Poliomyelitis is a highly infectious disease caused by poliovirus (PV). It can result in paralysis and may be fatal. Integrated global immunization programs using live-attenuated oral (OPV) and/or inactivated (IPV) PV vaccines have systematically reduced its spread and paved the way for eradication. Immunization will continue posteradication to ensure against reintroduction of the disease, but there are biosafety concerns for both OPV and IPV. They could be addressed by the production and use of virus-free virus-like particle (VLP) vaccines that mimic the “empty” capsids (ECs) normally produced in viral infection. Although ECs are antigenically indistinguishable from mature virus particles, they are less stable and readily convert into an alternative conformation unsuitable for vaccine purposes. Stabilized ECs, expressed recombinantly as VLPs, could be ideal candidate vaccines for a polio-free world. However, although genome-free PV ECs have been expressed as VLPs in a variety of systems, their inherent antigenic instability has proved a barrier to further development. In this study, we selected thermally stable ECs of type 1 PV (PV-1). The ECs are antigenically stable at temperatures above the conversion temperature of wild-type (wt) virions. We have identified mutations on the capsid surface and in internal networks that are responsible for EC stability. With reference to the capsid structure, we speculate on the roles of these residues in capsid stability and postulate that such stabilized VLPs could be used as novel vaccines.IMPORTANCE Poliomyelitis is a highly infectious disease caused by PV and is on the verge of eradication. There are biosafety concerns about reintroduction of the disease from current vaccines that require live virus for production. Recombinantly expressed virus-like particles (VLPs) could address these inherent problems. However, the genome-free capsids (ECs) of wt PV are unstable and readily change antigenicity to a form not suitable as a vaccine. Here, we demonstrate that the ECs of type 1 PV can be stabilized by selecting heat-resistant viruses. Our data show that some capsid mutations stabilize the ECs and could be applied as candidates to synthesize stable VLPs as future genome-free poliovirus vaccines. Poliomyelitis (polio) is an incapacitating human disease caused by three structurally similar but antigenically distinct serotypes of poliovirus (PV) termed PV-1, PV-2, and PV-3 13. Live-aPV has a 7.5-kb positive-sense RNA genome enclosed in a 30-nm icosahedral capsid that comprises 60 copies each of viral proteins VP1, VP2, VP3, and VP4 , 11. The12–Structural proteins VP1, VP2, and VP3 differ in amino acid sequence but form structurally similar wedge-shaped, 8-stranded β-barrels, comprising two antiparallel β-sheets essential for virion stability . Within In the course of infection, attachment of a 160S PV particle to the CD155 receptor triggers conformational changes in the virion capsid that result in formation of 135S particles  and rele20–Saccharomyces cerevisiae, or human cells) (–Empty PV capsids (ECs) with intact VP0 have been isolated from infected cells , 20. Then cells) –27; howe cells) –. Althoug cells) –, their u cells) –, 31.An alternative strategy could involve the generation of stabilized capsids that undergo antigenic conversion less readily, e.g., by introduction of mutations. RNA viruses are prone to continual mutagenesis due to the nonproofreading RNA-dependent RNA polymerase (RdRp), which allows rapid evolution under selection pressure , 33. Thi10-unit reduction in infective titers even after further selection for two rounds (data not shown), due to selection pressure at 57°C. These 3 pools of thermally selected viruses, VS51, VS53, and VS57, were further characterized.The construction of a genome-free PV VLP vaccine has been hindered in part by the instability of wt PV-1 genome-free ECs. Therefore, we first sought to select PV mutants with increased thermal stability using repeated thermal inactivation followed by passage of surviving virus, focusing on PV-1. At 51°C, the infectivity of wt PV-1 was reduced by 99.99% compared to the unheated control, and it was chosen for the initial selection temperature . Heat-reP < 0.0001) from those for the wt.The ability of VS51, VS53, and VS57 to withstand elevated temperatures was assessed using two methods: thermal inactivation  and particle stability thermal-release assay (PaSTRy) . Thermal-inactivation assays showed that the selected viruses  maintained infectivity at higher temperatures than the wt. Complete thermal inactivation of the wt occurred at 52°C, while the selected viruses were inactivated at higher temperatures . Data foP < 0.0001) from those for the wt.PaSTRy can directly determine the thermal stability of virus particles without the need for virus growth . The assSYPRO orange had two peaks. One indicated conformational change associated with capsid expansion and exposure of hydrophobic residues at 48°C (wt), 51°C (VS51), 53°C (VS53), and 59°C (VS57). The second peak, at around 85°C in all the virus samples, has been previously associated with denaturation . The datRT) and the first protein melting event (mT), which may coincide with thermal inactivation. The data further show that for all the selected viruses, an increase in RT was matched by an increase in mT. In contrast, hepatitis A virus (HAV), a more thermally stable picornavirus, was shown to have a RT higher than its mT by PaSTRy  were employed for immunoprecipitation, MAb 234, which recognizes native (N/D) virions, and MAb 1588, which recognizes nonnative (H/C) particles  to a nonnative or heated (termed H or C) form due to conformational changes in capsid proteins , 40. ECsarticles , 41. Thetibodies . Wt PV-1tibodies . In conttibodies . AntigenP < 0.05) and VS51 (P < 0.001); however, VS51 and VS53 were not significantly different  corresponded. Nucleotide sequence alignment of the thermally selected viruses, VS51, VS53, and VS57, against wt PV-1 (Mahoney) (NCBI GenBank reference sequence V01149.1) (1ASJ)  sequences were determined. The nucleotide sequences of PCR amplicons and individual clones (01149.1)  showed v01149.1) . The pos) (1ASJ)  using Py) (1ASJ)  Table 2n = 3 cor) (1ASJ) . PV-1 (M) (1ASJ)  and comp) (1ASJ) . These mThe VP1 pocket mutation V1087A was reported previously to increase the thermal stability of PV-1 and PV-2 . In ordeP < 0.0001). PV51 was significantly more stable than the wt (P < 0.05) and PV53 (P < 0.001); however, PV53 was not significantly different from the wt. PV57 was significantly (P < 0.05) more stable than all of the viruses tested (P < 0.0001) withstand higher temperatures than the corresponding nondelta mutant (PV53), while PV57 and its corresponding delta mutant had similar inactivation temperatures (P < 0.001), decrease of PV53, respectively. Removal of I1194V from VS57, however, resulted in a 40-fold, significant (P < 0.05), increase in the virus titer  was used to generate clones PV51, PV53, and PV57, both with and without (δ) the mutation I1194V , and thes tested . PV53δ ceratures  and B. Rus titer . Althougus titer .P < 0.001) more thermally stable than PV51, and both were significantly (P < 0.00001) more thermostable than the wt virion. The antigenic switches of virions occurred at 44°C (PV51), 49°C (PV53δ), and 50°C (PV57δ). EC antigenicity profiles (P < 0.05) and PV51 (P < 0.0001). For the corresponding ECs of PV51, PV53δ, and PV57δ, antigenic switches occurred at 38°C, 42°C, and 45°C, respectively (P < 0.001) less stable than BRP, both PV53δ and PV57δ were as stable as BRP , 53°C (VS53), and 57°C (VS57). This was confirmed by thermal-inactivation assays, which measured virus infectivity and fitness. We also measured the capsid flexibility under continuously increasing thermal stress by PaSTRy. We were able to capture the melting points of the virus capsids, which increased upon selection. We also showed that ECs generated from selected virions were antigenically more stable, withstanding temperatures up to 45°C. Through reverse genetics, we identified the mutations responsible for the increased thermal stabilities and confirmed them by reintroducing individual mutations into an infectious clone, with similar results.The two key VP1 mutations (V1087A and I1194V) that evolved in early cycles of selection were maintained as a consistent pair in all subsequent selected candidates. In 2004, a study reported that V1087A is a heat-resistant mutation able to withstand 50°C , althougVP1 residues 87 and 194 both lie within the pocket that is known to play a role in capsid stability. These residues are located on the B and G strands of the VP1 β-barrel, respectively, and may play vital roles in EC stability. Based on the proximity of the B and G strands to antigenic binding sites on the BC loop , interacIn the native conformation, residues 95 to 104 around the VP1 BC loop of the viral capsid contribute to antigenic sites , 49. Of Recently, it has been postulated that heating 160S particles results in capsid expansion that creates a greater distance between adjacent antigenic BC loops in an event that coincides with loss of native antigenicity , 52. In Further selection of VS51 generated a virus with two internal mutations, one in VP4 (F4046L) and a second in VP1 (T1026A). Further selection led to reversion of these mutations and identification of additional internal mutations, such as an interpentamer VP3 mutation (T3175A) that, in combination, stabilized the EC structure further. Owing to high rates of mutation, RNA virus populations comprise a quasispecies of genetically linked viruses distributed around a consensus sequence . As a poOwing to the inability of the I1194V mutation to assemble particles, we assessed the effect of omitting the mutation in PV53 and PV57 mutants . Our findings showed that in the absence of I1194V, PV53δ and PV57δ had greater capsid stabilities. This may further suggest the destabilizing effects of mutating the highly conserved VP1 residue 194 on virus fitness.It has been shown that stabilized N-specific ECs of PV are recognized by all known neutralizing monoclonal antibodies , 55, as Through thermal selection, we have shown that the PV-1 EC can be stabilized by mutations, e.g., in the VP1 pocket and at the 5-fold vertex. Selected variants were infectious and had thermally and antigenically stabilized ECs (stabilized PV-2 and PV-3 VLPs are in development). These capsid mutations can now be introduced into recombinant expression systems to synthesize antigenically stable potential PV VLP vaccines for a polio-free world.in vitro RNA synthesis. A hammerhead ribozyme was included at the 5′ end, resulting in production of an authentic infectious PV-1 RNA  used in this study was sourced from Bert Semler, University of California, and was cloned downstream of a T7 RNA promoter to allow PV-1 RNA . To initPV-1 RNA , 58. VirPV-1 RNA . Virus t35S]Cys/Met 2.75 h postinfection/transfection. At 3.25 h postinfection/transfection, the cells were treated with GuHCl at a final concentration of 2 mM to inhibit RNA replication and stimulate accumulation of genome-free ECs (Virus was propagated in cysteine/methionine-deficient medium and radiolabeled with [free ECs . The ECsfree ECs  and detefree ECs .g (using a Beckman SW55 Ti rotor) for 3.5 h at 4°C. The virus pellet was resuspended in phosphate-buffered saline (PBS) and clarified by differential centrifugation. The supernatant was purified through a 15 to 45% (wt/vol) sucrose density gradient by ultracentrifugation at 300,000 × g (using a Beckman SW55 Ti rotor) for 50 min at 4°C  sucrose cushion at 300,000 × n at 4°C . The peaVirus samples were heated for 30 min at temperatures resulting in 99.99% loss in titer. The surviving virus was propagated at 37°C in HeLa cells by standard methods, and the infectivity titers of pre- and postheating samples were determined by plaque assays . ThermalMutants were serially passaged five times without selection pressure. Serially passaged samples were then heated at the respective selection temperatures . Preheating and postheating virus samples were assayed for infectivity.Virus samples were incubated at a range of temperatures for 30 min within a thermal cycler and immediately cooled to 4°C for 5 min. The heat-treated samples were titrated by plaque assays .The thermal stability of the virus particles was further characterized by a thermofluorometric dual dye-binding assay using the nucleic acid dye SYTO9 and the protein-binding dye SYPRO orange (both from Invitrogen). Reaction mixtures of 50 μl containing 1.0-μg double-purified and desalted virus fractions, 5 μM SYTO9, 150× SYPRO orange, and PaSTRy buffer  were mixed and ramped from 25 to 95°C, with fluorescence reads taken at 1°C intervals every 30 s within the Stratagene MX3005p quantitative-PCR (qPCR) system .Radiolabeled virions and ECs were purified by sucrose density gradients as previously described  and incuImmunoprecipitated samples were boiled in SDS-loading loading buffer . Proteins were separated through a 12% polyacrylamide gel by electrophoresis .2O (ddH2O). The radioisotope signal was amplified using a fluorometric solution , and gels were vacuum dried onto filter paper. The dried gels were exposed to X-ray film at −80°C prior to developing (Proteins separated by SDS-PAGE were fixed in a solution of 25% (vol/vol) isopropanol, 10% (vol/vol) acetic acid, and 65% (vol/vol) double-distilled Hveloping .Cell-cultured viruses and intermediates were harvested as previously described. Virus proteins were dissociated and separated by SDS-PAGE using standard protocols  and immuPurified virions and ECs were dialyzed from sucrose fractions into dialysis buffer  and visualized by negative-staining transmission electron microscopy according to standard protocols .Native virion particles were immunoprecipitated, and total RNA was extracted with guanidinium thiocyanate-phenol-chloroform . The strEscherichia coli DH5α cells were transformed with mutagenized plasmid constructs and selected for antibiotic resistance according to standard protocols .Where appropriate, data were analyzed by one- and two-way analysis of variance (ANOVA) for multitest analysis of multiple groups, while Student


Avian H9N2 and 2009 pandemic H1N1 (pH1N1) influenza viruses can infect pigs and humans, raising the concern that H9N2:pH1N1 reassortant viruses could emerge. Such reassortants demonstrated increased replication and transmissibility in pig, but were still inefficient when compared to pH1N1. Here, we evaluated if a reassortant virus containing the hemagglutinin and neuraminidase of A/quail/Hong Kong/G1/1997 (H9N2) in the A/California/04/2009 (pH1N1) backbone could become better adapted to pigs by serial passaging. The tropism of the original H9N2:pH1N1 (P0) virus was restricted to the nasal mucosa, with no virus detected in the trachea or lungs. Nevertheless, after seven passages the H9N2:pH1N1 (P7) virus replicated in the entire respiratory tract. We also compared the transmissibility of H9N2:pH1N1 (P0), H9N2:pH1N1 (P7) and pH1N1. While only 2/6 direct-contact pigs showed nasal virus excretion of H9N2:pH1N1 (P0) ≥five days, 4/6 direct-contact animals shed the H9N2:pH1N1 (P7). Interestingly, those four animals shed virus with titers similar to those of the pH1N1, which readily transmitted to all six contact animals. The broader tissue tropism and the increased post-transmission replication after seven passages were associated with the HA-D225G substitution. Our data demonstrate that the pH1N1 internal-protein genes together with the serial passages favour H9N2 virus adaptation to pigs. Since the late 1990s, H9N2 viruses have been occasionally isolated from humans and swine in China, leading to increasing concerns about their pandemic potential8. However, all reported human or swine H9N2 virus infections were dead-end events and the virus failed to spread within the human or swine population9.H9N2 avian influenza viruses are currently endemic in poultry in South East Asia and the Middle Eastα2,6-linked sialic acids  and to improve the replication of H9N2 viruses in human airway epithelial cells in vitro, supporting concerns about their pandemic potential12. The binding of the viral HA to sialyloligosaccharides, present on the host cell surface, is the first step for influenza virus infection. Because of the predominant expression of Siaα2,6Gal in human and swine upper respiratory tract14, most human and swine influenza viruses more willingly bind to receptors that contain Siaα2,6Gal15. In contrast, avian influenza viruses preferentially bind sialic acids linked to galactose by an α2,3 linkage . Therefore, a switch from Siaα2,3Gal to Siaα2,6Gal receptor-binding preference is considered an important step for avian influenza virus adaptation to mammals16. Nevertheless, enhanced Siaα2,6Gal binding is not entirely sufficient to make H9N2 viruses transmissible between mammals18. This suggests that other amino acid substitutions and/or an adequate constellation of internal genes is also required for efficient transmission of H9N2 viruses between mammals.A significant proportion of poultry H9N2 field isolates contains a leucine instead of glutamine residue at position 226 in the hemagglutinin (HA) receptor-binding site (RBS) . This mutation has been shown to enhance HA binding to terminal 19. In that sense, the sporadic isolation of avian H9N2 influenza viruses from pigs and humans in China increases the possibility of co-infections with endemic swine or human influenza viruses, which could result in the generation of novel reassortant viruses with enhanced transmissibility in mammals.Due to the segmented nature of the influenza viruses, the RNA segments of two (or more) different influenza viruses can “reassort” when co-infecting the same host cell, thereby generating progeny viruses with properties different from the parental viruses20. After its first appearance in 2009, the virus has spread around the world in both human and swine populations21. Moreover, novel reassortant viruses containing pH1N1 internal-protein and surface-protein genes from other endemic swine influenza virus have been isolated frequently from pigs worldwide25. In addition, H3N2 swine influenza viruses containing the pH1N1-derived matrix (M) protein gene (H3N2 variant) were isolated from 364 human cases in the US since 201126. This suggests that pH1N1 internal-protein genes are compatible with several surface-protein gene combinations and allow for robust replication in mammals.The 2009 pandemic H1N1 virus (pH1N1) is a swine-origin reassortant virus containing human, swine and avian influenza virus genes27. In pigs similar reassortant viruses also showed increased transmission when compared to the parental H9N2. However, their transmission was still not as efficient as that of swine-adapted viruses like pH1N128. We previously demonstrated that serial passages of A/quail/Hong Kong/G1/1997 (H9N2) virus in pigs slightly enhanced replication and transmission in swine . Although the resulting pig-adapted virus carried a number of mutations compared to the parental virus, transmission was still less efficient than that of naturally circulating viruses. In the present study, we have examined the replication and transmissibility after ten serial pig passages of a reassortant virus containing A/quail/Hong Kong/G1/1997 (H9N2) surface-protein genes and NP gene within the A/California/04/2009 (pH1N1) background to evaluate if the pH1N1 backbone confers an advantage in H9N2 adaptation to pigs. This serial passaging of the H9N2:pH1N1 reassortant resulted in a virus that replicated in and transmitted between pigs at high rates. The predominant mutation in the passaged reassortant virus was an aspartic acid to glycine at position 225 in the HA RBS. Therefore, our results showed that the combination of reassortment and mutations induced by the serial passages generated a virus with a predominant mutation at position 225 in HA RBS that replicated and transmitted at high rates in pigs.In ferret transmission studies, a reassortant H9N2 virus containing pH1N1 internal-protein genes readily transmitted to all contact animals by respiratory droplets6,5 TCID50 of H9N2:pH1N1 (P0). To determine nasal virus excretion, individual pigs were swabbed daily from 0 to four days post-inoculation (dpi). None of the pigs showed clinical signs of disease. The reassortant H9N2 virus was detected in nasal swabs of all experimentally infected pigs. The highest amount of virus was excreted during passages three and eight  virus and its descendent isolated after seven passages in pigs (H9N2:pH1N1 (P7)). The latter virus was chosen because virus titers in nasal swabs and the respiratory tract samples were highest at this passage, suggesting a substantial degree of swine-adaptation. As a positive control, we used a representative swine-adapted virus, the A/California/04/2009 (pH1N1) virus As expected, all six A/Cal/04/09 direct-contact pigs excreted high amounts of virus (AUC >17) and seroconverted with VN titers ≥192. While all six direct-contact animals in the H9N2:pH1N1 (P0) group also seroconverted and excreted virus, only two shed virus with an AUC >6. In the H9N2:pH1N1 (P7) group, in contrast, just four out of six direct-contact animals excreted virus but all of them showed an AUC >21, similar to the A/Cal/04/09 group. The remaining two H9N2:pH1N1 (P7) direct-contact pigs did not excrete any virus and failed to seroconvert. VN titers in the six H9N2:pH1N1 (P0) and four out of six H9N2:pH1N1 (P7) direct-contact pigs were <256.de novo assembled consensus sequence of the H9N2:pH1N1 (P7) stock virus are shown in Table de novo assembled consensus sequence of the H9N2:pH1N1 (P7) stock virus and the reference sequence for the A/quail/Hong Kong/G1/1997(H9N2) virus. The nonsynonymous variants detected at a frequency ≥0.5% in the H9N2:pH1N1 (P7) genome are shown in Supplementary Table We next compared the variants present in the H9N2:pH1N1 (P0) virus population and the H9N2:pH1N1 (P7) virus isolated from the nasal mucosa of a directly inoculated pig and the direct-contact pig with the highest nasal excretion in the transmission study. By comparing the virus population from the directly-inoculated and the direct-contact pigs we aimed to determine if the mutations selected during the serial passaging were maintained after transmission or if the transmission itself favored the acquirement of a different set of mutations. In addition, the H9N2:pH1N1 (P0) virus stock was included to determine whether the detected variants in the passage seven virus were already present in the virus stock before pig adaptation. Illumina MiSeq deep sequencing was performed on full genome RT-PCR products of the samples. Sequencing of H9N2:pH1N1 (P0) virus revealed contamination of the H9N2:pH1N1 virus stock with H9N2 internal-protein genes, which were most pronounced for the PB1, NS and NP gene segments (see Table i.e. the substitution of aspartic acid by glycine at position 225 of the HA RBS (HA-D225G). This mutation was absent  stock virus showed variation above 5% at positions 535 and 615 of the basic polymerase 2 protein (PB2), position 119 of the HA and position 179 of the non-structural 1 protein NS1) Table . Nonethe Table . 30. However, the continuous exposure of humans and pigs to avian H9N2 viruses might pose a real threat for public health if these viruses would acquire the genetic changes that allow them to transmit efficiently between mammals. In this study, we evaluated the efficiency of direct-contact transmission between pigs of a reassortant H9N2 virus containing pH1N1 internal-protein genes, and the effect of serial pig passages on transmission efficiency. We demonstrated, for the first time, that a pig-passaged reassortant H9N2:pH1N1 virus was excreted by more than half of the direct-contact pigs at titers similar to those of the parental pH1N1 virus.Since the late 1990s, avian H9N2 viruses have been isolated from pigs and humans, so far always as dead end events28. In fact, virus excretion from the nasal cavity and replication in the respiratory tract were present at variable rates during the ten serial pig passages. This finding contrasts with a previous study from our group using the same parental H9N2 virus (A/quail/Hong Kong/G1/1997), however without reassortment with pH1N1 internal-protein genes  virus did not show replication in the lungs during passage one, it replicated homogeneously in the entire respiratory tract during passage two. Strikingly, the virus replication in the lungs then progressively decreased during passages four, five and six to increase again at passage seven. This suggests the presence of a selection bottleneck due to the genetic pressure generated by the experimental set up31. However, a deeper analysis of the viral population would be needed to elucidate the possible cause of the decrease in the replication rates.Our data confirm the ability of the H9N2:pH1N1 reassortant viruses to infect pigs as was reported in previous studies28. In the aforementioned studies ≤50% of the direct-contact pigs excreted virus. This higher transmission rate suggests that the serial passaging in pigs contributed to the adaptation of the reassortant H9N2 virus. Yet of the six direct-contact pigs two animals that were housed in the same isolator did not excrete virus at all. This is most likely due to the low amount of virus excreted by the direct-inoculated pig (AUC 17.1), which not have reached a critical threshold to allow virus transmission. Further detailed genetic analyses of the virus excreted by the donor pig would be needed to better understand if there is, besides the low titer, also a genetic basis for the lack of transmission.We consider an influenza virus as swine-adapted when it succeeds to transmit between pigs with a similar efficiency as described for the endemic swine influenza viruses. Therefore, we compared pig-to-pig transmission of the seventh passage virus with that of the original reassortant and pH1N1 viruses. Whereas both reassortant viruses transmitted to direct-contact pigs, the seventh passage virus was clearly shed at higher titers by four out of six direct-contact pigs. This virus also showed higher transmission efficiency when compared with previous studies describing H9N2:pH1N1 reassortant viruses in pigs36, those studies focused either on the role of the PB2 and the M-coding genes or on the complete internal gene cassette. Not much is known about the exact role of pH1N1-origin NP-protein gene in the adaptation of influenza viruses to mammals. However, it has been described that the interaction between the NP and importin-α can influence the influenza virus host range. For example, the presence of the NP-N319K substitution was described as an important factor for the replication of H7N7 viruses in mice37. Nevertheless, both the H9N2 and the pH1N1 NP-protein genes lack that mutation. Thus, further analysis with reverse genetic generated reassortants would be necessary to elucidate if the combination of H9N2-origin NP-protein gene and pH1N1-origin internal genes has an effect in the replication and transmission of influenza viruses in mammals. Regarding the mutations detected during the analysis, only one of the mutations that appeared in the virus after seven passages and was maintained even after transmission, the HA-D225G substitution in the RBS. This mutation was therefore likely associated with the higher replication and transmission efficiency of the H9N2:pH1N1 (P7) virus. Interestingly, this mutation was present in all forty-seven swine and seventeen human H9N2 field isolates available in GenBank in November 2016. This mutation was also associated with enhanced replication and transmission of parental H9N2 after four passages in pigs . In the 1918 and 2009 pandemic H1N1 viruses this mutation has been associated with increased Siaα2,3Gal tropism39. In pigs and humans Siaα2,3Gal receptors are mainly found in the lungs13. The presence of the HA-D225G mutation after seven passages may therefore explain the consistent replication in the lungs during passage eight. Although specific receptor-binding tests would be necessary to elucidate the exact role of the HA-D225G mutation within the HA-RBS, our data suggest that it may be a marker for adaptation of H9N2 viruses to swine.To elucidate the effects of the serial passaging and the pig-to-pig transmission on the virus population, we compared the viral diversity in the original reassortant virus with the virus isolated after seven passages in pigs and the virus excreted after direct-contact transmission. It should be mentioned here that we detected H9N2 internal-gene segments in our original reassortant virus stock. Interestingly, the serial passaging resulted in the purification of the original virus and, after seven passages only the NP-coding gene was completely of H9N2-origin. As in the original stock the H9N2-derive NP gene segment was present at 91%, this high percentage might have been the reason for the purification towards the H9N2-origin instead of towards the pH1N1-origin as seen for the other gene segments. However, the NS-coding gene showed the same percentage of contamination and was completely selected upon passaging. Although previous studies demonstrated that pH1N1-origin internal genes conferred an advantage in replication and transmission of influenza viruses in mammals28, this report demonstrated that avian H9N2 influenza viruses can reassort with pH1N1 internal genes, resulting in enhanced virus transmission in pigs when compared to the parental, non-reassorted H9N2 virus. The present study also underscores that repeated introduction of reassortant H9N2 viruses into the swine or humans may result in the selection of virus variants with a transmission efficiency close to that of the endemic swine or human influenza viruses. However, the lack of transmission detected in one of the contact groups used for transmission of passage seven virus, emphasizes the complexity of the adaptation process of an avian virus to a mammalian species and the need for additional research to better understand this crucial step in cross-species transmission of influenza viruses.In line with previous studies17. Another passage in MDCK cells was made at the arrival of the virus to the Laboratory of Virology at Ghent University to generate a stock to be used in this study.The reassortant virus containing A/California/04/2009 (pH1N1) internal genes and A/quail/Hong Kong/G1/1997 (H9N2) HA and NA was produced by reverse genetics at the Department of Diagnostic/Pathobiology at Kansas State University. The virus was plaque purified, passaged three times in MDCK cells, characterized and experimentally validatedA/California/04/2009 (pH1N1) was kindly provided by the Centers for Disease Control and Prevention (US CDC). The virus underwent three passages in MDCK cells at the US CDC. Later one passage in eggs and one in MDCK cells were performed in the Laboratory of Virology at Ghent University before use.6.5 TCID50 of virus. Pigs were clinically monitored daily for general  and respiratory  symptoms. Nasal swabs were also collected daily from 0 to four dpi. At four dpi both pigs were euthanized with a lethal dose of pentobarbital sodium (≥100 mg/kg) in the jugular vein. The following tissue samples were collected for virus titrations: nasal mucosa respiratory part , nasal mucosa olfactory part (i.e. ethmoid labyrinth), trachea  and five different samples representative of the entire lung. The nasal mucosae (respiratory part) were pooled and a 20% (w/v) tissue homogenate was prepared and used to inoculate two pigs with this passage one virus inoculum. Ten blind  serial passages were performed in this way. Nasal swabs from all pigs, tissue samples and the inocula used for the subsequent passages were titrated on MDCK cells.Forty-seven three-week-old piglets were purchased from a commercial herd free of swine influenza and porcine reproductive and respiratory syndrome virus. Upon arrival, pigs were confirmed seronegative to influenza virus with a commercial blocking anti-influenza A nucleocapsid ELISA (ID-VET) and by virus neutralization (VN) tests. Consecutive passages started with the housing of two pigs in a biosafety level-3 (BSL-3) HEPA filtered isolator, and their intranasal inoculation with three ml of phosphate-buffered saline (PBS) containing 106.5 TCID50 of the respective virus. Two dpi, two pigs were introduced in each isolator allowing direct contact with the inoculated pig. All animals were clinically monitored and nasal swabs for virus isolation were collected daily from all pigs during eleven days after cohousing. Sixteen dpi, the animals were relocated to a BSL-2 HEPA filtered isolation unit. Serum samples were collected at 0, 16, 23 and 30 dpi and 0, 14, 21 and 28 days post-contact.For each transmission experiment nine three-week-old pigs were used. At 0 dpi three pigs were housed in three separate BSL-3 HEPA filtered isolators (one index pig per isolator) and intranasally inoculated with 10All experimental procedures were conducted in accordance with the E. U. Animal Welfare Directives, and were authorized and supervised by the Local Ethical and Animal Welfare Committee of the Faculty of Veterinary Medicine, Ghent University (EC2013/63 number).2, virus positive MDCK cells were visualized by immunoperoxidase staining. The cells were fixed with 4% paraformaldehyde for ten minutes at room temperature and subsequently incubated with mouse anti-NP monoclonal HB-65 antibody  for two hours. Incubation with horseradish peroxidase-conjugated goat anti-mouse polyclonal antibody  for one hour was followed by a development step with H2O2 as substrate and 3-amino-9-ethyl-carbazole (AEC) as precipitating agent. Virus titers were calculated by the method of Reed and Muench (1938) and expressed as log10 50% tissue culture infective doses (TCID50) per 100 milligram  or per gram (tissues).Nasal swabs from both nostrils were suspended in 1 ml PBS supplemented with 10% foetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin and mixed vigorously at 4 °C for 1 hour. Tissue samples were weighed and grounded in PBS with 10 IU/ml penicillin and 10 µg/ml streptomycin to obtain 20% (w/v) tissue homogenates. Briefly, confluent monolayers of cells were inoculated with 10-fold serial dilutions of the sample. After five days of incubation at 37 °C with 5% CO50 of virus per well as previously described40. Briefly, 2-fold serum dilutions were incubated  with 100 TCID50 of MDCK cell-grown virus. MDCK cells (800.000 cells per ml) were incubated with the virus-serum mixture for 24 h, after which the virus positive cells were visualized by immunoperoxidase staining. Antibody titers were expressed as the reciprocal of the highest serum dilution that completely inhibited virus replication in MDCK cells.VN tests were performed on MDCK cells in 96-well plates, using 100 TCIDNasal virus shedding of individual pigs was quantified by calculation of the AUC. The means of the AUCs of different viruses were compared using standard two–sample Mann-Whitney U tests. Differences were considered significant when p < 0.05. GraphPad Prism Software, version 5, was used for statistical analyses.41. Two separate reactions were performed, using primers specific for the influenza A vRNAs: CommonUni12G (GCCGGAGCTCTGCAGATATCAGCGAAAGCAGG) and CommonUni12A (GCCGGAGCTCTGCAGATATCAGCAAAAGCAGG). Subsequently, all eight genomic segments were amplified in one PCR reaction containing an 1:1 mix of CommonUni12G and CommonUni12A cDNA, primer CommonUni13 (GCCGGAGCTCTGCAGATATCAGTAGAAACAAGG) (200 nM) and the Phusion High Fidelity polymerase (Thermo Scientific)43. RT-PCR was performed as described41, with the first 5 PCR cycles performed with an annealing temperature of 45 °C (instead of 72 °C). PCR products were purified using the High Pure PCR Product Purification Kit (Roche) and elution of the DNA in sterile ultrapure water.The RNA from the virus samples was isolated using the QIAamp Viral RNA Mini Kit . cDNA was synthesized with the Transcriptor First Strand Synthesis kit (Roche), already described500 ng of each purified RT-PCR sample was sheared with an M220 focused-ultrasonicator (Covaris) set to obtain peak fragment lengths of 400 bp. Next the NEBNext Ultra DNA Library Preparation kit (New England Biolabs) was used to repair the ends and to add the Illumina MiSeq-compatible barcode adapters to 100 ng of fragmented DNA. The resulting fragments were size-selected at 300 to 400 bp using Agencourt AMPure XP bead sizing (Beckman Coulter). Then, indexes were added in a limited-cycle PCR (10 cycles), followed by purification on Agencourt AMpure XP beads. The fragments were analysed on a High Sensitivity DNA Chip on the Bioanalyzer (Agilent Technologies) before loading on the sequencing chip. After the 2 × 250 bp MiSeq paired-end sequencing run, the data were base-called and converted to Illumina FASTQ files (Phred +64 encoding).41. The processed sequencing reads were subsequently mapped to the de novo assembled consensus sequence of the H9N2:pH1N1 (P7) stock virus sequence. This consensus sequence is available through GenBank accession number (KY785904-KY785911). The processed sequencing reads for the H9N2:pH1N1 (P7) stock virus were mapped to a reference genome composed of the H9N2:pH1N1 (P7) stock virus sequence and the A/quail/Hong Kong/G1/1997(H9N2) virus genome. The raw sequencing data were submitted to the NCBI Sequence Read Archive where they can be found under project number: SRP095510.Sequence data analyses were performed on the resulting Illumina FASTQ files (Phred 64+ encoding) using CLC Genomics Workbench (Version 7.0.3) following the analysis pipeline as describedSupplementary Table S1: Variants present in H9N2:pH1N1(P0) virus stock.


To investigate changes to health insurance costs for post-discharge postpartum care after the introduction of a midwife-led coordinated care model.The study included mothers and their newborns insured by the Helsana health insurance group in Switzerland and who delivered between January 2012 and May 2013 in the canton of Basel Stadt (BS) (intervention canton). We compared monthly post-discharge costs before the launch of a coordinated postpartum care model  to those after its introduction . Costs in the intervention canton were also compared to those in five control cantons without a coordinated postpartum care model .The average monthly post-discharge costs for mothers remained unchanged in the seven months following the introduction of a coordinated postpartum care model, despite a higher use of midwife services (increasing from 72% to 80%). Likewise, monthly costs did not differ between the intervention canton and five control cantons. In multivariate analyses, the ambulatory costs for mothers were not associated with the post-intervention phase. Cross-sectionally, however, they were positively associated with midwifery use. For children, costs in the post-intervention phase were lower in the first month after hospital discharge compared to the pre-intervention phase (difference of –114 CHF [95%CI –202 CHF to –27 CHF]), yet no differences were seen in the cross-sectional comparison.The introduction of a coordinated postpartum care model was associated with decreased costs for neonates in the first month after hospital discharge. Despite increased midwifery use, costs for mothers remained unchanged. There is a growing trend towards earlier hospital discharge after birth ; a trend3457410121314In countries where diagnosis-related group reimbursement (DRG) is in place, care provision and respective costs have shifted from the in-hospital to the ambulatory sector 20212223.202122Shifting early postnatal care from in-patient to community-based settings requires intensified network building between inpatient and outpatient care providers, which is seen as an intermediate position between full service segregation and full integration . Yet, fr262728293135In Switzerland, average postnatal hospital stays have more than halved over the last 50 years, from nearly two weeks to 5.7 days in 2012 38. With 42What was unclear, however, was whether the model increased or lowered costs covered by the mandatory health insurance. For this reason, a cost analysis was conducted to investigate health insurance costs for the post-discharge phase of deliveries before and after the introduction of Familystart.The midwife-led coordinated postpartum care model, Familystart, guarantees postpartum care at home for each mother- child pair discharged from either of the two obstetric clinics in Basel Stadt (the University Women’s Clinic and the Bethesda private clinic). Familystart’s integrated care strategy is based on a needs analysis of new parents  and on tWe used the database of the largest health insurer in Switzerland, the Helsana Group, to recruit the study population. The study included mothers insured by the Helsana Group and who delivered between January 2012 and May 2013 in the canton of Basel Stadt (BS), the intervention canton where Familystart began in November 2012, and their newborn children. We also included mothers and their newborns from five control cantons without a coordinated postpartum care model . Mothers with a basic compulsory insurance coverage were identified through a claim with a delivery-related DRG starting in this period. Newborns were identified either through the unique family number at Helsana or by combining the mother’s address and the date of birth of the child from the hospital where the mother had delivered.In a pre-/post design, post-discharge costs covered by the Helsana Group were analysed, first for a 10-month period before the introduction of Familystart and then for a seven-month period after the introduction of Familystart. The shorter period of measurement in the post-intervention phase was due to time restrictions imposed by the research programme funding the study. Based on a hospital code, data could also be analysed separately for the two hospitals participating in Familystart. A subsequent cross-sectional comparison was made between mothers and their newborns delivered in BS and those from the five control cantons for the period 1 January 2012 to 31 May 2013.0 and 30-day periods after hospital discharge were labelled as month1, month2, etc. up to the end of the treatment period. In case of overlap of treatment between these monthly periods, costs were assigned proportionally to the duration of the treatment. Of the 236 women having delivered in BS, the exact date of delivery was unavailable for 56, thus they were excluded from the post-delivery cost calculation. Subsequently, monthly costs of the intervention canton (intervention group) were compared to those in the five control cantons . For this analysis, costs were compared per calendar month, from January 2012 to May 2013. Furthermore, the duration of pre- and post-delivery hospital stay was calculated and analysed analogously. DRG codes were grouped into four categories: vaginal delivery without (V) and with complications (Vk) and caesarean section without (C) and with complications (Ck). Additional information was extracted from the Helsana Group database to control for possible confounding factors: mother’s age, insurance type , amount of the deductible , length of hospital stay, and use of midwifery service. Being insured in a managed care model was included in the models for ambulatory costs as well.For the study period, costs could be retrieved for 97% of all bills relating to mothers. Costs were calculated as the average (and median) monthly post-discharge costs for mothers and for those children also insured with Helsana Group. As the data for costs were skewed, boot strap estimation was used to derive 95% confidence intervals of mean cost differences between the groups . Costs fMultivariable regression analyses were used to determine whether the coordinated care model was associated with an increase or decrease of costs for mothers and children when controlling for other cost determinants. These analyses were conducted for costs for mothers and costs for children in the first month after hospital discharge. A sensitivity analysis was also conducted for costs occurring in first two months after hospital discharge.All statistical analyses were performed using R version 3.1.0 (2014–04–10)  47.There were 16, 424 women throughout Switzerland insured at the Helsana Group and who had delivered within the study period. Of them, 236 mothers had delivered in BS and 7, 767 had delivered in one of the five control cantons. Overall, 46% of mothers had an uncomplicated vaginal delivery, 19% had a vaginal delivery with complications, 21% had an uncomplicated caesarean section and 14% had a caesarean section with complications. Table mothers within the six months following discharge . Re-hospitalisations of children were more frequent, overall 10% were re-hospitalised (24/236), of whom 17/141 (14%) were re-hospitalised before and 7/95 (8%) after the introduction of Familystart .There were only a few re-hospitalisations of Of the 16 registered emergency consultations with children, 10 occurred before and six after November 2012 . Of the 129 emergency consultations with mothers, there was no significant difference before and after the introduction of Familystart (70 versus 59 consultations).mothers (before and subsequent to the introduction of Familystart in BS) remained unchanged for six months  as the main determinant of interest (Table children during the first month yielded no significant associations (data not shown).st Table . There wFurther multivariate regression analyses were conducted to investigate possible reasons underlying the different length of hospital stays in BS and in the control cantons (data not shown). In the longitudinal analysis (before/after start of Familystart), duration of hospital stay was not associated with delivery under the integrated care model, only with type of delivery  and with supplementary private hospital insurance coverage.In the cross-sectional analysis, a lower mean duration was associated with delivering in BS compared to delivering in a control canton, and also with a higher deductible. Higher mean length of hospital stay was associated with higher maternal age, the type of delivery  and having private/semiprivate insurance coverage.In our study, average monthly costs for mothers remained unchanged in the seven months after the introduction of a coordinated postpartum care model, despite a higher use of midwifery services, which increased from 72% to 80%. Likewise, costs did not differ between the intervention canton and the five control cantons (average use of midwives 68 %). The longitudinal comparison for the first month after hospital discharge showed lower costs for children after the introduction of Familystart compared to the pre-intervention phase, with a difference of –114 CHF (95%CI –202 to –27) per person, whereas the cross-sectional comparisons with five control cantons showed no differences.Cost reductions associated with postnatal home care programmes have been reported by previous studies on postnatal care for a low risk population in Switzerland  and for 16It is noteworthy that costs for mothers did not increase despite an increase in the use of midwifery services from 72% to 80%. In multivariable analyses, the post-discharge costs for mothers did not differ between the pre- and post-intervention phases. However, in the cross-sectional analysis, they were associated with midwifery use. Health cost savings may have occurred simultaneously with some cost increases associated with the greater use of midwives. Higher quality of care may have been achieved by a range of measures to foster inter-professional collaboration, such as the inter-professional experts’ board, a mixed working group of midwives and health visitors, or the guideline on collaborative care provision and continuing education. The increased access to midwifery care after hospital discharge is in line with previous studies showing an association between the degree of inter-professional collaboration and accessibility of services 53. In boThe reduction in costs for children may have occurred indirectly as midwives can charge services only to the mother’s bill and not to the child’s. Midwives may have prevented emergency consultations or paediatric consultations for children by informing and reassuring parents. The number of re-hospitalisations was very low, both among mothers and children, and unlikely to account for the observed cost difference for children. The longitudinal comparison of paediatric costs did not reveal any difference. To substantiate the claim that families used paediatric services less frequently after the introduction of Familystart, more data on children would be needed, such as their age at discharge.Overall costs may vary with the length of hospital stay, thus, determinants of hospital-stay length are of interest. A complicated mode of delivery was expected to be associated with longer hospital stays . Yet, hi1714The study has several limitations and strengths worth mentioning. The intervention group was small, limiting the power of detecting differences, for instance, for hospital re-admissions. Also, we cannot exclude some residual confounding by information not assessed in the database, such as parity or age of children at discharge. If mothers insured by the Helsana Group differ from mothers insured elsewhere with regard to risks in pregnancy and delivery, this might affect the findings. We have, however, no evidence that there is a positive or negative risk selection associated with insurance from the Helsana Group.A strength of the study is that we could take into account costs after hospital discharge for mothers and infants over a period of six months and costs could be retrieved for 97% of all claims relating to the study period. In addition to before/after comparisons, we could also conduct cross-sectional comparisons between the intervention canton and five cantons without a coordinated care offer and triangulate the respective findings. We did not restrict our analysis to healthy full-term children.Although the introduction of a coordinated postpartum care model increased the access and use of midwifery home care, there was no cost increase for mothers but a cost reduction for neonates during the first month after hospital discharge. Further research should include a continuous evaluation over extended periods of time, and enable the assessment of both economic as well as health outcomes.


Mycobacterium abscessus is a reemerging pathogen that causes pulmonary diseases similar to tuberculosis, which is caused by Mycobacterium tuberculosis. When grown in agar medium, M. abscessus strains generate rough (R) or smooth colonies (S). R morphotypes are more virulent than S morphotypes. In searching for the virulence factors responsible for this difference, R morphotypes have been found to form large aggregates (clumps) that, after being phagocytozed, result in macrophage death. Furthermore, the aggregates released to the extracellular space by damaged macrophages grow, forming unphagocytosable structures that resemble cords. In contrast, bacilli of the S morphotype, which do not form aggregates, do not damage macrophages after phagocytosis and do not form cords. Cording has also been related to the virulence of M. tuberculosis. In this species, the presence of mycolic acids and surface-exposed cell wall lipids has been correlated with the formation of cords. The objective of this work was to study the roles of the surface-exposed cell wall lipids and mycolic acids in the formation of cords in M. abscessus. A comparative study of the pattern and structure of mycolic acids was performed on R (cording) and S (non-cording) morphotypes derived from the same parent strains, and no differences were observed between morphotypes. Furthermore, cords formed by R morphotypes were disrupted with petroleum ether (PE), and the extracted lipids were analyzed by thin layer chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry. Substantial amounts of trehalose polyphleates (TPP) were recovered as major lipids from PE extracts, and images obtained by transmission electron microscopy suggested that these lipids are localized to the external surfaces of cords and R bacilli. The structure of M. abscessus TPP was revealed to be similar to those previously described in Mycobacterium smegmatis. Although the exact role of TPP is unknown, our results demonstrated that TPP are not toxic by themselves and have a function in the formation of clumps and cords in M. abscessus, thus playing an important role in the pathogenesis of this species. Mycobacterium genus other than the Mycobacterium tuberculosis complex and Mycobacterium leprae. The overall prevalence of lung disease related to NTM is increasing worldwide and is caused by Mycobacterium avium complex, Mycobacterium kansasii, and increasingly, Mycobacterium abscessus, which is the species most commonly implicated in human pulmonary disease  or rough (R) colonies when grown on agar medium. S colonies exhibit a bright and moist texture related to the presence of cell surface-exposed glycopeptidolipids (GPL), whereas R colonies are characterized by an irregular dry surface with many wrinkles and crests and are devoid of GPL  and S (non-cording) morphotypes that were derived from the same parent strain. The localization of candidate lipids on the surfaces of cords and bacilli was accomplished by TEM.The objective of this work was to identify the compounds responsible for cording in M. abscessus 390, M. abscessus type strain DSMZ 44196T, and a clinical isolate of M. abscessus BE48. S and R morphotypes of M. abscessus 390 and DSMZ 44196T were obtained in previous studies  for pellicle formation and in Middlebrook 7H9 agar  for colony morphology observation and mycolic acid extraction. One liter bottles with 100 ml of Middlebrook 7H9 broth were inoculated with 1 ml of bacterial suspension, adjusted to No. 1 of the McFarland standards of turbidity. Pellicles of M. abscessus grew at the air-medium interface of Middlebrook 7H9 broth at 37°C.The bacterial strains used in this study were  studies . For then-hexane. The n-hexane extracts that contained the methyl mycolates were evaporated to dryness at 40°C under nitrogen stream. The mycolates were concentrated by precipitation in cold methanol  and analyzed by conventional TLC on silica gel-coated plates . The 10 μl of each sample was loaded to the TLC and plates were developed with n-hexane/diethyl ether . The mycolates were observed as dark spots after the plates were sprayed with phosphomolybdic acid  and charred at 120°C. Purification of mycolates was performed by using preparative TLC plates with a concentration zone  that were developed as described above. Mycolates were visualized with iodine vapors, scraped from TLC plates and recovered with diethyl ether. The purification steps were monitored by TLC as described above.For extraction and methylation of mycolic acids, bacteria scraped from Middlebrook 7H9 agar plates were subjected to an acid methanolysis procedure . Briefly3  and transferred to 5-mm-diameter NMR tubes. NMR experiments were recorded on a Bruker Avance II 600  equipped with a 5 mm TBI probe with Z-gradients that operated at a 1H NMR frequency of 600.13 MHz and at 298.0 K. 1D 1H NMR spectra were acquired using a standard 90° pulse sequence with an acquisition time of 1.71 s and a relaxation delay of 3 s. Data sets were collected as 32k data points with a spectral width of 9590 Hz and as the sum of 128 transients. The resulting free induction decays were Fourier transformed, manually phased, and baseline corrected. All spectra were calibrated using the residual solvent signal (CHCl3) at a chemical shift (δ) of 7.27 ppm. The relative molar ratios of characteristic molecular moieties were determined by the integration of representative resonances.Purified dried mycolates were dissolved in 600 μl of CDCl4 and 0.7% ferrocyanide in phosphate buffer and washed with water. This process was followed by dehydrating the samples in an ascending ethanol series  and subsequently critical-point drying the samples with CO2. Finally, the samples were coated with gold and observed using an SEM EVO  at 15 kV.The spreading pellicles that formed on the surface of the liquid medium were collected with a 0.2 μm nuclepore membrane  and processed for analysis by scanning electron microscopy (SEM) as previously described . BrieflyM. abscessus formed a consistent pellicle at the air-medium interface. A superficial lipid extract of these pellicles was performed by using a modification of Bloch’s protocol  and agitated them for 5 min. After that, the PE extracts were filtered and evaporated. Then, one part of the cellular residue formed by the treated bacteria was recovered in a tube with glass beads, which was shaken for 10 min. Then, 2 ml of PBS was added, the tube was shaken for 10 s, and three drops of the suspension were placed in a slide and left to dry. As a control, bacteria were treated with the same protocol but without the PE extraction step. All the samples were stained using Ziehl-Neelsen stain. Another part of the cellular residue was plated in agar plates to test the viability of the treated bacteria by CFUs count and to observe the colonial morphology. After growing, these treated bacteria were inoculated in Middlebrook 7H9 broth and the biopellicles formed were extracted with PE as described above.After 2 weeks of growing in Middlebrook 7H9 broth, protocol . BrieflyA series of TLCs were performed to study the lipidic profile of the PE extracts, using 10 μl of all samples for each TLC. For unidimensional TLCs, the mobile phases that were used were PE 60–80°C/diethyl ether , chloroform/methanol , and chloroform/methanol/water . For bidimensional TLCs, the mobile phase used were first direction PE 60–80°C/ethyl acetate , second direction PE 60-80°C/acetone ; first direction PE 60-80°C/acetone , second direction toluene/acetone ; first direction chloroform/methanol , second direction toluene/acetone ; and first direction chloroform/methanol/water , second direction chloroform/acetone/methanol/water . Compounds in the TLC plates were revealed by using anthrone  or phosphomolybdic acid  and then heating the plate at 120°C or by using molybdenum blue  without heat.Compounds X, Y, Z and TAGs were purified by column chromatography. Approximately 50 mg of PE extract obtained as described above was added to a Silica Gel 60  column. A series of solvent mixtures of PE 60–80°C with increasing concentrations of diethyl ether was used for the elution of the PE-extracted components.3  and analyzed by NMR spectroscopy. The equipment described operating at 1H and 13C NMR frequencies of 600.13 and 150.90 MHz, respectively, and at 298.0 K, was used. 1D 1H spectra of the three compounds were acquired and processed using the same parameters previously described. 2D 1H,1H-COSY; 1H,13C-HSQC; 1H,13C-HMBC; and 1H-DOSY experiments were performed using standard Bruker pulse sequences and acquired under routine conditions. Chemical shifts were referenced to the residual solvent signals . Proton signal multiplicity is indicated in the text by d (doublet), t (triplet), dd (double doublet) and m (multiplet). In the case of compounds Y and Z, standard 1D 31P spectra were also recorded.Purified compounds were dissolved in 600 μL of CDClFor MS, purified compounds X, Y, and Z were dissolved in 50 μl of chloroform/methanol . This suspension was mixed with a matrix made of 10 mg/ml 1,8,9-anthracenetriol (dithranol) at a 1:1 ratio, and 1 μl of the mix was deposited on a ground steel plate. The sample was analyzed using a negative polarity reflectron and an acceleration voltage of 25 kV in a MALDI-TOF UltrafleXtreme . The calibration was performed using external calibrators .Mycobacterium abscessus pellicles were collected in sterilized filters, passed to a tube and processed for TEM observation. Two processes were used to observe the samples: the conventional preparation, as described by 4 in PBS for 1 h at room temperature. Then, the samples were dehydrated in an ascending ethanol series and post-stained with saturated uranyl acetate at the 90% ethanol step for 30 min at 37°C. Bacteria were finally embedded in epoxy resin, and ultrathin sections were made (4 but were not post-stained with uranyl acetate were exposed to a 1% hot aqueous solution of TCH (thiocarbohydrazide) for 1 h at 50°C, and this was followed by four to eight washes with hot water. Then, the sections were again exposed to OsO4 (ere made . For the to OsO4 . This me to OsO4 . All sam10 beads/ml were transferred to a tube and these beads washed twice with 0.05 M carbonate-bicarbonate buffer (pH 9.6) and coated with purified TPP obtained from the R morphotype of the M. abscessus 390 strain and maintained in DMSO . Beads were incubated in 8% DMSO in PBS with 0.8 mg/ml of TPP for 2 h at 37°C with agitation. Control uncoated beads were treated with 8% DMSO in PBS without TPP. After the 2 h incubation, the beads were washed twice with PBS and blocked with 5% BSA  in PBS for 1 h at 37°C with agitation to prevent non-specific binding. After that, beads were washed once with 0.5% BSA in PBS.Amine-modified yellow–green latex beads  with an average diameter of 1 μm were used and coated with TPP A by using variations of the protocols described by The successful coating with TPP was demonstrated by staining the beads with Nile Red . Briefly, 1 μl of 0,5 mg/ml Nile Red in ethanol was added to 50 μl of beads, which were incubated for 10 min at 37°C. Then, the beads were washed three times with PBS , and 10 2, between 3 and 6 μm2, and more than 6 μm2 . The analysis described three types of aggregates, depending on their area: fewer than 3 μman 6 μm2 . The are2 in Dulbecco’s modified Eagle’s medium with L-glutamine and high glucose (DMEM) . This media was supplemented with 10% heat-inactivated fetal bovine serum (FBS) , 100 U/ml penicillin G , and 100 μg/ml streptomycin , which was considered to be CM.J774 murine macrophage cell line was used for the experiments and maintained at 37°C in a humidified atmosphere of 5% CO4 cells per well in CM and incubated for 24 h. Then, TPP-coated and uncoated beads were added on the macrophage cultures at an optimized ratio of 30 beads per macrophage  at a density of 1.5 × 10crophage . The medcrophage . ViabiliM. abscessus 390R strain as previously described  and IL-6  were determined, following the manufacturer instructions.5 cells per dish. Those macrophages were cultured in DMEM with LysoTracker Red DND-99  at a concentration of 1:10000. The 24 h after the seeding, cells were placed in contact with beads in order to compare the phagolysosome fusion between macrophages with uncoated beads versus macrophages with TPP-coated beads. After 24 h, dishes were observed using a TCS-SP5 CLSM  with a PlanApo 63  oil objective, operating at a zoom of 2.5. One hundred macrophages were counted for each sample, and colocalization between LysoTracker and the green fluorescence produced by the beads was calculated using Pearson’s correlation coefficient.J774 macrophages were seeded onto CLSM culture dishes  at a concentration of 2.5 × 10t-tests. Statistical significance was determined using the Holm–Sidak method, an extension of the Holm–Bonferroni method, in Prism 6 . Differences were considered to be significant at p < 0.05.Analysis of the percentage of aggregates and comparison of the macrophage viability with TPP-coated and uncoated beads were made using multiple M. abscessus 390 and 44196 were determined by TLC, no differences were detected in the ratios of α-mycolic and α’-mycolic acids. In both strains, the two morphotypes produced similar amounts and ratios of the mycolic acids . As shown in Figure 1B, the NMR analysis indicated that both strains had a very similar spectrum for purified mycolic acid methyl esters. In a comparison of the relative molar ratios, samples from the R and S morphotypes showed the same average of cis and trans double bonds, and cyclopropane rings were detected only in trace amounts .When the mycolic acid profiles of S and R morphotypes of Figure 2A). In contrast, all S morphotypes  produced a thin pellicle with a flat surface. No organization of the bacilli in clumps or cords was observed when the ultrastructure of these pellicles was observed by SEM .All R morphotypes , when grown on liquid media, produced thick and wrinkled pellicles with structures that resembled macroscopic cords. When observed with SEM, pellicles from the R morphotype showed an organization of their bacilli that was typical of that in cord-forming mycobacteria . However, disintegration of clumps and cords was observed in samples treated with PE. In these samples, higher amounts of free bacteria in all the fields monitored from each preparation were clearly observed. Although in some points there were still some aggregates, the disorganization of the clumps and cords was evident .Pellicles of R morphotypes that were untreated or treated with PE were observed by using optical microscopy after they were stained with the Ziehl-Neelsen method. Bacteria in the untreated samples were aggregated, forming clumps and cords, and only a few bacteria were solitary . After PE extracts were fractionated on a silica gel column, a third compound that was retained at the point of sample application in TLC was purified (compound Z) , and none of the compounds were detected in the 44196S extract. Trace amounts of TAG were detected in all the morphotypes. TAG of the 390R strain were purified and identified by NMR  and bidimensional TLC eluted with PE 60–80°C/ethyl acetate  and PE 60–80°C/acetone . When PE extracts were monitored with other TLC developing systems, no organic material was observed. In samples from R morphotypes, two relevant compounds (X and Y) were observed when unidimensional TLC plates were revealed with phosphomolybdic acid . The identification was made on the basis of comparison with previous data , 5.03 (dd), 4.20 (m), 3.98 (m) and 3.97 (m) ppm corresponding to H4, H2, H6, H6’, and H5, respectively, were identified. 1H,1H-COSY correlations, as well as carbon resonances of glucosyl units, obtained from 1H,13C-HSQC and 1H,13C-HMBC spectra, were in accordance with the TPP A structure and with previously described values. The intense signals corresponding to the polyunsaturated fatty acyl substituents are indicated in the figure. The MALDI-TOF MS analysis further confirmed the identity of the molecule    . Residua1H and 13C NMR characterization , as well as methylene (δH 1.2–1.3 ppm) and methyl groups (δH 0.9 ppm) characteristic of alkyl chains were observed. The same 1H and 13C NMR signals corresponding to the TPP A trehalose unit were exhibited in spectra of Y and Z, thus suggesting that they have a common glucosyl residue. In the case of compound Y, H3 (3.54 ppm) and H4 (3.82 ppm) of the second glucosyl residue were strongly shielded compared with those of TPP A , which suggests that position 3 and/or 4 are not acylated. Similarly, H4 (3.82 ppm), H6 (3.58 ppm) and H6’ (3.53 ppm) of the second glucosyl residue of Z were strongly shielded compared with analogous protons of TPP A , which suggests that position 4 and/or 6 are not acylated. These results were supported by MS analyses , whereas this material did not accumulate in the S morphotype cell wall . When a minor magnification of the TEM images was analyzed, the cord organization in the R morphotype and the accumulation of this electrodense material outside the cord were observed .When bacilli of the R and S morphotypes of 4 deposit on these lipids. An important accumulation of OsO4 was observed outside the bacilli in R morphotypes from the samples treated with this stain, and no accumulation was observed in S morphotypes .The OTO stain provides a major contrast of the lipidic components because it augments the OsOFigures 5A,B), and these beads appeared to be surrounded by red fluorescence. When observed by CLSM, the TPP A-coated beads presented more aggregation than uncoated beads. Statistical analysis showed significant differences between the areas of the aggregates of the two samples, thus indicating that TPP A-coated beads produced larger aggregates than uncoated beads . The percentage of aggregates that were larger than 6 μm2 was 20.4% ± 5.7 (mean ± SD) for the uncoated beads and 38.9% ± 6.2 (p < 0.05) for the TPP A-coated beads. The difference in area between aggregates of uncoated beads that were smaller than 3 μm2 (57.0% ± 2.3 of the area covered) and aggregates of TPP A-coated beads of the same size (38.8% ± 7.5 of the area covered) was also significant . These results are obtained from beads coated with TPP A. However, beads were coated also with a mix of the three compounds described by NMR , all TPP molecules, and no differences were observed between the results obtained with the beads coated with TPP A and those obtained with the beads coated with the mix of TPP molecules  (data not shown).The coating of beads by TPP A was confirmed by CLSM, because Nile Red stained the lipids coating the beads . Both types of beads were found inside the cells and in a similar ratio. J774 macrophages with phagocytized uncoated beads represented a percentage of 32.39% ± 5.08 from the total of counted macrophages, and macrophages with phagocytized TPP A-coated beads represented the 37.69% ± 9.73. Moreover, macrophage viability was not altered after the interaction with TPP A-coated beads. No significant differences were observed in the viability of macrophages in contact with uncoated beads or TPP A-coated beads . When cytokine production was analyzed, no production of TNF-α and IL-6 was detected in macrophages treated either with uncoated or TPP A-coated beads (data not shown). Also related with the possible effect of TPP A on the viability of macrophages, no differences were observed when analyzing the colocalization of the beads inside the macrophages with acidic vesicles. Pearson’s correlation coefficient was of 0.40 ± 0.16 for uncoated beads and of 0.54 ± 0.15 for TPP A-coated beads (the results represent the mean ± SD of triplicate preparations) . Similar results were obtained when macrophages were in contact with beads coated with the mix of TPP molecules , that is no effect on the viability of the macrophages was observed  and no production of TNF-α and IL-6 was detected (data not shown). Bacteria from R morphotype had the expected action in front of macrophages, as it has been already published .As observed with CLSM, macrophages did not distinguish between uncoated and TPP A-coated beads in phagocytosis  and cording morphotypes . Both acids from all sources possessed approximately the same amounts of cis-double bonds, and cyclopropane rings were nearly absent. Recently, a cord-deficient M. abscessus mutant has been generated, and no differences have been found in the cyclopropanation of mycolic acids between the mutant and the parent strains   . Recentlcterized . In this species .4, a commonly used stain for lipids in electron microscopy, makes the lipids appear as an electrodense zone  the confirmation that in natural mutants of M. abscessus no differences exist in mycolic acid composition between cording and non-cording morphotypes; (iii) the structural characterization of TPP in M. abscessus strains; (iv) the location of lipidic compounds on the surface of cording R morphotypes of M. abscessus but not in the surface of non-cording S morphotypes; (v) the determination of the aggregative capacity of TPP; and (vi) the determination of the no toxicity of TPP for macrophages. All these findings allow us to propose TPP as candidate molecules responsible for the formation of clumps and cords in R M. abscessus strains.In summary, the novel findings reported in this work are: (i) the description for the first time of the fine structure of mycolic acids of cording and non-cording morphotypes derived from the same parent strain of papA3, pks, fadD23, and mmpL10 genes have recently been found to be involved in the biosynthesis and transport of TPP  are present in the cell walls of this species and related with hydrophobicity and pathogenicity (M. tuberculosis.Because the t of TPP , it willgenicity , 2017. FConceived and designed the experiments: ML-F, EJ, and ML. Performed the experiments: ML-F, MP-T, and CB. Analyzed the data ML-F, MP-T, EJ, and ML. Contributed reagents/materials/analysis tools TB and FA. Contributed to the writing of the manuscript: ML-F, MP-T, EJ, CB, TB, FA, and ML.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our overall aim is to develop epitope-based assays for accurate differential diagnosis of B virus zoonotic infections in humans. Antibodies to cross-reacting epitopes on human-simplexviruses continue to confound the interpretation of current assays where abundant antibodies exist from previous infections with HSV types 1 and 2. To find B virus-specific epitopes we cloned ten monoclonal antibodies (mAbs) from the hybridomas we produced. Our unique collection of rare human sera from symptomatic and asymptomatic patients infected with B virus was key to the evaluation and identification of the mAbs as reagents in competition ELISAs (mAb-CE). The analysis of the ten mAbs revealed that the target proteins for six mAbs was glycoprotein B of which two are reactive to simian simplexviruses and not to human simplexviruses. Two mAbs reacted specifically with B virus glycoprotein D, and two other mAbs were specific to VP13/14 and gE-gI complex respectively. The mAbs specific to VP13/14 and gE-gI are strain specific reacting with B virus isolates from rhesus and Japanese macaques and not with isolates from cynomolgus and pigtail macaques. The mAb-CE revealed that a high proportion of naturally B virus infected rhesus macaques and two symptomatic humans possess antibodies to epitopes of VP13/14 protein and on the gE-gI complex. The majority of sera from B virus infected macaques and simplexvirus-infected humans competed with the less specific mAbs. These experiments produced a novel panel of mAbs that enabled B virus strain identification and confirmation of B virus infected macaques by the mAb-CE. For human sera the mAb-CE could be used only for selected cases due to the selective B virus strain-specificity of the mAbs against VP13/14 and gE/gI.To fully accomplish our aim to provide reagents for unequivocal differential diagnosis of zoonotic B virus infections, additional mAbs with a broader range of specificities is critical. Macacine herpesvirus 1) is a member of the family Herpesviridae in the genus Simplexvirus within the subfamily Alphaherpesviridae .The mAb competition ELISA (mAb-CE) identifies antibodies to epitopes recognized by mAbs following natural infection. This test was performed essentially as previously described . BrieflyRhesus sera, positive and negative and a mouse negative serum were tested for neutralizing antibodies at 3-fold dilutions, 1:10 to 1: 2430. Some mAbs that were not concentrated or purified were tested at a 1:2 dilution and others that were concentrated or purified were tested at 3-fold dilutions (1:2 to 1:54). Serum dilutions were performed in the BSL2 lab. The 48-well Vero cell plate and the serum dilutions were transferred to the BSL-4 to for the addition of 25 μl containing 50 PFU (plaque forming units) B virus to 25 μl of the diluted serum.2 for virus adsorption. After additional 1 hr incubation, the virus+ serum inoculum was removed and 1%-methylcellulose was added to each well. The plate was incubated for 48 hours in a 37°C incubator to allow for plaque formation. After 48 hours, the methylcellulose from each well was removed and the wells were washed with PBS and fixed with 100% methanol. The plate was dunked out of BSL-4, washed, and then the cell cultures in the wells were stained with crystal violet (0.2%) to count the number of plaques. Percent neutralization of the virus was calculated relative to the virus + PBS control that measured the amount of plaques used in the test. The following formula was used: 100x[1-(S/B)], where S represents the number of plaques obtained from the serum + virus mixtures and B represents the number of plaques obtained with the virus + PBS control mixtures.The 50 μl virus + serum mixtures were incubated at 37°C incubator for 1 hour. A virus + PBS control was also introduced. After incubation 50 μL of virus + serum or virus + PBS mixtures were added to each well of the 48-well plate and incubated for 1 hr at 37°C in a humidified chamber containing 5% COThe WBA was performed according to standardized, CLIA-approved, diagnostic laboratory protocols as previously described , 13, 15.http://www.thermoscientific.com/pierce).Immunoprecipitation was performed using the “Thermo Scientific Pierce Crosslink Immunoprecipitation Kit #26147  per manufacturer’s protocol , and washing solution II . The washed gel pieces were finally dehydrated with 100% acetonitrile and dried under speed-vac. The dried gel pieces were either underwent trypsin digestion or kept at -80°C until they were treated with trypsin for the MS -peptide analysis. In brief, the gel pieces were incubated with appropriate amount of trypsin  in proteaseMAX surfectant  at 37°C for 2~ 3 hours. After incubation, the digested peptides were extracted with 2.5% of trifluoroacetic acid. The extracted peptides were further purified and concentrated by ZipTip, a C18 micro-reverse phase resin tip  according to the manufacturer’s protocol.Protein bands of interest were excised from the gel. The excised gel pieces were washed first with dd-HExtracted peptides were then analyzed by 4800 MALDI Tof/Tof tandem mass spectrometer  with MS/MS tandem mode. Protein identifications were performed using Mascot search engine  against Swiss Pro or NCBI protein database.http://www.graphpad.com.One-way ANOVA with Bonferroni's Multiple Comparison Test was performed to evaluate differences in mAb reactivity using GraphPad Prism™ version 5.00 software, San Diego California USA, ELISA results of mAbs 1–8 (Summarized in The majority of mAbs (#3–8) reacted with recombinant gB proteins. The mAbs #9 and #10 reacted with B virus gD Tables , while mBy WBA, mAb #1 reacted with a 75 kDa protein  and the Based on the reactivity and specificity of the mAbs each was classified into one of four categories:Category-I includes mAbs #1 and #2 that recognized rhesus B virus isolates and Japanese B virus isolates, but not B virus isolates from pigtail or cynomolgus macaques, or any other tested simplexvirus.Category-II includes mAbs #3 and #4 reactive with B virus gB, but non-reactive or poorly reactive with HSV-1 gB. Monoclonal antibody #3 reacted to each B virus isolate, however, mAb #4 did not react with isolates from pigtail macaques, although it did react with the others. These two mAbs reacted with some of the simian simplexviruses, but did not react with the human simplexviruses (HSV-1 and HSV-2)  were tested by the mAb-CE against two “Category-I mAbs (12F5.C1 and 12G9.G5), one Category-II mAb (7F7.G7), and against three Category-III mAbs .The percent of individual B virus positive rhesus and cynomolgus macaques that were positive by the mAb-CE was calculated and presented in Approximately 80–100% of the rhesus and cynomolgus macaque sera tested competed against the Category-II and Category-III mAbs. As expected , the CatTwenty-two individual human serum samples and three pooled control serum samples  were tested by the mAb-CE against two mAbs of Category-I and mAb #6 of Category-III . Twenty-Of the 21 individuals, 12 were monitored post macaque exposure and seroconverted (asymptomatic) while seven were from patients with clinically apparent disease (symptomatic), and two had no laboratory or clinical signs of infection (negative). Of the seven with acute disease four succumbed as a result of B virus zoonotic infection .The confirmatory laboratory results of “B virus or HSV antibodies” are shown in the last four columns of Eleven of 12 B virus antibody positive, post-exposure sera competed only with the 7H1.G5 mAb. Of the 11 human sera, three  had no detectable HSV antibodies, thus, competition in the absence of HSV antibodies suggests B virus infection. The mAb-CE competition results obtained with the other eight B virus positive sera that were also positive for either HSV-1 or HSV-2 antibodies could not be interpreted as a confirmation for a B virus infection. Three of the seven symptomatic patients recovered from disease. No serum samples from the four deceased patients competed with Category-I mAbs, but three competed with the 7H1.G5. One of the three sera was HSV antibody-negative and therefore the competition likely resulted from the presence of B virus antibodies. Of the three sera from symptomatic patients who recovered form zoonotic B virus disease, one failed to block all three mAbs. Serum samples from the two other patients competed with all three mAbs. The competition observed with the two B virus specific Category-I mAbs was interpreted as further evidence of B virus specific antibodies in these two serum samples. In addition, the competition of these two serum samples with mAb #6, indicates that the B virus infection in these individuals also induced antibodies to group-common epitopes.To rule out the possibility that a negative competition against the Category-I mAbs is due to coexisting HSV-1 or HSV-2 antibodies, antibody-negative sera were spiked with HSV-1 and/or HSV-2 antibody positive sera. Analysis of mAb-CE data indicate that HSV antibodies did not interfere with the B virus antibody-specific competition with the Category-I mAbs .The current diagnostic tests are sufficient for B virus diagnosis in macaques, however, in humans; B virus diagnosis is confounded because of the cross-reacting human simplexviruses (HSV-1 & -2). Therefore, there is a need to develop improved assays for differential B virus diagnosis, which requires B virus specific reagents. To achieve this goal we produced mAbs to B virus antigen. Our present study resulted in the production of a novel panel of mAbs, which was used to identify specific immunoreactive epitopes of B virus proteins for the first time since the discovery of B virus over 80 years ago. The mAbs identified epitopes of five B virus proteins: gD, gI, gE gB and VP13/14. Neutralizing experiments revealed that none of the panel of mAbs neutralized B virus. Envelope virus gD binds to host cell receptors and triggers B virus and HSV entry into target cells –30. The The mAbs 12F5.C1, 2G12.D12.D4, 6E10.D7 appeared to recognize linear epitopes, while the other mAbs likely recognized conformational epitopes. In comparison with others, we obtained a greater proportion of mAbs reactive with conformational epitopes possibly because we used immunogens in which protein conformation was partially or fully preserved . B virusB virus gE and gI were identified as target proteins for mAb 12G9.G5. Because this mAb did not react with recombinant gE or gI and because glycoproteins gE and gI are known to form complexes, this mAb is likely directed to an epitope located at the junction of the gE/gI complex , 36. BotThe B virus stain specificity of the Category-I mAbs was also confirmed by the mAb-CE test in which these mAbs were competed by rhesus macaque B virus antibodies and not by cynomolgus macaque B virus antibodies or human anti HSV antibodies. Category-II mAbs reacted with each B virus isolate as well as with certain non-human primate simplexviruses, but did not react with the human simplexviruses. Interestingly, however, when tested by the mAb-CE test, these mAbs were competed by HSV-1/HSV-2 antibodies. Blocking binding of the Category-II mAbs to their B virus-specific epitopes may be caused by steric hindrance due to binding of the HSV-common antibodies to a nearby cross-reacting epitopes , 21. HowCategory-III mAbs recognize simplexvirus group-common epitopes, facilitating studies to better understand simplex virus conserved epitopes that are found in human and nonhuman primate herpesviruses Tables  and 3. THere we show that the mAb-CE can be used to confirm the presence of B virus antibodies in macaques by employing Category-II and Category-III mAbs since there is no evidence, thus far, of the presence of antibodies to other simplexviruses in macaques that may confound the diagnosis by steric hindrance , 12, 22.In conclusion, a novel panel of mAbs was described that demonstrated B virus specific reactivity differentiable fromll simplexvirus group reactivity.A rare collection of patient sera enabled us to perform this study to better understand antibody responses to zoonotic B virus infections and to potentially enhance identification of these deadly infections.We have shown that the Category-I mAb can be used in competition ELISAs to identify zoonotic B virus infections in humans even in the presence of HSV antibodies.The selective strain specificity of these mAbs limit their general use for diagnosis of B virus infections in humans currently, but may be of value for strain identification of field isolates and for studying strain-specific pathogenesis.These findings further our aim of developing assays that enable unequivocal differential diagnosis of zoonotic B virus infection. To fully achieve our aim more mAbs have to be produced to complete the range of desired specificities. In addition, epitope mapping of these mAbs and of those that will be produced in the future will have to be carried out. B virus specific epitopes, thus identified, will be used for the development of accurate peptide-based assays for differential serological diagnosis of B virus infections in humans.


Viruses are involved in various interactions both within and between infected cells. Social evolution theory offers a conceptual framework for how virus-virus interactions, ranging from conflict to cooperation, have evolved. A critical examination of these interactions could expand our understanding of viruses and be exploited for epidemiological and medical interventions. Viruses are involved in various interactions both within and between infected cells. Social evolution theory offers a conceptual framework for how virus-virus interactions, ranging from conflict to cooperation, have evolved. A critical examination of these interactions could expand our understanding of viruses and be exploited for epidemiological and medical interventions. Virus-virus interactions are pervasive and highly diverse . Some viDespite this growing body of empirical evidence suggesting virus-virus interactions, we lack a well-founded conceptual framework that provides an understanding of how these interactions have evolved and how they could shape viral pathogenesis. Social evolution theory was originally developed to explain animal behavior, but has since been extended to microorganisms, including bacteria and unicellular eukaryotes. Yet this social perspective has not been embraced in the study of viruses.Potential misgivings with this social-evolution approach include the idea that viruses are too simple to interact socially, or that to infer traits like cooperation would be anthropomorphic. On the flip side, social interactions have driven the evolution of life at all levels of complexity and thus could have a role in viral evolution as well: genes cooperate to form genomes, cells cooperate to form multicellular organisms, and multicellular organisms form complex cooperative societies. Conflict is also ubiquitous: “selfish” genes drive their transmission at a cost to the genome, cells exploit collectively produced goods like nutrient-scavenging molecules, and organisms compete with each other for resources. Since no complex phenotypes are necessary for the evolution of social traits, the appearance of anthropomorphism or the “simple” organization of viruses may not pose problems for the application of sociobiology models in virology.We argue for a social evolution approach to understand and predict virus-virus interactions. This framework can clarify unexplained phenomena in virus-virus interactions by identifying evolved viral social traits, their genetic and mechanistic basis, and the selective pressures underlying these traits. We outline how this approach could lead to new breakthroughs in both fundamental and applied virology.Social interactions take place when the traits of one individual influence the fitness of another individual. As such, evolutionary analysis of social interactions relies fundamentally on natural selection. The only major difference with non-social models is that two or more interacting individuals are involved. For example, cooperative traits, which benefit others, will only be selected for by natural selection if they provide a fitness benefit to the individual performing them A. CooperOne way cooperation can be evolutionarily favored is when it is directed toward genetically identical individuals or relatives. From an evolutionary perspective, helping a genetically identical individual reproduce is the same as reproducing yourself. By extension, helping relatives reproduce provides a fitness benefit to the actor, as relatives share a fraction of the actor’s genes. This process by which individuals increase their fitness by helping relatives reproduce is termed kin selection.An important potential difficulty in applying social evolution to virology resides in the definition of an individual. One may consider the virion as an individual. Yet in some cases, including paramyxoviruses, birnaviruses, filoviruses, retroviruses, and inoviruses, a virion can carry multiple genome copies (n-ploidy). Functionally, these n-ploid virions should be similar to multiple virions entering the same cell, because they would both lead to infections with multiple viral genomes. As such, we suggest that the definition of an individual should be set at the level of the single infectious viral genome.Another potential complication stems from the fact that some viruses, notably RNA viruses, show extremely high mutation rates, leading to the suggestion that the minimal level at which an individual RNA virus can be defined is a sequence cloud, or quasispecies . In thisIf we think of a genome as an individual, then the natural history of viruses is filled with opportunities for social interactions , even thEvidence in favor of viruses acting as cooperative social agents is, paradoxically, provided by the occasional spread of uncooperative “cheats.” If individuals are not genetically identical, they will not necessarily have the same evolutionary interests, creating the potential for conflict. Social evolution theory predicts that if multiple genomes infect a cell, one that invests less in cooperative traits such as transcription and invests more in its own replication will be favored, because it will benefit from cooperation without paying as much of the cost . This isTwo important variables determine the extent to which multiple genomes infect a cell: the multiplicity of infection (MOI) and the rate of spontaneous mutation. High mutation rates such as those exhibited by RNA viruses result in extremely diverse populations, whereas a high MOI can bring genomes from different lineages into the same host cells. These variables can enable genomes to interact in coinfection, as long as there is sharing of viral products within cells.A clear experimental test of the association between MOI and cheater evolution was generated with bacteriophage phi6 . Serial in vivo and to determine the ensuing levels of intra-host genetic relatedness in viruses.Defective interfering particles (DIPs) appear to be “cheats.” DIPs usually have a large portion of their genome deleted, including essential proteins, and hence can only reproduce in the presence of completely functional “helper” viruses. However, their smaller genomes provide a replication advantage, outcompeting helper viruses in mixed infections of cells and severely reducing viral population fitness. In the laboratory, artificially high MOIs are well-known to promote the emergence of DIPs . HoweverIn turn, the association between mutation rate and cheating has been shown in experimental populations of RNA viruses treated with base analog mutagens. This selected for a fraction of viral genomes with DIP-like behavior, greatly reducing population fitness  can also evolve. All that is required is that the individuals have some shared interest, such that they have higher fitness if they cooperate.Cooperation between different genetic variants is a familiar concept in virology, but rigorous evidence supporting it remains scarce. The finding that population genetic diversity correlated with the ability of poliovirus to cause disease in mice led to the suggestion of cooperation, with similar processes postulated for hepatitis B virus, among others  and that the trait has evolved at least partially due to this benefit . This laThus, the challenge resides in explaining cooperative traits that evolved to benefit others, which becomes difficult with complex interactions. Intercellular virus-virus interactions mediated through changes in the host, such as breakage of physical barriers or immune suppression, could easily fall into the elephant-dung beetle category. Similarly, some of these synergistic interactions reported as heterotypic cooperation between virus variants may be transient.The designation of byproduct rather than cooperation does not imply that the observed phenomena are not important. Rather, they raise different questions about why they are favored by natural selection, which can be crucial for understanding and manipulating these viral social interactions. The utility of identifying cooperation or cheating, as opposed to a transient interaction, is that they represent evolved social interactions. This identification has two consequences. First, we can uncover the genetic underpinnings of the trait. For instance, the specific mechanism underlying cheating by DIPs has led to DIP-derived treatments that depend only on the gene, rather than the entire viral particle. Second, we can establish the environmental and social pressures that led to selection of that trait. This knowledge of the selective regime can lead to generalizable principles, such as low-MOI vaccine propagation to avoid the appearance of DIPs.In practice, the key first step will be to test whether evolved social interactions are really occurring between strains, and what form they take B. First,The recently discovered arbitrium system, whereby bacteriophages produce virus-encoded small peptides that are detected by specific virus-encoded receptors in neighboring cells, provides a good test case . This syVirulence can be a social trait. In the simplest scenario, greater pathogen growth will increase transmission, but also increase host mortality (virulence). Consequently, overall viral transmission can be maximized at an intermediate growth rate, where the host is prudently exploited, with the associated virulence an unavoidable consequence. Myxoma virus provides a potential example of prudent exploitation in the field. Myxoma virus causes a lethal disease known as myxomatosis in the European rabbit and was released as a means to control rabbit populations in Australia and France. However, after years of natural infection, the virus evolved toward slightly reduced fatality rates and prolonged host survival times, increasing opportunities for both vector-borne and direct transmission .Virus or host dispersal can also influence the social evolution of virulence. Consider a spatially structured population with limited dispersal of both virus and hosts. In this case, if a viral strain grows quickly, then it can deplete the local supply of susceptible hosts and hence slow transmission of that strain in the long run. Theoretical models predict that such spatially structured populations will select for lower virulence and hence more prudent host exploitation, as first experimentally verified in an insect virus .In contrast to the above example, low virulence can also evolve in response to decreased relatedness. When host exploitation relies on intra- and intercellular cooperation between individual genomes, coinfection can select for cheats, leading to the breakdown of cooperation and, consequently, lower parasite growth and reduced virulence .A social perspective on virus evolution can open new research and treatment possibilities. Social thinking can cast viruses in a new light and question fundamental assumptions to advance basic virology. Similarly, by focusing on virus-virus antagonism and alliances, we may be able to uncover new weapons in our own virus-host battle.The central role of genetic relatedness and kin selection in the evolution of cooperation can help us better understand key processes in virology, such as superinfection exclusion, which makes the cell refractory to entry of other, potentially non-identical genomes, and why some viruses tend to be transmitted as groups (collective infectious units). The social evolution perspective also draws attention to certain long-held assumptions regarding standard virological practices. For example, studies have found that plaque assays may not yield clonal populations (reviewed in The social evolution perspective also opens the door to engineering of new, better viral interventions by deploying our knowledge of social evolution. New attenuated vaccines using DIPs could be generated by using social principles underlying the emergence of cheats . Theory In sum, there is growing evidence that virus-virus interactions are pervasive and that viruses have mechanisms to mediate these interactions. We suggest that these traits are best understood and predicted using a social evolution approach. As such, sociovirology holds promise for breakthroughs in fundamental and applied virology.