Complete Genome Sequences of a Hantavirus Isolate from New York.

We report here the complete genome sequences for all three segments of the New York hantavirus (New York 1). This is the first reported L segment sequence for hantaviruses maintained in Peromyscus spp. endemic to the eastern United States and Canada.

Genomic analysis of filoviruses associated with four viral hemorrhagic fever outbreaks in Uganda and the Democratic Republic of the Congo in 2012.

In 2012, an unprecedented number of four distinct, partially overlapping filovirus-associated viral hemorrhagic fever outbreaks were detected in equatorial Africa. Analysis of complete virus genome sequences confirmed the reemergence of Sudan virus and Marburg virus in Uganda, and the first emergence of Bundibugyo virus in the Democratic Republic of the Congo.

Characterization and template properties of RNA dimers generated during flock house virus RNA replication.

Flock house virus (FHV) is the best studied member of the Nodaviridae, a family of small, nonenveloped, isometric RNA viruses of insects and fish. Nodavirus genomes comprise two single-stranded positive-sense RNA segments (RNAs 1 and 2) that encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The RdRp replicates both genomic RNAs and also generates a subgenomic RNA (RNA3) that is not encapsidated. Although genomic RNAs replicate through negative-sense intermediates, little is known about these RNAs or the details of the replication mechanism. Negative-sense RNAs 1, 2, and 3, as well as putative dimers of RNAs 2 and 3, have been detected in previous studies. In this study we detected dimers of RNAs 1, 2, and 3 by Northern blot analyses of RNA samples from FHV-infected Drosophila cells, as well as from mammalian and yeast cells supporting FHV RNA replication. Characterization of these RNA species by RT-PCR and sequence determination showed that they contained head-to-tail junctions of FHV RNAs. RNAs containing the complete sequence of RNA2 joined to RNA3 were also detected during replication. To examine the template properties of these dimeric RNAs, we made corresponding cDNAs and transcribed them from a T7 promoter in mammalian cells constitutively expressing T7 RNA polymerase, together with RNA1 to provide the RdRp. Although heterologous terminal extensions inhibit FHV RNA replication, monomeric RNA2 was resolved and replicated from complete or partial homodimer templates and from an RNA2-RNA3 heterodimer.

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: impact on hormone secretion.

OBJECTIVE: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/beta-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli beta-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). DESIGN: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/beta-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. METHODS: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for beta-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. RESULTS: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/beta-gal showed widespread expression of the beta-galactosidase transgene around the injection areas. CONCLUSIONS: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.

Generation of a recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus expressing a foreign gene under the control of a very late promoter.

A recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) expressing beta-galactosidase under the control of the polyhedrin promoter was generated in our laboratory. To this end, we cloned the AgMNPV-2D genomic DNA fragment containing the polh gene and subcloned and sequenced the polyhedrin gene and its flanking regions. Based on this sequence information, sets of primers were designed to amplify the flanking regions by PCR, including appropriate restriction sites. The transfer vector (pAgPHZ) was constructed by the consecutive cloning of these PCR fragments flanking the Escherichia coli LacZ gene, in place of the polh gene. pAgPHZ was used for cotransfection of UFL-AG-286 insect cells with AgMNPV-2D DNA and the required recombinant, generated by homologous recombination with the polh locus, was identified by its polh(-)/LacZ+ plaque phenotype. Its genome structure was confirmed by PCR, restriction digestion and Southern blot analyses. The kinetics and levels of expression of beta-galactosidase in UFL-AG-286 cells infected with the recombinant were tested by SDS-PAGE and enzymatic activity assays.

Arenavirus nucleocapsid protein displays a transcriptional antitermination activity in vivo.

RNA polymerase pausing and transcriptional antitermination regulates gene activity in several systems. In arenavirus infected cells the switch from transcription to replication is subjected to a hairpin-dependent termination and requires protein synthesis to bypass this signal. The transcriptional antitermination control by Junín virus nucleocapsid protein N, has been demonstrated in vivo by infecting BHK-21 cells expressing this viral protein in the presence of translation inhibitors. This is the first demonstration in vivo of a transcriptional antitermination control in arenavirus-infected cells.

Arenavirus phylogeny: a new insight.

Arenaviridae is a worldwide distributed family, of enveloped, single stranded, RNA viruses. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as the geographic distribution. In this study the phylogenetic relationship among the members of the Arenaviridae was examined, using the reported genomic sequences. The comparison of the aligned nucleotide sequences of the S RNA and the predicted amino acid sequences of the GPC and N proteins, together with the phylogenetic analysis, strongly suggest a possible kinship of Pichindé and Oliveros viruses, with the Old World arenavirus group. This analysis points at the evolutive relationships between the arenaviruses of the Americas and can be used to evaluate the different hypotheses about their origin.

Molecular characterization of attenuated Junin virus strains.

The Junin virus strain Candid #1 was developed as a live attenuated vaccine for Argentine haemorrhagic fever. In this paper we report the nucleotide sequences of S RNA of Candid #1 and its more virulent ancestors XJ#44 and XJ (prototype). Their relationship to Junin virus wild-type MC2 strain and other closely and distantly related arenaviruses was also examined. Comparisons of the nucleotide and amino acid sequences of N and GPC genes from Candid #1 and its progenitor strains revealed some changes that are unique to the vaccine strain. These changes could be provisionally associated with the attenuated phenotype.

Characterization of arenaviruses using a family-specific primer set for RT-PCR amplification and RFLP analysis. Its potential use for detection of uncharacterized arenaviruses.

Arenaviruses are enveloped viruses with a genome composed of two ssRNA species, designated L and S. The arenaviruses were divided in two major groups (Old World and New World), based on serological properties and genetic data, as well as geographic distribution. A sequence alignment analysis of all reported arenavirus S RNAs yielded 17 conserved regions in addition to a reported conserved region at the end of both RNAs. The consensus sequences of these regions were used to design generalized primers suitable for RT-PCR amplification of a set of overlapping nucleotide sequence fragments comprising the complete S RNA of any arenavirus. A restriction analysis (RFLP) was designed to rapidly typify the amplified fragments. This RT-PCR-RFLP approach was tested with Old World (LCM) and New World (Junin and Tacaribe) arenaviruses. Furthermore, using this procedure the whole S RNA of a novel arenavirus isolate obtained from a rodent trapped in central Argentina, was amplified and characterized. Partial nucleotide sequence data were used for phylogenetic analyses that showed the relationships between this arenavirus and the rest of the members of the family. This relatively simple methodology will be useful both in basic studies and epidemiological survey programs.

The glycoprotein precursor gene of the attenuated Junin virus vaccine strain (Candid #1).

A live attenuated virus vaccine has been recently developed to prevent Argentine hemorrhagic fever. In this paper, we report the nucleotide sequence of the glycoprotein precursor gene (GPC) of the Junin virus vaccine strain (Candid #1) and its flanking untranslated regions. The untranslated regions flanking the GPC genes of different arenaviruses are variable in length, sequence, and secondary structure. However, when this highly attenuated Junin virus strain is compared with the MC2 strain, which is of intermediate virulence, one nucleotide insertion and four nucleotide substitutions are found at positions that do not affect the predicted secondary structure. When Candid #1 and MC2 RNAs are compared, the nucleotide sequence changes in the GPC open reading frame are concentrated in the amino-proximal and the carboxy-proximal regions. The comparison of the amino acid residues shows that the major changes are located in the amino-proximal region of the GPC.

PCR screening for carriers of bovine leukocyte adhesion deficiency (BLAD) and uridine monophosphate synthase (DUMPS) in Argentine Holstein cattle.

BLAD (Bovine Leukocyte Adhesion Deficiency) and DUMPS (Deficiency of Uridine Monophosphate Synthase) are monogenic autosomal, recessive inherited diseases of Holstein cattle. Single nucleotide changes (point mutations) responsible for the genetic disorders were detected by polymerase chain reaction coupled with restriction fragment length polymorphism assays (PCR-RFLP). Using oligonucleotide primers, DNA fragments of predicted sizes were amplified, and the products' specificity was assessed by nucleotide sequencing. Mutations were detected in DNA samples from bovine blood and semen by the presence or absence of restriction sites within the PCR amplification products (Taq I, Hae III for BLAD, Ava I for DUMPS). The test included 104 bulls and 950 cows of Argentinean Holstein breed. Defective alleles frequencies were as follows: 2.88% BLAD in bulls used in artificial insemination, 1.79% in cows; 0.96% DUMPS in bulls and 0.11% in cows.

Phenol extraction revisited: a rapid method for the isolation and preservation of human genomic DNA from whole blood.

We report a fast and simple DNA isolation method from whole blood. It avoids cell separation and lysis steps and consists of three successive solvent extractions and an ethanol precipitation. All the steps are carried out at room temperature. The main advantage of this method is the immediate sample inactivation achieved by mixing the blood sample with Tris-HCl (pH 8.0) saturated phenol, thus minimizing the biohazard involved in the subsequent manipulation of the samples potentially contaminated with infectious agents (the procedure has been called SP for 'straight phenol'). In addition, extensive field sample collections are facilitated by the fact that the SP procedure can be stopped right after the simple manipulation of mixing the blood sample with the phenol; neither freezing nor refrigeration of the sample proved to be required. At this stage, the nucleases as well as infectious agent are inactivated and the rest of the protocol can wait to be carried out in the laboratory. In fact, the DNA preparation can be resumed after prolonged storage of the blood-phenol mix (up to 72 days has been checked in our laboratory) at room temperature without affecting the yield. The SP protocol may be scaled up, when large quantities of DNA are needed, or scaled down to smaller volumes, such as fingerprick blood samples.