A large part of the world’s architectural heritage is composed of masonry buildings located in seismic areas, and its vulnerability has been shown by the damage caused by the last earthquakes. Meeting the safety demands of cultural heritage buildings according to the performance-based seismic codes requires a deep knowledge of the mechanical properties of material components. Traditional mortars are among these. However, significant samples of structural mortars cannot be taken from existing masonry walls to perform mechanical tests, but tests can, alternatively, be conducted on samples realized according to traditional instructions for composition. Based on a historical study of mix proportions, this paper presents the results of a mechanical test campaign of traditional mortars. The samples were obtained combining lime and pozzolan according to the proportions derived from ancient treatises. The laboratory tests were performed taking into account three different types of limes, and a discussion involving the results presented in the literature is provided. Besides the contribution to fulfilling the lack of knowledge about the mechanical properties of traditional lime mortars, the test results are good references for on-site preparation of mortars for use in restoration. There is a focus on natural pozzolanic lime mortars, widely used in the Neapolitan area and, in general, in the whole Italian territory. During the centuries, mortar has been one of the most used building materials, and it was used in combination with natural stones, bricks or artificial blocks to raise the complex buildings of today’s world cultural heritage. The mix of natural pozzolan and lime has been the base of the mortars used in the course of history, and the interest in the field is testified to by several experimental investigations present in the literature ,2. Good A large part of the world’s architectural heritage is composed of masonry buildings placed in seismic areas, and its vulnerability has been shown by the damage caused by the last earthquakes. Meeting the safety demands of cultural heritage buildings according to the performance-based seismic codes requires a deep knowledge of the mechanical properties of material components ,6,7. TraTests on small dimension samples of existing mortars and non-destructive tests can give reliable information about the chemical properties of the in-situ materials . The reaThe first Italian codes were based on experimental tests by Baronio and Binda , which uIn the last decades, the use of lime mortar as a building material has been strongly reduced in favor of cement mortars, even in the restoration field, where severe damage can be observed after several years of such interventions . The corA large part of Southern Italy’s cultural heritage involves masonry buildings in which pozzolanic mortar plays a key role, so this paper presents a study on its mechanical properties. The first step in the study was the examination of architectural treatises in order to acquire information about the true mix proportions corresponding to the “the rules of the art” at the time of the printed document. The result of a large campaign of laboratory tests on lime mortars is presented in this paper. Based on the historical survey, three distinct lime types were employed to realize the samples .A discussion of the laboratory outcomes obtained by the authors and those presented in the relevant literature is provided ,27,28,29Natural pozzolan can be used in the mortar as it comes from the quarry, while the artificial pozzolan needs industrial treatment to acquire the necessary characteristics ,32, in pEnabling cultural heritage buildings to meet safety demands, according to the seismic codes, requires a deep knowledge of the mechanical properties of the manufact as well as the restoration materials ,14. RefeAs previously noted, the large number of tests performed by Vicat on lime mortars is not useful for this work because the pozzolan is not present as an aggregate in the examined samples ,53. The Since recent Italian building codes do not consider pozzolanic mortars, specimen dimensions, curing and testing methods have been derived from an Italian set of laws dated to 1939, those currently in force [All the testing operations were performed at room temperature . The pozThe mixture was placed in the molds at 80% relative humidity, and this last level was maintained until the demolding after 24 h curing. The process was completed by placing the specimens in water to have three different curing times: 28, 60, and 180 days. The geometry and total number of the specimens for every shape are reported in 2. The compressive tests on cubic specimens were performed by means of a universal testing machine, C1 class, with a servo-controlled hydraulic actuator with 200 kN force rating  has a low value so that reference for the discussion is made to the mean values. The details of the single test are reported in the Although the number of tests is lower than the minimum necessary to make a complete statistical study, the relative frequency distribution reported in Italian codes  considerThe variation of the corresponding data for water cured and air cured mortars is strongly sensible too. The importance of water curing for natural pozzolanic mortars has been well known since the Vitruvius treatise , which sThe A-type mortar is made, in distinction to the other two types, with powdered lime. Its strength rate increment is faster with respect to the other two binders since lime putty needs several months to complete curing. According to the indications of Plinius , “In antIn the literature, there is a lack of direct tensile test results, due perhaps to the complex realization of specimens without damage or disturbance. The tests referred to in the literature are mainly standard three-point flexural tests and compressive tests on small cubes, which are obtained as a result of the flexural test, so that a comparison is possible with only these types of tests performed by the authors, reported in The three-point test gives values of the mortar’s indirect tensile strength higher than those of the direct test. Suitable corrective factors should be involved in the numerical constitutive models when indirect tensile strength is taken into account since direct tensile strength ranges from 40% to 60% of the indirect strength. 2 [2 .It must be noted that the mechanical performances of the three types of mortar tested by the authors are somewhat similar, with small differences due to the lime type. In particular, the laboratory slaked putty and the manufactured mortar, independently of the production process, give comparable results in compression and tension range. This feature can be useful in the professional field since the yard realization of a lime putty can be avoided by choosing a manufactured product resulting from a quality process.This paper presents the results of a large laboratory test campaign performed by the authors on three different types of pozzolanic lime mortars, whose compositions are derived from architectural treatises. Standard three-point tests, together with compressive tests on small and large size cubes, as well as direct tensile tests have been performed on every type of lime mortar.The results show that long-term curing increases the mechanical performance of mortars based on lime putty more than those based on powdered lime; this is different from the weeks immediately following the preparation, when a reverse behavior is observed. In general, water cured natural pozzolanic mortars show better mechanical performance compared with mortars made with the same mix proportions but cured in air.The analysis of the test data encourages the use of lime mortars in the restoration field and the development of suitable guidelines for the relevant professionals. These guidelines should respect “the rules of the art” since the curing conditions, the pozzolan quality and the mix proportions strongly influence the quality of the results.


Background: To slow the increasing global spread of the SARS-CoV-2 virus, appropriate disinfection techniques are required. Ultraviolet radiation (UV) has a well-known antiviral effect, but measurements on the radiation dose necessary to inactivate SARS-CoV-2 have not been published so far.Methods: Coronavirus inactivation experiments with ultraviolet light performed in the past were evaluated to determine the UV radiation dose required for a 90% virus reduction. This analysis is based on the fact that all coronaviruses have a similar structure and similar RNA strand length.Results: The available data reveals large variations, which are apparently not caused by the coronaviruses but by the experimental conditions selected. If these are excluded as far as possible, it appears that coronaviruses are very UV sensitive. The upper limit determined for the log-reduction dose (90% reduction) is approximately 10.6 mJ/cm2 (median), while the true value is probably only 3.7 mJ/cm2 (median).Conclusion: Since coronaviruses do not differ structurally to any great exent, the SARS-CoV-2 virus – as well as possible future mutations – will very likely be highly UV sensitive, so that common UV disinfection procedures will inactivate the new SARS-CoV-2 virus without any further modification. The newest coronavirus disease COVID-19 is a highly transmittable and pathogenic viral infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged in Wuhan, China and spread around the world. So far, more than 3.8 million infections have led to more than 265,000 fatalities worldwide [The responsible pathogen, SARS-CoV-2, is an enveloped single-stranded RNA virus and member of the family coronavidae of the order nidovirales. All viruses of this family exhibit very similar features. They have a spheroid shape with a diameter of about 100–150 nm, are covered with spike proteins on the outside, and have an RNA strand length of 27–32 kb on the inside [To stop the spread of SARS-CoV-2, several measures such as containment, social distancing and wearing face masks have been taken. Among other steps, hygiene procedures have been intensified. The employment of liquid disinfectants is one procedure that has been successful against the 6 older coronaviruses . ThermalRadiation disinfection, especially ultraviolet (UV) radiation, is another well-known inactivation approach for all known microorganisms and viruses that offers some advantages over liquid disinfectants and heat sterilization. It can be performed automatically and employed to disinfect surfaces, liquids, air and rooms, and it is also very energy-efficient.The ultraviolet spectrum is divided in 4 sections: Radiation with a wavelength between 100 and 200 nm is called vacuum ultraviolet radiation (VUV). It is usually not applied for disinfection purposes because it is strongly absorbed by air. The better-known ultraviolet ranges are UVC, UVB, and UVA with spectral ranges of 200–280 nm, 280–315 nm, and 315–380 nm, respectively. Among these last three UV ranges, UVC is the one with the strongest antimicrobial/antiviral properties . For RNAThe most common UVC light sources for many decades now have been mercury discharge lamps, especially low-pressure mercury vapor lamps, with a strong emission peak at 254 nm, which is near the RNA absorption maximum at about 260 nm, as depicted in Figure 1B 2 of 254 nm UVC radiation from a mercury discharge lamp for a 3-log reduction, but for adenovirus (Type 40), the value is approximately 6 times higher (140 mJ/cm2) [Although it is known that this kind of UVC radiation has an inactivating effect on all microorganisms and viruses, all pathogens require different UVC irradiation doses for successful inactivation. For instance, the rotavirus requires about 25 mJ/cm mJ/cm2) .To answer the important question regarding SARS-CoV-2 and other coronaviruses as to which irradiation doses are needed for inactivation, the existing coronavirus photoinactivation results of the last 60 years have been reviewed and analyzed in this study.Google Scholar and Pubmed were searched for different combinations of the following terms: coronavirus, inactivation, photoinactivation, disinfection, ultraviolet, radiation, and light. In addition, a number of individual viruses that belong to the families of coronaviruses although the term “coronavirus” does not occur in their name were also searched for, such as porcine epidemic diarrhea virus, transmissible gastroenteritis virus, feline infectious peritonitis virus, mouse hepatitis virus, sialodacryoadenitis virus, hemagglutinating encephalomyelitis virus, and infectious bronchitis virus.The retrieved sources were evaluated according to the type of sample irradiated , the type of light source (including emission peak wavelength), the inactivation effect achieved, and the applied irradiation dose.If information on disinfection results for different irradiation doses could be found in a single article, those describing a virus reduction by approx. 3–4 log levels were selected. Results that were only displayed as illustrations without the corresponding values in the text or tables were read from previously enlarged figures.If the necessary information was incomplete, e.g., because of missing irradiation details, the irradiation was estimated by available lamp information, provided that lamp type and distance were given or by other available means. Publications in which radiation was combined with photosensitizers or other chemical or biochemical agents were excluded.Subsequently, these data were employed to calculate the log-reduction dose, i.e., the irradiation dose required for a 90% virus reduction.About 30 publications of scientific investigations regarding photoinactivation of coronaviruses were identified. This included studies on CoV, SARS-CoV or MERS-CoV. An overview of the results is presented by sample condition and wavelength in Table 1 Almost all experiments were performed with mercury vapor lamps, with a peak emission at 254 nm (UVC), which is near the RNA absorption peak in Figure 1 In most studies, the authors did not intend to investigate the log-reduction doses of coronaviruses, but rather virus inactivation in various applications. In all experiments and for all coronaviruses, a successful virus inactivation was observed. However, because of the different objectives of the studies, the experimental conditions to determine the specific log-reduction doses were often difficult to identify. In many cases, information important for the present study’s analysis was missing.To evaluate photoinactivation results, the basic inputs were the virus reduction and the applied irradiation dose. Not all authors provided the applied irradiation dose, but at least for some studies this value could be calculated as the product of irradiation duration and irradiation intensity, or it could be estimated. For some investigations, it was impossible to quantify the disinfection success exactly; in these cases, the values were estimated based on the information given in Table 1 2 (bovine corona virus [2 (SARS (CoV Urbani) [The calculated and estimated results, given as log-reduction doses, exhibit extreme variability, even within the 254 nm results, ranging from 0.6 mJ/cmna virus ) to 11,7 Urbani) ). Even tPossible reasons for this observation might be biological and biochemical differences between the coronavirus strains. However, comparing the experimental details, two other potential dominant factors attract attention:The necessary irradiation doses are lower for viruses on surfaces, aerosols and pure salt solutions. When irradiation experiments were performed with the virus in different solutions, it is important to bear in mind that the solutions contain organic materials, e.g., blood products or residue from cell culture medium. These solutions exhibit very high absorption of the applied UVC radiation, resulting in much lower irradiances for viruses that are deeper inside the sample. This effect is clearly illustrated in Figure 1B Most authors did not measure the UVC absorption properties of their biological materials because it was of no importance for their research task; thus, it is almost impossible to extract the role of the absorption in the calculation of the necessary irradiation doses for a 90% virus reduction. In consequence, the lower values for the log-reduction doses, mostly from viruses in salt solutions, surfaces or aerosols, might be a more realistic approach to determine the true virus log-reduction dose.Several investigators performed their irradiation experiments in microtiter plates (MTPs). This might become a problem if the plates are too close to the irradiation source, e.g., in the range of only a few centimeters. Besides the risk of heating the sample and subsequently increased sample evaporation, determination of the irradiation intensity inside the MTP wells becomes difficult. The MTP well walls shade the virus-containing samples from irradiation that does not originate directly above the well. Hence, the true irradiation intensity within a well is probably quite low compared to the intensity measured by a photodetector at the same distance.Table 1 All coronaviruses exhibit a similar structure and a single-stranded RNA length of about 30 kb, allowing the conclusion that they also feature very similar UVC absorption and UVC disinfection properties. It is therefore justified to consider all coronaviruses alike in terms of the investigated UVC-based log-reduction and to summarize the individual results.2 (average 11.9±11.4 mJ/cm2) These values were calculated without the torovirus data and outliers, but the input included viruses in media that probably had higher UVC absorption, leading to reduced photoinactivation. Therefore, this 10.6 mJ/cm2 is probably not the real value for the log-reduction dose, but instead could be considered as an upper limit.This leads to a total UVC median log-reduction dose of 10.6 mJ/cm2 (average 5.8±5.5 mJ/cm2).Recalculation of the UVC log-reduction dose without using results from higher-absorption media should lead to more realistic values. In this case, the total median log-reduction dose would be 3.7 mJ/cm2 [2 [This overview covers all coronaviruses and both UVC wavelengths (222 nm and 254 nm), but without results obtained from studies with probably high absorption media. The obtained results agree well with UVC inactivation data for other ssRNA viruses, such as influzenza A with log-reduction doses around 2 mJ/cmSo far, most instances of successful coronavirus inactivation have been performed using mercury vapor lamps with peak emission at 254 nm. To reduce the use of the toxic mercury, it seems possible that these vapor lamps will be replaced in the future by 222-nm excimer lamps or by 270-nm LED. Since the RNA absorption strengths are similar, the disinfecting effect at these wavelengths will probably be approximately the same as with mercury vapor lamps. However, this should be investigated in more detail in the future, since absorptions in the lipid envelope might have a larger influence on virus inactivation than currently assumed.Because there are only single results available for the effect of 365 nm (UVA) and daylight, the focus of this analysis is on UVC coranavirus inactivation. Both irradiation methods demonstrated a virus reduction, albeit seemingly much less effective than that achieved with 254 nm irradiation. Nevertheless, these longer wavelengths might also be of future interest because their absorption in samples with organic materials is much lower, resulting in higher penetration depths which may allow virus inactivation of larger volumes.2, but the probably more precise estimation is 3.7 mJ/cm2.To date, UVC radiation has been effective against all coronaviruses in all published investigations, although the absorption properties of the sample media reduced inactivation success. The calculated upper limit for the log-reduction median dose (in low-absorbance media) is 10.6 mJ/cmThese results were obtained by investigations on many different coronaviruses, including SARS-CoV and MERS-CoV, but not SARS-CoV-2. Nevertheless, it can be assumed that they are also applicable for SARS-CoV-2 and all future mutations. RNA mutations might have a strong influence on the pathogenicity of a virus, but they do not result in larger structural differences, especially concerning the UV absorption properties of the RNA, which are the main cause for the antiviral effect of ultraviolet radiation.Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia or Candida albicans [2. Therefore, it can be concluded that existing UVC disinfection systems and procedures will be sufficient to deal with all coronaviruses, including SARS-CoV-2.The above-mentioned log-reduction doses are in the same order of magnitude or even smaller than log-reduction doses for other important pathogens, such as albicans . They aralbicans  with itsThe authors declare that they have no competing interests.


Cajal bodies (CBs) are nuclear membraneless bodies composed of proteins and RNA. Although it is known that CBs play a role in RNA metabolism and the formation of functional ribonucleoprotein (RNP) particles, the whole breadth of CB functions is far from being fully elucidated. In this short review, we will summarize and discuss the growing body of evidence pointing to an involvement of this subnuclear compartment in plant-virus interactions. Cajal bodies  are membraneless subnuclear organelles first described in 1903 by the cytologist Santiago Ramon y Cajal ) Figure.The defining component of CBs is a protein named coilin, considered the signature marker of this organelle. Strikingly, and despite the conservation of coilin across vertebrates and in plants, functional information about this protein is limited ; it has In recent years, multiple works have drawn a connection between plant CBs and responses to abiotic and biotic stresses (reviewed in ). NevertRice stripe virus (RSV) ssRNA genome), the nuclear inclusion protein A (NIa) from Potato virus A (PVA) ssRNA genome), and the protein encoded by open reading frame 3 (ORF3) from Groundnut rosette virus (GRV) ssRNA genome) interact with the CB and nucleolar component fibrillarin [Poa semilatent virus ssRNA genome) and the 16K protein from Tobacco rattle virus , on the other hand, interact with the signature CB protein coilin [Tomato yellow leaf curl virus (TYLCV)  interacts with AGO4 mostly in CBs, where it accumulates [Grapevine red blotch-associated virus  can also be detected in CBs [Citrus tristeza virus (CTV)  [In recent years, the CB has started to gain visibility in the plant virology field: multiple independent studies of the viral infection by both DNA and RNA viruses have unbiasedly converged on this subnuclear compartment. To begin with, a number of virus-encoded proteins have been found to interact with CB components and/or localize to CBs . For exarillarin ,22,23,25n coilin ,26. The umulates . The V2 d in CBs , but wherovirus) .In some instances, a specific function of CBs (or the nucleolus) in the viral cycle has either been demonstrated or can be hypothesized. For some RNA viruses, the association with CBs/nucleolus is essential for systemic infection; that is the case for the umbravirus GRV, which does not encode a canonical capsid protein (CP). In the absence of a CP, it is the virus-encoded ORF3 protein that associates with the viral genome in order to protect it, forming filamentous ribonucleoproteins (RNPs) that move long-distance in the plant ,31. DuriBarley stripe mosaic virus, might promote viral movement through a strategy similar to that used by the ORF3 protein, involving fibrillarin and the nucleolus [N. benthamiana plants, in which no CBs are detected, retain post-transcriptional gene silencing (PTGS) activity in local transient assays [Other proteins encoded by RNA viruses, e.g. P2 from RSV and TGB1 from ucleolus ,35. The ucleolus . P23, houcleolus ; whetherucleolus , suggestucleolus . Of noteArabidopsis thaliana displays lower levels of AGO4 [A different scenario is that of the infection by DNA viruses. The genomes of DNA viruses are targeted by DNA methylation, which is considered an effective anti-viral defense . In plan of AGO4 . Other c of AGO4 ,18,38,39 of AGO4 ; whetherCotton leaf Multan virus can also interact with AGO4 [In order to achieve a successful infection, DNA viruses have evolved TGS suppressors. This is the case of V2 from TYLCV which, among other functions, can interact with AGO4, suppressing its binding to and subsequent methylation of the viral genome ,40. The ith AGO4 .N. benthamiana and tobacco plants in which coilin has been silenced [Tobacco rattle virus (TRV) or Tomato black ring virus , coilin-silenced plants do not show recovery from the infection, as opposed to wild-type plants; along the same lines, infection by Barley stripe mosaic virus (BSMV)  and Tomato golden mosaic virus  results in the development of stronger symptoms in coilin knocked-down plants. These results suggest that CBs/coilin play a crucial role in anti-viral defense against these viral species. The exact underlying mechanism, however, is unclear: coilin silencing does not affect neither TRV nor BSMV replication in protoplasts; and while for TRV differences in viral accumulation are found in the young tissues where recovery takes place, the increased BSMV accumulation can be detected locally and systemically at early stages of the infection only [Potato virus Y (PVY)  and Turnip vein clearing virus , pointing to a positive role of coilin in the viral cycle. In the case of PVY, such a role might be related to viral replication, since knock-down of coilin leads to reduced viral accumulation both in protoplasts and inoculated leaves, although not in systemic leaves [The functional relevance of the CB  has been assessed in an unbiased manner using transgenic silenced ,24. In tsilenced . Followiion only . In contc leaves .Subsequent investigations shed light on the effect of coilin on the infection by TRV : the TRVA growing body of work is making the CB emerge as a relevant player in plant-virus interactions, based on localization and interactions of adapted virus-encoded proteins, as well as on functional genetic data. However, considering the results currently available in the literature, general patterns have not so far become evident; the nature of the involvement of CBs in the infection by a particular virus might depend on biological properties of the species, hence its requirements and evolved strategies for replication and spread. Although an involvement of CBs in the assembly of viral RNPs, and hence systemic movement, has been clearly demonstrated for the RNA virus GRV, and a link between CBs and RdDM-mediated methylation of the viral genome seems likely for the DNA virus TYLCV, CBs may exert a plethora of other functions during the viral subversion of the host cell. A broad and comprehensive analysis of the role of CBs across diverse plant-virus interactions will likely be required for commonalities to arise.


McDonald  for his In regard to Dr. McDonald’s comments about virus size and whether it will be filtered by N95 respirators, we would like to point out that we issued a correction of the paper accepting that what is important here is the size of particles containing the virus and not the size of the virus itself.Second, we would like to address Dr. McDonald’s claim that installing HEPA filters in homes will “eliminate the risk of air duct transmission.” HEPA filters are very good at removing particles in a broad spectrum of sizes from the air. However, no filter or mechanical system is perfect , as demoWe thank Dr. McDonald for assessing our review so critically. We do believe, however, that the current body of knowledge supports our statements.


Neuronal excitability is classified as type I, II, or III, according to the responses of electronic activities, which play different roles. In the present paper, the effect of an excitatory autapse on type III excitability is investigated and compared to type II excitability in the Morris-Lecar model, based on Hopf bifurcation and characteristics of the nullcline. The autaptic current of a fast-decay autapse produces periodic stimulations, and that of a slow-decay autapse highly resembles sustained stimulations. Thus, both fast- and slow-decay autapses can induce a resting state for type II excitability that changes to repetitive firing. However, for type III excitability, a fast-decay autapse can induce a resting state to change to repetitive firing, while a slow-decay autapse can induce a resting state to change to a resting state following a transient spike instead of repetitive spiking, which shows the abnormal phenomenon that a stronger excitatory effect of a slow-decay autapse just induces weaker responses. Our results uncover a novel paradoxical phenomenon of the excitatory effect, and we present potential functions of fast- and slow-decay autapses that are helpful for the alteration and maintenance of type III excitability in the real nervous system related to neuropathic pain or sound localization. Neurons have different expressions of ion channels, and can produce action potentials via different responses to stimulations1. Based on the responses of the resting state to constant depolarization current stimulations, excitability was classified into three types by Hodgkin3. Neurons with type III excitability cannot generate repetitive firing even for large depolarization currents within a biophysically relevant range, and they fail to form a well-defined frequency-current curve5, which is different from repetitive firing for type I and II excitabilities6. For type I excitability, the resting state changes to repetitive firing with an arbitrarily low firing frequency7. For type II excitability, the resting state switches to repetitive firing with a certain non-zero frequency7. Depending on their excitabilities, neurons exhibit different firing frequency responses to periodic stimulus, distributions of interspike intervals to noise, properties of stochastic resonance, and precision of spike timing10, which is important for neural information processing. For example, different biophysical basis such as internal and external currents and dynamical behaviors to sustained stimulations of three types of excitability have been identified7. The transition between types of excitability can be induced by changes of ionic currents13, external synaptic inputs7, and autaptic currents16. There have been many investigations of the physiological significance14 and dynamics20 of different excitabilities. For example, a type I neuron is similar to a low-pass filter tuned to lower frequencies, and a type III neuron to a band-pass filter tuned to higher and lower frequencies2. From the theory of nonlinear dynamics, type I and II excitability correspond respectively to saddle-node bifurcation on an invariant circle and Hopf bifurcation23. Complex bifurcations25, including high-codimension bifurcations19 related to the transition between type I and type II excitability, have been identified.Action potentials related to ionic currents play important roles in neural information transmission and coding27. Neurons with type III excitability only fire a spike or a few spikes at the onset of step depolarization current stimulation, which is also known as phasic firing10. Type III excitability has been observed in neurons such as the spinal cord neuron6, dorsal root ganglion neuron28, and auditory brain stem neuron31. Neurons with type III excitability exhibit extraordinary temporal precision for phase locking32 and enhanced coincidence detection27. These properties are related to some physiological33 and pathological functions34. For instance, for dorsal root ganglion neurons, cellular changes induce a change from type III to type II excitability, which may result in neuropathic pain and muscle spasm28. The nucleus laminaris neurons in the auditory brain stem exhibit type III excitability and spike with high timing precision33, which contributes to sound localization, whereas repetitive firing will degrade sound localization. For nucleus laminaris neurons, brief and repetitive excitatory stimulations can elicit repetitive firing, but constant depolarization current evokes only a single spike31. In addition, the transition between types of excitability can be induced by changes of ionic currents. For example, type III excitability can transition to type II excitability, and to type I excitability by adding low-threshold outward currents such as M-type potassium currents, or blocking low-threshold inward currents such as L-type calcium currents and persistent sodium currents6. However, when adding low-threshold outward currents such as M-type potassium currents or blocking low-threshold inward currents such as calcium currents, type I excitability is changed to type II excitability, and to type III excitability6. Type II excitability is changed to type I excitability, and back to type II excitability by enhancing the conductance of A-type potassium current and non-inactivating calcium current13. From the viewpoint of nonlinear dynamics, type III excitability corresponds to a stable steady state, and no bifurcations appear for any constant depolarization currents in the physiological range10, which is different from type I and II excitability. With application of any constant currents within the physiological range to a steady state, neurons with type III excitability initiate to fire a spike and change to a different steady state corresponding to the constant stimulation current. For that reason, the steady state is stable and no bifurcations occur for type III excitability, and bifurcations have not been identified in the transition between type III excitability and the other two types of excitability10. A fast excitatory autapse has recently been identified as inducing type III excitability to change to type II excitability26, which implies that the fast autapse may be a negative factor for the maintenance of type III excitability, and can largely counterbalance the distinction between type II and III excitability. The results show that type II and III excitability have similar responses to the fast excitatory autapse.Compared with type I and II excitability, there are far fewer investigations of type III excitability35, and has been identified in many types of neurons in brain regions, including the hippocampus36, cerebral cortex35, and neocortex38. The autapse plays important roles in modulating firing activities. For example, an inhibitory autapse can promote spike-timing precision38 and suppress repetitive firing37, and an excitatory autapse can elicit persistent firing39. The effect of excitatory autapses on bursting patterns have been reported in biological experiments40. In theoretical models51, the autapse is identified as influencing electronic activities of single neurons such as an excitability switch16 or resonance53, and spatiotemporal behaviors of neuronal networks such as spiral waves56 and synchronization58. For example, an inhibitory autapse with time delay, which corresponds to a slow autapse, can enhance firing frequency, which is a novel phenomenon different from the common viewpoint that inhibitory effects should induce the reduction of firing frequency59. An inhibitory autapse can induce the enhancement of signal transmission in neuronal networks60, and the enhancement of bursting61 has been simulated and analyzed in theoretical models. An excitatory or inhibitory autapse can induce a transition between type I and II excitability16. An excitatory autapse can induce the reduction of the number of spikes within a burst, in contrast to the traditional viewpoint that an excitatory effect should induce the enhancement of firing frequency62. Slow- and fast-decay inhibitory autapses can induce a depolarization block  to change to firing and subthreshold oscillation64, respectively, which shows that slow- and fast-decay autapses have different effects on electronic activities. These phenomena are abnormal behaviors induced by excitatory or inhibitory autapses. Except for the autapse, excitatory or inhibitory effects such as external stimulation or coupling are identified to induce abnormal phenomena. For example, inhibitory stimulation can enhance neuronal firing66, and inhibitory coupling can enhance the firing frequency of a neuronal network69. Such results of abnormal activities of neural firings enrich the knowledge of neurodynamic and nonlinear dynamics.The autapse of a neuron is a special synapse that connects to the neuron itself69, fast-decay autapses may have similar effects to type II and III excitability26, and slow-decay autapses may have effects on neuronal ring patterns different from fast ones64, the influences of slow-decay excitatory autapses on firing dynamics of type II and III excitability are investigated in the present paper and compared with fast-decay excitatory autapses. Four important results are obtained. First, based on a previous study indicating that the dynamical properties of type II and III excitability respond to sustained stimulation6, similar and different responses to current stimulations with brief or long durations between type II and III excitability are acquired and compared with Hopf bifurcation or nullclines. Second, the fast-decay excitatory autapse, which plays a role similar to periodic stimuli of brief duration, can induce a resting state to change to repetitive firing for both type II and III excitability. This resembles the result of a recent investigation26 and is consistent with the common viewpoint that excitatory effects can promote neuronal firing activities. Such a result shows that there is no obvious difference in the effects of a fast-decay autapse on type II and III excitability. Third, a slow-decay excitatory autapse can induce a resting state for type II excitability changing to repetitive firing, and for type III excitability changing to a resting state following a transient spike instead of repetitive firing, which shows that a slow-decay autapse has different effects on type II and III excitability. Last, the slower the excitatory autapse the stronger the excitatory effect of the autaptic current. However, for type III excitability, a slow-decay excitatory autapse cannot induce the resting state to change to a stable firing pattern, which is different from the common viewpoint that an excitatory effect should enhance neural firing activities. Such a result shows that the dynamical behavior of type III excitability with a slow-decay excitatory autapse is paradoxical, in contrast to the traditional viewpoint. This phenomenon is explained by the responses of the resting state for type III excitability to the autaptic current of a slow-decay autapse.Based on the three viewpoints that an excitatory autapse can induce abnormal dynamical behaviorsIpulse = 40 μA∕cm2 with duration 1000 ms, lower line in the bottom panel of Fig. Ipulse = 50 μA∕cm2 and 60 μA∕cm2 with duration 1000 ms, top two lines in the bottom panel of Fig. For the Morris-Lecar (ML) neuron with type II excitability, a subthreshold stimulation  are applied, as shown in the bottom panel of Fig. Ipulse = 60 μA∕cm2), not a spike but a subthreshold oscillation is induced, as depicted in the third panel of Fig. Ipulse increases to become suprathreshold (80 μA∕cm2 and 100 μA∕cm2), a spike is induced at the onset of the pulse, as shown in the second and top panels, respectively, of Fig. For the ML neuron with type III excitability, three stimulation current pulses with different intensities (Iapp) are shown in Fig. IH = Iapp ≈ 42.797 μA∕cm2 (red circle), by which a stable focus (solid black line) changes to an unstable focus (solid red line), and an unstable limit cycle (black hollow cycle) appears. In the present paper, IH is the current point at which the Hopf bifurcation occurs. The unstable limit cycle and stable limit cycle (black solid cycle) collide and disappear at Iapp ≈ 42.179 μA∕cm2 (green dot) to form a fold (saddle-node) bifurcation of the limit cycle. The stable limit cycle corresponds to firing. The upper (lower) solid circle corresponds to the maximal  value of the membrane potential of the stable limit cycle. The red hollow circle and green solid circles correspond to the subcritical Hopf bifurcation point and the fold bifurcation point of the limit cycle. The three stimulation (Ipulse) values in Fig. Ipulse) value corresponding to the first star, and the behaviors are stable firing for the stimulation (Ipulse) values corresponding to the second and third stars.For type II excitability, bifurcations of the membrane potential with increasing depolarization current (Iapp) values are stable, and no spiking or bifurcation occurs for any fixed applied depolarization current (Iapp) values in a physiological range, as shown in Fig. Ipulse) values used in Fig. Ipulse) value, the behavior is stable equilibrium.For type III excitability, the resting states for all applied depolarization current  is the stable focus for the applied depolarization current Iapp = 0 μA∕cm2, and RS2 (red hollow dot) is the unstable focus for the applied depolarization current Iapp = 100 μA∕cm2, as shown in Fig. Iapp = 100 μA∕cm2. The nullcline Iapp = 0 μA∕cm2 is the same as for the applied depolarization current Iapp = 100 μA∕cm2, as shown by the gray solid curve, since Iapp. The gray dashed curve and dotted curve represent the nullclines of Iapp = 0 μA∕cm2 and Iapp = 100 μA∕cm2, respectively, which shows that the position of the nullcline Iapp increases. The results show that the resting state changes to the stable limit cycle for type II excitability as the applied depolarization current Iapp switches from 0 μA∕cm2 to 100 μA∕cm2.For neurons with type II excitability, due to the Hopf bifurcation occurring at the applied depolarization current cm2 Fig. , the poi1 (gray solid dot) and RS2 (black solid dot) are stable equilibria for the applied depolarization current Iapp = 0 μA∕cm2 and the applied depolarization current Iapp = 100 μA∕cm2, respectively, as shown in Fig. 1 is the intersection point between nullclines Iapp = 0 μA∕cm2. When Iapp = 100 μA∕cm2, the nullcline 2 is the intersection of nullclines Iapp = 100 μA∕cm2. The equilibria RS1 for the applied depolarization current Iapp = 0 μA∕cm2 and RS2 for the applied depolarization current Iapp = 100 μA∕cm2 are stable because no bifurcations occur with respect to Iapp for type III excitability. The results show that the steady-state membrane potential increases for type III excitability as the applied depolarization current Iapp switches from 0 μA∕cm2 to 100 μA∕cm2.For type III excitability, the points RSIpulse > IH) of long duration. For example, the repetitive firing and dynamical behaviors in the -plane are shown in Fig. Ipulse = 100 μA∕cm2 of duration 60 ms. Before the pulse stimulation, the membrane potential stays at the resting state corresponding to the stable equilibrium RS1 for Iapp = 0 μA∕cm2. After the application of pulse current, the dynamical behavior corresponding to Iapp = 100 μA∕cm2 with duration 60 ms begins from RS1, evolves to the stable limit cycle (red circle with counterclockwise arrows), and stays at the limit cycle until 60 ms have elapsed, as shown by the red lines in Fig. 1 for Iapp = 0 μA∕cm2, as shown by the blue curves in Fig. For type II excitability, repetitive firing can be induced by suprathreshold pulse current stimulation  which takes about 20 ms. In the second phase (green curve), the trajectory stays at RS2 because the equilibrium RS2 is stable, as shown in Fig. 2, recovers to the equilibrium RS1, and stays at RS1, which is the last phase (blue curve), as depicted in Fig. Iapp switches from 0 μA∕cm2 to 100 μA∕cm2.For type III excitability, only a single spike can be induced by a suprathreshold stimulation with long duration, e.g., the depolarization pulse current with strength Ipulse = 100 μA∕cm2 of a brief duration of 1.5 ms, a single spike is elicited for both type II and III excitability, as shown in Fig. V, w)-plane, as shown in Fig. 1) for Iapp = 100 μA∕cm2, runs across the middle branch of the nullcline A (square) when the pulse stimulation is terminated, as shown by the red curve in Fig. A is located to the right of the middle branch of the nullcline A can run across the right branch of nullcline Iapp = 0 μA∕cm2 and Iapp = 100 μA∕cm2 to form a spike, and then it evolves to and remains at the stable equilibrium RS1, as depicted by the blue curves in Fig. 2 for type III excitability because the pulse duration of 1.5 ms is too short.The responses of the resting state to a suprathreshold pulse current with brief duration are similar for both type II and III excitability, as shown in Fig. Ipulse = 100 μA∕cm2, brief duration 1.5 ms, and period 11.5 ms are applied to the resting state, a repetitive spiking pattern is induced, and each pulse can induce a spike for both type II and III excitability, as shown in Fig. From Fig. The interspike interval of the repetitive firing induced by the periodic pulse currents is nearly equal to the period of the pulse currents, which is different from the repetitive firing induced by the suprathreshold current of long duration, where the interspike interval is nearly equal to the period of the limit cycle for type II excitability.IH ≈ 42.797 μA∕cm2 can induce repetitive firing whose interval equals the period of the limit cycle bifurcated from the Hopf bifurcation. However, for type III excitability, a suprathreshold current of long duration cannot induce repetitive firing, and it induces just a single spike. Such results can be used to recognize the autapse-induced dynamical behaviors of the ML model with type II or III excitability. For convenience, the repetitive firing induced by periodic pulse currents of brief duration is called case-1 firing, and that induced by current higher than IH ≈ 42.797 μA∕cm2 (stimulation with long duration) is called case-2 firing, and this appears for only type II excitability. The results also show that responses of the resting state to current stimulation of long duration are different for type II and III excitability, while current stimulations of brief duration are similar for both.Comparing Figs. Another issue should be emphasized. For a neuron with type II excitability, a pulse current of long duration Fig.  providesβ, excitatory autapses can induce different behaviors for both type II and III excitability with time delay τ = 15 ms and autaptic conductance gsyn = 3.0 mS∕cm2. The behaviors of the ML neuron induced by an excitatory autapse when β = 1, β = 0.1, and β = 0.01 are shown in Figs. Ipulse = 100 μA∕cm2 and a brief duration of 1.5 ms, as shown in the bottom panels of Figs. β = 1, the repetitive firing induced by the excitatory autapse is similar for type II and III excitability, as shown in the third panels of Fig. Isyn = − gsyns(t − τ)(V − Esyn)), which is delayed τ = 15 ms and can evoke the second spike. The autaptic current induced by the second spike increases quickly and decreases relatively quickly to form a pulse-like current, which is mainly determined by the fast decay (β = 1) of the variable s, as shown in the top panels of Fig. k-th spike can induce the (k + 1)-th spike , which leads to repetitive firing. In the process, each pulse-like autaptic current can induce a spike, which resembles the periodic pulse currents with brief duration depicted in Fig. τ.By varying the decay rate β decreases to 0.1, i.e., the excitatory autapse becomes relatively slow, the excitatory autapse can induce repetitive firing for type II and III excitability, as shown in Fig. β = 0.1) of the variable s, the autaptic current (red curve) decays relatively slowly. For type II excitability, the first spike can activate the autaptic current, which is delayed 15 ms and can induce the second spike. Then the autaptic current  for type II and III excitability are as shown in Fig. As the decay rate β = 1, the distributions of the resting and firing states for type II excitability resemble those for type III excitability, as shown in the top panels of Fig. For a relatively large decay rate, e.g., β decreases to a relatively small value, e.g., β = 0.1, the parameter region of firing enlarges for type II excitability and narrows for type III excitability, as shown in the middle panels of Fig. gsyn < 2.7 mS∕cm2 and time delay τ > 5 ms, the autaptic current exhibits a periodic pulse-like characteristic, and the interspike interval of the firing pattern nearly equals the time delay τ. The firings resemble case-1 firing. (2) For the remaining parameter region of firings, the autaptic current is greater than IH ≈ 42.797 μA∕cm2, and the firings resemble case-2 firing. When gsyn > 2.7mS∕cm2 and τ > 5 ms, the firings resemble those shown in Fig. τ < 5 ms, a representative example of case-2 firing is shown in Fig. As the decay rate β decreases for type II excitability. The increase is seen in both the time delay τ and autaptic conductance gsyn. However, comparing Fig. β increases for type III excitability, and is induced by the increase of the time delay threshold for firing.Comparing Fig. β = 0.01, the dynamical behavior for type II excitability is different from that for type III excitability in the parameter region shown in Fig. τ < 30 ms). For type II excitability, the firing behavior covers most regions of the plane, as shown in Fig. As the decay rate further decreases, e.g., β for type II excitability, which is induced by the decrease of gsyn for firing. However, comparing Fig. τ < 30ms.Comparing Fig. Isyn for the decay rate β = 0.01  of the autaptic current Isyn cannot induce repetitive firing, while a weaker excitatory effect can do so, which differs from the traditional viewpoint that a strong excitatory effect should enhance firing activities.Comparing the autaptic current between the black dashed lines in the second panels of Figs. .01 Fig.  is much = 1 Fig. , no actiIsyn, the average autaptic current within two black dashed lines in Figs. β = 1  and Fig. gsyn = 3.0mS∕cm2, τ = 15 ms), respectively. With decreasing β, the autapse becomes slower and the average autaptic current increases, which means a stronger excitatory effect of autaptic current Isyn for both type II and III excitability. However, the excitatory effect of the autaptic current induces different responses between type II and III excitability. For example, for type II excitability, the excitatory effect of the autaptic current can induce firing when the decay rate β ∈  for gsyn = 3.0 mS∕cm2 and τ = 4 ms. Such a result for type II excitability is consistent with the common viewpoint that the excitatory effect usually facilitates neuronal firing activities. However, for type III excitability, the excitatory effect of autaptic Isyn can induce firing only when β ∈ , and cannot induce firing for β ∈  when gsyn = 3.0 mS∕cm2 and τ = 4 ms. For type III excitability, a stronger excitatory effect cannot induce firing, while a weaker excitatory effect can. The border between firing and non-firing is depicted by the vertical dashed line in Fig. To quantitatively measure the excitatory effect of = 1 Fig.  to 167.2= 1 Fig.  to 182.3.01 Fig. . The cha35, and it plays important roles in modulation of neuronal electronic activities51, which are complex due to the nonlinearity of neurons and autapses. Based on bifurcation or nullcline, similar or different responses of the resting state to current stimulations of brief or long duration between type II and III excitability are identified. Comparing dynamical characteristics of autaptic currents with those of current stimulations, the effects of fast- and slow-decay excitatory autapses on the firing activities of the ML model with type II and III excitability are identified in the present paper.The autapse is a self-feedback connection of neurons26. These results are consistent with the common viewpoint that the excitatory effect should enhance neural firing activities. In biological experimental studies, nucleus laminaris neurons exhibit type III excitability, and can generate repetitive firing under brief and repetitive excitatory stimulations31. For a fast-decay excitatory autapse, the autaptic current exhibits a pulse-like characteristic, which induces similar responses for both type II and III excitability. Furthermore, the slow-decay excitatory autapse has different effects on type II and III excitability26 because type II excitability corresponds to Hopf bifurcation, while no bifurcations correspond to type III excitability. The current value corresponding to Hopf bifurcation points for type II excitability is IH. For an excitatory autapse with a slow decay characteristic, the autaptic current Isyn is larger than IH in most time durations, which leads to repetitive firing. For type III excitability, a slowly changing autaptic current induces a transient spike instead of repetitive firing. Specifically, the autaptic variable s activated by a spike remains at a high level before being reactivated by another spike, hence the variable s changes little. The small change of autaptic current influenced by the variable s cannot induce action potentials, which is the dynamical mechanism of the resting state following a transient spike induced by a slow-decay autapse for type III excitability. The result of the present paper is largely consistent with the result of different responses of type II and III excitability to sustained stimulation reported in a previous study6. For a fast decaying autapse, the autaptic currents are similar to periodic stimuli of short duration. However, a slowly decaying autapse behaves as sustained stimulation. Thus, understanding the dynamics of the responses to periodic and sustained stimulus suffices to understand the responses of two types of excitability to autapses with different decaying kinetics, at least for the simplistic model utilized in the present study. The dynamical mechanisms that explain the different responses to slow-decaying autaptic stimulation of type II and III excitability are similar to that explain the different responses to sustained stimulation: destabilization of a fixed point through a subcritical Hopf bifurcation for type II excitability and a fixed point remaining stable (a quasi separatrix crossing mechanism of spike initiation) for type III excitability6. The results of the present paper also show that slow- and fast-decay autapses have different effects on neural firing or oscillations, which is consistent with previous studies64. One property of type III excitability is coincident detection. That is, type III neurons respond only to temporally coincident stimulations (with large amplitudes). Our results indicate that a slow-decay autapse can maintain the property of type III excitability, which is consistent with experimental studies showing that excitatory autapses can enhance coincident detection in a neocortical pyramidal neuron40.For both type II and III excitability, a fast-decay excitatory autapse can induce changes from the resting state to repetitive firing, similar to the findings of a previous report62. Therefore, a novel example in contrast to the common viewpoint of the excitatory effect is given in the present paper. The novel cases of abnormal phenomena induced by excitatory or inhibitory effects should be investigated.The slow-decay excitatory autapse-induced resting state following a transient spike for type III excitability is a paradoxical phenomenon. The smaller the decay rate the slower the autaptic current decays. That is, a slow-decay excitatory autapse provides a stronger excitatory effect than a fast one. However, a fast-decay autapse can induce repetitive firing, while a slow-decay autapse instead induces a resting state following a transient spike. Such a result is different from the traditional viewpoint that strong excitation should induce enhancement of firing activities. In a recent report on abnormal phenomena induced by an excitatory autapse, the excitatory effect was identified to reduce the number of spikes within a burst33 and diseases70 of the nervous system. The cellular excitability changes from phasic firing corresponding to type III excitability to repetitive firing corresponding to type II excitability, which is related to neuropathic pain in dorsal root ganglion neurons70. The excitatory autapse with slow decay characteristic is helpful for the maintenance of type III excitability, which may alleviate muscle spasm and neuropathic pain. In fact, for neurons with type II or III excitability and without autapses, the equations to describe the autapse can be taken as an effective feedback modulation measure, which can be achieved in biological experiments with a dynamic patch clamp or in a circuit implementing the nervous system. The results of the present paper reveal that different effects of fast- and slow-decay excitatory autapses or feedback to the resting state of type II and III excitability are important to adjust neural dynamical behaviors, or may be useful for neuronal information processing.Type III excitability has been found to be involved in some functionsIn the present paper, a single-compartmental phenomenological model neuron with different types of excitability, which receives a non-plastic excitatory synaptic input via autaptic connection or feedback current, is used to simulate biological phenomena, and is simple enough to recognize complex dynamical behaviors. However, to further identify how biophysically different neurons operate under temporally complex, natural synaptic excitation is important to recognize biophysically complex and diverse biological neurons. The behavior of the theoretical model  under more complex, aperiodic stimuli and/or incorporating analysis of the effects of synaptic plasticity should be investigated.6 is described as V is membrane potential; w is the activation of the delayed rectifier potassium channel; V and w, respectively; gNa and ENa are the maximum conductance and reversal potential, respectively, of the sodium current; gk and EK are respectively the maximum conductance and reversal potential of the delayed rectifier potassium current; gL and EL are respectively the maximum conductance and reversal potential of the leakage current; C is the membrane capacitance; and Iapp is the applied depolarization current.The modified ML modelIapp is constant and is a bifurcation parameter, and Iapp is pulse current stimulation with strength Ipulse and duration Δt to induce the changes of dynamical behavior of the ML model. For example, if a pulse with Ipulse = 100 μA∕cm2 and duration Δt = 60 ms is applied to the ML model, then the dynamical behavior corresponding to Iapp = 0 μA∕cm2 is changed to that corresponding to Iapp = 100 μA∕cm2 for 60 ms, and then it returns to behavior corresponding to Iapp = 0 μA∕cm2.In the present paper, two kinds of applied current are chosen. Isyn mediated by an autapse is introduced to Eq. Isyn is described by gsyn is the autaptic conductance, Esyn is the reversal potential of the autapse, Vpos is the postsynaptic membrane potential, τ is the time delay due to the time lapse occurring in synaptic processing, and s is the activation variable of the synapse, determined as Vpre is the presynaptic membrane potential; Γ(Vpre) is the sigmoid function of Vpre; and α and β are the rise and decay rates, respectively, of synaptic activation. In the nervous system, the deactivation time of the NMDA receptors of an excitatory autapse ranges from  ~ 10 to  ~ 100 ms, and can be modulated by the values of β. The smaller the β value the slower the decay of the autapse. Γ(Vpre − θsyn) = 1∕(1 + exp(−10(Vpre − θsyn))), where θsyn is the synaptic threshold, which is set at a suitable value to ensure that the synaptic transmitter release occurs only when the presynaptic neuron generates a spike, i.e., when Vpre > θsyn. In Eqs. Vpre = Vpos = V to ensure the synapse is an autapse.When the current Esyn = 30 mV to ensure that the autapse is excitatory, θsyn = 10 mV, and α = 12. The parameters gsyn, τ, and β are chosen as control parameters to modulate the effects of the excitatory autapse.The parameter values of the autapse are set as follows: βw6 and βm18. In the present study, the values of βw are selected to ensure that the model exhibits type II and III excitability. βw = − 25 mV for type III excitability and βw = − 13 mV for type II excitability. Other parameters for both type III and II excitability are set as: gNa = 20 ms∕cm2, gK = 20 ms∕cm2, gL = 2 ms∕cm2, ENa = 50 mV, EK = − 100 mV, EL = − 70 mV, C = 2 μF∕cm2, βm = − 1.2 mV, γm = 18 mV, γw = 10 mV, and ϕw = 0.15. Iapp is chosen as the control parameter to modulate the dynamical behavior of the model.The ML neuron model can exhibit firing properties with different excitabilities by modulating its intrinsic parameters, including 71, which is freely available at http://www.math.pitt.edu/bard/xpp/xpp.html.The equations are integrated using the fourth-order Runge-Kutta method with integration step 0.01 ms, and bifurcation diagrams are obtained using XPPAUT 8.0


Nucleocytoviricota). Beyond the practical difficulties of establishing reliable homology relationships from extremely divergent sequences, I present here conceptual arguments highlighting several fundamental limitations plaguing the reconstruction of the deep evolutionary history of viruses, and even more the identification of their unique or multiple origin(s). These arguments also underline the risk of establishing premature high level viral taxonomic classifications. Those limitations are direct consequences of the random mechanisms governing the reductive/retrogressive evolution of all obligate intracellular parasites.The extension of virology beyond its traditional medical, veterinary, or agricultural applications, now called environmental virology, has shown that viruses are both the most numerous and diverse biological entities on Earth. In particular, virus isolations from unicellular eukaryotic hosts (heterotrophic and photosynthetic protozoans) revealed numerous viral types previously unexpected in terms of virion structure, gene content, or mode of replication. Complemented by large-scale metagenomic analyses, these discoveries have rekindled interest in the enigma of the origin of viruses, for which a description encompassing all their diversity remains not available. Several laboratories have repeatedly tackled the deep reconstruction of the evolutionary history of viruses, using various methods of molecular phylogeny applied to the few shared “core” genes detected in certain virus groups (e.g., the  Since the serendipitous discovery of the tobacco mosaic virus by Dmitri Ivanovsky in 1892 . About fAll viruses are unconventional, in the sense that this category of microorganisms lacks a set of universal homologous components that they all share, and that could be used to define the “norm” that any virus should obey to be recognized as such. To clarify the significance of this unique feature of viruses, I will take the counterexample of the cellular world which encompasses an enormous diversity of cell types, from the already highly diverse prokaryotes (Archaea and Bacteria domains), to the eukaryotes. Yet, all the “conventional” cell types will share homologs of the enzymes and structural proteins required to replicate their DNA genome, to express their genes, synthetize their proteins, together with a number of metabolic pathways to synthetize amino-acids, nucleotides, and generate ATP.Escherichia coli or yeast) has thus become a founding stone of modern Biology. This is well summarized by the aphorism: “what is true for E. coli is true for the elephant”, attributed to Jacques Monod and Francois Jacob [Babela massiliensis) [Rickettsia) [Chlamydiae or Tremblaya princeps) [There are two fundamental consequences to the existence of such common set of components and subsystems. First, except for the smallest details, what is learned on a given cell type can usually be transposed to many others. Ribosomes, for instance, work the same way in all cells. The notion of “model” systems  , ATP synkettsia) , and/or rinceps) ,40.Bamfordvirae” kingdom [If we now try to do the same exercise for viruses, i.e., identify a “model” species and then define “unconventional viruses” versus “regular ones”, one soon realizes that it is impossible. Amazingly, this is not even possible among members belonging to the same Baltimore’s classification, such as the dsDNA viruses (on which I will focus for the sake of clarity), or even within the same kingdom. For instance, Mimivirus (with a 1.2 Mb genome)  is by no kingdom  that als kingdom  or a new kingdom   of which a model can be built, but a conceptual process. What makes viruses alike is not what they are made of, but the cyclic scenario they use to reproduce themselves. In the most general terms, this scenario is as follows: transported in a molecular box, a genome of some kind (RNA or DNA) gain access to a cellular system that is used to produce more copies of itself, and package them into neo-synthetized boxes that are then released in the environment. This abstract scenario can be materially implemented in many different ways, many of which may not have been discovered yet, making environmental virology one the frontiers of the Terra Incognita of basic Biology ,43. AmazOne of the main properties that is common to all viruses is that the “active” part of their replication cycle can only happen inside a cell. They are “obligate” intracellular parasites, a property that most non-specialists believe is unique to viruses. However, we now know that this property alone is not sufficient to discriminate viruses from the cellular world, as modern microbiology revealed a fascinating underworld of “unconventional” parasitic cells that can only live within other cells  ,38,39,40Tropheryma) [Tremblaya) [At this point, it is interesting to notice that although “obligate” parasitism sounds like a qualitative character , it actually covers a whole gradation of dependency toward the host cell. In some cases, for instance, supplementing a culture medium with a specific metabolite was found sufficient to turn an absolute intracellular parasitic bacterium into a free-living one  . In otheemblaya) . AbsolutViruses can actually be ranked relative to each other in a similar way, from minimal viral genomes merely encoding the blueprints of their particle  ,47, to gBamfordvirae kingdom (that include a whole spectrum of virus families with and without virus-encoded DNA/RNA polymerases) (Coccolithovirus and the Marseilleviridae) encode a DNA-dependent RNA polymerase that, most surprisingly, is not packaged in their particles, forcing them to initiate their cytoplasmic replication cycle by first recruiting nuclear functions [Even if the gene contents of viruses (including those infecting the same host) appear largely uncorrelated, there is some order in this apparent chaos. For instance, there seems to be a strict hierarchy governing the presence of encoded DNA-dependent DNA polymerases and of DNA-dependent RNA polymerases in viral genomes . As of tmerases) . Furtherunctions ,53.However, the reason why the loss of the RNA polymerase should always precede that of the DNA polymerase is not clear, as the absence of any of these genes will force a previously cytoplasmic virus to become dependent of cellular functions located in the nucleus. Once evolved to gain access to the nucleus, a virus devoid of its own DNA polymerase could use that of the cellular host, independently of the presence/absence of its own RNA polymerase. Thus, no basic biological rule would be violated by the eventual discovery of such “unconventional” dsDNA virus. Interestingly, the presence of a DNA-dependent RNA polymerase strictly conditioned to that of a DNA polymerase is also observed in all known dsDNA bacteriophages, which suggests that it is not linked to the presence of a nucleus.Finally, another “unconventional” type of large dsDNA virus is represented by the polydnaviruses of parasitic wasps. Amazingly, if their particles could package up to 800 kb of DNA, it does not contain any of the genes required for its replication or the production of virions (reviewed in ). In thiThree main scenarios have been proposed to explain the origin of viruses. The “virus-first” theory states that viruses predated the emergence of cells. At the opposite, the “reduction hypothesis” states that viruses evolved as reduced parasitic forms of early cellular organisms. The third one, “the escape hypothesis”, is a variation of the later stating that ancestral viral genomes were constituted of subsets of cellular genes that escaped cell control (reviewed in ).I never personally understood how the first hypothesis could even be proposed, since it is properly absurd if we respect the precise meaning, accepted by all, of the word "virus": an obligate intracellular parasite. This mere definition immediately implies that the first virus(es) had to emerge in the context of preexisting cell-like organisms . The first viruses— in the sense that we give it today—could not precede the emergence of their hosts. Furthermore, the ancestor of the first virus(es) could not be one itself, but had to be an  unknown free-living cell-like organism(s). From this point on, only some sort of reduction hypotheses should constitute the theoretical context on which to base the comparison of extant viral genomes and the reconstruction of their phylogeny.There is, however, a fundamental difficulty in reconstructing the evolutionary history of obligate intracellular parasites by comparing them without reference to the free-living organisms from which they originated. For tree-based phylogenetic approaches to deliver a sensible scenario, all protagonists of the evolutionary game must be included in the analysis. This difficulty is illustrated in Chlamydiae being a noticeable exception). In contrast to These conclusions are of course totally erroneous, as shown in Alphaparasites/Rickettsiales in Nucleocytoviricota, zero within kingdom Bamfordvirae) [In the context of the reduction hypothesis, the simulated analysis in rdvirae)   , by imprIf the loss of viral genes duplicating cytoplasmic functions  probably can happen quickly, in an almost random manner (the virus benefiting from a free lunch within the cell), the loss of the functional virus-encoded DNA replication or transcription machineries must be concomitantly compensated by an access to the cellular ones, in the host nucleus. The viruses must thus evolve a strategy to either transport their genomes to the intact nucleus ,65, or mTo illustrate two main points, a diagram of virus evolution through genome reduction from a cell-like (non-virus) ancestor is represented in Second, even when similar genotypes  are recognized in different viruses they cannot be used as reliable evidence of common ancestry as they could originate from totally unrelated evolutionary pathways from which intermediate viral forms have disappeared or are not yet discovered. In absence of such intermediates, polyphyletic viral clades might erroneously appear monophyletic . Such cases are illustrated by the genotypes indicated in red in In this conceptual article, I showed that the phylogenetic reconstruction of the evolution of viruses suffers from several fundamental limitations, in the hypothesis that they were derived from cell-like microorganisms, the only logically sound scenario if we respect the definition of viruses as “obligate intracellular parasites”. One limitation is due to the lack of free-living lineage(s) against which to compare the various virus families . The othIn this paper, I voluntarily neglected two additional confounding evolutionary processes: (i) the acquisition of genes by horizontal transfers from cells or other viruses, (ii) the de novo creation of genes by the viruses themselves . It is nI made no hypothesis on the very nature of the ancestral microorganism(s) at the origin of viruses, but it was in all likelihood equipped with a DNA transcription/replication machinery, and protein synthesis. I proposed that the loss of the later is the evolutionary event that initiated all dsDNA virus lineages, for which there is no evidence—or logical need—that it only happened once.Phycodnaviridae family into that of Mimiviridae [Viruses are traditionally classified into families on the basis of common infection and intracellular replication strategies, overall particle structures, and large, specifically shared gene contents attesting their descent from a common (quasi-extant) viral ancestor. Cladistics  is often a convenient and sufficient method to delineate families that should clearly appear as monophyletic clades . It is iviridae  (which civiridae ) is a goMimiviridae, Adenoviridae, Phaeovirus, and virophages , or the Herpesviridae and a large number of phages (now included in the Heunggongvirae kingdom). While I fear that this new taxonomical scheme will be taken as a word of the gospel by the incoming generation of virologists, I am also confident that the future discovery and characterization of many more unconventional viruses will quickly convince them that any attempt to lock viruses into such a deep and rigid classification does not make any biological sense.Having listed the fundamental limitations plaguing the deep phylogenetic reconstruction of viruses beyond the family level, I can only wonder about the merits of a spectacular fifteen-rank classification hierarchy recently adopted by the ICTV . Familie


We sequenced the genomes of our stock of HSV-1 strain 17syn+ and seven derived viruses and found nonsynonymous mutations in many viral proteins that had no impact on skin infection. In contrast, deletions in the LAT locus severely impaired HSV-1 replication and lesion formation in skin. However, skin replication was not affected by impaired intron splicing. Moreover, although the LAT locus has been implicated in regulating gene expression in neurones, we observed only small changes in transcript levels that were unrelated to the growth defect in skin, suggesting that its functions in skin may be different from those in neurones. Thus, although the LAT locus was previously thought to be dispensable for lytic infection, we show that it is a determinant of HSV-1 virulence during lytic infection of human skin.Herpes simplex virus 1 (HSV-1) infects skin and mucosal epithelial cells and then travels along axons to establish latency in the neurones of sensory ganglia. Although viral gene expression is restricted during latency, the latency-associated transcript (LAT) locus encodes many RNAs, including a 2 kb intron known as the hallmark of HSV-1 latency. Here, we studied HSV-1 infection and the role of the LAT locus in human skin xenografts  Herpes simplex virus type 1 (HSV-1) infects and destroys the outer layer of skin cells, producing lesions known as cold sores. Although these lesions heal, the virus persists in the host for the lifetime and can reactivate to cause new lesions. This is possible because the virus enters the axons of neurones in the skin and moves to their cell bodies located in spinal or cranial nerve bundles called ganglia, where the virus becomes dormant (latent). The most abundant viral RNAs expressed during this state are the latency associated transcripts (LATs), which have been considered a hallmark of HSV-1 latency. Here, we studied HSV-1 infection and spread in human skin. Unexpectedly, we found that the LAT locus is necessary for lesion formation in skin. HSV-1 viruses that were genetically mutated to delete the start of the locus could not spread in skin, whereas viruses with many other genetic mutations had this capacity. Our results suggest that an antiviral drug that inhibits transcripts from this region of the viral genome could block viral spread in skin, or a vaccine could possibly be produced by genetically modifying the virus at the LAT locus and by doing so, limit the virus’ ability become latent in neurones. Furthermore, many HSV-1 transcripts are expressed in a cell-type specific manner, and their functions may be related to the cells in which they are expressed  A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Paul D. Ling, Ph.D.Associate EditorPLOS PathogensBlossom DamaniaSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: Figures 3D-G are numbered correctly in figures and legends but incorrectly in the body of the Results.Please clarify that the significant results at 4dpi which become insignificant at 7 (?not 8) dpi are due to lack of residual keratinocytes destroyed by cytopathic spread of wt virus?Figure 3E adds little as the results are not significant for the reasons explained. Either further replicates are needed for even nonparametric statistics or the results deleted or presented as supplementary figures. The two other LATS mutants are sufficiently convincing.Reviewer #2: The revised manuscript, titled “The Latency-Associated Transcript Locus of Herpes Simplex Virus 1 is a Virulence Determinant in Human Skin”, reports that HSV-1 viruses mutated to interfere with viral transcription in the LAT locus can’t spread in human skin xenografts and cultured skin explants. The observation is interesting and potentially significant in the HSV-1 pathogenesis. The careful analysis and comprehensive comparison on wide-type and mutant viral genomes is to be praised in the detailed characterization of virus phenotypes.Prior to this study, LAT has not been implied in the lytic infection of HSV life cycle. The authors showed that deletions of the upstream of LAT promoter and 5’ LAT region, as depicted in the strain 17ΔN/H, severely impaired HSV replication and lesion formation in skin. However, this defect in virus spread was caused neither by the defect in 2kb LAT production, which is the hall mark of HSV latency, nor by many nonsynonymous mutations found in the viral coding regions. Based on detailed mapping on the transcripts produced in the LAT locus, it seems like the 0.7 kb transcript upstream of LAT promoter and/or the 5’ exon sequences in the mLAT could be responsible for the observed phenomenon. The author added KOSΔPST mutant and confirmed the defective phenotype. But the mutation in ΔPST overlaps with 0.7kb transcript, so as 17ΔN/H. Since the 0.7kb transcript has been suggested in the virulence of HSV-1 17syn+, further evaluation using mutants separating in the two regions might be needed in providing clarity on the role of LAT in HSV-1 skin pathogenesis.Reviewer #3: The authors have made significant improvements to this paper, which now provides compelling evidence that the HSV-1 latency associated transcripts act as a virulence determinant in human skin. The inclusion of additional LAT mutant viruses and, where available, revertant viruses strengthens their message. The inclusion of full sequence data for the mutant viruses will also act as a wake-up call to research groups working in this complex field. This is an important body of work that adds important new information concerning the role of LATs in pathogenesis outside of known functions in neuronal cells.The authors should carefully review Figure 3, and the text describing this Figure in the results - it appears that some panels are miss-labelled/missing.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: (No Response)Reviewer #2: (No Response)Reviewer #3: (No Response)**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: (No Response)Reviewer #2: There is mixed use of LATs with LAT locus in the Author Summary, starting on line 44. Since the LAT splicing mutant have no effect in skin virulence, plus it is unknown whether other transcripts in the LAT promoter region express in the latency, it is more precise to use LAT locus than LATs, to avoid misleading.Fig. 3C, Fig. 3D, 3E, 3F, 3G, seem shifted, the text and figure don’t match. There is one panel missing in Fig. 3.Line 357-359 needs modification. Fig. 1 and 2C didn’t verify ICP0 retained its ubiquitin ligase function. Fig.2C only showed ICP0 expression.Line 411-412 needs modification. Three references  only used 17 and KOS strain in cell culture. No mention on F strain.Reviewer #3: The authors should carefully review Figure 3, and the text describing this Figure in the results - it appears that some panels are miss-labelled/missing.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. 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For instructions see  3 Nov 2020Attachment2020-11-03 PPATHOGENSD2001788 response to the reviewers.docxSubmitted filename: Click here for additional data file. 12 Nov 2020Dear Dr Vanni,We are pleased to inform you that your manuscript 'The Latency-Associated Transcript Locus of Herpes Simplex Virus 1 is a Virulence Determinant in Human Skin' has been provisionally accepted for publication in PLOS Pathogens.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Paul D. Ling, Ph.D.Associate EditorPLOS PathogensBlossom DamaniaSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************************************************Reviewer Comments : 17 Dec 2020Dear Dr Vanni,We are delighted to inform you that your manuscript, "The Latency-Associated Transcript Locus of Herpes Simplex Virus 1 is a Virulence Determinant in Human Skin," has been formally accepted for publication in PLOS Pathogens.We have now passed your article onto the PLOS Production Department who will complete the rest of the pre-publication process. All authors will receive a confirmation email upon publication.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064