Detection of bovine immunodeficiency-like virus infection in experimentally infected calves

Summary.  Detection of BIV virus infection by serological means, PCR and virus isolation in experimentally infected calves is described. Viral sequences were specifically detected by PCR in peripheral blood mononuclear cells (PBMCs), with primer systems located in the gag, pol and tat regions of the viral genome. An enzyme-linked oligosorbent assay (ELOSA) in microtiter plates is described, for the detection of PCR products, the sensitivity of which was shown to be comparable to that of membrane hybridization detection. Serological response of the animals against the BIV p26 protein was shown, using a recombinant fusion protein ((His)₆p26) expressed in E. coli and purified by metal affinity chromatography, in ELISA and Western blot studies. The presence of infectious virus was demonstrated by its rescue, by virus isolation in cell cultures, from PBMCs during a one year follow-up. Accepted August 14, 1997 Received November 28, 1996     

Comparison of the RNA polymerase genes of marine birnavirus strains and other birnaviruses

Summary.  The cDNA nucleotide sequence of the genome segment B encoding the VP1 protein, the putative RNA-dependent RNA polymerase (RdRp), was determined for 5 marine birnavirus (MABV) strains from different host or geographic origins and 1 infectious pancreatic necrosis virus (IPNV) strain AM-98. Segment B of the IPNV AM-98 strain and 4 MABV strains, Y-6, YT-01A, H1 and NC1, contained a 2535 bp ORF, which encoded a protein of 845 amino acid residues with a predicted MW of 94.4 kDa. Only the MABV AY-98 RdRp had 1 amino acid shorter RdRp. Pairwise comparisons were made among our data and 4 other known IPNV sequences. The nucleotide sequences of the 5 MABV strains were very similar each other, with identities of 98.3–99.7%. The highest divergence of the nucleotide level was between MABV strains and IPNV SP strain (serotype A2), with 20.4–20.8% divergences in the coding region, which gave 10.1–11.3% divergence in the amino acid level. The aquabirnavirus RdRp was noticeably conserved in amino acid sequences. Though the identities of the nucleotide sequences of encoding region were 85.1–85.9% between MABV strains and IPNV serotype A1 strains, they shared as high as 95.1–95.9% identities in amino acid level. A phylogenetic tree was constructed based on the amino acid sequences of the RdRp gene from different birnaviruses including avibirnavirus and entomobirnavirus. Ten aquabirnavirus strains were clustered into 3 Genogroups. The Genogroup I consisted of four IPNV A1 serotype strains. All MABV strains were clustered into Genogroup II. Only IPNV SP strain was clustered into an independent Genogroup III. Received August 19, 2002; accepted October 30, 2002     

Isolation and establishment in in vitro culture of a Theileria annulata– infected cell line from Spain

The isolation of Theileria annulata-infected lymphocytes using blood from an animal suffering from Mediterranean theileriosis as a source of parasites is described. The present work reports the first isolation and establishment in in vitro culture of a T. annulata-infected cell line from southwestern Europe, where Mediterranean theileriosis causes important economic losses, especially in southern Spain. The parasite was identified by staining of cells from culture with Giemsa, by immunofluorescent antibody techniques (IFAT), and by isoenzyme characterization. The possibility of using this T. annulata-infected lymphoblastoid cell line to obtain an antigen for diagnosis of Mediterranean theileriosis by IFAT and to develop a tissue-culture vaccine against this disease in our geographic area shows the significance of this isolation and culture. Received: 21 September 1996 / Accepted: 1 October 1996     

An Anti-apoptosis Gene of the Bcl-2 Family from Marine Birnavirus Inhibiting Apoptosis of Insect Cells Infected with Baculovirus

VP5 is a 15-kDa nonstructural protein encoded by a small open reading frame in 5′-terminal of segment A of the Marine Birnavirus (MABV) (strainY-6) genome. Comparisons of the amino acid sequence of the VP5 with other Bcl-2 family member proteins indicated that the VP5 protein contains Bcl-2 homology (BH) domains BH1, BH2, BH3, and BH4, but without the transmembrane region. The VP5 gene from MABV was fused to enhancing green fluorescence protein (eGFP) gene and inserted into the baculovirus genome under the control of polyhedrin gene promoter, and then was highly expressed in insect cells. The expressed VP5 was capable of enhancing insect cell viability, prevented membrane blebbing and delayed DNA internucleosomal cleavage when cells were infected with the recombinant virus. The results suggested that the VP5 of MABV is a novel anti-apoptosis gene, which could regulate the cell apoptosis-off system.     

Comparison of Immunofluorescence and Isolation Techniques in the Diagnosis of Respiratory Viral Infections of Children

The immunoflourescent antibody technique (IFAT) and cell culture isolation procedures were compared for their efficiency in the etiological diagnosis of viral respiratory illness in children. Before the IFAT was incorporated as a routine procedure, antisera used in the test were carefully calibrated to insure specificity. A study was then conducted in which 375 nasopharyngeal suctions were investigated by both IFAT and isolation for the presence of parainfluenza virus types 1, 2, and 3, respiratory syncytial, influenza A, and influenza B viruses. Methods already established in our hospital for patient management and specimen collection were not altered for the purposes of the study. The IFAT, as conventionally practiced in the detection of respiratory virus antigens, requires adequate numbers of ciliated epithelial cells. There were 68.5% specimens which contained cells suitable for IFAT, whereas 31.5% had either an insufficient number or inappropriate types of cells and could be used only for virus isolation. Cell-associated immunoglobulins were detected in 16% of those specimens with adequate cells. When all specimens were considered regardless of their cell population, IFAT was inferior to isolation in diagnostic efficiency. However, isolation complemented by IFAT resulted in a statistically significant increase in number of positive virus identifications. Under routine working conditions in a large pediatric hospital, it was found that IFAT could not replace isolation techniques but could, if used in conjunction with isolation, provide a significant overall increase in number of positive diagnoses. The time that the specimen was taken in relation to first symptoms was found to be an important variable with respect to the method most likely to succeed in virus identification.     

Genetic and phenotypic characterization of the newly described insect flavivirus,Kamiti River virus

Summary.  We have described in the accompanying paper by Sang, et al., ([57], Arch Virol 2003, in press) the isolation and identification of a new flavivirus, Kamiti River virus (KRV), from Ae. macintoshi mosquitoes that were collected as larvae and pupae from flooded dambos in Central Province, Kenya. Among known flaviviruses, KRV was shown to be most similar to, but genetically and phenotypically distinct from, Cell fusing agent virus (CFAV). KRV was provisionally identified as an insect-only flavivirus that fails to replicate in vertebrate cells or in mice. We report here the further characterization of KRV. Growth in cell culture was compared to that of CFAV; although growth kinetics were similar, KRV did not cause the cell fusion that is characteristic of CFAV infection. The KRV genome was found to be 11,375 nucleotides in length, containing a single open reading frame encoding 10 viral proteins. Likely polyprotein cleavage sites were identified, which were most similar to those of CFAV and were comparable to those of other flaviviruses. Sequence identity with other flaviviruses was low; maximum identity was with CFAV. Possible terminal secondary structures for the 5′ and 3′ non-coding regions (NCR) were similar to those predicted for other flaviviruses. Whereas CFAV was isolated from insect cells in the laboratory, the isolation of KRV demonstrates the presence of an insect-only flavivirus in nature and raises questions regarding potential interactions between this virus and other mosquito-borne viruses in competent vector populations. Additionally, this virus will be an important tool in future studies to determine markers associated with flavivirus host specificity. Received October 23, 2003; accepted January 6, 2003 Published online April 9, 2003     

Mitotic activity of the hemocytes in the tick Ixodes ricinus (Acari; Ixodidae)

 The blood cells, or hemocytes, of Ixodes ricinus have been shown to recognize, attack, and phagocytose microorganisms invading the body cavity, or hemocoel, of this tick. Regulated proliferation and differentiation of hemocytes, also referred to as immunocytes, is basic to an effective immune response to invading microorganisms. Therefore, this study dealt with hemopoiesis in I. ricinus, the vector tick of the Lyme disease spirochete Borrelia burgdorferi. Histological evidence for the presence of hemopoietic tissue, a preferential proliferation site of hemocytes, is presented. Mainly the mitotic activity of free-floating hemocytes was examined. By means of microscopical photometry and flow cytometry, all three types of hemocytes in engorging female I. ricinus were found in different stages of the cell cycle. In the engorging tick, up to 40% of the hemocytes counted were in the S phase or the G₂/M phase. From this study we conclude that the differentiated hemocyte types do not differentiate from stem cells in the adult tick. Moreover, microorganisms entering the hemocoel of engorging ticks are confronted with high numbers of hemocytes and, therefore, with an effective cellular immune response. Received: 20 October 1995 / Accepted: 30 January 1996     

Detection of SRV/D shedding in body fluids of cynomolgus macaques and comparison of partial gp70 sequences in SRV/D-T isolates

We previously reported the isolation of a novel subtype of SRV/D-Tsukuba (SRV/D-T) from two cynomolgus monkeys (Macaca facicularis) in the breeding colony of Tsukuba Primate Research Center (TPRC). We surveyed for SRV/D infection in the TPRC cynomolgus colony using SRV/D-specific PCR primer sets designed based on the entire gag region sequence. The only SRV/D subtype detected in the colony was SRV/D-T with a positive infection rate of 22.4% (n = 49). It has been reported that the mode of transmission of SRV/D is via contact with virus shed in the body fluids. In this report, to investigate the infection route of SRV/D-T in monkeys at TPRC, we performed virus isolation and PCR for detection of the SRV/D genome from peripheral blood mononuclear cells (PBMCs), plasma, saliva, urine, and feces. Virus isolation and PCR detection were positive in plasma, saliva, urine, and fecal samples from all monkeys on which virus was isolated from PBMCs. This suggests that the spread of SRV/D-T infection in TPRC is via contact with virus shed in saliva, urine, and/or feces. Also, comparison of sequences of gp70 on multiple SRV/D-T isolates revealed that there was little intra- and inter-monkey variation, suggesting that SRV/D-T is fairly stable.     

Characterization of petunia flower mottle virus (PetFMV), a new potyvirus infecting Petunia x hybrida

Summary.  With the introduction of cutting-grown Petunia x hybrida plants on the European market, a new potyvirus which showed no serological reaction with antisera against any other potyviruses infecting petunias was discovered. Infected leaves contained flexuous rod-shaped virus particles of 750 – 800 nm in length and inclusion bodies (pinwheel structures) typical for potyviruses in ultrathin leaf sections. The purified coat protein with a M_r of approximately 36 kDa could be detected in Western immunoblots with a specific antibody to the coat protein of the petunia-infecting virus. The 3′ end of the viral genome encompassing the 3′ non-coding region, the coat protein gene, and part of the NIb gene was amplified from infected leaf material by IC/PCR using degenerate and specific primers. Sequences of PCR-generated cDNA clones were compared to other known sequences of potyviruses. Maximum homology of 56% was found in the 3′ non-coding region between the petunia isolate and other potyviruses. A maximum homology of 69% was found between the amino acid sequence of the coat protein of the petunia isolate and corresponding sequences of other potyviruses. These data indicate that the petunia-infecting virus is a previously undescribed potyvirus and the name petunia flower mottle virus (PetFMV) is suggested. Accepted November 5, 1997 Received July 25, 1997     

Molecular evidence that sugarcane yellow leaf virus (ScYLV) is amember of the Luteoviridae family

Summary.  A previously uncharacterized virus was reported in southeast Brazil causing a yellowing leaf disease in sugarcane. The virus, termed sugarcane yellow leaf virus (ScYLV), shares features typical of the luteoviruses. To start the molecular characterization of ScYLV, the nucleotide sequence of the coat protein (CP), 17 kDa protein and C-terminus of the RNA-dependent RNA polymerase coding regions was determined from an RT-PCR amplification product. Comparisons showed that the deduced amino acid sequences share a considerable degree of identity and similarity with corresponding sequences of known luteoviruses, thus clearly establishing ScYLV as a member of the family Luteoviridae. The authenticity of the CP open reading frame was confirmed by its expression in Escherichia coli. The recombinant CP positively reacted in immunoblot assays with polyclonal antibodies raised against native ScYLV. Furthermore, phylogenetic analyses also suggest that the 5′ and 3′ coding blocks of the ScYLV genome possess different taxonomic affinities within the Luteoviridae family, as does also the genome of soybean dwarf virus. Received July 2, 1999/Accepted November 19, 1999     

NTS influence on transgene expression from mono- and bicistronic plasmid vectors after lipofection in a human fibroblast cell line

 We evaluated the survival, transgene production, and copy numbers of integrated plasmid units per host genome after lipofection with mono- and bicistronic plasmid vectors in different cell lines and under various conditions. The addition of an integration enhancing murine sequence nontranscribed spacer (NTS) to the plasmids increased transfection efficiency, survival, and transgene expression. However, in human fibroblast cells this sequence had only marginal effects on overall plasmid copy number in bulk cultures. Clones producing the highest amounts of the transgene contained only one or two copies of plasmid per genome, independent of cell type and plasmid design. Received: 1 October 1996 / Accepted: 27 November 1996     

宁夏HFRS疫源地宿主动物汉坦病毒的首次分离和鉴定

目的　对宁夏肾综合征出血热（ ＨＦＲＳ） 新发疫源地进行宿主动物汉坦病毒的分离和鉴定。方法　采用幼龄长爪沙鼠接种和ＶｅｒｏＥ６ 细胞培养的方法进行汉坦病毒分离。用单克隆抗体间接免疫荧光试验,属特异性逆转录聚合酶链反应（ＰＣＲ） 结合限制性内切酶分析和型特异性逆转录ＰＣＲ 进行分离株的鉴定。结果　从宁夏黑线姬鼠肺成功地分离了２ 株汉坦病毒。单克隆抗体间接免疫荧光试验表明２ 株病毒与汉滩型病毒反应谱相同。逆转录ＰＣＲ 试验表明,２ 株病毒均可被汉坦病毒属特异性引物以及汉滩型特异性引物扩增,属特异性逆转录ＰＣＲ 产物的限制性内切酶分析表明２株病毒与汉滩型病毒酶切图谱相同。结论　首次在宁夏疫区成功分离了汉坦病毒并鉴定其属于汉滩型病毒,为进一步研究和控制该地区ＨＦＲＳ 提供了科学依据。     

An enzyme-linked immunosorbent assay for detection of Theileria parva antibodies in cattle using a recombinant polymorphic immunodominant molecule

Field and experimental bovine infection sera were used in immunoblots of sporozoite and schizont lysates of Theileria parva to identify candidate diagnostic antigens. Four parasite antigens of Mr 67,000 (p67), 85,000 (the polymorphic immunodominant molecule, PIM), 104,000 (p104), and 150,000 (p150) were selected for a more detailed analysis. The p67 and p104 antigens were present only in the sporozoite lysates, whereas PIM and p150 were found in both sporozoite and schizont lysates. The four antigens were expressed as recombinant fusion proteins and were compared with each other in an enzyme-linked immunosorbent assay (ELISA) and in the whole-schizont-based indirect fluorescent antibody test (IFAT) in terms of their ability to detect antibodies in sera of experimentally infected cattle. The PIM-based ELISA provided a higher degree of sensitivity and specificity than did the ELISA using the other three recombinant antigens or the IFAT. Further evaluation of the PIM-ELISA using experimental sera derived from cattle infected with different hemoparasites and field sera from endemic and nonendemic T. parva areas showed that the assay had a sensitivity of >99% and a specificity of between 94% and 98%. Received: 19 October 1997 / Accepted: 19 November 1997     

Ultrastructural localization of a sialic acid-specific hemolymph lectin in the hemocytes and other tissues of the hard tick Ixodes ricinus (Acari; Chelicerata)

 Lectins have been suggested to function as pattern-recognition molecules in invertebrate immune mechanisms. A lectin from the hemolymph of the tick Ixodes ricinus with main specificity for sialic acid was characterized and antibodies directed against this lectin were prepared. In this study, these antibodies were used to localize the lectin in the tissues of I. ricinus. Immunoreactivity with poly- and monoclonal antibodies was detected in the granules of both types of granular hemocytes, at the membrane of hemocytes, and at the basal laminae surrounding the hemocoel. Furthermore, cells attached to the midgut, invaginations of Géné’s organ, and granular inclusions of nephrocytes were labeled. The immunoreactivity detected in hemocytes and the hemocoel lining supports the idea that the hemolymph lectin may function as a recognition molecule in the immune system of I. ricinus. Another function could be protection of eggs that are coated with secretions by Géné’s organ. The lectin activity could also be involved in transmission of Borrelia burgdorferi, the causative agent of Lyme borreliosis, and the tick-borne encephalitis virus. Received: 7 June 1995 / Accepted: 29 August 1995     

Defective RNA packaging is responsible for low transduction efficiency of CAEV-based vectors}

Summary.  Replication defective retroviral vectors are regularly used for transfer and expression of exogenous genes into dividing cells and in animals. Since lentiviruses are able to infect terminally differentiated and non-dividing cells, their use to produce replication defective vectors may overcome this limitation. We developed two replication-defective lentiviral vectors based on the genome of Caprine Arthritis Encephalitis Virus (CAEV). The first vector (pBNL2) carries the neo and lacZ marker genes. Neo gene is expressed from a genomic RNA and lacZ gene from a subgenomic RNA. The second vector (pCSHL) carries a single fusion gene encoding both phleomycin resistance and β-galactosidase activity. Replication-competent CAEV was used as helper virus to provide the viral proteins for transcomplementation of these vectors. Our data demonstrated that the genomes of both vectors were packaged into CAEV virions and transduced into goat synovial membrane cells following infection. However, the vector titers remained 3 to 4 logs lower than those of CAEV. Further analysis showed a lack of accumulation of unspliced pBNL2 RNA into the cytoplasm of producer cells resulting in the packaging of pBNL2 sub-genomic RNA only. In contrast, RNA produced from pCSHL vector was correctly transported to the cytoplasm and more efficiently packaged than the pBNL2 sub-genomic RNA as revealed by slot-blot and quantitative RT/PCR analyses. However this higher packaging efficiency of pCSHL genome did not result in a higher transduction efficiency of lacZ gene. Accepted November 26, 1997 Received August 8, 1997     

Molecular characterisation of Banana mild mosaic virus, a new filamentous virus in Musa spp.

Summary.  Banana mild mosaic virus (BanMMV) is a previously undescribed filamentous virus infecting Musa spp. The complete genome has been sequenced from PCR clones and consists of 7352 nucleotides, encoding five open reading frames (ORFs) of 205, 25.5, 12.4, 8.0 and 26.8 kDa, respectively. BanMMV was most closely related to fovea- and carlaviruses, based on phylogenetic analysis using a portion of the viral replicase. Analysis of other parts of the genome revealed similarities with fovea-, carla- and potexviruses, but the virus was not clearly aligned to any previously recognised genus. Received June 21, 2000 Accepted February 28, 2001     

A 1-year investigation of the parasite Haplosporidium nelsoni (MSX) in the Pacific oyster Crassostrea gigas from Dayaowan Bay, China

The infection prevalence of the protozoan parasite Haplosporidium nelsoni (MSX) in Pacific oysters (Crassostrea gigas), collected from Dayaowan Bay on the north coast of the Yellow Sea, China, was investigated in 2007. The traditional histological method of diagnosing H. nelsoni infection in oysters was compared to that of polymerase chain reaction (PCR). Histology and the first PCR analysis detected infection in 21 (a total of 240 oysters) (8.75%) oysters, and the second PCR revealed infection in 26 oysters (10.83%). Only local or epithelial infections were found; no systemic infections were detected. Infection by H. nelsoni mostly occurred from April through October, and the monthly prevalence ranged from 5% to 25%, with a peak in August. These results suggest that the prevalence of the parasite is low in Dayaowan Bay. The prevalence of H. nelsoni is thought to be controlled in some way by temperature and salinity.     

Nucleotide sequence and genome organisation of cherry mottle leaf virus and its relationship to members of the Trichovirus genus

Summary.  The nucleotide sequence of cherry mottle leaf virus (CMLV) was determined and compared to sequences of a number of plant viruses including the type member of the Trichovirus genus (apple chlorotic leafspot virus, ACLSV), and members of the Vitivirus genus including grapevine virus B, (GVB). The CMLV genome was determined to consist of 8003 nt excluding the poly(A) tail at the 3′ end of the genome. The overall A+U content of CMLV genomic RNA was 59. 1%, which is similar to ACLSV, but significantly different from GVB. Four putative open reading frames were identified (ORFs 1, 2, 3, and 4) encoding proteins of M_r 215. 8 kDa, 47 kDa, 21.6 kDa, and 15. 3 kDa, respectively. This differs from ACLSV which has 3 ORFS, and GVB which has 5 ORFs. Protein database searches showed no matches of CMLV ORF4 with ACLSV sequences, but found similarities between ORF4 of CMLV and ORF5 of GVB, suggesting that this may be a nucleic acid-binding protein. CMLV and ACLSV formed a common virus clade in phylogenetic analysis of the coat protein amino acid sequence and except for CMLV’s ORF4, these viruses show high levels of similarity throughout the genome. CMLV appears to be a member of the Trichovirus genus. Accepted November 19, 1999/Received August 12, 1999     

Molecular characterization and interviral relationships of a flexuous filamentous virus causing mosaic disease of sugarcane (Saccharum officinarum L.) in India

Summary.  A virus isolate causing mosaic disease of commercial sugarcane was purified to homogeneity. Electron microscopy revealed flexuous filamentous virus particles of ca 890 × 15 nm. The virus isolate reacted positively with heterologous antiserum to narcissus latent virus form UK, but failed to react with potyvirus group specific antiserum. N-terminal sequencing of the intact coat protein (CP) and the tryptic peptides indicated that the virus was probably a potyvirus but distinct from several reported potyviruses. Comparison of the 3′-terminal 1084 nucleotide sequence of the RNA genome of this virus revealed 93.6% sequence identity in the coat protein coding region with the recently described sugarcane streak mosaic virus (Pakistani isolate). The molecular weight of the coat protein (40 kDa) was higher than that deduced from the amino acid sequence (34 kDa). The apparent increase in size was shown to be due to glycosylation of the coat protein which has not been reported thus far in the family, Potyviridae. This is the first report on the molecular characterization of a virus causing mosaic disease of sugarcane in India and the results demonstrate that the virus is a strain of sugarcane streak mosaic virus, a member of the Tritimovirus genus of the Potyviridae. We have named it sugarcane streak mosaic virus – Andhra Pradesh isolate (SCSMV-AP). Received October 14, 1997 Accepted August 7, 1998