Homopteran Vector Biomarkers for Efficient Circulative Plant Virus Transmission are Conserved in Multiple Aphid Species and the Whitefly Bemisia tabaci

Plant viruses in the families Luteoviridae and Geminiviridae are phloem restricted and are transmitted in a persistent,circulative manner by homopteran insects.Using fluorescence 2-D difference gel electrophoresis to compare the proteomes of F2 genotypes of Schizaphis graminum segregating for virus transmission ability,we recently discovered a panel of protein biomarkers that predict vector competency.Here we used aphid and whitefly nucleotide and expressed sequence tag database mining to test whether these biomarkers are conserved in other homopteran insects.S.graminum gene homologs that shared a high degree of predicted amino acid identity were discovered in two other aphid species and in the whitefly Bemisia tabaci.Selected reaction monitoring mass spectrometry was used to validate the expression of these biomarkers proteins in multiple aphid vector species.The conservation of these proteins in multiple insect taxa that transmit plant viruses along the circulative transmission pathway creates the opportunity to use these biomarkers to rapidly identify insect populations that are the most efficient vectors and allow them to be targeted for control prior to the spread of virus within a crop.

Studies on Mucosal Immunity Induced by Transmissible Gastroenteritis Virus Nucleocapsid Protein Recombinant Lactobacillus casei in Mice and Sow

Mucosal immunity plays an important role in protecting pigs against transmissible gastroenteritis virus （TGEV） infection. To elicit mucosal immune response against TGEV, we developed a surface antigen display system using the poly-γ- glutamate synthetase A （pgsA） protein of Bacillus subtilis as an anchoring matrix to express recombinant fusion proteins of pgsA and nucleocapsid protein of TGEV in Lactobacillus casei. Surface location of fusion protein was verified by ELISA and indirect immunofluorescence test. Oral and intranasal inoculations of pregnant sow and mice with recombinant L. casei resulted in high levels of serum immunoglobulin G （IgG） and secretory immunoglobulin A （slgA） against recombinant N protein as demonstrated by ELISA. More importantly, the level of specific slgA in colostrum significantly increased compared with that of IgG. The serum IgG levels of the piglets increased after suckling colostrum produced by sows was previously inoculated with recombinant L. casei. These results indicate that immunization with recombinant L. casei expressing TGEV N protein on its surface elicited high levels of specific slgA and circulating IgG against TGEV N protein.

A viral movement protein targets host catalases for 26S proteasome-mediated degradation to facilitate viral infection and aphid transmission in wheat

The infection of host plants by many different viruses causes reactive oxygen species(Ros)accumulation and yellowing symptoms,but the mechanisms through which plant viruses counteract RoS-mediated immunity to facilitate infection and symptom development have not been fully elucidated.Most plant viruses are transmitted by insect vectors in the field,but the molecular mechanisms underlying virus-host-insect interactions are unclear.In this study,we investigated the interactions among wheat,barley yellow dwarf virus(BYDV),and its aphid vector and found that the BYDV movement protein(MP)interacts with both wheat catalases(CATs)and the 26S proteasomeubiquitin receptor non-ATPase regulatorysubunit2homolog(PSMD2)to facilitate the 26S proteasome-mediateddegradation of CATs,promotingviral infection,disease symptom development,and aphid transmission.Overexpression of the BYDV MP gene in wheat enhanced the degradation of CATs,which leading to increased accumulation of ROS and thereby enhanced viral infection.Interestingly,transgenic wheat lines overexpressing BYDV MP showed significantly reduced proliferation of wingless aphids and an increased number of winged aphids.Consistent with this observation,silencing of CAT genes also enhanced viral accumulation and reduced the proliferation of wingless aphids but increased the occurrence of winged aphids.In contrast,transgenic wheat plants overexpressing TaCAT1 exhibited the opposite changes and showed increases in grain size and weight upon infection with BYDV.Biochemical assays demonstrated that BYDV MP interacts with PSMD2 and promotes 26S proteasome-mediated degradation of TaCAT1 likely in a ubiquitination-independent manner.Collectively,our study reveals a molecular mechanism by which a plant virus manipulates the Ros production system of host plants to facilitate viral infection and transmission,shedding new light on the sophisticated interactions among viruses,host plants,and insect vectors.

Towards unfolding the prion misfolding mystery: Protein free radical chemistry in transmissible spongiform encephalopathies

Owing to the high oxygen-respiration in the brain of mammals, oxidative damage to prion protein hasbeen suggested to be an additional factor. A large body of intriguing features of scrapie and prion diseases haveprovided multiple lines of indirect chemistry evidence, suggesting that the infectious agents may be putative forms ofsequence-specific prion radicals (SSPR) and/or their immediate precursors in the transmissible spongiform encepha-lopathies (TSE). Here a molecular mechanism corresponding to the self-replication of scrapie protein mediated byprion free-radical processes, consonant with "protein-only" hypotheses is proposed. This new theory may not onlyaid our understanding of the occurrence of prions, but also provides new insight into the possible chemistry principlesunderlying the neurodegenerative disorders. It is anticipated that future studies based on this suggestion and chem-istry principles of genetic diseases may allow us to determine an effective approach to stop mad cow disease and itshuman version, new variant of Creutzfeldt-Jakob disease (v CJD).

Research progress toward the influence of mosquito salivary proteins on the transmission of mosquito-borne viruses

Mosquito-borne viruses(MBVs)are a large class of viruses transmitted mainly through mosquito bites,including dengue virus,Zika virus,Japanese encephalitis virus,West Nile virus,and chikungunya virus,which pose a major threat to the health of people around the world.With global warming and extended human activities,the incidence of many MBVs has increased significantly.Mosquito saliva contains a variety of bioactive protein components.These not only enable blood feeding but also play a crucial role in regulating local infection at the bite site and the remote dissemination of MBVs as well as in remodeling the innate and adaptive immune responses of host vertebrates.Here,we review the physiological functions of mosquito salivary proteins(MSPs)in detail,the influence and the underlying mechanism of MSPs on the transmission of MBVs,and the current progress and issues that urgently need to be addressed in the research and development of MSP-based MBV transmission blocking vaccines.

Insect-specific RNA virus affects the stylet penetration activity of brown citrus aphid(Aphis citricidus)to facilitate its transmission

Sap-sucking insects often transmit plant viruses but also carry insect viruses,which infect insects but not plants.The impact of such insect viruses on insect host biology and ecology is largely unknown.Here,we identified a novel insect-specific virus carried by brown citrus aphid(Aphis citricidus),which we tentatively named Aphis citricidus picornavirus(AcPV).Phylogenetic analysis discovered a monophyletic cluster with AcPV and other unassigned viruses,suggesting that these viruses represent a new family in order Picornavirales.Systemic infection with AcPV triggered aphid antiviral immunity mediated by RNA interference,resulting in asymptomatic tolerance.Importantly,we found that AcPV was transmitted horizontally by secretion of the salivary gland into the feeding sites of plants.AcPV influenced aphid stylet behavior during feeding and increased the time required for intercellular penetration,thus promoting its transmission among aphids with plants as an intermediate site.The gene expression results suggested that this mechanism was linked with transcription of salivary protein genes and plant defense hormone signaling.Together,our results show that the horizontal transmission of AcPV in brown citrus aphids evolved in a manner similar to that of the circulative transmission of plant viruses by insect vectors,thus providing a new ecological perspective on the activity of insect-specific viruses found in aphids and improving the understanding of insect virus ecology.

Plants affect the horizontal transmission of a new densovirus infecting the green peach aphid Myzus persicae by modulating honeydew production

In a tritrophic context of plant-insect-entomopathogen,plants play important roles in modulating the interaction of insects and their pathogenic viruses.Currently,the influence of plants on the transmission of insect viruses has been mainly studied on baculoviruses and some RNA viruses,whereas the impact of plants on other insect viruses is largely unknown.Here,we identified a new densovirus infecting the green peach aphid Myzus persicae and tested whether and how host plants influence the transmission of the aphid densovirus.The complete single-stranded DNA genome of the virus,M.persicae densovirus 2,is 5727 nt and contains inverted terminal repeats.Transcription and phylogenetic analysis indicated that the virus was distinct from other a few identified aphid densoviruses.The virus abundance was detected highly in the intestinal tract of aphids,compared with the lower level of it in other tissues including head,embryo,and epidermis.Cabbage and pepper plants had no obvious effect on the vertical transmission and saliva-mediated horizontal transmission of the virus.However,the honeydew-mediated horizontal transmission among aphids highly depended on host plants(65%on cabbages versus 17%on peppers).Although the virus concentration in the honeydew produced by aphids between 2 plants was similar,the honeydew production of the infected aphids reared on peppers was dramatically reduced.Taken together,our results provide evidence that plants influence the horizontal transmission of a new densovirus in an aphid population by modulating honeydew secretion of aphids,suggesting plants may manipulate the spread of an aphid-pathogenic densovirus in nature.

Aphids and their transmitted potato viruses: A continuous challenges in potato crops

Aphid is one of the most destructive insect pests on cultivated plants in temperate regions.Their piercing-sucking mouthparts and phloem feeding behavior directly damage crops and deplete plant nutrients.Potato(Solanum tuberosum L.)is one of the most important food sources on the planet,and several aphid species,e.g.,Myzus persicae(Sulzer)(green peach aphid)and Macrosiphum euphorbiae(Thomas)(potato aphid)(Hemiptera:Aphididae)colonize potato and transmit several economically important viruses.Aphid-transmitted potato viruses have been emerging all over the world as a very serious problem in potato production,inducing a wide variety of foliar and tuber symptoms,leading to severe yield reduction and loss of tuber quality.In this review,recent advances in understanding the interactions of potato viruses with their hosts,aphid vectors and the environment are described.

Identification of a protein associated with circulative transmission of Barley yellow dwarf virus from cereal aphids, Schizaphis graminum and Sitobion avenae

Using 2-D electrophoresis and virus overlay assay, a 50-kDa protein (P50) exhibiting specific binding to purified virus particles of BYDV-GAV was found in the protein extracts from Schizaphis graminum and Sitobion avenae, two aphid species transmitting BYDV-GAV. P50 in the extracts of S. graminum was isolated by preparation electrophoresis and electro-eluted proteins from the gel slices for antiserum preparation. After feeding the antiserum through membrane, the transmission efficiencies of S. graminum and S. avenae for BYDV-GAV decreased significantly. It was suggested that P50 should be related with transmission pro- cess. Location of P50 was found at the plasma membrane surrounding the accessory salivary gland (ASG) in the head tissues of S. graminum by immunogold-labelling experiment. The ascertainment of the protein associated with virus transmission has a significance influence on further understanding the transmission mechanism and genetic engineering for resistant to vector transmission.

Effects of perinatal protein deprivation and recovery on esophageal myenteric plexus

AIM:To evaluate effects of preand postnatal protein deprivation and postnatal recovery on the myenteric plexus of the rat esophagus. METHODS: Three groups of young Wistar rats (aged 42 d) were studied: normalfed (N42), proteindeprived (D42), and proteinrecovered (R42). The myenteric neurons of their esophagi were evaluated by histochemical reactions for nicotinamide adenine dinucleotide (NADH), nitrergic neurons (NADPH)diaphorase and acetylcholinesterase (AChE), immunohistochemical reaction for vasoactive intestinal polypeptide (VIP), and ultrastructural analysis by transmission electron microscopy.RESULTS: The cytoplasms of large and medium neurons from the N42 and R42 groups were intensely reactive for NADH. Only a few large neurons from the D42 group exhibited this aspect. NADPH detected in the D42 group exhibited low reactivity. The AChE reactivity was diffuse in neurons from the D42 and R42 groups. The density of large and small varicosities detected by immunohistochemical staining of VIP was low in ganglia from the D42 group. In many neurons from the D42 group, the double membrane of the nuclear envelope and the perinuclear cisterna were not detectable. NADH and NADPH histochemistry revealed no group differences in the prof ile of nerve cell perikarya (ranging from 200 to 400 μm2).CONCLUSION: Protein deprivation causes a delay in neuronal maturation but postnatal recovery can almost completely restore the normal morphology of myenteric neurons.

柑桔衰退病毒相关研究进展

柑桔衰退病毒引起的柑桔衰退病是一种广泛分布于世界各柑桔主产区的重要柑桔病毒病害,对柑桔产业的危害极大。随着我国柑桔产业的快速发展,近年来柑桔衰退病在我国多个柑桔产区暴发,造成了严重的经济损失。为了给深入开展柑桔衰退病毒研究和有效防控柑桔衰退病提供参考,根据已有研究文献报道,就柑桔衰退病毒的起源、类型、传播方式、蛋白功能、病毒与寄主的互作关系、茎陷点症状形成、蚜传机理,以及防治方法进行了综述。

Evidence for transmission of aphid-pathogenic fungi by migratory flight of Myzus persicae alates

The alates of the green peach aphid, Myzus persicae, were daily trapped from the air from late October through early January and carried to laboratory for determination of fungal infection by individually rearing them for 7 d on detached cabbage leaves in Petri dishes. Among 760 alates trapped, 266 (35%) were found carrying various fungal pathogens, 87.3% of them died due to mycosis during the first 3-day period of rearing and the rest died in the following two days. Most of the deaths of the alates were attributed to entomophthoralean fungi, taking 94.4%, and the rest were the hyphomycetous fungus Beauveria bassiana. Among the Entomophthorales-killed alates, P. neoaphidis took a proportion of 66.1%, Z. anhuiensis 22.6%, E. planchoniana 9.7%, and N. fresenii 1.6%, respectively. Two alates were found suffering from cross infection of two fungal species, i.e. P. neoaphidis with Z. anhuiensis and N. fresenii, respectively. The results represent the first report on transmission of aphid-pathogenic fungi

Phylogeography,Transmission,and Viral Proteins of Nipah Virus

Nipah virus (NiV), a zoonotic paramyxovirus belonging to the genus Henipavirus, is classified as a Biosafety Level-4 pathogen based on its high pathogenicity in humans and the lack of available vaccines or therapeutics. Since its initial emergence in 1998 in Malaysia, this virus has become a great threat to domestic animals and humans. Sporadic outbreaks and person-to-person transmission over the past two decades have resulted in hundreds of human fatalities. Epidemiological surveys have shown that NiV is distributed in Asia, Africa, and the South Pacific Ocean, and is transmitted by its natural reservoir, Pteropid bats. Numerous efforts have been made to analyze viral protein function and structure to develop feasible strategies for drug design. Increasing surveillance and preventative measures for the viral infectious disease are urgently needed.

烟草扭脉病毒云南分离物rtp基因克隆及序列分析

探究来自云南不同时间和不同地区的烟草扭脉病毒(TVDV)各分离物间rtp基因序列差异,分析烟草丛顶病近年在田间发生趋势减缓的原因.以2020—2022年从云南楚雄、大理、保山及红河采集的染病烟草样品为材料,对TVDV的rtp基因进行扩增和序列分析,共得到TVDV 7个不同分离物的rtp基因序列,长度均为2172 nt,包含618 nt的cp基因及1554 nt的rtd区;推导的TVDVRTP蛋白共编码722个氨基酸,其中包含CP蛋白的205个氨基酸,RTD的517个氨基酸.多重序列比对结果发现,获得的7个TVDV分离物与GenBank上登录的其他TVDV分离物间cp基因核苷酸序列一致性为98.71%~100%,不同TVDV分离物间CP蛋白的氨基酸序列一致性为98.05%~100%,存在4个氨基酸的差异.获得的7个TVDV分离物与GenBank数据库登录的其他TVDV分离物间rtd区核苷酸序列一致性为96.68%~99.81%,RTD蛋白的氨基酸序列一致性为96.52%~100%,存在39个氨基酸的差异.这些TVDV分离物rtp基因的变异可能会对TVDV的蚜虫传播效率产生影响,进而影响到烟草丛顶病在田间的发生与流行.

实现生物单分子成像的液相扫描电镜新技术

细胞膜蛋白的定位和分布在疾病发生发展中起着重要的作用,而原位表征纳米级膜蛋白需要单分子水平技术。为实现生物样品的原位液态环境高分辨成像,发展了一套基于扫描电镜液体环境的单分子成像技术。该技术采用扫描透射成像装置和商业化液体芯片装置组合,利用该技术观察了三阴性乳腺癌细胞膜蛋白表皮生长因子受体的精确定位、分布和聚合态。结果表明,采用高真空环境大约10~(-4) Pa、加速电压30 kV、束流约10~(-10) A实验参数,液体环境扫描透射成像模式,图像分辨率达到2~3 nm。标定膜蛋白的量子点ZnS@CdSe和Au纳米颗粒,具有相对于生物和液体更高的原子序数,因此在成像中形成了更好的衬度和信噪比。该技术为解决细胞生物学领域的高分辨单分子成像提供了一种先进的原位研究方法和直观观测平台。

PEDV、TGEV与PDCoV S蛋白表位基因三联疫苗的构建及其鉴定

[目的]构建一种同时预防猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪德尔塔冠状病毒(PDCoV)的三联表位疫苗,以预防上述病原导致的猪的相关疾病。[方法]本研究运用免疫信息学在线软件对3种猪肠道冠状病毒(SeCoVs)的S蛋白B、T细胞表位进行预测分析,构建新的表位肽段,命名为PPT,通过ExPASy、VaxiJen、TMHMM、SOPMA、SOLpro和AlphaFlod2等在线软件对PPT进行生物信息学分析,并利用C-IMMSIM在线软件对其免疫反应进行模拟。通过T2A将PPT与PEDV的COE序列、TGEV的SAD序列及PDCoV的CTD序列连接并构建至真核表达载体pEGFP-N1,经PCR和双酶切鉴定正确后,将获得的重组质粒转染HEK293A细胞,经DAPI染色、CCK8、RT-PCR及Western blotting试验验证重组质粒在体外表达情况。[结果]构建的表位蛋白PPT由17条表位肽段组成,经生物信息学软件分析,该蛋白为非跨膜蛋白,结构稳定,具有抗原性和可溶性,亲水性高,无过敏性。C-IMMSIM结果显示,PPT能引起机体树突状细胞(DC)增加,B、T细胞免疫反应,刺激免疫球蛋白IgG、IgM和细胞因子γ-干扰素(IFN-γ)、白细胞介素-2(IL-2)水平升高。重组质粒经PCR和双酶切鉴定结果显示,分别在3359、2375、1064、944、764和467 bp出现特异性目的条带,与预期相符;重组质粒转染HEK293A细胞后,可见绿色荧光;RT-PCR扩增分别在3359、2375、1064、944、764和467 bp处获得与目的基因大小相符的条带;CCK-8检测结果表明,重组质粒对细胞均无明显毒性作用;Western blotting检测结果显示,分别在31.7、16.1、37.9和27.5 ku处出现与目的蛋白分子质量大小一致的条带,重组质粒成功在HEK293A细胞中表达。[结论]本研究基于计算机软件分析设计的PPT表位蛋白成功构建三联疫苗,且在体外表达,为评价PEDV-TGEV-PDCoV三联表位疫苗的免疫效果提供依据。

Leafhopper salivary carboxylesterase suppresses JA-Ile synthesis to facilitate initial arbovirus transmission in rice phloem

Plant jasmonoyl-L-isoleucine(JA-Ile)is a major defense signal against insect feeding,but whether or how insect salivary effectors suppress JA-Ile synthesis and thus facilitate viral transmission in the plant phloem remains elusive.Insect carboxylesterases(CarEs)are the third major family of detoxification enzymes.Here,we identify a new leafhopper CarE,CarE10,that is specifically expressed in salivary glands and is secreted into the rice phloem as a saliva component.Leafhopper CarE10 directly binds to rice jasmonate resistant 1(JAR1)and promotes its degradation by the proteasome system.Moreover,the direct association of CarE10 with JAR1 clearly impairs JAR1 enzyme activity for conversion of JA to JA-Ile in an in vitro JAIle synthesis system.A devastating rice reovirus activates and promotes the co-secretion of virions and CarE10 via virus-induced vesicles into the saliva-storing salivary cavities of the leafhopper vector and ultimately into the rice phloem to establish initial infection.Furthermore,a virus-mediated increase in CarE10 secretion or overexpression of CarE10 in transgenic rice plants causes reduced levels of JAR1 and thus suppresses JA-Ile synthesis,promoting host attractiveness to insect vectors and facilitating initial viral transmission.Our findings provide insight into how the insect salivary protein CarE10 suppresses host JA-Ile synthesis to promote initial virus transmission in the rice phloem.

Change of cholinergic transmission and memory deficiency induced by injection of β-amyloid protein into NBM of rats

The change of cholinergic transmission of p-amyloid protein (P-AP) treated rats was studied by intracerebral microdialysis sampling combined with HPLC analysis. β-AP1-40 was injected into nucleus basalis magnocellularis (NBM). Passive avoidance response test (step-down test) and delayed alternation task were used for memory testing. The impairment of memory after injection of β-AP1-40 into NBM exhibited mainly the deficiency of short-term working memory. One week after injection of β-AP1-40 the release of acetylcholine (ACh) from frontal cortex of freely-moving rats decreased significantly, and the response of cholinergic nerve ending to the action of high [K+] solution was rather weak. In control animals the percentage of increase of ACh-release during behavioral performance was 57%, while in β-AP1-40-treated rats it was 34%. The temporary increase of the ACh-release of the rat put into a new place was also significantly diminished in β-AP1-40 -treated rats. The results show that the injection of β-AP1-40 into NBM impairs the cholinergic transmission in frontal cortex, and the impairment of cholinergic transmission may be the main cause of the deficit of working memory.

DEAD-box解旋酶21对猪传染性胃肠炎病毒复制的调控作用

旨在探究DEAD-box解旋酶21(DDX21)对猪传染性胃肠炎病毒(TGEV)复制的调控作用。首先通过蛋白免疫印迹(Western Blot)分析了TGEV感染对DDX21表达的影响;进一步构建了猪DDX21真核表达质粒以及建立稳定敲低DDX21的PK-15细胞系,运用荧光定量PCR(RT-qPCR)、蛋白免疫印迹、间接免疫荧光(IFA)和半数细胞培养物感染量(TCID 50)探究外源过表达DDX21及敲低DDX21对TGEV体外复制的调控作用;构建一系列DDX21截短突变体真核表达质粒,鉴定了DDX21调控TGEV增殖的关键功能域。Western Blot分析显示,在TGEV WH-1株感染早期,PK-15细胞内源性DDX21蛋白的表达水平显著上调;RT-qPCR、Western Blot、IFA和TCID 50试验显示,超表达DDX21可以显著提高TGEV N基因的mRNA水平、N蛋白的表达及病毒滴度,且呈剂量依赖性,其601-784 aa区域是其影响TGEV复制的关键;与阴性对照相比,在相应DDX21敲低细胞系中TGEV的增殖显著被抑制,而回补试验逆转了DDX21敲低细胞系中TGEV的滴度。该研究首次揭示了猪DDX21对TGEV增殖的促进作用并鉴定了其调控TGEV复制的关键结构域,为今后研究DDX21蛋白的功能及TGEV的致病机理提供了理论依据。

Interspecies transmission and evolution of the emerging coronaviruses: perspectives from bat physiology and protein spatial structure

Emergent coronaviruses(CoVs)such as SARS-CoV and MERS-CoV have posed great threats to public health worldwide over the past two decades.Currently,the emergence of SARS-CoV-2 as a pandemic causes greater public health concern.CoV diversity is due to the large size and replication mechanisms of the genomes together with having bats as their optimum natural hosts.The ecological behavior and unique immune characteristics of bats are optimal for the homologous recombination of CoVs.The relationship of spatial structural characteristics of the spike protein,a protein that is critical for recognition by host receptors,in different CoVs may provide evidence in explaining the coevolution of CoVs and their hosts.This information may help to enhance our understanding of CoV evolution and thus provide part of the basis of preparations for any future outbreaks.