Abdominal-B contributes to abdominal identity in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

The homeotic complex gene Abdominal-B (Abd-B) is involved in regulating the development of posterior abdomens and has been extensively studied in holometabolous insects. However, the function of Abd-B in hemimetabolous insects is not fully understood. Here, we functionally characterize an Abd-B homologue in the brown planthopper (BPH), Nilaparvata lugens. The full-length cDNA of the N. lugens Abd-B homologue (NlAbd-B) is 2334 nt, with an open reading frame of 1113 bp. NlAbd-B has the highest expression level at the egg stage relative to the nymphal and adult stages and is mainly expressed in the fourth to the ninth abdominal segment of embryos. RNA interference (RNAi)-mediated knockdown of NlAbd-B in nymphs disrupted the development of genitalia both in females and males and caused a genitalia-to-leg transformation. Parental RNAi of NlAbd-B in both female and male adults caused an extra abdominal segment in offspring nymphs, while parental RNAi of the N. lugens abdominal-A homologue in both female and males adults led to embryos with leg-like appendages on the second to the eighth abdominal segment. These findings suggest that NlAbd-B plays a pivotal role in genital development and posterior abdominal patterning and thus highlight the conservational role of Abd-B in holometabolous and hemimetabolous insects.

A SARS-CoV-2 variant with the 12-bp deletion at E gene.

The coronavirus disease 2019 (COVID-19) pandemic is still ongoing and has become an important public health threat. This disease is caused by a new coronavirus named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, and so far, little is known about this virus. In this study, by using plaque purification, we purified two SARS-CoV-2 virus strains from the same specimen, one named F8 containing a 12-bp deletion in the E gene and the other named 8X containing the wild-type E gene. There was no significant difference in the viral titer and infectivity of these two strains. The S protein content of the F8 viral culture was 0.39 µg/ml, much higher than that of 8X. An inactivated vaccine made from the F8 strain could trigger high levels of the IgG titer and neutralizing antibody titer, which could last for at least 6 weeks and were significantly higher than those from the 8X strain at 1 and 3 weeks post vaccination, respectively. In conclusion, we reported that both the E gene mutant and wild-type SARS-CoV-2 strains were isolated from the same clinical sample by plaque purification. A 12-bp deletion in the E gene was important for SARS-CoV-2 replication and immunogenicity.

An adult gerbil model for evaluating potential coxsackievirus A16 vaccine candidates.

A suitable animal model of CVA16 infection is crucial in order to understand its pathogenesis and to help develop antiviral vaccines or screen therapeutic drugs. The neonatal mouse model has a short sensitivity period to CA16 infection, which is a major limitation. In this study, we demonstrate that adult (60-day-old) gerbils are susceptible to CVA16 infection at high doses (108.0 TCID50). A clinical isolate strain of CVA16 was inoculated intraperitoneally into adult gerbils, which subsequently developed significant clinical symptoms, including hind limb weakness, paralysis of one or both hind limbs, tremors, and eventual death from neurological disorders. Real-time RT-PCR revealed that viral loads in the spinal cord and brainstem were higher than those in other organs/tissues. Histopathological changes, such as neuronal degeneration, neuronal loss, and neuronophagia, were observed in the spinal cord, brainstem, and heart muscle, along with necrotizing myositis. Gerbils receiving both prime and boost immunizations of alum adjuvant inactivated vaccine exhibited no clinical signs of disease or mortality following challenge by CVA16, whereas 80% of control animals showed obvious clinical signs, including slowness, paralysis of one or both hind limbs, and eventual death, suggesting that the CVA16 vaccine can fully protect gerbils against CVA16 challenge. These results demonstrate that an adult gerbil model provides us with a useful tool for studying the pathogenesis and evaluating antiviral reagents of CVA16 infection. The development of this animal model would also be conducive to screening promising CVA16 vaccine candidates as well as further vaccination evaluation.

Global Gene Expression Analysis of the Brainstem in EV71- and CVA16-Infected Gerbils.

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two most important pathogens of hand, foot, and mouth disease (HFMD). However, the neuropathogenesis of EV71 and CVA16 has not been elucidated. In our previous study, we established gerbils as a useful model for both EV71 and CVA16 infection. In this work, we used RNA-seq technology to analyze the global gene expression of the brainstem of EV71- and CVA16-infected gerbils. We found that 3434 genes were upregulated while 916 genes were downregulated in EV71-infected gerbils. In CVA16-infected gerbils, 1039 genes were upregulated, and 299 genes were downregulated. We also found significant dysregulation of cytokines, such as IP-10 and CXCL9, in the brainstem of gerbils. The expression levels of 10 of the most upregulated genes were confirmed by real-time RT-PCR, and the upregulated tendency of most genes was in accordance with the differential gene expression (DGE) results. Our work provided global gene expression analysis of virus-infected gerbils and laid a solid foundation for elucidating the neuropathogenesis mechanisms of EV71 and CVA16.

Risk Factors and Preventions of Breast Cancer.

Breast cancer is the second leading cause of cancer deaths among women. The development of breast cancer is a multi-step process involving multiple cell types, and its prevention remains challenging in the world. Early diagnosis of breast cancer is one of the best approaches to prevent this disease. In some developed countries, the 5-year relative survival rate of breast cancer patients is above 80% due to early prevention. In the recent decade, great progress has been made in the understanding of breast cancer as well as in the development of preventative methods. The pathogenesis and tumor drug-resistant mechanisms are revealed by discovering breast cancer stem cells, and many genes are found related to breast cancer. Currently, people have more drug options for the chemoprevention of breast cancer, while biological prevention has been recently developed to improve patients' quality of life. In this review, we will summarize key studies of pathogenesis, related genes, risk factors and preventative methods on breast cancer over the past years. These findings represent a small step in the long fight against breast cancer.

Coxsackievirus A16 induced neurological disorders in young gerbils which could serve as a new animal model for vaccine evaluation.

Coxsackievirus A16 (CA16) is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD) in the Asia-pacific region. Although CA16 infections are generally mild, severe neurological manifestations or even death has been reported. Studies on CA16 pathogenesis and vaccine development are severely hampered because the small animal models that are currently available show major limitations. In this study, gerbils (Meriones unguiculatus) were investigated for their suitability as an animal model to study CA16 pathogenesis and vaccine development. Our results showed that gerbils up to the age of 21 days were fully susceptible to CA16 and all died within five days post-infection. CA16 showed a tropism towards the skeletal muscle, spinal cord and brainstem of gerbils, and severe lesions, including necrosis, were observed. In addition, an inactivated CA16 whole-virus vaccine administrated to gerbils was able to provide full protection to the gerbils against lethal doses of CA16 strains. These results demonstrate that gerbils are a suitable animal model to study CA16 infection and vaccine development.

Insight into the Dual Functions of Bacterial Enhancer-Binding Protein Rrp2 of Borrelia burgdorferi.

UNLABELLED: It is well established that the RpoN-RpoS sigma factor (σ(54)-σ(S)) cascade plays an essential role in differential gene expression during the enzootic cycle of Borrelia burgdorferi, the causative agent of Lyme disease. The RpoN-RpoS pathway is activated by the response regulator/σ(54)-dependent activator (also called bacterial enhancer-binding protein [bEBP]) Rrp2. One unique feature of Rrp2 is that this activator is essential for cell replication, whereas RpoN-RpoS is dispensable for bacterial growth. How Rrp2 controls cell replication, a function that is independent of RpoN-RpoS, remains to be elucidated. In this study, by generating a series of conditional rrp2 mutant strains, we demonstrated that the N-terminal receiver domain of Rrp2 is required for spirochetal growth. Furthermore, a D52A point mutation at the phosphorylation site within the N terminus of Rrp2 abolished cell replication. Mutation of the ATPase motif within the central domain of Rrp2 did not affect spirochetal replication, indicating that phosphorylation-dependent ATPase activity of Rrp2 for σ(54) activation is not required for cell growth. However, deletion of the C-terminal domain or a 16-amino-acid truncation of the helix-turn-helix (HTH) DNA-binding motif within the C-terminal domain of Rrp2 abolished spirochetal replication. It was shown that constitutive expression of rpoS is deleterious to borrelial growth. We showed that the essential nature of Rrp2 is not due to an effect on rpoS These data suggest that phosphorylation-dependent oligomerization and DNA binding of Rrp2 likely function as a repressor, independently of the activation of σ(54), controlling an essential step of cell replication in B. burgdorferi IMPORTANCE: Bacterial enhancer-binding proteins (bEBPs) are a unique group of transcriptional activators specifically required for σ(54)-dependent gene transcription. This work demonstrates that the B. burgdorferi bEBP, Rrp2, has an additional function that is independent of σ(54), that of its essentiality for spirochetal growth, and such a function is dependent on its N-terminal signal domain and C-terminal DNA-binding domain. These findings expand our knowledge on bEBP and provide a foundation to further study the underlying mechanism of this new function of bEBP.

Enterovirus 71 infection causes severe pulmonary lesions in gerbils, meriones unguiculatus, which can be prevented by passive immunization with specific antisera.

Neurogenic pulmonary edema caused by severe brainstem encephalitis is the leading cause of death in young children infected by Enterovirus 71 (EV71). However, no pulmonary lesions have been found in EV71-infected transgenic or non-transgenic mouse models. Development of a suitable animal model is important for studying EV71 pathogenesis and assessing effect of therapeutic approaches. We had found neurological disorders in EV71-induced young gerbils previously. Here, we report severe pulmonary lesions characterized with pulmonary congestion and hemorrhage in a gerbil model for EV71 infection. In the EV71-infected gerbils, six 21-day-old or younger gerbils presented with a sudden onset of symptoms and rapid illness progression after inoculation with 1×105.5 TCID50 of EV71 via intraperitoneal (IP) or intramuscular (IM) route. Respiratory symptoms were observed along with interstitial pneumonia, pulmonary congestion and extensive lung hemorrhage could be detected in the lung tissues by histopathological examination. EV71 viral titer was found to be peak at late stages of infection. EV71-induced pulmonary lesions, together with severe neurological disorders were also observed in gerbils, accurately mimicking the disease process in EV71-infected patients. Passive transfer with immune sera from EV71 infected adult gerbils with a neutralizing antibody (GMT=89) prevented severe pulmonary lesion formation after lethal EV71 challenge. These results establish this gerbil model as a useful platform for studying the pathogenesis of EV71-induced pulmonary lesions, immunotherapy and antiviral drugs.

The composition and transmission of microbiome in hard tick, Ixodes persulcatus, during blood meal.

The tick Ixodes persulcatus is the predominant tick species in Northeastern China, and it is a major vector in transmission of tick-borne diseases. By 16S rRNA Illumina sequencing, we investigated the microbiome of I. persulcatus and assessed the variation of the microbiome before and after blood feeding. The prolonged blood meal dramatically altered the composition of the microbiome but did not influence the bacterial diversity. Overall, 373 and 289 bacterial genera were assigned to unfed and fed ticks, respectively. To investigate microbes that were potentially transmitted to vertebrate hosts during a blood meal, we examined the microbiome in rat blood after tick bites. Our data showed that 237 bacterial genera were suspected to be pathogens of vertebrates because they were commonly detected in unfed ticks, fed ticks, and rat blood samples after tick bites. Additionally, the prevalence survey on Borrelia burgdorferi s.l., Ehrlichia chaffeensis, Anaplasma phagocytophilum and Yersinia pestis was performed. We found that B. garinii and B. afzelii are the predominant genospecies of the Lyme disease spirochete in I. persulcatus ticks. This is the first time that the microbial composition in this tick species and in rat blood transmitted via tick bites has been reported. These data may ultimately assist in identification of novel pathogens transmitted by I. persulcatus ticks.

Enterovirus 71-induced neurological disorders in young gerbils, Meriones unguiculatus: development and application of a neurological disease model.

A reliable disease model mimicking Enterovirus 71 (EV71) infection in humans is essential for understanding pathogenesis and for developing a safe and effective vaccine. Commonly used rodent models including mouse or rat models are not suitable for vaccine evaluation because the rodents are resistant to EV71 infection after they reach the age of 6 days. In this study, 21-day-old gerbils inoculated intraperitoneally (IP) with a non mouse-adapted EV71 strain developed neurological lesion-related signs including hind limb paralysis, slowness, ataxia and lethargy similar to those of central nervous system (CNS) infection of EV71 in humans. The infected gerbils eventually died of the neurological lesions and EV71 could be isolated from lung, liver, spleen, kidney, heart, spinal cord, brain cortex, brainstem and skeletal muscle. Significantly high virus replication was detected in spinal cord, brainstem and skeletal muscle by cellular analysis, real-time quantitative PCR (RT-PCR) and immunohistochemical staining. Histopathologic changes such as neuronal degeneration, neuronal loss and neuronophagia were observed in spinal cord, brain cortex, brainstem, and skeletal muscle along with necrotizing myositis and splenic atrophy. Gerbils that received two doses of inactive whole-virus vaccine showed no EV71-specific symptoms after challenged with EV71. In contrast, gerbils that received mock vaccination died of EV71-induced neuropathology after challenged with EV71. The result indicates that gerbils can serve as a reliable disease model for evaluating safety and efficacy of EV71 vaccine.

[Genotype of hantavirus in Zhejiang province].

OBJECTIVE: To identify the genotype and clades of hantavirus (HV) in Zhejiang province. METHODS: The partial S and M segment of the HV in Zhejiang province were amplified with RT-PCR using genotype-specific primers, and then were sequenced and compared with other known hantaviruses. RESULTS: The genotype of 11 strains were HTNV and other 7 strains were SEOV by homology and phylogenesis analysis, yet the clade distribution was significantly different among foci of Zhejiang with 5 clades of HTNV and 3 clades of SEOV. There also existed special clade of HTNV named ZNB-1, ZNB-2, A3 and of SEOV named Gou3, ZJ5. The homology of M segments of ZNB-1 and ZNB-2 with other HTNV clades were 69.7%-74.0% except Nc167, A3 with other HTNV clades were 73.6%-76.3% except B78. CONCLUSION: Zhejiang province is co-circulating with HTN and SEO. Say the least of the clades are 5 of HTNV and 3 of SEOV and there also existed special clade of HTNV and SEOV.

[The expression and bioactivity of S gene in Vero-E6 cells using baculovirus vector].

OBJECTIVE: The S gene of a Hanta Virus (HV) Z10 strain was cloned into a baculovirus shuttle bacmid pDual-CMV contained a CMV promoter to generated a recombinant baculovirus BAC-pDual-CMV-HVS, then the recombinant baculovirus was transfected into Vero-E6 cell. The cells with recombinant baculovirus were applied to the detection of HV antiserum. METHODS: To generate the recombinant baculovirus BAC-pDual-CMV-HVS, the sequence of CMV promoter was obtained from the plasmid pEGFP-N1 by PCR, and subsequently cloned to the baculovirus shuttle bacmid pFastBacDUAL resulting the recombinant plasmid pDual-CMV. Then the sequence of HV-S gene was inserted to the plasmid pDual-CMV, to generate the plasmid pDual-CMV-HVS. Plasmid pDual-CMV-HVS was transformed into the DH10BAC competent cells to get the recombinant baculovirus BAC-pDual-CMV-HVS. The antigen substrate slides were made by transfecting the recombinant virus into Vero-E6 cells. RESULTS: The plasmid pDual-CMV-HVS was verified by sequencing. The recombinant virus BAC-pDual-CMV-HVS was generated according to the protocol of the baculovirus and transfected into Vero-E6 cells. The expression of the HV-S gene was verified by positive HV antiserum. CONCLUSION: [corrected] The recombinant virus were successfully generated and applied to prepare the antigen substrate slides. The antigen substrate slides was conveniently prepared without special equipments, and can be used to detect the antiserum of HV virus.

[Development and evaluation of TaqMan-based one-step reverse transcription-polymerase chain reaction assay for the detection of Japanese encephalitis virus].

OBJECTIVE: To establish a TaqMan based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of Japanese encephalitis virus. METHODS: The gene sequences of Japanese encephalitis virus downloaded from the GenBank was aligned, using the biologic software. Specific primers and probes were designed in the conserved region of the C gene for Japanese encephalitis virus. The real-time RT-PCR reactive condition was optimized and the sensitivity, specificity and the stability of the assay were evaluated. Mosquitoes collected from Zhejiang province were detected by this assay. RESULTS: Mg2+, primer and probe were optimized at 5 mmol/L, 0.2 micromol/L and 0.1 micromol/L respectively. The specificity of the assay was high and there were no cross reactions with dengue virus, rabies virus, seoul virus or hantan virus. The detection limits of the assay was 0.1 TCID50. Results from preliminary application showed that TaqMan RT-PCR for Japanese encephalitis virus was sensitive, easier and faster to perform the process of traditional virus isolation and identification. It took only three hours to extract viral RNA and perform the real-time RT-PCR. CONCLUSION: This TaqMan-based one-step RT-PCR assay was a quick, sensitive and specific tool for molecular diagnosis of Japanese encephalitis virus.

Bombyx mori nucleopolyhedrovirus ORF9 is a gene involved in the budded virus production and infectivity.

The ORF9 of Bombyx mori nucleopolyhedrovirus (BmNPV) (Bm9) is conserved in all completely sequenced lepidopteran nucleopolyhedroviruses. RT-PCR analysis demonstrated that Bm9 is an early and late transcribed gene that is initiated at 3 h post-infection, and immunofluorescence microscopy showed that Bm9 is localized mainly in the cytoplasm of infected cells. To determine the role of Bm9 during virus infection, Bm9 was knocked out by recombination in a BmNPV genome propagated as a bacmid in Escherichia coli. The budded virus (BV) production of Bm9-deleted bacmids was reduced more than 10-fold compared with wild-type (wt) bacmid; however, the kinetics of viral DNA replication were unaffected. The defect in BV production was recovered by the Bm9 rescue bacmid. In addition, electron microscope observations revealed that polyhedra formation was not affected by the deletion of Bm9. Bioassays showed that the Bm9-deleted bacmid took approximately 14-22 h longer to kill fifth instar B. mori larvae than wt bacmid, and the LD(50) was about 15 times higher than that of the wt bacmid. In conclusion, Bm9 is an important but not essential factor in virus production and infectivity in vivo and in vitro.

[Complete genome sequence analysis of Japanese encephalitis virus newly isolated in China].

OBJECTIVE: To study the complete genome sequence of Japanese encephalitis virus (JEV) strain XJ69 isolated in ZheJiang province and explore its evolution. METHODS: Overlapping primers were designed according to the full-length genomes from GenBank. RT-PCR was used to amplify the fragments and RT-PCR products were cloned T vector, sequenced and analyzed. RESULTS: The genome of strain XJ69 and XJP613 were 10 964 nucleotides in length with a single open reading frame encoding 3432 amino acids. Comparison of the complete genome sequences of different JEV isolates showed XJ69 and XJP613 were 83.5%-99.2% and 83.4%-99.4% nucleotide sequence homology among them respectively, which resulted in 94.8%-99.7% amino acid sequence homology. Phylogenetic analysis through PrM/C,E and full-length genome showed that the XJ69 and XJP613 strain belonged to genotype I. CONCLUSION: The nucleotitede sequence and deduced amino acid sequence of XJ69 and XJP613 strain were similar to that of those of genotype I of Japanese encephalitis virus. It belonged to genotype I and were close to the isolates SH17M-07.

Characterization of Bombyx mori nucleopolyhedrovirus with a deletion of bm118.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF118 (bm118) is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF142, one of the core genes existing in all baculovirus genomes sequenced to date, suggesting that Bm118 plays a critical role in viral infection. In this study, the primary role of Bm118 was investigated by using homologous recombination in Escherichia coli to generate a bm118 knockout bacmid containing the BmNPV genome. In addition, the bm118 rescue bacmid was constructed by transposing a bm118 gene cassette into the polh locus of the bm118 knockout bacmid. Transfection assays demonstrated that the bm118 knockout bacmid was incapable of producing budded virion (BV). Nevertheless, this defect could be partially recovered by a rescue bacmid. Electron microscopy analysis revealed that the bm118 knockout produced aberrant capsids characterized by translucent, elongated nucleocapsids present as bundles within the nuclei. This construct also produced polyhedra lacking virions. These results reveal that Bm118 is essential for BV production and nucelocapsid maturation.

Bombyx mori nucleopolyhedrovirus ORF56 encodes an occlusion-derived virus protein and is not essential for budded virus production.

Bombyx mori nucleopolyhedrovirus ORF56 (Bm56) is a baculovirus core gene that is highly conserved in all baculoviruses that have had their genomes sequenced to date. Its transcripts in BmNPV-infected cells could be detected from 12 h post-infection (p.i.) and the encoded protein could be detected at 16 h p.i. by using a polyclonal antibody against glutathione S-transferase-Bm56 fusion protein. Western blot analysis showed that Bm56 is a structural component of the occlusion-derived virus nucleocapsid. Subsequent confocal microscopy revealed that Bm56 was distributed in the outer nuclear membrane and the intranuclear region of infected cells. To investigate the role of Bm56 in virus replication, a Bm56-knockout bacmid of BmNPV was constructed via homologous recombination in Escherichia coli. The Bm56 deletion had no effect on budded virus (BV) production in cultured cells; however, the deletion affected occlusion-body morphogenesis. A larval bioassay demonstrated that the Bm56 deletion did not reduce infectivity, whereas it resulted in a 50 % lethal time that was 16-18 h longer than that of the wild-type bacmid at every dose used in this study. These results indicate that Bm56 facilitates efficient virus production in vivo; however, it is not essential for BV production in vitro.

Characterization of a nucleopolyhedrovirus with a deletion of the baculovirus core gene Bm67.

Open reading frame (ORF) 67 (Bm67) of the Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that is found in all completely sequenced baculoviruses; its function is unknown. In the present study, a Bm67-knockout virus was generated for studying the role of Bm67 in the BmNPV infection cycle. Furthermore, a Bm67-repair bacmid was constructed by transposing the Bm67 native promoter-promoted Bm67 ORF into the polyhedrin locus of the Bm67-knockout bacmid. After these recombinant bacmids were transfected into BmN cells, the Bm67-knockout bacmid caused defects in the production of infectious budded viruses. However, the Bm67-repair bacmid could rescue the defect, and budded virus titres reached wild-type levels. Quantitative real-time PCR analysis indicated that Bm67 is required for normal levels of DNA synthesis or for the stability of nascent viral DNA at the early stage. Electron microscopic analysis revealed that the formation of normal-appearing nucleocapsids is reduced in Bm67-knockout bacmid-transfected cells, and nucleocapsids are rarely found in the cytoplasm. The presence of 'enveloped' nucleocapsids at the nucleoplasm bilayer indicated that they are enveloped abnormally. These results indicated that Bm67 is required for the production of infectious budded viruses and for assembly of envelope and nucleocapsids.

Genome sequence and organization of a nucleopolyhedrovirus that infects the tea looper caterpillar, Ectropis obliqua.

The complete nucleotide sequence of Ectropis obliqua nucleopolyhedrovirus (EcobNPV), which infects the tea looper caterpillar, was determined and analyzed. The double stranded circular genome is composed of 131,204 bp and is 37.6% G+C rich. The analysis predicted 126 putative, minimally overlapping open reading frames (ORFs) with 150 or more nucleotides that together compose 89.8% of the genome. The remaining 10.2% constitute non-coding and three homologous regions. Comparison with previously sequenced baculoviruses indicated that three ORFs were unique to EcobNPV, while the remaining 123 ORFs shared identity with other baculovirus genes. In addition to two bro homologues, three other repeat ORFs, including dbp, p26, and odv-e66, were identified. Phylogenetic analysis indicated that each member of the paired ORFs was acquired independently. Gene parity plot analysis and percent identity of gene homologues suggested that EcobNPV is a Group II NPV, although its genomic organization was highly distinct.

Characterization of ORF39 from Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus, the gene containing RNA recognition motif.

In the genome of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus, open reading frame 39 (Ha39) is the only gene predicted to encode an RNA recognition protein. Computer analysis revealed that Ha39 homologues were found in 15 NPVs, but not in GVs. Its transcripts were detected from 3 through 72 hours post infection (h p.i.) using RT-PCR and Northern blot analysis. The protein was detected in infected-cell lysates from 6 h p.i. Western blot assay of ODV and BV preparations revealed that Ha39 encodes a structural protein associated with BVs.Additionally, immunofluorescence microscopy demonstrated that the protein was present within cytoplasm in virusinfected cells, but not in the nuclear region.