Oscar Grau

Detection of Tospovirus species by RT-PCR of the N-gene and restriction enzyme digestions of the products.

Tomato spotted wilt is a serious disease that affects several economically important crops. From the epidemiological point of view and for the development of a successful plan for transgenic resistance plants, the four known Tospovirus species must be discriminated at the molecular level. A RT-PCR assay using primers complementary to the N gene was used to detect and differentiate fourteen Argentinian isolates of Tospovirus from different crops and geographical areas. Extracts were reverse transcribed using a thermo-resistant reverse transcriptase and PCR reactions were performed for 30 min in a capillar thermo-cycler. The products were digested with restriction enzymes and three of the four described species were identified. Additionally, the results were confirmed by DAS-ELISA. The method described here is rapid and reliable.

Detection of Citrus Psorosis Virus by ELISA, Molecular Hybridization, RT-PCR and Immunosorbent Electron Microscopy and its Association with Citrus Psorosis Disease

Psorosis is a citrus disease of undemonstrated etiology that can be diagnosed by biological indexing on sweet orange seedlings followed by a cross protection test. Its presumed causal agent is Citrus psorosis virus(CPsV), type species of the genus Ophiovirus. We compared detection of CPsV by ELISA, RT-PCR, molecular hybridization and immunosorbent electron microscopy, and examined its association with psorosis disease in 11 biologically characterized isolates and in 47 uncharacterized field sources by observation of field symptoms and by biological indexing including the cross protection test. Detection of CPsV by any of the four procedures always coincided with diagnosis of psorosis by cross protection, but it did not always correlate with observation of symptoms thought to be specific, in field trees or in graft-inoculated indicator plants. Trials to detect CPsV by ELISA, molecular hybridization and RT-PCR in citrus sources from different geographical origins, presumed to be psorosis-infected on the basis of field symptoms or reaction of indicator plants, were sometimes unsuccessful, indicating that psorosis symptoms may be induced by causes other than CPsV.

The complete nucleotide sequence of a Spanish isolate of Citrus psorosis virus: comparative analysis with other ophioviruses.

Summary.The complete genomic sequence (11278 nt) of Citrus psorosis virus (CPsV), isolate P-121 from Spain, was determined and compared with those from isolate CPV-4 and from other ophioviruses. The three RNAs of P-121 had similar size and identical organization as those of CPV-4. The 24K and the RdRp proteins were potentially encoded in the viral complementary (vc) strand of RNA 1, the 54K protein potentially encoded in vcRNA 2 and the coat protein encoded in vcRNA 3. These four proteins from P-121 and CPV-4 had 87, 92, 93 and 94% amino acid identity, respectively, but only 22, 38, 25 and 33% identity with their homologous proteins from Mirafiori lettuce big vein virus (MLBVV), the only other ophiovirus completely sequenced. Biological and genetic differences between CPsV and MLBVV (and the other ophioviruses), would support their future allocation in different genera within a tentative family Ophioviridae.

Population structure of Citrus tristeza virus from field Argentinean isolates

We studied the genetic variability of three genomic regions (p23, p25 and p27 genes) from 11 field Citrus tristeza virus isolates from the two main citrus growing areas of Argentina, a country where the most efficient vector of the virus, Toxoptera citricida, is present for decades. The pathogenicity of the isolates was determinated by biological indexing, single-strand conformation polymorphism analysis showed that most isolates contained high intra-isolate variability. Divergent sequence variants were detected in some isolates, suggesting re-infections of the field plants. Phylogenetic analysis of the predominant sequence variants of each isolate revealed similar grouping of isolates for genes p25 and p27. The analysis of p23 showed two groups contained the severe isolates. Our results showed a high intra-isolate sequence variability suggesting that re-infections could contribute to the observed variability and that the host can play an important role in the selection of the sequence variants present in these isolates.

A highly sensitive heminested RT-PCR assay for the detection of citrus psorosis virus targeted to a conserved region of the genome.

Psorosis is a widespread and damaging disease of citrus in many parts of the world. The causal agent is a multipartite virus with RNA genome present in very low concentration in infected citrus tissue. Diagnosis is made by biological indexing on indicator citrus seedlings, but it is a slow and costly procedure and therefore it is not used generally. No sensitive wide-spectrum assay for Citrus Psorosis virus (CPsV) has been reported based on RT-PCR. A highly sensitive heminested RT-PCR assay is described for the detection of CPsV. Fragments of 313 bp amplified from RNA 1 of different isolates were cloned and sequenced. Very high homology was found among six isolates from the citrus producing region of Argentina: 96.6-100% in nucleotide sequence. The consensus sequence obtained was used for the design of the primers for heminested PCR assay. It has been tested on different Argentine isolates, employing various methods for RNA extraction from infected tissue. This test is able to detect CPsV in dilutions of 10(10) of the original sample.

Citrus psorosis is probably caused by a bipartate ssRNA virus

Isolate 90-1-1 Concordia (Argentina) of the citrus psorosis agent was graft-transmitted to citrus and mechanically transmitted to Chenopodium quinoa, which was used as a local lesion assay host. Infected citrus and C. quinoa plant lesions were used as starting materials for the purification of the psorosis-associated agent. In extracts partially purified by differential centrifugation, infectivity was abolished by RNase treatment, even in 0.3 M NaCl, indicating that ssRNA is required for biological activity. The total loss of infectivity produced by proteinase K treatment and the decline in infectivity caused by phenol extraction indicated that protein may be essential for infectivity. When partially purified extracts were subjected to sucrose density gradient centrifugation, infectivity on C. quinoa from certain 2-fraction combinations was higher than expected, compared to the infectivity of the individual fractions. Therefore, infectivity was not associated with a single component but with the combination of at least two components which were distinguishable on sedimentation. The infectious material was present in the top and bottom zones of a sucrose gradient, which on further purification by a second gradient and agarose gel electrophoresis, revealed the presence of a 50-kDa protein. This protein was absent in comparable gradient fractions from healthy plants, and therefore most likely represented the capsid protein of both the top and bottom sucrose gradient zone components. Taken together, these results led to the conclusion that the citrus-psorosis-associated virus (CPsAV) is a multipartite virus, containing ssRNA and a 50-kDa coat protein.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple nucleic acid amplification assay for the rapid detection of Junín virus in whole blood samples.

Argentine hemorrhagic fever (AHF) is an endemoepidemic disease with cardiovascular, renal and neurologic alterations acquired in the richest farming land in Argentina. It is caused by Junín virus, one of the few human pathogenic arenaviruses. The S RNA of Junín virus has been molecularly cloned and its nucleotide sequence determined in our laboratory. This information was used to develop a rapid nucleic acid-based diagnostic test commensurate with the low viraemia detected in AHF patients. Junín virus-specific cDNA probes labeled using various methods proved insensitive in dot-hybridizations. Therefore, a RT polymerase chain reaction (PCR) was developed using a pair of oligonucleotide primers to reverse-transcribe and amplify the viral S RNA. The amplification of the target sequences was measured by ethidium bromide staining of the DNA fragments after agarose gel electrophoresis. This type of assay allowed the specific detection of Junín virus RNA sequences present in a single infected BHK21 cell over a background of 10(4) uninfected cells. Control reactions were performed on RNA samples extracted from uninfected cells or cells infected with a high multiplicity of LCMV, another arenavirus present in the AHF endemic area. The PCR was first adapted to detect viral RNA in peripheral blood mononuclear cells, described to harbor most of the virus. A simplification of this assay allows the detection of Junín virus in RNA extracted from 100 microliters of whole blood using guanidium thiocyanate disruption and acid phenol extraction. Under the conditions described in this paper, it is possible to detect up to 0.01 pfu of Junín virus in a blood sample. An early and rapid laboratory diagnostic test for AHF is important since the only effective therapy that reduces the mortality rate from 30% to less than 1% consists of early treatment with immune plasma.

Molecular Identification of Cinara cupressi and Cinara tujafilina (Hemiptera, Aphididae)

ABSTRACT A condition called “cypress mortality” affects forest of Austrocedrus chilensis (D. Don) Pic. Ser et Bizarri in Argentina. Their classic groups of symptoms has been described as a slow process of defoliation that culminating in death of the tree; nevertheless, dying and recently dead trees with abundant foliage are frequently observed in which foliage changes to red. Cinara (Cupressobium) cupressi (Buckton) is considered the agent responsible for reddening this indigenous conifer in Chile. Therefore, the relationship between the presence of C. cupressi and the new aerial symptoms in A. chilensis from Argentina required evaluation. However, Cinara (Cupressobium) tujafilina (del Guercio) also has been reported from this host, and the differentiation of both species of Cinara is time consuming and requires a great expertise because they share many morphologic and microscopic characters. A rapid molecular method of identification of C. cupressi and C. tujafilina is desirable to detect and differentiate them. We report the development and evaluation of a polymerase chain reaction-restriction fragment length polymorphism method based on the mitochrondial cytochrome oxidase I gene to identify C. cupressi and C. tujafilina in colonies of aphids. The first detection of C. cupressi from A. chilensis in Argentina, is reported based on the new method.

A fast one-step reverse transcription and polymerase chain reaction (RT-PCR) amplification procedure providing highly specific complementary DNA from plant virus RNA.

Reverse transcription and polymerase chain reaction (RT-PCR) are being used increasingly for detection and typing RNA viruses. For this purpose, metal block thermal cyclers (MBTC) are considered to provide higher DNA yield, whereas air thermal cyclers (ATC) allow PCR amplification in a much shorter time. A fast ATC protocol (0 s denaturation, 0 s annealing, and 4-8 s elongation) was developed to amplify genomic segments from two RNA viruses, which allowed increasing the number of cycles without a parallel increase of non-specific DNA fragments. Under these conditions, 80-90 cycles with the ATC provided a DNA yield close to that of a standard 40-cycles MBTC protocol in about half the time. The DNA synthesised by the new procedure was highly specific and could be cloned readily.

Effect of actinomycin D on arenavirus growth and estimation of the generation time for a virus particle

In an attempt to define the involvement of host transcription in arenavirus growth, a study was made of the effect of actinomycin D (AMD) on the yields of infectious Pichinde, Tacaribe and Junin viruses. The drug was added either immediately after virus adsorption or later after infection, at the stationary phase of virus growth. The time of exposure of the infected cells to the inhibitor was so chosen that the generation and release of virus into the medium took place in the presence of AMD. A double label technique was used to estimate the generation time of an arenavirus particle. This was found to be 6 h or less for all of the arenaviruses examined. The results indicated that treatment of the host cells with AMD, either immediately after virus adsorption or later after infection, does not affect the yield of infectious Pichinde, Tacaribe or Junin viruses, thus implying that continuous host transcription is not required for the replication cycle of these arenaviruses.