Development of an ELA-DRA gene typing method based on pyrosequencing technology.

The polymorphism of equine lymphocyte antigen (ELA) class II DRA gene had been detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and reference strand-mediated conformation analysis. These methodologies allowed to identify 11 ELA-DRA exon 2 sequences, three of which are widely distributed among domestic horse breeds. Herein, we describe the development of a pyrosequencing-based method applicable to ELA-DRA typing, by screening samples from eight different horse breeds previously typed by PCR-SSCP. This sequence-based method would be useful in high-throughput genotyping of major histocompatibility complex genes in horses and other animal species, making this system interesting as a rapid screening method for animal genotyping of immune-related genes.

DNA profile of dog feces as evidence to solve a homicide.

Dog fecal samples were collected at the crime scene and from the shoes of the suspect to see whether they could be linked. DNA was genotyped using a 145bp fragment containing a 60bp hotspot region of the mitochondrial DNA (mtDNA) control region. Once the species origin was identified, sequences were aligned with the 23 canine haplotypes defined, showing that evidence and reference had 100% identity with haplotype 5. The frequency of haplotype 5 and the exclusion power of the reference population were 0.056 and 0.89, respectively. The forensic index showed that it was 20 times more likely that the evidence belonged to the reference dog than to some other unknown animal. The results support that the mtDNA hypervariable region 1 (HV1) is a good alternative for typing in trace or degraded casework samples when the STR panel fails, and demonstrate the utility of domestic animal samples to give additional information to solve human legal cases.

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.

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.

Characterization and validation of bovine gonadotripin releasing hormone receptor (GNRHR) polymorphisms.

Gonadotropin releasing hormone and its receptor (GNRHR) play a critical role in sexual differentiation and reproduction. Available evidence shows a strong genetic component in the timing of puberty. In bovines, there are significant differences within and among beef breeds in the time when bulls reach puberty. Despite its economic importance, there are not many SNPs or genetic markers associated with this characteristic. The aims of the study were to identify DNA polymorphism in the bovine GNRHR by re-sequencing analysis, determine haplotype phases, and perform a population study in a selected tag SNP in six breeds. Eight SNPs were detected, including: one in the Upstream Regulatory Region (URR), five in the coding regions, and two in non-coding regions. This polymorphism level corresponds to one variant every 249.4bp and a global nucleotide diversity of 0.385. Two haplogroups comprising nine haplotypes and two linkage blocks were detected. Despite 5 tag SNPs were required to capture all variability, just one SNP allowed to define both haplogroups, and only two SNPs were needed to differentiate the most common haplotypes. An additional taq SNP was necessary to identify both URR variants. Allele-frequency analysis of a selected taq SNP among breeds showed a geographical cline. European Bos taurus breeds had lower frequencies of the C allele than B. indicus type cattle, while Creole cattle and Wagyu breeds had intermediate frequency. There was a significant correlation between frequency profile and timing of puberty among the studied breeds, which seems to suggest that genetic variation within bovine GNRHR gene could explain at least part of the reported variability.

South American camelid illegal traffic detection by means of molecular markers.

South American camelids comprise the wild species guanaco and vicuña and their respective domestic relatives llama and alpaca. The aim of the present study was to determine by DNA analysis to which of these species belong a herd of camelids confiscated from a llama breeder but alleged to be alpacas by the prosecution, and to evaluate the usefulness of mitochondrial and autosomal DNA markers to solve judicial cases involving camelid taxa. Cytochrome b and cytochrome oxidase I mitochondrial genes and 7 STR were analyzed in 25 confiscated samples. Mitochondrial results were inconclusive because 18 of the sequestered samples presented haplotypes that corresponded to the guanaco haplogroup and the remaining seven belonged to a vicuña linage. Microsatellite data of casework samples and llama reference samples revealed different genetic profiles by the presence of private alleles at two microsatellites suggesting that the confiscated animals could be alpaca, or at least alpaca hybrids instead of pure llama.

Effect of consanguinity on Argentinean Angus beef DNA traceability.

Since the 1990s several authors have envisaged the use of DNA to certify meat origin. Two major parameters must be assessed before a DNA based traceability protocol can be implemented in the food chain: (i) the information content of a DNA marker set in a specific livestock breed or group of breeds; (ii) the minimum number of DNA markers needed to obtain a statistically acceptable match probability. The objective of the present work was to establish the effect of different levels of inbreeding in the matching efficiency, and the minimum number of microsatellite markers needed, in a DNA based meat traceability program, starting from an 11-microsatellite marker panel. Samples were obtained from beef production farms in South America, where animals are typically bred under pasture-based extensive conditions. Three groups of animals with different consanguinity rates were sampled. Exclusion power (Q) was higher than 0.999998 and match probability lower than 3.01E-08, for the whole set of markers within each group. Both values were affected by consanguinity. To reach a two mismatch criteria exclusion power (Q(2)) of 99.99, six markers were needed in unrelated animals whereas seven markers were needed in related animals. To reach Q(2)=99.9999, 8 and 10 microsatellite markers, respectively, were needed. In general, one or two more microsatellite markers were needed to identify consanguineous animals. This study proved the DNA marker set used to be suitable for the identification of the meat from all slaughtered animals in Argentina, per week, month, and year.

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.