The microRNA pathway activation in insect cell model upon Autographa californica multiple nucleopolyhedrovirus infection

Background:The immune system of insects exerts fundamentally different antiviral mechanisms than mammals.MicroRNAs(miRNAs)play vital roles in developing insect antiviral immunity.MiRNAs expression profiles of insects changed significantly during baculovirus infection.Methods:Differential expression profiles of miRNAs in Spodoptera frugiperda were monitored by next-generation sequencing(NGS)and RT-qPCR during Autographa californica multiple nucleopolyhedrovirus(AcMNPV)infection.The transcription levels of genes were detected by RT-qPCR.The 50%tissue culture infective dose(TCID_(50))endpoint dilution assay was used to determine the proliferation of progeny virus.Results:NGS revealed that 49 miRNAs were differentially expressed in Sf9 cells,and 10 of them were significantly up-or down-regulated.Though RT-qPCR analysis,we observed the similar trends for the expression patterns of significantly differentially expressed miRNAs from NGS.Moreover,the transcription levels of core genes,Exportin5,Dicer1,and Argonaute1,in miRNA biogenesis pathways were significantly increased after AcMNPV infection.For five selected miRNAs,miR-34-5p could regulate the proliferation of baculovirus progeny virus and energy metabolism.Conclusion:The miRNAs biogenesis pathway in Sf9 cells plays an important role and may be stimulated to resist AcMNPV infection.This work provides evidence for the molecular mechanism of baculovirus-insect interaction and offers novel ideas and directions for green pest control technology.

Application of Spodoptera litura Nucleopolyhedrovirus for Crop Pest Control

The laboratory bioassay and field control efficacy of Spodoptera litura nucleopolyhedrovirus（Spli NPV） Chenzhou strain were preliminarily examined. The efficient artificial propagation method was to feed the host larvae with virus suspension,and the average mortality of the insects was 65.0%. The death peak of the pests appeared 4-8 d after virus infection. The high temperature, high humidity and poor light could help the virus infection and propagation. Filed control efficacy of Chenzhou strain was 86.6% in laboratory, which was better than of another commercial strain. The corrected control efficacy of this strain was 88.4% the field, which was higher than that of avermectin pesticide significantly. It was detected that the occlusion body（OB） concentration of the initial virus＇ s stock solution was 1.03×1011OBs/ml,and it was a strong SpliNPV strain, as it showed an excellent efficacy to control the pest Spodoptera litura, and thus there will be a good prospect of application and development of this SpliNPV strain.

AcMNPV e18基因酵母双杂交诱饵载体构建和转录自激活检测

用PCR方法扩增苜蓿银纹夜蛾核多角体病毒(Autographa californica multiple nucleopolyhe-drovirus,AcMNPV)被膜蛋白ODV-E18基因,克隆至酵母双杂交诱饵载体pGBKT7构建诱饵质粒pG-BKT7-e18。将诱饵质粒分别转化酵母菌株Y187和AH109感受态细胞,被转化细胞在涂有X-gal的SD/-Trp营养缺陷型固体培养基上形成白色菌落;在SD/-Trp/-His和SD/-Trp/-Ade固体培养基上均不形成菌落,表明诱饵基因表达产物BD-E18在这两种细胞中都不能激活报告基因转录。pGBKT7-e18转化的Y187细胞在SD/-Trp营养缺陷型液体培养基中的生长速度与空载体转化细胞相同,显示BD-E18对酵母细胞无细胞毒性。结果表明,AcMNPV ODV-E18可能不直接参与对宿主细胞或病毒基因表达的调节,其编码基因可以作为诱饵基因通过筛查病毒宿主cDNA文库识别与其相互作用的蛋白质。

Baculovirus per os Infectivity Factors Are Involved in HearNPV ODVs Infection of HzAM1 Cells in vitro

Baculoviruses produce two viral phenotypes, the budded virus （BV） and the occlusion-derived virus （ODV）. ODVs are released from occlusion bodies in the midgut where they initiate a primary infection. Due to the lack of an in vitro system, the molecular mechanism of ODV infection is still unclear. Here we present data demonstrating that Helicoverpa armigera nucleopolyhedrovirus （HearNPV） ODV infected cultured Hz-AM1 cells in a pH dependent manner. The optimal pH for ODV infection was 8.5, which is same to that in the microvilli of midgut epithelial cells, the ODV native infection sites. Antibodies neutralization analysis indicated that four HearNPV oral infection essential genes p74, pif-l, pif-2 and pif-3 are also essential for HearNPV ODV infection in vitro. Thus, HearNPV-HzAM1 system can be used to analyze the mechanism of ODV entry.

Encyclopedia of Autographa californica Nucleopolyhedrovirus Genes

The Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) was the first baculovirus for which the complete nucleotide sequence became known. Since then 15 years lapsed and much research has been performed to elucidate putative functions of the annotated open reading frames of this virus and this endeavour is still ongoing. AcMNPV is the most well-known and well-studied baculovirus species, not in the least for its application as a vector for the high-level expression of foreign genes in insect cells. This article is the first monograph of a single baculovirus and gives a current overview of what is known about the 151 AcMNPV ORFs, including (putative) function and temporal and spatial presence of transcripts and protein. To date 60 ORFs have a proven function, another 19 ORFs have homologs for which functions are known in other baculoviruses and 72 ORFs are still enigmatic. This paper should assist the reader in quickly finding the essentials of AcMNPV.

The ORF 113 of Heliocoverpa armigera Single Nucleopolyhedrovirus Encodes a Functional Fibroblast Growth Factor

Fibroblast growth factor (FGF) is a key regulator of developmental processes. A FGF homolog (vFGF) is found in all lepidopteran baculoviruses. Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) vFGFs are chemotactic factors. Here we analyzed the vfgf of Helicoverpa armigera NPV (HearNPV), a group II NPV. The HearNPV vfgf transcripts were detected from 18 to 96 h post-infection (hpi) of Hz-AM1 cells with HearNPV and encoded a 36 kDa protein, which was secreted into the culture medium. HearNPV vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. Unlike its AcMNPV homolog, HearNPV vFGF specially chemoattracted Hz-AM1, but not other insect cells such as Sf9 and Se-UCR and not the mammalian cells 293 and HepG2. HearNPV vFGF is also associated with the envelope of BV but is absent in occlusion-derived virus, which coordinated to the chemotatic activity analysis.

Involvement of Lipid Rafts and Cellular Actin in AcMNPV GP64 Distribution and Virus Budding

GP64 is the major envelope glycoprotein associated with the budded virus (BV) of Autographa californica nucleopolyhedrovirus (AcMNPV) and is essential for attachment and budding of BV particles. Confocal microscopy and flotation assays established the presence of lipid raft domains within the plasma membranes of AcMNPV-infected Sf9 cells and suggested the association of GP64 with lipid rafts during infection. GP64 and filamentous actin (F-actin) were found to co-localise at the cell cortex at 24 and 48 hpi and an additional restructuring of F-actin during infection was visualised, resulting in a strongly polarised distribution of both F-actin and GP64 at the cell cortex. Depletion of F-actin, achieved by treatment of Sf9 cells with latrunculin B (LB), resulted in the redistribution of GP64 with significant cytoplasmic aggregation and reduced presence at the plasma membrane. Treatment with LB also resulted in reduced production of BV in Sf9 cells. Analysis of virus gene transcription confirmed this reduction was not due to decreased trafficking of nucleocapsids to the nucleus or to decreased production of infectious progeny nucleocapsids. Reduced BV production due to a lack of GP64 at the plasma membrane of AcMNPV-infected Sf9 cells treated with LB, suggests a key role for F-actin in the egress of BV.

Entry into Midgut Epithelial Cells is a Key Step in the Selection of Genotypes in a Nucleopolyhedrovirus

An isolate of the Spodoptera frugiperda multiple nucleopolyhedrovirus comprises a stable proportion of deletion genotypes （e.g., SfNIC-C）, that lack pifl and pif2 rendering them noninfectious per os, and that survive by complementation with a complete genotype （SfNIC-B） in coinfected cells. To determine whether selection for particular ratios of complete and deletion genotypes occurs mainly during the establishment of the primary infection in insect midgut cells or during subsequent systemic infection, we examined genotype frequencies in insects that fed on OBs comprising different co-occluded mixtures of genotypes. Dramatic changes in genotype frequencies were observed between the OB inoculum and budded virus （BV） samples taken from larvae inoculated with OBs comprising 10% SfNIC-B ＋ 90% SfNIC-C indicating that a marked reduction of SfNIC-C genotype had occurred in the insect midgut due to the immediate elimination of all OBs that originated from cells that had been infected only by SfNIC-C. In contrast, immediate changes were not observed in OBs comprising mixtures of 50% SfNIC-B ＋ 50% SfNIC-C or those comprising 10% SfNIC-B ＋ 90% SfNIC-C as most of the OBs in these mixtures originated from cells that had been infected by both genotypes. Subsequent changes in genotypic frequencies during five days of systemic infection were fairly small in magnitude for all genotypic mixtures. We conclude that the prevalence of defective genotypes in the SfNIC population is likely determined by a balance between host selection against OBs produced in cells infected by SfNIC-C alone and within-host selection for fast-replicating deletion genotypes. The strength of intra-host selection is likely modulated by changes in MOI during the infection period.

Putative Phosphorylation Sites On WCA Domain of HA2 Is Essential For Helicoverpa armigera Single Nucleopolyhedrovirus Replication

Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality.The Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) orf2-encoded nucleocapsid protein HA2 participates in orchestration of virus-induced actin polymerization through its WCA domain,in which phosphorylation status are supposed to be critical in respect to actin polymerization.In the present study,two putative phosphorylation sites (232Thr and 250Ser) and a highly conserved Serine (245Ser) on the WCA domain of HA2 were mutated,and their phenotypes were characterized by reintroducing the mutated HA2 into the HearNPV genome.Viral infectivity assays demonstrated that only the recombinant HearNPV bearing HA2 mutation at 245Ser can produce infectious virions,both 232Thr and 250Ser mutations were lethal to the virus.However,actin polymerization assay demonstrated that all the three viruses bearing HA2 mutations were still capable of initiating actin polymerization in the host nucleus,which indicated the putative phosphorylation sites on HA2 may contribute to HearNPV replication through another unidentified pathway.

Localization and Functional Analysis of SeMNPV IE1 in Mammalian Cells

In this paper, the function of the iel gene from baculovirus Spodoptera exigua multiple nucleopolyhedrovirus （SeMNPV）, belonging to group II nucleopolyhedrovirus, was studied in mammalian cells We amplified the SeMNPV iel gene and expressed it by fusing to the C terminal of enhanced GFP protein in HEK 293 cells. Confocal microscopy revealed that the IE1-GFP fusion protein was localized in the nucleus of the mammalian cells. The promoter sequences of AcMNPV gp64, SeMNPV F protein and Drosophila hsp70 were also analyzed, to further study the function of SeMNPV IE1. The results showed that, in the absence of the hr sequence, IE1 improved the expression of the F promoter but didn＇t influence the gp64 promoter significantly, but IE1 moderately stimulated the hsp70 promoter.

Polyhedrosis Virus in Bacillus thuringiensis

This study was conducted to build a recombinant strain with highly insecticidal activity and a wide host range by using the crylAc and p74 gene. Firstly, the p74 gene was amplified from the genosome ofAutographa californica multicapsid nucleopolyhedrovirus. The crylAc gene and the terminator gene of crylAc, named crylAct, were amplified from the plasmid of Bt 4.0718 strain. Three T vectors, named pTp74, pT1Ac, and pT1Act which held the aimed gene p74, cry1Ac, and crylAct, respectively, and two middle vectors, named pTp74Act and pTIAcp74 which held the aimed fusion gene p74-crylAct and cry lAc-p74, respectively, were built by using pMDI 8-T. Then pTiAcp74 and the shuttle plasmid were digested and linked and an expressing-vector pH1Acp74 was built. Finally, pH1Acp74 was transformed into the acrystalliferous strain XBU001 and the aimed recombinant strain XBU-H1Acp74 was obtained. The expression of Bt transformant XBU-H1Acp74 was analyzed by SDS-PAGE which showed XBU-H1Acp74 could produce 130 kDa CrylAc protein and 50 kDa P74 protein. The insecticidal activity of transformant against Spodoptera exigua was evaluated compared with the contrast strains HTX-42 （only crylAc gene was transformed into XBU001） after autolysis. The LCs0 of HTX-42 was higher than that of the XBU-H IAcp74＇s, which implied that P74 could increase the efficacy and range of Bt Cry toxins in insect control. The fusion gene of crylAc and p74 were constructed successfully which will be served as the foundation lbr constructing the fusion genes of Bt cry gene and other foreign genes.

BmCH25H,a vertebrate interferon-stimulated gene(ISG)homolog,inhibits BmNPV infection dependent on its hydroxylase activity in Bombyx mori

Cholesterol-25-hydroxylase(CH25H)has been identified as an interferon-stimulated gene(ISG)in mammals that exerts its antiviral effects by catalyzing the conversion of cholesterol to 25-hydroxycholesterol(25HC).However,invertebrates lack an antiviral system homologous to vertebrate interferons(IFNs)because the genomes of invertebrates do not encode IFN-like cytokines.Nevertheless,CH25H is present in insect genomes and it therefore deserves further study of whether and by which mechanism it could exert an antiviral effect in invertebrates.In this study,the Bombyx mori CH25H(BmCH25H)gene,of which the encoded protein has high homology with other lepidopteran species,was identified and located on chromosome 9.Interestingly,we found that the expression of BmCH25H was significantly upregulated in B.mori nucleopolyhedrovirus(BmNPV)-infected BmN cells and silkworm(B.mori)larvae at the early infection stage.The inhibitory effect of BmCH25H on BmNPV replication was further demonstrated to depend on its catalytic residues to convert cholesterol to 25HC.More importantly,we demonstrated that during BmNPV infection,BmCH25H expression was increased through the Janus kinase–signal transducer and activator of transcription(JAK–STAT)pathway,similar to the induction of ISGs following virus infection in vertebrates.This is the first report that CH25H has antiviral effects in insects;the study also elucidates the regulation of its expression and its mechanism of action.

The piRNA pathway is required for nucleopolyhedrovirus replication in Lepidoptera

The Piwi-interacting RNA(piRNA)pathway has been shown to be involved in the antiviral defense against RNA viruses,especially in mosquitoes,but its universality has been questioned.Here,we used the Bombyx mori nucleopolyhedrovirus(BmNPV)-infected silkworm as a model to explore the effects of the key factors of piRNA pathway,BmAgo3 and Siwi,on replication of a large DNA virus(belonging to the family of Baculoviridae).We demonstrated that BmAgo3 and Siwi could promote the replication of BmNPV through both overexpression and knockdown experiments in BmN cell lines and silkworm larvae.In addition,we also studied the effect of PIWI-class genes on Autographa californica nucleopolyhedrovirus(AcMNPV)replication in the Spodoptera frugiperda cell line Sf9.By knocking down the expression of PIWI-class genes in Sf9,we found that Piwi-like-1 and Piwi-like-2-3 could inhibit AcMNPV replication,while Piwi-like-4-5 promoted virus replication.Our study provides compelling evidence that the piRNA pathway affects host infection by exogenous viruses in Lepidoptera.Also,our results reflect the diversity of the roles of PIWI-class genes in virus infection of the host across species.This study is the first to explore the interaction of PIWI-class proteins with DNA viruses,providing new insights into the functional roles of the piRNA pathway.

Antiviral function of peptidoglycan recognition protein in Spodoptera exigua(Lepidoptera:Noctuidae)

Peptidoglycan recognition proteins(PGRPs)are a class of molecules that play a critical role in insect immunity.Understanding the function of PGRPs is important to improve the efficiency of microbial insecticides.In this study,we investigated the role of PGRP-LB(a long type PGRP)in insect immunity against viruses using Spodoptera exigua and Spodoptera exigua multiple nucleopolyhedrovirus(SeMNPV)as an insect-virus model.We cloned and identified a PGRP-LB gene from S.exigua;the gene consisted of 7 exons that encoded a polypeptide of 234 amino acids with a signal peptide and a typical amidase domain.Expression analysis revealed that the abundance of SePGRP-LB transcripts in the fat body was greater than in other tissues.Overexpression of SePGRP-LB resulted in a significant decrease of 49%in the rate of SeMNPV-infected cells.In addition,the multiplication of SeMNPV was significantly decreased:a decrease of 79%in the production of occlusion-derived virion(ODV),and a maximum decrease of 50%in the production of budded virion(BV).In contrast,silencing of SePGRP-LB expression by RNA interference resulted in a significant 1.65-fold increase in the rate of SeMNPV-infected cells,a significant 0.54-fold increase in ODV production,a maximum 1.57-fold increase in BV production,and the larval survival dropped to 21%.Our findings show that SePGRP-LB has an antiviral function against SeMNPV,and therefore this gene may provide a target for lepidopteran pest control using virus insecticides.

表达蝎毒素的重组斜纹夜蛾核型多角体病毒(SpltMNPV)的构建及毒力

【目的】开发高毒力的重组斜纹夜蛾核型多角体病毒(Spodoptera litura multicapsid nucleopolyhedroviruse,SpltMNPV)杀虫剂。【方法】构建编码蜕皮激素UDP-葡萄糖基转移酶(ecdysterioid UDP-glucosyl transferase gene,egt)基因缺失并插入东亚钳蝎神经毒素(B.martensi Karsch,BmK ITa1)基因的重组转移载体,重组转移载体与SpltMNPVⅡ基因组DNA共转染斜纹夜蛾细胞,通过荧光斑法与有限稀释法相结合筛选重组病毒。【结果】成功筛选出缺失egt基因的、早期启动子(ie-1)启动的、表达BmK ITa1成熟肽的重组病毒SpltMNPV-Δegt-Pph-egfp-ie-1-BmK ITa1。生物测定结果显示,重组病毒的杀虫速度(LT50)较野生病毒提前0.7-0.8 d。【结论】通过在SpltMNPV病毒基因组中插入外源毒素基因可明显增强病毒的杀虫效果,结果说明开发高毒力SpltMNPV生物杀虫剂具有可行性。

草地贪夜蛾Sf9细胞系Arp2/3-p40/ARPC1亚基基因的克隆与功能研究

杆状病毒感染诱导的昆虫宿主细胞肌动蛋白聚合主要由病毒编码的Wiskott Aldrich综合征蛋白(Wiskott Aldrichsyndrome Protein,WASP)同源蛋白P78/83激活宿主细胞内的Arp2/3复合物,进而引起肌动蛋白单体(G-actin)聚合成为纤维状肌动蛋白(F-actin)。为了研究Arp2/3复合物在病毒感染过程中所发挥的作用,我们克隆了昆虫Sf9细胞的Arp2/3复合体P40亚基基因,并对其预期产物进行了生物信息学与功能分析。我们发现在病毒感染过程中,P40亚基由细胞质转移到核膜的内缘,与本实验室过去报道的病毒感染后期核内F-actin的分布相吻合,提示P40可以很好地用于研究Arp2/3复合体的亚细胞定位情况;通过免疫共沉淀(Co-IP)实验,还证实病毒感染可以促进P40和P78/83的相互作用,提示某些未知病毒因素参与调控了由P78/83和Arp2/3复合体介导的肌动蛋白聚合过程。

Improving Baculovirus Transduction of Mammalian Cells by Incorporation of Thogotovirus Glycoproteins

Baculovirus can transduce a wide range of mammalian cells and is considered a promising gene therapy vector. However,the low transduction efficiency of baculovirus into many mammalian cells limits its practical application. Co-expressing heterologous viral glycoproteins(GPs), such as vesicular stomatitis virus G protein(VSV G), with baculovirus native envelope protein GP64 is one of the feasible strategies for improving virus transduction. Tick-borne thogotoviruses infect mammals and their GPs share sequence/structure homology and common evolutionary origins with baculovirus GP64.Herein, we tested whether thogotovirus GPs could facilitate the entry of the prototype baculovirus Autographa californica multiple multiple nucleopolyhedrovirus(AcMNPV) into mammalian cells. The gp genes of two thogotoviruses, Thogoto virus and Dhori virus, were inserted into the AcMNPV genome. Both GPs were properly expressed and incorporated into the envelope of the recombinant AcMNPVs. The transduction rates of recombinant AcMNPVs expressing the two thogotovirus GPs increased for approximately 4–12 fold compared to the wild type AcMNPV in six of the 12 tested mammalian cell lines. It seemed that thogotovirus GPs provide the recombinant AcMNPVs with different cell tropisms and showed better performance in several mammalian cells compared to VSV G incorporated AcMNPV. Further studies showed that the improved transduction was a result of augmented virus-endosome fusion and endosome escaping, rather than increased cell binding or internalization. We found the AcMNPV envelope protein GP64-mediated fusion was enhanced by the thogotovirus GPs at relatively higher p H conditions. Therefore, the thogotovirus GPs represent novel candidates to improve baculovirus-based gene delivery vectors.

Expression and immunocytochemical analysis of Autographa californica nucleopolyhedrovirus （AcMNPV） orf74 gene

Autographa californica nucleopolyhedrovirus off74 （Ac74） is located between 62 311 and 63 108bp in the AcMNPV genome, which encodes 265 amino acid residues with a predicted 31 kDa molecular weight. The homologues of Ac74 were searched using BLASTP in protein databases, GenBank/EMBL and SWISS-PROT. The result revealed that deduced Ac74 protein was homologous to the predicted products from 10 lepidoptera NPV ORFs. The multiple sequence alignments of Ac74 and its 10 homologues manifested only one amino acid residue was completely conserved. The transcript analysis revealed that the transcript of Ac74 was detected from 24-72 hours post-infection （hpi）. The product of Ac74 was detected at 24 hpi and lasted until 72 hpi by Western blot using anti-Ac74 antiserum, consistent with reverse transcriptase polymerase chain reaction results. These results suggested Ac74 was expressed during the later stages of infection. The product of Ac74 was 31kDa in size, consistent with predicted molecular weight. The subcellular localization of Ac74 proteins manifested Ac74 protein in the cytoplasm, and was hardly present in the nucleus at 24 hpi. The fluorescence was also observed in polyhedra, except cytoplasm at 72 hpi. Together, Ac74 is a functional protein with 31kDa molecular weight and is located in the cytoplasm and the polyhedra.

Host permissiveness to baculovirus in flue nces time-dependent immune responses and fitness costs

Insects possess specific immune responses to protect themselves from different types of pathogens.Activation of immune cascades can inflict significant developmental costs on the surviving host.To characterize infection kinetics in a surviving host that experiences baculovirus inoculation,it is crucial to determine the timing of immune responses.Here,we investigated time-dependent immune responses and developmental costs elicited by inoculations from each of two wild-type baculoviruses,Autographa californica multiple nucleopolyhedrovirus(AcMNPV)and Helicoverpa zea single nucleopolyhedrovirus(HzSNPV),in their common host H.zea.As H.zea is a semi-permissive host of AcMNPV and fully permissive to HzSNPV,we hypothesized there are differential immune responses and fitness costs associated with resisting infection by each virus species.Newly molted 4th-instar larvae that were inoculated with a low dose(LD15)of either virus showed signify icantly higher hemolymph FAD-glucose dehydrogenase(GLD)activities compared to the corresponding control larvae.Hemolymph phenoloxidase(PO)activity,protein concentration and total hemocyte numbers were not increased,but instead were lower than in control larvae at some time points post-inoculation.Larvae that survived either virus inoculation exhibited reduced pupal weight;survivors inoculated with AcMNPV grew slower than the control larvae,while survivors of HzSNPV pupated earlier than control larvae.Our results highlight the complexity of immune responses and fitness costs associated with combating different baculoviruses.

Analysis of a late gene, orfl01 from Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirns open reading frame 101 （ha101） is 762 nts in length and encodes a 254 amino acid peptide with predicted 29 kDa molecular weight. The homologues of ha101 were explored using BLASTP searching tool in the updated GenBank/EMBL and SWlSS-PROT databases. The results showed that the homologues of ha101 were present in all the completely sequenced lepidopteran nucleopolyhedrovirnses and granulovirnses, suggesting that ha101 might be a functional gene associated with their lepidopteran hosts. Sequence alignment of ha101 and its homologues revealed that 10 amino acids were completely conserved. RT-PCR analysis of ha101 manifested that the transcript of ha101 was first detected at 24 hpi and remained detectable at up to 122 hpi, suggesting that ha101 was transcribed during late stages of infection. Ha101 was expressed using Bac to Bac system in Tn5B-1-4 cells. The product of ha101 expressed in Tn5B-1-4 cells was approximately 29kDa, consistent with the predicted molecular weight, and the results were confirmed by western blot analysis. The subcellular localization indicated that ha101 was aggregated along nuclear envelope during the early stages of infection and spread out to the entire nucleus including virogenic stroma in late stages of infection, suggesting that ha101 may play a specific role in virion assembly process or virogenic stroma arrangement.