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.     

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).     

An improved bioacoustic method for monitoring of respiration.

Reliable monitoring of respiration plays an important role in a broad spectrum of applications. Today, there are several methods for monitoring respiration, but none of them has proved to be satisfactory in all respects. We have recently developed a bioacoustic method that can accurately time respiration from tracheal sounds. The aim of this study is to tailor this bioacoustic method for monitoring purposes by introducing dedicated signal processing. The method was developed on a material of ten patients and then tested in another ten patients treated in an intensive care unit. By studying the differences in the variation of the spectral content between the different phases of respiration, the described method can distinguish between inspiration and expiration and can extract respiration frequency, and respiration pause periods. The system detected 98% of the inspirations and 99% of the expirations. This method for respiration monitoring has the advantage of being simple, robust and the sensor does not need to be placed closed to the face. A commercial heart microphone was used and we anticipate that further improvement in performance can be achieved trough optimization of sensor design.     

An improved method for the detection of DNA fragmentation.

An application of the Southern blot technique is described which permits the detection of DNA fragmentation due to cell death by apoptosis. DNA fragments were isolated from cell suspensions and tissues, separated on agarose gel, transferred by Southern blot and hybridized with a radiolabeled total cellular DNA probe. The application of this procedure to thymus cell samples, revealed the distinct ladder pattern of DNA fragments in multiples of about 180-200 base pairs, a characteristic feature of DNA fragmentation. In comparison to conventional DNA visualization with ethidium bromide staining, the radiolabeled probe improved the detection of DNA fragments at least eight-fold. This method detects low levels of DNA fragments, as well as physiological tissue DNA fragmentation, while avoiding cell damage due to DNA radiolabeling.     

An improved method for the detection of peroxidase-conjugated antibodies on immunoblots

A sensitive method for the detection of antigen-antibody complexes on immunoblots is described which employs a modified substrate for peroxidase-conjugates. Dengue virus proteins were chosen for study and were separated by SDS-PAGE followed by electrophoretic transfer onto sheets of nitrocellulose. Virus-specific antigens bound to the nitrocellulose paper were then probed with both mouse monoclonal antibodies and human convalescent sera. Antigen-antibody complexes were detected with anti-species specific IgG-peroxidase conjugates followed by incubation in five different enzyme substrates. By far the most sensitive substrate was found to be a simple mixture of 4-chloronaphthol and diaminobenzidine (CND). A dot-immunobinding assay employing a purified viral protein and a specific monoclonal antibody was able to detect as little as 0.1 ng of antigen using this mixture. The increased sensitivity obtained involves no additional 'enhancement' steps in the procedure, the substrates are inexpensive and the product of the enzyme reaction is black, making the immunoblots ideal for photographic reproduction. Optimization of additional parameters involved in the immunoblot procedure is also described.     

An improved method for detection of ganglionic latency in herpes simplex virus type-1 infected guinea pigs

A simple method was developed which increased the sensitivity and reliability of detecting latent herpes simplex virus type-1 (HSV-1) in trigeminal ganglia of guinea pigs. Animals were infected with the Shealey strain of HSV-1 immediately following scarification of the cornea and maintained for 30-40 days to ensure that true latency was established. Viral latency was defined as the appearance of infectious virus in ganglia only upon cultivation in vitro. Thus, ganglia from similarly infected animals, homogenized immediately upon removal, did not contain infectious virus. Excised ganglia were incubated intact in high glucose medium and yielded maximal positive results (90-100%) by the twelfth day of incubation. This method was compared with the standard cocultivation technique in which minced fragments of ganglionic tissue were explanted onto Vero cell cultures. Cocultivation yielded a considerably lower latency rate and was more variable (29-57%) than the whole ganglion culture method.     

An improved method for detection and titration of antibodies cytophilic for macrophages

A new microtechnique for detection and titration of antibodies cytophilic for homologous macrophages is introduced and experiments designed to determine the optimum conditions for its performance reported. The technique employs a series of small chambers formed on a microscope slide by the application of a plastic film in which holes have been punched. The procedure uses 5 x 10⁴ cells and 25 μl of serum per chamber, and each test can be completed in 2 h. A permanent preparation results which can be examined as and when convenient.     

Comparison of the coat protein, movement protein and RNA polymerase gene sequences of Australian, Chinese, and American isolates of barley yellow dwarf virus transmitted by Rhopalosiphum padi

Summary.  Barley yellow dwarf luteovirus-GPV (BYDV-GPV) is a common problem in Chinese wheat crops but is unrecorded elsewhere. A defining characteristic of GPV is its capacity to be transmitted efficiently by both Schizaphis graminum and Rhopaloshiphum padi. This dual aphid species transmission contrasts with those of BYDV-RPV and BYDV-SGV, globally distributed viruses, which are efficiently transmitted only by Rhopaloshiphum padi and Schizaphis graminum respectively. The viral RNA sequences encoding the coat protein (22K) gene, the movement protein (17K) gene, the region surrounding the conserved GDD motif of the polymerase gene and the intergenic sequences between these genes were determined for GPV and an Australian isolate of BYDV-RPV (RPVa). In all three genes, the sequences of GPV and RPVa were more similar to those of an American isolate of BYDV-RPV (RPVu) than to any other luteovirus for which there is data available. RPVa and RPVu were very similar, especially their coat proteins which had 97% identity at the amino acid level. The coat protein of GPV had 76% and 78% amino acid identity with RPVa and RPVu respectively. The data suggest that RPVu and RPVa are correctly named as strains of the same serotype and that GPV is sufficiently different from either RPV strain to be considered a distinct BYDV type. The coat protein and movement protein genes of GPV are very dissimilar to SGV. The polymerase sequences of RPVu, RPVa and GPV show close affinities with those of the sobemo-like luteoviruses and little similarity with those of the carmo-like luteoviruses. The sequences of the coat proteins, movement proteins and the polymerase segments of BYDV serotypes, other than RPV and GPV, form a cluster that is separate from their counterpart sequences from dicot-infecting luteoviruses. The RPV and GPV isolates consistently fall within a dicot-infecting cluster. This suggests that RPV and GPV evolved from within this group of viruses. Since these other viruses all infect dicots it seems likely that their common ancestor infected a dicot and that RPV and GPV evolved from a virus that switched hosts from a dicot to a monocot. Accepted December 19, 1997 Received September 9, 1997     

An improved method for the detection of Down's syndrome aneuploidy in uncultured amniocytes

We report a modified method for the rapid detection of aneuploidies directly on human uncultured amniocytes that simplifies and shortens the entire experimental procedure, yielding signals which allow correct diagnosis of trisomy 21 in 97% of cases. The improvement is based on two points: 1) use of cosmid pockets specific for the Down's syndrome minimal region as FISH probes, and 2) a modified protocol for the fixation and preparation of amniocytes.     

An improved method for the detection of HIV antigen in the blood of carriers

The measurement of HIV antigen levels in sera or plasma of HIV-infected individuals is critical for determining the existence of antigen or infectious virus before seroconversion and for prognosis. Pretreatment of sera or plasma of HIV carriers by heating at 70 degrees C for 10 min at an acidic pH enabled us to estimate antigens efficiently in immune complexes. This procedure will also be useful in determining antigen levels in HIV carriers more precisely.     

An improved radioimmunoassay method for the detection of IgG antibodies against cytomegalovirus

The non-specific binding seen with human sera in a radioimmunoassay for the detection of IgG antibodies specific for CMV can be reduced greatly by using a murine monoclonal antibody as a radiolabelled detecting antibody. Such non-specific binding formerly obtained with a polyclonal detecting antibody was due to the binding of the polyclonal reagent to factors on the solid phase other than IgG molecules.     

An improved ELISA and serum neutralisation test for the detection of turkey rhinotracheitis virus antibodies

The development of a double-well ELISA test for the detection of turkey rhinotracheitis (TRT) virus antibody in turkey and chicken sera, utilising a streptavidin-biotin detection protocol is described. This test was compared with a conventional ELISA, which detects antibody using a peroxidase-labelled anti-turkey IgG. The double-well streptavidin-biotin ELISA was more sensitive than the conventional ELISA, and, due to the consistently low background values obtained, was further modified to a single-well ELISA test. Antibody titres obtained using the single-well streptavidin-biotin ELISA were similar to those obtained using the conventional ELISA. A microtitre serum neutralisation test for TRT virus read using an indirect immunoperoxidase (IIP) detection protocol after 48 h was also developed and compared to the standard neutralisation test employing a 7 day incubation and read by CPE. The specificity of the single-well streptavidin-biotin ELISA was confirmed using the rapid (IIP) serum neutralisation test.     

Expression of the barley yellow dwarf virus-GAV movement protein and its detection in the infected and transgenic plants

Movement proteins (MPs) that facilitate virus movement in the plants were identified in a number of plant viruses. In this study, full-length MP gene of the Chinese isolate Barley yellow dwarf virus-GAV (BYDV-GAV) was cloned and expressed in Escherichia coli. About 32% of the expressed MP was soluble providing the concentration of isopropyl-beta-D-galactopyranoside (IPTG), time of the induction, temperature and shaking speed were optimized. The soluble MP was purified using nickel-affinity column. Immune serum prepared against purified MP was used for the detection of MP in the BYDV-GAV infected leaves of oat and in the leaves of transgenic wheat plants expressing the full-length and truncated MP gene.