Ana Maria Sandino

Characterization of rotavirus guanylyltransferase activity associated with polypeptide VP3

Rotaviruses transcribe mRNA containing a 7mGpppGmp cap at the 5 end in vitro. Guanylyltransferase activity associated with the viral particle was detected by SDS-PAGE due to the formation of a nucleotide-enzyme complex when the virus was incubated with [alpha-32P]GTP. Using purified viral particles it was shown that only the core polypeptide VP3 exhibits the ability to form a complex with the nucleotide. The reaction is specific for GTP or dGTP when Mg2+ is used as a cofactor. The reaction also depends on the incubation temperature and the pH, as described for other guanylyltransferases. The GMP-VP3 complex transfers the GMP to pyrophosphate, synthesizing GTP or GDP, resulting in the formation of a GpppG cap. These properties of the complex allowed the core polypeptide VP3 to be identified as the rotavirus guanylyltransferase.

Polyacrylamide gel electrophoresis of viral genomic RNA as a diagnostic method for infectious pancreatic necrosis virus detection

A rapid and simple technique for the diagnosis of IPNV from cell culture and infected fish tissues has been developed. It is based on the observation of IPNV bisegmented double-stranded RNA genome in silver stained polyacrylamide gels after electrophoresis. The method is highly specific and can detect as little as 1 ng of viral RNA, which corresponds to 1 x 10(5) pfu, making it possible to visualize the viral genome as soon as the initial cythopatic effect appears. Furthermore, the RNA viral genome could be detected directly from fish tissues when fish showed clear clinical signs of the disease. The method has the advantage of detecting any strain of IPNV. An acrylamide concentration of 6% and bisacrylamide concentration of 0.13% give a rapid and definitive result in less than 6 h.

In vitro reconstitution of rotavirus transcriptional activity using viral cores and recombinant baculovirus expressed VP 6

SummaryPurified baculovirus-expressed group A rotavirus VP 6 polypeptide was shown to be active in the recovery of the transcriptase activity associated with the reconstitution of the single-shelled rotavirus particle. Recombinant VP 6 polypeptide was able to restore the transcriptional activity in purified viral cores from both SA-11 and RF rotavirus strains. Recombinant group C VP 6 (Cowden strain) is capable of binding as a trimer to group A viral core particles but unable to restore the transcriptase activity, suggesting that the binding of the polypeptide to cores is not the only requirement to restore the transcriptase activity. The VP 6 group A polypeptide was shown to bind as a monomer to viral cores, indicating that trimerization of VP 6 may be not required for reconstitution of the polymerase activity.

Inhibition of in vitro reconstitution of rotavirus transcriptionally active particles by anti-VP6 monoclonal antibodies.

SummarySix monoclonal antibodies specific for the major capsid protein of rotavirus, VP6, previously characterized, were tested in a biological assay for their capacity to block the transcriptase activity associated with the single-shelled particles. The results showed that two MAbs (RV-50 and RV-133), specific for distinct antigenic sites, were able to block the transcription when they were incubated with a purified baculovirus-expressed group A VP6, prior to the reconstitution of the single-shelled particles from the cores, suggesting that at least two domains are involved in active single-shelled particle reconstitution. The results obtained previously from immunochemistry of synthetic peptides did not allow us to attribute this biological activity to a particular linear sequence of the protein, the domain involved being probably complex and dependent on the folding of the protein. However, the C-terminal end, which is necessary for binding into single-shelled particles could be necessary but not sufficient to restore the transcription, since neither of these two MAbs reacted significantly with peptides of this region. These two MAbs will be useful reagents to study the interactions between VP6 and the cores.

Function of rotavirus VP3 polypeptide in viral morphogenesis

The phenotype of the rotavirus SA-11 mutant tsB carrying a thermosensitive mutation in gene 3, which encodes VP3, was characterized further from both infected cells and purified viral particles. The mutant phenotype was initially identified as negative for in vivo double- and single-stranded RNA synthesis. Our results show that the in vitro transcriptional properties of the tsB mutant at the restrictive temperature were identical to those of the wild-type strain. Similar results were obtained with respect to the VP3-associated guanylyl-transferase activity. Analysis of viral particles made by mutant-infected cells at the restrictive temperature showed that only empty single-shelled particles were assembled. This indicates that viral morphogenesis is halted after the initial viral transcription and before RNA replication, suggesting that VP3 may be required as part of the replicase system but not for subviral particle assembly. These data suggest that such a phenotype is not due to alteration of a VP3 function related to transcription.

Effect of nucleotide analogues on rotavirus transcription and replication

The effects of several nucleoside and nucleoside triphosphate analogues were studied on rotavirus replication and transcription. Nucleoside triphosphate analogues modified at sugar residues were capable of inhibiting in vitro transcription, including adenosine-9-beta-D-arabinofuranoside 5-triphosphate, 3-deoxyadenosine 5-triphosphate, adenosine 5-triphosphate 2,3-dialdehyde, guanosine 5-triphosphate 2,3-dialdehyde, and cytosine-9-beta-D-arabinofuranoside 5-triphosphate. Two dialdehyde derivatives, adenosine 5-triphosphate 2,3 dialdehyde and guanosine 5-triphosphate 2,3-dialdehyde, were irreversible inhibitors, forming a stable complex with the viral polypeptide VP3. The effect of the corresponding nucleosides of the inhibitory analogues was studied in SA-11 rotavirus-infected MA-104 cells. Adenosine-9-beta-D-arabinofuranoside and 3-deoxyadenosine were effective inhibitors of RNA synthesis, an effect that could be due to their inhibition of viral transcription.

Characterization of rotavirus electropherotypes excreted by symptomatic and asymptomatic infants.

Human rotavirus isolates from 1100 stool samples were analyzed by polyacrylamide gel electrophoresis, and 48 different migration patterns were detected. Heterogeneity in the migration of segment 10 was observed in both long and short electropherotypes in which three long and two short patterns were identified. In spite of these variations all short and long electropherotypes were subgrouped by enzyme immunoassay as subgroups I and II respectively. Mixed infections were detected in 17% of cases and the subgrouping correlated with the corresponding electropherotypes. The same electropherotypes were present in severe, mild and asymptomatic cases and no electropherotype was particularly associated with greater virulence. Furthermore, the electropherotypes isolated from nosocomial asymptomatic cases were the same as those detected from those admitted with severe diarrhea. It seems unlikely that electropherotyping can be used to identify more virulent strains of rotavirus.

Detection of the infectious hematopoietic necrosis virus directly from infected fish tissues by dot blot hybridization with a non-radioactive probe

A method was developed for the rapid diagnosis of the infectious hematopoietic necrosis virus (IHNV) based on dot blot hybridization with a non-radioactive probe. When the assay was developed through a color reaction both biotin- and alkaline phosphatase-labeled probes were highly specific for IHNV and the sensitivity reached to 20 pg of viral RNA. When the alkaline phosphatase-labeled probe was developed by a chemiluminiscent reaction, the sensitivity showed a five-fold increase to 4 pg of viral RNA. The procedures successfully detected IHNV directly from infected symptomatic and asymptomatic fishes exhibiting higher sensitivity than the traditional approaches involving viral propagation in cell cultures. Additional advantages of the method are its simplicity and it takes only about 6 h to carry out the procedures from the initial processing of the tissues to diagnosis.

Rotavirus detection by dot blot hybridization assay using a non-radioactive synthetic oligodeoxynucleotide probe

A synthetic oligodeoxynucleotide of 40 nucleotides corresponding to nucleotides 33-72 of the gene coding for the viral protein VP7 of rotavirus, was used as a nucleic acid probe to develop a non-radioactive hybridization method for rotavirus detection. The probe was labelled at the 3 end with biotin-7-dATP. The sensitivity and specificity of the dot blot hybridization assay for rotavirus detection was evaluated with 303 stool specimens. The results indicate that the hybridization assay has a higher sensitivity than both PAGE and EIA. Among the rotavirus strains tested 37 different electropherotypes were found. The results suggest that rotavirus diagnosis by dot hybridization using a non-radioactive probe may become routine laboratory procedure because it is simple, highly specific and very sensitive.