers for 1 week, one at 25 to 28°C and one at 35 to 37°C. Fungal isolates included in this study were recultured from glycerol stocks, and samples were transferred to tubes containing Universal Transport Medium . A total of 75 isolates were included, split into two batches (Table S1). Batch one samples (61 diverse isolates) were utilized in virus discovery and PCR screening experiments, while batch two samples (14 isolates of g) to pellet solid matter and cellular debris. Supernatant was treated with 20 μL TURBO DNase  for 30 min at 37°C to remove naked DNA. Nucleic acids were extracted using the Boom method  was transferred to a reaction tube and centrifuged , and those containing visible misassemblies were discarded. Genome completeness was assessed by the presence of both Rep and Cap genes plus genome circularity . Circular overlap was trimmed from complete genomes, and they were rotated to begin with the Rep gene. For all RNA and DNA genomes, inspection was performed by mapping quality controlled reads to respective sequences using BWA MEM v0.7.17 . A 40-cycle first round was performed, with nested PCR carried out if this was negative, and PCR products were Sanger sequenced. For ssDNA viruses, only index samples were screened due to nondetection. Both the original unamplified samples and an aliquot with RCA repeated were screened. To explore the possibility that they represented contaminants of reagents, we also examined ssDNA virus read distribution across all RCA libraries. Since reagents were shared between samples, we expected that contaminants would be detected in all or most sequencing libraries. This was tested by mapping reads from all RCA libraries to the set of cressdnaviral sequences using BWA and examining their read distributions per sample, using 50 reads per million (RPM) as the cutoff for positive detection.https://www.ncbi.nlm.nih.gov/orffinder), though the yeast mitochondrial genetic code was applied for Rhizopus microsporus mitovirus 1 (LC671615), since its UGA codon encodes tryptophan rather than a translation termination signal (http://imed.med.ucm.es/Tools/sias.html). For cressdnaviruses, Rep protein sequences were aligned alongside a database covering recognized and proposed lineages collated for a previously published phylogeny  or polyprotein sequences were gathered using the International Committee on Taxonomy of Viruses (ICTV) website and GenBank, and alignment was performed using MAFFT v7.490  with then signal . Maximumn signal  with authylogeny , now wits 1 LC6715, since oviridae . PhylogeLC671611–LC671637. Raw Illumina reads are available under European Nucleotide Archive project accession PRJEB49942. Alignments, tree files, and one hybrid sequence are available from https://figshare.com/projects/Viruses_infecting_clinical_mycology_cultures/128186.Assembled genomes are available from INSDC databases under accessions


Varroa destructor is an ectoparasitic mite associated with significant losses of honeybee colonies globally. The mite vectors a range of pathogenic viruses, the most important of which is the Deformed wing virus (DWV). In the absence of Varroa, DWV exists as a low-level, highly diverse virus population. However, when transmitted by Varroa, certain variants become highly elevated, and may become near-clonal and cause symptomatic infections. Mite transmission between colonies can occur when parasitised workers drift from or rob adjacent hives. These activities can result in elevated mite levels, but the resulting change in the DWV population, the primary determinant of winter colony losses, has not been determined. In reciprocal studies, we investigated the influence of the removal of mites, or their acquisition, on the DWV population. When mites were removed from heavily infested colonies, there was a striking and rapid reduction in virus load. Conversely, siting Varroa-naïve colonies in a mite-infested apiary resulted in the acquisition of mites and concomitant changes in the virus population. We observed both near-clonal and highly divergent virus populations regardless of titre, suggesting changes were stochastic and colony-specific. Our findings have implications for the outcome of strategies in areas with total or patchy implementation of Varroa control plans. Varroa destructor was originally a parasite of the Asian honeybee (Apis cerana), before jumping hosts to infest European honeybee (Apis mellifera) colonies about a century ago. Since then, Varroa has become near-globally distributed with the movement of managed hives [6 genome copies/bee, levels which rarely, if ever, cause symptomatic disease [Varroa drastically alters the dynamics of infection [Varroa feed on developing pupae, causing a dramatic increase in viral load and symptomatic infections , and inpies/bee ,9,10. Coter bees .Varroa infestations significantly reduces the incidence of DWV-mediated pathogenesis and markedly reduces colony losses. However, the remaining Varroa reproduce, necessitating annual, or more frequent, mite management. In addition, mite levels may be further amplified through the acquisition of ‘phoretic’ mites from workers drifting during orientation flights, or by the activities of foragers robbing nearby collapsing (mite-infested) colonies [Varroa infestation show an increased acceptance of drifting workers, allowing uptake of other associated pests and pathogens to occur [Controlling colonies ,13. An acolonies , and thicolonies . Acceptacolonies . Additioto occur .Effective miticide treatments exist, and the use of tau-fluvalinate strips to treat mite-infested colonies with high DWV resulted in a 1000-fold virus reduction compared with neighbouring, untreated colonies . HoweverVarroa infestations have been reported to be associated with a reduction in DWV diversity [Varroa alter the virus transmission route, something which can be replicated by injection [Varroa is not a binary event and that other factors can influence the DWV population dynamic [In addition to significant increases in viral load, iversity ,6. Low-lnjection ,20, sugg dynamic .Varroa. Using an engineered recombinant DWV, Gusachenko et al. (2020) demonstrated reporter gene expression, and hence, virus replication in mites fed virus using an artificial feed packet system. Other studies have suggested that DWV Type-A does not replicate in mites and is instead vectored in a non-propagative manner [DWV exists as two frequently reported variants, DWV Type-A  and DWV e manner .Varroa and measured subsequent changes in the DWV population in the colonies to determine improvements in colony health. A ‘shook swarm’ is a standard beekeeping technique that separates the adult workers and the queen from the brood, comb, and stores. It is used to encourage the colony to draw fresh comb and may help eliminate brood diseases such as European foulbrood [It has been shown that symptomatic DWV infections, which cause over-wintering colony losses, can persist after mite removal. Additionally, it was found that low mite levels may not translate to low DWV levels, meaning some colonies could still collapse over winter after mite removal . To inveoulbrood . We reasoulbrood , a shookVarroa levels in treated colonies. More importantly, within one brood cycle, the residual DWV level was reduced to a very low level and remained low for the remainder of the season. In reciprocal studies, we demonstrated that healthy Varroa-naïve colonies become rapidly infested when placed near mite-infested colonies, resulting in changes to the virus population and high colony losses. We observed mixed populations of high-titre DWV infections in symptomatically infected workers, indicating that not all symptomatic infections are caused by clonal viral populations. Despite differences in DWV levels between seasons, the colonies invariably died after a period of high DWV infections and no Varroa treatment interventions. These studies demonstrate a novel method of significantly reducing mite and virus loads rapidly to improve colony health. In parallel, we demonstrate the speed with which Varroa are acquired and how their introduction alters DWV levels in infested colonies. These studies highlight the importance of using timely and effective interventions to improve or maintain honeybee colony health and can be implemented using standard beekeeping methods.We show that this treatment combination significantly reduces The emerging brood was sampled from each of ~12 colonies and analysed by qPCR to determine which colonies in the infested apiary had the highest DWV levels and, therefore, presumably high mite levels. Those with the most consistently elevated DWV yields were selected for the experiment (data not shown).The shook swarm process was conducted after locating and caging the queen for safety. The hive was moved to an adjacent stand and replaced with a brood box containing frames with foundation and two amitraz-impregnated Apivar strips . All bees from the original hive were shaken into the new brood box, which was sealed with the caged queen, and moved to a quarantine apiary approximately 130 km away. The queen was released the following day, and the colony fed one gallon of thin syrup to encourage the drawing of new comb. Apivar strips were removed after six weeks. The apiary housing the newly positioned colonies had very low mite levels and was monitored throughout the study. Mite counts were performed by recording the number of dead mites observed on the floor of each colony.Each year, four colonies were treated with an approved miticide in the University of St Andrews apiary in 2017 and 2019. The dead mites were counted for each hive after the treatments to establish infestation levels. A worker brood sample was taken to analyse the virus levels for each colony before they were transferred to the mite-infested apiary. The colonies were placed in the centre of the apiary, which contained between 15–18 colonies each year. No colonies in the infested apiary received any miticide treatments, although drone trapping was occasionally used to gather mites. Emerging worker brood was sampled approximately every brood cycle until the queens had stopped laying eggs for winter .Varroa were collected in 50 mL falcon tubes 24 h after the first treatment and frozen at −80 °C. Frozen samples were homogenized using the Precellys Evolution tissue homogenizer  for 3 × 15 s at 10,000 rpm with 20 s pauses between each pulse. RNA extractions from whole individual bees were performed using the GeneJET RNA purification kit  as per the manufacturer’s instructions. Total RNA from individual workers was quantified and quality assessed based on spectra cleanliness and a 260/230 ratio using the Nanodrop-1000 and stored at −80 °C. Total cDNA was synthesized from 1μg RNA per sample using the qScript cDNA synthesis kit  following the manufacturer’s protocol using oligo(dT) and random primers.All emerging brood samples were snap-frozen and stored at −80 °C. For the Shook Swarm experiment, dead 10–103 GE/μg.In measuring absolute viral load, quantitative polymerase chain reactions (qPCRs) were performed for DWV in a Bio-Rad CFX96 Touch Real-Time PCR Detection System . Reactions consisted of 1 × Luna Universal qRT-PCR master mix , 0.25 µM forward and reverse primers, and 100 ng of cDNA in a final volume of 20 µL. The DWV forward primer was 5′-ATATAGGTTCGGCTGGATCTCC-3′, and the reverse primer was 5′-TTCCAGATGCACCACACATGC-3′, amplifying a region of 150 bp in the helicase. Amplification was performed using the following thermal profile: 1 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, and 30 s at 60 °C. Post amplification melting curve analysis was used to check for non-specific amplification (60–95 °C increasing 0.5 °C every 5 s). Negative template controls and a serial dilution of a positive control standard were included in each run. DWV genome equivalents were calculated from the standard curve generated from a serial dilution of a cDNA clone control obtained from 1µg of DWV VVD RNA transcript as per , with a DWV and actin end-point PCR assays were carried out in 25 µL reactions containing 10 × PCR buffer, 0.2 mM dNTPs, 0.4 µM forward and reverse primer, 2U Taq DNA polymerase  and ~100 ng of cDNA template. The DWV forward primer was 5′-CAGTAGCTTGGGCGATTGTTTCG-3′, and the reverse primer was 5′-CGCACTTAACACACGCAAATTATC-3′, amplifying a region of 1510 bp in the helicase. The actin PCR assay was performed as per . An initLarge DWV Amplicons (>10 KB) were purified, and each sample was barcoded (NCBI BioProject: PRJNA811438). Illumina Hi-seq (2 × 300 bp) paired-end reads were processed from these amplicons at the University of St Andrews. Illumina reads were then extracted and trimmed using Geneious Prime (v.2019.1.3) and extracted as a single fasta file for each honeybee sample. Haplotype diversity was calculated using ShoRAH (Short read assembly into haplotypes) with all settings run as default  and all p-values < 0.05 in the final analysis of SNVs. Based on this method, combined with the probabilistic clustering method implemented by ShoRAH, SNVs can be called from deep sequenced viral populations with high accuracy [Single nucleotide variants (SNVs) were called using statistical modelling at a frequency lower than the error rate of sequencing. ShoRAH analysis includes a statistical test of strand bias by utilising a Fisher’s exact test and a Benjamini-Hochberg correction process, which rejects any accuracy . We utilnlme package in R [Varroa-infested apiary. First, we used a linear model with the DWV level as a response variable and sampling month as the independent variable. Second, we created a mixed effect model by extending the linear model with the colony as a random effect. Model fit was compared using the Akaike Information Criterion (AIC) and 95% confidence intervals of the estimates calculated using the nlme: intervals function.All models were implemented using the age in R ,37,38. WVarroa control was not used. The queen was caged, and workers and nurse bees were shaken into a new hive with foundation only, containing two strips of amitraz-containing miticide. The queen was returned to the colony, which was relocated to a quarantine apiary and fed ad libidum with sugar syrup to encourage the production of a fresh comb.In May 2018 and 2019, we conducted shook swarms on a total of six colonies of equal strength (based on the number of brood frames and colony size) and mite burden (three in each season) from a mite-infested apiary in which The mite drop was recorded 24 h post-treatment and periodically over subsequent weeks until the colony stopped rearing brood in October. In both seasons, the mite drop immediately after treatment was high but rapidly decreased and then remained low for the remainder of the season A. The miVarroa load in the screened colonies, being higher in 2019 than in 2018, with an average virus level of >106 genome equivalents (GE)/μg RNA observed in all colonies. In 2018 the DWV level ranged from 7 × 105–1 × 109 GE/μg RNA across the three colonies, but in 2019 the range was 2 × 105–4 × 1010 GE/μg RNA. Post-treatment, no emerging brood showed DWV disease symptoms, and average DWV levels in the samples analysed were all <106 GE/μg RNA for the remainder of the season during which brood was present. Prior to conducting the shook swarm experiments, a screen of colony DWV levels was carried out using qRT-PCR analysis of 12 emerging brood samples per colony in the infested apiary. The DWV copy numbers were broadly reflective of the Both the simple linear models and the mixed models indicated a significant decrease in the log levels of DWV in sampled bees over time in 2018 and 2019 B. The loVarroa clearance and the consequent marked reduction in DWV levels (Varroa for each colony collected 24 h after treatment), June (TP2), and September (TP5) of 2019 were prepared and sequenced, with the resulting Illumina reads analysed using ShoRAH  [Honeybees with low DWV levels have previously been shown to have divergent populations of DWV ,6. To deV levels B, sampleplotype) . ShoRAH plotype) ,24,39.Varroa sample. In colony 19-S2, the virus population at TP1 is also Type B only, but the pooled mite samples for 19-S2 and 19-S3 contained mixed populations of A and B variants. The TP2 workers for 19-S2 contained a mix of Type A and B variants. However, the population was still predominantly (>80%) Type B. Different regions of the genome aligned with different variants in colony 19-S2 and 19-S3, with the RdRp containing Type A variants, but other regions containing only Type B, suggesting viral recombinants are potentially present. By TP5, very little diversity was observed at an individual level in the samples from all three colonies.The haplotypes were assigned to ‘clusters’ based on the branches they formed during the phylogenetic analysis. These clusters were subsequently compiled as bar charts showing the percentage composition of each variant to visualise changes in diversity over time A. Colonyp-values of each SNV were used to generate plots for each time point  caller . This meme point B. The laVarroa levels in an apiary already containing highly infested colonies, in which the mite population was unmanaged, to determine what changes occur in the virus population of Varroa-naïve colonies post-Varroa infestation. Prior to the movement, the introduced ‘naïve’ colonies were treated with a miticide, and DWV levels were quantified. Only colonies with consistently low DWV (<106 GE/bee) by qPCR analysis, no symptomatic workers, and very low mite levels  were used as test colonies in this part of the study.We installed colonies with very low In two seasons, 2017 and 2019, colonies were placed within 10 metres of 15–18 mite infested colonies and the brood was sampled every 21–28 days. All changes in colony health were measured through changes in DWV level and sequence diversity. Additionally, qPCR was used to quantify the DWV in the emerging brood from each of the test colonies and from one infested neighbouring (IN) colony at the end of each season.4 GE/μg RNA in July increased 100,000-fold to 5 × 109 GE/μg RNA by October  colony were sequenced from the four time points in year one (2017). Haplotypes were assigned to clusters, as described above, and compiled into bar plots to visualise changes over time   . The oneAs per Additionally, Sanger sequencing was used to examine the virus population in the year one samples (2017) . The domApis mellifera) colony losses, which can exceed 30% of all managed colonies [Varroa destructor and the consequent transmission of Deformed wing virus (DWV). Varroa feeding results in direct systemic transmission of DWV, an endemic virus that is usually spread horizontally in the colony during larval feeding and trophallaxis [Varroa-transmitted virus replicates to significantly elevated levels, causing overt disease in recipient pupae, and may exhibit a marked reduction in DWV population diversity [The majority of overwintering honeybee  or heather.Beekeeping in the UK is unregulated, and consequently, colony management techniques and ntrolled ,13. To cVarroa-naïve colonies, when co-located with mite-infested colonies, rapidly exhibit elevated DWV levels and changes in their virus population as a consequence of acquiring mites from nearby colonies. In two repeated seasons, the introduced colonies either died within 14 months or exhibited the very high virus levels associated with symptomatic disease and overwintering colony losses  and mixed populations of Type A and B variants of DWV . In addition, two-thirds of the workers analysed contained a single dominant DWV variant that had not exceeded the 3% detection threshold in the initial colonies , and they differed in the titre of this dominant virus, which varied from ~107-1011 GE/μg RNA (Varroa) may result in markedly different outcomes. However, the dominance of cluster-21 by the final time points in colony 1-1 is similar to one sample from the infested neighbouring colony data .The SNV analysis B indicatVarroa-infested) brood, coupled with miticide application would achieve the maximum impact on the mite population and enable us to investigate the post-treatment consequences for the virus population. The latter is an important consideration; if virus levels remain high, colony survival may still be threatened, as observed by Highfield et al., 2009, where some colonies with low mites had high DWV and died over winter. Furthermore, such colonies may be particularly susceptible to damaging levels of virus transmission should they become re-infested with Varroa following environmental exposure, as observed by [Unmanaged mite populations can rapidly expand and threaten colony health. Mite daily reproductive rates are reported as 0.017–0.021, and the population doubling time has been estimated at ~33–41 days, meaning a population of ~100 mites in spring could reach >3000 mites by autumn assuming uninterrupted brood rearing . To comberved by .Varroa levels counted on the trays underneath the colonies declined rapidly to <100 mites total within five days and <10 mites total within nine days . Like the infested colonies , this suAs not all the mid-season brood were sequenced, when this virus population shift took place is unclear. However, it likely occurred during feeding/trophallaxis, where a high threshold for virus transmission to larvae meant only a subset of a low-level DWV population was transmitted. In turn, variants were lost from generation to generation. This high transmission threshold supports the findings that the highly infected adults did not transmit high virus numbers to the first brood samples post-treatment. The sample size in both experiments was relatively small and may only represent a snapshot of the true virus population changes in these colonies. However, these changes could still be reflective of stochastic changes occurring at the colony level.Recent reports, based on field study sample analysis, have suggested that DWV Type B may be increasing in prevalence and, in some cases, superseding Type A variants. Based on the sequencing analysis from the infested colonies , the proVarroa infestation on the DWV load and population at an individual and colony level when they are left untreated and the consequences of this on the colony’s health. Analysis of viruses following the placing of Varroa-naïve colonies in a mite-infested environment shows the dramatic influence infesting mites have on both the level and the composition of the virus population. This is further emphasised when we investigated what happens to the virus population after the targeted removal of Varroa by a colony management intervention that should remove up to 90% of Varroa coupled with application of an approved miticide. This combination effectively and rapidly reduced Varroa and DWV levels to those associated with a ‘healthy’ colony within a single brood cycle, and no symptoms or large increases in virus levels were observed for the rest of the study. These experiments, while providing insights into dynamic virus population changes, further emphasize the importance of good beekeeping practices and mite management techniques to improve and maintain honeybee colony health.The results of this study clearly demonstrate the rapid impact of


This study aimed to develop improved multi-component beads with controlled, sustained delivery of antibiotics, such as gentamicin (GM). Antibiotic-loaded beads with rapid-release and the sustained-release system can be used for bone restoration. Single and multi-component beads were prepared by gelation using various combinations of chitosan and calcium chloride as cationic components and alginate and citric acid as anions. GM release was also controlled by crosslinking using citric acid. The optimum beads were obtained using 5% or 2% sodium alginate, 3% chitosan, and 0.1 mol/L citric acid. The beads were characterized by FTIR, TG-DTG, swelling behavior, and SEM. All GM-loaded beads revealed good antimicrobial activity. The rate and kinetics of release in the phosphate buffer solution were controlled by changing the amount of chitosan in the calcium chloride solution and using citric acid as the crosslinking agent. Crosslinked beads were prepared for the release of about 80% of the loaded drug within 24 h. The study concluded that the chitosan-alginate beads provided faster GM release but crosslinking with citric acid was efficient for sustained-release beads containing gentamicin. Drug delivery systems frequently use biodegradable, biocompatible, and natural biopolymers to control the kinetics of drug release . Chitosaand C2b) . QuantitRecently, polyelectrolyte complexes have been used for designing drug delivery systems ,17,18,19Sionkowska et al. reported that gentamicin release from complexes of chitosan and collagen could be controlled and localized by variations in the chitosan content ,23. Drug2 were used for alginate gelation [Daly and Knorr reported that macromolecular chitosan rapidly binds onto the surface of alginate droplets, but there was a limitation of diffusion from the inner core. Chitosan solution and CaClgelation  to increE. coli) and some strains of Gram-positive  bacteria; moreover, it shows cost-efficacy and stability in thermal properties. The surface morphology of the beads was characterized by scanning electron microscopy (SEM), and loss of weight, swelling behavior, and the release profile using UV/Vis spectrophotometer and LC-tandem mass spectrometer (LC/MS/MS) were determined.In this study, we prepared alginate-chitosan-gentamicin beads and alginate-gentamicin beads crosslinked with citric acid (CA) to develop a stable, non-toxic polymer complex to control drug release properties. CA can form crosslinked beads by ionic interaction with positively charged Ca-alginate. Gentamicin, a broad-spectrum antibiotic used in this study, has effective antibacterial activity against many strains of Gram-negative , calcium chloride dihydrate, gentamicin sulfate (GM), citric acid (CA), and chitosan  were obtained from Sigma-Aldrich . Phosphate buffer saline (PBS) was purchased from Gibco . Acetonitrile and trifluoroacetic acid were purchased from Biosolve , and formic acid was obtained from Merck .w/v) was used as dope. Chitosan  was dissolved in 2 % (v/v) acetic acid at 60 °C and named 3C. Homogenous aqueous solutions of 3C and calcium chloride dihydrate  with different chitosan solution content were used as coagulation solutions. The mixing of the solution was performed for 2 h. Alginate solution (50 mL) was added dropwise through a 21 G needle into the coagulation solution (200 mL) while stirring at 150 rpm. The obtained spherical beads remained for 6 h in the coagulation solution with stirring. Then, beads were filtered, washed ten times with distilled water, and freeze-dried for 24 h. The beads were prepared by two methods, as shown in 2·2H2O solution (named 5A). For preparing the 5A/GMCa beads, 50 mL of 5% sodium alginate solution was dropped into 200 mL of 0.1 N CaCl2·2H2O solution mixed with 0.05 g of gentamicin powder, and 5A/GMCa/CA was prepared from 5A/GMCa beads by 5 h post-crosslinking treatment in citric acid. The prepared beads were washed 10 times with distilled water. Alginate-gentamicin (5A/GMCa) conjugates and alginate-chitosan-gentamicin (2A/x%3CGMCa) beads were prepared by gelation . A 50 mL2·2H2O solution to make 2A/0.1%3C (including 0.1 wt.% of 3% chitosan solution), 2A/0.5%3C (including 0.5 wt.% of 3% chitosan solution), and 2A/1.0%3C (including 1.0 wt.% of 3% chitosan solution). Likewise, alginate-chitosan beads loaded with gentamicin (0.05 g) were named 2A/0.1%3CGMCa, 2A/0.5%3CGMCa, and 2A/1.0%3CGMCa. The alginate-chitosan beads were rinsed with distilled water, filtrated, and then freeze-dried for 24 h.Alginate-chitosan beads were produced by dropping 2% sodium alginate into 3% chitosan solutions of different chitosan contents in 200 mL of 0.1 N CaCl−1 range with a resolution of 1 cm−1. The thermal properties of the sodium alginate, gentamicin sulfate, alginate-chitosan-gentamicin (2A/x%3CGMCa), and alginate-gentamicin (5A/GMCa) beads were examined by thermogravimetric analysis  in the range from 30 °C to 800 °C with a heating rate of 5 °C/min in nitrogen atmosphere (flow rate of 40 mL/min).The surface morphologies of the beads were studied using a field-emission scanning electron microscope . The beads size distribution was calculated using an optical analyzer . The FTIR spectra for the beads were obtained using an FTIR spectrometer equipped with an attenuated total reflection module  in a 500–4000 cmFirst, 10 mg of beads were placed in 6 wells containing 2 mL of PBS solution at 37 °C with 150 rpm shaking. For measuring the swelling ratio, the beads were immersed in PBS solution. The wet weight of the swollen beads was measured at periodic intervals immediately after removing excess water with filter paper. The swelling ratio was calculated by the following Formula (1) :(1)SR , in which The PBS content of beads after being soaked in PBS for 24 h was calculated by the following Formula (3):The experiments were performed in triplicate.Escherichia coli (E. coli) obtained from Korean Collection for Type cultures  and Staphylococcus mutans (S. mutans) from the American Type Culture Collection . Briefly, 17 mg of beads were transferred into a transwell, and the beads were immersed in each well of 24 plates containing 1 mL of E. coli-inoculated PBS (1 × 105/mL). This plate was incubated for 24 h at 37 °C. Then, aliquots of 100 uL were collected from each supernatant, plated on nutrient agar plates, and incubated for 24 h at 37 °C. After 24 h, colony formation was examined to determine antibacterial activity. Meanwhile, 5% alginate (5A) beads and alginate-chitosan beads without gentamicin-loading (2A/x%3C) were used as the control. Antibacterial activity was also performed for S. mutans plated on BHI agar plates using the same procedure. All experiments were performed in triplicate.The antibacterial activity of gentamicin-loaded beads was evaluated following the viable-cell count method . The teso-phthal aldehyde solution, 1% (w/v) o-phthal aldehyde solution was prepared by dissolving in 5 mL of methanol. Then, 20 μL of 1% o-phthal aldehyde solution was mixed in 2 mL of released GM solution. The released GM solution was examined using a UV/Vis spectrophotometer at a wavelength of 230 nm [To study the in vitro release of GM from alginate beads and alginate-chitosan beads, the beads were soaked in PBS and incubated at 37 °C. At various time points, 1 mL of extract was taken to measure the amount of released GM. After each measurement point, the wells were refilled with fresh PBS solution. To measure the spectrophotometric value after color reaction with an f 230 nm . ® HSS T3 column , a column temperature of 40 °C, a flow rate of 0.35 mL/min, an autosampler temperature of 15 °C, and an injection volume of 1 μL. The mobile phase was composed of 0.1% (v/v) aqueous trifluoroacetic acid  and 100% acetonitrile with 0.1% formic acid (solvent B), using the following gradient elution program: 100% solvent A maintained for 0 to 6 min; solvent B increased linearly to 100% for 6 to 10 min; 100% solvent B held for 10 to 14 min; solvent A immediately returned to 100% and re-equilibrated for 15 to 20 min. Tandem MS (MS/MS) data were obtained from ionization in the electrospray ionization (ESI) positive mode, a capillary voltage of 2.5 kV, source temperature of 130 °C, desolvation temperature of 250 °C, desolvation gas flow of 600 Lh−1, cone gas flow rate of 50 Lh−1, cone voltage of 40 V, and multiple reaction monitoring (MRM) transitions as m/z 478.3 → 322.2/157.3 for Gentamicin C1, m/z 450.3 → 322.2/160.3 for Gentamicin C1a, and m/z 464.3 → 322.2/160.3 for Gentamicin C2, C2a, and C2b.The total amount of released gentamicin was evaluated with an LC/MS/MS. An ACQTTY ultra-performance liquid chromatography (UPLC) H-Class system , coupled to a quadrupole time-of-flight tandem mass spectrometer , was used. The separation was performed on a UPLC with an ACQTTY UPLC−1 overlapped by N–H stretching of chitosan. The C–H stretching peaks of sodium alginate and chitosan at 2935 cm−1 are shown. The absorption peak of C=O of sodium alginate and amide I band of chitosan appeared at 1630 cm−1. The amide II band, due to N–H bending, appeared at 1640 cm−1, which is overlapped by the amide I band. The carboxyl group of sodium alginate shows another C–O absorption at 1430 cm−1. The N–H bending vibration peak was observed at 832 cm−1. FT-IR spectra of alginate-chitosan beads confirmed a bipolymeric formation. −1 could be assigned as the amide I, amide II, and amide III bonds of gentamicin, respectively. The peak observed at 1108 cm−1 was due to the HSO4−1 group. The peak observed at 617 cm−1 was due to the SO2 band. The peak of gentamicin at 1541 cm−1 was present with low intensity, suggesting successful loading of gentamicin into alginate beads.In the FTIR spectra of gentamicin sulfate b, the tyThe TGA curves are shown in S. mutans and E. coli. Chitosan has been widely used as antibacterial material owing to its positively charged moiety. However, the chitosan-alginate beads exhibited no bacteria inhibition capacity, as illustrated in S. mutans and gram-negative E. coli. The neat alginate  did not exhibit any antibacterial killing ability against both tested bacteria, S. mutans and E. coli. All gentamicin-loaded beads  showed suitable antibacterial activity against E. coli and S. mutans. 2, and -COOH groups of alginate or alginate-chitosan beads.2 and -OH groups compared to GM-loaded alginate beads and crosslinked 5A/GMCa beads. Especially, 5A/GMCa/CA showed the lowest SR due to a strong chemical network formed after crosslinking.The swelling ratios (SR) of the 2A/x%3C and 2A/x%3CGMCa beads in 150 min are presented in 2 and -OH groups in the structure. The electrostatic interactions between the alginate and GM delay the release of GM from alginate-GM beads. The release rate of GM was slow, and 90% of GM was released after 9 h. The release of GM from 5A/GMCa beads was higher than that from the 5A/GMCa/CA because of the strong crosslinking function of citric acid between alginate and GM.GM release from the swollen beads was induced by the interaction between PBS and gentamicin in the polymer in p > 0.05). The 2A/0.1%3CGMCa beads had the highest GM release profile. These results demonstrate that the GM release is attributed to the difference in surface roughness and chemical structure of chitosan, alginate, and GM conjugates.Liquid chromatography-mass spectrometry (LC-MS) was very efficient for identifying and quantifying GM release from the beads. Using the standard concentrations of 3.125, 6.25, 12.5, 25, 40, 100, 250, and 500 mg/L, the concentration of GM in extract solution after 0 to 24 h was quantified. As shown in Gentamicin was successfully loaded into the alginate and alginate-chitosan beads. The surface irregularity and size of the beads varied according to the chitosan content. All the GM-loaded alginate-chitosan and alginate beads had obvious antibacterial activity. Alginate-chitosan beads loaded with GM reached about 95% GM release within 3 h, while alginate beads loaded with GM reached about 85% of GM release after 9 h. The amount of GM release was less for crosslinked alginate-GM beads than for uncross-linked beads. The release time and release rate of GM could be manipulated by the chemical structure modulation between alginate, chitosan, gentamicin, and citric acid and the difference in surface roughness and degree of swelling. Controlled gentamicin release through biopolymer conjugation is expected for local disinfection reducing the side effects of systemic antibiotics delivery.


Viral disease in potatoes has been a major problem in potato production worldwide. In addition to the potential risk of introducing new diseases in new areas, viral-disease epidemics/pandemics can be initiated by “spillover” of indigenous viruses from infected alternative hosts into introduced cultivars. To investigate the tendency of potential viral infection/resistance, we analyzed the viromes of introduced and indigenous varieties of potatoes among different tissues using RNA-seq libraries. Bioinformatics analyses revealed that potato viruses PVM, PVY, and PVS were dominant and the most frequently identified viruses infecting potato virus-free plants in the field, and showed an infection bias between introduced and indigenous cultivars. PVY and PVS were the major viruses in introduced varieties, whereas PVM showed an extraordinarily high percentage in the indigenous variety. Other three common viruses, PVH, potato mop-top virus, and potato leafroll virus were identified specifically in the indigenous variety. There was a tendency for tissue-specific infection and sequence variation in viruses: underground parts  harbored more unusual viruses, and tubers harbored relatively more variation with a high frequency of single nucleotide polymorphisms than other tissues. Taken together, our study provides a comprehensive overview of the composition, distribution, and sequence variation of viruses between introduced and indigenous varieties of potatoes. More than 100 countries cultivate potatoes. It is an essential staple food and vegetable crop and ranked fourth in production after rice, wheat, and maize . China hSpecial interest has arisen from the study of potato-virus interactions that occur with landraces or introduced species worldwide. Potatoes were first brought from Andean crop domestication center to Europe in the 16th century, after which the crop was taken to other continents . UnknowiStudying the exchange of viruses between native landraces and introduced cultivars is a neglected area that has the potential to provide information on threats to introduction cultivation and biodiversity, as well as virus mutation and evolution. Liangshan Yi autonomous prefecture, a remote, impoverished, and mountainous area in southern China, has been planting potatoes for more than 100 years and nurtured many unique potato landraces because of its unique natural environment . What exIn recent years, various detection approaches have been established for identification of plant viruses (including potato viruses) . In addiHere we identified potato viromes using total RNA-seq. Twenty-two libraries were prepared from the leaves, stems, roots, stolons, and tubers collected from five cultivars in the native landrace of Liangshan and introduced varieties from Russia, of which potato plants from virus-free tissue-culture breeding systems re-infected and co-infected by indigenous viruses after transplantation to the field. We wished to address the impact of indigenous viruses on native and introduced potato germplasm through comparing the viromes of different varieties among different tissues, and characterize the host-specific viral communities in a tissue-level atlas. Comprehensive bioinformatics analyses revealed the complexity and unique viral communities associated with various factors, which will lay the foundations of virus disease-control strategies for future production of introduced cultivated potatoes.We collected totally five potato varieties: four introduced cultivars from Russia named “Rosa,” “Nevsky,” “Talovskij,” and “Pioneer,” respectively, and one native landrace “S24” from Liangshan Yi autonomous prefecture, the southwest mountainous area in China. The germplasms introduced from Russia were obtained from the China National Potato Improvement Center . They passed rigorous quarantine and were in the form of virus-free tissue-cultured small plants. Similarly, virus-free plants of “S24” were obtained by culture of shoot tips. The original potatoes grown from these virus-free plantlets in the substrate pot were planted in the field, and the recovery of plants from virus infection among different varieties had been detected. In the harvesting period, we collected five tissues involving in leaves, stems, stolons, roots, tubers from each cultivar. Unexpectedly, one of the cultivars, Pioneer, did not develop tubers, and the stolons and roots of Talovskij were rotted upon sampling. As a result, 22 samples were used for RNA extraction.Fresh tissues from each sample were ground in Hanks’ buffered salt solution (1:10) with four ceramic beads using a tissue homogenizer . Then, total RNA was extracted using the RNeasy Plant Mini Kit  in accordance with manufacturer instructions. The quantity of extracted RNAs was detected using a spectrophotometer  and the quality was measured by gel electrophoresis. For library construction of RNA sequences, the NEBNext Ultra RNA Library Prep Kit for an Illumina platform was used according to manufacturer instructions . Transcriptome sequencing was done on a HiSeq 2500 system (Illumina).1  using phyML 3.09 , with pa9 . The genTo identify the mutation positions of identified viruses, we detected SNPs for the identified viral genomes among different cultivars and tissues. The assembled genome sequences of six virus PVS, PVY, PVM, PMTV, PLRV, and PVH were used as reference virus genome sequences. We carried out SNP analysis as described previously . In brieTo quantify the virus-related RNAs associated with identified viruses in each library, we calculated the number of sequenced reads associated with respective viruses contigs via MEGABLAST search . At the For the confirmation of the viruses obtained by the NGS analysis and for the future application of the virome results, we conducted RT-PCR for three viruses PVS, PVY and PVM with specific primers . Total RPRJNA760655.The raw data of RNA-seq in this study have been deposited in the NCBI database under BioProject number We wished to identify viral populations inhabiting potato varieties from different germplasm sources and the established viromes of multiple tissues. Introduced and indigenous potato plants were grown in the same field. Potatoes showing disease symptoms were sampled. Finally, we collected the leaves, stems, roots, stolons, and tubers from four cultivars introduced from Russia  and one native landrace (“S24”) from Liangshan. Samples were pooled based on the varieties and tissues. Twenty-two libraries for RNA-seq were prepared and paired-end sequenced by the HiSeq150 system . Three libraries  were absent due to a failure of tissue sampling. The raw sequence data for RNA-seq are listed in de novo using Trinity11 with the assembled contigs (transcripts) ranging from 98,039 to 144,588 and contig N50 ranging from 1,584 to 1,862  was the dominant virus, followed by PVY (304 contigs) and PVS (206 contigs) . In consNext, we examined the number of samples for each individual identified virus. Among 22 libraries, the most frequently identified virus was PVY , followed by PVM and PVS (both of which were present in 18 libraries). According to virus-associated contigs, the virus composition was essentially identical among different cultivars, and PVY, PVS, and PVM were dominant. However, the proportion of viruses showed significant differences between introduced and indigenous varieties . PVY wasWith regard to virus distribution among tissues, the three dominant viruses  comprised the whole viromes of leaves, stems, and stolons. PVM was the dominant virus in stems (80.4%) and PVY was the major virus in stolons (67.85%). The proportions of the three viruses in leaves were distributed relatively evenly, with PVS reaching 41.85%. In addition to PVY, PVS, and PVM, some unusual viruses, such as PLRV, papaya ringspot virus (PRSV) and leek yellow stripe virus (LYSV), were identified in roots. As expected, tubers harbored more unusual viruses, in which PLRV, tobacco necrosis virus A (TNVA), PMTV, and PVH were identified, although the levels were very low .To assemble the complete (or nearly-complete) genomes of identified viruses, virus-associated contigs were mapped on each reference viral genome. Contigs associated with PVM, PVY, PLRV, PMTV, PVS, and PVH covered nearly the complete genomes, whereas TNVA, PRSV, LYSV, and XMRV-1 were partially mapped by the virus-associated contigs .Representative viral sequences of PVS, PVM, and PVY were then examined for phylogenetic anomalies using the full suite of options in RDP. Nine PVS sequences gave significant evidence of recombination. PVM had rarely recombinants and only one recombinant sequence was found. PVY exists as a complex of strains, including a growing number of recombinants. We identified twelve PVY sequences in RDP analyses which were recorded as significant recombinants . After rTo determine virus variation among different cultivars and tissues, we mapped raw sequence reads on the six viral genomes in which nearly complete genomes were assembled from our study. Simultaneously, raw sequence reads from each transcriptome were aligned to the assembled viral genomes for Single-Nucleotide Polymorphism (SNP) calling.First, we examined the mapping patterns of sequence reads on an individual virus. The whole regions of most viral genomes were mapped by reads without gaps. Interestingly, for most viruses identified from tubers, the number of mapped reads increased from the 5’ region to the 3’ region of the viral genome . Next, wTo identify co-infections of viruses among different cultivars and tissues, we built a matrix to calculate the correlation coefficient between different viruses through normalizing the number of reads related to different viruses in each library because the number of sequenced reads associated with identified viruses in each transcriptome differed. Interestingly, the leaves, stems, and tubers of Rosa and S24 accumulated many virus-associated reads (31.08–69.78%), whereas the proportion of other cultivars and tissues was relatively low (0.002–8.28%).In addition, we calculated the copy number  of each identified virus based on the number of virus-associated reads. The results for the copy number were similar to those for virus accumulation, and the infective patterns of different viruses were identified readily. PVM was enriched specifically in S24, whereas virus accumulation decreased by three orders of magnitude in the introduced varieties. PVM in S24 was co-infected among different tissues, of which the copy numbers ranged from 93,389 in roots to 253,408 in leaves. Cultivar-specific infection was more prominent for PVS because only Rosa had extremely high virus accumulation among all three introduced varieties, the copy number of which ranged from 179,764 in roots to 230,648 in leaves. Nevertheless, PVS co-infected S24 relatively mildly, with the copy numbers ranging from 643 in roots to 5,349 in leaves. However, PVY infection seemed to be tissue-specific instead of cultivar-specific because the virus co-infected almost all varieties but had only a high degree of accumulation in leaves and stems, therefore it did not infect a certain variety in a concentrated manner.Virome research has gradually become a hot spot in virus research and has been reported in a number of crops and fruits . Althougin vitro culture methods have been applied for production of virus-free plants  to north (Inner Mongolia) and from east (Zhejiang) to west (Tibet). Potato viral diseases, particularly PLRV, PVA, PVM, PVS, PVX, and PYV, are a limiting factor for sustainable production of potato in China because of the significant tuber yield losses they cause. Due to potatoes have been spread to Liangshan for more than 100 years, and there is not much communication with the outside due to traffic congestion, the evolution and mutation of local viruses are worthy of in-depth study. From the results of this study, the native landrece S24 was mainly infected by PVM, PVS, and PVY, and showed significant abundance differences among tissues. As reported by former researchers, the detection rates of PVX and PVA viruses were relatively high, reaching 17.74 and 16.41%, while the detection rates of PVY were below 10% in the main potato producing area of Sichuan Province, southwest China . HoweverThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/LY, YZ, and XL designed the research. XL, CW, and WZ prepared the plant sample. XL and HW performed the transcriptome sequencing, assembly, and annotation. XL and JL performed the virus identification and variation analysis. YZ and HW supervised the experiments and analysis and revised the manuscript. LY and XL wrote the initial draft of the manuscript. All authors read and approved the final manuscript.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.