The p.A2215D Thyroglobulin Gene Mutation Leads to Deficient Synthesis and Secretion of the Mutated Protein and Congenital Hypothyroidism with Wide Phenotype Variation

Context: Thyroglobulin (TG) is a large glycoprotein and functions as a matrix for thyroid hormone synthesis. TG gene mutations give rise to goitrous congenital hypothyroidism (CH) with considerable phenotype variation. Objectives: The aim of the study was to report the genetic screening of 15 patients with CH due to TG gene mutations and to perform functional analysis of the p. A2215D mutation. Design: Clinical evaluation and DNA sequencing of the TG gene were performed in all patients. TG expression was analyzed in the goitrous tissue of one patient. Human cells were transfected with expression vectors containing mutated and wild-type human TG cDNA. Results: All patients had an absent rise of serum TG after stimulation with recombinant human TSH. Sequence analysis revealed three previously described mutations (p. A2215D, p. R277X, and g. IVS30 + 1G > T), and two novel mutations (p. Q2142X and g. IVS46-1G > A). Two known (g. IVS30 + 1G/p. A2215D and p. A2215D/p. R277X) and one novel (p. R277X/g. IVS46-1G > A) compound heterozygous constellations were also identified. Functional analysis indicated deficiency in TG synthesis, reduction of TG secretion, and retention of the mutant TG within the cell, leading to an endoplasmic reticulum storage disease, whereas small amounts of mutant TG were still secreted within the cell system. Conclusion: All studied patients were either homozygous or heterozygous for TG gene mutations. Two novel mutations have been detected, and we show that TG mutation p. A2215D promotes the retention of TG within the endoplasmic reticulum and reduces TG synthesis and secretion, causing mild hypothyroidism. In the presence of sufficient iodine supply, some patients with TG mutations are able to compensate the impaired hormonogenesis and generate thyroid hormone. (J Clin Endocrinol Metab 94: 2938-2944, 2009)

Transient expression of a novel serine protease in the ectoderm of the ascidian Herdmania momus during development

We have studied gene expression during ascidian embryonic development using the technique of differential display and isolated partial cDNA sequences of 12 genes. Developmental regulation of these genes has been confirmed by northern hybridization analysis. Further cDNA cloning and sequence analysis of an mRNA that is present during gastrulation, neurulation and tailbud formation reveals that it encodes a novel serine protease containing a single kringle motif and catalytic domain. The spatial expression of this gene, designated Hmserp1, is restricted to precursor cells of the epidermis. The structure and expression of Hmsery1 is discussed in relation to possible functions during development.

Lipopolysaccharide biosynthesis genes in koala type I Chlamydia: Cloning and characterization

We showed in 1988 that there are two strains of Chlamydia psittaci which infect the koala (Phascolarctos cinereus). In order to further investigate the role of these chlamydial strains in pathogenesis, we have attempted to identify genes of koala type I strain chlamydial which are involved in the immunogenic response, Transformation of Escherichia coli with a plasmid containing a 6.3-kb fragment (pKOC-10) of C. psittaci DNA caused the appearance of a specific chlamydial lipopolysaccharide (LPS) epitope on the host strain. The smallest DNA fragment capable of inducing the expression of chlamydial LPS was an Xbal fragment, 2.4 kb in size (pKOC-5). DNA sequence analysis of the complete fragment revealed regions of high identity, at the amino acid level, to the gseA genes of C. pneomoniae, C. psittaci 6BC and C. trachomatis, and the kdtA gene of E. coli which code for transferases catalysing the addition of 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A. Two open reading frames (ORFs) of 1,314 and 501 nucleotides in size, within the 2.4-kb fragment, were evident, and mRNA species corresponding to these ORFs were detected by Northern analysis. Both ORF1 and ORF2 are required for the appearance of chlamydia-specific LPS on the surface of recombinant E. coli.

Differential expression of mitochondrial NADH dehydrogenase in ethanol-treated rat brain: Revealed by differential display

The technique of polymerase chain reaction (PCR) differential display was used to detect alterations in gene expression after chronic alcohol administration. Male Wistar rats were treated with ethanol vapor for 14 days. The cDNA generated from mRNA isolated from the hippocampi of ethanol-treated and control animals was compared by PCR differential display. A differentially expressed cDNA fragment was used to screen mRNA samples by Northern analysis. The level of a mRNA was significantly elevated (x 2.5) in the hippocampus, but not the cortex of alcohol-treated rats up to 48 hr after withdrawal. Sequence analysis of the cDNA fragment revealed an almost perfect homology to rat mitochondrial NADH dehydrogenase subunit 4 mRNA. The selective induction of this mRNA in alcohol-treated rat brain areas suggests altered metabolic processes and possible dysfunction of the mitochondria. The technique of PCR differential display may prove useful in further analysis of gene expression during alcohol dependence and withdrawal.

Molecular characterization of the Hepatitis B virus genotypes in Colombia: A Bayesian inference on the genotype F

Hepatitis B is a worldwide health problem affecting about 2 billion people and more than 350 million are chronic carriers of the virus. Nine HBV genotypes (A to I) have been described. The geographical distribution of HBV genotypes is not completely understood due to the limited number of samples from some parts of the world. One such example is Colombia, in which few studies have described the HBV genotypes. In this study, we characterized HBV genotypes in 143 HBsAg-positive volunteer blood donors from Colombia. A fragment of 1306 bp partially comprising HBsAg and the DNA polymerase coding regions (S/POL) was amplified and sequenced. Bayesian phylogenetic analyses were conducted using the Markov Chain Monte Carlo (MCMC) approach to obtain the maximum clade credibility (MCC) tree using BEAST v.1.5.3. Of all samples, 68 were positive and 52 were successfully sequenced. Genotype F was the most prevalent in this population (77%) - subgenotypes F3 (75%) and Fib (2%). Genotype G (7.7%) and subgenotype A2 (15.3%) were also found. Genotype G sequence analysis suggests distinct introductions of this genotype in the country. Furthermore, we estimated the time of the most recent common ancestor (TMRCA) for each HBV/F subgenotype and also for Colombian F3 sequences using two different datasets: (i) 77 sequences comprising 1306 bp of S/POL region and (ii) 283 sequences comprising 681 bp of S/POL region. We also used two other previously estimated evolutionary rates: (i) 2.60 x 10(-4) s/s/y and (ii) 1.5 x 10(-5) s/s/y. Here we report the HBV genotypes circulating in Colombia and estimated the TMRCA for the four different subgenotypes of genotype F. (C) 2010 Elsevier B.V. All rights reserved.

Molecular Characterization, Distribution, and Dynamics of Hepatitis C Virus Genotypes in Blood Donors in Colombia

Hepatitis C virus (HCV) is a frequent cause of acute and chronic hepatitis and a leading cause for cirrhosis of the liver and hepatocellular carcinoma. HCV is classified in six major genotypes and more than 70 subtypes. In Colombian blood banks, serum samples were tested for anti-HCV antibodies using a third-generation ELISA. The aim of this study was to characterize the viral sequences in plasma of 184 volunteer blood donors who attended the ""Banco Nacional de Sangre de la Cruz Roja Colombiana,`` Bogota, Colombia. Three different HCV genomic regions were amplified by nested PCR. The first of these was a segment of 180 bp of the 5`UTR region to confirm the previous diagnosis by ELISA. From those that were positive to the 5`UTR region, two further segments were amplified for genotyping and subtyping by phylogenetic analysis: a segment of 380 bp from the NS5B region; and a segment of 391 bp from the E1 region. The distribution of HCV subtypes was: 1b (82.8%), 1a (5.7%), 2a (5.7%), 2b (2.8%), and 3a (2.8%). By applying Bayesian Markov chain Monte Carlo simulation, it was estimated that HCV-1b was introduced into Bogota around 1950. Also, this subtype spread at an exponential rate between about 1970 to about 1990, after which transmission of HCV was reduced by anti-HCV testing of this population. Among Colombian blood donors, HCV genotype 1b is the most frequent genotype, especially in large urban conglomerates such as Bogota, as is the case in other South American countries. J. Med. Virol. 82: 1889-1898, 2010. (C) 2010 Wiley-Liss, Inc.

Recessive COL6A2 C-globular Missense Mutations in Ullrich Congenital Muscular Dystrophy ROLE OF THE C2a SPLICE VARIANT

Ullrich congenital muscular dystrophy (UCMD) is a disabling and life-threatening disorder resulting from either recessive or dominant mutations in genes encoding collagen VI. Although the majority of the recessive UCMD cases have frameshift or nonsense mutations in COL6A1, COL6A2, or COL6A3, recessive structural mutations in the COL6A2 C-globular region are emerging also. However, the underlying molecular mechanisms have remained elusive. Here we identified a homozygous COL6A2 E624K mutation (C1 subdomain) and a homozygous COL6A2 R876S mutation (C2 subdomain) in two UCMD patients. The consequences of the mutations were investigated using fibroblasts from patients and cells stably transfected with the mutant constructs. In contrast to expectations based on the clinical severity of these two patients, secretion and assembly of collagen VI were moderately affected by the E624K mutation but severely impaired by the R876S substitution. The E624K substitution altered the electrostatic potential of the region surrounding the metal ion-dependent adhesion site, resulting in a collagen VI network containing thick fibrils and spots with densely packed microfibrils. The R876S mutation prevented the chain from assembling into triple-helical collagen VI molecules. The minute amount of collagen VI secreted by the R876S fibroblasts was solely composed of a faster migrating chain corresponding to the C2a splice variant with an alternative C2 subdomain. In transfected cells, the C2a splice variant was able to assemble into short microfibrils. Together, the results suggest that the C2a splice variant may functionally compensate for the loss of the normal COL6A2 chain when mutations occur in the C2 subdomain.

Isolation and partial characterization of Brazilian samples of feline immunodeficiency virus

Feline immunodeficiency virus (FIV) causes a slow progressive degeneration of the immune system which eventually leads to a disease comparable to acquired immune deficiency syndrome (AIDS) in humans. FIV has extensive sequence variation, a typical feature of lentiviruses. Sequence analysis showed that diversity was not evenly distributed throughout the genome, but was greatest in the envelope gene, env. The virus enters host cells via a sequential interaction, initiated by the envelope glycoprotein (env) binding the primary receptor molecule CD134 and followed by a subsequent interaction with chemokine co-receptor CXCR4. The purpose of this study was to isolate and characterize isolates of FIV from an open shelter in Sao Paulo, Brazil. The separated PBMC from 11 positive cats were co-cultured with MYA-1 cells. Full-length viral env glycoprotein genes were amplified and determined. Chimeric feline x human CD134 receptors were used to investigate the receptor utilization of 17 clones from Brazilian isolates of Fly. Analyses of the sequence present of molecular clones showed that all clones grouped within subtype B. In contrast to the virulent primary isolate FIV-GL8, expression of the first cysteine-rich domain (CRD1) of feline CD134 in the context of human CD134 was sufficient for optimal receptor function for all Brazilian FIV isolates tested. (C) 2011 Elsevier B.V. All rights reserved.

Genotyping of potentially zoonotic Giardia duodenalis from exotic and wild animals kept in captivity in Brazil

We have studied the variability of glutamate dehydrogenase (gdh) and small subunit ribosomal (SSU) rRNA coding genes of Giardia species in fecal samples isolated from wild and exotic animals in Brazil, and compared with homologous sequences of isolates from human and domestic animals characterized in previous studies. Cysts of Giardia duodenalis were obtained from feces of naturally infected monkeys (Alouatta fusca) (n = 20), chinchillas (Chinchilla lanigera) (n = 3), ostriches (Struthio camelus) (n = 2) and jaguar (Panthera onca) (n = 1). Assemblage AI was assigned to the unique isolate of jaguar. All the samples from monkeys, chinchillas, and ostriches were assigned to Assemblage B. There was little evolutionary divergence between the referred isolates and isolates described elsewhere. The Assemblage B isolates identified in this study were closely related to Assemblage BIV isolated from humans. The molecular identification of Assemblages A and B of G. duodenalis isolates from exotic and wild animals demonstrates that such hosts may be a potential reservoir for zoonotic transmission of G. duodenalis. (C) 2011 Elsevier B.V. All rights reserved.

Avian Metapneumovirus Subtypes Circulating in Brazilian Vaccinated and Nonvaccinated Chicken and Turkey Farms

Avian metapneumovirus (AMPV) causes turkey rhinotracheitis and is associated with swollen head syndrome in chickens, which is usually accompanied by secondary infections that increase mortality. AMPVs circulating in Brazilian vaccinated and nonvaccinated commercial chicken and turkey farms were detected using a universal reverse transcriptase (RT)-PCR assay that can detect the four recognized subtypes of AMPV. The AMPV status of 228 farms with respiratory and reproductive disturbances was investigated. AMPV was detected in broiler, hen, breeder, and turkey farms from six different geographic regions of Brazil. The detected viruses were subtyped using a nested RT-PCR assay and sequence analysis of the G gene. Only subtypes A and B were detected in both vaccinated and nonvaccinated farms. AMPV-A and AMPV-B were detected in 15 and 23 farms, respectively, while both subtypes were simultaneously found in one hen farm. Both vaccine and field viruses were detected in nonvaccinated farms. In five cases, the detected subtype was different than the vaccine subtype. Field subtype B virus was detected mainly during the final years of the survey period. These viruses showed high molecular similarity (more than 96% nucleotide similarity) among themselves and formed a unique phylogenetic group, suggesting that they may have originated from a common strain. These results demonstrate the cocirculation of subtypes A and B in Brazilian commercial farms.

Epidemiological survey and molecular characterization of avian infectious bronchitis virus in Brazil between 2003 and 2009

As part of an epidemiological study of infectious bronchitis virus (IBV) in Brazil, 252 samples from IBV-suspect flocks were tested and the IBV-positive samples were analysed by sequencing of hypervariable regions 1 and 2 of the S1 gene. A high prevalence of IBV variants was found and the sequence analysis of 41 samples revealed a high molecular similarity among the Brazilian isolates (from 90.2 to 100% and from 85.3 to 100% nucleotide and amino acid identity, respectively). The Brazilian isolates showed low genetic relationship with Massachusetts (63.4 to 70.7%), European (45.9 to 75.6%), American (49.3 to 76.4%) and other reference serotypes (67.5 to 78.8%). The Brazilian isolates branched into one unique cluster, separate from the reference serotypes used for infectious bronchitis control in other countries. The variants analysed in this work had a high similarity with all previously published Brazilian IBV isolates, suggesting the presence and high prevalence of a unique or predominant genotype circulating in Brazil. In addition, the virus neutralization test showed that the three Brazilian isolates analysed in the present study are antigenically related to one another but are different from the Massachusetts serotype. The present study shows that IBVs of a unique genotype can be associated with different clinical diseases, and that low genetic variation was detected in this genotype over a long period of time. The molecular characterization of the Brazilian variants isolated from 2003 to 2009 from different geographic regions of the country shows that only one predominant genotype is widespread in the Brazilian territory, denominated in this study as BR-I genotype.

Detection and Molecular Characterization of Gene 3 and 5 of Turkey Coronavirus from Turkeys with Severe Enteritis in Brazil

Turkey coronavirus (TCoV) is a causative agent associated with poult enteritis and mortality syndrome (PEMS) in turkeys worldwide. The disease is an acute, highly contagious enteric disease that is characterized by depression, anorexia, diarrhea, and high mortality in commercial turkey flocks. The presence of TCoV in 12 intestinal-content samples, from turkey flocks aged between 10 and 104 days and exhibiting severe enteritis, was monitored during the period of 2004 to 2006. TCoV detection was accomplished by a reverse transcriptase-polymerase chain reaction (RT-PCR) through amplification of the 3` UTR region, followed by amplification of genes 3 and 5. Molecular characterization of the viruses was done through amplification of genes 3 and 5 and showed evidence of genetic similarity between them, although they differed from sequences of other TCoVs described in the literature. In relation to gene 3, samples showed a greater relationship with chicken infectious bronchitis virus (IBV), while gene 5 showed greater identity with pheasant coronavirus (PhCoV). Our results suggest that the strategy of amplification of the 3` UTR region, followed by sequencing of genes 3 and 5, has proven to be an effective means of detecting TCoV in intestinal contents.

GP5+/6+ SYBR Green methodology for simultaneous screening and quantification of human papillomavirus

Background: Detection and quantification of human papillomavirus (HPV) may help in predicting the evolution of HPV infection and progression of associated lesions. Objectives: We propose a novel protocol using consensus primers GP5+/6+ in a SYBR Green quantitative real-time (Q-RT) polymerase chain reaction (PCR). The strategy permits screening for HPV infection and viral load quantification simultaneously. Study design: DNA from 153 archived cervical samples, previously tested for HPV detection by GP5+/6+ PCR and typed by EIA-RLB (enzyme immunoassay-reverse line blot) or sequence analysis, was analysed using SYBR Green Q-RT PCR. Melting temperature assay (T(m)) and cycle threshold (C(t)) were used to evaluate HPV positivity and viral load. The T(m) in the range of 77-82 degrees C was considered to be positive for HPV-DNA. HPV results generated through GP5+/6+ conventional PCR were considered the gold standard against which sensitivity and specificity of our assay were measured. Results: Out of 104 HPV positive samples, 100 (96.2%) were also determined as positive by SYBR Green Q-RT PCR; of the 49 HPV-negative samples, all were determined as negative. There was an excellent positivity agreement (K = 0.94) between the SYBR Green Q-RT and the previous methods employed. The specificity and sensitivity were 100% and 96.2%, respectively. Comparison of SYBR Green Q-RT and TaqMan oligo-probe technologies gave an excellent concordance (pc = 0.95) which validated the proposed strategy. Conclusions: We propose a sensitive and easy-to-perform technique for HPV screening and viral load quantification simultaneously. (C) 2009 Elsevier B.V. All rights reserved.

Mutation rates in the dihydrofolate reductase gene of Plasmodium falciparum

A new method has been established to define the limits on a spontaneous mutation rate for a gene in Plasmodium falciparum. The method combines mathematical modelling and large-scale in vitro culturing and calculates the difference in mutant frequencies at 2 separate time-points. We measured the mutation rate at 2 positions in the dihydrofolate reductase (DHFR) gene of 3D7, a pyrimethamine-sensitive line of P. fulciparum. This line was re-cloned and an effectively large population was treated with a selective pyrimethamine concentration of 40 nM. We detected point mutations at codon-46 (TTA to TCA) and codon-108 (ACC to AAC), resulting in serine replacing leucine and asparagine replacing serine respectively in the corresponding gene product. The substitutions caused a decrease in pyrimethamine sensitivity. By mathematical modelling we determined that the mutation rate at a given position in DHFR was low and occurred at less than 2(.)5 x 10(-9) mutations/DHFR gene/replication. This result has important implications for Plasmodium genetic diversity and antimalarial drug therapy by demonstrating that even with lon mutation rates anti-malarial resistance will inevitably arise when mutant alleles are selected under drug pressure.

Development of disabled, replication-defective gene transfer vectors from the Jembrana disease virus, a new infectious agent of cattle

Jembrana disease virus (JDV) is a newly isolated and characterised bovine lentivirus. It causes an acute disease in Ball cattle (Bos javanicus). which can be readily transmitted to susceptible cattle with 17% mortality. There is as yet no treatment or preventive vaccine. We have developed a gene transfer vector system based on JDV that has three components. The first of the components is a bicistronic transfer vector plasmid that was constructed to contain cis-sequences from the JDV genome, including 5 '- and 3 ' -long terminal repeats (LTRs), 0.4 kb of truncated gag and 1.1 kb of 3 ' -env, a multiple cloning site to accommodate the gene(s) of interest for transfer, and an internal ribosome entry site plus the neomycin phosphotransferase (Neo) gene cassette for antibiotic selection. The second element is a packaging plasmid that contains trans-sequences. including gag, pol. vif, tar and rev: but without the env and packaging signals. The third is a plasmid encoding the G glycoprotein of vesicular stomatitis virus (VSV-G) to supply the vector an envelope for pseudotyping. Cotransfection of 293T cells with these three plasmid components produced VSV-G pseudotyped. disabled, replication defective, bicistronic JDV vectors encoding the green fluorescent protein (EGFP) and the Neo resistance selection maker simultaneously with a titre range of (0.4-1.2) x 10(6) CFU/ml. Transduction of several replicating primary and transformed cells from cattle, primate and human sources and importantly growth-arrested cells with the JDV vectors showed high efficiency of EGFP gene transfer at 35-75%, which was stable and the expression of EGFP was long term. Furthermore, these JDV vectors were designed to suit the inclusion and expression of genes corresponding to JDV specific proteins, such as gag or env, for the development of vaccines for Jembrana disease. This strategy should also be applicable to other bovine diseases as wall. The design and construction of the JDV vector system should facilitate the study of the lentivirology and pathogenesis of the diseases associated with JDV or other bovine virus infections. To our knowledge, this is the first such vector system developed from a cattle virus. (C) 2001 Elsevier Science B.V. All rights reserved.