Cleavage activity of the sapovirus 3C-like protease in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Summary. We recently determined the ORF1 cleavage map of Mc10, a human sapovirus (SaV) strain, as follows: NH₂–p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)–COOH. This cleavage was dependent on the viral encoded 3C-like protease. To identify the cleavage site of SaV ORF1, putative p70 (Pro-Pol) and p14-p70 (VPg-Pro-Pol) were expressed as N-terminal GST and C-terminal 6 × His-tag fusion proteins in Escherichia coli, and the expressed products were analyzed by SDS-PAGE and Western blotting. Our results indicated that the efficient proteolytic cleavage occurred between p14 (VPg) and p70 (Pro-Pol), and N-terminal amino acid sequencing revealed that the cleavage site was between E¹⁰⁵⁵ and A¹⁰⁵⁶. In contrast, the p70 (Pro-Pol) was not further cleaved. We also found that SaV protease cleaved the Q/G site within the rhinovirus 3C protease recognition site. Site-directed mutagenesis in a conserved GDCG motif of the protease completely abolished these proteolytic activities. This is the first report to identify the cleavage site of the SaV ORF1 polyprotein.     

Sequence and phylogenetic analyses of highly virulent infectious bursal disease virus

Summary.  The nucleotide sequences of the genome segment A and B encoding the precursor polyprotein (NH₂-VP2-VP4-VP3-COOH) and VP1 were determined for a highly virulent strain of infectious bursal disease virus (IBDV). The precursor polyprotein and VP1 coding regions of highly virulent OKYM strain consisted of 3 039 nucleotides (1 012 deduced amino acids) and 2 640 nucleotides (879 deduced amino acids), respectively. Comparison of the deduced amino acid sequences of the highly virulent IBDV (HV-IBDV) with other serotype 1 and 2 sequences revealed 17 amino acid residues which were conserved only in the HV-IBDV. Among the 17 unique amino acid differences, 8 were in VP1, 4 were in VP2, 3 were in VP3 and 2 were in VP4. Although it is impossible to predict the effect of the unique amino acid residues without detailed knowledge of the three-dimensional structure and function of the proteins, they could affect the virulence of HV-IBDV. Alignment of the nucleic acid sequences of precursor polyprotein, VP1, VP2, VP3 and VP4 coding regions followed by distance analysis allowed the generation of phylogenetic trees. The same tree topology was obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. On the other hand, the tree topology of VP1 was quite different from that obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. These findings indicate that not a genetic recombination but a genetic reassortment may play an important role in the emergence of HV-IBDV. Accepted January 16, 1997; Received October 25, 1996     

Biological activity of FGF-23 fragments

The phosphaturic activity of intact, full-length, fibroblast growth factor-23 (FGF-23) is well documented. FGF-23 circulates as the intact protein and as fragments generated as the result of proteolysis of the full-length protein. To assess whether short fragments of FGF-23 are phosphaturic, we compared the effect of acute, equimolar infusions of full-length FGF-23 and various FGF-23 fragments carboxyl-terminal to amino acid 176. In rats, intravenous infusions of full-length FGF-23 and FGF-23 176–251 significantly and equivalently increased fractional phosphate excretion (FE Pi) from 14 ± 3 to 32 ± 5% and 15 ± 2 to 33 ± 2% (p < 0.001), respectively. Chronic administration of FGF-23 176–251 reduced serum Pi and serum concentrations of 1α,25-dihydroxyvitamin D. Shorter forms of FGF-23 (FGF-23 180–251 and FGF-23 184–251) retained phosphaturic activity. Further shortening of the FGF-23 carboxyl-terminal domain, however, abolished phosphaturic activity, as infusion of FGF-23 206–251 did not increase urinary phosphate excretion. Infusion of a short fragment of the FGF-23 molecule, FGF-23 180–205, significantly increased FE Pi in rats and reduced serum Pi in hyperphosphatemic Fgf-23 ^−/− knockout mice. The activity of FGF-23 180–251 was confirmed in opossum kidney cells in which the peptide reduced Na⁺-dependent Pi uptake and enhanced internalization of the Na⁺-Pi IIa co-transporter. We conclude that carboxyl terminal fragments of FGF-23 are phosphaturic and that a short, 26-amino acid fragment of FGF-23 retains significant phosphaturic activity.     

3CD, but not 3C, cleaves the VP1/2A site efficiently during Aichi virus polyprotein processing through interaction with 2A

Picornavirus genomes are translated into a single large polyprotein, which is processed by virus-encoded proteases into individual functional proteins. 3C of all picornaviruses is a protease, and the leader (L) and 2A proteins of some picornaviruses are also involved in polyprotein processing. Aichi virus (AiV), which is associated with acute gastroenteritis in humans, is a member of the genus Kobuvirus of the family Picornaviridae. The AiV L and 2A proteins have already been shown to exhibit no protease activity. In this study, we investigated AiV polyprotein processing by 3C and 3CD using a cell-free translation system. 3C and 3CD were capable of processing the polyprotein in trans; 3C, however, cleaved the VP1/2A site inefficiently, while 3CD cleaved this site almost completely. Mammalian two-hybrid and coimmunoprecipitation assays showed an interaction between 2A and 3CD. Using a 3CD mutant and various 2A mutants of substrate proteins, we showed a clear correlation between the 2A-3CD interaction and the VP1/2A cleavage by 3CD. Thus, this study suggests that tight interaction of 3CD with the 2A region of a precursor protein is required for efficient cleavage at the VP1/2A site.     

Infectious bursal disease virus polyprotein expression arrests growth and mitogenic stimulation of B lymphocytes

Summary. Infectious bursal disease virus (IBDV) causes lymphocytolysis and immunosuppression in infected poultry. The IBDV genome encodes a polyprotein VP243 that is post-translationally cleaved by the VP4 protease into the two structural proteins pVP2 and VP3. The objective of the present study was to determine if IBDV polyprotein induced suppression of bursal B lymphocyte growth and their capacity for proliferation. Bursal B cells were examined both for chickens infected with IBDV and for chickens orally inoculated with a DNA construct expressing IBDV VP243 polyprotein. Bursae were collected at 0, 12, 24 and 48 hours after inoculation. Proliferation of bursal B cells (purified AvBu1⁺ cells) in response to concanavalin A mitogenic stimulation was significantly suppressed by infection at 1 day old with either the classical STC or variant E strains of IBDV. Oral administration of DNA constructs expressing the IBDV VP243 polyprotein from either the classical STC or variant E strains in the pCR3.1 vector resulted in persistent, moderate levels of construct in the bursa until at least 48 hours after inoculation. The VP243 DNA construct similarly induced suppression of proliferation for bursal lymphocytes independently of the virus infection. Expression of VP243 polyprotein in transiently transfected DT40 B lymphocyte culture also suppressed cell growth and proliferative responses to mitogen stimulation. Polyprotein expression did not affect cell viability and suppression of proliferation probably occurred by means of cell cycle arrest. The expression of the mature viral proteins VP2, VP4 or VP3 did not change the rate of cell proliferation or response of B cell cultures to mitogen. The results suggested that IBDV polyprotein is a mediator of immunosuppression.     

The rubella virus nonstructural protease recognizes itself via an internal sequence present upstream of the cleavage site for trans-activity

Summary. The substrate requirement for rubella virus protease trans-activity is unknown. Here, we analyzed the cleavability of RV P200-derived substrates varying in their N-terminal lengths (72–475 amino acids) from the cleavage site by the RV protease trans-activity. Only substrates with at least 309 amino acid residues N-terminal to the cleavage site were able to undergo cleavage. Further, rubella sequence was found to be necessary in the N-terminal region of the substrate, whereas a heterologous sequence C-terminal to the cleavage site was tolerated. These results demonstrated a requirement for residues located between amino acids 994–1102 of the RV P200 polyprotein, besides its cleavage site for RV protease trans-activity. This region overlaps with the starting site of the essential cis-protease activity of RV P200 polyprotein. This is a novel observation for a viral protease of the family Togaviridae.     

Identification and characterization of Iflavirus 3C-like protease processing activities

Viral replication and capsid assembly in the viruses in the order Picornavirales requires polyprotein proteolytic processing by 3C or 3C-like (3CL) proteases. We identified and characterized the 3CL protease of Ectropis obliqua virus (EoV) of the newly established family Iflaviridae (order Picornavirales). The bacterially expressed EoV 3CL protease domain autocatalytically released itself from larger precursors by proteolytic cleavage, and cleavage sites were determined via N-terminal sequencing of the cleavage products. This protease also mediated trans-proteolytic activity and cleaved the polyprotein at the same specific positions. Moreover, we determined the critical catalytic residues (H2261, D2299, C2383) for the protease activity, and characterized the biochemical properties of EoV 3CL and its responses to various protease inhibitors. Our work is the first study to identify an iflaviral 3CL protease and further characterize it in detail and should foster our understanding of EoV and other iflaviruses.     

Complete nucleotide sequence of Sesbania mosaic virus: a new virus species of the genus Sobemovirus

Summary.  The complete nucleotide sequence of the Sesbania mosaic virus (SeMV) genomic RNA was determined by sequencing overlapping cDNA clones. The SeMV genome is 4149 nucleotides in length and encodes four potential overlapping open reading frames (ORFs). Comparison of the nucleotide sequence and the deduced amino acid sequence of the four ORFs of SeMV with that of other sobemoviruses revealed that SeMV was closest to southern bean mosaic virus Arkansas isolate (SBMV-Ark, 73% identity). The 5′ non-coding regions of SeMV, SBMV and southern cowpea mosaic virus (SCPMV) are nearly identical. However ORF1 of SeMV which encodes for a putative movement protein of M_r 18370 has only 34% identity with SBMV-Ark. ORF 2 encodes a polyprotein containing the serine protease, genome linked viral protein (VPg) and RNA dependent RNA polymerase domains and shows 78% identity with SBMV-Ark. The N-terminal amino acid sequence of VPg was found to be TLPPELSIIEIP, which mapped to the region 326–337 of ORF2 product and the cleavage site between the protease domain and VPg was identified to be E³²⁵-T³²⁶. The cleavage site between VPg and RNA dependent RNA polymerase was predicted to be E⁴⁴⁵-T⁴⁴⁶ based on the amino acid sequence analysis of the polyprotein from different sobemoviruses. ORF3 is nested within ORF2 in a − 1 reading frame. The potential ribosomal frame shift signal and the downstream stem-loop structure found in other sobemoviruses are also conserved in SeMV RNA sequence, indicating that ORF3 might be expressed via − 1 frame shifting mechanism. ORF4 encodes the coat protein of SeMV, which shows 76 and 66% identity with SBMV-Ark and SCPMV, respectively. Thus the comparison of the non-coding regions and the ORFs of SeMV with other sobemoviruses clearly revealed that it is not a strain of SBMV. Phylogenetic analysis of six different sobemoviruses, including SeMV, suggests that recombination event is not frequent in this group and that SeMV is a distinct member of the genus sobemovirus. The analysis also shows sobemoviruses infecting monocotyledons and dicotyledons fall into two distinct clusters. Received April 20, 2000 Accepted August 28, 2000     

Identification of the 3C-protease-mediated 2A/2B and 2B/2C cleavage sites in the nonstructural polyprotein precursor of a dicistrovirus lacking the NPGP motif

Dicistroviruses have motifs for picornavirus 2C, 3C, and 3D proteins in their nonstructural polyprotein C-terminal region. The proteins from the nonstructural, N-terminal region of the polyprotein remain to be characterized. We have identified 3C-mediated cleavage sites in the N-terminal region of the nonstructural polyprotein of the dicistrovirus Plautia stali intestine virus (PSIV). The 2B/2C cleavage site mapped to amino acids (aa) 408–409 (QD). 2B/2C cleavage sites were suggested to be conserved in dicistroviruses. The most N-terminal PSIV cleavage site was aa 286–287 (QS). Including previous results, the polyprotein contains nine proteins arranged as follows: 2A, 2B, 2C, 3A, 3B1, 3B2, 3B3, 3C, and 3D.     

Molecular epidemiology of enterovirus 71 in Taiwan

Summary.  Taiwan suffered a severe and widespread outbreak of enterovirus infection in 1998. More than 400 children were hospitalized, with seventy-eight fatalities due to central nerve system (CNS) involvement and cardiopulmonary collapse. Enterovirus 71 (EV71) was incriminated as the causative agent for the fatal cases. To understand the viral molecular epidemiology in this outbreak, fragments of 207-bp length of the VP4 region in 23 Taiwanese EV 71 isolates were sequenced. Pair-wise comparison revealed a 17.5–24.4% difference between the isolates and the prototype BrCr. However, all the changes in the VP4 region of the isolated strains were synonymous substitutions. Phylogenetic analysis was performed on these 23 isolates and 21 others deposited in GenBank. In this study, forty-four EV71 isolates from the world were separated into three distinct genotypes: A, B and C. The EV71 prototype strain, BrCr/70, is the only strain of genotype A. Group B included strains from the United States, Japan and Taiwan. Most strains in genotype B were isolated prior to 1990. Group C consisted of strains from Japan and Taiwan. Most strains of genotype C were isolated after 1990, they were further divided into 3 clusters: i.e. C-1, C-2 and C-3. In Taiwan, two genotypes, B and C-3, were co-circulating during the outbreak in 1998, although a minor group of genotype B may have appeared in Taiwan before 1986. The majority of the isolates clustered in genotype C-3. Genotype C showed a higher evolutionary rate than genotype B (3.9 × 10⁻³vs. 1.4 × 1010⁻³) in the VP4 region. There seems to be a worldwide trend with strains of genotype B appearing earlier than strains of genotype C which took over later in the dominance. Received June 13, 2000 Accepted July 29, 2000     

Expression of CD62L on Donor CD4<Superscript>+</Superscript> T Cells in Allografts: Correlation with Graft-Versus-Host Disease after Unmanipulated Allogeneic Blood and Marrow Transplantation

Introduction  The aim of this study was to investigate the association of donor CD4⁺ T cells expressing CD62L with transplant outcomes. Materials and Methods  We report a prospective analysis of 31 patients who were treated with a Bu/Cy regimen, followed by unmanipulated blood and marrow transplantation. Results  Median number (range) of CD4⁺CD62L⁺, CD4⁺CD45RA⁺CD62L⁺, and CD4⁺CD45RO⁺CD62L⁺ cells infused were 0.31(0.05–1.10)×10⁸/kg, 0.22(0.03–0.95)× 10⁸/kg, and 0.17(0.01–0.81)×10⁸/kg, respectively. The incidence of grades II to IV aGVHD was 36%. In a multivariate analysis, infusion of >0.22 × 10⁸ CD4⁺CD45RA⁺CD62L⁺ cells infused/kg increased the risk of grades II to IV aGVHD (HR = 4.741, 95% CI = 1.037–21.662, P = 0.045). Thirteen of 31 patients experienced cGVHD, the risk of cGVHD was increased in patients receiving >0.45 × 10⁸ CD4⁺CD45RA⁺ cells infused/kg (HR = 4.614, 95% CI = 1.265–16.829, P = 0.021). Conclusion  Our results suggest that a high cell dose of CD4⁺CD45RA⁺CD62L⁺ cells increase the incidence of grades II–IV aGVHD. A high number of CD4⁺CD45RA⁺ cells infused were associated with increased risk of cGVHD in our transplant settings. Ying-Jun Chang: performed research, analysis and interpretation of data, and drafting of the article, and gave final approval of the version to be published; Xiang-Yu Zhao: performed research, analysis and interpretation of data, and drafting of the article and gave final approval of the version to be published; Ming-Rui Huo: performed research, analysis and interpretation of data, and drafting of the article and gave final approval of the version to be published; Xiao-Jun Huang: involved in conception and design, revising the article critically, and final approval of the version to be published.     

Assessment of Leishmania promastigote growth in vitro by means of nucleoside hydrolase activity determination

Nucleoside hydrolases (NH) are involved in the purine salvage pathway of protozoan cells for the biosynthesis of nucleic acids. We developed a simple and reliable microassay based on N-ribohydrolase dosage using 4-nitrophenyl-β-d-ribofuranoside (NPR) substrate for the quantification of Leishmania infantum. The free promastigote stage of L. infantum contains high amounts of NH capable of cleaving NPR, but the parasitic amastigote does not. The method allows reliable quantification of viable parasites over a wide range of concentrations (5 × 10⁴–2 × 10⁸ parasites ml⁻¹) in a single assay. No difference in NH activity was observed between various strains at equivalent concentrations and growth curves determined with NH dosage were similar to optical counts. Samples can be stored at −20 °C for weeks before use in this unique assay without significant loss of NH activity. The method is particularly simple and versatile and proves well adapted for the determination of growth characteristics and drug screening studies of L. infantum promastigotes in vitro. Received: 4 August 2000 / Accepted: 4 August 2000     

Co-ordinated changes in cAMP, phosphorylated phospholamban, Ca2+ and contraction following β-adrenergic stimulation of rat heart

 Concentration-dependent changes in cyclic AMP (cAMP), site-specific phosphorylation of phospholamban, the intracellular calcium ([Ca²⁺]_i) transient and contraction were measured in isolated rat ventricular myocytes exposed to the β-adrenoceptor agonist isoprenaline. Cyclic AMP was measured by [¹²⁵I]-cAMP scintillation proximity assay, phosphorylation of phospholamban at Ser¹⁶ and Thr¹⁷ was assessed using a pair of site-specific polyclonal antibodies, and [Ca²⁺]_i was monitored with the fluorescent dye fura 2. Cyclic AMP rose to twice basal levels in the presence of 10^–6 M isoprenaline. The maximum increase in phosphorylation at Ser¹⁶ and Thr¹⁷ of phospholamban was seen at 10^–7 M isoprenaline. At this stage Ser¹⁶ phosphorylation was six times higher, and Thr¹⁷ phosphorylation was three times higher than that recorded in the absence of isoprenaline. Phosphorylation at Ser¹⁶ correlated more closely with changes in the [Ca²⁺]_i transient and contraction than did phosphorylation at Thr¹⁷. This is the first study of its kind to measure simultaneous changes in cAMP, the phosphorylation of phospholamban, the [Ca²⁺]_i transient and contraction over a range of concentrations of β-agonist. The results suggest that phosphorylation of phospholamban at Thr¹⁷ is of lesser physiological relevance to the effects of β-adrenergic stimulation on the heart than phosphorylation at Ser¹⁶. Received: 9 June 1998 / Received after revision and accepted: 16 July 1998     

Micropatterned surfaces of PDMS as growth templates for HEK 293 cells

In this paper the easy and reliable preparation of precise micropatterns on PDMS surfaces is described and the growth of HEK 293 cells on those patterns during culture over several days is examined. The first patterning approach described is based on soft-lithography and polyelectrolyte multilayer deposition. Two different soft-lithographic techniques are employed for creating surface patterns of PAH, PSS, untreated and oxidized PDMS. The growth behavior of HEK 293 cells is investigated on all the dual combinations of the four surfaces, and decreasing preference of the cells for the surfaces in the order PAH (–NH₂) > ox-PDMS (–OH) >> PSS (–SO₃ ⁻) > PDMS (–CH₃) is revealed. As the second patterning approach a method is introduced, which allows the deposition of gel droplets in a microarray format utilizing differences in the surface wettability. This concept is new and expected to be very useful for various applications. Finally, a speculative explanation for the different cell spreading behavior is provided considering the interplay between individual cell–surface interactions and a permanent cell tractional force.     

Genetic diversity in the VP1 gene of foot-and-mouth disease virus serotype Asia 1

Summary.  Complete nucleotide sequence of the 1D (VP1-encoding) gene of 61 foot-and-mouth disease (FMD) serotype Asia 1 virus isolates recovered from different outbreaks in India between 1985 and 1999 including two vaccine strains currently used were determined. The sequences were compared with each other and those from other Asian countries. On the basis of phylogenetic analysis the viruses could be grouped into four genotypes (genotypes I–IV). All the 61 isolates from India belong to a single genotype (genotype-II) which is further subdivided into three lineages (B1, B2 and B3) under the same genotype. The viruses of the lineage B1 and B3 were found to be more prevalent before 1996 while the viruses of lineage B2 appeared to be new variants responsible for most of the recent outbreaks. Most of the isolates of lineage B1 lack one amino acid in the VP1 protein (position 44) whereas most of the isolates of lineage B2 and B3 contain it which indicates the possibility of these lineages having evolved independently. The rate of evolution of FMDV Asia 1 virus was also estimated and found to be 2.7 × 10⁻² synonymous substitutions per nucleotide per year. Received May 7, 2001 Accepted August 1, 2001     

Carbonylation of oxyhemoglobin solution (HbO2→ HbCO) using a membrane oxygenator

 In the purification process of hemoglobin (Hb) from red blood cells, we stabilized Hb as carbonylhemoglobin (HbCO) against pasteurization at 60°C. In this study, the process of carbonylation (HbO₂→ HbCO) was tested with a membrane oxygenator (CX-II08; membrane area, 0.8 m²; maximum circulation rate, 1.2 l/min) under the conditions of a solution flow rate of 100–1000 ml/min and a CO gas flow rate of 30–100 ml/min. Comparing the overall O₂ transfer coefficient of carbonylation with that of deoxygenation (N₂ flow) revealed that the resistance to O₂ transfer of carbonylation was about 35 times smaller, indicating that carbonylation hindered O₂ rebinding (deoxyHb → HbO₂). On the other hand, the O₂ released in the course of carbonylation hindered carbonylation at the beginning, because rebinding of O₂ is competitive with carbonylation. The time required for carbonylation was significantly shortened from 1000 to 150 s when the solution flow rate was increased from 50 to 400 ml/min; however, the CO gas flow rate did not affect it very much. Increasing the Hb concentration from 7.5 to 15 g/dl accelerated carbonylation by 1.3 times. Even though further study is necessary to select a suitable polymer membrane to avoid protein adsorption, a membrane oxygenator will be effective for the large-scale carbonylation of Hb as a starting material of HbV in the production process. Received: August 3, 2001 / Accepted: December 27, 2001     

Complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus,a member of a new species in the genus <Emphasis Type="Italic">Kobuvirus</Emphasis>, family <Emphasis Type="Italic">Picornaviridae</Emphasis>

Kobuvirus is a new genus in the family Picornaviridae. Two species are currently known: Aichi virus (human kobuvirus) and Bovine kobuvirus (U-1). In this study, the complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus (Kobuvirus/swine/S-1-HUN/2007/Hungary, EU787450) were determined. The structure of the S-1-HUN genome, VPg–5′UTR–leader protein–structural proteins (VP0, VP3, VP1)–non-structural proteins (2A–2C, 3A–3D)–3′UTR–poly(A) tail, was found to be typical of picornavirus. The 8210-nucleotide (nt)-long RNA genome contains a large open reading frame (7467 nt) encoding a potential polyprotein precursor of 2488 amino acids (aa) that has 57/56% and 63/64% nt/aa identity with Aichi virus and U-1, respectively. The 5′UTR contains a hepacivirus/pestivirus-like internal ribosomal entry site (IRES type IV group-B-like) with conserved pseudoknot, II and IIIa–f domains. A tandem repeat (a 30-amino-acid-long motif) was detected in 2B. Thirty-nine (65%) of the 60 fecal samples from pigs under the age of 6 months at the tested farm were positive (the incidence was 90% under the age of 3 weeks). Porcine kobuvirus belongs to a potential new species—the third—in the genus Kobuvirus. Nucleotide sequence data reported are available in the GenBank database under accession number EU787450.     

Detection and characterisation of a second potyvirus from Thunberg fritillary in China

Summary. Plants of Thunberg fritillary (Fritillaria thunbergii Miq.) from Zhejiang Province, were found to be co-infected with two distinct potyviruses. One was an isolate of the recently reported Thunberg fritillary mosaic virus (TFMV; Wei et al., (2005) Arch Virol 150: 1271–1280), while the other was a distinct virus that did not react with TFMV antiserum nor with antisera to 17 other potyviruses, except for a weak reaction with antibodies produced to soybean mosaic virus (SMV) Pinellia strain. Both viruses could be transmitted mechanically to their original host but not to any of a range of commonly used indicator plants. No local lesion host was identified that would enable the viruses to be propagated independently. The complete sequences of both viruses were determined; that of the new virus (9656 nt) had the typical genome organisation and recognised sequence motifs of a potyvirus, encoding a putative polyprotein of 351 kDa. Phylogenetic analysis, sequence comparisons, and the pattern of polyprotein cleavage sites all indicated that it was a member of the Bean common mosaic virus subgroup. The most closely related species are Soybean mosaic virus and Wisteria vein mosaic virus, with 68–69% amino acid identity between their polyproteins. This is sufficiently different for the new virus to be regarded as a distinct species, which we have tentatively named Fritillary virus Y.     

Cloning and expression of yellowtail ascites virus segment A

Summary.  cDNA of yellowtail ascites virus (YAV) segment A encoding a polyprotein of VP2, NS, and VP3 has been cloned. Comparison of the nucleotide and the deduced amino acid sequences showed very high homology between YAV and other aquatic birnaviruses. The two small open reading frames (VP5) besides the 5′ terminus of the VP2 gene were found on segment A of YAV. Proteins encoded by cDNAs from segment A and the serotype-specific epitope region on VP2 were expressed using a baculovirus vector. Western blot analysis confirmed that a polyprotein was expressed and processed into VP2 and VP3 in insect cells infected with the recombinant baculovirus containing the complete polyprotein coding region. In the case of expression in silkworm larvae, only VP3 was detected in hemocytes and fat body of silkworm larvae infected with the recombinant virus. The recombinant fusion protein consisting of VP2 epitope region and polyhedrin was expressed in insect cells and cross-reacted with a mouse monoclonal antibody against VP2 which had a neutralizing activity to YAV. Received June 3, 1998 Accepted February 28, 1999