Viral Sense Research Articles

Biochemical characterization of C4 protein of Cotton Leaf Curl Kokhran Virus-Dabawali

BackgroundCotton leaf curl Kokhran Virus-Dabawali (CLCuKV-Dab) is a monopartite begomovirus encoding two proteins V1 and V2 in the virion sense and four proteins C1, C2, C3 and C4 in the complementary sense. The C4 protein of monopartite begomoviruses has been implicated to play a role in symptom determination and virus movement. The present work aims at the biochemical characterization of this protein. MethodsThe C4 protein of CLCuKV-Dab was purified in fusion with GST and tested for the ability to hydrolyze ATP and other phosphate containing compounds. ATPase activity was assayed by using radiolabeled γ-[32P]-ATP and separating the product of reaction by thin layer chromatography. The hydrolysis of other compounds was monitored by the formation of a blue colored phosphomolybdate complex which was estimated by measuring the absorbance at 655nm. ResultsThe purified GST-C4 protein exhibited metal ion dependent ATPase and inorganic pyrophosphatase activities. Deletion of a sequence resembling the catalytic motif present in phosphotyrosine phosphatases resulted in 70% reduction in both the activities. Mutational analysis suggested arginine 13 to be catalytically important for the ATPase and cysteine 8 for the pyrophosphatase activity of GST-C4. Interaction of V2 with GST-C4 resulted in an increase in both the enzymatic activities of GST-C4. ConclusionsThe residues important for the enzymatic activities of GST-C4 are present in a motif different from the classical Walker motifs and the non-classical ATP binding motifs reported so far. General significanceThe C4 protein of CLCuKV-Dab, a putative natively unfolded protein, exhibits enzymatic activities.

Molecular characterization of the full-length L and M RNAs of Tomato yellow ring virus, a member of the genus Tospovirus

Tomato yellow ring virus (TYRV), first isolated from tomato in Iran, was classified as a non-approved species of the genus Tospovirus based on the characterization of its genomic S RNA. In the current study, the complete sequences of the genomic L and M RNAs of TYRV were determined and analyzed. The L RNA has 8,877 nucleotides (nt) and codes in the viral complementary (vc) strand for the putative RNA-dependent RNA polymerase (RdRp) of 2,873 amino acids (aa) (331 kDa). The RdRp of TYRV shares the highest aa sequence identity (88.7 %) with that of Iris yellow spot virus (IYSV), and contains conserved motifs shared with those of the animal-infecting bunyaviruses. The M RNA contains 4,786 nt and codes in ambisense arrangement for the NSm protein of 308 aa (34.5 kDa) in viral sense, and the Gn/Gc glycoprotein precursor (GP) of 1,310 aa (128 kDa) in vc-sense. Phylogenetic analyses indicated that TYRV is closely clustered with IYSV and Polygonum ringspot virus (PolRSV). The NSm and GP of TYRV share the highest aa sequence identity with those of IYSV and PolRSV (89.9 and 80.2-86.5 %, respectively). Moreover, the GPs of TYRV, IYSV, and PolRSV share highly similar characteristics, among which an identical deduced N-terminal protease cleavage site that is distinct from all tospoviral GPs analyzed thus far. Taken together, the elucidation of the complete genome sequence and biological features of TYRV support a close ancestral relationship with IYSV and PolRSV.

Sequences within the Spinach curly top virus virion sense promoter are necessary for vascular-specific expression of virion sense genes

Sequences necessary for activity of the Spinach curly top virus virion sense promoter have been identified within an 84 bp region upstream of two transcription start sites located at nt 252 and 292. RNAs initiating at these sites are expressed at equivalent levels in SCTV-infected Arabidopsis and from promoter-reporter constructs. The promoter is capable of directing expression of all three virion sense genes, although not to the same degree. While CP and V3 expression are similar, expression of V2 is elevated. The promoter is active in transient leaf infusion assays in the absence of C2. In Nicotiana benthamiana plants the promoter is active in vascular tissue and under no conditions did we detect promoter activity in the mesophyll. This is in contrast to begomoviruses where the virion sense promoter is dependent on AL2, a positional homolog of C2, and the promoter is functional in both vascular and mesophyll tissue.

Molecular comparative analysis of component 1 (DNA-R) of an Egyptian isolate of banana bunchy top nanovirus isolated from banana aphid (Pentalonia nigronervosa)

We are reporting a molecular comparative analysis of component 1 BBTV-DNA-R of an Egyptian isolate of (BBTV) and 30 different geographical isolates. DNA was extracted from BBTV-infected adult banana aphids collected from El-Qalubia Governorate, Egypt. Using specific primers the BBTV-DNA-R was amplified, cloned into a prokaryote vector, sequenced and a molecular comparative analysis of BBTV-DNA-R of this study and some overseas isolates of BBTV-infected banana plants was determined. Results showed that the component 1 consists of 1108 nts and contains a sequence of 69 nts representing the CR-SL of 31 nts. A CR-M (90 nts) at the position (972–1062) characterized with GC-rich sequence from nts 76 to 90 (average of 80% G + C) was found. Alignment results of BBTV DNA-R confirmed the presence of a number of conserved regions in all isolates. Large ORF of 861 nts at position 102 to 962 in the virion sense were detected. The predicted protein of this ORF consisted of 286 amino acids and had a molecular weight of 33.8 kDa. The DNA-phylogenetic analysis showed a percent identity of 98.0 and 97.9 between BBTV DNA-R and isolates of Pakistan (isolate TJ1) and Australia (isolate V1), respectively. The similarities between the gene product of Egyptian BBTV DNA-R and the 30 overseas isolates ranged from 93.7 to 99.0%. Differences in phylogenetic trees based on the entire sequence of BBTV DNA-R, CR-M and amino acid sequences confirmed the existence of two taxonomic groups of BBTV and the Egyptian isolate belongs to the south pacific group.

Molecular identification of a new begomovirus associated with yellow mosaic disease of Jatropha gossypifolia in India

Yellow mosaic disease was observed on Jatropha gossypifolia plants growing in Kathaupahadi, Madhya Pradesh, India, and whiteflies (Bemisia tabaci) were found in the vicinity. Association of a new begomovirus with yellow mosaic disease of J. gossypifolia has been detected by PCR using begomovirus DNA-A-specific primers. The complete DNA-A genome (~2.7 kb) of this virus isolate was amplified by rolling-circle amplification (RCA) followed by digestion with Bam HI. The ~2.7-kb amplicons was cloned and sequenced, and the data obtained were submitted to GenBank under accession numbers FJ177030. The genome of the virus isolate consisted of six open reading frames (ORFs): V2 (pre-coat protein) and V1 (coat protein) in the virion sense and C3 (REn protein), C2 (TrAP protein), C1 (replication-associated protein) and C4 (C4 protein) in the complementary sense. BLASTn analysis of the nucleotide sequence (2757 nt) of the viral genome (FJ177030) showed 84-85% identity and a distinct phylogenetic relationship with DNA-A of tomato leaf curl virus-Bangalore II (U38239) and tomato leaf curl Karnataka virus (AY754812). Based on its 85% sequence identity to all other begomoviruses known to date and ICTV species demarcating criteria (< 88% identity), the name Jatropha yellow mosaic India virus (JYMIV) is proposed. JYMIV is considered to be monopartite, as neither DNA-B nor DNA-β components associated with begomoviruses were detected.

English

Morning glory (Ipomoea indica) plants showing yellow mosaic and vein yellowing symptoms were collected from&nbsp;Jiangsu and Yunnan province of China. The amplified DNA-A components of two isolates named JS1 and Y338 were comprised of 2827 and 2801 nucleotides, respectively, in length with genome organization similar to that of begomoviruses. Complete nucleotide sequence revealed the presence of two ORFs (AV1 and AV2) in the virion sense and four ORFs (AC1, AC2, AC3 and AC4) in the complementary sense. Comparison analysis showed that DNA-A sequence of JS1 isolate was closely related to that of sweet potato leaf curl virus (SPLCV) from United States with nucleotide sequence identity of 97.0% and DNA-A of Y338 showed&nbsp;highest sequence identity at 97.8% with an isolate of SPLCV from China. Phylogenetic analysis showed that JS1 clustered together with SPLCV-US and Y338 showed close relationship with SPLCV-CN. Sequence analysis indicated that isolates JS1 and Y338 were strains of SPLCV. &nbsp; Key words:&nbsp;Sweet potato leaf curl virus, begomoviruses,&nbsp;Ipomoea indica, sequence comparison.

Genomic characterization of Calla lily chlorotic spot virus and design of broad‐spectrum primers for detection of tospoviruses

Calla lily chlorotic spot virus (CCSV) collected from calla lily plants showing symptoms in Taiwan has been identified as a tentative species of the genus Tospovirus based on the comparison of S RNA sequences. In this investigation, the complete sequences of its L and M RNAs were determined. The L RNA contains 8911 nucleotides (nt) and encodes a putative RNA‐dependent RNA polymerase of 2882 amino acids (aa) (332 kDa) in the viral complementary (vc) sense. The M RNA was shown to be 4704 nt in length, encoding a nonstructural NSm protein of 309 aa (35 kDa) in the viral sense and a Gn/Gc glycoprotein precursor (GP) of 1123 aa (128 kDa) in the vc sense. Phylogenetic analysis of the individual tospoviral proteins indicated that CCSV is closely related to Tomato zonate spot virus, a new tentative tospovirus isolated from tomato in southern China. Two degenerate primer pairs gL2740/gL3920c and gM410/gM870c, designed, after sequence comparison, from the highly conserved regions of the L and NSm genes, respectively, were successfully used to detect 12 greenhouse‐cultured tospoviruses, including six formal species and six tentative species, by reverse transcription‐polymerase chain reaction. They were also used to detect Melon yellow spot virus and Watermelon silver mottle virus from field cucurbit samples in Taiwan. The results indicate that the two degenerate primer pairs can be used for prompt detection of most tospoviruses and exploration of unknown tospovirus species.

Genome organisation and sequence comparison suggest intraspecies incongruence in M RNA of Watermelon bud necrosis virus

Watermelon bud necrosis virus (WBNV), a member of the genus Tospovirus, family Bunyaviridae is an important viral pathogen in watermelon cultivation in India. The complete genome sequence properties of WBNV are not available. In the present study, the complete M RNA sequence and the genome organisation of a WBNV isolate infecting watermelon in Delhi (WBNV-wDel) were determined. The M RNA was 4,794 nucleotides (nt) long and potentially coded for a movement protein (NSm) of 34.22 kDa (307 amino acids) on the viral sense strand and a Gn/Gc glycoprotein precursor of 127.15 kDa (1,121 amino acids) on the complementary strand. The two open reading frames were separated by an intergenic region of 402 nt. The 5' and 3' untranslated regions were 55 and 47 nt long, respectively, containing complementary termini typical of tospoviruses. WBNV-wDel was most closely related (79.1% identity) to Groundnut bud necrosis virus, an important tospovirus that occurs in several crops in India, and was different (63.3-75.2% identity) from the other cucurbit-infecting tospoviruses known to occur in Taiwan and Japan. Sequence analysis of NSm and Gn/Gc revealed phylogenetic incongruence between WBNV-wDel and another isolate originating from central India (WBNV-Wm-Som isolate). The Wm-Som isolate showed evolutionary divergence from the wDel isolate in the Gn/Gc protein (74.6% identity) potentially due to recombination with the other tospoviruses that are known to occur in India. This is the first report of a comparison of complete sequences of M RNA of WBNV.

Nucleotide sequence and genome organization of the medium RNA of Iris yellow spot virus from the United States

Iris yellow spot tospovirus (IYSV) of the family Bunyaviridae causes a serious disease in onion in the USA and other parts of the world. Inspite of its economic importance, the complete genomic sequence of IYSV from the USA is not available. The genome structure and organization of the medium (M) RNA of a Washington (WA) isolate of IYSV were determined and compared to the corresponding region of two isolates previously described from Brazil and The Netherlands. Sequence analysis showed that the M-RNA was 4,817 nucleotides long and potentially coded for the movement protein (NSm) in the viral sense and the glycoprotein precursor (Gn and Gc) in the viral complementary sense. The predicted sizes of NSm and Gn/Gc precursor were 34.7 and 128.84 kDa, respectively. The two open reading frames are separated by a 380 nucleotide intergenic region. Phylogenetic analysis of the NSm and Gn/Gc genes from the WA isolate showed grouping that reflected their respective serogroups. The WA isolate formed a close cluster with the two previously reported IYSV isolates and the IYSV cluster was distinguishable from other tospovirus species. This is the first report of complete genomic sequence of the M-RNA of IYSV from the US.

Identification of Critical Molecular Determinants of West Nile Virus PrM Protein: A Potential Site for Antiviral Targeting

get. This involves both oligomerization of the capsid (C) protein and packaging of the viral RNA. The RNA binding properties of the C protein is poorly understood. This study aims to characterize the C and RNA interaction. Method: Co-localization study was performed by transfecting in vitro transcribed WN virus RNA and C protein clones into BHK cells and visualized under fluorescence microscopy. RNA binding properties of C protein were further investigated with a Northwestern Blot assay and RNA pull-down assay. Synthesized C protein peptides were used to map out the RNA binding regions on C protein. In addition, C protein immunopurified from BHK cells were used to investigate the effect of phosphorylation of C protein on its RNA binding properties. Results: RNA and C protein have failed to show colocalization in BHK cells by immuno-fluorescence but interactions were observed at the molecular level. It showed that the first 465 and last 693 nucleotides of the WN virus RNA had specific affinity for the full length C protein. In addition, the aminoand carboxy-terminal of the C protein were shown to bind to the virus RNA. It was also found that the C protein had affinity for viral anti-sense RNA. Phosphorylated peptides of C protein and C protein expressed in BHK cells show attenuated binding to viral RNA. Conclusion: C protein interaction with anti-sense indicates that its interaction with viral sense RNA may not be specific. Phosphorylation of C protein could play a role in regulating C and RNA interaction and allow time for viral assembly. Understanding the interaction of C protein with viral RNA and role of phosphorylation in nucleocapsid assembly could help develop anti-viral strategies aimed at disrupting viral assembly.

Molecular characterization of sweet potato leaf curl virus isolate from China (SPLCV-CN) and its phylogenetic relationship with other members of the Geminiviridae

A Sweet potato-infecting sweet potato leaf curl virus (SPLCV) isolated in China was detected by Polymerase Chain Reaction (PCR). PCR products amplified from DNA-A were cloned and sequenced. The isolates of SPLCV from China(SPLCV-CN)has a genome organization similar to that of monopartite begomoviruses. The DNA-A had two ORFs (AV1 and AV2) in the virion sense and four ORFs (AC1, AC2, AC3, and AC4) in the complementary sense, separated by an intergenic region (IR) containing a conserved stem-loop motif. Three incomplete direct repeat iterons were also found within the IR. The presence of AV2 ORF supports the relationship of SPLCV-CN to the Old World gemimiviruses. Sequence comparisons showed that the DNA-A sequence of SPLCV-CN were closely related to those of sweet potato leaf curl Georgia virus-[16] (SPLCGV-[16]), Ipomoea yellow vein virus (IYVV-SI), and sweet potato leaf curl virus (SPLCV) with nucleotide sequence identity ranging from 88% to 91%. Comparison of individual encoded proteins between SPLCV-CN and that of three other SPLCV isolates showed the coat protein (AV1) shared the highest amino acid sequence identity (93%-96%), suggesting the coat protein of these viruses may have identical ancestor. The relationships between SPLCV-CN and other whitefly-transmitted geminiviruses were investigated by using phylogeny of derived AV1, AC1, and AV2 amino acid sequences. In all phylogenetic trees, SPLCV-CN clustered with three other isolates of SPLCV. The analyses revealed that the four isolates of SPLCV have coat proteins which are unique from its counterparts from both the Old World and New World. The present of AV2 and phylogenic analysis of AC1 suggest that SPLCV is more close to begomoviruses from the Old World but isolates of this virus seems to form a separate subset.

Transcriptional Analysis of Complementary Sense Genes in Spinach curly top virus and Functional Role of C2 in Pathogenesis

Spinach curly top virus (SCTV), the fifth characterized Curtovirus species belonging to the family Geminiviridae, is an agriculturally significant plant pathogen representing an emerging disease threat in the southern United States. The SCTV genome comprises a single DNA chromosome of approximately 3.0 kb, with the potential to code for seven proteins larger than 10 kDa but which relies extensively on the host for replication and transcription of its genome. In this study, we have identified viral and complementary sense transcripts in SCTV-infected plants, confirming a bidirectional transcription strategy for SCTV. The most abundant RNA maps to the virion sense (1.1-kb transcript) and is comparable in size and location to that observed in Beet curly top virus (BCTV). Two complementary sense transcripts (1.7 and 0.7 kb) were identified in SCTV-infected plants. The large, 1.7-kb transcript is comparable in size and position to that identified in BCTV and several begomoviruses and most likely encodes the C1 protein. Both complementary sense RNAs could potentially direct expression of C2 and C3 from polycistronic mRNAs. A mutation in the C2 gene of SCTV results in expression of a truncated protein of 38 amino acids that is capable of interacting with two cellular kinases, AKIN11 and ADK2, and the resulting mutant virus remains highly infectious. A second mutant virus can only express the first three amino acids of the C2 protein and is unable to interact with the same kinases. However, this mutant virus still remains infectious, although a reduction in infectivity and symptom severity was seen in both Arabidopsis and spinach. A possible relationship between the interaction of C2 with AKIN11 and ADK2 and disease severity is presented.

Mapping the 5′ ends of banana bunchy top virus gene transcripts

Banana bunchy top virus (BBTV), a multi-component circular ssDNA virus, replicates via a dsDNA intermediate that also serves as a template for virion sense transcription. Seven virus-derived transcripts have been previously identified and analysed in BBTV-infected bananas by northern analysis and 3' rapid amplification of cDNA ends (3' RACE). In this study, we have used RNA ligase-mediated rapid amplification of 5' cDNA ends (RLM-RACE) to complete the mapping of the BBTV gene transcripts and have now fully mapped the transcribed regions of each BBTV component and effectively defined the upstream regulatory region.

Differential roles of C4 and βC1 in mediating suppression of post-transcriptional gene silencing: Evidence for transactivation by the C2 of Bhendi yellow vein mosaic virus, a monopartite begomovirus

Bhendi yellow vein mosaic disease (BYVMD) is caused by the association of a DNA β satellite with a begomovirus component. The begomovirus component has two promoters, one in the virion sense (V-sense) and the other in the complementary sense (C-sense) in the intergenic region (IR). To study the promoter activities of V-sense and C-sense promoters, mGFP gene fusion was made downstream to the promoters. Transient and stable expressions in N. benthamiana leaves showed significant GFP expression under C-sense promoter whereas the expression under the V-sense promoter was very weak in the absence of the transactivator C2. Untransformed N. benthamiana plants were agroinfiltrated with binary vector constructs containing V-sense-GFP alone or along with C1, C2, C4, V1, V2 or β C1 (in both sense and antisense orientations) to understand the roles of these gene products in transactivation and/or suppression of post-transcriptional gene silencing (PTGS). The results showed strong suppression of gene silencing activities for C4 and βC1 but a weak activity for C2. The suppression activities were also confirmed using gfp-silenced GFP16c/GFPi plants by agroinfiltration and agroinoculation. The expression of C4 and βC1 as transgenes produced abnormal phenotypic growth compared to the other viral genes mentioned above, further supporting their suppressor function.

Studies on the activity of a bidirectional promoter of Mungbean yellow mosaic India virus by agroinfiltration

The AV promoter expressing AV1 and AV2 genes and AC1 promoter expressing AC1 gene are present in opposite orientation in the intergenic region of Mungbean yellow mosaic India virus (MYMIV). Transient Agrobacterium-mediated delivery of putative promoter constructs into Nicotiana benthamiana and different legumes, followed by reporter gene (β- d-glucuronidase, GUS) assay, identified the promoter region of both AC1 and AV genes that is necessary for transcriptional initiation. Transcription activator protein-independent activity of AV promoter and differential regulation of AC1 promoter are unique to MYMIV. The AV promoter is a composite core promoter having both TATA box and Initiator elements (TATA +Inr +). Many transcription factor binding sites were identified in the upstream promoter sequences of both virion and complementary sense genes, which might be used in the transcription regulation studies of the host plant as well as the virus.

Molecular characterization of tomato-infecting begomoviruses in Thailand

Bipartite geminiviruses infecting tomatoes in Thailand were detected by polymerase chain reaction (PCR) using CPA5/CPA2 primers. Products derived from PCR-amplified full-length DNA-A and DNA-B of TYLCV collected from Chiang Mai, Nong Khai, and Sakon Nakhon were cloned and sequenced. DNA-A from Chiang Mai was 2747 nts long; Nong Khai, 2744 nts; and Sakon Nakhon, 2747 nts, and those of DNA-B from Chiang Mai were 2750 nts long; Nong Khai, 2749 nts; and Sakon Nakhon, 2749 nts. The genomes of these virus isolates were organized like those of other begomoviruses. The DNA-A had two ORFs in the virion sense and four ORFs in the complementary sense. The DNA-B had two ORFs in the virion sense and one ORF in the complementary sense. Nucleotide sequences of DNA-A of TYLCV from Chiang Mai, Nong Khai, and Sakon Nakhon were closely related to those of Tomato yellow leaf curl Thailand virus (TYLCTHV) and Tomato yellow leaf curl Thailand virus -[Myanmar] (TYLCTHV-[MM]) with nucleotide sequence identity ranging from 89% to 95%. Based on sequence comparisons and phylogenetic analyses, these three virus isolates studied were identified as new strains of TYLCTHV and named Tomato yellow leaf curl Thailand virus-Chiang Mai (TYLCTHV-[CM] 1 1 The GenBank accession codes for DNA-A of TYLCTHV-[CM], -[NK], and -[SK] are AY514630, AY514631 and AY514632, respectively. The GenBank accession codes for DNA-B of TYLCTHV-[CM], -[NK], and -[SK] are AY514633, AY514634, and AY514635, respectively. ), Nong Khai (TYLCTHV-[NK] and Sakon Nakhon (TYLCTHV-[SK]).

Diversity of DNA 1: a satellite-like molecule associated with monopartite begomovirus–DNA β complexes

DNA 1 components are satellite-like, single-stranded DNA molecules associated with begomoviruses (family Geminiviridae) that require the satellite molecule DNA β to induce authentic disease symptoms in some hosts. They have been shown to be present in the begomovirus–DNA β complexes causing cotton leaf curl disease (CLCuD) and okra leaf curl disease (OLCD) in Pakistan as well as Ageratum yellow vein disease (AYVD) in Singapore. We have cloned and sequenced a further 17 DNA 1 molecules from a diverse range of plant species and geographical origins. The analysis shows that DNA 1 components are associated with the majority of begomovirus–DNA β complexes, being absent from only two of the complexes examined, both of which have their origins in Far East Asia. The sequences showed a high level of conservation as well as a common organization consisting of a single open reading frame (ORF) in the virion sense, a region of sequence rich in adenine and a predicted hairpin structure. In phylogenetic analyses, there was some evidence of grouping of DNA 1 molecules according to geographic origin, but less evidence for grouping according to host plant origin. The possible origin and function of DNA 1 components are discussed in light of these findings.

Phloem specific expression driven by wheat dwarf geminivirus V-sense promoter in transgenic dicotyledonous species.

Wheat dwarf geminivirus (WDV) is a single-stranded DNA Mastrevirus. The large intergenic region (LIR) of WDV contains cis-acting elements essential for the replication of the genome as well as for the bidirectional transcription of virus genes. The LIR was fused to the GUS (uidA) reporter gene and the WDV viral sense (V-sense) promoter activity derived from the stable integration of that promoter was analysed in transgenic dicot plants. Various dicot species were tested, including Nicotiana tabacum, Nicotiana benthamiana, Arabidopsis thaliana and Cucumis melo. The GUS activity driven by the WDV promoter was also compared to that obtained in plants transformed with the GUS gene controlled by the CaMV 35S promoter as well as two phloem-specific promoters derived from the Arabidopsis thaliana AtSUC2 and AtAHA3 genes. Histochemistry showed that the WDV V-sense promoter consistently induced an expression pattern restricted to the vascular tissues, predominantly in the phloem of all organs. This promoter exhibited levels of GUS activity comparable to that driven by AtSUC2 and AtAHA3 promoters. A vascular expression pattern was observed in the four dicots tested. This was stable during plant development and was not altered following viral infection by an unrelated geminivirus. The uses of such a promoter are discussed regarding the targeting to the phloem of molecules active against vascular pests or pathogens.

Influenza vaccines generated by reverse genetics.

Influenza viruses cause annual epidemics and occasional pandemics of acute respiratory disease. Vaccination is the primary means to prevent and control the disease. However, influenza viruses undergo continual antigenic variation, which requires the annual reformulation of trivalent influenza vaccines, making influenza unique among pathogens for which vaccines have been developed. The segmented nature of the influenza virus genome allows for the traditional reassortment between two viruses in a coinfected cell. This technique has long been used to generate strains for the preparation of either inactivated or live attenuated influenza vaccines. Recent advancements in reverse genetics techniques now make it possible to generate influenza viruses entirely from cloned plasmid DNA by cotransfection of appropriate cells with 8 or 12 plasmids encoding the influenza virion sense RNA and/or mRNA. Once regulatory issues have been addressed, this technology will enable the routine and rapid generation of strains for either inactivated or live attenuated influenza vaccine. In addition, the technology offers the potential for new vaccine strategies based on the generation of genetically engineered donors attenuated through directed mutation of one or more internal genes. Reverse genetics techniques are also proving to be important for the development of pandemic influenza vaccines, because the technology provides a means to modify genes to remove virulence determinants found in highly pathogenic avian strains. The future of influenza prevention and control lies in the application of this powerful technology for the generation of safe and more effective influenza vaccines.

日本の周辺国で問題となっているウイルス感染症 ３．リフトバレー熱ウイルス

Rift Valley fever virus (RVFV) causes massive mosquito-borne epidemics among humans and decimates ruminants in which the mortality rate is about 1% and 10-30%, respectively. Morbidity in RVFV-infected humans is high largely due to the effects of hemorrhagic fever and encephalitis. This virus is native to sub-Saharan Africa; yet if this virus is introduced into the environment, virus transmission appears to occur whenever sheep and cattle are present with abundant mosquito populations. RVFV is a negative-strand RNA virus which belongs to the family Bunyaviridae, genus Phlebovirus, and contains tripartite-segmented genomes (S, M, and L). S-segment is the ambisense genome, where N and NSs genes are coded in an antiviral-sense and viral sense S-segment, respectively. The inhibition of host mRNA synthesis, which is induced by the binding of NSs protein to RNA polymerase II transcription factor TFIIH, is the primary reason for the host-protein shut-off in RVFV-infected cells. Development of a RVFV reverse genetics system, which has not been accomplished yet, is important for the study of viral replication mechanisms, host virus interaction, viral pathogenicity as well as vaccine evaluation and development.