Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. IBDV has a bi-segmented double-stranded RNA genome. Segments A and B encode the capsid, ribonucleoprotein and non-structural proteins, or the virus polymerase (RdRp), respectively. Since the late eighties, very virulent (vv) IBDV strains have emerged in Europe inducing up to 60% mortality. Although some progress has been made in understanding the molecular biology of IBDV, the molecular basis for the pathogenicity of vvIBDV is still not fully understood.in vivo reassortant or mosaic recombinant viruses derived from the 88180 and the attenuated Cu-1 IBDV strains. The reassortant virus rescued from segments A of 88180 (A88) and B of Cu-1 (BCU1) was milder than mc88180 showing that segment B is involved in 88180 pathogenicity. Next, the exchange of different regions of BCU1 with their counterparts in B88 in association with A88 did not fully restore a virulence equivalent to mc88180. This demonstrated that several regions if not the whole B88 are essential for the in vivo pathogenicity of 88180.Strain 88180 belongs to a lineage of pathogenic IBDV phylogenetically related to vvIBDV. By reverse genetics, we rescued a molecular clone (mc88180), as pathogenic as its parent strain. To study the molecular basis for 88180 pathogenicity, we constructed and characterized in vivo pathogenicity of IBDV, independently of the replication efficiency of the mosaic viruses.The present results show that different domains of the RdRp, are essential for the  Infectious bursal disease virus (IBDV) is a pathogen of worldwide significance to the poultry industry. IBDV destroys the immature B lymphocytes in the bursa of Fabricius (BF) of chickens, thus causing immunosuppression, and clinical disease mainly occurs between 3 and 7 weeks of age Birnaviridae, genus Avibirnavirusi) the outer capsid, VP2, the maturation, tri dimensional structure and major immunogenic determinants of which have been described ii) the virus protease, VP4 iii) an internal protein, VP3, that binds to viral dsRNA to form ribonucleoprotein complexes in vitroi) an N-terminal domain (residues 1–167) ii) the central polymerase domain (residues 168–658) which contains the “Fingers”, “Palm” and “Thumb” subdomains and iii) a C-terminal domain (residues 659–878) Birnaviridae as compared with other viral RdRps 2+ dependence of this polymerase IBDV is a bisegmented double stranded (ds) RNA virus belonging to the family Although some progress has been made in understanding the molecular biology of IBDV, the molecular basis for the pathogenicity of vvIBDV is still not fully understood. The study by reverse genetics of the genes or genomic regions of vvIBDVs involved in pathogenicity is particularly challenging because these viruses are not adapted to cell culture. Currently, the vvIBDV phenotype is still largely defined by a significantly increased mortality following challenge. Segment A-encoded proteins do play a role, as demonstrated by reverse genetics studies where a mosaic classical virus containing the VP2 of a vvIBDV strain did induce lesions of the BF. However the mosaic virus failed to induce either morbidity or mortality in young chickens, thus demonstrating that VP2 of vvIBDV was not the sole determinant for their pathogenicity. In addition, the introduction of VP4 or VP3 of a vvIBDV into the genetic background of a classical virus did not confer any pathogenicity The literature regarding the influence of segment B on IBDV phenotype reveals somewhat conflicting results. It was suggested in an early study that segment B of vvIBDV has a limited influence on pathogenicity In the present study, we investigated the molecular basis for the pathogenicity of IBDV strain 88180, previously characterized in our laboratory as the only known representative of a lineage of pathogenic IBDV with significant phylogenetic relationships to vvIBDV Because this study provided the first full length sequences of an IBDV isolate belonging to an original genetic lineage In segment A, the sequence of the 5′ NCR of 88180 exhibited 4 or 5 nt changes as compared with Cu-1 or D6948, respectively. The overall predicted secondary structure of the A5′NCR was more similar with that of Cu-1 and one nucleotide change found in 88180 (44C) restored a predicted stretch of paired nucleotides  also predicted in D6948 (data not shown). The predicted VP5 protein of 88180  was four aa shorter than in D6948 at its N-terminal extremity, as also found in Cu-1, and exhibited several aa changes in its putative cytoplasmic domain. In the predicted polyprotein, the VP2 precursor (pVP2) of 88180 differed from Cu-1 and D6948 at 13 and 6 aa positions, respectively. All but one of these changes were located in the so-called “antigenic domain” of VP2, and 3 of the differences observed with D6948 affected aa positions conserved in typical vvIBDV . The only other change  affected the pep46 peptide released upon the maturation process of VP2. The deduced VP4 protein of 88180 was much similar with VP4 of D6948 (only one aa change), but exhibited 6 aa changes as compared with Cu-1, two of these changes (aa 541 and 751) occurring in regions of the protease possibly important for the cleavage at the VP4-VP3 and pVP2-VP4 junctions, respectively. VP3 of 88180 exhibited 1 aa change as compared to its D6948 counterpart in the putative RNA-fixation site (aa 990) and 3 aa changes as compared with Cu-1. Two of the 3 latter changes (aa 981 and 990) were present in the putative RNA-fixation site and one (aa 1005) in the VP1 interaction site. Finally, nts changes occurring in the 3′NCR of segment A did not appear to cause drastic changes in the predicted secondary structure of the NCR (data not shown).In segment B, the 5′ NCR of 88180 exhibited 6 and 3 nt changes as compared with Cu-1 and D6948, respectively. None of these combinations of changes modified the predicted secondary structure of the NCR. Interestingly, this structure included the base-pairing of nts 53–56 with nts 62–65, hence forming a hairpin structure close to a terminal loop, and base-pairing was maintained at these positions although both nts of the 55–63 pair were modified in Cu-1 . This suggests that the secondary structure at this position could be important for the function of the B5′ NCR. Comparison of the VP1 aa sequence of the three viruses revealed 23 aa differences between Cu-1 and 88180, and 11 differences between 88180 and D6948. In both comparisons, most amino acid changes were quite evenly distributed along the ORF, however both pairs of viruses exhibited aa differences located close to the catalytic domain of VP1 . Out of the 8 aa positions that had been previously reported to be unique and common to all vvIBDV The nt sequences of the A and B genome segments of 88180 were finally compared, using phylogenetic methods, with their counterparts in a panel of vvIBDV and classical strains retrieved from databanks The antigenicity of mc88180 was first compared with that of the 88180 virus. Both viruses exhibited the same antigenic profile as previously reported  8.2 EID50/g whereas the three other viruses yielded comparable and significantly lower virus titers . None of the other observed criteria , ACU1B88 5% and mcCu-1 0%. The two latter groups did not differ significantly with respect to morbidity, but differed significantly (p≤0.05) from both mc88180 and A88BCU1 (data not shown). Only mc88180 induced mortality, although at a lower rate than in the previous experiment (5%) .5.3±1.6 EID50/g detected in qRT-PCR, 2, 5.8±0.3 EID50/g detected in qRT-PCR) and proved both highly pathogenic  and immunogenic (mean VN titer 11.6±1.3 log2). The two reassortant viruses exhibited markedly different properties. On the one hand, ACU1B88 replicated to a significantly lower extent in the bursa , it hardly induced any bursal lesion  and proved poorly immunogenic . These results suggested an infectivity even lower than that of mcCu-1 which induced significantly more bursal atrophy, histological lesions and antibodies at 20 DPI . On the other hand, A88BCU1 replicated as well as the mc viruses (105.5±0.3 EID50/g detected in qRT-PCR) and induced marked bursal atrophy (mean b/B ratio at 20 DPI = 0.7+0.2‰), severe histological lesions  and high levels of antibodies (geometric mean VN titer = 9.2±2.3 log2).Analysis of the other criteria see  confirmeTaken together, our results showed the low compatibility of the segment A of CU1 with the segment B of 88180 and also confirmed the paramount role of segment A in the pathogenicity. However, all criteria but the virus titer indicated also a slight but significant (p≤0.05) reduction of the pathogenicity of A88BCU1 as compared with mc88180 , in agrein vivo for their pathogenicity as before. As in the previous experiments, the mock-inoculated group kept uninfected throughout the experiment. Calculation of a newly designed mean symptomatic index  only, a mixed virus population was detected. Indeed, at aa position 281, the virus pool contained the Glutamic acid used as a genetic tag for mc88180 as well as the Glycin corresponding to the aa found in wild-type 88180. This result shows that mc88180 partly reverted to the wild-type sequence in experiment i), which could explain the highest pathogenicity of mc88180 observed in this specific experiment. Further studies would be required to assess precisely the role of aa 281 in 88180 pathogenicity. Whatever this may be, the intensity of the clinical signs induced by mc88180 in the successive experiments was enough to investigate the role of the segments A and B in the pathogenicity of 88180, by comparison with the attenuated Cu-1 virus.The mc88180 virus was shown to exhibit the same phenotype as its parental 88180 strain, as demonstrated by a similar antigenic reactivity and ability to induce morbidity and mortality. However, some variation in the mortality induced by mc88180 was observed in our three animal experiments, as the mc88180-induced mortality ranged 5 to 27% . Although mortality in the first experiment (27%) did not differ significantly from that induced by the 89163 vvIBDV control virus (30%), the percent mortality observed in subsequent experiments was lower than would have been expected in our experimental model for a typical vvIBDV Complete sequencing of genome segments A and B of IBDV strain 88180 revealed that both genome segments are related to their counterparts in vvIBDV but are nevertheless significantly different. This result confirmed previous analysis performed on partial sequences from this virus strain Reassortant viruses derived from mcCu-1 and mc88180 were constructed in order to check whether some genome segments or the compatibility between the segment A- and segment B- encoded proteins could be major determinants of the 88180 pathogenic phenotype. Although A88BCU1 apparently replicated to the same extent as mc88180, we observed in two independent animal experiments that this virus was significantly less pathogenic than mc88180 . This suggests that BCU1 played a role in the reduced pathogenicity of this reassortant virus. This hypothesis is in agreement with a previous study by our group showing that a naturally occurring reassortant virus, with a vvIBDV-derived segment A and an unrelated segment B, had a reduced pathogenicity in spite of a replication ability at 4 DPI similar to that of pathogenic IBDVs mc88180 and A88BCU1 but not ACU1B88 induced morbidity, mortality (at least for mc88180), and severe bursal lesions. This suggests that segment A of 88180 played a critical role in determining the pathogenicity of the reverse genetics-generated viruses. This is unlikely to be linked to bursa tropism only, as ACU1 has also been shown previously to confer the ability to replicate in bursal cells ii) was performed with the original low-titer virus stock. As both the mcCu-1 and mc88180 replicated well in the bursa of the inoculated chickens in the same experiment, the lower replication of ACU1B88 cannot be attributed to a single genome segment, but rather to a low segment compatibility. A lower or delayed replication capacity has also been described in laboratory constructed reassortant IBDV, either derived from the Cu-1 and the serotype 2 23/82 strains trans-activate the synthesis of VP1 In addition to the predominant role of segment A88 in pathogenicity, our results also demonstrated the limited compatibility of segments ACU1 and B88. Indeed, reassortant virus ACU1B88 was not pathogenic  and replicated poorly as compared with all other viruses inoculated in the same experiment. This low replication capacity was already apparent at the rescue step. It could not be improved by one further passage of this virus on SPF chickens (data not shown), which explains why the ACU1B88 challenge in experiment Quite discrepantly as compared with ACU1B88, segment incompatibility was not observed with the other reassortant virus, A88BCU1, which induced bursal damage and the same virus titer as mc88180 in BF at 4 DPI . Hence, a reassortant virus with a segment B derived from a cell-culture-adapted IBDV strain (mcCu-1) replicated well in the bursa. This is in contrast with previous reports studying reassortant viruses derived from bursa- or cell-culture- propagated viruses in vivo pathogenicity. The exchanged regions within segment B of Cu-1 and 88180 were defined based on the previously determined putative functional motifs within the IBDV RdRp  and the pathogenicity of the mosaic viruses. The low fitness and pathogenicity of the reassortant ACU1B88 virus suggested that these two traits are both affected in this virus. However, for A88BCU1 and the mosaic viruses their fitness and virulence seem to be two separate traits. This is somewhat comparable with what has been described for the foot-and-mouth disease virus (FMDV), for which viral fitness and virulence are two different virus traits Our mosaic segment B experiments showed that the D1 and especially the D2 domains play an important role in pathogenicity. The reduced pathogenicity in viruses with heterologous D1 and D2 domains  suggests that the compatibility between D1 and D2 of the RdRp, , which is affected when one out these two domains comes from a different genetic background, is required for pathogenicity, although the disruption does not interfere critically with the ability of the viruses to replicate. Interestingly, the recombinant virus incorporating only domain D2 of Cu-1 was milder than its D1 counterpart as it induced significantly less bursal atrophy and histological lesions than B88[D1CU1] or mc88180. This suggests that the Palm 2 and Thumb subdomains of the central domain and C-terminal domain of the polymerase could be involved in functions or interactions more important for pathogenicity. Whether the more important role of D2 could be related to one of the D2 aa differences between Cu-1 and 88180 that is located in motif E of the RdRp is not yet known.in vitro replication ability but only had a limited influence on bursal lesions. The different impact of the NCR exchange is most probably due to the secondary structures of the exchanged NCRs being very different in the two studies .The same level of replication of the mosaic viruses compare to mc88180 suggests that irrespective of their mosaic structure, all RdRps interacted efficiently with the 3′ and 5′ NCRs originating either from Cu-1 or from 88180. Hence, incompatibility of the segment A- and segment B- NCRs was not the only cause for the reduced pathogenicity of A88BCU1. However, the mirror experiment remains to be performed and it cannot be excluded that introducing partial or full length NCRs of Cu-1 in the B88 segment would also result in attenuation. Altogether, these results can hardly compare with a previous study by Schröder et al. Finally, our results also showed that restoring the catalytic domain Dc of 88180  either the incorporation of both the D1 and D2 of B88 , or ii) possibly the restoration of the whole interaction domain of the RdRp with VP3 iii) even the restoration of the whole B88 segment. This demonstrates that different regions of the polymerase are essential for IBDV pathogenicity, even without any impact on virus replication. This is evocative of results reported in Picornaviruses, in which complex interactions involving different domains of the polymerase  critically contribute to the control of the fidelity of the polymerase, Altogether, our results suggest that restoration of the full pathogenicity of A88BCU1 could require All experiments were performed in agreement with national regulations on animal welfare and animal experiments, according to authorisations N°22-4 and B-22-745-1, by Prefecture des Cotes-d'Armor, France. The rescue of IBDV recombinant viruses and their in vivo characterization (see below) were performed according to authorization n° 3569 CA-I by the French Commission for Genetically Modified Organisms.50).IBDV strain 88180 Transfection experiments were performed using CEF derived from SPF hen eggs (Anses-Ploufragan/Plouzané laboratory) grown in BHK-21 medium (Eurobio) supplemented with 0.3% Tryptose Phosphate Broth (TPB), 5% foetal calf serum (FSC), 200 U/ml antibiotics (streptomycin and penicillin) and 2 µg/ml fungizone. Animal experiments were performed using five-to-seven-week-old SPF White Leghorn chickens (Anses-Ploufragan/Plouzané laboratory) maintained in level 3 containment facilities in filtered-air negative-pressure isolation units.E.coli (Invitrogen) of the resulting plasmids followed by plasmid extraction (Qiagen) were performed according to the manufacturer's protocol. Sequencing of the resulting cDNA clones was repeated until at least five clones with the longest and same nucleotide sequence had been analysed.Virus RNA was extracted from infected bursae as described previously EcoRI or the XbaI restriction site in primers designed for segments A or B, respectively. The resulting cDNA was amplified in PCR reactions performed as described above and based on primer pairs consisting of the same sense primer as used for RT, combined with an antisense primer with a 5′ extension corresponding to a cloning site  followed by a linearisation site . The conditions for the amplification of the full-length segments were 35 cycles with denaturation at 95°C for 30 sec, annealing for 45 sec  and extension at 72°C for 3 min. The PCR products were purified as above. After enzymatic digestion of the PCR products with EcoRI only (segment A) or XbaI and SmaI (segment B), each segment was ligated to the similarly cleaved pUC18 vector. The resulting plasmids containing the segment A or B sequence, designated as pA88 and pB88 for segments A and B, respectively, were transformed into E. coli, purified and sequenced on both strands as described above. Comparison of the full-length A and B segment sequences derived from the bursa propagated 88180 virus (used as the reference sequences) with the corresponding sequences of full-length cloned segment A and B  was performed. Three unintended nt mutations  and one (nt 2828) were present in pA88 and pB88, respectively. Mutation at nt 972 of pA88 was non-silent and induced the mutation from a Glycine residue to a Glutamic acid at position 281 and was conserved as a genetic tag to differentiate the molecular clone mc88180 to 88180 virus. The three other mutations, nts 3261, 3262 in pA88 and nt 2828 in pB88, were missing cytosine nts in the 3′ extremity of the 3′ NCRs and were corrected by site directed mutagenesis (Stratagene) with specific primers as described in Full-length single stranded cDNA copies of the A and B segments of the 88180 strain were produced by RT as described above, using oligonucleotide primers complementary to the 3′ end of the non-coding strand of segments A and B ( = “sense” primers) . The twoSspI, BstXI, PmLI and BsRGI conserved and unique restriction sites (position shown at i) NCRs, ii) catalytic domain (Dc) , iii) both the NCRs and the Dc iv) the NCRs, the Dc and the 5′ coding part of VP1 (D1) and v) the NCRs, the Dc and the 3′ coding part of VP1 (D2)  .i) To construct a clone of segment B of Cu-1 with the NCRs of 88180, the SspI-BsRGI fragment (approximately 3.2 kbp) was first excised from pB88 and replaced by its counterpart similarly derived from pBCU1, to obtain pBCU1[NCR3′ 88]. Then, the Ssp1-BstXI fragment of pB88 containing the 5′NCR of 88180 was subcloned into the similarly cleaved pBCU1[NCR3′ 88] to obtain pBCU1[NCRs88], corresponding of the B segment of Cu-1 with both the 5′ and 3′ NCRs of 88180. ii) and iii) The two mutations typical of Dc of 88180 (iv) To construct a clone of segment B of 88180 with D2 of Cu-1, the BstXI-PmLI fragment of pB88 was subcloned into the similarly cleaved pBCU1[NCRsDC88] to obtain pB88[D2CU1]. v) Finally, to construct a full- length clone of segment B of 88180 with D1 of Cu-1, the PmLI-BsRGI fragment of pB88 was subcloned into the similarly cleaved pBCU1[NCRsDC88] to obtain pB88[D1CU1]. The sequences of the cloned or mutated regions in pBCU1[NCRs88], pBCU1[Dc88] and pBCU1[NCRsDC88] were checked and the sequences of the two last constructs, plasmids pB88[D2CU1] and pB88[D1CU1], were completely determined as described above.of 88180  were intof 88180 , resultiPstI restriction site whereas the antisense primer carried an XmaI site. After PstI-XmaI digestion, the PCR product was cloned into the similarly cleaved POP24+(100A) containing a polyA EcoRI restriction site. In order to use the pUC18 plasmid  the PstI-EcoRI fragment of POP24+GFP100A was subsequently ligated into the similarly cleaved pUC18 plasmid to obtain the plasmid pT7GFPpolyA with the GFP gene under the control of the T7 promotor and with the EcoRI site used as linearisation site. Upon in vitro transcription this plasmid generates a 1047 nt- long GFP cRNA ending with a stretch of 100 A nucleotides, thus mimicking a polyadenylated GFP mRNA.Two primers (sequence available upon request) complementary to the 3′ extremities of both the coding and non-coding strands of the Green Florescent Protein gene  were used to amplify the GFP gene from the pcDNA3.1/Zeo/GFP plasmid, in a PCR as described above. The sense primer contained a 5′ extension carrying the T7 promoter preceded by a in vitro transcription as described by Mundt and Vakharia (1996) et al. (2004) in vitro transcription reaction was incubated for 1 h at 37°C. Synthesis of cRNA was confirmed by electrophoresis of 2 µl of the reaction mixture in a 1% agarose gel and visualisation under UV light.The plasmids containing the full-length cDNA copies of the native or chimeric IBDV genome segments or the GFP gene were linearised with the restriction enzymes specific for the restriction sites introduced at their 3′ ends by the primers listed in in vitro transcription/transfection was checked using pT7GFPpoly100A by visualizing fluorescence under UV light in the transfected cells at 48 h post transfection. Forty eight hours after transfection, the cells were scraped and harvested with supernatants and inoculated by the intramuscular route to SPF chickens . Four days after inoculation, the chickens were humanely killed and their bursae were harvested and processed for virus isolation as described previously The capped cRNAs were used, in various combinations, for transfection of CEF, as described previously The parent virus and its molecular clone were antigenically characterized using a previously described antigen capture ELISA (AC-ELISA) based on a panel of 8 different neutralizing anti-IBDV Mabs detecting at least 6 epitopes of the VP2 protein i) 88180 and mc88180, ii) mc88180, mcCu-1 and the corresponding reassortant viruses (A88 BCU1 and ACU1 B88) and iii) mc88180 and the recombinant viruses derived from mosaic B segments. A summary of each experimental design is presented in supplementary 5 EID50 by bird except in experiment ii) where the ACU1 B88 virus was inoculated with 103.6 EID50 by bird). In each experiment a group was kept as a mock-inoculated control receiving PBS only. Clinical signs until 10 days post inoculation (DPI) and cumulated mortality rates were measured. Bursae and blood were sampled at different times post infections. At each sampling date  and a symptomatic index that we specifically designed for the quotation of IBD symptoms. This index ranges from 0 to 3, with 0 meaning “lack of signs”, 1 meaning “typical IBD signs (ruffled feathers) conspicuous in quiet bird only, the bird stimulated by a sudden change in environment  appears normal, motility is not reduced”; 2 meaning “typical IBD signs conspicuous even when bird is stimulated, dehydration is apparent, motility may be slightly reduced” and 3 standing for “typical severe IBD signs with prostration or death”. In the third experiment, the mean symptomatic index (MSI) of the surviving chickens was calculated daily from days 1 to 10 PI for each inoculated virus.All histological examinations were performed by Dr M. Lagadic . Bursae of Fabricius from the different groups and from the different sampling times were examined individually. The severity of the IBD-induced histological lesions was quantified according to Skeeles' scale for bursal lesion scores (BLS) 50) per well of the CT IBDV strain (serotype 1), as described previously 2 of the last dilution resulting in 100% neutralization of the cytopathogenic effect. VN titers higher than 3.3 were considered as positive.Virus neutralization (VN) tests were performed in CEF, using 100 median tissue infective doses  for IBDV segment A. A pair of suitable primers and a FAM-labelled TAMRA-quenched probe  were def50) using a standard curve prepared from serial 10 fold dilutions of aliquoted and repeatedly titrated reference suspensions of the F52/70, 89163, mc88180 or mcCu-1 viruses.Briefly, a sample of BF (approximately 250 mg) was first ground weight/vol in PBS. The supernatant harvested after centrifugation at 12500 rpm for 10 min at +4°C was used for virus RNA extraction . The “high capacity cDNA archive kit” (Applied Biosystems) was used for RT. The RT mix  was incubated at 37°C for 1 h. The PCR was then performed according to the manufacturer's protocol in a 96-well format using the ABI Prism® 7000 Sequence Detection System (Applied Biosystems). The reaction mixture  was subjected to the following thermocycling conditions: 50°C, 2 min; 95°C, 10 min; 40 cycles of 95°C, 15 sec, 60°C 1 min. All cDNA samples were tested in duplicates. Data were analysed using the Sequence detection version 1.2.3 software (Applied Biosystems). The final results were expressed as cycle threshold (Ct) value, which is the cycle when the change in the emission of fluorescence by the reporter dye significantly passes a background threshold calculated by the software. Ct values were converted into virus titers  were compared using the Chi squared test. Small series of quantitative values were compared using the non parametric Kruskal-Wallis test for the one-way analysis of variance. All statistical analysis were performed using version 9.0 of the Systat software .The full-length nt sequences for segments A and B of IBDV strain 88180 were submitted to the EMBL database with accession numbers AM111353 and AM111354, respectively.Table S1References of the viruses rescued from the different combinations of cloned segments A and B. pACU1 and pBCU1 correspond to plasmids pUCCu-1A and pUCCu-1B, respectively, described by Zierenberg et al. (2004) (DOC)Click here for additional data file.Table S2Sequence of the oligonucleotide primers and probe used in this study.(DOC)Click here for additional data file.Table S3Summarized protocol of the three animal experiments. The three animal experiments were designed to: i) compare the pathogenicity of 88180 with mc88180, ii) compare the pathogenicity of mc88180, mcCu-1 and reassortant viruses derived thereof and iii) compare the pathogenicity of mosaic segment-B derived viruses.(DOC)Click here for additional data file.


Myrmeleon hyalinus and the habitat specialist Cueta lineosa. Previous research has shown that the foraging performance of the generalist is better than that of the specialist, even in the latter’s characteristic habitat. We illustrate that this apparent superiority of the habitat generalist does not manifest itself along other niche axes; rather, the habitat specialist holds a set of traits that provide an advantage under harsh environmental conditions. Specifically, C. lineosa has an advantage over M. hyalinus at high temperatures, exhibiting a higher survival rate and improved foraging success . C. lineosa is also more efficient in its energy budget, losing less mass during starvation and gaining mass more efficiently during feeding. This superior efficiency is a result of physiological adaptations as well as behavioural responses to harsh conditions. In conclusion, our results imply that the habitat specialization of C. lineosa has not led it towards an evolutionary dead-end.Classical ecological studies discussing specialization usually focus on species’ performance along one niche axis. This approach may overlook niche differentiation evident in another dimension which could explain species co-occurrence. The present research exemplifies a comprehensive approach to examining local adaptation. Specifically, we examined multiple niche axes by subjecting a model organism to various experimental conditions to monitor responses to extreme stress associated with heat, desiccation and starvation. Our model system comprised two pit-building antlions: the habitat generalist  Adaptation to local biotic  and abiotic  conditions is important for the survival and reproduction of any organism Escherichia coli have demonstrated that experimental populations always became better adapted to the temperatures at which they evolved Temperature is one of the most important physical factors that define a species’ fundamental niche. Temperature limits some species’ distributions High temperatures may be associated with either low or high relative humidity. These differences may be apparent when contrasting different climatic regions, such as desert vs. tropical climates, or even at smaller scales, when contrasting adjacent microhabitats 2. Insects maintained in dry air experience starvation and desiccation, whereas those maintained at higher humidity levels experience mainly starvation.Water availability and temperature are the two most important abiotic variables influencing the distribution and abundance of insects Small body size is commonly accepted as disadvantageous for insects in terrestrial environments because of their small water storage capacity, combined with a relatively large surface area, that enables faster cutaneous water loss Myrmeleon crudelis and M. carolinus, occupying shaded and open habitats, respectively, and found evidence for physiological adaptations to harsh environments in M. carolinus, at the expense of a reduced survivorship when interacting with other species. However, despite their preferences for different soil types, pit-building antlions may also construct pits in less desirable habitats Myrmeleon hyalinus Olivier, 1811, inhabits and performs equally well in both sand- and loess-derived soils Cueta lineosa Rambur, 1842, inhabits only fine-grained soils such as loess M. hyalinus larvae originating from desert habitats exhibit strong selectivity to sand M. hyalinus’ superior performance in both soil types seemed to show only part of the picture. An additional important difference in these species’ natural occurrence is their microhabitat selection: M. hyalinus is usually found under trees or bushes , while C. lineosa is found in open areas, exposed to direct sunlight. Since niche theory predicts that co-occurring species should show some level of niche separation C. lineosa should perform better than M. hyalinus along other environmental axes, related to arid conditions – i.e., high temperature, low relative humidity, and poor food abundance.Different species of antlions show major differences in their microhabitat preferences – soil type, particle size, shade, etc. Low and stochastic food abundance are characteristic of desert conditions, and animals originating from such conditions are expected to handle starvation periods better than animals from more benign habitats e.g., . AnimalsM. hyalinus, inhabiting shaded microhabitats, to lose body mass more rapidly during starvation than C. lineosa, which inhabits open areas, characterized by higher temperatures, lower relative humidity, and a low and stochastic prey encounter rate. Physiological differences in the ability of individuals to cope with starvation may be attributed to genetic differences, phenotypic plasticity or a combination of both. To test the effect of these factors, individual antlions’ ability to handle starvation stress should be examined under several different habitat conditions . If each species’ response to starvation is affected mainly by its local conditions, then we expected both species to respond in similar fashions when exposed to the same climatic conditions. However, it should be mentioned that G×E interactions may effectively 'hide' genetic differences under certain environmental conditions We hypothesized that antlion species originating from harsher environments, which experience less favorable conditions and lower prey encounter rates, should be less sensitive to starvation. Specifically, we expected M. hyalinus larvae under different tamarisk trees located in Nahal Secher , a sandy area 15 km south of the city of Be'er-Sheva, Israel, and brought them to the laboratory. M. hyalinus is the most abundant pit-building antlion in Israel C. lineosa larvae from the loessial plains near Be’er-Sheva . Occurring mainly in the Israeli Negev desert, C. lineosa also exists in several small populations located in central and northern Israel, but is restricted to light soils, such as loess M. hyalinus prefers shaded microhabitats C. lineosa is mainly found in open microhabitats exposed to direct sunlight We collected M. hyalinus) or loess (for C. lineosa). Antlion larvae were fed with mealworms ad libitum until the beginning of the experiments (habituation period). Larvae were weighed throughout the experiments to ±0.1 mg .The study was comprised of three complementary experiments: 1) Thermal stress experiment, investigating the behavioral responses and growth efficiency of the two species when exposed to increased temperatures, while also testing if these effects are consistent when using more realistic prey items such as ants, under field-representative temperatures. 2) Gradual heating experiment, to test whether the antlions respond differently to gradual heating vs. sudden heating. 3) Starvation and humidity experiment, testing responses to starvation under different relative humidity levels. In all the experiments, fresh antlions were collected in the field and kept separately in round plastic cups  filled with 3 cm of sand  were starved for one week. This procedure was used to standardize the hunger level of the antlions. It ensures that the physiological state of all antlion larvae is approximately the same at the beginning of the experiment. Thereafter, we put the antlions into a controlled environmental chamber  for three days, after which they were each fed with a single mealworm. The antlions were then exposed to three days of high temperature , and then fed another mealworm. An exception is one treatment in which the antlions were exposed to 45°C for only 24 hours. A temperature of 45°C for three days was found to be a lethal treatment for M. hyalinus, but not for C. lineosa, therefore the shorter treatment (in which both species survived) was added. At the end of the period of thermal stress, the temperature was reduced to 25°C for three days, and the larvae were fed again. By the end of each of the three phases, we monitored pit building activity, pit diameter, response to a single mealworm prey (yes/no), response time to prey  within each treatment using Pearson’s Chi-square. Pit diameter, response time to prey and growth efficiency were similarly compared between periods using Wilcoxon’s signed-rank test.Pheidole pallidula and Messor aegyptiacus) from the same locations described above. After the habituation period, all individuals were starved for one week. Thereafter, we placed the antlions in a controlled environmental chamber  for three days of high temperature (30°C or 40°C), after which they were each fed with a single ant. We measured each antlion’s response time to prey, and whether it succeeded in capturing the ant prey. Response time was compared between species and temperatures using ANOVA GLM, using the residuals after regressing response times against antlion body mass. Prey capture success was compared between species and temperatures using logistic regression. While temperatures in the first phase of this experiment were selected to exert the highest stress possible and examine the lethal temperature limit, milder, more realistic field temperatures were chosen in the second phase: 30°C being close to the mean temperature near Be’er-Sheva during the summer months  were starved for one week. Thereafter, all antlions were placed into one of two controlled environmental chambers  for seven days. In the first chamber, the temperature was kept stable for 3.5 days, after which it was abruptly increased to 40°C for the remaining 3.5 days. In the second chamber, the temperature was gradually and linearly increased  to 40°C. We weighed the antlions at the beginning and end of the experiment and measured their pit diameter periodically. At the end of the experimental period each antlion was fed a single mealworm in order to measure response time to prey and growth efficiency. Antlions were kept in plastic cups (10.5 cm diameter) filled with 3 cm of sand  or loess (for C. lineosa). To achieve a better understanding of the changes in body mass throughout the experiment, while minimizing pit disturbance, an additional group of antlions was kept in separate cups, under the same experimental conditions. Antlions in this second group were weighed more frequently, but their pit diameter was not measured, because of the frequent disturbance caused by the weighing. Relative growth rate was calculated according to the common formula  into one of two climate chambers, both set to the same temperature of 30°C (day:night length 12∶12 h), but to different relative humidity levels –30% (9.52 mmHg) vs. 70% (22.22 mmHg). This temperature was selected as it is close to daily average temperatures during the summer months, and takes into account the larvae’s inability to escape warm temperatures by burrowing deeper into the soil. Although field temperatures may reach peaks of over 40°C, they also decrease dramatically during the night. Since we wished to impose a steady but survivable desiccation stress, we chose a temperature closer to the daily mean, rather than the daily maximum. The antlions were starved for 60 days, while control groups were fed one mealworm per week. We weighed the antlions and measured their pit diameter periodically throughout the experiment. Also, at the beginning and end of the starvation period, one pre-weighed mealworm was given to each antlion in order to measure response time to prey, and growth efficiency. Pit diameter, response time, growth efficiency and relative growth rate (or mass loss) were compared between species and treatments using ANOVA GLM.All statistical analyses were carried out using STATISTICA, ver. 8.0.M. hyalinus were 29/30 (96.7%), 30/30 (100%), and 29/30 (96.7%) for 30°C, 40°C and 45°C/24 h respectively, and for C. lineosa 28/30 (93.3%), 29/30 (96.7%) and 27/30 (90.0%) for 30°C, 40°C and 45°C/24 h respectively . In the harsher treatment of 45°C/72 h, survival rates were 24/30 (80.0%) in C.lineosa, but no M. hyalinus survived . In the 50°C treatment, no antlions survived from either species.Survival rate did not differ significantly between species in the milder treatments: survival rates for M. hyalinus, 45°C/24 h) was there a significant decrease in the proportion of antlions that constructed pits . When comparing pit-building proportions before and after the heat period, no significant differences were found , indicating that the heat period caused no lingering effects.The tendency to construct pits  was consM. hyalinus , and returned to its original level when the temperature was restored to 25°C. In M. hyalinus, the proportion decreased significantly in the 45°C/24 h treatment . The proportion increased back when the temperature was restored to 25°C, but remained lower than the original level .The proportion of antlions that responded to prey  was largM. hyalinus  decreased significantly during the heating period in the 30°C  and the 45°C/24 h  treatments, but returned to original levels when the temperature was restored to 25°C. In C. lineosa, growth efficiency increased significantly during heat in the 30°C treatment , and decreased significantly during heat in the 45°C/72 h treatment . In all the treatments, growth efficiencies returned to their original levels when temperature was restored to 25°C.In C. lineosa responded faster . Similarly, C. lineosa were more successful in capturing prey when exposed to 40°C, while M. hyalinus were more successful when exposed to 30°C. . No difference was found between species . M. hyalinus showed a slightly higher mass loss rate in the gradual heating treatment , while in C. lineosa this trend was statistically significant , leading to a marginally significant interaction term . However, when separating the mass loss rate into two equal periods: the earlier, cooler 3.5 days vs. the later, warmer 3.5 days . In the undisturbed cups , and no difference was found between treatments . Growth efficiency of M. hyalinus at the end of the experiment (0.48±0.03) was significantly higher than that of C. lineosa . No differences were found between treatments . Concerning response time, generally, M. hyalinus responded to prey faster (3.14±0.56 s) than C. lineosa . Response times in the gradual heating treatment decreased in C. lineosa (3.42±0.67 s), and increased in M. hyalinus (4.13±0.87 s), compared to the sudden heating treatment . Pit diameter of M. hyalinus (4.33±0.19 cm) was larger than that of C. lineosa . However, the pit diameter of M. hyalinus decreased over the time of exposure, while the pit diameter of C. lineosa remained unaffected .Antlions in the group that was weighed and disturbed more frequently  lost mas3.5 days , we founbed cups , M. hyalM. hyalinus (51.25±0.97 mm) was larger than that of C. lineosa . In general, pit diameter decreased over time , but this trend was less prominent in the humid-air treatment  and more prominent in M. hyalinus, as opposed to C. lineosa, which did not show significant changes in pit diameter over time . Pit diameter of M. hyalinus was larger in the humid-air treatment , while C. lineosa showed no significant difference between treatments . In the control groups (which were fed regularly), high variances prevented finding significant differences in pit diameter between species  or between treatments .In the starvation treatments, pit diameter of M. hyalinus responded to prey faster than C. lineosa , and response times in the humid-air treatment were longer than in the dry-air treatment . Among starved C. lineosa, response times were shorter after the starvation (5.49±1.10 vs. 7.26±1.11 s before starvation), while in M. hyalinus they were slightly longer . This trend was more prominent in the humid-air treatment. In the control (fed) groups, relative humidity was not found to affect response time , but, as in the starved groups, M. hyalinus responded faster to prey .1,66 = 2.605, P = 0.111) or between treatments . No such differences were found in the control group, either .We found no difference in growth efficiency between species . Both species lost mass faster in the dry-air treatment . In the control (fed) groups .In the starved treatments , C. line) groups , C. lineC. lineosa, has an advantage over the habitat generalist, M. hyalinus, at high temperatures, exhibiting a higher survival rate, increasing its pit size, and improving both its response time to prey and its prey capture success. From the perspective of thermal adaptation C. lineosa is a high-temperature specialist, relative to the low-temperature generalist M. hyalinus. C. lineosa is also more efficient in its energy use, losing less body mass than M. hyalinus during prolonged starvation, and gaining mass more efficiently than M. hyalinus when fed regularly. Remarkably, this advantage of the habitat specialist over the habitat generalist is evident at high and low levels of desiccation stress. These results indicate that the previously reported apparent superiority of the habitat generalist Classical ecological studies discussing specialization usually focus on species’ performance along one niche axis, overlooking underlying mechanisms of species co-occurrence and coexistence M. hyalinus suffered more severely from the heating in the warmer phase of the experiment, in terms of body mass loss. In the undisturbed cups, differences between the species were consistent with our other experiments, with M. hyalinus constructing larger pits, losing mass more rapidly, and decreasing its pit size over time, compared to the relatively stable pit size of C. lineosa. Since the gradual heating is experienced by the antlions as the harsher treatment (faster mass loss), response times also agree with other results: C. lineosa, which is better adapted to harsh conditions, responds faster in the gradual heating treatment, while M. hyalinus responds faster in the sudden heating treatment. These results reinforce the notion that C. lineosa is a high-temperature specialist, while M. hyalinus is a low-temperature generalist.Antlions seemed to experience gradual heating as a harsher treatment than sudden heating, losing more mass in the former. In their natural environment, antlions experience large day/night temperature fluctuations, causing behavioural changes as a response C. lineosa, the species usually exposed to high temperatures, also performs better under these conditions, and thus M. hyalinus loses its advantage. These findings point towards a separation between the two species along the thermal niche axis. The effect of temperature on foraging activity has also been demonstrated in other insects, most commonly in ants C. lineosa did not seem to suffer less at low relative humidity, as might be expected, but perhaps exposure to higher temperatures (for a shorter time period), combined with the low relative humidity, may have brought forth different results. Perhaps in a short-term experiment with high temperatures and high relative humidity, M. hyalinus would have performed better than under dry conditions. It is important to note that the antlions’ exposure to high temperature in the lab is different than field conditions - under lab conditions the sand in each antlion’s cup heats up in a relatively uniform fashion, and the antlions can’t escape the high temperature by burrowing deeper into the soil, as is evident in field studies Classical studies examining local adaptation usually compared organisms or populations originating from different climates C. lineosa was also found to be more efficient than M. hyalinus – under constant food supply, C. lineosa grows faster. Possible explanations for this may include a lower basal metabolic rate or better growth efficiency . Under starvation, C. lineosa lost mass slower than M. hyalinus, a result that may indicate a decreased metabolic rate, different metabolic fuel utilization (e.g., C. lineosa may be able to better handle sporadic feeding, and exploit prey efficiently even when it is encountered intermittently, thus exhibiting a better ability to persist in a variable environment. This ability is specifically important in sit-and-wait predators, which suffer from fluctuations in prey abundance much more than actively searching predators on e.g.,  or behavC. lineosa, originating from a harsher microhabitat, seems to have a lower basal metabolic rate than M. hyalinus, irrespective of food abundance. Note that the conditions antlions experienced in our experiments were extreme, and in some cases even higher than seasonal means in the field. However, we still find our results to be ecologically relevant, since natural selection exerts its most dramatic influence at the edges of the organism’s tolerance limits, even if exposure to these edges happens only once every few generations Like other sit-and-wait predators, such as spiders, antlions are capable of dramatically reducing their metabolic rates C. lineosa’s superior tolerance to high temperatures. These mechanisms could include cuticle lipid composition and permeability to water loss To conclude, this work exemplifies a comprehensive approach to examining local adaptation, bringing forth the importance of differences in microclimates in promoting species coexistence, examining several niche axes, and exposing model organisms to extreme experimental conditions, in order to examine the limits of each species’ niche. Future directions for this research should include an exploration of the physiological and molecular mechanisms underlying


Influenza viruses isolated during the 2009 H1N1 pandemic generally lack known molecular determinants of virulence associated with previous pandemic and highly pathogenic avian influenza viruses. The frequency of the amino acid substitution D222G in the hemagglutinin (HA) of 2009 H1N1 viruses isolated from severe but not mild human cases represents the first molecular marker associated with enhanced disease. To assess the relative contribution of this substitution in virus pathogenesis, transmission, and tropism, we introduced D222G by reverse genetics in the wild-type HA of the 2009 H1N1 virus, A/California/04/09 (CA/04). A dose-dependent glycan array analysis with the D222G virus showed a modest reduction in the binding avidity to human-like  receptors and an increase in the binding to avian-like  receptors in comparison with wild-type virus. In the ferret pathogenesis model, the D222G mutant virus was found to be similar to wild-type CA/04 virus with respect to lethargy, weight loss and replication efficiency in the upper and lower respiratory tract. Moreover, based on viral detection, the respiratory droplet transmission properties of these two viruses were found to be similar. The D222G virus failed to productively infect mice inoculated by the ocular route, but exhibited greater viral replication and weight loss than wild-type CA/04 virus in mice inoculated by the intranasal route. In a more relevant human cell model, D222G virus replicated with delayed kinetics compared with wild-type virus but to higher titer in human bronchial epithelial cells. These findings suggest that although the D222G mutation does not influence virus transmission, it may be considered a molecular marker for enhanced replication in certain cell types. The majority of studies have reported that presence of D222G is sufficient to enhance virus replication and lethality in mouse models, with this effect ranging from modest to pronounced Previous studies on highly pathogenic influenza viruses have contributed to the identification of numerous molecular markers associated with adaptation to human hosts and increased virulence and transmissibility, including key amino acids at the 627 and 701 positions in PB2 and the presence of full-length PB1-F2 and NS1 proteins The location of position 222 in the receptor binding site of HA predicts that alteration of this position would influence the binding specificity of a virus bearing this mutation Asp222 in the wild-type CA/04 HA is a characteristic residue predominantly found in human-adapted H1N1 viruses. This residue has been shown to play a key role in providing optimal contacts with the penultimate Gal sugar in glycans terminated by α2-6-linked sialic acid To investigate the effect of D222G mutation on glycan-receptor binding properties of CA/04 virus, we analyzed the binding of both wild-type and mutant virus in a dose-dependent fashion to representative α2-3 and α2-6 sialylated glycans on a glycan array platform . Our anaA recent study demonstrated that the introduction of D222G in a 2009 H1N1 virus did not alter the pathogenicity or transmissibility of a 2009 H1N1 isolate A/Netherlands/602/09 in ferrets 10 PFU/ml nasal turbinates) and the lower respiratory tract (6.3±0.6 log10 PFU/g lung tissue) at similar titers as 2009 H1N1 wild-type viruses day 3 p.i. 10 PFU/ml), and in rectal swabs collected on days 1 and 3 p.i. in 2/3 ferrets (average titer 1.4±0.2 log10 PFU/ml), comparable to other 2009 H1N1 isolates Ferrets inoculated with the D222G virus exhibited similar clinical signs as CA/04 wild-type virus and a reverse-genetics derived CA/04 virus, with ferrets exhibiting transient weight loss (mean maximum weight loss of 11.3% on day 6–7 p.i.) and fever (mean maximum fever 1.9°C over baseline day 2 p.i.) before returning to baseline levels 6 PFU of virus. Approximately 24 hours p.i., inoculated-contact pairs were established by placing naïve ferrets in each of three adjacent modified cages with a perforated side wall, allowing air exchange between ferrets in the absence of direct or indirect contact. Nasal washes (NW) were collected on alternate days p.i. or post-contact (p.c.) for virus titration. Detection of infectious virus in NW of contact ferrets and seroconversion of contact ferrets were considered evidence of virus transmission. Consistent with the experimental transmission data obtained with wild type 2009 H1N1 viruses 10 PFU/ml day 1 p.i., significantly higher than CA/04 virus at this time (p<0.03) 10 PFU/ml on day 3 p.c. . These results indicate that CA/04 virus is not well suited to infect mice by the ocular route, and the presence of D222G does not confer an ocular tropism in this model.A recent report indicated that the presence of D222G enhanced the ability of a 2009 H1N1 virus to cause ocular disease in mice, with virus detected in the eye of one mouse following intranasal inoculation 10 PFU/ml) through 72 hrs p.i. in this cell type. In contrast, both viruses replicated efficiently in the human bronchial epithelial cell line Calu-3  virus were constructed as described previously https://www.who.int/csr/resources/publications/swineflu/Laboratorybioriskmanagement.pdf).CA/04 and 222G viruses were rescued as described previously A streptavidin plate array comprising representative biotinylated α2→3 and α2→6 sialylated glycans as described previously 6 PFU of virus in a 1 ml volume, a dose reported to consistently infect ferrets with human or avian influenza viruses, was determined as previously described Male Fitch ferrets , 8–10 months of age and serologically negative by hemagglutination inhibition (HI) assay for currently circulating influenza viruses, were used in this study. Ferrets were housed for the duration of the experiment in cages within a Duo-Flow Bioclean environmental enclosure . The pathogenesis of each virus was assessed following intranasal (i.n.) inoculation of 105 PFU of CA/04 or D222G virus in a 50 µl volume as previously described 5 PFU in a 5 µl volume t test.Female BALB/c mice , 8–12 weeks of age, were inoculated i.n. with 10p-tosyl-L-phenylalanine chloromethyl ketone-treated (TPCK) trypsin  in cultures using primary human cells. Aliquots of culture supernatant taken post-infection (p.i.) from triplicate cultures were immediately frozen at −80°C until titrated for the presence of infectious virus by standard plaque assay.The bronchial epithelial cell line Calu-3  was cultured on membrane inserts and infected apically as previously described All animal research described in this study was specifically approved by CDC's Institutional Animal care and Use Committee (IACUC). The animal research was conducted under the guidance of CDC's IACUC and in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility.Table S1Expanded nomenclature of glycans used in the glycan array.a Neu5Ac: N-acetyl D-neuraminic acid; Gal: D-galatose; GlcNAc: N-acetyl D-glucosamine. α/β: anomeric configuration of the pyranose sugars. All the sugars are linked via a spacer to biotin .(DOC)Click here for additional data file.


Glyma_18_02680 on chromosome 18) was a receptor like kinase (RLK) encoded within the soybean (Glycine max L. Merr.) Rhg1/Rfs2 locus. The locus underlies resistance to the soybean cyst nematode (SCN) Heterodera glycines (I.) and causal agent of sudden death syndrome (SDS) Fusarium virguliforme (Aoki). Previously the leucine rich repeat (LRR) domain was expressed in Escherichia coli.The protein encoded by GmRLK18-1 (in vitro. Cross-linking experiments in vitro showed the H274N region was close (<11.1 A) to the highly conserved cysteine residue C196 on the second homo-dimer subunit. Binding constants of 20–142 nM for peptides found in plant and nematode secretions were found. Effects on plant phenotypes including wilting, stem bending and resistance to infection by SCN were observed when roots were treated with 50 pM of the peptides. Far-Western analyses followed by MS showed methionine synthase and cyclophilin bound strongly to the LRR domain. A second LRR from GmRLK08-1 (Glyma_08_g11350) did not show these strong interactions.The aims here were to evaluate the LRRs ability to; homo-dimerize; bind larger proteins; and bind to small peptides. Western analysis suggested homo-dimers could form after protein extraction from roots. The purified LRR domain, from residue 131–485, was seen to form a mixture of monomers and homo-dimers ab-initio modeling was partially validated by cross-linking. The effect of the 3 amino acid replacements present among RLK allotypes, A87V, Q115K and H274N were predicted to alter domain stability and function. Therefore, the LRR domain of GmRLK18-1 might underlie both root development and disease resistance in soybean and provide an avenue to develop new variants and ligands that might promote reduced losses to SCN.The LRR domain of the GmRLK18-1 protein formed both a monomer and a homo-dimer. The LRR domain bound avidly to 4 different CLE peptides, a cyclophilin and a methionine synthase. The CLE peptides GmTGIF, GmCLE34, GmCLE3 and HgCLE were previously reported to be involved in root growth inhibition but here GmTGIF and HgCLE were shown to alter stem morphology and resistance to SCN. One of several models from homology and  Glycine max L. Merr.) are the soybean cyst nematode  and sudden death syndrome (SDS) agent Fusarium virguliforme (Aoki) . CD. CD29]. in vivo as part of a signal cascade that alters plant development ethyl methanethiosulfonate,  reagent dissolved in dimethyl formamide was used to modify 15 μg (3.5 pM) of the LRR in the dark for 1hr at room temperature after , 60mM NaCl, 5mM MgCl A away) . The bioInitially, the LRR domains were digested with trypsin (dissolved in 1mM HCl), in a ratio of 1:5 of the enzyme: substrate, for 3 hours in 25mM ammonium bicarbonate at 37°C. Then the reaction was stopped with the protease inhibitor PMSF in (~20 fold molar excess to trypsin) before passing through a monomeric avidin column (with a binding capacity of biotinylated proteins to be ~1.2mg/ml) which was already blocked with blocking buffer (4mM d-biotin dissolved in 1x PBS) and also washed with 12 column volumes of elution buffer (0.4% (v/v) trifluoro-acetic acid and 40% (v/v) acetonitrile). The biotinylated peptides were eluted from the column using 5 column volumes of the elution buffer (200 μl). Samples were sent for peptide fragment size estimation to the MS facility at SIUC for MALDI analysis. Briefly, a 1μl drop of trypsinized protein was added to 1 μl of MALDI matrix  acetonitrile) and dried on a stainless steel plate. A Bruker Daltonics Microflex™  time of flight mass spectrometer was used to analyze the sample with a pulse nitrogen laser set at 337nm with a 20 kHz repetition rate and ions that resulted were observed in the positive ion mode as the sum of 500 individual mass spectra over an m/z range from 1–10 thousand.SDS–PAGE of total plant proteins from Essex and Forrest followed by Western hybridization was carried out by methods described previously ,7 with tFor Far-Westerns the LRR domain was used as a probe to filters transferred from 1 or 2 D gels. As the secondary probe, anti-his antibody conjugated to HRP  was used. Spots and bands were picked manually and the proteins in them digested with trypsin and identified by direct infusion MS/MS following the methods in .in vitro assays potential peptide ligands were labeled with fluorescein-5-isothiocyanate (FITC) in a molar ratio of 10:1 at room temperature for 60 mins . Labeled peptides were stored at −20 C in 10% (v/v) glycerol until use. For ligand binding assays peptides were diluted to 50nM/ml in 20 mM phosphate buffer (pH6.9) in a cuvette and the amount of polarized luminescence measured in a luminescence spectrometer scan from 500–600 nm with excitation energy of 494 nm and a detection wavelength 520 nm. The excitation slit was set to 4 nm and the emission slit was set at 4 nm with a scan speed of 100 nm/min. Ligands were added to the cuvette in excess (50 nM) Peptides were added in 5 nM aliquots and the increase in polarization measured as fluorescence units. Base-line binding to non-CLE and non-LRR peptides was subtracted from the polarization units. Experiments were repeated 3 occasions with 3 different protein preparations. Dissociation constants (Kds) were calculated from double reciprocal Scatchard plots [For rd plots .In planta assays used twelve plants of each of cultivar X5 and Westag 97 in two separate experiments over a month for a root dip assay [ip assay . SCN susrhg1 locus, was translated to its polypeptide sequence in-silico using the EXPASY translation tool (http://www.expasy.ch/tools/dna.html). The complete (855 amino acid) sequence obtained for GmRLK18-1 was analyzed for domain architecture (smart.embl-heidelberg.de). The LRR-domain (amino acid 52 to amino acid 440) was used for generation of models. The Apache server found at (supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY) was used to predict the superfamily, fold and class of the protein. The ratio of synonymous to non-synonymous substitutions was analyzed by the web based algorithm SNAP (http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html).The nucleotide sequence for GmRLK18-1, the RLK encoded within the http://compbio.washington.edu; http://protinfo.compbio.washington.edu). The initial template and the corresponding sequence alignments of GmRLK18-1 were chosen from the 3D-Jury server (http://BioInfo.PL). The best template was decided by inspection of the consensus sequence of the GmRLK18-1-LRR protein and all template sequences . The structures of GmRLK18-1 allotypes  were generated using SCWRL3. To evaluate the stability of the structures, their pseudo-energy was calculated using RAPDF.http://www.ebi.ac.uk/thornton-srv/software/PROCHECK/). Analysis of main chain, side chain parameters and Ramachandran plot analysis used the PROCHECK algorithm. The 3 amino acid changes from the SCN Type I resistance allotype to the susceptible allotypes were analyzed further for functional and structural importance using two prediction servers .Protein structure validation checks, mainly the stereo-chemical quality of protein structure were performed using PROCHECK  plants that Westag 97 (panel F-H) plants. Panels A and E were before depotting. Panels B and F were 2 min after dipping and repotting. Panels C and G were 15 minutes after treatment. Panel D and G were at root harvest 28 dai (42 dag). Cyst were abundant on the water treated plants but 10±6 were found on each of the 5 plants dipped in HgCLE or GmTDIF (Table 2).Effect of exogenous treatments of roots of X5 and Westag 97 with CLE peptides a root dip assay. SCN susceptible plants that had been infested with HgType 0 (isolate JB3) were depotted at 10 dai (24 dag) and a 0.5g root sample taken. Roots were then dipped in CLE peptides TGIF or HgCLE in water at 50 pM concentration and returned to the infested soil. Wilting and stem bending was more severe in X5  were in disallowed regions.Click here for fileRAPDF scores for 3 LRR mutants and the wild type GmRLK18-1-LRR. The RAPDF scores suggested that these mutations may affect the stability of the homodimeric protein although these residues were not directly implicated in the homodimer interface.Click here for fileSequence diversity among the seven GmRLK18-1 allotypes. Alanine at position 87 is only present in the ‘Peking’ sequence. Two additional changes Q to K at position 115 and H to N at position 274 are not exclusive to resistance type I.Click here for fileEffect of three non-synonymous substitutions on protein stability calculated from the Fold X algorithm (http://fold-x.embl-heidelberg.de). The computed free energy of folding and the change in free energy between the wild type protein  and the mutant proteins is shown. The H to N and Q to K change increase the free energy of folding, whereas the alanine to valine change results in a significant decrease in the free energy. (DOC 45 kb)Click here for filea) shows the crystal structure of BRI1 (pdb id: 3RGX). Brassinosteroid recognition is mediated through the LRR domain of BRASSINOSTEROID-INSENSITIVE 1 (BRI1). BR1I exists as a monomer. The protein exists as a helical solenoid structure. (b) crystal structure of the Polygalacturonase inhibiting protein (PGIP). The PGIP protein (PDB id: 1ogq) is a cell wall localized protein that interacts with fungal endopolygalacturonases. (c) X-ray structure of the porcine ribonuclease inhibitor (PRI). The Leucine rich repeat of PRI (PDB id: 2BNH) forms tight complexes with ribonucleases thereby regulating RNA levels. PRI adopts a horseshoe configuration with the LRR motif composed of repeat beta-loop helix units.The 3D structures of LRR containing proteins showing high sequence homology to RHG1. Panel (Click here for file


Central to the disparate adaptive immune systems of archaea and bacteria are clustered regularly interspaced short palindromic repeats (CRISPR). The spacer regions derive from invading genetic elements and, via RNA intermediates and associated proteins, target and cleave nucleic acids of the invader. Here we demonstrate the hyperactive uptake of hundreds of unique spacers within CRISPR loci associated with type I and IIIB immune systems of a hyperthermophilic archaeon. Infection with an environmental virus mixture resulted in the exclusive uptake of protospacers from a co-infecting putative conjugative plasmid. Spacer uptake occurred by two distinct mechanisms in only one of two CRISPR loci subfamilies present. In two loci, insertions, often multiple, occurred adjacent to the leader while in a third locus single spacers were incorporated throughout the array. Protospacer DNAs were excised from the invading genetic element immediately after CCN motifs, on either strand, with the secondary cut apparently produced by a ruler mechanism. Over a 10-week period, there was a gradual decrease in the number of wild-type cells present in the culture but the virus and putative conjugative plasmid were still propagating. The results underline the complex dynamics of CRISPR-based immune systems within a population infected with genetic elements. Adaptive immune systems of most archaea and many bacteria primarily target invading viruses and conjugative plasmids and have recently been classified into major classes, denoted types I, II, IIIA and IIIB  and locus E (seven spacers). Surprisingly, large numbers of newly incorporated, unique, spacer sequences were found at each locus  and D  and specific TCN or CCN PAM motifs respectively. Moreover, spacers showing one to four mismatches which may have been active in interference . Fourteen clones were selected that showed PCR evidence for new spacer uptake in the CRISPR loci C, D and E. Sequencing revealed that each of the clones carried new spacer inserts. Six clones contained new spacers in either locus C or D, seven clones had acquired spacers in both loci C and D, and one clone exhibited new spacers in all three loci . One-thiThree of the single clones, S11 with an insertion in locus C, S3 with an insertion in loci C and D and S10 with insertion in loci C, D and E , and an orf174 within an operon in viral contig 1 . However, exceptionally for crenarchaeal viruses, SIRV2 and ATV, in contrast to STSV2 and the Yellowstone virus, have been shown to induce cell lysis  the Sulfolobus subfamily I CRISPR arrays and (ii) a single genetic element in the mixture; moreover, no spacers derived from the S. solfataricus P2 chromosome   and virus particles were isolated by filtration of the supernatant through 0.2 µm pore filters . This virus mixture was then used to infect S. solfataricus P2 (DSM 1617) cultured in the Sulfolobus medium  in Monument Geysir Basin, Yellowstone National Park. One millilitre of this sample was added to 50 ml of S. solfataricus P2 were harvested from fresh culture by centrifuging  and resuspending in 1 ml of TYS medium. Twenty microlitres of virus mixture was added at 3 PFU µl−1 and after incubating for 2 h at 80°C, infected cells were transferred to 50 ml of pre-heated (78°C) TYS medium. Infected S. solfataricus P2 was then incubated at 78°C for 3–6 days before isolating infectious particles as described above for the enrichment culture. Infections with the single viruses SIRV2, ATV and STSV2 were performed using the same procedure.Cells of Sulfolobus solfataricus cells were infected and grown for 3 days in 2 l of TYS medium. Cells were separated by centrifugation  and virus particles were isolated from the supernatant by filtration using Vivaspin 20 columns  at 1500 g and dissolved in 10 mM Tris-HCl, pH 8.0. The virus mixture was then loaded onto 0.45 g ml−1 CsCl and centrifuged for 48 h at 38 000 r.p.m. in a SW41 rotor . The virus band was extracted from gradients and CsCl was removed by dialysis against 10 mM Tris-HCl, pH 8. DNA was isolated using DNeasy® Blood&Tissue Kit . Sequencing by Illumina sequencing and raw data treatment was performed by Beijing Genomics Institute . Clean data consisting of approximately 90 bp DNA fragments were assembled using the CLC genomics workbench . orf174 within an operon in viral contig 1 and the traG-like gene in contig 2 of the putative conjugative plasmid were selected to detect the appropriate genetic element in host cells. DNA was amplified by PCR using forward and reverse primers 5′-CCCACCTATATCGAATTC-3′ and 5′-GTGTCTCTCATATTTGCAATC-3′, respectively, for the virus and 5′-GCCTTAGCGAGGGCCCAGTTGAACCTGG-3′ and 5′- CTATCCTATCCCTGTCTATCCCTAG-3′, respectively, for the putative conjugative plasmid. DNA from the initial enrichment culture and the S. solfataricus P2 culture were used as positive and negative controls respectively.S. solfataricus cells was purified in a CsCl density gradient followed by dialysis against 10 mM Tris-HCl, pH 8. One hundred and fifty microlitres of purified virus mixture was digested with DNase I  at 37°C. DNA of the digested virus mixture and an undigested 150 µl sample was isolated using DNeasy® Blood&Tissue Kit (Qiagen). DNA concentration was measured using NanoDrop®.The virus mixture obtained from infected Virus particles were adsorbed onto carbon-coated copper grids for 5 min and stained with 2% uranyl acetate. Images were recorded using a Tecnai G2 transmission electron microscope , with a CCD camera, at an acceleration voltage of 120 kV.cas genes, were infected with virus mixture and cultivated, with an uninfected control sample, in 50 ml of TYS medium at 78°C. One millilitre of samples of each culture was taken every 6 h and optical densities were recorded at 600 nm. Two millilitres of samples of each culture was taken every 24 h and cells were harvested by centrifugation . DNA was isolated using DNeasy® Blood&Tissue Kit (Qiagen). The leader proximal regions of the six CRISPR loci were amplified by PCR using forward primers 5′-AGCTTCTGACCCGCTCCTGA-3′ for locus A, 5′-AGGGGTTTGTGGGATGGGTTGTG-3′, for locus B, 5′-TCGCTTATCTCTCTCATGCGCCATT-3′ for locus C, 5′-AGTTCCACCCCCGAAGCTCCT-3′ for locus D, 5′-ATAGGGAAAGAGTTCCCCCG-3′ for locus E, 5′-CGGCGTTATAATGGGTATCGGAATCGG-3′ for locus F and reverse primers 5′-GCACATCATCAAACAATGGTAAGCC-3′ for locus A, 5′-ACAACTACCACCACTACCACGG-3′ for locus B, 5′-TGTCCCGTTTTTGTAAGTGGGGG-3′ for locus C, 5′-AGCCGGGACAAGTTTCACAAATTGA-3′ for locus D, 5′-TGACTCTAGTGCAATCTTCGA-3′ for locus E, 5′-GCTCACTATCTCACCCCTATCAATACCC-3′ for locus F. Single clones of infected S. solfataricus cells were obtained on Gel-rite plates and cultured in TYS medium (Wild-type P2 strain and the mutant strain lacking CRISPR loci A to D and their associated PCR products were separated on 1.5% agarose gels and bands larger than the those of the uninfected control sample were excised from the gel and purified with QIAquick Gel Extraction Kit (Qiagen). Purified PCR products were cloned using InsTAclone™PCR Cloning Kit (Fermentas) following the manufacturers' protocols. Plasmid purification and sequencing were performed by GATC Biotech AG, .


The F virus presented faster growth kinetics and stronger induction of cytokines than M virus in human alveolar lung epithelial cells. In the murine model in vivo, the F virus showed a stronger morbidity and mortality than M virus. Remarkably, a higher proportion of mice presenting infectious virus in the hearts, was found in F virus-infected animals. Altogether, the data indicate that strains of pH1N1 virus with enhanced pathogenicity circulated during the 2009 pandemic. In addition, examination of chemokine receptor 5 (CCR5) genotype, recently reported as involved in severe influenza virus disease, revealed that the F virus-infected patient was homozygous for the deleted form of CCR5 receptor (CCR5Δ32).Pandemic 2009 H1N1 (pH1N1) influenza viruses caused mild symptoms in most infected patients. However, a greater rate of severe disease was observed in healthy young adults and children without co-morbid conditions. Here we tested whether influenza strains displaying differential virulence could be present among circulating pH1N1 viruses. The biological properties and the genotype of viruses isolated from a patient showing mild disease (M) or from a fatal case (F), both without known co-morbid conditions were compared  Influenza A viruses are endemic in many wild avian species and can produce infections in mammals with varying morbidity and mortality rates. In humans they cause annual epidemics and occasional pandemics of respiratory disease with potentially fatal outcome. Since 1997, highly pathogenic avian H5N1 viruses have sporadically produced infections in humans but although this emerging virus presented a high mortality rate it showed poor transmissibility between humans and did not spread over human population The co-morbid conditions were similar for both the pH1N1 and the previous seasonal influenza viruses and include chronic metabolic disease, primarily diabetes mellitus and renal disease, chronic lung and cardiac disease, immunosuppressive conditions and neoplasms in vivo, compared with the virus obtained from the patient with mild disease. In addition, the deceased patient was homozygous for the CCR5Δ32 allele, a rare genetic background found in less than 1% of the population CCR5 allele.To analyze possible virulence differences, we have compared two contemporary human pH1N1 viruses from two patients without known co-morbid conditions, one that became fatal while the other showed only mild respiratory disease. The results indicate that the virus isolated from the fatal case replicates faster, induces higher levels of cytokines in human alveolar lung epithelial cells and is more pathogenic in a murine model The National Influenza Center in Madrid  and other regional laboratories from different Spanish regions, constituted the ReLEG network included in the Spanish Influenza Surveillance System (SISS), which monitored the circulation of influenza viruses each influenza season as a part of the countrywide surveillance. This study has been developed within this activity, which was approved by the institutional review board at the Instituto de Salud Carlos III.All the procedures that required the use of animals complied with Spanish and European legislation concerning vivisection and the use of genetically modified organisms, and the protocols were approved by the National Center for Biotechnology Ethics Committees on Animal Experimentation and the Consejo Superior de Investigaciones Científicas (CSIC) Bioethics Subcommittee. In particular, we follow the Guidelines included in the current Spanish legislation on protection for animals used in research and other scientific aims: RD 1201/2005, 10 October and the current European Union Directive 86/609/CEE, DOCE 12.12.86 (N.L358/1 to N.L358/28) on protection for animals used in experimentation and other scientific aims.Two distinct influenza viruses named A/CastillaLaMancha/RR5661/2009 (M) and A/CastillaLaMancha/RR5911/2009 (F), were isolated at the National Influenza Centre  from respiratory samples sent by the Spanish Influenza Surveillance System for virological characterization. Isolations were made at the middle stage of the 2009 pandemic. Both viruses belong to Caucasian individuals. Virus M was detected in a 23 years old man and virus F was isolated from a 35 years old woman. Both viruses were isolated from bronchoalveolar lavages, collected in 3 ml virus transport medium . Semi-confluent monolayers of MDCK cells were used for primary viral isolation. The monolayers were inoculated with 200 µl of homogenized samples and when the cytopathic effect was 75–100%, the cultures were harvested and the supernatants used for virus stock generation by inoculation of MDCK cells.For virus purification, culture supernatants of MDCK-infected cells were centrifuged for 10 min at 10,000 rpm and 4°C. The supernatants were sedimented through a sucrose step gradient  for 1 h at 40,000 rpm and 4°C in a SW41 rotor. The 50 to 33% interphase was collected, diluted in TNE buffer, and pelleted through a cushion of 33% sucrose in TNE for 1 h at 40,000 rpm and 4°C in a SW41 rotor. The isolation of total RNA from the pellet was carried out using RNAeasy isolation reagent (Quiagen) according to the manufacturer’s instructions. Appropriate amounts of each sample were analyzed by high-throughput sequencing as indicated below.Library preparation was performed using the Illumina mRNA seq sample preparation kit (Illumina kit RS-100-0801) as previously described −3 PFU/cell (low moi) or 3 PFU/cell (high moi), after 1 h the non-bound virus was rinsed off with acid PBS (pH 5.3) and at different hours post-infection (hpi), cell supernatants were collected and used for virus titration by plaque assay.Cultured human lung alveolar epithelial cells (A549) from the American Type Culture Collection (ATCC) were infected at 10The secretion of cytokines by human alveolar lung epithelial cells (A549) infected at 3 PFU/ml with different influenza viruses was measured 12 and 24 hpi in the culture supernatant using the Luminex 100 technology , following the manufacturer’s instructions. The following 15 mediators were tested: granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1α (IL-1α), IL-1β, IL-6, IL-8, IL-10, IL-15, alpha 2 interferon (IFNα), IFN-γ, interferon gamma-induced protein 10 (IP-10), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1β (MIP-1β), Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) and tumor necrosis factor α (TNF-α) and TNFβ.6 PFU of either M or F influenza viruses or were mock infected. The animals were monitored daily for clinical signs and body weights. On days 2, 4, 7 and 14 dpi mice were euthanized and necropsied. Clinical specimens from lungs, kidneys, hearts and brains were homogenized in phosphate-buffered saline-0.3% bovine serum albumin in a Dounce homogenizer and used for determination of viral titers by plaque assay.Female BALB/c AnNHsd mice (6–8 weeks old) were infected intranasally with 10g, 5 min). Virus titers of triplicate tissue samples or cell culture supernatants were determined by standard plaque assay on MDCK cells.Tissue samples were homogenized (10% [wt/vol]) in PBS and debris was pelleted by centrifugation  by conventional methods. To visualize influenza virus in animal lungs, a polyclonal nucleoprotein (NP) antibody CCR5 gene 5′-CATTATACATGCAGTCCTC-3′ and 5′-GATGGCAAAGATGAGCCTAC-3′ primers for mice DNA.Genotyping was performed by standard methods described elsewhere To determine possible differences in virulence, we compared the biological properties of two distinct influenza viruses named mild (M) and fatal (F), both isolated from young patients without known health risk conditions. Virus M was detected in a patient that presented mild respiratory symptoms, whereas virus F was isolated from a patient that developed severe pneumonia, was hospitalized in the intensive care unit and died several days after admission.Genetic characterization of M and F viruses was performed by ultrasequencing of purified virion RNAs obtained after two passages in MDCK cells. The consensus sequences obtained for the viral genomes represent more than 95% of the total sequence reads at each position. The two viruses differed in 29 nucleotides distributed over all the RNA segments, which produce 9 amino acids changes affecting PB2 (1 aa), PA (3 aa), NP (1 aa), HA (3 aa) and NA (1 aa). Three of these amino acid changes involve conservative substitutions of basic residues (aa 269 and 328 in PA and aa 400 in NP) . The amiTo analyze the properties of these viruses, cultures of human alveolar lung epithelial cells (A549) were infected with each virus at low multiplicity of infection and viral titers were determined at different hpi. A difference of more than 2 logs in the viral titers was observed shortly after infection (9–12 h). However, both viruses reached similar titers at later times . The virTo determine the capacity of the M and F viruses to induce cytokines, their profiles were evaluated in infected human alveolar lung epithelial cells. For comparison, the cells were also infected with previous seasonal H1N1 virus, the A/New Caledonia/20/99 (NC) strain. Samples of A549 cells were infected in triplicate and in all cases the cytokine induction was evaluated at 12 and 24 hpi using the Luminex 100 System. At 12 hpi, F virus-infected cells showed increased accumulation of pro-inflammatory cytokines such as IL-6 (8-fold) or IL-8 (65-fold), chemokines such as RANTES (20-fold) as well as MCP-1 (371-fold), colony-stimulating factor GM-CSF (4-fold) and IFN-α (2-fold) as compared with M-infected cells . These din vivo relevance and the consequences of this difference in the murine model. Six BALB/c mice were intranasally infected with 106 PFU of M or F viruses or were mock-infected. Survival and body weight were monitored daily for two weeks. No loss of body weight was observed in mock-infected mice whereas mice infected with M and F viruses had a peak of weight loss 5 to 7 days after inoculation. However, in the F virus-infected mice the weight loss was more pronounced and the recovery of body weight was slower  , Lung. AThe detection of pH1N1 influenza virus in organs different from the lung, such as kidney and brain has been reported in vivo infection and is summarized in In domestic animals influenza viruses produce bronchointerstitial pneumonia characterized by the appearance of both bronchiolar necrosis and diffuse alveolar damage Next, immunohistochemistry analysis was performed to detect the presence of NP viral protein in the lung cells of M and F virus-infected mice at different days post-infection. Animals inoculated with either virus showed very strong immunostaining reaction in bronchiolar epithelial cells at 2 dpi and in the interstitial cells at 2–4 dpi (data not shown). In M virus-infected mice the bronchiolar reaction was progressively reduced to become negative at 14 dpi  a–c. TheCCR5 is expressed on activated macrophages and plays an important role in the macrophage response to influenza virus infection since knock out mice for Crc5 gene display increased mortality rates CCR5 that prevents the surface expression of the protein (CCR5Δ32) CCR5 alleles. Bronchoalveolar lavages were used to isolate total nucleic acids that were used for RT-PCR reactions. RNA was amplified by using previously reported primers surrounding the 32-bp deletion in the CCR5 gene CCR5 RNA results in a 197-bp product, but the Δ32 allele yields a 165-bp product. The amplified products were analyzed in agarose gels together with the amplification products obtained from previously characterized CCR5 homozygous (wt) and heterozygous (ht) human samples. The results showed that the M virus-infected patient was homozygous for the wild type CCR5 allele, but the F virus-infected patient was homozygous for the CCR5Δ32 deletion  and the other resulted in a fatal case (F).Recognition and rapid clearance of pathogens by the innate immune system provides the first line of defense upon infection. However, pathological inflammation is produced when excessive activation of the innate immune system occurs. Several reports pointed out the role of an excessive cytokine response (known as a cytokine storm) as a key contributor to morbidity and mortality of highly virulent 1918 and avian H5N1 influenza virus infections Analysis of cytokines induction in infected human alveolar epithelial cells with the M and F viruses as well as the seasonal A/New/Caledonia/20/99 (NC) strain revealed that F virus infection induces earlier and higher levels of IL-6, IL-8, MCP-1, GM-CSF and RANTES than M or NC infections . Besides6 PFU were required to reach a 50% lethal dose or even the mice survived at the highest dose used 6 PFU with the F virus but not with the M virus died, indicating that F virus is more pathogenic than other pH1N1 viruses. Moreover, infectious virus was recovered in higher proportion of animals and remained at later days post-infection in the hearts of F virus-infected mice , the hemagglutinin (HA), neuraminidase (NA), and non-structural protein 1 (NS1) CCR5 allele that contains a 32 bp deletion (CCR5Δ32) has been correlated with more severe course of the pH1N1 infection in a small cohort CCR5Δ32 mutation has been found . Around 15–20% of the Caucasian population is heterozygous for the CCR5Δ32 allele, whereas the proportion of CCR5Δ32 homozygous is below 1% CCR5 receptor. Influenza virus infected Ccr5 knock-out mice display an accelerated macrophage accumulation in the lungs that is likely to be linked to enhanced expression of MCP-1 and RANTES and increased mortality rates associated with acute, severe pneumonitis CCR5Δ32 homozygosis for influenza virus infection is required, our data with those of other laboratories support a CCR5 role in the course of influenza virus infection. Other potential influenza virus genetic determinants have been recently described as an increase on a single nucleotide polymorphism allele of the tumor necrosis factor Potential influenza virus host genetic determinants have been described and a significant increase on heterozygosis of a in vivo, the F virus showed a stronger morbidity and mortality than M virus and moreover higher proportion of mice having viral particles in the hearts of F-virus infected mice was found. Therefore the F patient was infected with a potential highly pathogenic virus and its genetic background of CCR5Δ32 homozygous could contribute to the severity of the infection.In summary, the F (A/CastillaLaMancha/RR5911/2009) virus replicates and induces cytokines in human alveolar epithelial cells very fast, including the pro-inflammatory IL-6 and IL-8 cytokines and the chemokine RANTES that seem to play an important role in influenza virus infection. In the murine model Figure S1t-test was performed to determine the P value. *P<0.05, **P<0.01, ***P<0.001,Profile of cytokines induction on A549 infected cells at 24 hpi. Cultured cells were infected at 3 PFU/cell with the A/New Caledonia/20/99 (NC) and the H1N1 pandemic M and F isolates. At 24 hpi cell supernatants were taken and used to determine the concentration of the indicated cytokines using the Luminex 100 System. Error bars indicate the standard deviation of the mean. Student’s (PDF)Click here for additional data file.Figure S2Ccr5 allele determination. Samples of cultured A549 (top) and kidneys from infected or uninfected mice (bottom)were used to isolate total RNA that was used for RT-PCR reactions and DNA sequencing to detect the CCR5 region that comprises the 32-bp deletion. The sequence of the CCR5 allele from human (CCR5 H. sapiens) or mice (Ccr5 M. musculus) have been used for comparison. The red line corresponds to the 32 bp deletion found in the CCR5Δ32 samples.(PDF)Click here for additional data file.Table S1Nucleotides at the indicated positions of M and F RNAs.(PDF)Click here for additional data file.Table S2Frequency of different residues at PB2 221, PA 529 and HA 127 positions in human, swine and avian viruses.(PDF)Click here for additional data file.


This first report suggests that dogs can be asymptomatically infected with Ebola virus. During the 2001–2002 outbreak in Gabon, we observed that several dogs were highly exposed to Ebola virus by eating infected dead animals. To examine whether these animals became infected with Ebola virus, we sampled 439 dogs and screened them by Ebola virus–specific immunoglobulin (Ig) G assay, antigen detection, and viral polymerase chain reaction amplification. Seven (8.9%) of 79 samples from the 2 main towns, 15 (15.2%) of 14 the 99 samples from Mekambo, and 40 (25.2%) of 159 samples from villages in the Ebola virus–epidemic area had detectable Ebola virus–IgG, compared to only 2 (2%) of 102 samples from France. Among dogs from villages with both infected animal carcasses and human cases, seroprevalence was 31.8%. A significant positive direct association existed between seroprevalence and the distances to the Ebola virus–epidemic area. This study suggests that dogs can be infected by Ebola virus and that the putative infection is asymptomatic. Ebola virus causes fulminant hemorrhagic fever in both humans and nonhuman primates  of the 284 cases in the 1976 Sudan outbreak  Dogs in Libreville and Port Gentil were sampled in a veterinary clinic. Blood was collected in 5-mL dry Vacutainers , and serum was prepared by centrifugation. Serum specimens were stored at –20°C until they were sent to the Centre International de Recherches Médicales de Franceville (CIRMF), Gabon, where they were stored at –80°C until testing. 2) Dogs from the Ebola virus–endemic area were sampled in the villages. An experienced veterinary team was located at Mekambo, where field laboratory facilities were set up; blood samples were collected on a daily basis in the vicinity of the village by using 5-mL dry Vacutainers and medetomidine anesthesia. The tubes were then transported to Mekambo each evening, and serum was decanted from whole blood. Serum samples were kept in liquid nitrogen in 1-mL aliquots at Mekambo until they were transported to CIRMF. Serum samples were then stored at –80°C until serologic testing, antigen detection, and RNA amplification were carried out.Dog owners were interviewed on their pets’ activities  and health history. The focus of the interviews was on potential Ebola virus–exposure events, including human cases that occurred in the village and among dog owners.Ebola virus–specific immunoglobulin (Ig) G was detected by using a standard enzyme-linked immunosorbent assay (ELISA) method as previously described  of the 102 blood samples from dogs living in France had detectable Ebola virus–reactive IgG . Seven oDuring the 2001–2002 Ebola outbreak in Gabon, human cases of Ebola virus infection appeared only in certain villages within the Ebola virus–epidemic area . The preThe seroprevalence rate was significantly lower in France (2.0%) than in Gabon . In partThe seroprevalence rates in dogs increased linearly as the sampling area approached the sites of human cases, as confirmed by the highly significant Cochran-Armitage test for trends in proportions (p < 0.0001), which used a score of 1 for France, 2 for major towns, 3 for Mekambo, 4 for villages from the disease-epidemic area without human cases, and 5 for villages from the Ebola virus–epidemic area with human cases . The resIn parallel, the seroprevalence rates in dogs increased linearly as the sampling area approached animal sources, as confirmed by a significant Cochran-Armitage test (p < 0.0001), using a score of 1 for France, 2 for major towns, 3 for Mekambo, 4 for villages where no animal source was observed (with or without human cases), and 5 for villages where an animal source was observed (with human cases) . Again, Neither Ebola virus antigens nor nucleotide sequences were detected in any of the positive or negative dog blood samples. We also failed to isolate the virus cells from 3 positive and 3 negative samples on Vero-E6.We investigated the potential involvement of domestic dogs in the occurrence or dissemination of Ebola virus hemorrhagic fever in humans. Based on a large serologic survey of dogs in the 2001–2002 Ebola outbreak area in Gabon, we found evidence that dogs can be infected by Ebola virus, a finding that raises important human health issues. The ELISA method was based on the use of Ebola virus–Z antigens. Although cross-reactions can occur with antibodies to other subtypes, the presence of these subtypes in our samples is unlikely because only the Zaire subtype circulates in the study area: all patients and nonhuman primates tested in this part of central Africa were infected by the Zaire subtype alone. The 2 positive dogs in France, an apparently Ebola virus–exempt part of the world, could be attributed to false-positive reactions due to the calculation of the positivity cut-off and the 1:400 serum dilution step used in the tests.We found that 40 of 159 dogs living in the 2001–2002 Ebola virus–epidemic area had detectable Ebola virus–specific IgG, indicating either true infection or simple antigenic stimulation. All the tests were standardized at the 1:400 serum dilution, and most serum specimens had high OD values even at higher dilutions, confirming the specificity of the reactions. These data are consistent with observations we made during the different Ebola outbreaks that occurred in Gabon and the Republic of Congo in recent years. We observed that some dogs ate fresh remains of Ebola virus–infected dead animals brought back to the villages, and that others licked vomit from Ebola virus–infected patients. Together, these findings strongly suggest that dogs can be infected by Ebola virus, and that some pet dogs living in affected villages were infected during the 2001–2002 human Ebola virus outbreak. No circulating Ebola antigens or viral DNA sequences (tested for by PCR) were detected in either positive or negative serum specimens, and attempts to isolate virus from these samples failed. These findings indicate either old, transient Ebola infection of the tested dogs, or antigenic stimulation.Symptoms did not develop in any of these highly exposed animals during the outbreak, a finding that tends to support antigenic stimulation, asymptomatic, or very mild Ebola virus infection. Wild animals, especially gorillas and chimpanzees, can also be infected by Ebola virus, but the infection is highly lethal and causes huge outbreaks and massive population declines , and that they had never visited the Ebola virus–epidemic area, in theory ruling out true infection. They may therefore have come into contact with free viral antigens, transmitted by aerosol or, to a lesser extent, experienced conjunctival exposure to virus-laden droplets of urine, feces, or blood of the unknown natural host. Ebola virus has been shown to be experimentally transmissible to rhesus monkeys by inhalation , to major towns (8.9%), then to Mekambo (15.2%), and then to villages in the Ebola virus–epidemic area (25.2%). This trend is supported by the increasing seroprevalence as the sampling area approached human cases and animal sources . These findings suggest that canine seroprevalence could reflect contact with the virus and, thus, virus activity in a given area and also the risk for human infection.The virus appears to jump from its natural host to humans only in specific, but unknown, conditions. Seroprevalence rates in dogs might serves as an indicator of Ebola virus in regions in which no animal deaths or human cases have been observed.In conclusion, this study offers the first evidence that dogs might be asymptomatically infected by Ebola virus in the wild. This finding has potential implications for preventing and controlling human outbreaks. The increasing canine seroprevalence gradient from low-risk to at-risk Ebola virus–endemic areas indicates that this seroprevalence might be used as an epidemiologic indicator of virus circulation in regions where no other means of virus detection are available.