Emerging diseases are a major challenge to public health. Revealing the evolutionary processes that allow novel pathogens to adapt to new hosts, also the potential barriers to host adaptation, is central to understanding the drivers of disease emergence. In particular, it is unclear how the genetics and ecology of pathogens interact to shape the likelihood of successful cross-species transmission. To better understand the determinants of host adaptation and emergence, we modelled key aspects of pathogen evolutionary dynamics at both intra- and inter-host scales, using parameter values similar to those observed in influenza virus. We considered the possibility of acquiring the necessary host adaptive mutations both before (‘off-the-shelf’ emergence) and after (‘tailor-made’ emergence) a virus is transmitted from a donor to a new recipient species. Under both scenarios, population bottlenecks at inter-host transmission act as a major barrier to host adaptation, greatly limiting the number of adaptive mutations that are able to cross the species barrier. In addition, virus emergence is hindered if the fitness valley between the donor and recipient hosts is either too steep or too shallow. Overall, our results reveal where in evolutionary parameter space a virus could adapt to and become transmissible in a new species. Despite the high morbidity and mortality due to some emerging infections, the evolution of sustained transmission cycles in a novel host following a species jump is a relatively rare occurrence. Rather, many, if not most, emergence events in reality reflect isolated ‘spillover’ events in which a pathogen causes infection in a new host but without large-scale onward transmission. An informative example is provided by the highly pathogenic H5N1 subtype of avian influenza A virus. Although this virus is primarily associated with outbreaks in wild and domestic birds (i.e. poultry), since 2003 it has caused over 800 laboratory-confirmed cases in humans in 16 countries, with more than 400 deaths [a) [figure 1b):Panel 1. The genetic and ecological barriers to successful emergence are seemingly low in the case of influenza A virus in pigs and humans. The relationship between swine and human influenza was noted during the global influenza pandemic of 1918–1919 [918–1919 , and ser918–1919 . Importa918–1919 , althoug918–1919  and henc918–1919 . Success918–1919 .Panels 2 and 3. While birds are likely the natural reservoir for influenza A viruses, transmission from birds to mammalian species, including humans, is relatively rare. Although occupational exposure to poultry seemingly provides ample ecological opportunity for adaptation, avian influenza virus generally results in dead-end spillover events in which onward transmission among humans is limited (or non-existent) because the virus lacks the necessary mammal-specific mutations. The recent appearance of highly pathogenic H5N1 and H7N9 influenza viruses, characterized by multiple spillovers into humans but little or no onward transmission, present informative cases of this effect [s effect .Panel 4. Equine-derived H3N8 canine influenza virus (CIV) is an example of a virus that appears to be genetically well adapted to transmission among dogs, but ecologically constrained due to the lack of sufficient host contact networks. This has resulted in a highly patchy distribution, characterized by sporadic and short-lived outbreaks in the United States, often confined to animal shelters [shelters , but a mshelters . Whethershelters .An additional factor that may act as a barrier to viral emergence is that most forms of transmission involve a major population bottleneck, in which a limited number of randomly sampled individuals (virions) create a new founding (inoculum) population. Such a sharp reduction in population size can severely reduce genetic variation and hence is likely to have profound effects on the population structure of the evolving virus [transmitted virions in natural infections, as opposed to the number of transmitted genotypes, remains largely unknown. Somewhat more is known about bottleneck size during virus transmission among plants, where experimental manipulation is easier. In these cases, estimates also predict an extremely narrow bottleneck of between 0.5–3.2 virions for insect-borne transmission [To establish a productive infection in a new host a virus must overcome multiple evolutionary and ecological barriers . From anfigure 1a) ,7. As a a) . Althouga) ,8,9, theng virus , and betng virus . These esmission  and 1.3–smission .Figure Transmissibility is a key component of host adaptation, and hence virus fitness, that should be optimized by natural selection. However, whether the mutational changes that underpin this trait are fixed before or after a virus enters a new host species is unclear, particularly given the nature of fitness landscapes described above. Here, we explore the likelihood of successful adaptation of a virus that jumps from a donor to a recipient species, incorporating a variety of fitness landscapes and population bottlenecks of differing magnitude. In particular, we consider two specific models of viral emergence. In the first, termed the ‘off-the-shelf’ model ,25, host2.(a)To determine the likelihood of a virus accumulating the necessary mutations to successfully cross a species barrier, and hence the probability of disease emergence in a novel host, we used an agent-based model (ABM) with a genetic algorithm. Our ABM simulates the intra- and interhost evolutionary dynamics of a virus population through time using parameters broadly analogous to those from H5N1 influenza virus. Our model assumes that a donor host is infected with a virus (agents) and experiences approximately 5 days of infection before the virus is transmitted to a recipient host of a different species. This recipient host itself then experiences an infection also lasting approximately 5 days, before we quantify the status of the infection (i.e. the number of virions and their level of adaptation).(b)et al. [m; all parameters and variables are given in electronic supplementary material, table S1) is equal across virions, loci and states. We use a mutation rate that is typical for influenza viruses, at 10−5 mutations per site, per genome replication [5 = 32 possible genotypes exist. However, for simplicity, we assume that virions with n mutations have equivalent fitness regardless of their position in the genome. A virion's fitness is defined by its probability of replicating and producing progeny, which is a function of the number of mutations carried and the host in which a virus finds itself. We explored eight different functions of genotype (number of mutations) on fitness . In terms of the fitness landscape described earlier, this can be envisaged as a measure of the slope of the fitness valley, in which a severe fitness effect reflects a steeper fitness valley between hosts. The fitness functions, therefore, ranged from very steep to progressively more shallow, which are referred to as f1 to f6. We also considered two additional fitness functions, f7 and f8, that reflect differences in fitness effects between the hosts . Under f7, viral mutations in the donor host have very little effect on fitness , but a large effect once transmitted to the recipient host . By contrast, f8 represents the opposing case (i.e. f1 in the donor and f6 in the recipient).We assume a haploid virus genome with host adaptation determined by five sites . Thereflication . Analogo(c)αS is the log odds probability of surviving when the density is 0 (i.e. the intercept in the regression); βS is the effect of density on the log odds of survival (i.e. the slope) and P is the population density . In all simulations we assume a negative slope, such that the probability of survival decreases as P increases. The survival probability of all virions is equally affected by density, and all parameters relating to density dependence were fixed throughout model runs. We selected parameters that allow steady population growth and then keep the population size at approximately 108 after the replication step once the carrying capacity is reached.The model is initialized with a (founding) population of 10 virions, comprising only the wild-type genotype. The model is then run for 20 iterations in this host, analogous to a typical round of influenza replication lasting 6 h over a 5-day infection. Although there is a broad consensus that duration of replication for influenza is approximately 6–8 h ,29, we rr new virions, with r set to 16 here . Each virion arising from a replication inherits the ‘parent's' genotype, with mutation. The probability of a virion replicating on an iteration in the donor is a function of the number of mutations carried. Again, we implement this as a logistic regression, as given by:n is the number of mutations the virion carries. We explored negative n increases the virion has a reduced probability of replicating, and we varied the severity of this effect , equivalent to assuming that virus genomes with no mutations are better suited to the donor host.Replication involves a ‘parental virion’ giving rise to (d)b randomly selected virions were chosen as founders for the new recipient host and the remaining population is disregarded. Because formal estimations of bottleneck size for virus transmission between animal hosts in nature are limited, we explored a wide range of values for b .After 20 generations in the donor  the host changes, analogous to a shift in host species. We assume that the population experiences a bottleneck during this transmission event. Hence, from the existing virus population (e)a)). Thus, the probability of replication in the recipient host is the same as equation (2.2), but As above, the virus infects a new host species following a population bottleneck. Again, the model iterates over 20 viral generations. The probability of a virion surviving generation-to-generation is identical in the two hosts (equation (2.1)). However, we assume that the effect of mutation on the probability of replication is inverted in the recipient host . Thus,b). First, we investigated ‘off-the-shelf’ adaptation in which all the mutations necessary for adaptation to a novel host must be transmitted from the donor host; hence, new mutations are unable to arise during replication in the recipient host. This is equivalent to saying that a virus population in the recipient is subject to selection but no new mutation. Second, we explored the ‘tailor-made’ model in which virions are able to accrue new mutations through replication in the recipient host (at mutation rate m).We also explored two modes of host adaptation b. First,(f)b values  with fitness functions f1 through f8, and assuming both off-the-shelf and tailor-made adaptation. We ran the model 200 times for each parameter combination. From each model run we recorded the diversity during the bottleneck, the dynamics of population growth in the recipient immediately following transmission, and the population size and distribution of genotypes after the specified period of infection in the recipient host.The model was programmed in C++. We explored a range of 3.(a)The probability of establishing infection in a new host is largely driven by the fitness effect, which dictates the probability that a specific virion type will reproduce. For the purposes of this study, we only evaluate if an infection becomes established in a new host and define an ‘established’ infection as one in which the virus population at the peak of infection (i.e. after 5 days of infection) in the recipient reaches > 1% of the carrying capacity (i.e. >1 000 000 virions).a,b). For instance, under a strong fitness effect (i.e. f1), the fitness valley between two host species is very steep, such that there is a very low probability of establishing an infection. However, as the fitness valley becomes shallower, infection in the recipient host becomes more likely. In addition, the probability of infection is also increased with a more relaxed (i.e. looser) population bottleneck at transmission. An important difference between the two models was observed when the bottleneck b > 100 and when the fitness valley was moderately steep (i.e. f3): under this parameter space there was an increased probability of established infection under the tailor-made model. Overall, however, it is notable that there is a high probability of infection in the recipient host for a wide range of parameters, even if it is only transient (i.e. that the virus is not fully adapted to the new host).Both the off-the-shelf and tailor-made models showed very similar results under each parameter combination a,b. For n mutations, where n = 0–5) over the first 10 generations following transmission to the recipient host . Notably, under fitness function f2 the number of wild-type virions decreased while the number of mutated virions increased; hence, this might represent the point in parameter space that best facilitates adaptation and in turn the best chance of sustained emergence in a new host species.We observed the population growth of each type of virion (i.e. virus genomes with (b)c,d). Specifically, in the absence of strong positive selection (i.e. f6) or under a very steep fitness valley (i.e. f1), only wild-type variants were present in the population at all bottleneck sizes. Similarly, when the bottleneck was severe (b ≤ 100), the vast majority of genomes contained only wild-type variants. However, when selection was neither too weak nor too strong (f2–f5), a larger proportion of virions contained a higher number of mutations for a wide range of bottleneck sizes, such that this represented the optimal conditions for host adaptation. Under the tailor-made model, there were, on average, more mutations per virion for each parameter combination than under the off-the-shelf model.The mean number of mutations per genome in the virus population was determined for different fitness effects and transmission bottlenecks. Under both models, the extent of positive selection (adaptive evolution) and bottleneck size had a considerable effect on the number of mutations per genome c,d. Spec(c)n mutations have equivalent fitness and thus phenotype. As expected, there are more distinct virions in the bottleneck population when the transmission bottleneck is wide (e). The maximum number of distinct virions under these relaxed parameters was, on average, approximately 2. Surprisingly, diversity in the bottleneck population was largely unaffected by the fitness effect. This lack of diversity is likely due to the very small proportion of the population being sampled, as well as selection acting against mutations in the donor.Owing to the large number of possible genotypes, we define genetic diversity simply as the number of distinct phenotypes in the population, assuming that virions possessing  is wide e. The maf,g), which revealed major differences between the tailor-made and off-the-shelf models. In particular, more diversity was present after infection under the tailor-made model as the virus can adapt to its new host. Under this model, a greater number of distinct virion types were present when the fitness valley was not too steep and the bottleneck was loose. By contrast, the diversity under the off-the-shelf model largely resembled that present in the bottleneck population, such that very little adaptation has occurred as transmission, thereby highlighting the limits of this model of viral emergence.We compared the number of distinct virions in the recipient host f,g, whic(d)a). By contrast, when viral replication took place over 12 h we found that there was (i) a decreased probability of infection for a wider range of parameters, (ii) fewer mutations and thus less adaptation to the recipient host and (iii) less overall diversity . Very similar results were obtained for 12 h replication under both the off-the-shelf and tailor-made models.The duration of viral replication was varied from 6 h  to both 8 and 12 h . Extending the duration of viral replication to 8 h had very little overall effect for either model f7 and f8, that reflect differences in fitness effects between the hosts (a). Under f7, viral mutations in the donor host have very little effect on fitness , but a large effect once transmitted to the recipient host . By contrast, f8 represents the opposing case (i.e. f1 in the donor and f6 in the recipient). Under fitness scenario f7, we found that an infection is never established under either emergence model and for all parameter combinations (f8), there is an increased probability of infection, a higher number of mutations and a greater number of different virus types for both models , regardless of how constrained they might be in the original host. We, therefore, investigated two additional fitness functions, he hosts a. Under inations , likely h models . In fact4.We investigated the likelihood of emergence under two models of host adaptation, nicknamed the ‘off-the-shelf’ and ‘tailor-made’ models. In both scenarios, the virus population in the donor host was subject to purifying selection such that any mutations that arose within this host had negative fitness effects. Nevertheless, error-prone virus replication may provide opportunities for multiple mutations to accumulate within a single viral genome even when they are deleterious. When transmitted from a donor species to a new recipient species, new genetic variants may result in a virus that is better adapted to the new host. Similar to previously proposed frameworks ,30, viriOverall, our analysis clearly shows that the interactions between within-host and between-host fitness landscapes play a central role in determining the probability of emergence, analogous to earlier findings . Under oThe size of the transmission bottleneck has a dramatic effect on the population dynamics of the evolving virus –33. Atteet al. [In the case of influenza, data regarding viral aerosol shedding from infected individuals may be especially informative. Milton et al.  sampled As few as five mutations may be required for avian H5N1 influenza virus to evolve airborne transmission in mammals . Using HAn additional concept to consider in this context is that the mutations required to enable mammalian transmission vary in their fitness effect. In particular, it is possible to conceive a two-step process in which a mutation of major effect enables a leap across a steep fitness valley, followed by the later accumulation of additional mutations of minor effect that marginally optimize or reduce viral fitness in the recipient and donor, respectively . Under tIt is unclear which model described here most accurately describes the process of pathogen emergence in nature. Indeed, it is important to note that little is known about where in parameter space the real-world settings of virus evolution and transmission lie. In addition, ecological factors too broad to parametrize here, such as increasing land-use, urbanization and climate change, will, unquestionably, have a considerable influence on the emergence of new diseases , particu


Further, the W452W149PB2 virus also induced the highest virus titers in lungs at all time points and the highest levels of inflammatory cytokine responses among all viruses tested. This high virulence phenotype was also confirmed by high viral titers in the respiratory tracts of infected ferrets. Further, a mini-genome assay revealed that W452W149PB2 has significantly increased polymerase activity (p < 0.001). Taken together, our study demonstrates that a single gene substitution from other avian influenza viruses can alter the pathogenicity of recent H5N8 viruses, and therefore emphasizes the need for intensive monitoring of reassortment events among co-circulating avian and mammalian viruses.Recently identified highly pathogenic avian influenza (HPAI) H5N8 viruses (clade 2.3.4.4) are relatively low to moderately pathogenic in mammalian hosts compared with HPAI H5N1 viruses. In this study, we generated reassortant viruses comprised of A/MD/Korea/W452/2014(H5N8) with substitution of individual genes from A/EM/Korea/W149/2006(H5N1) to understand the contribution of each viral gene to virulence in mammals. Substituting the PB2 gene segment or the NA gene segment of the H5N8 virus by that from the H5N1 virus resulted in significantly enhanced pathogenicity compared with the parental H5N8 virus in mice. Of note, substitution of the PB2 gene segment of the H5N8 virus by that from the H5N1 virus resulted in a 1000-fold increase in virulence for mice compared with the parental virus (MLD Since the mid-1990s highly pathogenic avian influenza (HPAI) A(H5N1) viruses have ravaged domestic poultry in Asia. The unprecedented spread of these viruses toward Africa and Europe since 2003 has given rise to at least 10 distinct phylogenetic clades based on the antigenicity of their hemagglutinin (HA) genes.A newly emerged, HPAI A(H5N8) virus caused poultry outbreaks in the Republic of Korea in mid-January 2014.Although HPAI H5N8 is highly pathogenic in chickens, it has thus far been extremely poor at inducing remarkable clinical illness in domestic ducks and ferrets, which may allow establishment of the virus in these hosts.50 of 105.8 EID50).Although most HPAI H5N1 viruses cause severe clinical symptoms and high mortality in experiment animal models including mice, the recent H5N8 viruses showed only moderate virulence in mice, similar to the 2009 pH1N1 viruses  virus were altered in mammals following reassortment events with the HPAI H5N1 (W149. Clade 2.2) virus, variants were plaque purified in Madin-Darby canine kidney (MDCK) cells following co-infection and were subsequently genotyped by RT-PCR using custom-designed gene-specific primers. Reassortants were then confirmed by full-length sequencing. The resulting plaque-purified reassortant viruses and selected single gene-reassortants used in this study are listed in 50) end point. Each reassortant virus containing the PB1, HA, NP, M, or NS gene of W452 showed similar growth properties to the parental W452 virus in embryonated chicken eggs  substitution virus (W452W149NA) exhibited reduced viral titers compared with the parental W452 virus (107 vs. 108.8 EID50/ml).To make the virus stock, each virus was propagated into 10-day-old embryonated chicken eggs and titered via the 50% egg infectious dose (EIDken eggs . Insertiin vitro growth properties of these viruses correspond with the pathogenic properties of H5N8 (W452) in mammalian hosts, their 50% mouse lethal doses were determined  , while all mice infected with parental H5N1 (W149) virus succumbed to death or were killed due to more than 25% weight loss and/or severe clinical signs within 8 dpi  (W149PB2 and W452W149NA) were included in the highly pathogenic group. Notably, mice infected with the W452W149PB2 virus experienced rapid weight loss of more than 25%, 100% mortality, and an MLD50 of 102.5 EID50, which is even lower than that of the wild-type H5N1(W149) virus (103.3 EID50 of MLD50)  in infected mice, in spite of the fact that it showed a reduced yield in embryonated eggs (107.0 EID50/ml). Reassortant viruses including W452W149PA and W452W149HA induced body weight loss and mortality in a manner similar to the wild-type H5N8 virus with moderate virulence (ranging from 105.7 to 105.8 EID50 of MLD50)  (To verify whether the different termined . In addi3 EID50) . For the3 EID50) , (ii) mo3 EID50)  and (iii3 EID50) . To compf MLD50) . The W45f MLD50) , Table 15 EID50) , Table 1W149PB2 virus peaked at 3 dpi (106.7 EID50/g) and was at least as high as that of the H5N1 (W149) virus (W149NA virus showed a low (104.8 EID50) MLD50  virus . Althoug0) MLD50 , the lun0) MLD50  and B. Til 9 dpi . In addiil 9 dpi . In contil 9 dpi .Figure W149PB2 virus was detected in all organs tested. These results clearly demonstrate that the PB2 gene from the HPAI H5N1 (W149) virus substantially alters virus replication and expand the tissue tropism in the infected host.Multiple organ failure is a typical feature of HPAI H5N1-infected mice and is the cause of the high mortality rate.W149PB2, and W452W149NA) compared with the other 2 groups (W149PA and W452W149HA) induced low to intermediate expression of cytokines with relatively delayed kinetics (5 to 7 dpi) with the expression levels gradually increasing up to 7 dpi  showed low levels of cytokine expression compared with W452 at 5 dpi  from 3 dpi until death . Moreover, the expression of IL-15 and MIP1β were highest at 3 dpi in the highly pathogenic viruses while the moderately and low-pathogenic virus-infected mice showed mildly elevated to normal expression levels . Further, expression of IL-2, IL-5, and IL-13 showed increases during the virus infections in each group . Together these data suggest that elevated expression of pro-inflammatory cytokines and chemokines is closely associated with the high virulence of the W452W149PB2 and W452W149NA reassortants.To elucidate the relationship between cytokine production and viral pathogenicity, we measured induction of cytokines in the bronchoalveolar lavage BAL) fluid of infected mice at 1, 3, 5, 7, and 9 dpi  showed histopathological damage, including serious exudate-filled alveolar spaces (W149NA demonstrated the most extensive infiltration of inflammatory cells (W149PA and W452W149HA) demonstrated inflammation in limited areas of the lungs , and normal human bronchial epithelial (NHBE) cells (W149PB2 viruses showed at least 100-fold higher mean peak titers (p < 0.05) than W452 in all infected cells. In MDCK cells, W452W149PB2, and to a lesser extent the viruses possessing the PA or HA of W149 replicated more efficiently than parental W452 virus  (To examine the E) cells . Viral tk titer) A-C.FiguW149PB2, and W452W149PAviruses reached higher mean peak titers (106.5 to 109.25 EID50/ml) than the W452 virus (105.25 EID50/ml) at 72 hours post infection (hpi) (W149HA and W452W149NA demonstrated similar peak titers to W452 (P>0.05), and the other viruses showed reduced mean peak titers (102.75 to 4.25 EID50/ml) (W149NA demonstrated higher titers (103.5 EID50/ml) at 24 h p.i. Overall, these results demonstrate that in the W452 backbone a single gene substitution from W149 can alter the in vitro growth characteristics of H5N8 (W452) virus in the 3 mammalian cell lines examined.In 2 systems of human cells , these viruses demonstrated delayed and reduced titers compared with those of MDCK cells D to I. Ion (hpi) D-E. MoreID50/ml) F. Viral ID50/ml)  G-I. IntW149PB2, W452W149HA, and W452W149NA viruses demonstrated increased virulence with low MLD50 compared with the other reassortant viruses. Since the ferret is recognized as the most suitable host for influenza virus studies, the W452W149PB2, W452W149HA and W452W149NA viruses were intranasally inoculated into groups (n = 15) of ferrets at 106 EID50 to investigate whether these viruses could efficiently replicate and be pathogenic in this model. One day after virus infection, 3 naïve ferrets were added into cages adjacent to infected animals to evaluate any possible indirect droplet transmission. For comparison, we also tested W149 and W452 viruses. Nasal washes from each group of ferrets were collected at 1, 3, 5, and 7 dpi for virus titration. In addition, 3 ferrets from each group were killed on 1, 3, 5, and 7 dpi (3 ferrets/dpi) to assess the viral titers and tissue distribution of the virus.In mice the W4525.8 EID50/Swab at 5 dpi, while the W452 virus showed the highest virus mean peak titer at 1 dpi (103.1 EID50/Swab), which gradually decreased until 7 dpi (only one ferret showed a titer of 10l.5 EID50/Swab(1/3))  with levels at 7 dpi of 102.8 EID50/Swab )  and B. Fab(1/3))  and G. ID50/Swab . FurtherD50/Swab  and G. H52 virus  and E. Fecovered  and GFiNone of the nasal washes from contact ferrets in any group were positive for virus during the duration of experiment (18 days) . MoreoveW149PB2, W452W149HA, and W452W149NA viruses could also influence virus replication properties outside of the respiratory organs. In agreement with the previous study, the H5N1(W149) virus was recovered from organs outside the respiratory tract, such as spleen, kidney and intestine  with comparable or decreased titers compared with W452 virus  viruses without any genetic incompatibility since all 8 combinations of single-gene reassortants were selected from virus co-infections of MDCK cells by plaque purificationAn outbreak of novel reassortant HPAI H5N8 virus, which is a member of clade 2.3.4.4, was reported in 2014 in South Korea and subsequently spread into nations in Asia (China and Japan), Europe , and North America (Canada and the United States).in vitro, increased virulence, an expanded range of tissue tropism, and in vivo growth properties at levels similar to those of H5N1(W149). This was especially true of the H5N8 (W452) virus containing the PB2 gene from W149 virus. In fact, the W452W149PB2 virus showed 1,000-fold higher virulence than W452 virus  and 10–100 fold higher viral lung titers at all-time points in mice with systemic infection. Reciprocally, the W149W452PB2 virus showed more than 10-fold attenuation in virulence compared with W149 virus   . Many stW149NA, showed relatively low viral titers in eggs (10–100 fold lower than the other reassortant) and viral growth, but was categorized into the highly pathogenic group due to the low MLD50 (4.8 log10EID50). Further study will be necessary to verify this, but it appears likely that HPAI H5 viruses belonging to clade 2.3.4.4 may begin to switch their neuraminidase gene with that of other viruses to obtain one more suitable for their HA and NA balance, such as H5N8.In China, an H5N1 inactivated vaccine has been compulsorily used to control widespread HPAI H5N1 throughout the country.cytokine storm and is followed by massive innate immune responses associated with acute respiratory distress syndrome, a typical medical condition of highly pathogenic influenza infection that results in high morbidity and mortality.The unbalanced production of inflammatory cytokines and chemokines observed during influenza virus infections has been suggested to be a mechanism of pathogenesis for these viruses.W149PB2 virus in mammalian hosts, we performed ferret infection and indirect transmission studies comparing these to wild-type W149 and W452 viruses. Similar to the results of the mouse study, the W452W149PB2 virus exhibited increased virus titers in nasal washes compared with the W452 virus; however, there was no evidence of aerosol transmission to naive contact ferrets (W149PB2 virus induced significantly high and prolonged virus titers in infected ferrets as well as wide organ distribution compared with the W452 virus (W149PB2 and W452 viruses (HA clade 2.3.4.4) were (101.2 to 102.8 EID50/g)  suggesting that such a reassortant is a potential threat to public health (To verify the increased virulence and growth properties of the W452 ferrets . Further52 virus . InteresEID50/g) . These rc health .Trojan horse and mixing vessel for the creation of novel reassortants with pandemic potential. Furthermore, the rapid and wide spread of H5N8 virus to Asia and Europe and the first outbreak of these viruses in North America has the potential for the creation of novel viruses with newly-introduced genes.The current study focused on the investigation of virulence properties of the reassortant H5N8 viruses in mice and ferrets, which are appropriate models for the evaluation of viral pathogenesis.50 end point. The handling of viruses was performed in an enhanced biosafety level 3 (BSL3) containment laboratory as approved by the Korean Centers for Disease Control and Prevention (KCDC-14–3–07).To obtain reassortant viruses containing single gene changes from A/EM/Korea/W149/06[W149(H5N1)] into the A/MD/Korea/W452/2014[W452(H5N8)] backbone, the 2 viruses were mixed (1:3 ratio) and was used to infect MDCK  cells, and then plaque purification was conducted. Briefly, a monolayer of MDCK cells in a 6-well plate were infected with serially diluted viruses for 1 hour, washed twice with phosphate buffered saline  and overlaid with a 0.7% agarose-EMEM media with or without L-1-tosylamide-2-phenylmethyl chloromethyl ketone (TPCK)-treated trypsin . After 3 d of incubation, plaque colonies were picked and resuspended in medium and injected into 10-day-old embryonated chicken eggs . After 30 hours of incubation, viruses were harvested and full-length gene segments sequenced to confirm the origin of each segment. Confirmed viruses were stored at -80°C and titrated via the EID50) of each virus, 8-week-old female BALB/c mice  for each virus were anaesthetized and inoculated intranasally with 10-fold serially diluted virus in a 30 ul volume. The mice were monitored daily for 14 d and checked for changes in body weight. The infected mice were killed if they lost more than 25% of their initial body weight.To determine the 50% mouse lethal dose . Tissues were homogenized with an equal volume (ml/g) of PBS containing antibiotics . Homogenates were cleared by centrifugation, and then viral titers in the 10-fold serial dilutions of supernatants were measured by determining the EID50. The remaining mice were used for H&E staining at 6 dpi.Additionally, groups of mice (n = 38 per group) were inoculated intranasally with 10BAL fluid from mouse lungs was collected at 1, 3, 5, 7, and 9 d after each virus infection or from the uninfected control group. The BAL solution was then centrifuged at 12,000 rpm for 5 min at 4°C and stored at −80°C until the analysis.A multiplex biometric immunoassay was adopted for cytokine measurement according to the manufacturer's instructions , The concentrations of TNF-α, lL-1β, IL-6, IL-12p70, IL-15/IL15R, INF-γ, IL-18, IL-2, IL-5, IL-13, GM-CSF, and MIP-1β were measured. Briefly, BAL fluid samples were incubated with antibody-coupled beads corresponding to the various cytokines. Complexes were washed, then incubated with biotin-conjugated secondary antibody, followed by another wash and incubation with phycoerythrin reporter conjugated-streptavidin. Assays were measured by detection of the fluorescent reporter signal and identification of beads on a Bio-Plex 200 system and data were analyzed using the associated Bio-Plex Manager software version 4.1.1 .Luciferase mini-genome reporter assays were performed using a luciferase reporter plasmid (pHW72-Luci) containing the influenza A virus M – driven noncoding region.50.MDCK, A549  and NHBE cells were cultured in 6-well plates and then infected with reassortant viruses at a multiplicity of infection (MOI) of 0.01 or 0.1 for 1 hour, washed and incubated at 35°C in a 5% CO2 atmosphere in the presence of TPCK-treated trypsin (2 ug/ml). Cell culture supernatants were collected at 12, 24, 48, and 72 hpi and virus titers were determined by EID6.0 EID50 of W452, W149 and W452W149PB2 viruses under anesthesia. Three ferrets per group were killed at 1, 3, 5, and 7 dpi for virological and pathogenesis experiments. After 24 h, naïve ferrets (n = 3) were co-housed in the other half of a cage  with a ferret in each inoculated group, with the ferrets only separated by 2 stainless steel grids allowing for airborne virus transmission. Ferret body weight and temperature were monitored every other day until 7 dpi. Nasal washes were collected every other day from the inoculated ferrets and daily from 1 day post-exposure from the contact ferrets until 18 d post contact (dpc) and blood was collected from contact groups at 18 dpc. Virus titrations of nasal washes and various tissue organs were determined by EID50 assays.Female ferrets, 18- to 20- weeks old and sero-negative for influenza A viruses (ID Bio Corporation) (n = 15/group), were intranasally inoculated with 10W149PB2 W452W149HA, and W452W149NA viruses were harvested at 6 dpi or 5 dpi, respectively. The samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. Histological assessment was conducted using standard hematoxylin and eosin staining and light microscopy (magnification x400) or stained with NP polyclonal antibodies. The slides were viewed using an Olympus η 71  microscope and DP controller software to capture images.Mouse lungs infected with each virus and ferret lungs infected with W452, W149, W452p < 0.05, **or †† indicates ** p < 0.01, and *** or ††† indicates p < 0.001). All statistical analyses were performed with using GraphPad Prism version 5.00 for Windows .To assess significant differences in viral titers and cytokine expression levels between groups or across time points W452-infected samples were compared. Asterisks indicate the statistical significance between W452 and other viruses determined by One-way ANOVA and subsequent Dunnett's test. Further, daggers indicate the statistical significance across time points. * or † indicates All animal experiments, including use of embryonated chicken eggs, were approved by the Medical Research Institute, a member of Laboratory Animal Research Center of Chungbuk National University (LARC)  and were conducted in strict accordance and adherence to relevant policies regarding animal handling as mandated under the Guidelines for Animal Use and Care of the Korea Center for Disease Control (K-CDC).


The incidence of ductal carcinoma in situ (DCIS) is increasing with the use of screening mammography, and approximately 30% of all women diagnosed with DCIS are treated by mastectomy. There is increasing use of a skin-sparing mastectomy (SSM) approach to surgically excise DCIS as this facilitates immediate breast reconstruction. The rates of locoregional recurrence (LRR) after simple mastectomy performed for pure DCIS are historically reported as 1%; however, international data suggest that LRR after SSM may be higher.To determine our rates of LRR and compare the effect of the type of mastectomy performed, we undertook a retrospective review of all patients who underwent a mastectomy for pure DCIS at our institution between 2000 and 2010. p = 0.012), log-rank. Univariate analysis showed the two factors that predicted the risk of recurrence were a young age at mastectomy and close or involved margins.In total, 199 patients underwent a mastectomy for pure DCIS , all of which were invasive ductal carcinoma. The recurrences all occurred after SSM, which was associated with a higher 5-year LRR of 5.9% (5/102) compared with 0% in the simple mastectomy group  contains supplementary material, which is available to authorized users. The Second All Breast Cancer Report, 38% of non-invasive breast cancers were treated by mastectomy in 2007.Ductal carcinoma in situ (DCIS) is a pre-invasive form of breast cancer where malignant cells are confined within the ductal basement membrane.A simple mastectomy was traditionally performed whereby the entire breast is removed with a large ellipse of overlying skin. Increasing use of skin-sparing mastectomy (SSM) facilitates immediate breast reconstruction by preserving the patients’ skin coverage with improved aesthetic and psychological outcome.The larger surface area of SSM skin flaps increases the potential to leave residual breast tissue. Achieving the ideal mastectomy flap that is thin enough to remove all breast tissue but thick enough to keep subdermal vessels and support an adequate blood supply is difficult.LRR after mastectomy for DCIS has historically been demonstrated as low, with the UK SLOANE audit reporting a 1% LRR,We aimed to determine our LRR after mastectomy for DCIS, comparing SSMs and simple mastectomies and evaluating which factors predicted LRR.We undertook a retrospective review of all patients who had a mastectomy for pure DCIS at the University Hospital of South Manchester between 2000 and 2010, after hospital ethical approval was obtained.The operation notes were reviewed to collect data on the type of mastectomy, the reconstructive strategy used, and the type of axillary surgery.Pathological reports were reviewed and data collected on histological type, grade, size of DCIS, and margin status. The presence or absence of microinvasion, lymphovascular invasion, and comedonecrosis was recorded, and the available molecular phenotype information, including estrogen receptor (ER) status, progesterone receptor (PR) status, and human epidermal growth factor receptor 2 (HER2) status, for those patients recruited to clinical trials was also recorded.Clinical notes were reviewed to evaluate the method of presentation, use of adjuvant therapy, and follow-up data. When recurrences did occur, we gathered information on the location of recurrence, which was recorded as local, regional or metastatic, and subsequent treatment and histopathological data.All patients underwent clinical examination, as well contralateral mammography, annually for a minimum of 5 years before returning to the National Health Service (NHS) breast screening program.Local recurrence was classified as ipsilateral skin, subcutaneous or chest wall recurrence, while contralateral recurrence was defined as contralateral breast parenchyma disease, both of which were proven by histopathological biopsy. Regional recurrence was classified as ipsilateral regional lymph node recurrence, either axillary, supraclavicular or internal mammary clearance, while metastatic recurrence was defined as any recurrence distant to the aforementioned sites. We included all patients who underwent a mastectomy for pure DCIS, and those individuals who had microinvasion or lobular carcinoma in situ (LCIS) were also included. All patients who had a definite invasive element and lymph involvement were excluded.t test was used to compare continuous variables between two groups, and the Chi-square test was used to compare categorical variables. Survival was evaluated using Kaplan–Meier survival curves and the log-rank test was used to compare survival between the two groups. As there were only eight recurrences, we had insufficient numbers for a robust regression analysis. The conventional 5% significance level was used . The Student’s sed Fig. .Fig. 1KaIn total, 199 patients underwent a mastectomy for DCIS between January 2000 and December 2010, with a median follow-up time of 65 months (range 0–152).SSM was undertaken on 102 patients and 97 had a simple mastectomy. Table p < 0.01). No difference in the size of DCIS excised, the percentage of high-grade DCIS, or margin involvement was found between the SSM (31%) and simple mastectomy (26%) groups  underwent immediate one-stage reconstruction, and 37 (36.3%) had insertion of a tissue expander followed by definitive reconstruction .During the 10-year analysis period, eight LRRs were noted, all in the SSM group. There were no local recurrences after simple mastectomy. Kaplan–Meier analysis demonstrated that overall 5-year LRRs were 3.1% at 5 years and 5.6% at 8 years. LRR rates were higher in the SSM group, which had a 5.9% 5-year LRR compared with 0% in the simple mastectomy group  and close (<2 mm) or involved margins. Screen-detected LRR was 4.5% (6/132), similar to 3.4% (2/59) for symptomatic presentation. In general, high-grade and ER-negative tumors were more likely to recur, however there were insufficient events to confirm this.The 5-year contralateral recurrence rate was 4.2%, rising to 8.5% at 8 years. Interestingly, the 5-year ipsilateral recurrence rate following SSM was higher at 5.9% than the 3.9% contralateral recurrence rate in the SSM group, suggesting that adequacy of excision played a key role.All eight recurrences were IDC and presented as a lump either on clinical follow-up or symptomatically. Invasive recurrence represents a loss of local control and therefore potentially increases patient mortality. Median disease-free survival time was 55 months (range 15–106 months). Four of the eight recurrences had surrounding DCIS alongside the invasive component.Of the eight recurrences, seven patients had immediate reconstruction at the time of their SSM. All eight of the recurrences had re-excision in the form of a WLE and axillary surgery (see Electronic Supplementary Table 1). Following recurrence, seven patients had adjuvant radiotherapy and seven had adjuvant chemotherapy (five with trastuzumab). Only three patients required endocrine treatment. One patient died after recurrent disease at 74 months post-surgery.In this large UK series evaluating LRR after mastectomy for DCIS we found a 3.1% 5-year LRR, consistent with US results highlighting a higher LRR than the 1–2% historically quoted.A retrospective review of 223 patients with DCIS treated by SSM, with a mean follow-up of 82.3 months, reported a 5.1% LRR.After mastectomy, many surgeons in the UK are resistant to re-excision if margins, including the anterior (skin) margin, are involved. Our results highlight the importance of achieving clear margins and, if close, further surgical excision is required, even if this is overlying skin.p = 0.09).n = 116) of patients with a close margin of <3 mm, undergoing intraoperative re-excision, a practice not used in the UK.Fitzsullivan et al. reported 810 patients who underwent mastectomy for DCIS at the MD Anderson Cancer Center, with a median follow-up time of 75.6 months and demonstrating a 1% overall LRR, with a 5% LRR in those with a margin <1 mm.Our unit had high rates of SSM use compared with the national average and this raises the question as to whether higher rates of SSM and immediate reconstruction have led to increased LRR. When undertaking SSM and immediate reconstruction, surgeons have to balance the further concern of pressure from the reconstruction on skin flaps and potential necrosis. Kim et al. reviewed recurrence excision specimens from 10 patients who developed LRR after SSM for DCIS. Five of the seven patients who underwent immediate reconstruction had residual breast tissue.Post-mastectomy radiotherapy (PMRT) in patients who had a margin of <2 mm was recommended because of a 16% LRR in one study.Half of the recurrence samples had DCIS associated with IDC, suggesting that DCIS had been left behind or that within residual tissue after mastectomy further DCIS had developed and the absence of radiological follow-up allowed invasive foci to supervene.The median disease-free survival time in this study was 55 months, similar to previous reportsThere is no accepted national surveillance policy for the ipsilateral chest wall or reconstructed breast following mastectomy for DCIS. The use of screening mammography when following up patients who have had an SSM with a transverse rectus abdominis muscle flap reconstruction has been shown to detect recurrences earlier while the disease is still in situ.Audits of LRR after mastectomy for DCIS have been large, retrospective, single-institution reviews; however, a large, prospective, multicenter audit of LRR after SSM is required. Repeated audit of post-mastectomy LRR has reduced LRR across all Dutch hospitals to less than 5% at 5 years, and a similar system of surgical quality control of LRR would likely reduce LRR in the UK.Univariate analysis demonstrated higher LRR in women under 40 years of age, a finding reported by others, with a 7.5% LRR <40 years of age as opposed to 1.5% >40 years of age.Surgeons must achieve clear margins and consider re-excision, including overlying skin, following SSM, particularly in young women with high-grade and ER-negative DCIS, in order to prevent LRR. Women undergoing mastectomy for DCIS should be counseled as to the potential increased need for further surgical excision which affects the final aesthetic outcome but lowers LRR. Further multicenter studies are necessary to evaluate LRR after SSM and the role of post-reconstruction mammography to aid earlier detection of LRR.Supplementary material 1 (DOCX 14 kb)Below is the link to the electronic supplementary material.


We propose that viruses with geometric defects are not necessarily flawed viruses. A geometric defect may be a reactive site. Defects may facilitate assembly, dissociation, or accessibility of cellular proteins to virion components. In single molecule studies of hepadnavirus assembly, defects and overgrowth are common features. Icosahedral alphaviruses and flaviviruses, among others, have capsids with geometric defects. Similarly, immature retroviruses, which are non-icosahedral, have numerous “errors”. In many viruses, asymmetric exposure of interior features allows for regulated genome release or supports intracellular trafficking. In these viruses, the defects likely serve a biological function. Commonly used approaches for spherical virus structure determination use symmetry averaging, which obscures defects. We suggest that there are three classes of asymmetry: regular asymmetry as might be found in a tailed phage, irregular asymmetry as found, for example, in defects randomly trapped during assembly, and dynamic asymmetry due to Brownian dynamics of virus capsids. Awareness of their presence and recent advances in electron microscopy will allow unprecedented investigation of capsid irregularities to investigate their biological relevance. Viruses come in a variety of shapes and sizes: rod-like, spherical, elliptical, and pleomorphic. From a virus’ perspective, the most efficient way to enclose the large volume need for the viral genome is to make a large capsid from many copies of relatively small protein . From an4 particles. But selection and averaging come at a cost.Viral symmetry has also played a critical role in structure determination. The realization that non-crystallographic icosahedral symmetry could be utilized to improve phases made the determination of virus crystal structures possible , a turniSymmetry averaging leads to a loss of asymmetric detail. Some features, such as the organization of the virus genome, though found in every particle, are expected to be obscured by averaging . HoweverAn icosahedral virus is by definition symmetric. Asymmetry can be imposed by several mechanisms that we term: regular asymmetry, irregular asymmetry, and dynamic asymmetry. In regular asymmetry, particles have a well-defined modification to their symmetry; examples are the preferred attachment of the transferrin receptor to canine parvovirus , the polTo demonstrate how averaging can act as a means of generating structure while losing information about symmetry defects, we built an incomplete virus capsid, generated a series of projections, and then calculated an averaged structure . In 2D cSymmetry contributes to capsid stability. Capsids must balance between a stable entity that protects its genomic material and a labile shell that disassembles at a precise time and cellular location to initiate an infection. When every subunit in the capsid interacts with its full complement of neighbors, the capsid is at an energy minimum. Icosahedral symmetry represents a realization of that energy minimum . The uniIt is possible to make a spherical capsid without symmetry. Computational studies show that when the number of subunits is relatively small, e.g., 12 subunits, there is a substantial energetic difference favoring the symmetrical 12-mer over incomplete/over-complete complexes; however, for larger oligomers, e.g., > 40-mers, there is a negligible difference between similarly sized complexes that do and do not satisfy icosahedral symmetry ,32. PartViruses with geometric defects are not necessarily defective or “flawed” particles. We define defective particles as ones that are generally not infectious, possibly a result of misassembly or damage during purification. Conversely, virions with geometric defects may be the infectious particles.Geometric defects can contribute to, and maybe required for, biological function. Hypothetically, a defect may be a scar from nucleation, an effect of the unique features of the genome, or a site for binding to a receptor or initiating dissociation. Below we give some examples from the literature of particles with defects and how the lack of an icosahedral structure may be biologically advantageous.Retroviruses, particularly human immunodeficiency virus (HIV), are a well characterized, biological example of irregularity. Tomographic structures of immature HIV reveal a partial shell with numerous holes in the Gag protein lattice . Such anUnlike pleomorphic HIV, alphaviruses show T = 4 icosahedral symmetry in 3D image reconstructions. Alphaviruses are enveloped viruses where the capsid and envelope proteins both appear to be well ordered. However, recent work indicates that, in vitro and in vivo, a substantial fraction of the alphavirus RRV has geometric defects in its capsids  camera, asymmetric reconstructions have required substantial asymmetry, such as a phage tail  or MS2 pFrom a biological perspective: Identifying the selective advantage of defects will require formulation of new hypotheses. We have presented two in this perspective: (i) that defects in a symmetric capsid allow the virus to be more responsive to environment and (ii) that defects provide access to buried viral components, as in HIV. In both cases, defects may facilitate capsid structural transitions and uncoating. Defects effectively provide a labile “end” to a closed polymer, while still providing enough structural integrity to protect the viral genome. There is nuance between a particle with geometric defects and a defective particle. Defects may arise as scars from initiating or completing assembly; they may be incorporated stochastically during assembly; they may be imposed by interactions with virus- or host-derived molecules. Studies of the physics of defects in virus capsids, whether the defect leaves a large gap or a mismatch/disclinations, have shown that defects do not need to be irretrievably destabilizing , analogo


To the Editor: Zika virus infection is an emerging arboviral disease linked to an increased risk for severe neurologic outcomes and devastating adverse fetal complications, including pregnancy loss and congenital microcephaly (A 51-year-old man (patient 1) who regularly traveled to Samoa, a Pacific island with known Zika virus transmission, returned to New Zealand in late January 2016. He reported having been sexually abstinent while overseas. After 1 week he experienced onset of fever, rash, arthralgia, and ankle edema. Aware of the symptoms of mosquito-borne illnesses prevalent in his region of travel, he assumed he had a mild case of chikungunya and sought no medical attention.A 53-year-old woman (patient 2) who was the sexual partner of patient 1 experienced onset of sore throat, fever, arthralgia, and rash 17 days after her partner’s return to New Zealand (10 days after he had onset of symptoms). She had had no recent overseas travel, had no blood transfusions, and was unaware of having any mosquito bites. Four days after symptom onset, she visited a general practitioner, whose investigations revealed mild neutropenia, normal liver and renal function, no evidence of streptococcal throat infection, and a demonstrated immunity to measles.The rash subsequently spread, and small joint arthralgia worsened. The patient returned to the general practitioner 9 days after symptom onset. Disclosing her partner’s travel history and symptoms and reporting having unprotected sexual intercourse with him after his return to New Zealand (before and during the time he was symptomatic), she enquired about the possibility of Zika virus infection. After discussing the matter with a clinical microbiologist and securing the consent of the woman and her partner, the general practitioner ordered tests for arboviruses common in the male partner’s region of travel. All laboratory tests for chikungunya and dengue viruses were negative for both patients. Testing for Zika virus RNA was performed by using real-time reverse transcription PCR (rRT-PCR) in accordance with published methods  (9). Sert] 25). Semen collected on days 35 and 76 also tested positive . Semen samples collected on days 99 and 117 tested negative for Zika virus RNA. Attempts at virus isolation from the semen sample collected on day 23 failed to cultivate infectious particles.Laboratory investigations for Zika virus RNA in patient 1 demonstrated negative rRT-PCR results for serum (first tested 19 days after symptom onset) and for urine (first tested 21 days after symptom onset). Serologic testing performed on day 21 detected Zika IgM and IgG (IgG titer 1:320). Semen collected on day 23 tested positive for Zika virus RNA (cycle threshold [CAedes species of mosquito capable of transmitting Zika virus infection is established in the country (It is very unlikely that transmission of Zika virus infection to patient 2 occurred through a mosquito bite. Although occasional interceptions of exotic mosquito species have occurred at international ports of entry into New Zealand, neither of the


Multipartite viruses have one of the most puzzling genetic organizations found in living organisms. These viruses have several genome segments, each containing only a part of the genetic information, and each individually encapsidated into a separate virus particle. While countless studies on molecular and cellular mechanisms of the infection cycle of multipartite viruses are available, just as for other virus types, very seldom is their lifestyle questioned at the viral system level. Moreover, the rare available “system” studies are purely theoretical, and their predictions on the putative benefit/cost balance of this peculiar genetic organization have not received experimental support. In light of ongoing progresses in general virology, we here challenge the current hypotheses explaining the evolutionary success of multipartite viruses and emphasize their shortcomings. We also discuss alternative ideas and research avenues to be explored in the future in order to solve the long-standing mystery of how viral systems composed of interdependent but physically separated information units can actually be functional. The architecture, organization, and packaging of viral genetic information can be divided into three categories: monopartite, segmented, and multipartite viruses. Monopartite viruses have a single nucleic acid molecule protected in a shell made of proteins  forming the virus particle. The genome of segmented viruses is divided into two or more nucleic acid segments that are all encapsidated together in a single virus particle. Multipartite viruses  have their genome divided into two or more nucleic acid segments, just as the segmented type, but these segments are each packaged into separate virus particles. This latter peculiar organization is the only one resulting in viral transmissible entities that do not contain the entire genetic information, and in which the co-transmission of several virus particles to a new cell or host appears mandatory to maintain the integrity of the viral genome. The biology of multipartite viruses challenges some basic concepts of virology and evolution, and, at this point, it remains hard  to conceive how they have evolved and how they can actually be functional.Tobacco rattle virus ..15]. ThiA main concern in this field of research is that most of the above cited studies are theoretical and that experimental support for the proposed benefits and costs is rare, if available at all. When considering the common wisdom on viruses, it is clear that a conceptual frame derived from the understanding of canonical monopartite viruses largely dominates. Possibly, this frame biases the conception of theoretical models intended to explain the evolution of multipartite viral systems and hampers the design of experiments that would relevantly address biological processes specifically adapted to the way of life of multipartite viruses.Below, we briefly review the successive steps of the life cycle of viruses, trying to extract and highlight empirical or theoretical data specifically relevant for the biology of multipartite viruses. We thereby identify major gaps and future research lines that would allow a better comprehension of these intriguing biological systems.Multipartite viruses, just as monopartite and segmented viruses, are replicated through a diversity of mechanisms depending partly on their genome nature. Whatever the molecular details or the cellular location of viral replication, two specific benefits have been proposed and modeled to explain the evolution of genome segmentation, thus of both segmented and multipartite viruses. The first putative replication-related benefit is that splitting a genome into several smaller segments should result in faster replication ,16,17].,17.16,17The second replication-related benefit proposed for segmented genomes is that smaller segments are smaller targets for mutations ,13. In tAltogether, it seems reasonable to conclude that the currently available data do not corroborate the hypotheses explaining the evolution of genome segmentation by a faster replication and/or an easier mutational escape of smaller genome segments. In addition, whatever the future outcome of this debate, the arguments apply to both segmented and multipartite viruses and, thus, cannot explain the separate encapsidation of segments in multipartite viruses.As opposed to the above arguable benefits, one replication-associated constraint for segmented genomes is the need to bear similar origins of replication and regulatory elements on all segments in order to be efficiently recognized and processed by the replication complex. The existence of conserved origins of replication among genome segments has been demonstrated for a number of segmented and multipartite viruses, as, for example, bromoviruses ,28, begoThe genetic exchanges between viral genomes can depend directly on the replication mechanisms. However, they can also be replication-independent, through genome break/repair processes and through reassortments in segmented and multipartite viruses.A reassortment can be defined as an exchange of homologous segments between two related virus isolates or species; thus, a form of genetic exchange not involving intramolecular crossovers. Reassortment had been suggested as a substitute for intramolecular recombination in segmented RNA genomes at a time when RNA crossovers were believed to be rare, if not impossible ,32. ThusInfluenza virus, Bluetongue virus, Tomato spotted wilt virus, Cucumber mosaic virus, and several nanoviruses . Su. Su49]. Faba bean necrotic stunt virus (FBNSV) ). For monopartite viruses, the independent action hypothesis (IAH) stipulates that infection of a cell and/or host can be initiated by a single infectious unit, and that each infectious unit can act independently   and Nanoviridae (6–8 ssDNA segments) or of the genus Benyvirus (4–5 ssRNA segments).MOI values have been experimentally estimated in a number of monopartite viruses infecting bacteria , insectsanalyzed . The estPotato virus Y (PVY) was shown to result from the transfer of as few as one or two genomes per aphid vector  and and99] a


Crystallinity and ferroelectric domain structures were investigated for the PZT/LSMO heterostructure. Interestingly, relatively high non-volatile magnetoelectric coupling effects were observed in both heterostructures at room temperature. The change of chemical valence for Mn and Ti at the PZT/MgO/LSMO interface may play a dominant role rather than external strain or orbital reconstruction, which lead to a large modulation of the magnetization. Correspondingly, the transport behavior of the PZT/MgO/LSMO heterostructure is investigated to confirm the role of oxygen vacancies motion. Our result indicates that the PZT/MgO/LSMO heterostructure have a promising application for future high-density non-volatile memories.Multiferroic hybrid structures PbZr Moreover, magnetoelectric (ME) coupling is a key effect to realize magnetoelectric random access memories employing electric writing and magnetic reading5. However, most of the reported achieved ME effects are weak and volatile, which limited the practical application severely. For example, single-phase magnetoelectrics such as BiFeO3, HoMnO3, TbMnO3, etc., face severe limitations due to the weak intrinsic ME coupling8. As an alternative, composite multiferroic systems with stronger ME coupling and utilizing a wider range of materials can extend the possible applications9. It is known that the mechanisms of ME coupling include strain10, exchange bias11, charge (carrier) modulation3, and orbital reconstruction12. Among those approaches, the coupling via charge carrier modulation is particularly effective to achieve a relatively strong and non-volatile ME coupling13.Multiferroic materials which combine multiple ferroic orders such as ferroelectricity and ferromagnetism, have attracted much interests for their promising applications0.2Ti0.8O3/La0.7Sr0.3MnO3 is one particular system that has received much attentions13. La0.7Sr0.3MnO3 (LSMO) is a half-metallic ferromagnetic material with a high spin polarization of nearly 100% and a high critical temperature Tc = 350 K14. PbZr0.2Ti0.8O3 (PZT) is a ferroelectric material with a high remnant polarization (Pr ~ 71.9 μC/cm2)15 and a high Curie temperature (TC = 320 °C)16. The ME coupling between LSMO and the PZT interface is complicated and interesting17. However, until now, there still exist disputes concerning the primary mechanisms of ME coupling in the PZT/LSMO heterostructure. Moreover, few investigations have been concerned the conductive mechanisms of PZT/LSMO heterostructures to determine its activation energy.The composite multiferroic heterostructure PbZr18. Moreover, the modulation of magnetic moments through the polarization field is a non-volatile effect, which is suitable for memory applications. Chemical state change of Mn and Ti at the interface of PZT/LSMO may play a dominant role as opposed to the extrinsic strain or intrinsic orbital reconstruction. Furthermore, an insulating MgO layer was introduced between the LSMO and PZT as a barrier to suppress the orbital reconstruction between Mn and Ti, which results in a maximum ME modulation of 85% at room temperature. Our result indicated the PZT/MgO/LSMO heterostructure may be a promising candidate in future high-density non-volatile memories.In this work, the modulation of magnetic moments under a non-volatile polarization field in a PZT/LSMO multiferroic heterostructure were investigated. Interestingly, a strong ME modulation up to 63% was achieved in a PZT/LSMO heterostructure room temperature, which is higher than other reported worksl) (l is the Miller index) peaks of PZT, MgO and LSMO layers were observed without any impurity phases, indicated all layers were grown with the c-axis normal to the surface of the substrate. An obvious Laue oscillation is shown in the inset of Fig. 19, the PZT unit cell fits well to the corresponding MgO and LSMO unit cells20. Figure Figure rms of the PZT layer is 0.5 nm which is sufficiently smooth to deposit the LSMO layer. Figure up and Pdown), respectively. It is noted that the ferroelectric domains of PZT cannot be switched under ±6 V, while the ferroelectric domains can be switched at ±10 V. Moreover, the spontaneous polarization state of NSTO//PZT is downward, which can be attributed to the differences between the work functions of PZT and NSTO23. Figure 2, which is consistent with the reported work22. Figure Figure NPL − MNL)/MNL)] under Pdown and decreased to 47% under Pup. Assuming this change is due to a change in the effective ferromagnetic thickness with each layer completely modulated and the average magnetic moments of Mn cations doesn’t change, the modulated thickness of LSMO layer is estimated to be 2.5 nm (~6 ML). Accordingly, the relative change of Ms between Pup and Pdown, η = [(M(Pup) − M(Pdown)]/M(Pdown), is estimated to be 63%. Interestingly, the value of η in our NPL sample is higher than other reported works18. Moreover, compared with MNL of pure LSMO, the value of Ms of NPL heterostructure at an initial state is increased up to 129%. This phenomenon may be attributed to the downward spontaneous polarization states of the PZT layer23. However, the change of Ms between Pdown and Pup for the NPL heterostructure is bidirectional, which is different from the unidirectional strain loops of PZT film as shown in Fig. To investigate the modulation of magnetic moments, the magnetic hysteresis (M-H) and XPS measurements of the NPL heterostructure were performed under different polarization fields at room temperature. As shown in Fig. 4+ content (CMn4+) of NL is estimated to be 30.1%, which is consistent with the stoichiometric proportion of LSMO target. However, the content of Mn4+ at the PZT/LSMO interface can be estimated to be increased up to 41.2% without electric field, down to 23.1% under Pup and further increased to 46.0% under Pdown, respectively. Accordingly, the relative change of Mn4+ contents between Pup and Pdown is estimated to be 50%. Moreover, as presented in Eq.  and 18.9% @+9V (Pdown), respectively. It is known that the magnetization of LSMO can be modulated by the changing of the ratio of Mn3+/Mn4+. As a result, the total magnetization of NPL heterostructure can be modulated accordingly. As shown in Fig. 4+ and Ti3+. Thus, the motions of oxygen vacancies at the PZT/LSMO interface due to the polarization field, would play a dominant role in modifying the chemical state of Mn and Ti, resulting in a high modulation of the magnetization.At the PZT/LSMO interface, those dynamic process would play a key role in the motions of the oxygen vacancies. Figure et al.12, an insulating MgO layer was introduced between the LSMO and PZT layers of the NPML heterostructure. Thus, the Ms of NPML heterostructure should be mainly dominated by the MgO/LSMO interface instead of the PZT/MgO interface26. As shown in Fig. down and decreased to nearly 70% for Pup at room temperature. Thus, the relative change of Ms between Pup and Pdown is 85% (compared to Pdown). The modulation trend of the NPML heterostructure is very similar to that of the NPL heterostructure presented in Fig. To exclude the orbital reconstruction effect proposed by Cui 4+ in the NPML heterostructure is 19.3% (Pup), 45.9% (0 V) and 50.0% (Pdown), respectively. Accordingly, the relative change of Mn4+ contents between Pup and Pdown is estimated to be 61.4%. Correspondingly, the contents of Ti3+ are estimated to be 11.5% (Pup) and 21.9% (Pdown). Figure + of NPML heterostructure is consistent with the ratios of Mn4+ and Ti3+. Since the possible orbital reconstruction between Mn and Ti is suppressed by the inserted MgO layer, the higher ME coupling of the NPML heterostructure may be mainly originated from the charge effect rather than orbital reconstruction. Furthermore, it is worth noting the modulation coefficients of ME coupling in NPML (η = 85%) is higher than that in NPL (η = 63%). Since the MgO layer was deposited in an anaerobic atmosphere27, then more oxygen vacancies defects would be formed in the PZT film of NPML heterostructure.As shown in Fig. up and Pdown states are about 30% (with 3 nm MgO) and 10% (with 4.2 nm MgO), respectively. Compared with NPML of 2.1 nm MgO layer (~85%), the changes of magnetic moments with thicker MgO layers show a decreasing tendency. A possible reason is that the MgO thickness is thicker than the designed barrier28. The ME coupling between LSMO and PZT becomes weaker with the increasing of MgO layer thickness.MH measurements for NPML heterostructure with different thickness of MgO layer were performed in Fig. I-V and J-E) characteristics of NPL heterostructures measured at various temperatures. The J-E characteristics were carried out using a Keithley 2430 pulsemeter, and a staircase-shaped dc bias voltage with a step of 0.05 V and span of 3 sec was used. Figure 290 is the permittivity of free space, μ is the charge carrier mobility, d is the thickness of the film, εr is the low-frequency permittivity of the film and θ is the ratio of the free carriers to the trapped carriers. As shown, under low electric fields and in the high temperature region (>160 K), the fitting slopes of the curves are close to 1, indicating Ohmic behaviour in this region. Moreover, the values of the slopes approach 2 for lower temperatures (<160 K), suggesting a SCLC conduction mechanism in the NPL heterostructure.Figure 30C is a constant, K is the Boltzmann’s constant, T is the temperature, ε0 is the permittivity of vacuum, ∅t is the trap ionization energy, e is the electron charge and ∈r is the optical dielectric constant. Figure 1/2 for negative bias and the temperature dependence on permittivities εr. As shown in the figure, the plot exhibits a linear relationship, which is associated with a thermal electron emission to overcome the Coulomb barrier31. To determine the value of the trap ionization energy, the relationships between the leakage current and temperature were also determined for the NPL heterostructure. Figure t0 of 0.61 eV. It is known that oxygen vacancies are one of the mobile ionic defects in an oxide film32. Specifically, in Pb-based ferroelectrics, the Et0 of oxygen vacancies is usually in the range from 0.56 eV to 1.58 eV36. Thus, the dc conductivity in the NPL heterostructure can be attributed to the electric field induced migration of oxygen vacancies.Alternatively, the current behavior can also be associated with a Poole–Frenkel mechanism according toI-V and J-E) characteristics of NPML heterostructure measured at various temperatures. Figure 29. Alternatively, the current behavior can be associated with a Poole–Frenkel mechanism30. Figure 1/2 for negative bias and the temperature dependence on permittivities εr. The linear relationship between ln(J/E) and E1/2 indicated a thermal electron emission to overcome the Coulomb barrier31. Figure t0 of 1.17 eV. Thus, the dc conductivity in the NPML heterostructure can be attributed to the electric field induced migration of oxygen vacancies37. It should be noted that NPML show an obvious asymmetric I-V behavior than that of NPL which may be attributed to the asymmetric structure and changed barrier height by MgO layer39.Figure 3+-O2−-Mn4+ and superexchange (SE) from Mn3+-O2−-Mn3+ exist in LSMO films17. Specifically, the ferromagnetism of LSMO could be enhanced by a DE effect, as shown in Fig. 17. As reported, the oxygen vacancies existed in MgO layer can be controlled by external electric filed40. Thus, the oxygen vacancies accumulated at the PZT/MgO and MgO/LSMO interfaces can pass through MgO layer with each other. Furthermore, due to the existence of a built-in electric field inside the PZT layer, the oxygen vacancies in the heterostructure could move between PZT and LSMO through the MgO thin layer. Therefore, the movements of oxygen vacancies at the PZT/LSMO interface would modulate the valence of Mn and Ti in the heterostructure, as presented in Eqs , the modulated thickness in the LSMO layer can be estimated to be about 2.5 nm. Meanwhile, the formation of Ti4+, the ferromagnetic DE effect from Mn3+-O2−-Mn4+ is enhanced while the antiferromagnetic SE effect from Mn3+-O2−-Mn3+ is weakened, which leads to the enhancement of the total magnetization of the heterostructures. Our results indicate that the PZT/MgO/LSMO heterostructure is a promising candidate for future high-density non-volatile memories.In summary, the multiferroic NPL and NPML heterostructures were epitaxially grown on the conductive NSTO substrates. Interestingly, at room temperature, the non-volatile modulations of magnetic moments up to 63% and 85% were observed inside the NPL and NPML heterostructures, respectively. The motions of oxygen vacancies in the heterostructures play a dominant role in the modulation of magnetization rather than the external strain and orbital reconstruction. With the increase of Mn1.1Zr0.2Ti0.8O3 (PZT), MgO and La0.7Sr0.3MnO3 (LSMO) ceramic targets were prepared by conventional solid reaction methods. Corresponding PZT, MgO and LSMO layers were deposited in sequence on (100) Nb-SrTiO3 (NSTO) substrates by pulsed laser deposition with a KrF excimer laser (λ = 248 nm). After deposition, the samples were annealed at 700 °C under an oxygen pressure of 104 Pa for 30 min to reduce the number of oxygen vacancies. The heterostructures of NSTO//PZT/LSMO and NSTO//PZT/MgO/LSMO are henceforth abbreviated as NPL and NPML, respectively. The thickness of the PZT, MgO and LSMO layers were 150 nm, 2.1 nm and 4 nm, respectively. For a comparison, a LSMO single layer with the thickness of 4 nm was deposited on NSTO under the same deposition conditions. X-ray diffraction (XRD) was carried out using a TTRIII multifunction with monochromatic Cu K-alpha radiation. The surface morphology and ferroelectric domain structure were examined by atomic force microscope . To characterize the electrical properties of NPL and NPML, patterned Pt electrodes were deposited on the surface of films by dc sputtering. Ferroelectric hysteresis loops were obtained with a TF-Analyzer 1000 at 1 kHz. The magnetic properties were measured by a vibrating sample magnetometer (VSM) option in a physical property measurement system (PPMS-9). X-ray photoelectron spectroscopy (XPS) analyses were measured by using a ESCALAB MK II.Stoichiometric Pb


Neto1−/−, but not Neto2−/−, neurons. Correspondingly, the presynaptic GluK1 KAR activity that tonically inhibits glutamate release at immature CA3-CA1 synapses was completely lost in the absence of NETO1 but not NETO2. The deficit in axonal KARs at Neto1−/− neurons resulted in impaired synaptogenesis and perturbed synchronization of CA3 and CA1 neuronal populations during development in vitro. Both these Neto1−/− phenotypes were fully rescued by overexpression of GluK1c, emphasizing the role of NETO1/KAR complex in development of efferent connectivity. Together, our data uncover a novel role for NETO1 in regulation of axonal KARs and identify its physiological significance in development of the CA3-CA1 circuit.Kainate-type glutamate receptors (KARs) are highly expressed in the developing brain, where they are tonically activated to modulate synaptic transmission, network excitability and synaptogenesis. NETO proteins are auxiliary subunits that regulate biophysical properties of KARs; however, their functions in the immature brain are not known. Here, we show that NETO1 guides the development of the rodent hippocampal CA3-CA1 circuitry via regulating axonal KARs. NETO deficiency reduced axonal targeting of most KAR subunits in hippocampal neurons in a subtype independent manner. As an interesting exception, axonal delivery of GluK1c was strongly and selectively impaired in the  Neto1- and Neto2-deficient mouse models, we show that NETO proteins regulate axonal delivery of KARs. The deficit in axonal KAR at immature Neto1−/− neurons manifested as loss of presynaptic KAR activity and resulted in impaired synaptogenesis and perturbed development of the hippocampal CA3-CA1 circuit. These findings suggest novel roles for the NETO auxiliary subunits in orchestrating development and refinement of synaptic connectivity, a process increasingly implicated in developmentally originating neurologic disorders.Kainate-type glutamate receptors (KARs) are highly expressed in the developing brain where they modulate synaptic transmission and synaptogenesis. NETO proteins act as auxiliary subunits for KARs; however, their functions at the immature circuit are not known. Using a combination of cell biology and electrophysiology in Ionotropic glutamate receptors are key signaling elements mediating and modulating fast excitatory neurotransmission in the brain. During development, glutamate receptors contribute to refinement of synaptic connectivity by triggering signals that regulate synaptic structure and function in response to patterned neuronal activity. In particular, the kainate-type glutamate receptors (KAR) are highly expressed in developing neuronal networks and have been implicated both in synaptogenesis , and actKARs are tetrameric receptors composed of five different subunits, GluK1-GluK5. In the hippocampus, GluK1 subunit containing receptors act presynaptically to modulate glutamate release  and at iNETO proteins are auxiliary subunits that critically modulate the biophysical properties of KARs  mice (Experiments were performed using wild-type (WT), rl) mice  of eithe18 primer. Real-time quantitative PCR (RT-qPCR) was performed using CFX96 Maxima SYBR Green qPCR Master Mix (Thermo Scientific) according to manufacturer’s recommendation and Real-Time PCR Detection System (Bio-Rad). All samples were analyzed in triplicate using Gadph as a reference gene and primers listed in Neto expression at different developmental stages was analyzed using standard 2−ddCt method. The values are expressed as percentage (%) of postnatal day (P)4 (100% = gene expression level at P4).Total RNA was purified from hippocampal tissue or dispersed WT hippocampal neurons using RNeasy Micro kit (QIAGEN) with an on-column DNase digestion step. cDNA was synthesized from 1 µg of total RNA using RevertAid First Strand cDNA synthesis kit (Thermo Scientific) with oligo(dT)Neto1 and Neto2 expression in the dispersed hippocampal neuron culture. PCR products were visually confirmed by gel electrophoresis.Reverse transcription (RT-PCR) was performed using Phusion High-Fidelity DNA polymerase  according to manufacturer’s recommendation to test Neto1 and Neto2 , and an in-frame HA tag was added to C terminus of NETO1 and 2 from Addgene plasmid 10792 (a gift of William Sellers). The epitope tagged constructs were subcloned into lentiviral transfer vector under a CMV promoter. Plasmids encoding various KAR subunits were described previously (Epitope tagged plasmid constructs for NETO1 and NETO2 were generated as described previously . Brieflyeviously . The leneviously .in vitro (DIV3), the cell cultures were infected with lentiviral vectors  and triturated. Cells were plated with a density of 10,000 cells/µm on poly-L-lysin (Sigma) coated 24-well plates containing glass coverslips and grown in Neurobasal A (Gibco) medium containing 2% B27 supplement, 0.5 mM L-glutamine, and 1% penicillin/streptomycin . At 3 d  vectors  and fixeFor immunostainings, fixed neurons were permeabilized with 0.2% Triton X-100 and incubated with PBS based blocking solution containing 5% goat serum, 1% bovine serum albumin (BSA), 0.1% gelatin, 0.1% Triton X-100, and 0.05% Tween 20. Primary antibodies  were diluted in a solution containing 1% BSA and 0.1% gelatin. The following secondary antibodies were used: goat anti-mouse Alexa Fluor 647 , goat anti-rabbit Alexa Fluor 647 , goat anti-guinea pig Alexa Fluor 568 , and goat anti-chicken Alexa Fluor 405 . The stained samples were mounted on microscope slides using Prolong Gold antifade reagent . Confocal images were acquired using a LSM Zeiss 710 confocal microscope .2) were counted using SynD and presented per 100 µm of axon length. All images were coded and analyzed in a blind fashion. Brightness/contrast and levels were adjusted in Photoshop for final figures.NETO-HA  and synaptophysin  colocalization was analyzed by calculating Pearson’s correlation coefficient (PCC) using Coloc2 plugin in Fiji (ImageJ) software. PCC was calculated for each z-stack separately. Axonal processes were identified using MAP2 staining. Cells exhibiting one MAP2 negative axon and several MAP2-positive dendrites were included in the analysis. Axonal intensity of the staining was measured using SynD/MATLAB  and normNeto1−/− mice in ice cold Gey’s balanced salt solution  supplemented with glucose (6.5 mg/ml). The hippocampi were mounted on 2% liquid agarose and 400-µm-thick slices were cut using a McIlwain tissue chopper. After 1 h recovery in carbogenated (95% O2 and 5% CO2) GBSS, the slices were transferred to MED64-probe plates  coated with polyethylenimine solution (0.1% PEI in 25 mM borate buffer) or onto Millicell-CM 0.4-μm membrane inserts  in six-well plates. The slices were cultivated in 250 µl of culture medium  on MED64 probes or 1.2 ml of culture medium on the membrane inserts. Cultures were maintained in humidified CO2 incubator (+35°C), where MED64 probes were kept on a rocking stage. The culture media was changed every second day. In some of the WT and Neto1−/− slices, lentiviral particles encoding for GFP and GluK1c under separate syn1 promoters  was monitored, and cells were discarded if Rs varied more that 20%. EPSCs were evoked by afferent stimulation with a basal frequency of 0.05 Hz in the presence of picrotoxin (100 μM) and D-AP5 (50 µM) to antagonize GABAA- and NMDA receptors, respectively. For mEPSC recordings, 1 µM TTX was also included. Data were collected using Axoscope 9.2 (Molecular Devices) or WinLTP software  were prepared from brains of neonatal (P4-P6) or juvenile (P14-P16) mice using standard methods . Whole-csoftware . mEPSCs p = 3.5, and verified visually. Bursts were defined as a sequence of spikes with a minimum of three spikes with a maximum interspike interval of 10 ms. For calculation of spike time tiling coefficient (STTC), at least two CA3 and CA1 channel pairs separated by at least 300 µm were selected for each slice. Time stamps for detected spikes from 10s period with clearly visible activity and without long (>300 ms) bursts were exported to MatLab. STTC was calculated as described previously . The data were collected with the Mobius Software (Alpha MED Scientific) using a low cut frequency of 0.1 Hz, high cut frequency of 5000 Hz and a sampling rate of 20 kHz. For analysis, the data were exported to LabView environment and filtered using Savitzky-Golay filter . Spikes were detected using the LabView Peak Detector algorithm (quadratic fit on a window of width = 3 points). The spike detection threshold was calculated using the median absolute deviation–based method  with a meviously , and thet test, one-way ANOVA with Holm-Sidak post hoc comparison or Kruskal-Wallis test was then used accordingly. All data are presented as mean ± SEM; p < 0.05 was considered statistically significant. In figures, the significance levels are indicated by asterisks as follows: *p < 0.05, **p < 0.01, ***p < 0.001.All statistics were calculated on raw  data using Sigma Plot software. Shapiro-Wilk test was used to test for normal distribution and Student’s paired Neto1 and Neto2 in brain is developmentally regulated . During development, Neto1 expression was strongly upregulated, in contrast to Neto2, which was downregulated, resulting in prevalent expression of Neto1 in the juvenile (P14) and adult (P50) hippocampus . Both NETO1-HA and NETO2-HA were abundantly transported to MAP2-positive dendrites , with flag-GluK1c and myc-GluK4 showing the highest relative intensity in this region . When exot NETO2 D,E.Neto1−/−, but not Neto2−/−, mice.Together, these data suggest that NETO proteins promote trafficking of KAR subunits to axons. Interestingly, axonal delivery of GluK1c, the subunit that is highly expressed in the CA3 pyramidal neurons in the neonatal hippocampus  was seleNeto1−/−, and Neto2−/− mice. At immature CA3-CA1 synapses, presynaptic GluK1 subunit containing KARs are tonically activated by ambient glutamate to inhibit transmitter release  but had no effect on their amplitude (103 ± 13%) in the WT slices. A similar increase in mEPSC frequency was also observed in the Neto2−/− slices , in contrast to Neto1−/− slices where ACET had no significant effect on mEPSCs , and Neto2−/− (p = 0.006) slices as compared with WT, while the mEPSC amplitude was lower in Neto2−/− (p = 0.02), but not in Neto1−/− slices .To study the role of NETO auxiliary subunits in regulation of presynaptic KAR function, patch clamp recordings of mEPSCs were made from CA1 pyramidal neurons in acute hippocampal slices from neonatal (P4-P6) WT,  release . Thus, a release . As expeNeto1−/− slices , in contrast to WT and Neto2−/− slices . These data show that Neto1 deficiency impairs the tonic activation of presynaptic KARs at the immature synapses.The tonic inhibition of glutamate release by GluK1 subunit containing KARs also manifests as facilitation of transmission during high-frequency activity at immature CA3-CA1 synapses . ConsistNeto1−/− slices is that in the absence of NETO1, the presynaptic receptors cannot be activated by the ambient glutamate due to their lower affinity. To address this possibility, we tested the effect of ATPA, a selective agonist for GluK1 KARs  and Neto2−/− mice , but had no effect on transmission at Neto1−/− slices at P4-P6 ; however, this effect was significantly (p = 0.03) smaller to that observed at the WT , Neto1−/− (n = 10), and Neto2−/− (n = 10), respectively; data not shown]. These data suggest that NETO1 deficiency affects presynaptic KAR function predominantly during early postnatal development, when GluK1 subunit is highly expressed at CA3 pyramidal neurons and presynaptic KARs are physiologically activated.The subunit composition as well as the downstream signaling mechanism of the presynaptic KARs changes during development . To studn = 7, p = 0.03) but no change in their amplitude (102 ± 6%). ACET application had no effect on mEPSC frequency in GFP-expressing Neto1−/− cultures , while a significant increase in mEPSC frequency was observed in Neto1−/− cultures where GluK1c was overexpressed in the CA3 pyramidal cells  application in the GFP-expressing WT slice cultures was associated with an increase in the frequency of mEPSCs  contacts. The mean density of synaptophysin puncta in GFP-expressing Neto1−/− , but not Neto2−/− , axons was significantly lower as compared with WT  as previously shown , resulting in similar density of the synaptophysin puncta as in GluK1c-expressing WT axons. In contrast, overexpression of flag-GluK1b or myc-GluK2 had no significant effect on the density of axonal synaptophysin puncta in any of the genotypes  probes and the spontaneous network activity was recorded at DIV2 and DIV6 using channels located at the CA3 and CA1 pyramidal cell regions. During this period in culture, the network activity patterns in WT slices gradually transformed from asynchronous firing (DIV2) to highly correlated bursting activity that ultimately takes the form of ictal-like epileptiform bursts . Thus, in the WT slices, the mean frequency of population spikes and bursts  increased during development in vitro both in areas CA3 and CA1  when GluK1c expression is already downregulated, presynaptic KARs could be pharmacologically activated to depress synaptic transmission also at the NETO1-deficient slices, although the effect was smaller as compared with the WTs. Finally, overexpression of GluK1c in the CA3 pyramidal neurons was able to rescue the presynaptic KAR function at CA3-CA1 synapses in the NETO1-deficient slices. Together, these findings show that NETO1 deficiency leads to loss of presynaptic KAR function particularly at immature synapses, where GluK1c is endogenously expressed. NETO1 promotes but is not absolutely required for presynaptic KARs function, as its dependence on NETO1 is reduced on KAR overexpression and during development.GluK1c and GluK4 are the subunits previously implicated in tonic presynaptic KAR activity at the immature synapses . Thus, GNETO1 deficiency reduced the axonal levels of recombinant GluK1c to ∼60% of the control level, raising a question whether such a modest reduction in axonal targeting can explain the complete loss of functional presynaptic KAR at immature synapses. It is possible that the observed effects of NETOs on recombinant KARs underestimate the reliance of native KARs on auxiliary subunits, as the mechanisms responsible for subcellular segregation of KARs may be overloaded during elevated expression, leading to nonphysiological compartmentalization . On the Neto1−/−, but not Neto2−/−, neurons and fully rescued by GluK1c overexpression. These data support the hypothesis that NETO1 promotes presynaptic development during the time of intense synaptogenesis and circuit refinement via regulation of axonal GluK1 subunit containing KARs. Since our analysis of presynaptic puncta was done on isolated axons, these findings further confirm that axonal GluK1c containing KAR affects presynaptic differentiation directly, in the absence of postsynaptic contact. Intriguingly, in contrast to the present results in isolated axons, GluK2 overexpression led to a strong increase in Syn puncta in axons that grow in bundles in microfluidic grooves  of functional synaptic inputs. Based on the lower facilitation of EPSCs during 50-Hz stimulation, mean Pr in the Neto1−/− mice is higher as compared with WTs, consistent with the loss of tonic KAR mediated inhibition of release  in the NETO1-deficient neurons while GluK1c overexpression in the CA3 pyramidal neurons robustly increased CA3 population firing rate. Higher charge transfer through postsynaptic KARs is expected to amplify spike transmission due to their integrative properties on transmission . Thus, tThere is strong evidence suggesting that correlation between neuronal spike times is vital for organization of the neural circuits during development . Despite