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The complete nucleotide sequence of maize dwarf mosaic virus Bulgarian isolate (MDMV-Bg) was determined. The viral genome was 9515 nt and contained an open reading frame encoding 3042 amino acids, flanked by 3'- and 5'-UTRs of 139 and 250 nucleotides, respectively. MDMV-Bg was more conserved in the coding region (52.9%) than in the UTRs (45.8%) when compared to the 15 other potyviruses. Of ten putative gene products of MDMV-Bg, the P1 was the most variable protein (24.9%) while the NIb was the most conserved protein (67.3%). Several sequence variations were observed between MDMV-Bg and Johnson grass mosaic virus (JGMV), and more between MDMV-Bg and the dicot potyviruses. Phylogenetic analysis suggested that MDMV-Bg was the most closely related to JGMV.

The single-stranded DNA geminiviruses produce transcripts from both strands (virion- and complementary-sense) of a nuclear double-stranded DNA molecule. In maize streak virus (MSV)-infected maize plants, approximately 80% of the complementary-sense transcripts produce the C1 protein, whilst the remaining 20% are spliced to remove a 92 nt intron and produce a C1:C2 fusion protein (Rep). Disruption of the complementary-sense 3' splice site abolished virus replication. The majority of the virion-sense transcripts initiated one nucleotide upstream of the V1 (movement protein) gene and a minority a further 141 nucleotides upstream. A 76 nt intron, with features typical of plant introns, was identified within the V1 gene, upstream of the coat protein gene. Spliced and unspliced forms of each virion-sense transcript were produced, but they differed in splicing efficiency. Approximately 50% of the major transcript and less than 10% of the minor transcript were processed. Mutagenesis of the consensus 5' splice site in the V1 gene resulted in the use of alternative cryptic splice sites, confirming the importance of splicing for MSV infection. Spliced virion-sense transcripts were also identified in tissue infected with the closely-related Digitaria streak virus (DSV) but not with another subgroup I geminivirus, wheat dwarf virus. Collectively, the multiple transcript initiation sites and different splicing efficiencies suggest that splicing is an important feature in the regulation of both early and late gene expression in MSV and DSV.

We have examined the molecular epidemiology of the leafhopper-borne maize rayado fino virus (MRFV) in Latin America. The coat protein gene and 3' non-translated region of 14 isolates of MRFV collected from Latin America and the United States were sequenced and phylogenetic relationships examined. The nucleotide sequence revealed remarkable conservation, with a sequence similarity of 88-99%. Phylogenetic analysis of sequence data obtained from a 633 bp fragment showed that MRFV has diverged into three main clusters, i.e. the geographically distinct northern and southern isolates and the Colombian isolates. Significant differences between the isolates collected from Colombia, previously named maize rayado colombiana virus, based upon differences in symptomatology and serological relationships to MRFV, and the other MRFV isolates, provides additional evidence supporting its designation as a unique strain of MRFV.

Maize streak virus (MSV) coat protein (CP) is required for virus movement within the plant. Deletion or mutation of MSV CP does not prevent virus replication in single cells or protoplasts but leads to a loss of infectivity in the inoculated plant. The mechanism by which MSV CP mediates the transfer of MSV DNA from cell to cell and through the vascular bundle is still unknown. Towards understanding the role of MSV CP in virus movement, the interaction of the CP with viral DNA was investigated using the 'south-western' assay. Wild-type and truncated MSV CPs were expressed in E. coli and the expressed CPs were used to investigate interactions with single-stranded (ss) and double-stranded (ds) DNA. The results showed that MSV CP bound ss and ds viral and plasmid DNA in a sequence non-specific manner. The binding domain was mapped to within the 104 N-terminal amino acids of the MSV CP. We propose that the binding of CP to MSV DNA is involved in viral DNA nuclear transport as well as encapsidation and thus may have a role in intra- and inter-cellular movement as well as systemic infection.

The complete sequence of a Tennessee isolate of maize chlorotic dwarf virus (MCDV-TN) was determined from cDNA clones and by direct sequencing of the viral RNA. The genome is 11813 nucleotides (nt) in length and contains one large open reading frame between nt 435 and 10763 that encodes a polyprotein of 3443 amino acids. The N-terminal amino acid sequences were determined for the three capsid proteins. All three were adjacent, starting at nt 2526 and putatively ending at nt 3761. Comparison of the sequence of MCDV-TN with other viral sequences revealed similarities to several plant picorna-like viruses including members of the Sequiviridae, Comoviridae and Potyviridae. This work also provides evidence based on genome organization that MCDV-TN is a member of the genus Waikavirus within the family Sequiviridae.

Analysis of transposition products generated after Activator (Ac) excision from the P locus in maize suggest that Ac excises either during or after replication of the P locus. The frequency of excision of the non-autonomous Ac derivative, Dissociation (Ds), from extrachromosomal replicating and nonreplicating vector DNAs in transfected black mexican sweet maize protoplasts was compared to assess directly a role of extrachromosomal vector DNA replication in Ds excision. Replicating (rep+) and nonreplicating (rep-) vector DNAs comprised a Ds element that harbored a geminivirus, wheat dwarf virus (WDV), origin of replication and WDV genes required for viral DNA replication (rep+) or mutant, inactive derivatives of these genes (rep-). Excision of Ds was detected only in those cell nuclei co-transfected with the replicating Ds-vector DNA and a transposase expression vector. Quantitative reconstruction experiments showed that Ds excised at least 3 x 10(5)-fold more frequently from replicating vector DNA as compared with nonreplicating vector DNA. Therefore, these results provide direct evidence for a coupling of Ds excision from extrachromosomal vector DNA to vector DNA replication in maize.

We have used a polyclonal antiserum derived from a bacterially expressed viral fusion protein to investigate the expression and subcellular localisation of the maize streak virus V1 product (PV1). Western blot analysis of agroinfected tissue showed that PV1 was detectable from 10 days postinoculation, coinciding with the first appearance of chlorotic viral lesions. The viral protein was only detectable in cell wall fractions of plant protein extracts. PV1 migrated with an apparent size of 14 kDa on SDS-PAGE, larger than the 10.9 kDa predicted from the amino acid sequence and therefore suggestive of posttranslational modification. Immunogold labelling located PV1 to the cell walls within lesion tissue and demonstrated a close association between the viral protein and secondary plasmodesmata. These results are consistent with the V1 product of MSV playing a role in the cell-to-cell movement of the virus in infected plants.

We developed a sensitive procedure to investigate the kinetics of transcription of an Agrobacterium tumefaciens transferred (T)-DNA-encoded beta-glucuronidase gusA (uidA) gene soon after infection of plant suspension culture cells. The procedure uses a reverse transcriptase-polymerase chain reaction and enables detection of gusA transcripts within 18 to 24 hr after cocultivation of the bacteria with either tobacco or maize cells. Detection of gusA transcripts depended absolutely on the intact virulence (vir) genes virB, virD1/virD2, and virD4 within the bacterium. Mutations in virC and virE resulted in delayed and highly attenuated expression of the gusA gene. A nonpolar transposon insertion into the C-terminal coding region of virD2 resulted in only slightly decreased production of gusA mRNA, although this insertion resulted in the loss of the nuclear localization sequence and the important omega region from VirD2 protein and rendered the bacterium avirulent. However, expression of gusA transcripts in tobacco infected by this virD2 mutant was more transient than in cells infected by a wild-type strain. Infection of tobacco cells with an Agrobacterium strain harboring a mutant virD2 allele from which the omega region had been deleted resulted in similar transient expression of gusA mRNA. These data indicate that the C-terminal nuclear localization signal of the VirD2 protein is not essential for nuclear uptake of T-DNA and further suggest that the omega domain of VirD2 may be required for efficient integration of T-DNA into the plant genome. The finding that the initial kinetics of gusA gene expression in maize cells are similar to those shown in infected tobacco cells but that the presence of gusA mRNA in maize is highly transient suggests that the block to maize transformation involves T-DNA integration and not T-DNA entry into the cell or nuclear targeting.

Objective: To determine the survival of porcine reproductive and respiratory syndrome virus (PRRSV) on nonliving substances (fomites) at 25 to 27 C., Design: Prospective controlled study., Sample Population: 3 solid, 6 porous, and 7 liquid fomites., Procedure: The fomites were contaminated with known concentrations of PRRSV. Samples for virus isolation were obtained on day 0 through day 11, assayed in cell cultures, and stained with fluorescent antibody conjugate., Results: The virus was recovered only on day-0 samples of alfalfa, wood shavings, straw, plastic, boot rubber, and stainless steel. Virus was isolated from city water through day 11, from well water through day 9, and from 2 buffer solutions for 4 and 6 days. The virus was isolated only on day 0 from swine saliva, urine, and fecal slurry., Clinical Implications: Results indicated that PRRSV is a fairly labile virus, but because of its duration of viability in water, contamination of drinking water and lagoons by PRRSV-shedding swine would serve as sources of virus to infect susceptible swine.

A complete infectious genome of an isolate of maize streak subgroup 1 geminivirus from Reunion Island (MSV-R) was cloned and sequenced. Using an Agrobacterium tumefaciens Ti plasmid delivery system, the cloned 2.7 kb circular DNA was shown to be infectious in maize. The agroinfected virus could be transmitted by Cicadulina mbila, the most common vector species of MSV in Reunion. Analysis of open reading frames (ORFs) revealed seven potential coding regions including the 4 ORFs conserved in all geminiviruses infecting monocotyledonous plants, the 2 on the viral "+" strand (MP, CP), and the 2 on the complementary "-" strand (RepA, RepB). The nucleotide sequence of MSV-R was compared to previously determined sequence of three African clones from Nigeria (MSV-N), Kenya (MSV-K), and South Africa (MSV-S). More similarity was found between the African clones (97.0-97.3%) than between these and MSV-R (94.4-95.3%). Nucleotide substitutions were frequent in the large intergenic region, particularly in and around the most likely TATA box for the complementary sense genes, and in the 5' end of ORF V1. The comparison of the predicted peptide sequences of the proteins encoded by ORFs MP, RepA and RepB confirmed the higher similarity between the African clones (97.8-99.3%) than between these and MSV-R (95.1-97.1%). However the amino acid sequences of the protein encoded by ORF CP (capsid protein) were very conserved among all the 4 clones, suggesting a high selection pressure on this ORF.

Since removal of the exposed N-terminus of the coat protein of some potyviruses abolishes aphid transmission, the role of this coat protein region of maize dwarf mosaic potyvirus (MDMV) in aphid transmission was investigated. The viral cDNA encoding this region was cloned and expressed as a fusion protein in bacteria. The resulting purified N-terminus of the coat protein was used in controlled aphid transmission experiments in competition with MDMV. The results show that this region inhibits aphid transmission of MDMV, indicating a direct involvement of the N-terminal region of the coat protein in aphid transmission.

The genome of maize streak virus (MSV) consists of one molecule of circular, single-stranded DNA of 2.7 kb. A reporter gene (bar) coding for phosphinothricin acetyl-transferase was inserted into the small non-coding region of the MSV genome. The recombinant bar-containing MSV vectors were introduced into maize seedlings via agroinfection. The chimeric viral DNA was found to replicate to high copy numbers in maize leaves resistant to the application of the herbicide Basta. This establishes the usefulness of MSV as an efficient replicating vector in cells of maize plants.

The nucleotide sequences of the small intergenic region (SIR) and the gene encoding the coat protein of 12 maize streak virus (MSV) isolates from different geographic locations have been determined. These have been used to assess the variability of the virus and to construct evolutionary dendrograms. For the viruses analyzed, the maximum levels of sequence divergence were found to be 10.9% and 2.0% at the nucleotide and amino acid levels, respectively. A genetically distinct strain of MSV was collected from islands in the Indian ocean. The significance of these findings for detection of the virus in epidemiological studies and breeding of resistant plant varieties is discussed.

Maize mosaic virus (MMV), a rhabdovirus, was identified associated to maize field plants, showing stunting and continuous chlorotic stripes uniformly distributed over the leaf blade. The virus was detected in field samples by agar-gel immunodifussion. Enveloped, bacilliform virus particles were observed by electron microscopy in thin sections of naturally infected leaf tissue.

Illinois (IL) and Minnesota (MN) RMV-like strains of barley yellow dwarf virus (BYDV) were identified from maize displaying red leaf symptoms by enzyme-linked immunosorbent assay (ELISA) using antiserum against a New York strain (BYDV-RMV-NY). Some IL and MN strains, but not the NY strain, could be detected by ELISA with a monoclonal antibody raised against BYDV-RPV-NY. The region of the viral genome representing the coat protein gene was amplified by polymerase chain reaction, cloned and sequenced. The nucleotide sequences of the BYDV-RMV-IL and BYDV-RMV-MN coat protein genes differed at just five nucleotide positions while the BYDV-RMV-IL and BYDV-RMV-NY differed at 101 of the 591 positions. The predicted amino acid sequences of the coat proteins of RMV-like strains from IL, MN, and NY shared approximately 60% identify with those of the coat proteins of beet western yellows virus, BYDV-RPV-NY, and potato leafroll virus.