Rapid resolution of duck hepatitis B virus infections occurs after massive hepatocellular involvement.

A study was carried out to determine some of the factors that might distinguish transient from chronic hepadnavirus infection. First, to better characterize chronic infection, Pekin ducks, congenitally infected with the duck hepatitis B virus (DHBV), were used to assess age-dependent variations in viremia, percentage of DHBV-infected hepatocytes, and average levels of DNA replication intermediates in the cytoplasm and of covalently closed circular DNA in the nuclei of infected hepatocytes. Levels of viremia and viral DNA were found to peak at about the time of hatching but persisted at relatively constant levels in chronically infected birds up to 2 years of age. The percentage of infected hepatocytes was also constant, with DHBV replication in virtually 100% of hepatocytes in all birds. Next, we found that adolescent ducks inoculated intravenously with a large dose of DHBV also developed massive infection of hepatocytes with an early but low-level viremia, followed by rapid development of a neutralizing antibody response. No obvious quantitative or qualitative differences between transiently and chronically infected liver tissue were detected in the intracellular markers of viral replication examined. However, in the adolescent duck experiment, DHBV infection was rapidly cleared from the liver even when up to 80% of hepatocytes were initially infected. In all of these ducks, clearance of infection was accompanied by only a mild hepatitis, with no evidence that massive cell death contributed to the clearance. This finding suggested that mechanisms in addition to immune-mediated destruction of hepatocytes might make major contributions to clearance of infections, including physiological turnover of hepatocytes in the presence of a neutralizing antibody response and/or spontaneous loss of the capacity of hepatocytes to support virus replication.     

Rapid identification of infectious pancreatic necrosis virus in infected cell cultures by immunoperoxidase techniques.

Direct and indirect immunoperoxidase (IP) techniques were evaluated for their potential in identifying infectious pancreatic necrosis (IPN) virus. Both techniques were shown to offer a relatively simple, rapid and efficient means for the specific identification of IPN virus in infected cells. The direct IP method resulted in less nonspecific staining; however, the indirect method was clearly specific and utilized commercially available reagents.     

Rapid detection and identification of odontoglossum ringspot virus by polymerase chain reaction amplification

Abstract A hemolysis gene ( hlx ) which lyses sheep erythrocytes on blood agar plates when expressed in Escherichia coli was cloned from Vibrio cholerae . The cloned gene is predicted to encode a polypeptide of 92 amino acid residues with a deduced molecular mass of 10451. E. coli transformed with this gene lysed sheep, goose, horse and chicken erythrocytes but not those of guinea pig and human. The hlx gene was observed in classical- and El Tor-biotype V. cholerae O1, V. cholerae non-O1, and V. mimicus , but not in V. parahaemolyticus .     

Rapid virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing

Background

Newcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs.

Methods

An amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow.

Results

For all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 10¹ 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq.

Conclusion

The depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV.

     

Multiple virus infections on Heterobasidion sp.

Heterobasidion viruses have previously been shown to affect each other’s transmission and phenotypic effects on their hosts in a complex way. In this work, Heterobasidion parviporum strains hosting five coinfecting viruses simultaneously were constructed and used as donors in transmission experiments. They showed that viruses move more frequently between the mycelia of the same species than between the mycelia of H. parviporum and Heterobasidion annosum. One of the strains was used to show that coinfection of five viruses is relatively unstable in a natural environment and analyses of the growth rate and competitive ability of Heterobasidion strains hosting various virus combinations revealed that viral effects are not additive. The results also supported the view that the transmission of the promising virocontrol agent HetPV13-an1 may be enhanced by coinfecting viruses in the donor mycelium. However, its detrimental effects may be blocked by the presence of other viruses in the same mycelium.RepositoriesGenBank accession number MN058080.     

Rapid identification of microorganisms by circular-intensity differential scattering.

There is a pressing need in clinical medicine for rapid identification of microorganisms. We describe a method that has the potential for such rapid identification: circular-intensity differential scattering, which is based on the differential scattering of left and right circularly polarized light. The scanning time required to obtain the spectral signature of an organism is about 4 min. Using a commercial circular dichrograph modified to measure circular intensity differential scattering at 90 degrees, we obtained significantly different spectra for five different crude influenza viruses. Salmonella typhimurium TA98 and TA100, and Escherichia coli HB101, HB101(pBR322), and HB101(pMB9). Purified supercoiled plasmid pBR322 DNA was readily distinguishable from linear pBR322 DNA; such differences in nucleic acid packaging may be significant factors in the discriminatory power of this technique.     

Rapid identification of DNA-binding proteins by mass spectrometry.

We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The determined molecular masses are often sufficient for identification. If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on posttranslational modifications. The protocol was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA.