Sequence requirements for translation initiation of Rhopalosiphum padi virus ORF2.

Rhopalosiphum padi virus (RhPV) is an aphid-infecting virus with a 10-kb ssRNA genome that contains two large open reading frames (ORFs). ORF1 and ORF2 encode the nonstructural and structural polyproteins, respectively. Both ORFs are preceded by noncoding regions of 500 nt that could function as internal ribosome entry segments (IRESes). The sequence for ORF2 lacks an obvious initiation codon, but an out-of-frame AUG codon is present that could translate ORF2 through a +1 frameshift. To investigate the mechanisms of translation initiation of ORF2, a series of point and deletion mutations were constructed and transcribed and translated in vitro. A bicistronic plasmid containing two copies of the RhPV intergenic region translated both ORFs efficiently, indicating that the region functioned as an IRES in vitro. Deletion analysis showed that the region required for activity of the IRES extended from 178 nt upstream and 6 nt downstream of the 5' border of ORF2. Changes in the out-of-frame AUG codon had little effect on translation initiation, but mutations that included the first and second codons of ORF2 or that disrupted a putative pseudoknot structure near the 3' end of the IRES failed to initiate protein synthesis. Sequence analysis of the in vitro synthesized proteins showed that the first amino acid of the polyprotein corresponded to the second codon of ORF2. These results show that in vitro the noncoding region upstream of RhPV ORF2 functions as an IRES that contains a pseudoknot that directs translation initiation at a non-AUG codon. If the in vitro synthesized proteins have not been processed by an aminopeptidase, translation is initiated at the second (GCA) codon of ORF2.

Solanaceous Weeds as Possible Sources of Cucumber mosaic virus in Southern Illinois for Aphid Transmission to Pepper.

Over 5,000 individual plants representing approximately 55 species from an area in southern Illinois where Cucumber mosaic virus (CMV) has been a major problem in pepper (Capsicum annuum) were tested for the presence of CMV by enzyme-linked immunosorbent assay (ELISA). Representative ELISA-positive samples were checked by western blot tests to confirm virus-specific reactions. Nearly all of the infected plants detected were either Solanum ptycanthum (eastern black nightshade) or Physalis spp. (principally P. heterophylla, groundcherry). Over 1,000 pepper transplants and approximately 500 tomato transplants, collected prior to planting, were negative for CMV by ELISA. In aphid transmission (arena) experiments, all five aphid species tested were capable of transmitting CMV from nightshade to pepper: Aphis fabae subsp. solanella, Aphis gossypii, Myzus persicae, Rhopalosiphum padi, and Sitobion avenae. Aphis fabae subsp. solanella, A. gossypii, and A. nerii were able to transmit CMV from P. heterophylla to pepper. Aphis fabae subsp. solanella was commonly found colonizing nightshade from May through October in southern Illinois.

Nucleotide sequence analysis shows that Rhopalosiphum padi virus is a member of a novel group of insect-infecting RNA viruses.

Rhopalosiphum padi virus (RhPV) is an aphid virus that has been considered a member of the Picornaviridae based on physicochemical properties. The 10,011-nt polyadenylated RNA genome of RhPV was completely sequenced. Analysis of the sequence revealed the presence of two open reading frames (ORFs). The predicted amino acid sequence of ORF1, representing the first 6600 nt of the RhPV genome, showed significant similarity to the nonstructural proteins of several plant and animal RNA viruses. Direct sequence analysis of the RhPV capsid proteins showed that ORF2, which represents the last 2900 nt, encodes the three structural proteins (28, 29, and 30 kDa). The predicted amino acid sequence of ORF2 is very similar to the corresponding regions of Drosophila C virus, Plautia stali intestine virus, and to a partial sequence from the 3' end of the cricket paralysis virus genome. The site of initiation of protein synthesis for ORF2 could not be determined from the amino acid and nucleotide sequences. ORF1 is preceded by 579 nt of noncoding RNA and the two ORFs are separated by more than 500 nt of noncoding RNA. Like picornaviruses, these regions may function to facilitate the cap-independent initiation of translation of the two ORFs. These data suggest that RhPV, Drosophila C virus, Plautia stali intestine virus, and probably cricket paralysis virus are members of a unique group of small RNA viruses that infect primarily insects.

In Situ Localization of Barley Yellow Dwarf Virus-PAV 17-kDa Protein and Nucleic Acids in Oats.

ABSTRACT Barley yellow dwarf virus strain PAV (BYDV-PAV) RNA and the 17-kDa protein were localized in BYDV-PAV-infected oat cells using in situ hybridization and in situ immunolocalization assays, respectively. The in situ hybridization assay showed labeling of filamentous material in the nucleus, cytoplasm, and virus-induced vesicles with both sense and antisense nucleic acid probes, suggesting that the filamentous material found in BYDV-PAV-infected cells contains viral RNA. BYDV-PAV negative-strand RNA was detected before virus particles were observed, which indicates that RNA replication is initiated before synthesis of viral coat protein in the cytoplasm. The 17-kDa protein was associated with filamentous material in the cytoplasm, nucleus, and virus-induced vesicles. The labeling densities observed using antibodies against the 17-kDa protein were similar in the nucleus and cytoplasm. No labeling of the 17-kDa protein was observed in plasmodesmata, but filaments in the nuclear pores occasionally were labeled. Since BYDV-PAV RNA and 17-kDa protein colocalized within infected cells, it is possible that single-stranded viral RNA is always associated with the 17-kDa protein in vivo. The 17-kDa protein may be required for viral nucleic acid filaments to traverse the nuclear membrane or other membrane systems.

Influence of penning type and feeding level on sexual behavior and feet and leg soundness in boars.

Ninety-six Yorkshire males at 28 d of age were placed on a pre-test feeding regimen (22% CP) of either ad libitum (A) access to feed or a restricted (R, 85% of A) diet. All pigs were housed in groups of four until approximately 30 kg body weight. The boars were then placed under test and assigned to a group pen (GP) containing eight boards or to individual pens (IP) and fed a 16% CP diet at A or R levels. Feeding types during pre-test and test phases were, therefore, ad libitum followed by ad libitum (AA), ad libitum: restricted (AR), restricted: ad libitum (RA), and restricted: restricted (RR) for each of the two (IP and GP) housing types. All boars were weighed every 2 wk to determine the feeding level and ADG. The pen floors were partly slatted with water nipples in the slatted areas. Areas available for each board were 1.63 and 2.15 m2 in IP and GP, respectively. Sexual behavior, semen characteristics, and feet and leg scores were recorded between 150 and 240 d of age. The IP boards required longer (P < .05) contact with receptive gilts before mating, made more (P < .01) ano-genital sniffings, and attempted more (P < .10) incorrect mounts than GP boars. The GP boards had a higher (P < .01) mating score, younger (P < .001) age at completion of the mating test, and lower (P < .01) total sperm count and sperm concentration (P < .001) than IP boars. The AA boards exhibited less (P < .10) chomping and salivation than boars on other feeding regimens.(ABSTRACT TRUNCATED AT 250 WORDS)

The nucleotide sequence of apple mosaic virus coat protein gene has no similarity with other Bromoviridae coat protein genes.

A double-stranded cDNA was synthesized from in vitro polyadenylated apple mosaic virus (ApMV) RNA 3 using oligo(dT) and sequence-specific primers, and was cloned into plasmid vectors. A set of overlapping cDNA clones was used to determine the nucleotide sequence of RNA 4. ApMV RNA 4 was found to contain an open reading frame (ORF) of 666 nucleotides, which was flanked by 5' and 3' non-translated sequences of 55 and 264 nucleotides, respectively. The ORF encoding the coat protein was identified by comparing the predicted amino acid sequence with that obtained from direct protein microsequencing of the native viral coat protein. The ORF encodes a protein with an M(r) of 25,056. The nucleotide sequence of the ApMV coat protein gene showed no similarity to those of alfalfa mosaic virus, tobacco streak virus (TSV), brome mosaic virus or cucumber mosaic virus. The predicted amino acid sequence of the amino-terminal region of the ApMV coat protein is basic, rich in cysteine residues and may contain a zinc finger motif similar to that found in TSV.

Detection of the readthrough protein of barley yellow dwarf virus.

The single open reading frame (ORF) 5 encoding the 50-kDa protein of barley yellow dwarf virus PAV-IL (BYDV-PAV-IL) was expressed in bacteria, purified, and used as an immunogen/antigen to produce/screen antibodies specific to the 50-kDa protein. Two monoclonal antibodies (MAb PAV-IL-22 kDa and MAb PAV-IL-50 kDa) raised against BYDV-PAV-IL could specifically detect the presence of the 72-kDa readthrough protein in extracts from the BYDV-infected leaf tissue. The results suggest that ORF 5 (50-kDa protein) is translated by readthrough of ORF 3 (22-kDa coat protein) to produce the 72-kDa protein. The readthrough protein is thought to be a structural protein on the external surface of BYDV.

Coat protein sequences of RMV-like strains of barley yellow dwarf virus separate them from other luteoviruses.

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.

A rapid chemiluminescent detection method for barley yellow dwarf virus.

Barley yellow dwarf virus (BYDV-PAV-IL) was detected with biotinylated in vitro transcript cDNA using a chemiluminescent substrate on nylon membranes. Signals were detected on X-ray film and quantified using either a densitometer or an ELISA plate reader. The time required for sample preparation was reduced so that the entire protocol could be completed in two days. The in vitro transcript probes could detect 1 ng of purified virus and as little as 1 microliter of sap extracts prepared from infected oat shoots.

Influence of ELISA conditions on detection of serological relationships among luteoviruses.

Three variations in the ELISA procedure were used in an attempt to understand the basis for serological relationships among three isolates of barley yellow dwarf virus (BYDV-RPV-I from Illinois, BYDV-RPV-N from New York and BYDV-PAV from Illinois), beet western yellows virus (BWYV) and soybean dwarf virus (SDV). Detection of serological relationships was dependent on the state of the virus particle (e.g. dissociated or intact) and the method of detection (e.g. direct or indirect). In indirect ELISA, where virus particles were dissociated due to incubation in a high pH buffer, all five virus isolates were serologically related. In double antibody sandwich (DAS) ELISA, identification of serological relationships was based on detection of epitopes associated with intact virus particles, which resulted the detection of fewer serological relationships. Direct ELISA showed, depending on the Ig, that the state of the virus particle and/or the method of detection did effect the ability to detect some serological relationships.

Relationships among luteoviruses based on nucleic acid hybridization and serological studies.

The luteoviruses barley yellow dwarf virus (BYDV-PAV and BYDV-RPV), bean leaf roll virus (BLRV) beet western yellows virus (BWYV), carrot red leaf virus, potato leaf roll virus, and soybean dwarf virus (SDV) were compared by hybridization with random cDNA probes and serologically with polyclonal antisera. For hybridizations, filters had each RNA blotted in duplicate dots at 10 ng/dot. Random-primed cDNA probes were prepared from 300 ng of each RNA and used to probe filters at three levels of stringency. Homologies were observed between BLRV and SDV and between BWYV and BYDV-RPV at the lowest level of stringency (Tm-38). In double-antibody sandwich enzyme-linked immunosorbent assay using polyclonal antisera, two-way relationships were observed between BWYV and BYDV-RPV. In indirect enzyme-linked immunosorbent assay, where purified virus denatured in pH 9.6 carbonate buffer was coated directly onto microtiter plates, relationships between members of the luteoviruses were much more extensive. BLRV, BWYV, BYDV-PAV, carrot red leaf virus, potato leaf roll virus, and SDV antisera reacted with each of the six luteoviruses tested in the indirect test, while the BYDV-RPV antiserum reacted only with BYDV-RPV, BWYV, and BLRV. BWYV and BYDV-RPV are closely related in both tests and should be considered strains of one virus.

Current problems in the taxonomy of luteoviruses.

While the classification of luteoviruses was based initially on biological characteristics, principally host range and vector specificities, recent serological studies of luteoviruses have further increased our understanding of their taxonomic relationships. Serological, biochemical and genetic methods will be necessary to clarify relationships among luteoviruses.

Maximizing the detection capability of a beet western yellows virus ELISA system.

Conditions for maximizing detection of a California isolate of beet western yellows virus (BWYV) were investigated with the double-sandwich enzyme-linked immunosorbent assay (ELISA) system. Within-plate variability was found to account for less than 1% of the total variation observed on individual microtiter plates. Variability across plates was greater than within plates and accounted for less than 10% of the total variation. Significant reductions in absorbance were observed when either coating or conjugated IgGs were incubated for 2 h at 37 degrees C. Incubation of coating IgGs overnight at 4 or 22 degrees C gave good results. No differences in absorbance were observed when antigen sources were incubated at 37, 22, or 4 degrees C. Absorbance at 1, 3 or 6 h incubation of conjugated IgGs at 4 or 22 degrees C was low to moderate but after 18 or 24 h absorbance was markedly increased without increasing background. Efficient extraction of virus from host and vector tissues increased absorbance. Carborundum added to the extraction buffer worked well when tissue samples were ground in a mortar and pestle but highest absorbance resulted when test samples were prepared with a high-speed tissue homogenizer. Background levels were unacceptably high when 10 or more Myzus persicae were pooled for analysis. Since virus can be detected in 3 or even 1 aphid, pooling for detection is unnecessary. Optimization of the BWYV ELISA system made possible the detection of virus quantities less than 2 ng/ml.

Detection, biological effects, and transmission of a virus of the aphid Rhopalosiphum padi.

A virus infecting an Illinois colony of the aphid Rhopalosiphum padi (L.) was transmitted transovarially, and significantly decreased longevity of infected aphids. The virus was detected by serological assay in R. padi colonies from North Dakota, and in two other aphid species maintained at Illinois, R. rufiabdominalis (Sasaki) and Schizaphis graminum (Rondani).

Purification and characterization of a virus from the aphid Rhopalosiphum padi.

A 27-nm icosahedral virus was purified from the oat bird cherry aphid, Rhopalosiphum padi (L.). The virus had an s(20,w) of 162 +/- 2 S, and bouyant densities of 1.37 in CsCl and 1.35 in Cs2SO4. It contained one ssRNA of 31 +/- 2 S and three major proteins. The relationship of the R. padi virus to other small RNA invertebrate viruses is unclear.