Dengue and Zika viruses are closely related mosquitoborne flaviviruses with similar transmission cycles, distribution throughout the tropics and subtropics, and disease manifestations including fever, rash, myalgia, and arthralgia. For patients with suspected dengue or Zika virus disease, nucleic acid amplification tests (NAATs) are the preferred method of diagnosis. Immunoglobulin M (IgM) antibody testing can identify additional infections and remains an important tool for the diagnosis of these diseases, but interpreting the results is complicated by cross-reactivity, and determining the specific timing of infection can be difficult. These limitations are a particular challenge for pregnant women in determining whether Zika virus infection occurred during or before the pregnancy.This report summarizes existing and new guidance on dengue and Zika virus diagnostic testing for patients with a clinically compatible illness who live in or recently traveled to an area where there is risk for infection with both viruses. CDC recommendations for screening of asymptomatic pregnant women with possible Zika virus exposure are unchanged. For symptomatic nonpregnant persons, dengue and Zika virus NAATs should be performed on serum collected ≤7 days after symptom onset. Dengue and Zika virus IgM antibody testing should be performed on NAAT-negative serum specimens or serum collected >7 days after onset of symptoms. For symptomatic pregnant women, serum and urine specimens should be collected as soon as possible within 12 weeks of symptom onset for concurrent dengue and Zika virus NAATs and IgM antibody testing. Positive IgM antibody test results with negative NAAT results should be confirmed by neutralizing antibody tests when clinically or epidemiologically indicated, including for all pregnant women. Data on the epidemiology of viruses known to be circulating at the location of exposure and clinical findings should be considered when deciding which tests to perform and for interpreting results.Patients with clinically suspected dengue should receive appropriate management to monitor and treat shock and hemorrhage. Women with laboratory evidence of possible Zika virus infection during pregnancy and their infants should be evaluated and managed for possible adverse outcomes. Dengue and Zika virus disease are nationally notifiable conditions, and cases should be reported to public health authorities. Aedes mosquitoes in the tropics and subtropics, the recent emergence of Zika virus has complicated diagnostic testing considerations and interpretation. For patients with suspected dengue or Zika virus disease, nucleic acid amplification tests (NAATs) are the preferred method of diagnosis because they can provide confirmed evidence of infection and distinguish the specific virus  and Medline (n = 276) and references cited in relevant articles (n = 4). Unpublished data also were considered, including package inserts for products submitted to the U.S. Food and Drug Administration (FDA) for approval (n = 4) or Emergency Use Authorization (n = 19), and data derived from diagnostic testing performed at CDC   are quantitative assays that measure virus-specific neutralizing antibody titers for dengue, Zika, and other flaviviruses to which the patient might have been exposed  a NAAT for use on serum and whole blood, 2) an enzyme immunoassay that detects dengue virus NS1 antigen in serum, and 3) an enzyme immunoassay that detects dengue virus IgM antibodies in serum   against Zika and other flaviviruses is considered evidence of recent dengue virus infection . ConversNegative PRNT titers against dengue and Zika virus in a serum specimen collected >7 days after illness onset rule out infection with either virus . WithoutFor nonpregnant patients who live in or recently returned from an area where there is an ongoing outbreak in which only one virus has been detected to be circulating, jurisdictions might reasonably elect to only perform testing for the virus causing the outbreak and not confirm IgM results by PRNT. If the patient does not reside in and has not recently traveled to an area with circulation of both viruses, and the only potential epidemiologic exposure is sexual contact with a person who has recently traveled to those areas, then the patient should only be tested for Zika virus infection.Pregnant women with a clinically compatible illness and recent possible exposure to dengue and Zika virus should have concurrent diagnostic testing for dengue and Zika virus infection performed by NAAT and IgM antibody testing on a serum specimen and NAAT on a urine specimen to diagnose Zika virus infection . SpecimeIf both dengue and Zika virus NAATs are negative but either IgM antibody test is positive, confirmatory PRNTs should be performed against dengue, Zika, and other flaviviruses endemic to the region where exposure occurred. For indeterminate IgM antibody results, IgM antibody testing should be repeated on the same specimen or PRNTs performed. If IgM antibody results are positive for one virus but the assay for the other virus was not performed, the second assay should be performed to appropriately interpret results. If the second assay is not performed, the single positive result should be interpreted as “presumptive flavirivus infection.” The interpretation for IgM and neutralizing antibody test results in a symptomatic pregnant woman is similar to that for nonpregnant patients; however, because dengue and Zika virus IgM antibodies can be detected in serum for months after an infection, the specific timing of infection cannot be determined, and other factors  should be used to assess the likelihood that the current illness was due to either dengue or Zika virus and that exposure occurred during the pregnancy . PregnanZika virus testing recommendations for asymptomatic pregnant women are unchanged (Data on the epidemiology of viruses known to be circulating at the location of exposure and clinical findings should be considered in clinical and testing decisions and when interpreting results (,Dengue and Zika virus disease are nationally notifiable conditions. Health care providers are encouraged to report suspected dengue and Zika virus disease cases to their state, territorial, or local health departments to facilitate diagnosis and mitigate the risk for local transmission. State and territorial health departments should report cases to CDC according to the Council of State and Territorial Epidemiologists case definitions (


A serosurvey of 600 workers newly arrived in Taiwan from 4 Southeast Asia countries showed that 18 (3%) were positive for Zika virus IgM; 6 (1%) fulfilled the World Health Organization criteria for laboratory-confirmed recent Zika virus infection. The incidence of Zika virus infection in Southeast Asia might be underestimated. Approximately 690,000 migrant workers live in Taiwan; most are from 4 countries in Southeast Asia: Indonesia, the Philippines, Thailand, and Vietnam. Although these migrants are valuable to the workforce, they may also bring the risk of spreading mosquitoborne diseases. Most imported cases of Zika virus and dengue virus (DENV) infections in Taiwan come from Southeast Asia. Although screening for fever at international airports was implemented in Taiwan in 2003  to test for IgM and IgG against dengue virus, anti-Zika virus IgG ELISA  to test for IgG against Zika virus, and InBios Zika Detect IgM Capture ELISA (http://www.inbios.com) to test for IgM against Zika virus (because this assay seems to have higher sensitivity) (50) and 90% (PRNT90)  who received preemployment examinations at a regional hospital in Tainan, Taiwan, during June–August 2017. Workers who were >20 years of age and willing to participate were eligible without specific exclusion criteria. We used commercial ELISAs ((PRNT90) . For perMost migrant workers were young adults 20–39 years of age  Table 1.90 titers >20, and their Zika virus PRNT90 titer ratio was ≥4 compared with that of 4 serotypes of DENV  had IgG against both Zika virus and DENV, 6 (1.0%) had only Zika virus IgG, 257 (42.8%) had only DENV IgG, and 110 (18.3%) were negative for both . All 18  of DENV  Table 4.ka virus  Table 4.Aedes mosquitoes in Taiwan. In addition, Zika virus can be detected in semen up to 6 months after symptom onset  because of false positivity resulting from cross-reactivity; nevertheless, the observed seroprevalence is comparable with that in Martinique (42.2%) and French Polynesia (49%) but lower than that in Brazil (63.3%) and Micronesia (73%) (To our surprise, seroprevalence of DENV IgM was lower than that of Zika virus IgM. In DENV-hyperendemic countries, children may have been exposed to multiple DENV serotypes and then acquired immunity; therefore, the incidence of dengue in adults is relatively low. Also of note, we observed much lower seroprevalences of Zika virus and DENV among workers in Taiwan from Vietnam, which may be because most of these workers originally came from rural areas in subtropical northern Vietnam, where the population density and climate are not suitable for establishing endemic transmission cycles of mosquitoborne viruses (Our finding that 1% of migrant workers from Southeast Asia had laboratory-confirmed recent Zika virus infection suggests that the incidence of Zika virus infection in this region is underestimated. Given the convenience of flight for global travel, the risk for international dissemination of Zika virus by workers and travelers originating from Southeast Asia cannot be neglected.Plaque reduction neutralization test methods and additional results for study of seroprevalence of Zika and dengue virus infections among migrant workers, Taiwan, 2017.


For its assimilation by eukaryotes, N2 must be converted to a metabolically tractable form such as ammonium. Such conversion is catalyzed by nitrogenase, an enzyme produced by a select group of microorganisms called diazotrophs. Crop yields necessary to feed the world's population have critically depended on applying nitrogenous fertilizers. Incorporation of prokaryotic determinates required to produce active nitrogenase into crop plants would have enormous economic and environmental benefits. The active-site cofactors of all nitrogenases have a common metallocluster precursor synthesized by NifB. Here, we identify the genetic determinants for NifB function in mitochondria of Saccharomyces cerevisiae, thereby advancing prospects to generate N2-fixing crops.Nitrogen is a constituent of many essential biomolecules and plentiful on earth as inert N S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure–Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.The radical  NifB uses radical SAM chemistry to initiate NifB-co formation, which is accomplished by fusing its accessory clusters and inserting one S and one C atom   . A His-t monomer , similarli cells . These eli cells . In this2-fixing systems are very complex from both genetic and biochemical perspectives   . UltimatnifB gene products in aerobically cultured yeast. Through iterative testing involving 62 NifB pathway variants, we found that NifB from M. infernus and Methanothermobacter thermautotrophicus can be produced in yeast. In both cases, coexpression of A. vinelandii NifU, NifS, and FdxN synthesized NifB that, as purified, supported FeMo-co formation in vitro. Importantly, one M. thermautotrophicus NifB pathway generated NifX that contained in vivo-formed NifB-co.Here, we apply combinatorial pathway design and assembly to investigate the function of 28 selected The starting hypothesis was that genetic factors influencing NifB activity would include expression levels of NifB and the accessory proteins NifU, NifS, NifX, and FdxN, as well as intrinsic properties encoded in distinct NifB variants. Different combinations of these factors were tested by applying pathway library design , 26, whiA. vinelandii nifU, nifS, nifX, and fdxN genes postulated as necessary to determine NifB activity in yeast. For nifU and nifS, expression was controlled by promoter/terminator combinations previously used . The rationale was that NifX expression levels have been reported to be generally higher than other Nif proteins involved in FeMo-co synthesis (S-transferase (GST) to facilitate purification. All proteins were targeted to the mitochondria of S. cerevisiae using sequence variants of the SU9 signal selected to limit undesired homologous recombination . Parental strain 3 produced the most consistent protein expression and was used as foundation for the second library .First, we designed a library of 6 parental strains to optimize stable expression of sly used , whereasynthesis  and thatynthesis . FdxN caynthesis A, while nifB gene sequences and synthesized them with codon optimization for expression in S. cerevisiae. Because the ultimate goal is to engineer nitrogenase in plants, nifB genes were selected according to the following criteria: 1) those originating from or being associated to photosynthetic organisms, 2) those originating from organisms with aerobic lifestyle, and 3) those whose products have been previously shown to be functional in NifB-co formation . As it was previously shown that somewhat-soluble NifB protein from the archaeon M. infernus could be produced in S. cerevisiae  and 3 of archaeal origin. Eighteen harbored the C-terminal NifX-like domain present in NifB proteins with a 2-domain structure, while the remaining 10 candidates contained only the conserved SAM-radical domain   .nifB was tested at 2 expression levels predicted to differ by 9-fold , together with nifU, nifS, fdxN, and nifX as expressed in parental strain 3 . To facilitate NifB detection and isolation, a purification tag was placed between the amino (N)-terminal SU9 mitochondria targeting signal and the NifB-encoding region . Three different tags  were tested for their effect on expression and solubility of NifB from M. infernus, used as reference protein . Because the solubility of the Twin-Strep (TS)-tagged NifB protein was highest , and because Strep-Tactin–based purification procedures do not rely on metal-affinity resins that can remove the labile [4Fe-4S] clusters carried by NifB proteins, the TS-tag was chosen for the library screen . The enhanced solubility of M. infernus TS-NifB at moderate temperature was unexpected as purification of a C-terminally His-tagged version required prior heat treatment , suggesting that addition of a neutral N-terminal extension (such as TS) could prevent its aggregation and/or membrane sequestration.A library of 56 yeast strains was built by factorial design in which each reatment . TemperanifB genes generated polypeptides with migration in sodium dodecyl sulfate (SDS) gels that corresponded to those expected after mitochondrial import and SU9 processing . Six of these NifB candidates accumulated mainly as soluble proteins , and Western blot analysis showed that NifB levels were insufficient to support this screening assay . Sufficient biomass was obtained from aerobically grown fermenter cultures of strain SB187Y expressing M. infernus NifB, which permitted the purification of approximately 3 mg of NifB . As-isolated NifB lacked activity, as previously reported . It was observed that FdxN accumulation in fermented cells decreased over time and that GST-NifX suffered degradation, which precluded NifX purification . Differential expression of NifB and FdxN could be problematic as FdxN is important for in vivo NifB activity . The nifX gene was not included at this stage, because NifX is not required for NifB function and, in addition, it could trigger release of NifB-co from NifB. Expression of all genes was controlled by GAL regulatory elements previously used for effective production of NifU, NifS, FdxN, and NifB . NifB from Methanosarcina acetivorans produced significant levels of a faster migrating isoform indicating protein degradation, so this construct was excluded from further analyses. Aerobic fermenter cultures for the remaining 5 strains were prepared, and NifB proteins were purified. Only NifB from M. thermautotrophicus and M. infernus yielded mainly soluble protein when produced at the high levels required for their biochemical characterization . Notably, both M. thermautotrophicus and M. infernus are archaea, and their NifB proteins lack the C-terminal NifX-like domain . Ultraviolet (UV)-visible spectra indicated that both proteins accommodated [Fe-S] clusters . Because the cluster occupancy appeared higher in the M. infernus NifB and given previous experience with its analysis, this variant was selected for in-depth biophysical analysis and for testing the requirement of NifU, NifS, and FdxN for NifB function.Although NifB is not highly expressed under diazotrophic conditions in their natural hosts , in-deptand NifB . All Nifnif gene product. For example, the NifB species produced in combination with NifU, NifS, and FdxN is indicated as NifBUSF . Because M. infernus NifBUSF accumulated as a largely soluble protein, it could be purified as a dark brown protein . Various NifB species anoxically isolated from aerated fermenter cultures of yeast cells yielded about 6 mg of protein per 100 g of cells, and the metal content and UV-visible spectra of these species were determined .For clarity, mitochondria-targeted TS-NifB produced in yeast and in the absence of any other prokaryotic component is hereafter denoted as NifB*. NifB species produced in yeast in combination with mitochondrial NifU, NifS, and FdxN are denoted by a superscript of the corresponding coexpressed  protein A–D. The M. infernus NifBUSF and NifBUS species could have the highest cluster occupancy , it was of interest to examine the electron paramagnetic resonance (EPR) spectra of these proteins. The EPR spectrum of as-isolated M. infernus NifBUSF measured at 12 K exhibited S = 1/2 signals typical of reduced [4Fe-4S]+ clusters . This assignment was confirmed by temperature-dependent measurements, which showed signal disappearance above 70 K  , indicating that almost all Fe in NifBUSF was in the form of [4Fe-4S]+ clusters. In contrast, only one type of [Fe4S4]+ clusters , while the RS and AC2 clusters were missing.NifB is known to carry 3 distinct [4Fe-4S] clusters . One of ig. S17) . The sigulations E, havingM. infernus NifB* had no ability to support in vitro FeMo-co synthesis, and NifBF and NifBUS had only minimal capacity relative to NifBUSF  , demonstrating that preformed NifB-co was not contained within as-isolated NifB.The abilities of various NifB species to support FeMo-co synthesis was tested using the above-described in vitro FeMo-co synthesis and insertion assay . As-isol NifBUSF A. In vitig. S19)  confirmetivation B. These M. thermautotrophicus NifBUSF was also purified with [Fe-S] clusters and supported in vitro formation of FeMo-co and apo-NifDK activation . M. thermautotrophicus NifBUSF purifications had higher yields than those of M. infernus NifBUSF , while the Fe content was slightly lower  .M. thermautotrophicus and M. infernus NifB proteins lack the C-terminal NifX-like domain, and it is possible that they are incapable of accumulating NifB-co. As NifX has demonstrated affinity for NifB-co, it was built into the pathways to trap any in vivo-formed NifB-co. To overcome the instability of GST-NifX observed in the library screening, GST was replaced by a TS-tag–tobacco etch virus (TEV) site cassette . In addition, as accumulation of M. thermautotrophicus and M. infernus NifB proteins in yeast was very high, endogenous mitochondria SAM levels could be limiting. A mitochondria-targeted variant of the cytosolic SAM synthase (Sam1p) was therefore engineered following the strategy used by Marobbio et al. . This approach is similar to the overexpression in E. coli of the SAM synthase gene metK used to obtain functional M. infernus NifB .Both o et al.  . Both M. infernus NifBUSF+SAM and M. thermautotrophicus NifBUSF+SAM supported SAM-dependent FeMo-co formation and apo-NifDK reconstitution using purified protein components  in aerobically grown proteins . The stuproteins . This reatalysis . In anott” genes . Both stgenes  and Cyanothece sp. (ATCC 51142) showed more than 95% identity, NifB accumulation was only detected for the Gloeothece sp. (KO68DGA) protein. Notably, some NifB variants were detected only when expressed at high levels, while others worked only at low levels. Moreover, it was not known a priori that for accessory genes, some combinations of expression levels would result in complete abrogation of NifB expression. In addition, a surprisingly small number of NifB proteins were soluble. Although both M. thermautotrophicus and M. infernus NifB proteins were predicted to be stable (Dataset S1), expression of other NifB sequences with similar low instability index could not be detected, highlighting that NifB accumulation was not easily anticipated from sequence information or from prior observations. One important observation is that both nifB genes were sourced from extreme thermophiles, implying that such proteins may be more stable in this heterologous expression system. Overall, because a library-based approach revealed these kinds of dependencies, we anticipate that in the future, methods to facilitate simultaneous testing of multiple design factors will be important for the engineering of pathways of increasing complexity.Taking into consideration recent progress in overcoming the anticipated problem of OA requirement for NifU and NifS to produce active NifB was not expected because the maturation of [4Fe-4S]-containing NifH in yeast does not require NifU and NifS. Namely, for the assembly and delivery of the [4Fe-4S] cluster contained in yeast-expressed NifH, it appears that the function of NifU and NifS can be supplanted by the yeast-encoded scaffold (ISU) and cysteine desulfurase (NFS1). Why this is not the case for NifB [4Fe-4S] clusters could be related to the complexity of this enzyme containing 3 different [4Fe-4S] clusters compared to a single [4Fe-4S] in NifH.nifUS mutation in Klebsiellapneumoniae decreased NifB levels by 10-fold and abolished NifB-co activity in extracts  could activate apo-NifDK to similar levels, although overall NifB-co levels were much lower in extracts of the ΔnifUS strain. This result contrasts with our results in S. cerevisiae, where NifU and NifS are required to produce NifB protein with high [Fe-S] cluster occupancy and no bound NifB-co was found. It should be noted that while NifB was produced in K. pneumoniae under strict anaerobic conditions, the S. cerevisiae strains used here were cultured under aerobic conditions. In addition, the different tags used for protein purification can influence metal content and activity, and the properties of the as-isolated proteins may not necessarily reflect the characteristics of the native protein. This could, for example, explain the different levels of NifX-bound NifB-co in S. cerevisiae strains carrying nifB from M. infernus or M. thermautotrophicus.It had been shown that a Δextracts . By uncoUS-directed NifB-co formation and why FdxN is not strictly essential for A. vinelandii N2 fixation  is insufficient. In other words, isolated NifBUSF is likely to represent a mixed population including one fraction replete with [4Fe-4S] clusters poised for NifB-co formation and another fraction that has released NifB-co but has not yet been recharged with the accessory clusters. Release of NifB-co from NifB upon its completion is supported by the observation that no NifB-co capable of supporting FeMo-cofactor formation in the absence of SAM was detected in as-isolated NifBUSF.It should be noted that, based on elemental analysis and UV-visible and EPR spectroscopies, the M. thermautotrophicus NifBUSF+SAM but not from yeast expressing M. infernus NifBUSF+SAM. Like M. infernus, M. thermautotrophicus does not harbor an obvious nifX gene that could indicate that its NifB protein has evolved to interact with NifX. Instead, the difference in NifX-bound NifB-co could be explained by different NifBUSF+SAM expression, solubility or activity, or a combination of those. Another important question is whether NifB activity within mitochondria might be limited by the natural levels of SAM within that organelle. The S. cerevisiae SAM synthase was here engineered into a mitochondria-targeted variant because of the very high expression levels of M. infernus or M. thermautotrophicus NifBUSF proteins using the GAL-induced expression systems. The exact physiological parameters for SAM-dependent NifB activity when produced at lower levels in mitochondria will require further studies.It is intriguing that we could detect NifX-bound NifB-co from yeast expressing 2-fixing eukaryote: 1) an active form of NifB, required for the formation of the NifB-co precursor to the active-site cofactor of all nitrogenase types, can be produced in the mitochondria of a model eukaryotic organism such as S. cerevisiae; 2) NifB-co can be produced in the mitochondria of a eukaryotic cell grown under aerobic conditions; 3) factors such as FdxN, which are not necessarily essential for N2 fixation in prokaryotic cells, could be essential to produce a N2-fixing eukaryote; 4) genes and expression levels not necessary to produce active NifH in S. cerevisiae, such as NifU and NifS, are necessary to produce active NifB; and 5) complementary assembly factors produced by highly divergent bacteria can be combined and sorted to achieve the production of building blocks essential to formation of an active nitrogenase in eukaryotes.The present study leads to 5 important observations that are key to engineering a NS. cerevisiae CEN.PK113–7D (MATa URA3 TRP1 LEU2 HIS3 MAL2–8c SUC2) was the host strain for all library constructs and grown at 30 °C in yeast extract–peptone–dextrose media, with 200 μg/mL G418 added when appropriate. Yeast transformations were carried out according to the lithium acetate method (E. coli DH5α (New England Biolabs) was used as a cloning strain and grown at 37 °C in lysogeny broth media with appropriate antibiotics (100 μg/mL carbenicillin or 25 μg/mL kanamycin) and inducer  was spread and dried on plates for blue/white screening when appropriate.e method , 36. CheAll Sanger sequencing reactions were performed by Quintara Biosciences. Plasmid isolations were performed with Qiagen Qiaprep kits. Genomic DNA was isolated using the Promega Wizard Genomic DNA Preparation Kit. Gel electrophoresis was carried out using 1% agarose E-Gels according to the manufacturer’s instructions (Invitrogen). BsaI was purchased from New England Biolabs. BbsI was purchased from Thermo Fisher Scientific. High concentration T4 DNA ligase was purchased from Promega. All PCR primers were ordered from Integrated DNA Technologies. All PCRs used Q5 2× Master Mix from New England Biolabs. PCRs were performed on Eppendorf thermocyclers.nifB genes, SU9, and TS-tag were designed for expression in S. cerevisiae using the GeneOptimizer tool (ThermoFisher) and synthesized by ThermoFisher via the Engineering Nitrogen Symbiosis for Africa project. Other sequences have been described previously . Vectors were verified by Sanger sequencing (Quintara Biosciences).Genes encoding eviously , 26. OncSI Appendix, Fig. S1 A and B and Tables S1 and S2). To construct the library of NifB variants, promoters Ptef1 and PPXR1 from S. cerevisiae were used for the high- and low-expression levels, respectively, and both used terminator TKIURA3 from Kluyveromyces lactis. A kanamycin-resistance gene was cloned into pDSM-bc with the strong promoter Pagtef1 and the TagTEFI terminator, both from Ashbya gossypii. These transcription units can recombine and integrate into yeast chromosome XV between NRT1 and GYP1 . Integrations were performed using PCR-amplified transcription units  from the pDSM vectors, as well as PCR-amplified bridging DNA derived from the yeast genome. Parts were introduced to yeast by homologous recombination using the lithium acetate transformation method aminomethane (Tris)⋅HCl (pH 8.8), 300 mM NaCl, 10% glycerol—supplemented with 2 mM dithionite (DTH), 5 mM β-mercaptoethanol (β-ME), 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin, and 5 μg/mL DNase I. The cells were lysed in an Emulsiflex-C5 homogenizer (Avestin Inc.) at 25,000 pounds per square inch. Cell-free extracts (CFEs) were obtained by removal of cell debris and precipitated yeast proteins by centrifugation  and filtration through a 0.2-μm pore-size filter . All procedures were performed under anaerobic conditions.2) using an AKTA Prime fast protein liquid chromatography system  inside a glovebox (MBraun). All buffers were previously made anaerobic by sparging with N2. Before loading the CFE, the Strep-Tactin column was equilibrated with buffer B . A pH above 7.5 of the CFE was ensured before loading. Typically, anaerobic CFE from 100 g of cell paste was loaded at 2 mL/min and washed with 5 successive washes of 15 mL of buffer B. Bound protein was typically eluted with 12 mL of buffer B supplemented with 50 mM biotin and desalted using a HiPrep 26/10 Desalting column  equilibrated with buffer C . Purity of the NifB protein was verified by SDS/PAGE and Coomassie staining, concentrated using a 10-kDa cutoff pore centrifugal membrane device , and analyzed by UV-visible spectroscopy. Finally, pure TS-NifB was supplemented with 2 mM DTH, frozen, and stored in liquid N2.TS-NifB was purified by Strep-tag–binding chromatography using a 5-mL Strep-Tactin XT Superflow Cartridge (IBA Lifesciences) under anaerobic conditions , 100 μL of adenosine triphosphate (ATP) mix , and 200 μL of UW140 (A. vinelandii ΔnifB) CFE at 14.64 mg/mL total protein concentration supplemented with 2 μM NifH. Finally, 50 μL of buffer (25 mM Tris⋅HCl [pH 7.8]), or buffer supplemented with NifB , or purified NifB-co as positive-control reaction  was added. The N2 atmosphere was changed to argon (Ar), and vials were incubated for FeMo-co synthesis and insertion at 30 °C for 35 min.Assays were performed as described by Curatti et al. , with slA. vinelandii DJ (wild-type) CFE, A. vinelandii UW140 CFE complemented with NifB-co purified from K. pneumoniae strain UC32   by acetylene reduction assay at 30 °C for 15 min following standard procedures . Positiv2MoO4, 175 μM R-homocitrate, 125 μM FeSO4, 125 μM Na2S, 125 μM SAM, 1.23 mM ATP, 18 mM phosphocreatine disodium salt, 2.2 mM MgCl2, 3 mM DTH, 40 μg/mL creatine phosphokinase, 5.0 μM NifB, 3.0 μM NifX, 1.5 μM apo-NifEN, 3.0 μM NifH, 0.6 μM apo-NifDK, and 1 mg/mL bovine serum albumin in 22 mM Tris⋅HCl buffer (pH 7.5). FeMo-co synthesis and insertion into apo-NifDK was performed under N2 atmosphere at 30 °C for 45 min. NifX-dependent FeMo-co synthesis was performed in the absence of NifB and SAM, and with NifX isolated from yeast replacing the A. vinelandii NifX in the above reaction.Assays were performed as described by Curatti et al. , with sl2, 3 mM DTH, 40 μg/mL creatine phosphokinase, final concentrations in 22 mM Tris⋅HCl [pH 7.5] buffer) in 9-mL vials under Ar atmosphere. Acetylene reduction assays were performed at 30 °C for 15 min following standard procedures   .SI Appendix, Fig. S14 were recorded at 12 K. EPR spectral simulations were performed using the simulation software Spin Count , 300 mM NaCl, 10% glycerol, 2 mM DTH, and 5 mM β-ME were prepared for EPR analysis. X-band (9.64 GHz) EPR spectra were recorded on a Bruker E500A spectrometer equipped with an Oxford ESR 910 cryostat for low-temperature measurements. The microwave frequency was calibrated with a frequency counter and the magnetic field with an NMR gauss meter. The temperature of the X-band cryostat was calibrated with a carbon-glass resistor temperature probe . For all EPR spectra, a modulation frequency and amplitude of 100 kHz and 1 mT were used. The EPR spectra of in Count ; 1 mM CuSI Appendix, or Dataset S1.All data are available in the main text, Supplementary FileSupplementary File


Varroa destructor, is unarguably the leading cause of honeybee (Apis mellifera) mortality worldwide through its role as a vector for lethal viruses, in particular, strains of the Deformed wing virus (DWV) and Acute bee paralysis virus (ABPV) complexes. This multi-level system of host-parasite-pathogen interactions makes it difficult to investigate effects of either the mite or the virus on natural host survival. The aim of this study was to remove confounding effects of varroa to examine the role of virus susceptibility in the enhanced survival of a naturally adapted Swedish mite-resistant (MR) honeybee population, relative to mite-susceptible (MS) honeybees. Caged adult bees and laboratory reared larvae, from varroa-free colonies, were inoculated with DWV and ABPV in a series of feeding infection experiments, while control groups received virus-free food. Virus infections were monitored using RT-qPCR assays in individuals sampled over a time course. In both adults and larvae the DWV and ABPV infection dynamics were nearly identical between MR and MS groups, but MS adults suffered significantly higher mortality than MR adults. Results suggest virus tolerance, rather than reduced susceptibility or virus resistance, is an important component of the natural survival of this honeybee population.The ectoparasitic mite,  Two virus-complexes in particular, the Deformed wing virus (DWV) complex  and the Acute bee paralysis virus (ABPV) complex  and Israeli acute paralysis virus (IAPV)7), are transmitted highly efficiently by varroa mites, with devastating, though different, consequences8. Of the two virus complexes, DWV has become the most common and wide spread virus9 due to its superior adaptation to the combined selection pressures of varroa-mediated transmission, beekeeper mite management and colony winter survival8. Both DWV and ABPV are single-stranded RNA viruses that infect all stages of honeybee development10. In the absence of varroa mites, they are maintained in the colony at low levels as largely innocuous infections through a variety of horizontal and vertical transmission routes16. The characteristic symptoms of DWV are severe wing deformities resulting in flightless adults that die shortly after emerging10. However, these symptoms are almost exclusively associated with varroa-mediated transmission of DWV when the mite feeds on the bee during the pupal developmental stages17. ABPV, when transmitted by varroa mites, is characterized mostly by severe pupal mortality, and at elevated titres in adults by trembling, paralysis and behavioural inadequacies7.Despite the current popular and scientific focus on the possible role of agrochemical exposure on wild and managed bee health14. For this reason, beekeepers need active mite control strategies to keep honeybee colonies alive in almost all parts of the world where the mite exists3. However, different honeybee populations naturally have varying responses to the pressure of this parasite. While most honeybees are susceptible to varroa mite parasitism and will typically die within a few years of an uncontrolled mite infestation3, some populations have been documented to survive without mite control measures and without the harmful effects typically associated with varroa mite infestation18. For example, mite control is not required in Africa or in parts of South America where the local A. mellifera populations or sub-species are effectively mite-resistant and maintain lower mite infestation levels than their relatives in other parts of the world19. There are also a few unique populations of honeybees in Europe and North America that have survived for extended periods without mite control20. One of the most well studied varroa mite surviving honeybee populations is on the island of Gotland, Sweden24. Through a natural selection process this population has adapted mite-resistant traits that reduce the mite’s reproductive success24.The mite population in an infested honeybee colony can grow at an exponential rate, rapidly leading to an ABPV and/or DWV epidemic that ultimately results in the death of the colony23. Additionally, this population also appears to be resistant, at the colony level, to other viruses that are not necessarily directly transmitted by varroa mites, but that nevertheless harm honeybee health and reduce long-term survival23.Honeybee populations can likewise have varying responses to the virus infections vectored by the mite. A recent study on the Gotland mite-resistant population documented their survival with high levels of DWV infections while local mite-susceptible honeybee colonies, with similarly high DWV infections, all died25. Honeybees, the varroa mite, and honeybee-infecting viruses that are also vectored by the mite, form a complex multi-level system of host-parasite-pathogen interactions, making it difficult to separate specific effects on the host when all three occur together. In order to better understand natural host survival mechanisms to uncontrolled mite infestation it is important to differentiate the extent to which they are due to resistance/tolerance to the mite, the viruses transmitted by the mite, or both.Resistance is the ability of the host to reduce the parasitic/pathogenic burden, whereas tolerance is defined as the ability of the host to reduce the harm done by the parasite/pathogen The aim of this study was to assess the possible contribution of virus resistance/tolerance to the enhanced survival of the Gotland mite-resistant (MR) honeybee population independent of the confounding effects of varroa mite parasitism. We removed the direct influence of varroa on virus titres and compared the susceptibility of individual larvae and newly emerged adult bees from the Gotland mite-resistant (MR) population to oral ABPV or DWV infections, relative to that of bees from a mite-susceptible (MS) population, in laboratory virus infection time-course studies. Virus susceptibility was determined by comparing the virus titres of virus-inoculated bees relative to both the pre-experiment background virus titres and the natural infection development in uninoculated control bees, across the time-course. This formed the basis for comparing the virus susceptibilities of the MR and MS populations, which were each represented by bees from multiple, independent colonies. For the adult infection experiment, the mortality of the bees over time was also recorded, as well as the virus titres in dead bees.No differences were detected between the MR and MS larvae in either DWV or ABPV susceptibility at any time during the experimental time course Fig. ; Fig. S1Vice versa, there was no significant difference in ABPV titres between the uninoculated control larvae and the DWV-inoculated larvae  decrease over time from the zero time-point, with a low after 72 hpi, after which they increase again, more markedly for DWV, at 120 hpi Fig. .Whereas in the larval infection experiments the background titres of both DWV and ABPV in the uninoculated/alternate virus-inoculated controls tended to decrease with time (with a late upswing), in the adult infection experiments the reverse is true, in that the background DWV and ABPV titres in the controls tended to increase during the early part of the time-course, with a slight subsequent decrease Fig. . The bacAs with the larval infection experiments, there was no significant difference between MR and MS bees in any of these time-course patterns, for either the DWV infection experiment or the ABPV infection experiment Fig. ; Fig. S1The only aspect in these virus experiments where there is a clear difference between the MR and the MS bees is in the survival rates of the virus-inoculated adult bees Fig. . In bothThe main result of these experiments is that there is no difference in susceptibility to DWV or ABPV infection between bees from the MR population and those from the MS population, for both adult bees and larvae. However, there is a significantly lower mortality for virus-infected adults from the MR population, relative to virus-infected adults from the MS population, with no major differences between ABPV-infected or DWV-infected bees. There was no difference in mortality between uninfected bees from the MR or MS populations. Survivability in larvae was not possible to assess since the experimental virus dose was optimized to prevent larvae from dying so that infection dynamics could be monitored and any differences in virus susceptibility could be observed.vice versa, for both larvae and adults, i.e. the viruses did not appear to compete or interfere with each other. This supports earlier research showing that different bee viruses evoke entirely different transcriptional responses and biological processes after oral inoculation26, and that different viruses develop equally well in isolation or when co-inoculated with other viruses26. Many bee viruses also show high tissue-specificity27, such that any temporal-spatial isolation of the viruses reduces direct competition at the molecular level. Possible genetic changes to the inoculated viruses during the time course as well as any molecular interactions with the host or other inoculated or background viruses are the subject of ongoing investigations.Another major result from these experiments is that oral inoculation with DWV has no effect on the background infection dynamics of ABPV and 29 and were presumably DWV- and ABPV-free, given Ålands virus-free history . Since the 0 hpi material never was exposed to virus in the laboratory, our best explanation is that these colonies became contaminated during the 2 months that were needed to turn over the bee populations after introduction of the MR and MS queens. Given their origins30, these queens will undoubtedly have been infected with at least DWV, which is efficiently transmitted through the eggs to the resulting progeny31, as well as to the rest of the colony through larval care and social interactions33. However, any virus infections thus acquired would be similar for all bees and experimental groups in our study and part of the molecular background to which the experimental inoculations are applied. DWV-symptomatic adult bees were not observed during the experiments, since DWV symptoms develop during the pupal phase, generally due to transmission by varroa. However, some of the adult bees that were orally inoculated with APBV were observed to be trembling, which is a symptom of APBV infection.The final major result is the shape of the infection curves of the uninoculated/alternate virus-inoculated controls. These are very different for larvae and adults, and also slightly different for DWV- or ABPV-inoculated bees, but are highly similar for the different populations and between the uninoculated and alternate virus-inoculated groups, comprising furthermore data from many different colonies. Although there was variation across the time-course in the mRNA expression levels of our internal reference gene RP49, these were insignificant compared to the logarithmic-scale variation in the virus titres across the same time-frame, and were therefore not responsible for the shapes of the curves. Since these represent the infections in the least manipulated bees, they are of special interest for identifying the natural interaction between viruses and their host. We have currently no explanation for why the natural DWV and ABPV titres in larvae should go down, and then up, or why in adult bees they should go up, then down, before levelling off. This material is currently the subject of further investigation. One final point that requires clarification is the relatively high initial DWV titre in newly hatched adult bees of both the MR and MS populations, or for that matter any background DWV and ABPV at all, given that the original colonies were sourced from varroa-free Åland34. Laboratory experiments have shown that the DWV-B genotype is preferentially transmitted by varroa and through injection compared to the DWV-A genotype35. The persistence of both strains in natural colonies attests to the importance of other transmission routes, such as oral and sexual transmission in maintaining this balance36, together with the different consequences of virulence at individual bee and colony level. Similar differences in virulence and infectivity have also been described for the members of the ABPV-complex  in relation to the various transmission routes and different consequences for individual and colony-level mortality10. Although we cannot rule out possible interactions of our inoculated viruses with background virus infections, or the host molecular condition (which will be the subject of a separate investigation), the focus of this study is how these experimental virus infections differentially affect bees from MR and MS colonies, both through the infection time-course and through adult mortality.Differences in DWV virulence have been associated with different major DWV genotypes, with the DWV-A genotype being more wide-spread but less virulent than the DWV-B genotype23. In animal health, tolerance is a highly effective mechanistic response to disease but is often overlooked, especially when resistance is present25. Tolerance adaptations do not inflict harm on the parasite, unlike resistance, so it is expected to fix in the population rather than causing an open-ended antagonistic coevolution, as is the case with resistance evolution25. This tolerance to virus infections has been demonstrated on earlier generations within the Gotland MR honeybee population and in different geographical locations23, so it appears to have a genetic heritable component to its expression. It is unclear if there are host fitness costs associated with this virus tolerance and to what degree this virus tolerance has on the overall long-term survival of the population in relation to the resistant mechanisms that limit the population growth of varroa mites.The enhanced survival of virus-infected adult bees from the Gotland MR population, relative to those of the MS population, despite near identical susceptibility to virus infections across the DWV and ABPV infection time-courses, suggests that host tolerance, rather than resistance, is an important component of the naturally adapted survival mechanisms of this population and that their individual-level tolerance response to virus infections complements earlier work demonstrating a colony-level tolerance to DWVvice versa37. For example, despite the individual level mite resistance of inhibiting mite reproduction, colony level varroa infestation rates in the Gotland honeybee population can still be high23, suggesting other factors could contribute to the population’s natural long-term survival. The results of this study demonstrate that tolerance rather than resistance, plays an important role in defence against varroa-vectored virus infections, at both levels of colony organization for this population23. In contrast to our study, reduced susceptibility to virus infections has been observed in savannah honeybees in Africa38 and in selectively bred mite-resistant honeybees, compared to non-selected lines, in the U.S.39. However, these studies did not exclude confounding influences on virus infections by the virus-vectoring varroa mite, which likely could have impacted their results. The disparity between these studies only demonstrates the potential for varying adaptive responses of honeybee populations to virus infections, which would be expected under varying environmental conditions41. Current efforts are underway to explore virus resistance and tolerance defence mechanism in other unique naturally adapted mite-resistant populations and to assess the potential to include virus response mechanisms in breeding programs to improve bee health and sustainable beekeeping.The honeybee host has a unique social organization that presents its own complexity in this system with multiple levels of possible host-parasite interactions and adaptation to parasites and disease infections. Resistance at the individual level could translate to tolerance at the colony level and 30, but excluding the confounding influences of varroa mite infestation and environmental factors e.g. the different colony development dynamics between the MR and MS bees22. Moreover, the observed virus tolerance applies equally and similarly to DWV- or ABPV- infected bees, suggesting that the tolerance mechanism may be primarily generic, rather than virus specific.In conclusion, individuals from the Gotland MR honeybee populations survive better than MS honeybees having a higher threshold for virus infections before bee health is compromised. This elevated virus tolerance for MR bees at the individual level supports the previously inferred virus tolerance at colony level21 and has demonstrated a variety of adaptations that enable their long-term survival with varroa mite infestation23. The mite-susceptible (MS) honeybees originated from a local unselected honeybee population near Uppsala that required regular varroa mite control interventions to avoid colony death. Six open-mated queens from each population were established in small 6-frame experimental colonies stocked with equal amounts of honeybees from Åland, a group of islands between Sweden and Finland. Åland is still entirely free from varroa mites29 and have undetectable levels of both DWV and ABPV in general surveys  so these bees provided the ideal experimental background for virus infection experiments, having never been exposed to the virus or the vector. Experiments did not begin until all individuals in each colony were the offspring of the introduced queens; approximately 2 months after the queens were introduced.The mite-resistant (MR) honeybees originated from an isolated population on the island of Gotland, Sweden that has been surviving without mite control treatments since 1999. This population was established as a selection experiment on honeybee survival with a new invasive parasite3 virus genome copies/bee), according to standard pupal propagation procedures15. From these 50 pupae, a clarified crude extract was made by homogenizing the pupae in a blender with 10 mL 0.5 M Phophate Buffer, pH 8.0 (DWV) or 10 mL 0.01 M Phosphate Buffer, pH 7.0 (ABPV),and stored in 50 μl aliquots at −80 °C15. These crude extracts were used for the virus infection experiments. The virological composition of the propagated virus stocks was determined using RT-qPCR assays for seven common bee viruses that can be propagated through injection13: DWV-A, DWV-B, ABPV, IAPV, KBV, sacbrood virus (SBV) and black queen cell virus (BQCV). Using an MuMLV 1st strand cDNA kit , random hexamer-primed cDNA was prepared from 1 ug of RNA extracted from the propagated inoculum to which 25 ng of RNA250  was added, as an exogenous reference RNA for normalizing the relative titres of the viruses against a common fixed standard. A six-step 10-fold dilution series was made from each cDNA and used as templates for the virus-specific qPCR reactions. The dilution series were used to obtain the reaction efficiencies of the different assays. These reaction efficiencies were then used to estimate the relative amount of each virus in each cDNA dilution in relation to the common fixed RNA250 standard42. The average of these multiple estimates of the relative amounts of each virus in each inoculum was used to construct the pie-charts in Supplementary Fig. The DWV and ABPV inocula for the oral infection experiments were prepared by propagating reference DWV-A and ABPV virus stocks each in fifty white eyed pupae from the varroa-free colonies from Åland (see above). Each pupae was injected with 1 micro litre of a 1/10000 dilution of purified concentrated virus stock . This dose selection criteria was used so that early  virus infectivity dynamics could be studied, as well as possible subsequent differential mortality between mite-resistant and mite-susceptible bees. The optimum single inoculation dose for larvae was 1.5 ± × 10The infection experiments were conducted separately on newly emerged adult bees and on newly hatched larvae. Each infection experiment consisted of several replicate infection trials for each MR and MS colony included in the experiment Table . Where p45, following standard larval rearing methods46. Extra larvae were grafted in each cell to compensate for any mortality due to the grafting procedure. The larvae were pre-incubated for 24 hours at 35 °C with a relative humidity of 96%, after which all dead and excess larvae were removed, such that 48 living larvae, one per well, were retained for the infection experiment. Larval mortality due to grafting procedure generally did not exceed 20%. The viable larvae (approximately 48 hour old) were then fed with larval food. For the larvae cohorts to be inoculated with virus, the larval food was spiked with the optimum single infectious dose of DWV or ABPV, as determined above. The larvae were fed daily according to established protocols46 and any dead larvae were removed. On each sampling occasion (see above), 4 live larvae from each infection cohort were collected in microcentrifuge tubes and stored at −20 °C until further analysis47.The larval infection experiments were conducted on larvae from 4 MR colonies and 4 MS colonies. Each infection trial was replicated at least two times, depending on the availability of larvae from the colony concerned. Larvae of similar age were obtained by confining the queens of the experimental colonies to a single frame for 24 hours for egg-laying. First instar larvae (between 24–36 hours old) were grafted with a Chinese grafting tool  into individual wells of 48-well tissue culture plates , each containing 10 μL of pre-warmed larval food consisting of 50% royal jelly , and 50% aqueous solution of D-glucose (12%), D-fructose (12%), and yeast extract (2%)48. For each inoculation cohort fifty newly emerged adults from each colony were placed in separate Lyson queen cages , and fed over a 24-hour period the optimum DWV and ABPV inoculation dose (as described above) in 2 mL Bifor®, a 66% w/w commercial honeybee sugar solution with a 2/1/1 ratio of sucrose/fructose/glucose  with control bees receiving just Bifor®. After inoculation, all cohorts of bees were fed uncontaminated Bifor®ad libitum for the remainder of the time-course. On each sampling occasion (see above) and for each infection cohort, all dead bees were counted and removed, retaining 5 bees for analysis, while 5 live bees were also sampled. Both live and dead bee samples were stored at −20 °C until further analysis.The adult infection experiments were conducted on newly emerged adult bees from 5 MR colonies and 3 MS colonies. Each infection trial was replicated between 1–4 times, depending on the availability of newly hatched adult bees from the colony concerned. The bees were hatched on caged frames inside an incubator at a constant 35 °C temperature and 96% relative humidity47. Total RNA was extracted from 100 μl of this homogenate by a QiaCube robot following the RNAeasy protocol for plants (Qiagen). The RNA was eluted in 50-μl RNase-free water, the RNA concentration was estimated by NanoDrop and the purified RNA was stored at −80 °C until further processing.Each experimental time-course sample, containing either 4 larvae or 5 adult bees, was placed in a mesh bag and ground to powder using liquid nitrogen and a pestle. A primary homogenate was produced by adding 200 μl/bee sterile water to each ground sample and mixing vigorously15) were determined using reverse transcription quantitative PCR (RT-qPCR), using the iScript One Step RT-PCR kit (Bio-Rad) with SYBR Green as the detection chemistry and the Bio-Rad CFX connect thermocycler. The reactions were performed in 20 μl volumes containing 0.2 μM of the forward and the reverse primers , the treatment , and the hours post inoculation (hpi) were included as categorical fixed effects. Time was used as categorical variable in order to perform pair wise comparisons and because titres did not simply increase/decrease over the course of the experiments. To account for the hierarchical data structure (multiple samples of a colony), sample was included as a random effect nested within each colony. The Type III test was used in the analysis since the sampling was unbalanced (different number of colonies with different numbers of replications within colonies). In order to obtain normally distributed residuals , the function transformTukey was used from the package rcompanion, which loops over exponents that maximizes/minimizes the Shapiro-Wilks/Anderson-Darling statistic. After performing the statistic with the recommended transformation  and a generalized linear mixed model using the package Anova from the package car was used to stepwise remove non-significant variables until the minimal adequate model was obtained. Tukey method adjusted pair wise comparisons  were performed using the package multcomp. To obtain predicted marginal means from each model we used the packages lsmeans. Further we calculated the conditional R2 value, which accounts for the fixed and random model part, and the second-order AIC.For all models, the type III test with function Thaduri et al. 2019. Supplementary Figures and Tables


Arabidopsis thaliana and Cucumber mosaic virus (CMV), which is significant in nature. We studied the genetic diversity of A. thaliana for two defence traits, resistance and tolerance, to CMV. A set of 185 individuals collected in 76 A. thaliana Iberian wild populations were inoculated with different CMV strains. Resistance was estimated from the level of virus multiplication in infected plants, and tolerance from the effect of infection on host progeny production. Resistance and tolerance to CMV showed substantial genetic variation within and between host populations, and depended on the virus x host genotype interaction, two conditions for coevolution. Resistance and tolerance were co-occurring independent traits that have evolved independently from related life-history traits involved in adaptation to climate. The comparison of the genetic structure for resistance and tolerance with that for neutral traits  indicated that both defence traits are likely under uniform selection. These results strongly suggest that CMV infection selects for defence on A. thaliana populations, and support plant-virus coevolution. Thus, we propose that CMV infection reduces host fitness under the field conditions of the wild A. thaliana populations studied.It has been proposed that in wild ecosystems viruses are often plant mutualists, whereas agroecosystems favour pathogenicity. We seek evidence for virus pathogenicity in wild ecosystems through the analysis of plant-virus coevolution, which requires a negative effect of infection on the host fitness. We focus on the interaction between  Arabidopsis thaliana for two defence traits, resistance and tolerance, to Cucumber mosaic virus (CMV) at a regional scale in the Iberian Peninsula. Resistance and tolerance to CMV showed substantial genetic variation within and between host populations, and depended on the virus x host genotype interaction, two conditions for coevolution. Resistance and tolerance were independent traits that co-occurred at the population and regional scales, and that have evolved independently from other adaptive life-history traits. Analyses also indicated that resistance and tolerance are likely under selection, most likely due to virus infection. These results support a hypothesis of plant-virus coevolution and contribute to demonstrate that plant viruses may be virulent parasites of plants in wild ecosystems.Plant-virus coevolution has not been demonstrated in any wild system, and it has been proposed that viruses often would be mutualistic symbionts, rather than pathogens, in wild plant ecosystems. We analyse if viruses are virulent pathogens of plants in wild ecosystems and, consequently, plants have evolved defences against virus infection. To test this hypothesis, we studied the genetic diversity of  It is commonly accepted that hosts and pathogens coevolve . This coTo our knowledge, plant-virus coevolution has not been demonstrated in any wild system. In fact, it has been proposed that viruses often would be mutualistic symbionts, rather than pathogens, in wild plant ecosystems –12, and Arabidopsis thaliana L. Heynh. (Brassicaceae)-Cucumber mosaic virus , (CMV). A. thaliana is an annual semelparous species with two distinct developmental periods: the vegetative growth period, producing a rosette of leaves, and the reproductive period in which the inflorescence grows, new flowers are produced continuously, and older flowers develop into siliques =0.95 . Genetic.95  1/n) for multiple comparisons.(XLSX)Click here for additional data file.S5 TableQST of resistance and tolerance to CMV for twelve A. thaliana populations are also shown.Mean, minimum and maximum values, heritability and (XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 TableValues are the averages of 76 population.(XLSX)Click here for additional data file.


Varroa destructor, the key DWV vector, we found high genetic diversity of DWV in infested United States honey bee colonies. Phylogenetic analysis showed that divergent US DWV genotypes are of monophyletic origin and were likely generated as a result of diversification after a genetic bottleneck. To investigate the population dynamics of this divergent DWV, we designed a series of novel infectious cDNA clones corresponding to coexisting DWV genotypes, thereby devising a reverse-genetics system for an invertebrate RNA virus quasispecies. Equal replication rates were observed for all clone-derived DWV variants in single infections. Surprisingly, individual clones replicated to the same high levels as their mixtures and even the parental highly diverse natural DWV population, suggesting that complementation between genotypes was not required to replicate to high levels. Mixed clone–derived infections showed a lack of strong competitive exclusion, suggesting that the DWV genotypes were adapted to coexist. Mutational and recombination events were observed across clone progeny, providing new insights into the forces that drive and constrain virus diversification. Accordingly, our results suggest that Varroa influences DWV dynamics by causing an initial selective sweep, which is followed by virus diversification fueled by negative frequency-dependent selection for new genotypes. We suggest that this selection might reflect the ability of rare lineages to evade host defenses, specifically antiviral RNA interference (RNAi). In support of this hypothesis, we show that RNAi induced against one DWV strain is less effective against an alternate strain from the same population.The impacts of invertebrate RNA virus population dynamics on virulence and infection outcomes are poorly understood. Deformed wing virus (DWV), the main viral pathogen of honey bees, negatively impacts bee health, which can lead to colony death. Despite previous reports on the reduction of DWV diversity following the arrival of the parasitic mite  Deformed wing virus, a key pathogen of honey bees, shows rapid diversification after genetic bottlenecks; a novel reverse-genetic system provides insights into the forces that shape virus diversity, suggesting that virus quasi-species diversification may be driven by selection of genotypes capable of evading host RNAi defences. Varroa destructor virus 1 (VDV1 or DWV-B), and infrequently, DWV-C [V. destructor [Varroa invasion, DWV was transmitted vertically and orally [Varroa-free Hawaiian Islands [Varroa mite [Varroa-free and Varroa-infested colonies are genetically distinct and that mite vectoring drives evolutionary changes in DWV. However, it is not clear which genetic changes make Varroa-transmitted DWV more virulent. Previous reports suggested that the VDV1-DWV recombinants detected in the United Kingdom and France [Varroa-free honey bees [Varroa-infested honey bees, contrasting with high viral variability in asymptomatic bees with low DWV levels [Varroa in Hawaii also preceded a significant reduction of DWV diversity in the wasp Vespula pensylvanica, a honey bee predator [Varroa-associated DWV population currently circulating in the mainland US indicated high genetic diversity of DWV. To investigate the evolutionary dynamics of DWV, we designed a reverse-genetics system for a virulent DWV population by cloning variants coexisting in a typical US DWV-A quasispecies [Varroa-selected genotypes causes a selective sweep, after which diversification via negative frequency-dependent selection results in high genetic heterogeneity, potentially benefiting the virus’s ability to escape genotype-specific antiviral defenses.Population fluctuations affect virulence of invertebrate RNA viruses including the positve-strand RNA deformed wing virus DWV; , a majory, DWV-C . Prevalastructor , increasstructor ,8, therestructor . Prior td orally ,5, and v Islands . Indeed,roa mite . This sud France –14 were ney bees . The mosney bees . AdditioV levels . Interespredator . Togetheispecies , the firispecies , wherebyVarroa-infested colonies compared with prior population surveys of the Varroa-associated DWV carried out in the UK in 2013 [Varroa-infested colonies in the Hawaiian Islands [Varroa-infested colony sourced in July 2013 was nearly clonal (Varroa-infested colony sourced in October 2015 in Maryland (Honey bee viromes comprehensively characterized by next-generation sequencing (NGS)  had surp Islands  and UK [Varroa-infested bees across the mainland US and has existed since at least 2010.Indeed, analysis of the divergent position distribution in a virulent DWV population from a US colony that collapsed 3 months after sampling showed that it had 5.6% polymorphic nucleotides across the viral genome, in which the proportion of the alternate nucleotide exceeded 3%, generating 1.8% amino acid substitutions , V99. NGVarroa-infested Maryland colony  produced for this study from a d colony . All clod colony , which wd colony , furtherVarroa infested, contained honey bees showing wing deformities consistent with high DWV levels [n = 5) and parental population  had highly similar distributions of divergent nucleotides (n = 286) and amino acids (n = 36)  virus levels were observed in pupae injected with PBS or with the mutant transcripts 304Δ and 306Δ, in which essential viral genes required for replication were deleted  . Shannon(n = 36)  with thaed pupae  4 days p deleted . Becauseroa free , this vi7 genome copies of either clone-derived or wild-type DWV (P > 0.01). Consensus DWV sequences at 3 dpi were identical to their corresponding cDNA clones in all but one case, likely because of a background DWV infection in the injected pupae (P < 0.0001) than 0-dpi pupae sampled immediately after injection with the virus, which had the same levels as PBS-injected controls  between DWV levels in pupae infected with either individual clone-derived isolates, all possible pairwise combinations of these isolates, or a mixture composed of all five isolates (P = 0.9825). Such a tight correlation between theoretical and observed profiles for mixed infections suggests that each of five coinfected genotypes replicated to similar levels in the reassembled population. NGS analysis of infections induced by pairwise clone-derived isolate mixtures  with dS/dN < 1, which are potentially subjected to diversifying positive and/or relaxed selection. Interestingly, 12 of these putative positively selected codons were located in the LP region, a 10-fold higher density than in the rest of the genome  to nonsynonymous (dN) substitutions (dS/dN) in the tested infectious clones reaching 24.7, indicating strong purifying selection . Accumule genome .To estimate the potential of US DWV populations to evade specific RNAi established to a single given DWV strain, we analyzed DWV NGS libraries from a typical colony, a single honey bee sampled in 2015, and a pool of eight honey bees sampled in 2010 —i.e., thVarroa infestation levels were fed with 1 μg of the dsRNA (ds304 or ds422) in 5 μL of 50% sucrose either with or without the virus extract containing 108 genome equivalents of the clone-derived DWV-304 or DWV-422. In order to control for the impact of non-sequence-specific triggering of antiviral response by dsRNA, a phenomenon reported in honey bees [We sought to experimentally test whether natural variation between DWV genotypes coexisting in a single population was sufficient to significantly impact the effectiveness of antiviral RNAi and, indeed, could act as an RNAi evasion mechanism. It was shown previously that antiviral RNAi in honey bees could be induced by introduction of double-stranded RNA (dsRNA) molecules orally . To do tney bees ,32, the P < 0.05, ANOVA. This was observed in two independent experiments using honey bees from different colonies with different wild-type background DWV levels  compared with the group “DWV-304/ds422” (which received ds422 mismatching the inoculated virus), P = 0.000185  were equally capable to establish high-level DWV infection when injected into the honey bee [Varroa-adapted DWV variants might involve large numbers of the mite-infested bees and considerable time to allow emergence of mutant variants of DWV that are virulent. This is supported by another study, which found that Varroa was present in honey bee colonies on the island of Fernando de Noronha for over 30 years, but DWV in the islands’ 20–40 honey bee colonies replicated only to low levels. It was suggested that, because of the small number of colonies, there was no chance to generate a virulent DWV variant. Importantly, the islands’ bees were susceptible to European virulent Varroa-selected DWV [Varroa-mediated transmission, survival and propagation of DWV became less dependent on the survival of infected bees, thereby allowing for the propagation of viral variants with increased replication levels and virulence. It is unclear which genetic changes are associated with a Varroa-mediated transition, but selection of certain strains of DWV may be involved, such as recombinants between DWV-A and DWV-B [Varroa mites [Varroa mites  are very common in the US. The DWV populations in individual bees sampled in 2015 in Maryland showed significantly higher diversity than the DWV in the UK 2013 study [Analyses of DWV population genetics provides insights into factors that contribute to sharp increases in virulence. DWV populations with different degrees of virulence are available, and the emergence and spread of highly virulent DWV has been linked to the spread of the mite structor ,6. Varro in 1987  and is c in 1987 , which r in 1987 ,11. Alth in 1987 , the mit in 1987 ,13. As acted DWV . Throughnd DWV-B –14,37. Aoa mites , it is poa mites ,13. It we manner ); howeveganglion , as wellganglion , from 3%ganglion ,13. Indeivergent . These Divergent  and closivergent . This miP = 0.7408, US 2015: P = 0.326, ANOVA). This suggests that individual bees sampled in the UK were infected with the same or very similar DWV variants, whereas individuals in the US were infected with many divergent genetic variants of the virus. Indeed, similar distribution profiles of polymorphic positions were observed in the NGS libraries of individual bees and a colony-level population sampled in the US  in a viruses . This pr viruses  and Infl viruses , 50.Varroa to the US mainland occurred in the 1980s [Varroa introduction, and/or samples from Hawaii prior to the Varroa introduction, would serve to confirm the role of Varroa in the selective sweep and significantly reinforce our model.A rapid expansion of one of many low-frequency genetic variants of a virus, which occurs due to increased fitness, will results in the temporary reduction of overall virus population diversity . Indeed,he 1980s ,34. AlthOur study of DWV has provided new insights into the dynamics of the divergent DWV population structure using a reverse-genetics system and high-throughput sequencing approaches. This system was particularly well suited to investigate interactions between viral genotypes within an invertebrate RNA virus quasispecies. The high genetic diversity of DWV and its potential to instantly respond to the pressures of sequence-specific RNA-mediated antiviral defense should be taken into account for the further development of antiviral interventions, including those based on RNAi  or superVarroa-infested honey bee colonies maintained by the USDA-ARS Bee Research Laboratory , which had high Varroa infestation (15%–20% of pupae infested with mites), and bees showing wing deformities consistent with high DWV levels were used as the source of mite-exposed honey bee pupae for NGS analysis and isolation of DWV particles in October 2015. Unparasitized honey bee pupae from colonies with low Varroa infestation (with below 1% of mite-parasitized pupae) were used in the injection experiments. Newly emerged pupae from the Maryland colonies with low Varroa infestation were used in the oral infection experiments. A colony from Texas sourced in January 2017 was used in the in vitro RNA transcript and wild-type RNA injection experiment. A colony from Florida sourced in February 2017 was used in the experiment involving injection of virus extracts, both clone-derived and wild-type.−5. To quantitatively assess the degree of similarity between different DWV genetic diversity profiles, we calculated the Pearson’s correlation between the Shannon’s diversity index profiles averaged for 100-nt windows. For detection of recombination breakpoints in the honey bee pupae injected with the mixtures of the clone-derived DWV genotypes, cleaned data from each sample NGS library were individually aligned to the VDV1 and DWV references using SpeedSeq (version 0.1.2). Structural variants were detected using the lumpy smooth script from the LUMPY package (version 0.2.13), and additional genotype and coverage metrics for each variant were calculated using SVTyper, a script within the SpeedSeq package. Breakpoint positions representing a recombination between two different viral isolate sequences were considered for further analysis if they had evidence from more than 10 supporting events (split or discordant reads) and exceeded 0.02% of NGS coverage . Phylogenetic analysis was carried out by aligning the viral nucleotide sequences using CLUSTAL and producing maximum likelihood and neighbor-joining trees, which were bootstrapped using 1,000 replicates with the RAcML and PHYLIP packages, respectively. dS and dN variations in viral genomes were identified using all known full-length North and South American DWV-A sequences ; putatiAnalysis of DWV diversity in Hawaiian (Oahu Island) apiary-level samples collected in 2015 as a part of the USDA-APHIS honey bee survey  includedVarroa-infested colony at the Bee Research Lab in Beltsville, Maryland, USA, in October 2015 as described previously [PmeI restriction site  according to the manufacturer’s instructions. After 3 hours of incubation at +37 °C, the DNA templates were digested using TURBO DNase (Life Technologies). The RNA transcripts were purified using the RNeasy mini kit (Qiagen), eluted with RNAse-free sterile water, and stored at −80 °C prior to use.Viral particles were purified by caesium chloride gradient centrifugation from a pool of 50 pupae out of a single eviously . Viral Rion site . The cDNion site , and theion site  containieviously  and in tVarroa mite feeding exposure. These pupae were extracted from brood frames with low Varroa infestation (approximately one mite-infested cell per 500) from a Texas apiary in December 2016. The pupae at pink eye stage were injected intra-abdominally into the hemolymph with 5 μg (13.2 × 1012 copies) of the in vitro transcripts or wild-type DWV RNA in 10 μL of PBS or 10 μL of PBS control using syringes with a 0.3-mm needle G31 (BD Micro-Fine), essentially as described previously [12 copies of the control nonreplicating DWV in vitro transcripts p304Δ and p306Δ had between 1.3 × 105 and 1.7 × 107 genome copies detected (RNA transcripts were injected into honey bee pupae without detected . For comdetected  1.g for 5 minutes and filtration through a 0.22 μm nylon filter (Millipore). The DWV concentration in the extracts was quantified by qRT-PCR, and the extracts were stored at −80 °C prior to use. To confirm the identity of the clonal DWV in the extracts with their respective parental DWV cDNA clones, the DWV RNA region, positions 30–1,266 nt, was amplified by RT-PCR, sequenced, and also digested with PacI restriction enzyme.For preparation of the clonal DWV extracts containing infectious DWV virus particles, individual transcript-injected pupae were homogenized with 2 mL of PBS at 3 dpi. For each individual pupal extract, 1 mL was used for preparation of the DWV inocula, which included clarification by centrifugation at 3,000Varroa mite feeding, were extracted from a Florida brood frame with low Varroa infestation (1 infested cell per 500) in January 2017. Pupae were intra-abdominally injected into the hemolymph to introduce either the filtered DWV extracts (107 genome copies in 10 μL of PBS), which included the clone-derived recovered DWV variants that originated from transcript-injected pupae, the wild-type DWV, or buffer control (PBS). We sampled time series from “time 0” (immediately after injection) through to 3 dpi, and the DWV RNA copy numbers were quantified in individual bees by qRT-PCR as described previously [Pink eye–stage honey bee pupae, with no exposure to eviously .8 clone-derived virus particles, DWV-304 or DWV-422, or no virus in the case of control groups (Varroa infestation levels (less than 1% of pupae being infested with the Varroa mites) and were starved for an additional 3 hours before feeding. The bees were kept for 7 days essentially as described in [ad libitum sources of water and 1:1 sucrose/water syrup (w/v), prior to RNA extraction.dsRNAs specific to the DWV clones were produced in vitro with T7 RNA polymerase  using as the templates PCR fragments corresponding to the positions 1,242–1,524 of the clones DWV-304 and DWV-422  with T7 promoter sequences at both the 5′ and 3′ termini. These PCR fragments were amplified using the US DWV cDNA clones p304 and p422 as the templates and the primers shown in l groups . The honribed in  in cagesRaw sequence data and consensus DWV sequences have been deposited at DDBJ/EMBL/GenBank within BioProject PRJNA431793 under the Sequence Read Archive (SRA) study SRP135682 (accessions SRR6833910–SRR6833954) and Transcriptome Shotgun Assembly (TSA) study GGSE00000000 (accessions GGSE01000001–GGSE01000045), respectively. Cloned viral cDNA sequences are available in GenBank with accession numbers MG831200–MG831204, MH069503–MH069507, and MH594118–MH594121.S1 FigNGS DWV-A read coverage (left panels) and Shannon’s diversity profiles (right panels) for the nonstructural gene region and 3′ UTR regions analyzed in (PDF)Click here for additional data file.S2 FigVarroa-infested colony sampled in 2015 (“Clone-”). Stars indicate the DWV cDNA clones designed in this experiment and tested for infectivity. Suffix “-Cons” indicates consensus NGS sequences. Green nodes and labels indicate NGS consensus sequences for DWV from individual honey bee pupae. Scale shows genetic distance (%). Isolates are labeled with their clone identifier or country of origin as well as their NCBI accession. DWV, deformed wing virus; IRES, internal ribosome entry site; NCBI, The National Center for Biotechnology Information; NGS, next-generation sequencing.A maximum likelihood phylogenetic tree was generated for a 9.7-kb DWV genome section (position 472 in the 3′ IRES to the 3′ poly(A) sequence, position 10,162), which includes the entire protein-coding sequence. Bootstrap values for 1,000 replicates are shown for the groups with more than 50% bootstrap support. Red nodes connect the cloned sequences from a Maryland (TIFF)Click here for additional data file.S3 FigVarroa-infested honey bee pupae. (C-E) Clone-derived DWV-A virus particles in filtered tissue extracts from honey bee pupa injected with in vitro transcript from full-length DWV-A cDNA clone 304. The virus particles on carbon-coated grids were negatively stained with sodium 1% phosphotungstic acid and observed at 80 KV with a Hitachi HT-7700 transmission electron microscope. The arrows indicate some filled DWV-A particles in virus preparations  and all DWV-A particles in the filtered extracts (C-E). Bar, 100 nm. DWV, deformed wing virus. Purified wild-type DWV-A virus particles isolated from (TIFF)Click here for additional data file.S4 Fig6 copies of each, and analyzed by NGS at 3 dpi. Overall ratios between the clone-derived DWV isolates in the progeny, calculated as an average of the expected polymorphic nucleotide proportions ± SD, are shown for each combination. The top panels show recombination breakpoints, identified using the LUMPY package, and their proportions in relation to the NGS read coverage. Only the recombination sites with more than 10 supporting events exceeding 0.02% of the NGS coverage are shown; black and red diamonds show the recombination sites . In the lower panels, the orange bars show ratios between the expected polymorphic nucleotides for each pairwise combination along with trend lines. The black bars show the ratios of the second most abundant nucleotide to the total coverage at the positions, which were not polymorphic for a given genotype pair, indicative of the background wild-type DWV infection. For each graph, x-axes show nucleotide position in the DWV genome and y-axes show recombinant read proportions (top panes) or polymorphic nucleotide ratios (bottom panes). dpi, days postinoculation; DWV, deformed wing virus; NGS, next-generation sequencing.Individual honey bee pupae were injected with two separate clone-derived DWV isolates, 5 × 10(TIFF)Click here for additional data file.S5 FigAlignment shows divergent positions in the full-length North and South American DWV sequences and in the infectious US DWV cDNA clones. Codon numbers are indicated below. Positions marked with “+” correspond to putative codons under positive diversifying selection in DWV strain from Pennsylvania (AY292384), which is used as the reference genotype. DWV, deformed wing virus.(TIFF)Click here for additional data file.S6 FigVarroa-infested apiary on Oahu Island, Hawaii, in 2015;  an individual honey bee pupa injected with clone-derived DWV-304;  an individual honey bee pupa infected with Maryland wild-type DWV (2015 sample source). Divergent positions are indicated with arrows. DWV, deformed wing virus; LP, leader protein; RdRpol, RNA-dependent RNA polymerase; RT-PCR, reverse-transcription PCR.Electropherograms of the direct sequencing of RT-PCR fragments corresponding to sections of DWV RNA: (A-C) LP region  and (D-F) RdRpol fragment Click here for additional data file.S7 FigVarroa-infested colony sampled in 2015. Bootstrap values for 1,000 replicates are shown for the groups with more than 50% bootstrap support. Scale bar shows genetic distance (%). Isolates are labeled with their clone identifier or country of origin as well as their NCBI accession. DWV, deformed wing virus; IRES, internal ribosome entry site; LP, leader protein; NCBI, The National Center for Biotechnology Information.Maximum likelihood phylogenetic tree corresponds to the section of the DWV genome coding for the LP and adjacent parts of 5′ IRES and the structural gene block . Blue nodes connect the cloned sequences from a Hawaiian apiary (Oahu Island) sampled in 2015; red nodes connect the cloned sequences and the full-length DWV genomes from a Maryland (TIFF)Click here for additional data file.S1 TextNucleotide alignment of the 283-nt section of DWV isolates DWV-304 and DWV-422 (GenBank accession numbers MG831200 and MG83120), positions 1,242–1,524 in the DWV genomic RNA, which were used to generate dsRNA. dsRNA, double-stranded RNA; DWV, deformed wing virus.(PDF)Click here for additional data file.S1 Tablehttps://www.ncbi.nlm.nih.gov/sra/SRP135682. NCBI, The National Center for Biotechnology Information; NGS, next-generation sequencing.NCBI Sequence Read Archive accession SRP135682, (XLSX)Click here for additional data file.S2 TableDWV, deformed wing virus; NGS, next-generation sequencing; NS, nonstructural; RdRp, RNA-dependent RNA polymerase.(XLSX)Click here for additional data file.S3 TablePearson’s correlation for Shannon's diversity profiles for a 100-nt window. The degree of shade indicates the correlation coefficients: no shade, below 0.5; light shade, above 0.5–0.7; medium shade, from 0.7 to 0.9; and dark shade, above 0.9. DWV, deformed wing virus.(XLSX)Click here for additional data file.S4 TableDWV, deformed wing virus.(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 DataSeparate data sheets of this Excel document, which are accordingly named, contain data for Figs (XLSX)Click here for additional data file.