Fluoroquinolone-resistant Streptococcus agalactiae isolates from Argentina.

Fluoroquinolone resistance is a growing problem that has only recently emerged in S. agalactiae. Between 2005-2007, WHONET--Argentina network evaluated levofloxacin susceptibility in 1128 clinical S. agalactiae isolates, 10 (0.9%) of which proved to be resistant. Nine of them had come from 5 hospitals (in Buenos Aires City and 4 Argentinean provinces) and recovered from urine (n=7) and vaginal screening cultures (n=2). Three strains were also resistant to macrolides, lincosamides and B streptogramins due to the ermA gene. All nine fluoroquinolone-resistant isolates bore the same two mutations, Ser79Phe in ParC and Ser81Leu in GyrA proteins. Genetic relationships were analyzed by Apal-PFGE and two clones were determined, A (n=6) and B (n=3). To our knowledge, these are the first fluoroquinolone-resistant S. agalactiae isolates detected in Latin America.

Research efforts to control highly pathogenic arenaviruses: a summary of the progress and gaps.

Significant progress has been made in the past 10 years in unraveling the molecular biology of highly pathogenic arenaviruses that are endemic in several West African countries (Lassa fever virus) and in some regions of South America (Argentine and Bolivian hemorrhagic fever viruses). While this has resulted in proof-of-concept studies of novel vaccine candidates in non-human primates and in the discovery of several novel antiviral small molecule drug candidates, none of them has been tested in the clinic to date. The recent Ebola outbreak in West Africa has demonstrated very clearly that there is an urgent need to develop the prophylactic and therapeutic armamentarium against viral hemorrhagic fever viruses as part of a global preparedness for future epidemics. As it pertains to this goal, the present article summarizes the current knowledge of highly pathogenic arenaviruses and identifies opportunities for translational research.

Conserved sequences and coding of two strains of lymphocytic choriomeningitis virus (WE and ARM) and Pichinde arenavirus.

Analyses of the 3' end sequences of the small, S, and large, L, RNA species of lymphocytic choriomeningitis (LCM) virus isolates ARM and WE, and DNA clones of LCM-WE, have shown that there are extensive RNA sequence homologies between the 3' ends of the two RNA species of both LCM strains. Limited sequence data of DNA clones representing the LCM-WE L RNA species indicate that a gene product (presumably the minor 200 kdalton virion protein) is coded in a viral-complementary mRNA species. Sequence analyses of LCM-WE S DNA clones indicate that approximately 50% of the 2040 nucleotides representing the 3' half of the viral RNA species (and its encoded 558 amino acid gene product) are identical in type and position to those of Pichinde arenavirus (Auperin, D., et al. (1984a), Virology 134, 208-219). For Pichinde virus, it has been shown that the 3' proximal gene product (the nucleoprotein, N) is translated from a subgenomic, viral-complementary mRNA (Auperin et al., 1984a). Data have recently been obtained (Auperin, D., et al. (1984b) J. Virol., in press) that indicate that the Pichinde glycoprotein precursor, GPC, is coded in a viral-sense subgenomic mRNA species corresponding to the 5' half of the S RNA. The nucleotide sequence that immediately follows the N coding region of both LCM-WE and Pichinde viruses can be arranged in a hairpin configuration. In view of this, and if, like Pichinde virus, LCM has an ambisense S RNA coding strategy, then it is probable that the intergenic hairpins function as transcription terminators for the N and GPC mRNA species of both viruses.

Complete sequence of the S RNA of lymphocytic choriomeningitis virus (WE strain) compared to that of Pichinde arenavirus.

Previous studies have reported that the 3' half of the small, S, RNA species of the WE strain of lymphocytic choriomeningitis (LCM) virus codes for the viral nucleoprotein in a subgenomic, viral-complementary, mRNA species (Romanowski, V. and Bishop, D.H.L. (1985) Virus Res. 2, 35-51). The complete sequence of the LCM-WE S RNA has now been obtained, indicating that the 5' half of the RNA codes for the viral glycoprotein precursor in a viral-sense sequence that does not overlap the N gene. It is concluded that, like Pichinde virus (Auperin, D. et al. (1984) J. Virol. 52, 897-904), LCM has an ambisense S RNA coding strategy. The LCM-WE S RNA is 3375 nucleotides in length, has a size of 1.14 X 10(6) Da and base composition of 26.1% A, 23.2% C, 21.5% G, 29.2% U. The 3' and 5' end sequences of the S RNA are complementary for some 30 nucleotides, depending on the arrangement. The non-coding regions at the two ends are 77 (5') and 60 (3') nucleotides long. The glycoprotein precursor has a primary amino acid size of 56293 Da and is rich in potential glycosylation sites as well as histidine and cysteine residues. It has both amino and carboxy proximal hydrophobic regions. The LCM-WE S RNA and predicted protein sequence data have been compared to those of Pichinde arena-virus. Extensive RNA and protein sequence homology exists for the two S RNA species, although the homology for the glycoprotein sequences of the two viruses (39%) is less than the 50% observed for the two viral nucleoproteins.

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.

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.

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.

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.

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.

[The study of protective properties of attenuated strain of Argentinian hemorrhagic fever]. / Izuchenie protektivnykh svoistv attenuirovannogo shtamma argentinskoo gemorragicheskoi likhoradki.

Immunization of BALB/c, C57BL/6, CBA/calac mice with strain XJ44 of Argentine hemorrhagic fever resulted in changes of nonspecific immunity parameters, such as interferon, interleukin-1, tumor necrosis factor, and natural killers. The formation of a specific humoral and cellular immune response in BALB/c mice immunized with this strain has been demonstrated. Immunization of BALB/c mice with strain XJ44 protected the animals from infection with a heterogeneous strain Carvallo of Bolivian hemorrhagic fever.

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.

The formation of arenaviruses that are genetically diploid.

Analyses of RNA extracted from preparations of arenaviruses indicate that the relative molar proportions of the genomic L and S RNA species are frequently far from equal. In order to investigate the genetic significance of this observation temperature-sensitive (ts) mutants of two lymphocytic choriomeningitis (LCM) virus strains (ARM and WE) have been recovered and categorized into recombination groups (Groups I and II). Fingerprint analyses of wild-type progeny viruses obtained from dual infections with ARM Group II and WE Group I ts viruses indicate that they have L/S RNA genotypes of WE/ARM. It is concluded that the ARM Group II ts viruses have mutations in their L RNA species and that the WE Group I ts viruses have mutations in their S RNA species. Correspondingly it is deduced that the ARM Group I ts viruses have S RNA mutations and the WE Group II ts viruses mutations in their L RNA species. Cells coinfected with certain WE Group I mutants, or an ARM Group I and certain WE Group I ts mutants, have also yielded wild-type viruses. Fingerprint analyses have shown that the wild-type viruses obtained from the latter crosses are diploid with respect to their S RNA species. On subsequent passage these wild-type viruses shed high proportions of ts mutants. We interpret the data to indicate that the original Group I ts mutants that yielded the diploid viruses have mutations in different S RNA gene products so that the progeny produce plaques at the nonpermissive temperature by gene product complementation. No wild-type recombinant viruses have been obtained from crosses involving Pichinde and LCM ts mutants.

Computational characterisation of potential RNA-binding sites in arenavirus nucleocapsid proteins.

A nucleocapsid protein of an RNA virus was characterised using computational methods. Similarity searches using standard algorithms and more sensitive methods based on profiles were performed. Also, secondary structure prediction and statistical methods were used. The results show that the protein belongs to a unique well-characterised family, with three regions with potential RNA binding capacity. The amino-terminal region is found to contain a mixed-charge segment similar to proteins that bear nucleic acid-protein interaction capacity. The middle-region has a slight homology to the nucleolar protein Fibrillarin containing an atypical RNP-1 conserved octamer. Finally, the carboxyl-terminal region has a putative zinc-finger.

Analyses of the mRNA transcription processes of snowshoe hare bunyavirus S and M RNA species.

The time course of synthesis of snowshoe hare bunyavirus small (S)- and medium (M)-sized viral RNA (vRNA), viral cRNA (vcRNA), and mRNA species was analyzed by using single-stranded DNA probes representing the S- and M-coded gene products. In the presence of puromycin, an inhibitor of protein synthesis, the subgenomic S mRNA species were detected, but not full-length S vcRNA or S vRNA species. No M-related RNA species were identified in puromycin-treated cells. In the absence of puromycin, full-length M and S vRNA, S vcRNA, and subgenomic S mRNA species were observed, as well as apparently full-length M vcRNA species, presumably including the approximately similar-sized M mRNA species. The 5' ends of the S and M mRNA species have been shown to be heterogeneous and some 12 to 17 bases longer than the ends of their corresponding presumptive replicative vcRNA species, in agreement with an earlier report that they represent nonviral primer sequences (D. H. L. Bishop, M. E. Gay, and Y. Matsuoko, Nucleic Acids Res. 11:6409-6418, 1983). The 3' ends of the M and S mRNA species were found to be shorter by some 60 and 100 nucleotides, respectively, than those of their corresponding full-length vcRNA species. Comparison of the 3' noncoding regions of the S and M vcRNA species revealed that there are conserved sequences following the translation termination codons of the two RNA species. One of these conserved sequences is a pyrimidine-rich template sequence that is approximately 20 nucleotides beyond the deduced S mRNA transcription termination site.

Molecular organization of Junin virus S RNA: complete nucleotide sequence, relationship with other members of the Arenaviridae and unusual secondary structures.

In this study, overlapping cDNA clones covering the entire S RNA molecule of Junin virus, an arenavirus that causes Argentine haemorrhagic fever, were generated. The complete sequence of this 3400 nucleotide RNA was determined using the dideoxynucleotide chain termination method. The nucleocapsid protein (N) and the glycoprotein precursor (GPC) genes were identified as two non-overlapping open reading frames of opposite polarity, encoding primary translation products of 564 and 481 amino acids, respectively. Intracellular processing of the latter yields the glycoproteins found in the viral envelope. Comparison of the Junin virus N protein with the homologous proteins of other arenaviruses indicated that amino acid sequences are conserved, the identity ranging from 46 to 76%. The N-terminal half of GPC exhibits an even higher degree of conservation (54 to 82%), whereas the C-terminal half is less conserved (21 to 50%). In all comparisons the highest level of amino acid sequence identity was seen when Junin virus and Tacaribe virus sequences were aligned. The nucleotide sequence at the 5' end of Junin virus S RNA is not identical to that determined of the other sequenced arenaviruses. However, it is complementary to the 3'-terminal sequences and may form a very stable panhandle structure (delta G-242.7 kJ/mol) involving the complete non-coding regions upstream from both the N and GPC genes. In addition, a distinct secondary structure was identified in the intergenic region, downstream from the coding sequences; Junin virus S RNA shows a potential secondary structure consisting of two hairpin loops (delta G -163.2 and -239.3 kJ/mol) instead of the single hairpin loop that is usually found in other arenaviruses. The analysis of the arenavirus S RNA nucleotide sequences and their encoded products is discussed in relation to structure and function.

Rapid diagnosis of Argentine hemorrhagic fever by reverse transcriptase PCR-based assay.

Argentine hemorrhagic fever (AHF) is an endemo-epidemic disease caused by Junín virus. This report demonstrates that a reverse transcriptase (RT) PCR-based assay developed in our laboratory to detect Junín virus in whole blood samples is sensitive and specific. The experiments were conducted in a double-blinded manner using 94 clinical samples collected in the area in which AHF is endemic. The RT-PCR-based assay was compared with traditional methodologies, including enzyme-linked immunosorbent assay, plaque neutralization tests, and occasionally viral isolation. The calculated parameters for RT-PCR diagnosis, with seroconversion as the "gold standard," were 98% sensitivity and 76% specificity. It is noteworthy that 94% of the patients with putative false-positive results (RT-PCR positive and no seroconversion detected) exhibited febrile syndromes of undefined etiology. These results could be interpreted to mean that most of those patients with febrile syndromes were actually infected with Junín virus but did not develop a detectable immune response. Furthermore, 8 laboratory-fabricated samples and 25 blood samples of patients outside the area in which AHF is endemic tested in a similar way were disclosed correctly (100% match). The RT-PCR assay is the only laboratory test available currently for the early and rapid diagnosis of AHF. It is sensitive enough to detect the low viremia found during the period in which immune plasma therapy can be used effectively, reducing mortality rates from 30% to less than 1%.

Patterns of transient expression of the arenavirus nucleocapsid protein gene in transfected cells.

The cloned genes for the nucleocapsid proteins N of Junín and LCM (lymphocytic choriomeningitis) arenaviruses were inserted into the SV40-derived expression vector designated pKG4. When BHK-21 (baby hamster kidney fibroblasts) and CV-1 (African green monkey kidney fibroblasts) cell lines were transfected using these constructions, the transient expression yielded a polypeptide that could not be distinguished either by size nor by immunoreactivity from the N protein synthesized during the viral infection. The immunofluorescence analysis showed a pattern of intracellular localization similar to that observed in virus infected cells, i.e. varying from a diffuse cytoplasmic staining to granules, either distributed throughout the cytoplasm or concentrated in the perinuclear region. The association of the N protein with basophilic granules is similar to that observed in the cytopathic effect caused by arenaviruses, and could be related to the physicochemical properties of this polypeptide containing numerous basic amino acid sequences, that would allow for the interaction with cellular RNAs.

Characterization of a granulovirus isolated from Epinotia aporema Wals. (Lepidoptera: Tortricidae) larvae.

A granulovirus (GV) isolated from Epinotia aporema (Lepidoptera: Tortricidae)-a major soybean pest-was studied in terms of its main morphological, biochemical, and biological properties. The ovoidal occlusion bodies were 466 by 296 nm in size, and their most prominent protein had an apparent molecular mass of 29 kDa. Its amino-terminal sequence was remarkably homologous to that of the granulins of other GVs. The DNA genome size was estimated to be 120 kbp. The high specificity and pathogenicity of this newly described granulovirus (EpapGV) indicate that it is indeed a good candidate for the biological control of this pest.