Orthopoxvirus genus. It is known to affect the neurological, respiratory, and gastrointestinal systems. The past few decades have seen endemic outbreaks of this viral infection due to the eradication of smallpox and subsequent laxity in vaccination efforts. Since it was initially diagnosed in 1970 in the Democratic Republic of Congo, it has spread to many countries worldwide, including the United States of America, becoming a disease of significant epidemiological importance. The most recent outbreak occurred in 2022. Although this viral disease is considered self-limiting, it poses serious public health concerns due to its complications and pandemic potential. This review will introduce a general overview of MPXV and describe the epidemiology, clinical features, evaluation, and treatment of monkeypox patients. It will also provide a means to raise awareness among primary and secondary healthcare providers. Furthermore, our review focuses on the most up-to-date clinical information for the effective management, prevention, and counselling of monkeypox patients worldwide.Monkeypox virus (MPXV) is an enveloped double-stranded DNA virus that results in a smallpox-like human disease. This causative organism belongs to the  Poxviridae family [Poxviridae is subdivided into two subfamilies: Chordopoxvirinae and Entomopoxvirinae. The Chordopoxvirinae subfamily is known to infect vertebrates, and it is further differentiated into 18 genera, including Avipoxvirus, Capripoxvirus, Cervidpoxvirus, Leporipoxvirus, Orthopoxvirus, Parapoxvirus, Suipoxvirus, and Yatapoxvirus. The Entomopoxvirinae subfamily infects invertebrates, and it is grouped into four genera  [Orthopoxvirus genus, including variola  and monkeypox. MPXV is a DNA virus, but its entire life cycle occurs in the cytoplasm of the infected cells [Monkeypox is a zoonotic disease caused by the monkeypox virus (MPXV), which belongs to the e family . Furthered cells .MPXV was first discovered during an outbreak among monkeys at a laboratory in Denmark in 1958 . HoweverThe first MPXV outbreak to occur outside of Africa was reported in the United States of America after infected African mammals were shipped from Ghana, transmitting MPXV to native American prairie dogs and spreading the disease to five states, totaling 47 reported human cases .EpidemiologyMacaca fascicularis), which had been transferred from Singapore for polio vaccine research to an animal facility in Copenhagen, Denmark [The clinical entity of human monkeypox was first identified in the DRC, formerly known as Zaire, in a town called Basankusu, Equateur Province, in 1970 . It was  Denmark . There a Denmark . However Denmark . In 2017 Denmark . Outbrea Denmark .EtiologyOrthopoxvirus, which also includes cowpox (CPX), variola virus (VARV), and vaccinia virus (VACV) [Orthopoxvirus genus, characterized by a 6379-bp terminal inverted repetition, which possesses a putative telomere resolution sequence and short tandem repeats [MPXV belongs to a family of double-stranded DNA viruses in the genus s (VACV) . It posss (VACV) . The MPX repeats . MPXV ca repeats ,13. With repeats .As mentioned earlier, two phylogenetic clades of MPXV have been described in the literature, the Central African (Congo Basin) clade and the West African clade, with differences in genomic structures responsible for the greater virulence exhibited by the Central African clade, resulting in more severe disease with higher case fatality rates. This significant virulence is due to its ability to inhibit T cell receptor-mediated T cell activation and prevention of the production of inflammatory cytokines such as interferon-gamma (IFN-γ) and tissue necrosis factor-alpha (TNF-α) by human cells ,14. In aTransmissionAnimal-human (zoonotic) transmission and human-human transmission are the two means of spreading the MPXV. Zoonotic transmission occurs through direct contact, bite or scratch from an infected animal, or consumption of an animal host, which is usually a rodent or a primate. Risk factors for zoonotic transmission of MPXV include living in forested or recently deforested areas, no smallpox vaccination, handling or eating dead bushmeat or monkeys, and sleeping on the floor (in endemic areas) . Human-tClinical featuresMonkeypox is a self-limiting disease with symptoms lasting two to four weeks with an incubation period of 8 days (4-14 days) . The iniDiagnosis [Monkeypox can be suspected in an individual with the aforementioned symptoms, especially if there is a history of contact or travel to areas endemic to monkeypox . A suspe .Testing AlgorithmPCR is the gold standard for diagnosis and should be done first. After a negative PCR test, if monkeypox infection is still suspected, other tests listed in Table ManagementAt the moment, there are no specific treatments approved for monkeypox. Fortunately, the clinical course of monkeypox infection is usually mild and self-limiting. Therefore, it seldom warrants specific therapy, and treatment is often supportive ,9. SuppoPreventionOrthopoxvirus infections, including monkeypox. When administered early in the incubation period, it can prevent disease onset or mitigate the severity of the illness. However, there is a risk of severe adverse effects in immunocompromised patients. The eradication of smallpox in 1980 led to the termination of vaccination efforts against the viral illness, leaving many people susceptible to monkeypox. Next-generation smallpox vaccines, which include ACAM2000 (live vaccinia virus), Modified Vaccinia Ankara (attenuated vaccinia virus), and LC16m8 (attenuated vaccinia virus), with attenuated strains not only offer an improved safety profile compared to first- and second-generation smallpox vaccines but also adequately stimulate antibody production in atopic and immunocompromised patients [Studies have shown that smallpox vaccination effectively prevents other Hospitalized patients should be under strict airborne isolation in a negative pressure room. Health personnel should wear appropriately fitted N95 masks, gloves, and eye protection before coming in contact with these patients until the lesions have crusted and scabs have fallen off .It is pertinent to note that many countries with confirmed monkeypox cases in the May 2022 outbreak are not endemic to monkeypox, and the cases had no travel links to endemic areas. Nevertheless, monkeypox's public health implications and pandemic potential must be studied extensively in light of the devastation caused by the COVID-19 pandemic. A better understanding of factors affecting the spread of this virus will aid in ensuring public health preparedness and developing strategies against future threats. The possibility of underreporting must also be considered as most cases are in rural Africa, countries plagued by poor health infrastructure and scarce resources.


We propose and analyze a new class of three dimensional space models that describes infectious diseases caused by viruses such as hepatitis B virus (HBV) and hepatitis C virus (HCV). This work constructs a Reaction–Diffusion-Ordinary Differential Equation model of virus dynamics, including absorption effect, cell proliferation, time delay, and a generalized incidence rate function. By constructing suitable Lyapunov functionals, we show that the model has threshold dynamics: if the basic reproduction number  Today, viral infection is linked to global health problems. Many diseases caused by viruses, such as hepatitis B virus (HBV), human immunodeficiency virus (HIV), hepatitis C virus (HCV), dengue virus, zika virus, and Sars-Cov-2 virus have drawn the attention of researchers to the viral infection process within a host. The mathematical modeling in a patient allows a better understanding of the transmission of diseases and thus improves the strategies for their eradication. On the basis of the virus infection models proposed in , 35–37,  where the density of uninfected cells is represented by H at position x at time t, the density of infected cells is represented by I at position x at time t and the density of free virus particles by V at position x at time t. The uninfected target cells are produced at a constant rate D is the diffusion coefficient.The basic reaction-diffusion-ODE viral infection dynamics model consists of the following three-dimensional system V. In this case, the incidence function : : : In the mathematical model  –4). For. For4). F is locally Lipschitz in For any data F is Lipschitz continuous on bounded subsets of f, there exists P and Z given in theorem F is Lipschitz continuous on bounded subsets of Let Now, IBVP –4) can  can 4) cLet For any For From the main results of the literature and the previous lemmas, we can state this following result.For any the IBVP –2) defi defi\docT(t) on For ary 4 of , we concentclass1pt{minimay 2.5 of  ensures In this section, we establish the boundedness in time of the global solution of the IBVP –4) for  for 4) fentclass1pt{minimaH, I, V) of problem (For any solution ( problem –4),\doc,\docH, IH, I, V) be a solution of problem . Let. LetH, Iple (see ), we infH, I and V are bounded on From the above discussion, we deduce that The aim of this section is to study the local and global stability of the uninfected equilibrium.It is easy to verify that system  always hand Zhao , we defiThe objective of this section is to prove the local stability of the spatially homogeneous uninfected equilibrium entclass1pt{minimaThe spatially homogeneous uninfected equilibrium entclass1pt{minimaLet Consider the following direct sumentclass1pt{minimaandi = 0, 1, 2 Id be the z and X is an eigenvalue if the matrix For each s system , hence \When mentclasspt{minimaNow, let us consider the distribution of the roots of  when \doAssume that mentclasspt{minimamentclasspt{minimaNow as equation  does notroots of  with resroots of  has all Therefore, if entclass1pt{minimai specifies the diffusion eigenvalue Now we consider the case lds that  has at lf is under the following hypothesis:We discuss about the global stability of the uninfected equilibrium entclass1pt{minima problem  when \doSuppose that entclass1pt{minima problem  is globaf is a decreasing function with respect to second and third variable,H, I, VG of Let entclass1pt{minima problem . Define entclass1pt{minima problem , where trinciple , Theorementclass1pt{minimaThe previous results confirm that the infection always dies out. These results strictly extend those of , 11, 32 f defined as follows:In this section, we present the numerical simulations done by using Matlab software to confirm the theoretical results that we established in the previous section for a particular case of the incidence function function  where \dThe aim of this section is: first to determine the existence and the uniqueness of the infected equilibrium for the system , then toAssume that entclass1pt{minima From \documenttituting  yields\dNow let’s take a look at uniqueness.If F is a strictly monotonic function allows us to conclude that the point It is sufficient to show that  the Eq.  as26\docing Eqs.  and 19)\document-mτ.From , we haveEquation , allows  Investigation of the existence and stability of such spatially-inhomogeneous equilibria will be the concern of a forthcoming work via an in-depth analysis of the above system.Indeed, any spatially-inhomogeneous equilibrium point entclass1pt{minimahe model  must solIn this section we deal with the local stability of the infected equilibrium LetNote that this proof is analogous to the proof of Proposition The characteristic equation of system  at the iIn our case we haveIf conditions H(1) are satisfied, H(1) satisfies, note that  From \documentstic Eq.  is equivrding to  and (31)rom Ruan .:H(1)), If roots of  have negIf roots of  have negAssume , I and V be any positive solution of the delayed Reaction–Diffusion-O.D.E model problem (g defined by Let  problem –4). Def. DefH, hepatitis C virus and the hepatitis B virus (HBV). The infection transmission process is modeled by a general incidence function which includes several forms existing in the literature. In addition, the global analysis of the proposed model is rigorously investigated. Furthermore, biological findings of our analytical results are presented. Moreover, mathematical virus models and results presented in many previous studies are extended and generalized. To study the mechanism of viral infection and replication, we performed the mathematical analysis of a dynamic model of diffusive in-host virus with a general non-linear incidence function. The well-posedness and the stability of the equilibria of this model are examined. The basic reproductive number ented in .


Haloferax are interesting models, as a reliable genetic system and versatile microscopy methods are available. However, only one virus infecting the Haloferax species is currently available. In this study, we tested ~100 haloarchaeal virus isolates for their infectivity on 14 Haloferax strains. From this, we identified 10 virus isolates in total capable of infecting Haloferax strains, which represented myovirus or siphovirus morphotypes. Surprisingly, the only susceptible strain of all 14 tested was Haloferax gibbonsii LR2-5, which serves as an auspicious host for all of these 10 viruses. By applying comparative genomics, we shed light on factors determining the host range of haloarchaeal viruses on Haloferax. We anticipate our study to be a starting point in the study of haloarchaeal virus–host interactions.Viruses can infect members of all three domains of life. However, little is known about viruses infecting archaea and the mechanisms that determine their host interactions are poorly understood. Investigations of molecular mechanisms of viral infection rely on genetically accessible virus–host model systems. Euryarchaea belonging to the genus  Caudoviricetes [Sphaerolipoviridae) [Pleolipoviridae) [Halspiviridae) [Hafunaviridae is the largest family of archaeal tailed viruses and its myoviruses have a broad host range [Microbial viruses are widespread and able to infect members of all three domains of life, including archaea. Archaea are ubiquitous microorganisms that can be found in extreme environments, such as salt lakes as well as in mesophilic surroundings such as the oceans and the human body ,2. The siricetes ,10,11,12viridae) , pleomorviridae) , or spinviridae) . Archaeaviridae) . All isoviridae) . Howeverviridae) . The strviridae) . While tviridae) . Previoust range . Other hst range .Sulfolobales are currently the most common archaeal models with genetic systems available [Halorubrum and Haloarcula strains that are infected by a substantial number of known viruses [Haloferax has become increasingly popular in the archaeal scientific community, and it is the euryarchaeal model for which the most advanced tools for genetic engineering, imaging, and molecular biology are available [Haloferax tools entail a versatile genetic system for overexpression and genomic knock-out, the availability of several plasmids and different markers, and a CRISPR-based repression system to downregulate gene expression [Haloferax as a central euryarchaeal model, has led to a substantial increase in the understanding of its cell biology [Haloferax as a model, is of great added value in studies of viral infection mechanisms.The study of the virus–host relationships and infection mechanisms of archaeal viruses would greatly benefit from the availability of genetically accessible virus–host models, for which molecular biology tools are available. For crenarchaeal virus hosts, several members of the genus vailable ,22,23,24vailable ,26,27,28 viruses ,19,29. T viruses ,32,33,34vailable . The Halpression ,37,38,39pression . It is apression . The dev biology ,54,55,56Haloferax strains. Almost 30 years ago, the HF1 virus was reported to infect Haloferax lucentense and Hfx. volcanii, and a defective provirus of Haloferax mediterranei has been identified, both of which are no longer available  [Haloferax host, and it was recently isolated together with its host Haloferax gibbonsii LR2-5 from the hypersaline Lake Retba in Senegal [In contrast to some other haloarchaea, there are far less viruses known that infect ication) ,58. At t Senegal ,60.Haloferax for molecular studies, we aim to identify viruses that infect Haloferax strains. In this study, we used the largest available collection of isolated and characterized haloarchaeal virus isolates, and we tested the infectivity of 95 viruses on 14 Haloferax strains. The virus collection contains viruses from all current haloarchaeal virus families, and these viruses represent the majority of haloarchaeal virus isolates isolated to date. This endeavor resulted in an extended virus–host matrix for Haloferax from which we could identify a promising model that is the host to a substantial number of viruses, Haloferax gibbonsii LR2-5. In addition, we used comparative genomics to identify viral factors that allow for the infection of this Haloferax host. With this work, we pave the way to using Haloferax as a model in virus–host interactions.Due to the attractiveness of Haloferax strains  was diluted to obtain 18, 20, or 23% SW in the top-agar media, plates, and liquid media, respectively. MGM also contained 5 g of peptone (Oxoid), 1 g of Bacto yeast extract , and Bacto agar  per liter. Viruses were grown on their own host strain listed in All  strains , virus h strains , and vir strains  were groHaloferax strains , and for more complex motifs a manual search was carried out in Geneious v 8.1.9..The genomic sequences of the viruses were retrieved from NCBI . PhylogeThe phylogenetic analyses of the adhesin and tail fiber gene sequences were carried out as follows. For both datasets, sequences were aligned using MAFFT v. 7.450 , and a mHaloferax, a collection of 95 haloarchaeal viruses  or Retbasiphovirus   . It is idetected .Haloferax host, which is an indication of the efficiency of infection. As a summary, we did not find any true virus–host pairs on any of the Haloferax strains, except on strain Haloferax gibbonsii LR2-5, which was isolated a few years ago from Lake Retba in Senegal at the same time as HFTV1 [Halorubrum or Haloarcula as their own host and their titers varied between 103 and 1010 PFU/mL on LR2-5  system . These aOne strategy of viruses to escape host recognition is the avoidance of certain motifs in their genomes, which are the targets for the RM systems . In lineMethylated motifs in the genome of LR2-5 were previously predicted . We searHafunaviridae family members showed the same genome organization within their own group   . There wviruses) . Many ofCaudoviricetes [Another highly variable region in the viral genomes is located around genes encoding the viral tail fiber and adhesin . Adhesiniricetes . Long tairicetes ,80. Moreiricetes . All haliricetes . HFTV1 iHaloarcula, Halobacterium, Halobellus, Halorubrum, and Haloterrigena strains [A tree of the hafunaviruses  tail fiber adhesin proteins showed that they are divided in four clades , as also strains . We aimeWe investigated the hypothetical relationship between the susceptibility of the LR2-5 strain to infection and diversification of the hafunavirus tail fiber and adhesin genes by comparing the number of Markov jumps  through the adhesin and the tail fiber gene evolution . A “nullHfx. gibbonsii LR2-5. However, it should be emphasized that our dataset was limited to Haloferax hosts. Such a correlation between host range and adhesin type might only become apparent when a more diverse set of hosts is used, as the Haloferax strains are generally not very susceptible for viral infection. However, we did find a few case examples, which seems to pinpoint the adhesin as a host determining factor. First, HRTV-26, one of the few haloferacalesviruses that infects LR2-5, encodes a Clade 1 adhesin, similar to most mincapviruses. Since the adhesins of haloferacalesviruses that are closely related to HRTV-26 all belong to Clade 3, it is likely that HRTV-26 picked up an adhesin gene via horizontal gene transfer from one of the Clade 1 viruses. This might be the explanation for why HRTV-26, even though it is an haloferacalesvirus, is still capable of infecting LR2-5.Based on this analysis, we did not observe any clear correlation between the type of adhesin or tail fiber gene and the viral ability to infect Second, viruses encoding a Clade 3 adhesin do not infect LR2-5, with the exception of HRTV-10. To gain more insight into the HRTV-10 tail fiber adhesin, a multiple sequence alignment was performed. We identified one mutated codon resulting in an amino acid change (T380A) in a conserved motif at the 3′end of the adhesin gene . This suHaloferax strains, as the most advanced molecular and genetic tools are available for these organisms. Due to the low number of known viruses that infect Haloferax, we set up a large-scale assay to identify viruses capable of infecting Haloferax strains. Our extensive screening of approximately 100 isolated and mostly characterized haloarchaeal viruses showed that only a small subset was capable of infecting Haloferax. Specifically, Hfx. gibbonsii LR2-5 showed itself as an auspicious host, as we identified 10 virus isolates that can infect this host and all other hosts could not be infected. Previously, we studied potential factors that make Hfx. gibbonsii LR2-5 (DSM No. 112399) so much more receptive to virus infection in contrast to related strains. For its virus susceptibility, several possible factors were identified as potential explanations. The most prominent was an absence of CRISPR-cas virus defense systems in LR2-5 [Hfx. gibbonsii LR2-5 has a different RM system from some other Haloferax strains, and the cell surface might show a different composition than that of other Haloferax strains because LR2-5 encodes divergent surface proteins that might serve as viral receptors, such as pili and S-layer proteins [We aimed to develop an euryarchaeal virus–host model system with attractive molecular, genetic, and imaging tools to dive deeper into virus–host relationships in archaea. We focused on in LR2-5 . In addiproteins . These lproteins ,82.Hfx. gibbonsii LR2-5 and transferred to a lysogenic cycle. Consequently, these types of interactions would not show clear plaques, and they could have been missed by our approach. However, further experimental research on the host range implications of these proteins is needed.Because we used plaque assays for the host range determination, we cannot distinguish at which step of the infection cycle viruses are not successful in infecting a host: entry, replication, or release. However, the results of the present study might indicate that adhesins, which are required for entry, are an important factor determining host range specificity, whereas other factors such as the viral egress proteins and restriction–modification systems might also play a role. Moreover, it should be noted that one limitation of the approach utilized is the assumption that the successful infection of the host results in plaque formation. It could be that some of the viruses studied here might have integrated their genome when infecting Hfx. gibbonsii LR2-5 as the only virus susceptible Haloferax strain. This host can be infected by 10 different viruses with sequenced genomes [Hfx. gibbonsii LR2-5. We conclude that this species is an extremely promising model host to study virus–host interactions in haloarchaea, and we anticipate that this work serves as a stepping stone for future in-depth molecular characterization of archaeal viral infection mechanisms.Our analysis has identified  genomes , and it  genomes . We are


Winter oilseed rape accounts for about 90% of total oilseed rape planting area in China, with the majority of it concentrated in the Yangtze River basin. The remaining 10% is mostly found in the provinces of China’s northwest plateau. Winter oilseed rape areas in China have gradually expanded to the north in the last decade, resulting in cabbage aphids and turnip mosaic virus (TuMV). The aphid is a vector for TuMV and is gradually increasing on winter and spring oilseed rape. Quantifying the probing behaviors of the aphids on spring oilseed rape and winter oilseed rape helps us to understand TuMV regulation of the aphids. We found that compared to mock-inoculated plants, cabbage aphids on infected plants increased brief probing frequency, cell penetration frequency, intracellular probing time, decreased time to first probe and pathway duration, potentially promoting viral acquisition and minimizing viral loss and plant damage. Viruliferous aphids had reduced pathway duration, increased cell penetration frequency, increased intracellular probing time, increased salivation frequency, and ingested less sap compared with non-viruliferous aphids, primed for viral infection. TuMV infection also differentially modified aphid feeding behavior on winter and spring oilseed rape cultivars, primarily on uninfected plants.Direct and indirect effects of plant virus infection on vector behavior have been discovered to improve virus transmission efficiency, but the impact of plant cultivars in virus–vector–plant interactions has received little attention. Electropenetrography (EPG) allows real-time tracking and quantification of stylet penetration behaviors, pathogen transmission, and plant resistance mechanisms. Quantitative probing behaviors on a spring oilseed rape cultivar, ‘Xinyou17’, and a winter oilseed rape cultivar, ‘Zheping4’, were investigated using EPG to compare turnip mosaic virus (TuMV) regulation of cabbage aphid probing behavior. Results for indirect effects showed that compared to mock-inoculated plants, cabbage aphids on infected plants increased brief probing frequency, cell penetration frequency, intracellular probing time, and decreased time to first probe and pathway time, potentially promoting viral acquisition. TuMV also directly influences aphid probing behavior. Viruliferous aphids had reduced pathway time, increased cell penetration frequency, increased intracellular probing time, increased salivation frequency, and ingested less sap than non-viruliferous aphids, primed for viral infection. Although oilseed rape cultivars can also influence aphid behavior, the main effect of cultivars was not significant on TuMV-infected plants. Plant virus infection changes both plant characteristics and vector (often insect) behavior, the changes of which in turn affect virus transmission mode and efficiency . AccordiBemisia tabaci Gennadius (Hemiptera: Aleyrodidae) and resulting in B. tabaci having better-balanced nutrient absorption, higher survival and oviposition rates, and a faster population growth rate than it does on healthy tobacco , virR. padi L. [Non-viruliferous vectors tend to feed on infected plants, whereas viruliferous vectors, such as BYDV-vectoring  padi L. ,53, tend padi L. . The preM. persicae, for example, is less susceptible to host VOC than non-viruliferous M. persicae [According to Ingwell et al. (2012)  and Medipersicae . SimilarReduced pathway duration and increased short probes and intracellular punctures have a positive link with the acquisition and subsequent inoculation of non-persistent viruses because aphids must release viruses to live cells to ensure the survival of viral particles ,57 such B. tabaci vectoring TYLCCNV on cotton was only approximately one-third that of non-viruliferous whiteflies, and that the virus had a negative impact on whitefly feeding behavior, but the negative impact could aid viral transmission. Wan et al. (2015) [L. striatellus males compared to those who were not infected. Our results also showed that viruliferous aphids increased salivation frequency and decreased ingestion when compared to non-viruliferous aphids. As a result, TuMV infection may change directly vector preference for probing but not favor feeding. Meanwhile, viruliferous aphids significantly secreted more saliva than non-viruliferous aphids on ‘Xinyou17’ but not on ‘Zheping4’, implying that the direct modification for aphid feeding behavior in ‘Xinyou17’ may be more obvious than in ‘Zheping4’. TuMV compelled aphids to secrete more saliva and ingest less sap in order to improve transmission efficiency and reduce damage caused by aphid ingestion because the aphid-susceptible phloem of ‘Xinyou17’ is suitable for aphid feeding.Short- and long-ingestion probes are known to be damaging to plant tissues and thus unlikely to aid virus transmission ,59. He e. (2015)  discoverBased on the results of the two-way ANOVA , TuMV inM. persicae on potato leafroll virus (PLRV)-infected and sham-inoculated plants. The effect of PLRV infection on settling behavior was significant for two of the cultivars, Desiree and Russet Burbank, whereas the difference was slight and non-significant for another two of the cultivars, Chipeta and IdaRose. This implied that the effect of cultivars as well as virus infection status on vector behavior is different in different vector–virus–plant systems. In our study, only two oilseed rape cultivars were selected, a spring rape cultivar ‘Xinyou17’ and a winter rape cultivar ‘Zheping4’, on which non-significant effects of cultivars on some variables were found   reportedre found , suggestVirus-infected plants attract vector insects, and after acquiring viruses, vectors prefer healthy plants, resulting in virus transmission. As a result, viruses clearly influence vector probing behavior . Our fin


To the editor:The SARS-CoV-2 virus is responsible for causing the current global COVID-19 pandemic, which has lasted for over 2 years now. Since its first outbreak at the end of the year 2019, the SARS-CoV-2 virus has undergone multiple mutations, from the Alpha variant to the Omicron variant, which is currently the most prevalent one in the world. With each mutation, the virus tends to become further infectious, raising health-related concerns. Each mutation leads to a shorter infection time and increased occult. According to the global statistics, the number of people that have been infected with the SARS-CoV-2 virus to date is approximately 500 million, and the number continues to increase sharply to several millions of infections as time passes [Previous studies have confirmed that ACE2 is the “key” factor involved in the process of entry of SARS-CoV-2 into the host cells. In the initial step of viral infection, the virus binds to the ACE2 receptor via the RBD domain in the viral spike protein . This stLubin et al. reported that the RBD domain in the Omicron variant is highly flexible and unstable  and formPerhaps the only thing to be thankful for regarding these mutations is that the clinical manifestations of the Omicron variant are much less severe compared to the Delta variant. According to a study conducted in the USA on the Omicron infection in children under the age of 5 years, the emergence of the Omicron variant decreased the number of emergency visits by 29% compared to the Delta variant, while the number of hospitalizations decreased by 67% and the number of hospitalizations involving admission into the ICU decreased by 68% .The virus has been evading the resistance of the vaccine through continuous mutation, thereby troubling scholars as to how to prevent the virus from invading the human body. The development of specific drugs against the COVID-19 virus continues to be one of the most urgent problems to be resolved. Currently, Paxlovid, a small molecule drug developed by Pfizer, is exhibiting excellent therapeutic effect on COVID-19 patients. Paxlovid reduces the risk of hospitalization or death related to COVID-19 disease by 89% . PaxloviIn a previous article reported by our research group, the essential role of soluble ACE2 or ACE2-specific small molecular peptides targeting ACE2 in preventing the virus from entering the human body was summarized, thereby suggesting ACE2 as the potential target for the development of specific drugs against the COVID-19 virus . Althoug


Ilarvirus. Homologous antiserum was effective in the detection of the virus in plant extracts and no cross reactions with two other distinct members of subgroup 2 were observed. Overall, the biological features, phylogenetic relationships and serological data suggest that this virus is a new member of the genus, for which we propose the name hydrangea vein banding virus (HdVBV).In this study, a new virus was identified in French hydrangea plants, exhibiting chlorotic vein banding and necrotic ring spots on older leaves. The virus was mechanically transmitted to herbaceous hosts, in which it induced local and systemic or only local symptoms. The genome of the new virus was characterized and consisted of three RNA sequences that were 3422 (RNA 1), 2905 (RNA 2) and 2299 (RNA 3) nucleotides long, with five predicted open reading frames; RNA2 was bicistronic and contained conserved domains and motifs typical of ilarviruses. The phylogenetic analysis of the predicted proteins—p1, p2a, p3a and p3b—revealed its close relationship to recognized members of subgroup 2 within the genus  Hydrangea L. (family Hydrangeaceae Dumort) encompasses 80 species that are native to a wide range of regions and countries, including Japan, Asia, Indonesia, the Himalayan mountains and the Americas [Hydrangea macrophylla, also known as French hydrangea or bigleaf hydrangea and here referred to simply as hydrangea, is the most popular cultivated species, with over 1000 recognized cultivars and varieties with various shapes, sizes and bloom colors that are grown in both temperate and sub-tropical climates [The genus Americas . Of thesclimates .Viral diseases may have a significant impact on the appearance, health and market value of hydrangea, especially because it is propagated vegetatively and viruses are easily transmitted and accumulated over time through cuttings, causing a decline in the crop. Moreover, in recent years, emerging viral diseases in hydrangea with the capacity to cause more serious yield losses have been described; thus, in addition to phytoplasma diseases, they are considered a serious developing threat to hydrangea . In totaSince the spring of 2016, severe symptoms of leaf deformation, chlorotic vein banding and a mosaic of light and dark green colors A have beIlarvirus, which was provisionally designated as ‘hydrangea vein banding virus‘ (HdVBV), based on the main symptom observed on the leaves. Due to the molecular features of HdVBV and its phylogenetic relationships with the other members of the genus, it was assigned to subgroup 2, along with asparagus virus 2 (AV2), citrus leaf rugose virus (CLRV), citrus variegation virus (CVV), elm mottle virus (EMV), lilac ring mottle virus (LRMV), spinach latent virus (SpLV), Tulare apple mosaic virus (TAMV) and tomato necrotic spot virus (ToNSV) [Here, we will demonstrate that the disease observed in the hydrangea was caused by a novel  (ToNSV) .Chenopodium amaranticolor, symptoms included pinpoint chlorotic local lesions . No further symptoms were recorded in the other inoculated plants within 60 dpi, and no symptoms were induced in back-inoculated C. amaranticolor plants.The symptoms observed on infected hydrangea plants varied slightly depending on the time of observation. In the springtime, we observed light chlorotic vein banding on the youngest leaves A that be lesions A that apat 7 dpi B,C, foll tabacum D; light lutinosa E; mosaicthamiana F; and liOf the three plants grafted with buds taken from a symptomatic plant, only one reproduced similar foliar symptoms to those observed on the symptomatic donor plant (not shown). The symptomatic leaves of this plant produced positive results in RT-PCR with degenerate primers for ilarviruses, and the subsequent sequencing of the amplicon showed 100% nucleotide identity with the sequence of the new ilarvirus, indicating that Koch’s postulates were met.The complete genomic sequence of HbVBV, obtained from genome walking and RACE-PCR, consisted of three ssRNA segments of 3422 nucleotides (nt) (RNA 1), 2905 nt (RNA 2) and 2299 nt (RNA 3) in length ; the seqRNA1 contains a single ORF with the ATG codon at position 73 and ending with a TAG codon at position 3297; it encodes a 121 kDa protein (p1) consisting of 1074 amino acids (AAs). The predicted protein has two conserved domains: a methyltransferase domain between residues 59 and 428 and a helicase domain between residues 769 and 1020. The protein shares 71–91% AA identity and 80–95% similarity with ilarviruses belonging to subgroup 2 .RNA2 is bicistronic with the two ORFs overlapping by 274 nt. The first ORF at the 5′ end of RNA2 encodes for a 788 AA protein (p2a) with a predicted molecular mass of 90 kD, whereas the second ORF encodes a 189 AA protein (p2b) with a predicted molecular mass of 21 kDa. The p2a protein has signature RNA-dependent RNA polymerase (RdRp) motifs between residues 225 and 666 [Bromovirus movement protein family [RNA3 contains two ORFs: the ORF 3a encodes a 281 AA protein (p3a) with an estimated molecular mass of 31 kD and has a signature motif between residues 5 and 244 that identifies the n family . The p3an family . The p3bn family .Ilarvirus. These results were confirmed by a high bootstrap value in all instances .Here, we reported the characterization of a new quasi-spherical virus—including its complete nucleotide sequence—isolated from an Ilarvirus; however, no thresholds have been defined. Although the host range study involved a limited number of inoculated hosts, some differences were observed in the host range of HdVBV compared to that reported for other ilarviruses from subgroup 2  found between HbVBV and CVV . The varIlarvirus, along with asparagus virus 2 (AV-2), citrus leaf rugose virus (CLRV), citrus variegation virus (CVV), elm mottle virus (EMV), lilac ring mottle virus (LRMV), spinach latent virus (SpLV), Tulare apple mosaic virus (TAMV) and tomato necrotic spot virus (ToNSV) [According to the International Committee on Taxonomy of Viruses , the spe (ToNSV) . TherefoInterestingly, a BLAST analysis of HdVBV RNA3 identified a sequence  in the database showing 97% identity with the query sequence. This sequence is annotated as “Surrounding non-legume associated ilarvirus isolate Kreis” and was identified in Germany in 2016 from an unknown weed. This correspondence further confirms: (i) the new identity of the virus studied here, (ii) that the two sequences represent two isolates of the same viral species, and (iii) that this virus is also present in Germany and perhaps in other countries as well.Potexvirus, family Alphaflexiviridae), tomato ringspot virus , tobacco ring spot virus , cherry leaf roll virus , tomato spotted wilt virus  and hydrangea chlorotic mottle virus . Moreover, some bacterial and fungal infections can mimic the symptoms of ring spot virus on hydrangea [As shown in ydrangea ,17. To aHydrangea macrophylla plants of an unknown variety showing the symptoms described above were identified in a private garden in the municipality of Napoli in the Campania region of southern Italy. One symptomatic plant, referred to as HM1, was removed along with its root system and established in a pot under greenhouse conditions at the Institute for Sustainable Plant Protection of the National Research Council (IPSP-CNR), secondary section of Portici , where symptoms were recorded over the years.2HPO4 buffer containing 0.2% sodium diethyldithiocarbamate. Before inoculation, 75 mg mL−1 of carborundum and activated charcoal were added to the sap extract [Nicotiana tabacum ‘Xanthi nc’, N. glutinosa, N. rustica, N. benthamiana, Chenopodium amaranticolor and Phaseolus vulgaris. Five plants of each species/cultivar were inoculated and kept in an insect-proof greenhouse under natural light with the temperature controlled at 20–22 °C; the plants were checked daily for the appearance of symptoms during a period of one month. Asymptomatic inoculated plants were back-inoculated to C. amaranticolor to ascertain the presence or absence of latent infections.Virus transmission was performed by grinding 1 g of young tobacco leaves in 4 mL of 0.03 M Na extract . The sluThree buds from the symptomatic hydrangea plant were grafted onto three independent healthy hydrangea plantlets to confirm the infectivity in the original host.Symptomatic leaves harvested from the HM1 plant were further used to obtain a partial purified preparation of the virus as previously described in , with mi2H/KH2PO4 buffer (pH 7.0), 2% (w/v) polyvinyl pyrrolidone  and 0.2% (w/v) Na2SO3, were added. The leaves were gently cut into small pieces with a razor blade in the buffer and the plant sap was used to make adsorbed preparations. Carbon-coated grids of 400 mesh were incubated for 5 min with a drop of the homogenates prepared from virus-infected plant samples or from partially purified virus preparations and subsequently washed with a gentle stream of approximately 20 drops of deionized water (Milli Q) to remove buffer salts. For negative staining, 3–5 drops of a 1% uranyl acetate solution in sterile deionized water were applied. The grids were then dried by gentle tapping on a piece of Whatman paper and examined with electron microscope [Partially purified virus preparations and plant sap from symptomatic leaves were used for dip assays (dip method). A small piece of symptomatic tissue was placed on a glass slide and a few drops of sample buffer, composed of 0.1 M Nacroscope .CATATGCAACATGTCTGGTAATG-3′) and 3′ reverse (5′-CTCGAGGTCAATCCTCAACAACCAAG-3′) primers were synthesized and used to amplify the CP gene of the RNA extracted from the partially purified virus and infected plants via reverse transcription-polymerase chain reaction (RT-PCR). These primers resulted in the amplification of the complete CP open reading frame (ORF) and the incorporation of NdeI and XhoI restriction sites into the amplicon upstream and downstream from the CP gene, respectively. The purified PCR product was cloned into the plasmid pGEMT  and sequenced. The target fragment was released from the recombinant plasmid by NdeI and XhoI digestion and inserted into the corresponding sites of the pET-30a(+) plasmid . This procedure resulted in a His-tag incorporated at the carboxy terminus of the CP.Two oligonucleotide primers were designed according to the nucleotide sequence of the putative coat protein . The forward (5′-Escherichia coli BL21 (DE3) pLysS cells transformed with pET-30a-CP were inoculated into 3 mL of LB medium containing 100 µg/mL kanamycin and cultured at 37 °C until the OD600 reached 0.25–0.3, after which shaking was continued for an additional 30 min at 28 °C. Protein expression was then induced with 0.4 mM isopropyl-ß-d-thiogalactopyranoside (IPTG); the cell suspension was maintained for 12 h at 20 °C to increase the proportion of soluble proteins that could be recovered for affinity chromatography. The induced bacterial cells were harvested, and the pellet was resuspended in 25 mL of LB , 1 mM imidazole and 5 mM 2-mercaptoethanol), and 1 mL of 10 mM lysozyme, 50 µL of 20 mM PMSF and 20 µg of leupeptin were subsequently added to the bacterial suspension. Next, the mixture was shaken at 50× g for 20 min; then, 250 µL of 8% sodium deoxycholate (NaDC) was added to the suspension before shaking on ice for another 20 min. The bacterial suspension was then sonicated, and the mixture was clarified by centrifugation at 15,500× g for 30 min at 4 °C. The supernatant was then loaded on a 3 mL Ni-NTA affinity column  and washed three times with a total of 10 mL LB. Residual proteins were then removed by washing with 50 mL of WB , 10 mM imidazole, 5 mM 2-mercaptoethanol) and EB1 , 50 mM imidazole and 5 mM 2-mercaptoethanol), and the washing fractions were discarded. The His-tagged CP was then purified using EB2 , 200 mM imidazole, 5 mM 2-mercaptoethanol). The eluted protein was concentrated by centrifugation onto an Amicon® Ultra-PL30 filter unit, following the manufacturer’s instructions, and dialyzed against 0.85% NaCl overnight. The protein concentration was then determined by protein standard comparison in 12.5% SDS-polyacrylamide gel and stored at −80 °C.The expression and purification of the proteins was performed according to the method described in . BrieflyOne milligram of purified CP His-tagged protein was emulsified with Freund’s incomplete adjuvant for the immunization of a rabbit by intradermal and intramuscular injections at Bio-Fab research . One week after the first injection, the rabbit was further immunized with seven ear vein injections of His-tagged CP (100 µg per injection) at daily intervals. The blood was collected from the immunized rabbit 2 weeks after the final injection and the antiserum was decanted after clotting of the red blood cells.w/v). The samples were then separated using 12.5% SDS-PAGE [TM-C nitrocellulose membranes  using a Mini Trans-Blot  at 200 mA for 1 h at 4 °C. The membranes were blocked overnight at 4 °C in PBS-Tween 20 (PBS-T) containing 5% skimmed milk powder (blocking solution). The crude polyclonal CP antiserum was then added to the blocking solution  and the incubation was continued for 1 h at 37 °C. After three washes in PBS-T, the membranes were further treated for 1 h with protein A-alkaline phosphatase conjugate  at 37 °C. After another round of three washes with PBS-T, the membranes were incubated for 20 min with freshly prepared substrate solution .To assess the presence of the new ilarvirus by Western blotting (WB), symptomatic leaf samples were homogenized in liquid nitrogen and the proteins were extracted in 2 × SDS protein buffer  at a ratio of 1:2  and asparagus virus 2 , while the healthy and infected hydrangea plants were also tested in WB with a polyclonal antiserum raised against the same tomato isolate of SpLV, kindly provided by Dr. Piero Roggero  [The antiserum produced against HdVBV was tested in WB against a tomato isolate of spinach latent virus  . The res® Plant RNA Kit  using symptomatic H. macrophylla and C. quinoa leaves, following the manufacturer’s instructions. RNA extracted from the leaves of commercial and certified healthy H. macrophylla plants was used as negative control in all molecular assays. The presence of viruses in the samples was ascertained via reverse transcription (RT)-PCR using different sets of specific or degenerate primers for the detection of the following virus taxa: ilarviruses [TM Reverse Transcription System, Promega, Madison, WI, USA) with the following conditions: 1 cycle of 42 °C for 90 min followed by 35 cycles of 94 °C for 45 s, 52 °C for 1 min and 72 °C for 2 min, and, finally, 1 cycle of 72 °C for 10 min. Amplicons of the expected sizes were obtained using RT-PCR with most of the primer sets and were directly sequenced at Microsynth Seqlab GmbH  in both directions.Total RNA was extracted with the E.Z.N.A.rviruses , tospovirviruses , carlavirviruses , como/nerviruses , closterrviruses , flexivirviruses , tymo/marviruses , potyvirrviruses , cucumberviruses  and alfarviruses . PCR prorviruses . A BLASTBased on the nucleotide sequences that were obtained, specific primers  were redhttp://pfam.xfam.org/, accessed on 12 January 2022) [To determine the relationship between HdVBV and other ilarviruses, phylogenetic trees were constructed for each of the viral protein ORFs using representative sequences of all member species recognized by the International Committee on Taxonomy of Viruses . Multiplry 2022) . The homry 2022) , adoptin


Recilia dorsalis as a mediator of paternal transmission of the reovirus Rice gall dwarf virus (RGDV) and a previously undescribed symbiotic virus of the Virgaviridae family, Recilia dorsalis filamentous virus (RdFV). We show that HongrES1 mediates the direct binding of virions to leafhopper sperm surfaces and subsequent paternal transmission via interaction with both viral capsid proteins. Direct interaction of viral capsid proteins mediates simultaneously invasion of two viruses into male reproductive organs. Moreover, arbovirus activates HongrES1 expression to suppress the conversion of prophenoloxidase to active phenoloxidase, potentially producing a mild antiviral melanization defense. Paternal virus transmission scarcely affects offspring fitness. These findings provide insights into how different viruses cooperatively hijack insect sperm-specific proteins for paternal transmission without disturbing sperm functions.Arboviruses and symbiotic viruses can be paternally transmitted by male insects to their offspring for long-term viral persistence in nature, but the mechanism remains largely unknown. Here, we identify the sperm-specific serpin protein HongrES1 of leafhopper  Recilia dorsalis as a mediator of paternal transmission of the reovirus rice gall dwarf virus and of a previously undescribed symbiotic virus of the Virgaviridae family, Recilia dorsalis filamentous virus, via direct binding of virions to leafhopper sperm surfaces and subsequent paternal transmission via interaction with both viral capsid proteins.Arboviruses and symbiotic viruses can be paternally transmitted by male insects to their offspring, but the mechanism remains largely unknown. Here, the authors identify the sperm-specific serpin protein HongrES1 of the leafhopper  Insect symbiotic viruses are generally considered to be maternally or paternally transmitted to host offspring4. For example, Sigma virus in dipterous insects, cell fusing agent virus and Galbut virus in Aedes aegypti, Gypsy retrovirus in Drosophila, and Diaphorina citri reovirus (DcRV) in psyllid host can be paternally transmitted to offspring6. Pteromalus puparum negative-strand RNA virus 1 in its parasitoid wasp host can even infect the male reproductive system and transmit to offspring via mating, finally regulating the offspring sex ratio7. Vertical transmission of insect symbiotic viruses is an important persistence model in nature8. Currently, whether or how insect symbiotic viruses exploit sperms for paternal transmission remains poorly understood.Insects are the host and vector of diverse viruses including those that infect vertebrates, plants, and fungi. Insect symbiotic viruses reside inside their insect hosts and are passed vertically from parent to offspring, suggesting that the viruses could invade host germ cells11. For example, La Crosse virus and Zika virus can be paternally transmitted by male mosquitoes15. Maternal arbovirus transmission through transovarial passage has been extensively investigated21, but how sperm-mediated paternal virus transmission occurs is not completely clear. Considering the sperm structure, arbovirus infection is expected to impair normal functions of mammal host sperm23. We have revealed that rice gall dwarf virus (RGDV), a plant reovirus, can be paternally transmitted from male leafhopper vectors to offspring through a direct association of viral outer protein with the sperm surface without disturbing sperm functions24. During maternal transmission, RGDV propagation in the oocytes of the female ovary often causes cytopathologic changes to decrease insect offspring fitness25. By contrast, sperm-mediated paternal transmission of RGDV scarcely affects insect offspring fitness24. Thus, paternal arbovirus transmission may have evolved as a preferred mode of vertical transmission during the long-term virus-vector interaction.Arthropod-borne viruses (arboviruses) that cause substantial global health or agricultural problems can also be paternally transmitted to insect vector progeny population24. However, HSPG is composed of unbranched, negatively charged heparan sulfate polysaccharides attached to a variety of cell surface or extracellular matrix proteins26. Insect sperm- or semen-specific components potentially mediate paternal virus transmission, but the mechanism remains unrevealed. Many sperm surface proteins have critical roles in various reproductive processes such as sperm maturation, capacitation, and sperm-egg recognition27. In mammals, the epididymis is responsible for sperm transport, concentration, storage, and maturation28. HongrES1 and Spink13, the members of the serpin (serine proteinase inhibitor) family, are specifically expressed in the cauda epididymidis, deposited on the sperm surface, and involved in sperm capacitation and maturation31. Thus, serpin proteins appear to be necessary for the regulation of critical serine protease activity during sperm fertilization and maturation. In insects, serpin proteins are also specifically present in the seminal fluid of mating males, but their functions remain undescribed34. Insect serpin proteins are involved in antiviral melanization process by negatively regulating the clip-domain serine proteases activity, which mediating the conversion of the zymogen prophenoloxidase (PPO) into active phenoloxidase (PO)35. Subsequently, PO mediates the formation of melanin, which directly encapsulates and kills certain pathogens37. In this study, we report that RGDV and a symbiotic virus in rice green leafhopper Recilia dorsalis activate and hijack HongrES1 homolog on sperm surface for paternal virus transmission without disturbing sperm function. Furthermore, the elevation of HongrES1 expression during viral infection effectively suppresses the production of excessive PO contents to benefit viral infection in insect male reproductive system. Our findings reveal a new mode for arboviruses and symbiotic viruses to cooperatively hijack insect sperm-specific proteins for co-paternal transmission.We previously revealed that major outer capsid protein P8 of RGDV interacts with heparan sulfate proteoglycan (HSPG) to facilitate sperm-mediated paternal virus transmissionVirgaviridae family from field-caught R. dorsalis population in Guangdong Province, Southern China in 2018, based on analysis of RNA sequencing data  belonging to the ta Fig. S. Phylogeion Fig. . Immunofis Fig. S.Fig. 1SpR. dorsalis via transovarial or sperm-mediated paternal transmission.To explore whether RdFV can be vertically transmitted, the offspring of four mating pairings, including infected unfemales × uninfected males; infected females × uninfected males; uninfected females × infected males; and infected female × infected males, were collected and analyzed. We observed about 52%, 67%, and 85% positive for RdFV from the offspring of infected females mating with uninfected males, uninfected females mating with infected males, and infected females mating with infected males, respectively Fig. . Thus, pR. dorsalis population, the eggs produced by two mating pairings, including one RdFV-positive virgin female × one RdFV-positive male and one RdFV-free virgin female × one RdFV-free male, were collected and analyzed. It was found that RdFV prolonged adult longevity and promoted egg development . Sequencing of the positive clones identified HongrES1 homolog of R. dorsalis. The full-length ORF of HongrES1 contained 1,590 nt and encoded 415 amino acid residues including a serpin (serine protease inhibitor) conserved domain  pull-down assays confirmed that RdFV CP interacted with HongrES1  to investigate how HongrES1 promoted paternal virus transmission. RT-qPCR and western blot assays showed that the knockdown of HongrES1 expression effectively decreased RdFV CP or RGDV P8 accumulation in the male reproductive system  and RDV P8  significantly reduced RGDV and HongrES1 accumulation in the male reproductive system, finally decreasing paternal RGDV transmission rate by about 25%  also significantly reduced RdFV and HongrES1 accumulation in the reproductive system of RdFV and RGDV co-positive male R. dorsalis, and decreased paternal RdFV transmission rate by about 22%  into RGDV-positive males decreased PO accumulation and activity, ultimately promoting viral infection in the male reproductive system 45. Plant virgaviruses are non-replicative in the insects47, while insect-specific RdFV and Hubei virga-like virus are also non-replicative in the plants45, suggesting the existences of host-specific barriers for virgavruses. In general, insect-specific symbiotic viruses such as Nilaparvata lugens reovirus and DcRV can be horizontally transmitted to their herbivorous insect hosts through plants49. Although vertical transmission rate of RdFV by R. dorsalis population is not 100%, rice plants might also serve as the passive vectors for the horizontal transmission of RdFV, finally ensuring the effective spread of RdFV population in nature. Recent metagenomic studies have found that insect-specific virgaviruses are widespread in insects39, and may represent a major group of insect-specific paternally transmitted symbiotic viruses. More importantly, RdFV prolongs host adult longevity and promotes egg development, thereby benefiting the fitness of adults or their offspring. Thus, RdFV is beneficial to R. dorsalis population expansion, which explains the reason of mutualistic symbiosis between RdFV and R. dorsalis host.Phylogenetic analysis showed that the insect-specific RdFV, gramineous plants-infecting furovirues, and insect-specific Hubei virga-like viruses belong to 4. For example, cell-fusing agent virus negatively regulates the infection of both dengue and Zika viruses in mosquitoes50, and palm creek virus suppresses the infection of West Nile virus in mosquitoes52. However, many symbiotic viruses are considered to benefit insect host fitness52, and thus could affect the insect host’ ability to acquire and transmit arboviruses. In this study, we show that RdFV is beneficial to insect host and offspring fitness, and thus RdFV existing in R. dorsalis population could facilitate vector competence for RGDV. We further reveal a direct interaction of RdFV and RGDV during their co-paternal transmission by R. dorsalis. The outer capsid proteins of these two viruses interact with each other, and RGDV spherical particles and RdFV filamentous particles are accumulated together in the testis epithelium, which are then together released into sperm-accumulated testis lumen via the apical plasmalemma. More importantly, HongES1 is associated with RGDV and RdFV particles in the testis epithelium and sperm-accumulated lumen, and gathers at the contact sites between sperm and virions. Thus, RdFV, RGDV and HongES1 form a complex via the CP-HongrES1-P8 interaction. Taken together, RGDV and RdFV could activate and hijack HongrES1 for their simultaneous paternal transmission, potentially including the invasion of testis epithelium, release into sperm-accumulated lumen, and attachment of sperm surfaces. However, whether RdFV virions could directly carry RGDV virions from testis epithelium into sperm-accumulated lumen remains elusive. In summary, our findings reveal a new mode for arboviruses and symbiotic viruses to cooperatively activate and hijack insect sperm-specific proteins for co-paternal transmission.Another important question to be solved is whether or how insect symbiotic microorganisms facilitate paternal arbovirus transmission. Some symbiotic viruses suppress the infection of arboviruses in insect vectors53, RGDV-positive R. dorsalis population can keep up to two generations on the weeds such as Alopecurus aequalis24. A male has a huge number of sperms and can mate repeatedly with females to enhance virus spread. Furthermore, RGDV does not affect the fitness of male-transmitted offspring54, but causes significant fitness cost of female-transmitted offspring24. However, insect symbiotic RdFV is beneficial for adult host and offspring fitness, and thus facilitating vertical RGDV transmission. Thus, the high efficiency for paternal RGDV transmission through males and the low efficiency for transovarial RGDV transmission via females simultaneously ensure effective viral transmission and insect fecundity. We deduce that sperm-mediated paternal RGDV transmission is a powerful type of vertical virus transmission by insect vectors, and plays a vital role in the efficient maintenance of RGDV during the cold seasons in the field. Many arboviruses such as La Crosse virus and Zika virus can also be paternally transmitted by male mosquitoes15. Potentially, insect sperm-specific proteins or symbiotic viruses might also facilitate paternal arboviruses transmission ensure viral long-term persistence in nature.Over the past 40 years, viral disease caused by RGDV is always epidemic in the field in Southern China. During the winter months (November–March) in Guangdong, Southern China55. We found that the increased HongrES1 expression during RGDV infection effectively suppresses the expression of clip-domain serine proteases and the subsequent conversion of PPO to the active PO, ultimately producing a mild PO activity in the male reproductive system. Thus, viral infection potentially activates HongrES1 expression to modulate the balance of virus-induced melanization and effective viral infection, guaranteeing the normal role of HongrES1 in sperm maturation and paternal viral transmission.Finally, we reveal a new function of HongrES1, like other members of the serpin family, in mediating insect antiviral melanization defense. HongrES1 has the serine proteinase inhibitor activity to modulate PO activity to benefit viral infection in insect male reproductive system. Once pathogen-associated molecular patterns are recognized, the PPO is converted to active PO via serpin-regulated serine proteases, while PO catalyzes the formation of melanin to immobilize microbesR. dorsalis adults were collected from rice fields in Luoding city, Guangdong Province, China from 2018, and propagated on TN-1 rice seedlings in cages at 25 ± 1 °C with 75 ± 5% relative humidity and 16-h light/8-h dark. High-throughput sequencing was used for identification of insect-specific viruses from this R. dorsalis population. Total RNA was extracted from R. dorsalis adults in TRIzol reagent according to the manufacturer’s instructions. High quality RNAs were selected for construction of small RNAs and transcriptomic libraries, which were sequenced using an Illumina NovaSeq 6000 platform in Novogene Co., Ltd, China. Low-quality reads and adapter sequences were removed from the raw reads to obtain clean reads. The clean reads were assembled, and the assembled contigs were analyzed using BLASTx searches in the nonredundant protein database available in NCBI. BLAST results were then checked carefully to screen potential viral sequences.56. This model was determined using the Bayesian information criterion by ModelFinder57. Markov chains were run for 2,000,000 generations, sampling every 100 generations. The sufficient sampling and parameter convergence were checked using Tracer 1.71, after discarded the first 25% samples as burn-in. The Bayesian 50% majority rule consensus tree was visualized in FigTree 1.4.4.A new positive-sense single-stranded RNA virus was screened and identified using RT-PCR assays, and then named as RdFV. The primers used in RT-PCR assays were shown in Supplementary Table R. dorsalis population  reared under controlled greenhouse conditions had the transcript of RdFV CP  or female (♀)  CP Fig. . To estaR. dorsalis. To obtain RGDV-positive or RdFV and RGDV co-positive R. dorsalis population, the 2th instar nymph of RdFV-free or positive leafhoppers were fed on RGDV-infected rice plants for 2 day and then transferred to healthy rice seedlings. At 14-day post-first access to diseased plants (padp), the presence of RdFV or RGDV was identified using RT-PCR assays. The offspring produced by RGDV-positive or RdFV and RGDV co-positive parents were reared to establish RGDV-positive or RdFV and RGDV co-positive population. The primers used in RT-PCR assays were shown in Supplementary Table Rice plants infected with RGDV isolates were also originally collected from Luoding city, Guangdong Province, China and maintained on rice plants via transmission by Rabbit polyclonal antibodies against RdFV CP, HongrES1, RGDV P8 and PPO were prepared by Genscript Biotech Corporation, Nanjing, China. The process was approved by the Science Technology Department of Jiangsu Province of China. Specific IgG against RdFV CP or RGDV P8 was conjugated to rhodamine to generate CP-rhodamine or P8-rhodamine. Specific IgG against HongrES1, RGDV P8 or RdFV CP was conjugated to fluorescein isothiocyanate (FITC) to generate HongrES1-FITC, P8-FITC, or CP-FITC. Mouse monoclonal antibody against GST was purchased from Transgene Biotech (HT601). The actin dyes Alexa Fluor 647 Phalloidin, and the nuclear dye 4’,6-diamidino-2-phenylindole (DAPI) were purchased from Thermo Fisher Scientific . Rabbit polyclonal antibody against histone H3 was purchased from Abcam (ab1791).R. dorsalis, the total RNAs were extracted from tissues or whole bodies using TRIzol reagent . The relative quantification of genes was tested by RT-qPCR assay using the 2 × RealStar Fast SYBR qPCR Mix  in the QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). The primers used in RT-qPCR assays were shown in Supplementary Table elongation factor 1 alpha (EF1α) of R. dorsalis (Genbank accession number: AB836665) served as the internal reference for the normalization of gene expression levels. Relative gene expression levels were estimated by the −ΔΔCT (cycle threshold) method24. The CT value of 35 was served as a cutoff value, and the CT values lower than 35 were considered as effective data. The experiments were replicated at least three times, and a pool of 30 leafhoppers was used for each replicate.To analyze the expression levels of genes of viruses or R. dorsalis, the alimentary canal, salivary gland, and male or female reproductive organs were dissected from 30 RdFV-free or -positive R. dorsalis leafhoppers. The samples were fixed in 4% (v/v) paraformaldehyde in PBS for 2 h, and then permeabilized in 0.2% (v/v) Triton-X for 1 h. The samples were then immunolabeled with RdFV CP-rhodamine (0.5 μg/μl) and the actin dye Alexa Fluor 647 Phalloidin (0.1 μg/μl). Immunostained tissues were visualized using a Leica TCS SPE inverted confocal microscope.For visualizing RdFV infection to different tissues of For visualizing the association of HongrES1 with RdFV or RGDV in the male reproductive system, the reproductive organs were dissected from 30 RdFV-free, RdFV-positive, or RdFV and RGDV co-positive males. The samples were fixed, permeabilized, immunolabeled with HongrES1-FITC, CP-FITC, CP-rhodamine, or P8-rhodamine (0.5 μg/μl), and then processed for immunofluorescence microscopy.For visualizing virus or HongrES1 association with sperms, mature sperms were excised from the testes of 30 RdFV-free, RdFV-positive, or RdFV and RGDV co-positive males, and then smeared on poly-lysine-treated glass slides. The sperms were successively fixed, permeabilized, immunolabeled with CP-rhodamine, P8-rhodamine, P8-FITC, HongrES1-FITC (0.5 μg/μl), stained with DAPI (2.0 μg/ml), and then processed for immunofluorescence microscopy.For electron microscopy, the midgut or reproductive organs of RdFV-free, RdFV-positive or RdFV and RGDV co-positive male or female adults, or the spermatheca of virus-free females at different days after mating with RdFV-positive males were excised, fixed with 2% (v/v) glutaradehyde and 2% (v/v) paraformaldehyde in PBS for 2 h at room temperature, and then postfixed with 1% (w/v) osmium tetroxide in PBS for 1 h at room temperature. The fixed samples were dehydrated in grade series of ethanol up to 100% and embedded in Spurr’s resin (SPI Ltd). Samples were sectioned on an ultramicrotome (Leica) with a diamond knife. The ultrathin sections were observed with a transmission electron microscope .For immunoelectron microscopy, the samples were fixed with 2% (v/v) glutaradehyde and 2% (v/v) paraformaldehyde in PBS for 2 h at room temperature. Fixed samples were dehydrated through a graded ethanol series at −20 °C and embedded in LR gold resin (Bioscience). Polymerization was allowed to proceed for 72 h at −20 °C. Samples were sectioned on an ultramicrotome (LKB Nova) with a diamond knife and the ultrathin sections were then immunolabeled with HongrES1-, CP- or P8-specific IgG (0.5 μg/μl) as the primary antibody, and then treated with goat anti-rabbit IgG conjugated with 15-nm diameter gold particles  as the secondary antibody. Then the samples were examined under the transmission electron microscope.R. dorsalis colony were conducted as follows: (i) infected virgin female × infected male; (ii) infected virgin female × uninfected male; (iii) uninfected virgin female × infected male; and (iv) uninfected virgin female × uninfected male  infected virgin female ×infected male; and (ii) uninfected virgin female × uninfected male  was used according to the manufacturer’s instructions. The full-length ORFs of RdFV CP and RGDV P8 were separately inserted into bait vector pDHB1 to generate pDHB1-CP and pDHB1-P8, and the full-length ORFs of HongrES1, RdFV CP, RSMV G, and RDV P8 were separately inserted into prey vector pPR3-N to generate pPR3-N-HongrES1, pPR3-N-CP, pPR3-N-G and pPR3-N-P8 (RDV). The bait and prey were co-transformed the yeast strain NMY51. The pLargeT/p53 interaction served as a positive control, and the pDHB1/pRR3N served as a negative control. Transformants were subsequently screened on the QDO  culture medium, and β-galactosidase activity was detected in Z buffer with X-Gal. The primers used in Y2H were shown in Supplementary Table CP, HongrES1 and RGDV P8 were separately cloned into the pGEX-4T-3 vector to construct plasmids expressing the GST fusion protein as baits . The full-length ORFs of HongrES1, RGDV P8 and RdFV CP also were separately cloned into the pEASY-Blunt E1 Expression Vector  to construct plasmids expressing the His fusion protein as preys . The recombinant proteins fused with GST tag and GST protein were respectively expressed in E. coli strain BL21. Lysates were then subsequently incubated with glutathione-Sepharose beads  and expressed RdFV CP, HongrES1 or RGDV P8, respectively. The eluates were analyzed by western blot assays using antibodies against GST-tag, HongrES1, RGDV P8 and RdFV CP (0.5 μg/μl). The primers used in GST pull-down assays were shown in Supplementary Table The ORFs of RdFV R. dorsalis, the whole body, alimentary canal, reproductive organs and salivary gland were dissected from 30 RdFV-free males or virgin females at 5-days post eclosion. The relative expression of HongrES1 in different tissues was detected by RT-qPCR assays. To verify the expression patterns of HongrES1, the total proteins were extracted from various tissues of 30 RdFV-free males or females, and then analyzed by western blot assays. Antibodies against HongrES1 and histone H3 (0.5 μg/μl) served as the primary antibodies, and goat anti-rabbit IgG-peroxidase (0.5 μg/μl) served as the secondary antibody.To analyze the expression levels of HongrES1 in different tissues of HongrES1 was detected by RT-qPCR assays. In the corresponding western blot assay, antibodies against HongrES1, RdFV CP, RGDV P8, and histone H3 (0.5 μg/μl) served as the primary antibodies, and goat anti-rabbit IgG-peroxidase (0.5 μg/μl) served as the secondary antibody. At least three biological replicates were performed.We also detected the effects of RdFV or RGDV infection on the expression levels of HongrES1 in the male reproductive system. The reproductive organs were dissected from 30 RdFV-free, RdFV-positive, or RGDV and RdFV co-positive males. The relative expression of Escherichia coli strain Rosetta, and the proteins were purified using nickel-nitrilotriacetic acid resin (Qiagen). Sperm smears collected from testes of RdFV-free R. dorsalis were successively incubated with the purified CP (0.5 μg/μl) for 1 h, smeared on poly-lysine-treated glass slides, immunolabeled with CP-rhodamine (0.5 μg/μl), stained with DAPI (2.0 μg/ml), and then processed for immunofluorescence microscopy.To detect the binding of RdFV CP with sperms in vitro, His-tag-fused CP was expressed in R. dorsalis were pre-incubated for 30 min with pre-immune antibody (0.5 μg/μl) or HongrES1 antibody (0.5 μg/μl), and then the in vitro CP-sperm binding experiment was performed as described above.In neutralization experiments to test the direct interaction between RdFV CP and HongrES1, mature sperms excised from the testes of RdFV-free In neutralization experiments to test the direct interaction between RGDV particles and HongrES1, mature sperms excised from RGDV-free leafhoppers were pre-incubated for 30 min with pre-immune antibody (0.5 μg/μl) or HongrES1 antibody (0.5 μg/μl), incubated with the purified RGDV particles (1.0 μg/μl) for 1 h, smeared on poly-lysine-treated glass slides, immunolabeled with P8-FITC (0.5 μg/μl), stained with DAPI (2.0 μg/ml), and then processed for immunofluorescence microscopy.HongrES1, RdFV CP, RGDV P8, or GFP gene (Supplementary Table HongrES1 (dsHongrES1), RdFV CP (dsCP), RGDV P8 (dsP8), or GFP (dsGFP) according to the protocol for the T7 RiboMAX Express RNAi System kit .RNA inference was performed to knock down the expression of related genes. The T7 promoter with the sequence 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGG-3′ was added to the forward and reverse primers at the 5′ terminal to amplify a region of ~500–800 bp of HongrES1 expression on RdFV or RGDV infection in male reproductive systems, newly emerged male adults of RdFV-positive or RGDV-positive R. dorsalis population were microinjected with dsHongrES1 or dsGFP (approximately 200 ng/leafhopper) using a Nanoject II Auto-Nanoliter Injector (Spring), and then transferred to healthy rice seedlings. To test the knockdown of RdFV CP or RGDV P8 expression on viral infection and HongrES1 accumulation in male reproductive system, newly emerged male adults of RdFV and RGDV co-positive R. dorsalis population were microinjected with dsCP, dsP8 or dsGFP (~200 ng/leafhopper), and then transferred to rice seedlings. For each treatment, approximate 100 insects were microinjected, and three replicates were performed.To test the knockdown of The male reproductive organs were dissected to test the expression levels of RdFV CP, RGDV P8, or HongrES1 using RT-qPCR and western blot assays. A pool of 30 dsRNA-treated males was used for each replicate, and the experiment was conducted in three replicates for RT-qPCR assays. The total proteins from reproductive organs of 30 dsRNA-treated males were analyzed for the protein levels in western blot assays by using HongrES1-, CP- or P8-specific IgG (0.5 μg/μl). Experiment was conducted in three replicates in western blot assays. To determine the effect of dsHongrES1, dsCP or dsP8 treatment on paternal transmission of RdFV or RGDV, one dsHongrES1-, dsCP-, dsP8- or dsGFP-treated RdFV- or RGDV-positive male mated with one virus-free virgin female in a glass tube containing a rice seedling for 3 days . A pool of 30 RGDV-positive males was used for each replicate in RT-qPCR and western blot assays, respectively. The experiment was conducted in at least three replicates for RT-qPCR and western blot assays. To analyze effect of RGDV infection on PO activity, the reproductive organs dissected from approximate 100 newly emerged males were homogenized with the His-Mg buffer  buffer in liquid nitrogen. The supernatant was gently mixed with 1 mM dopamine in 10 mM Tris-HCl buffer (pH 8.0) in a 96-well plate at room temperature for 5 min. Enzyme activity was measured using the phenoloxidase kit  according to the manufacturer’s protocol. To analyze the effect of M. luteus infection on PO activity, freeze-dried M. luteus was dissolved in water, and then microinjected in dose of ~23 ng/leafhopper into newly emerged males. At 24-h post microinjection, the reproductive organs of approximate 100 RGDV-infected or M. luteus-treated males were dissected and tested for PO activity.The reproductive organs were individually dissected from the newly emerged male adults of RdFV and RGDV co-positive PPO or HongrES1 expression on PO activity and RGDV infection. The newly emerged male adults of RdFV and RGDV co-positive R. dorsalis population were microinjected with dsGFP, dsPPO or dsHongrES1 (~200 ng/leafhopper). The male reproductive organs of these tested leafhoppers were individually collected and dissected for RT-qPCR and western blot assays to determine the effect of dsRNAs on the expression levels of HongrES1, PPO, or RGDV P8, and the conversion of PPO to active PO, as well as PO activity. A pool of 30 males was used for each replicate in RT-qPCR and western blot assays, respectively. A pool of 100 males was tested for each replicate in PO activity. The experiment was conducted in three replicates for RT-qPCR and western blot assays, as well as PO activity tests.We then tested the effect of knockdown of To analyze the probability of discrepancy occurrence in a specific range, all quantitative data presented in the text and figures were analyzed with unpaired two-tailed t-test in GraphPad Prism 7 software .All micrographs of electron, immunoelectron, immunofluorescence and transmitted light were representative of the results of at least 3 biological experiments. Data showed in GST pull-down assays were representative of 3 biological experiments.Further information on research design is available in the Supplementary informationPeer Review FileReporting Summary


Orthopoxvirus genus of the Poxviridae family which includes both the variola  virus and the Vaccinia virus .1 Monkeypox was initially discovered in 1958 and occurs mostly in Central and West Africa. Monkeypox is a global public health concern because more than 120 confirmed or suspected cases of the disease were recently identified in at least 11 non-endemic countries such as North America and Europe in May 2022.3 Monkeypox is a rare zoonotic infectious disease. Monkeypox virus belongs to the 5 Several reasons have been suggested for the recent increase and spread of pandemics in the world. Based on current evidence environmental exploitation caused by land-use changes, deforestation, agriculture expansion, wildlife trade and consumption, and urbanization can disrupt natural interactions between wildlife and their microbes, as well as increase contact between wildlife, livestock, and humans, potentially resulting in new infectious diseases and pandemics around the world.1 Human-to-human transmission occurs through direct contact with lesions, body fluids, and respiratory droplets. Prolonged face-to-face contact is required because respiratory droplets can only reach a few feet. Although almost all recently diagnosed cases include men aged 20 to 50, many of whom are men who have sex with men, it is unclear at this time if monkeypox can be transmitted specifically through sexual transmission routes. Indirect contact with contaminated materials such as bedding has also been documented. The animal-to-human transmission may occur through a bite or scratch as well as direct or indirect contact with body fluids or lesion material.7 Monkeypox is less contagious than smallpox. The virus enters the body through broken skin, the respiratory tract, or the mucous membranes .7 Clinical presentations of monkeypox are similar to but less severe than smallpox which was eradicated in 1980. The incubation period varies from 6 to 13 days but can take from 5 to 21 days. The disease is characterized by fever, headache, myalgia, backache, chills, exhaustion, and lymphadenopathy that may initially appear similar to chickenpox, measles, and smallpox. The illness typically lasts for 2 to 4 weeks.7 What should we do now to prevent the spread of monkeypox and the possibility of an epidemic? Based on the limited information available at present, the risk of monkeypox to the public is very low. Although containment strategies are not necessary at present time, however, the following measures that can be taken to prevent the silent spread of the disease: (a) raising public awareness of risk factors and educating people about the measures they can take to reduce their exposure to the virus; (b) anyone who has clinical manifestations of monkeypox should contact a health care provider right away; (c) isolation of suspected or confirmed cases of monkeypox; (d) hand washing with soap and water or using an alcohol-based hand sanitizer immediately after unprotected contact with wild or sick animals or suspected patients or contaminated materials; (e) avoid contact with animals that could harbor the virus or direct contact with any contaminated materials such as bedding or laundry; (f) availability of laboratory equipment for confirmatory monkeypox virus-specific testing on lesion specimens that clinicians obtain from suspected patients; (g) feasibility of access to suitable vaccine especially for high-risk people such as laboratory workers and health professionals as well as general population if necessary.8 However, routine smallpox vaccination with vaccinia-based vaccines has been stopped by all countries at least 40 years ago. Therefore, unvaccinated populations, aged under 40, are now more susceptible to monkeypox virus infection. According to the current knowledge, vaccines used during the smallpox eradication program also provide protection against monkeypox. According to statistics from Africa, the smallpox vaccination is at least 85% effective at preventing monkeypox. None.


Plants are grown under adverse conditions of multiple plant viruses in the field, and inevitably, mixed infections are common. Several important virus diseases of plants are the outcomes of interactions between different viral agents. An example is maize lethal necrosis, a devastating disease caused by the synergistic combination of corn chlorotic mottle virus and potato Y virus  varieties from a Slovenian wine-growing region. A complete description of coinfection in each plant was well documented. Interestingly, they showed that no grapevine was found to be virus- and viroid-free, and no grapevine was found to be infected with only one virus or viroid, while the highest number of viral entities in a plant was eight, indicating that mixed infections of viruses and viroids is the rule. These results will improve the early diagnostics of preclonal candidates of grapevine varieties, which is especially important to control virus diseases in grapevines at their early growing stage.High-throughput sequencing (HTS) technologies have become indispensable tools to characterize plant virus diversity, being very powerful to characterize mixed infections as virtually all viruses present in a plant sample might be identified without prior sequence knowledge  in symptom induction and aphid transmission by successful construction of infectious clones. They showed clear evidence that only co-inoculation of these four causal agents to tobacco plants could cause typical TBTD symptoms. Moreover, they showed that the successful transmission of tobacco bushy top virus , TBTV satellite RNA, and tobacco vein distorting virus (TVDV) satellite RNA by Myzus persicae depended on the presence of TVDV (a polerovirus), while the presence of TBTV satellite RNA increased the aphid transmission efficiency of TBTV and TVDV, indicating a mutual interaction between different viruses and their associated satellites. Jia et al. studied another interesting field within the scope of this Research Topic. In addition to the synergistic effect of rice stripe mosaic virus (RSMV), a newly discovered plant cytorhabdovirus, and rice gall dwarf virus (RGDV) on viral replication potential and pathogenicity in rice plants, these authors also found that RGDV significantly promoted the propagation of RSMV when co-infecting the vector, Recilia dorsalis. Accordingly, co-infection significantly promoted the acquisition and transmission efficiencies of RSMV by R. dorsalis, Showing that the synergy between plant viruses also takes place in the transmitting vectors.In nature, most of the plant viruses are transmitted horizontally by insect vectors, and deciphering virus–vector interactions are an emerging research subject in plant virology (Whitfield et al., Tatineni et al. analyzed the results of infection in wheat with up to four viruses in different combinations. The outcome of the infections was evaluated by observations of symptoms as well as measurement of the amount of viral RNA and coat protein. Interestingly, for some virus combinations, stronger virus symptoms were not accompanied by increasing virus titers. This study shows that there are complex antagonistic and synergistic interactions between viruses in field-grown crops. Overall, the papers in this Research Topic reveal different perspectives of current research on mixed infections of plant viruses in nature, from staple crop studies to investigations into the important cash crops and then into the intricate synergistic effects on the tripartite interactions between viruses, plants, and vectors. Illuminating the mechanisms of these mixed infections is crucial for understanding viral pathogenesis and evolution and, consequently, developing efficient and stable control strategies in the field.A mixed virus infection that results in more severe symptoms is usually referred to synergistic interactions Syller, . TatinenYX and YL wrote the first draft of the editorial. MG revised the draft and added additional sections. All authors contributed to the article and approved the submitted version.This work was partially supported with funding provided by the National Natural Science Foundation of China Grant 32172376, the Startup Fund for Distinguished Scholars from Nanjing Agricultural University to YX.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.