Denaturing gradient gel electrophoresis (DGGE) as a powerful novel alternative for differentiation of epizootic ISA virus variants

Infectious Salmon Anemia is a devastating disease critically affecting world-wide salmon production. Chile has been particularly stricken by this disease which in all cases has been directly related with its causative agent, a novel orthomyxovirus which presents specific and distinctive infective features. Among these, two molecular markers have been directly associated with pathogenicity in two of the eight RNA sub genomic coding units of the virus: an insertion hot spot region present in viral segment 5 and a Highly Polymorphic Region (HPR) located in viral segment 6. Here we report the successful adaptation of a PCR-dependent denaturing gel electrophoresis technique (DGGE), which enables differentiation of selected reported HPR epizootic variants detected in Chile. At the same time, the technique allows us to distinguish one nucleotide differences in sequences associated with the intriguing, and still not well-understood, insertion events which tend to occur on RNA Segment 5. Thus, the versatility of the technique opens new opportunities for improved understanding of the complex biology of all ISA variants as well as possible applications to other highly variable pathogens.     

Localised Infection of Atlantic Salmon Epithelial Cells by HPR0 Infectious Salmon Anaemia Virus

Infectious salmon anaemia (ISA) is an important, systemic viral disease of farmed Atlantic salmon, Salmo salar L. Endothelial cells are the main target cells for highly virulent HPR-deleted ISA virus (ISAV) types. Here we examine the pathogenesis of non-virulent ISAV HPR0 infections, presenting evidence of an epithelial tropism for this virus type, including actual infection and replication in the epithelial cells. Whereas all HPR0 RT-qPCR positive gills prepared for cryosection tested positive by immunohistochemistry (IHC) and immunofluorescent labelling, only 21% of HPR0 RT-qPCR positive formalin-fixed paraffin-embedded gills were IHC positive, suggesting different methodological sensitivities. Only specific epithelial cell staining was observed and no staining was observed in endothelial cells of positive gills. Furthermore, using an ISAV segment 7 RT-PCR assay, we demonstrated splicing of HPR0, suggesting initial activation of the replication machinery in the epithelial gill cells. Immunological responses were investigated by the expression of interferon-related genes (e.g. Mx and γIP) and by ELISA for presence of anti-ISAV antibodies on samples taken sequentially over several months during an episode of transient HPR0 infection. All fish revealed a variable, but increased expression of the immunological markers in comparison to normal healthy fish. Taken together, we conclude that HPR0 causes a localized epithelial infection of Atlantic salmon.     

Ultra-Deep Pyrosequencing of Partial Surface Protein Genes from Infectious Salmon Anaemia Virus (ISAV) Suggest Novel Mechanisms Involved in Transition to Virulence

Uncultivable HPR0 strains of infectious salmon anaemia viruses (ISAVs) infecting gills are non-virulent putative precursors of virulent ISAVs (vISAVs) causing systemic disease in farmed Atlantic salmon (Salmo salar). The transition to virulence involves two molecular events, a deletion in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) gene and a Q₂₆₆→L₂₆₆ substitution or insertion next to the putative cleavage site (R₂₆₇) in the fusion protein (F). We have performed ultra-deep pyrosequencing (UDPS) of these gene regions from healthy fish positive for HPR0 virus carrying full-length HPR sampled in a screening program, and a vISAV strain from an ISA outbreak at the same farming site three weeks later, and compared the mutant spectra. As the UDPS data shows the presence of both HE genotypes at both sampling times, and the outbreak strain was unlikely to be directly related to the HPR0 strain, this is the first report of a double infection with HPR0s and vISAVs. For F amplicon reads, mutation frequencies generating L₂₆₆ codons in screening samples and Q₂₆₆ codons in outbreak samples were not higher than at any random site. We suggest quasispecies heterogeneity as well as RNA structural properties are linked to transition to virulence. More specifically, a mechanism where selected single point mutations in the full-length HPR alter the RNA structure facilitating single- or sequential deletions in this region is proposed. The data provides stronger support for the deletion hypothesis, as opposed to recombination, as the responsible mechanism for generating the sequence deletions in HE.     

Infectious salmon anaemia virus—molecular biology and pathogenesis of the infection

Aquaculture has a long history in many parts of the world, but it is still young at an industrial scale. Marine fish farming in open nets of a single fish species at high densities compared to their wild compatriots opens a plethora of possible infections. Infectious salmon anaemia (ISA) is an example of disease that surfaced after large-scale farming of Atlantic salmon (Salmo salar) appeared. Here, a review of the molecular biology of the ISA virus (ISAV) with emphasis on its pathogenicity is presented. The avirulent HPR0 variant of ISAV has resisted propagation in cell cultures, which has restricted the ability to perform in vivo experiments with this variant. The transition from avirulent HPR0 to virulent HPRΔ has not been methodically studied under controlled experimental conditions, and the triggers of the transition from avirulent to virulent forms have not been mapped. Genetic segment reassortment, recombination and mutations are important mechanisms in ISAV evolution, and for the development of virulence. In the 25 years since the ISAV was identified, large amounts of sequence data have been collected for epidemiologic and transmission studies, however, the lack of good experimental models for HPR0 make the risk evaluation of the presence of this avirulent, ubiquitous variant uncertain. This review summarizes the current knowledge related to molecular biology and pathogenicity of this important aquatic orthomyxovirus.     

Spontaneous phase variation in Bordetella pertussis is a multistep non-random process.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non-pathogenic upon culturing in vitro. Spontaneous variants of the Tohama and #165 pathogenic strains of B. pertussis were selected by their ability to grow on synthetic and semi-synthetic solid media. The frequency of these variants was between 10(-6) and 10(-7). About 250 variant strains were screened for the presence of virulence-associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7-11% of the variants displayed all traits, 17% of the variants carried the toxin and FHA, 5-11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin quantitatively affected the other traits. These results suggest that phase variation in B. pertussis is a non-random process, involving multistep disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. In contrast, all 300 variants of strain #18323 of B. pertussis, which were able to grow on the selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral growth capability.     

Primary structure of the two variants of a sperm-specific histone H1 from the annelid Platynereis dumerilii

The amino acid sequences of the two variants (H1a 121 residues and H1b 119 residues) of the sperm-specific histone H1 from the polychaete annelid Platynereis dumerilii have been completely established. Comparison of the sequences of these two variants shows one deletion of two residues in histone H1b and 22 substitents, of which most occur in the globular domain. The two variants differ highly in a sequence of nine residues adjacent to the conservative phenylalanine residue of histone H1 (64-72 in H1a, 62-70 in H1b) which makes H1a less hydrophobic than H1b. The small molecular size of Platynereis H1a and H1b is a unique feature among the histones H1 of which the size ranges between 189 residues (chicken erythrocyte H5) and 248 residues (sea urchin sperm H1). H1a and H1b have short N- and C-terminal basic domains but the size of the globular domain (approximately equal to 80 residues) is similar to that of other H1s. In the globular region the variant H1a exhibits a close relationship with somatic or sperm H1s whereas the variant H1b is more related to H5 histones.     

Extensive regions of homology in front of the two hsp70 heat shock variant genes in Drosophila melanogaster

The major heat shock protein of 70,000 M_r in Drosophila melanogaster is encoded by two variant gene types located, respectively, at the chromosomal sites 87A7 and 87C1. We present the DNA sequence of a complete hsp70² gene of the 87A7 type and of the adjacent regions from both variants, extending to 1·2 × 10³ bases upstream from the start of the messenger coding region. We find an untranslated region of 250 nucleotides at the 5′ end of the messenger coding sequence in both variants. There is only one open reading frame which allows coding of a 70,000 M_r protein within the 87A7 variant, as found for an 87C1 variant (Ingolia et al., 1980). We observe 4·2% nucleotide divergence between these two variants with complete conservation of the reading frame. There is a conserved sequence of 355 nucleotides in front of each hsp70 gene, which is 85% homologous between the two variants. The presence of the same sequence element in γ, in front of the αβ heat shock genes (R. W. Hackett & J. T. Lis, personal communication) suggests that this element contains the regulatory signals for the coordinate expression of both the hsp70 and the αβ heat shock genes. Finally we find a very A + T-rich sequence of 150 basepairs which is highly conserved (91·8%) 0·6 × 10³ bases upstream from two hps70 gene variants.     

Structure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant

Several variants of the serotonin 5-HT4 receptor are known to be produced by alternative splicing. To survey the existence and usage of exons in humans, we cloned the human 5-HT4 gene. Based on sequence analysis seven C-terminal variants (a-g) and one internal splice variant (h) were found. We concentrated in this study on the functional characterization of the novel splice variant h, which leads to the insertion of 14 amino acids into the second extracellular loop of the receptor. The h variant was cloned as a splice combination with the C-terminal b variant; therefore, we call this receptor 5-HT4(hb). This novel receptor variant was expressed transiently in COS-7 cells, and its pharmacological profile was compared with those of the previously cloned 5-HT4(a) and 5-HT4(b) isoforms, with the latter being the primary reference for the h variant. In competition binding experiments using reference 5-HT4 ligands, no significant differences were detected. However, the broadly used 5-HT4 antagonist GR113808 discriminated functionally among the receptor variants investigated. As expected, it was an antagonist on the 5-HT4(a) and 5-HT4(b) variant but showed partial agonistic activity on the 5-HT4(hb) variant. These data emphasize the importance of variations introduced by splicing for receptor pharmacology and may help in the understanding of conflicting results seen with 5-HT4 ligands in different model systems.     

Generation of seal influenza virus variants pathogenic for chickens, because of hemagglutinin cleavage site changes.

Influenza virus A/seal/Mass/1/80 (H7N7) was adapted to grow in MDCK cells and chicken embryo cells (CEC) in the absence of exogenous protease. The biological properties of the virus variants obtained coincided with intracellular activation of the hemagglutinin (HA) by posttranslational proteolytic cleavage and depended on the cell type used for adaptation. MDCK cell-adapted variants contained point mutations in regions of the HA more distant from the cleavage site. It is proposed that these mutations are probably responsible, through an unknown mechanism, for enhanced cleavability of HA in MDCK cells. Such virus variants were apathogenic in chickens. CEC-adapted variants, on the other hand, contained an insertion of basic amino acids at the HA cleavage site, in addition to scattered point mutations. The insertions converted the cleavage sites in the variant virus HAs so that they came to resemble the cleavage site found in highly pathogenic avian influenza viruses. CEC variants with such cleavage site modifications were highly pathogenic for chickens. The lethal outcome of the infection in chickens demonstrated for the first time that an influenza virus derived from a mammalian species can be modified during adaptation to a new cell type to such an extent that the resulting virus variant becomes pathogenic for an avian species.     

Identification and characterization of the compound heterozygous variants of TYR gene in a northern Chinese family with Oculocutaneous albinism type 1

Oculocutaneous albinism (OCA) is a genetically heterogeneous disease and is most inherited in an autosomal recessive manner. The characteristic manifestation of OCA is due to disfunction of melanin synthesis. OCA1 is the most severe subtype of OCA and is caused by homozygous or compound heterozygous variants in tyrosinase (TYR) gene, which is the key gene for melanin synthesis. This study aimed to identify the genetic variants of a northern Chinese family with OCA1. Clinical information and peripheral blood samples were collected. PCR amplification and Sanger sequencing were used to detect the entire exons and adjacent flanking sequences of TYR gene. Functional prediction of variants was performed by various bioinformatic analyses, while the pathogenicity classification of variants was evaluated according to ACMG standards and guidelines. A missense variant NM_000372.5:c.107G > C;NP_000363.1:p.C36S was discovered in TYR gene which converted cysteine to serine. Another variant in intron, NM_000372.5:c.1037–7 T > A, also affected the function of TYR gene. We verified the pathogenicity of the intron variant with a pCAS2 mini-gene based splicing assay and found that c.1037–7 T > A led to an insertion of 5 bp upstream from the common acceptor site of exon 3, which caused a frameshift TYR:c.1037–7 T > A:p.G346Efs*11. The results showed that the compound heterozygous variants c.107G > C:p.C36S and c.1037–7 T > A:p.G346Efs*11 of TYR gene were the pathogenic variants for this OCA1 family.     

Pathogenicity of live bacteria and extracellular products of motile Aeromonas isolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD₅₀ 10^5·4-10^7·6 cfu fish^-1) but no Aer. sobria , Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 10^8·4 cfu fish^-1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD⁵⁰ 1·0–3·2 μg (g fish)^-1) reproducing the symptoms associated with natural disease. ECP from non-pathogenic species were inactive on fish cell lines as well as being poorly lethal for eels (LD⁵⁰ > 9·2 μg (g fish)^-1). All these biological activities of Aeromonas ECP were lost after heat treatment. These findings indicate differences between pathogenic and non-pathogenic Aeromonas species with respect to the expression of virulence factors, and show that elastases, haemolysins and exotoxins play a leading role in the pathogenicity of motile Aeromonas for eels.     

The human lambda L chain Ig locus. Recharacterization of JC lambda 6 and identification of a functional JC lambda 7

The human lambda L chain Ig gene complex consists of multiple JC gene segments. A seventh human lambda C region gene segment, C lambda 7, was found 2.7 kb downstream of C lambda 6 in this gene complex. A J lambda gene segment, J lambda 7, was found 1.2 kb upstream of C lambda 7 and contains potentially functional nonamer and heptamer recombination sites, an RNA splice site and J coding region. C lambda 7 maintains an open reading frame and encodes a new lambda isotype. C lambda 7 encodes Kern+ and Oz- determinants, but does not encode any of the Kern+Oz- myeloma proteins published to date. Nevertheless, we present evidence that JC lambda 7 is transcribed in normal lymphocytes and is functional. In contrast, we present new data that the C lambda 6 gene segment, reported by others to encode the Kern+Oz- protein, is non-functional due to a 4-bp insertion in our cosmid clone. The 4-bp insertion was characterized further in 32 genomic DNA samples by producing a distinctive restriction fragment length and verified by the DNA sequences of the polymerase chain reaction products of two different cell lines. We discuss the possibility that the Kern+Oz- myeloma proteins do not define an isotype and are not encoded by JC lambda 7 nor other non-allelic genes, and we discuss the level of expression of JC lamba 7 as compared to that of JC lambda 2 and JC lambda 3.     

Identification of GB virus C variants by phylogenetic analysis of 5'-untranslated and coding region sequences.

Phylogenetic analysis of 44 GB virus C (GBV-C) 5'-untranslated region (5'-UTR) sequences from 37 individuals suggested the presence of GBV-C genotypes (A. S. Muerhoff, J. N. Simons, T. P. Leary, J. C. Erker, M. L. Chalmers, T. J. Pilot-Matias, G. J. Dawson, S. M. Desai, and I. K. Mushahwar, J. Hepatol. 25:379-384, 1996) that correlated with geographic origin: type 1, 2a and 2b, and 3 isolates are found predominantly in West Africa, the United States and Europe, and Japan, respectively. We have extended our analysis to include 5'-UTR sequences from 129 globally distributed GBV-C isolates and sequences from the second envelope protein (E2) gene and nonstructural (NS) regions 3 and 5b from a subset of these isolates. Bootstrap analysis of a 157-nucleotide segment of the 5'-UTR from 129 sequences provided weak support for the existence of the four major groups of GBV-C isolates previously described, although phylogenetic analysis of a 374-nucleotide segment of the 5'-UTR from 83 isolates provided stronger support. Thus, the groups of GBV-C variants previously identified upon analysis of the entire 5'-UTR can be distinguished by analysis of the shorter, 374-nucleotide region from the 5'-UTR. In contrast, independent analysis of the E2, NS3, or NS5b region sequences does not identify groups of GBV-C variants that correlate with geographic origin. However, bootstrap analysis of these coding sequences, when linked to form colinear sequences, demonstrates that longer coding regions can produce GBV-C groupings that are similar to that determined from 5'-UTR sequence analysis. The inability to distinguish between GBV-C variants by using small segments of coding sequence suggests that the GBV-C genome is constrained. As a result of these constraints, there is a high degree of nucleotide and amino acid sequence conservation between isolates from widely separated geographic areas. Hence, substitutions at many nucleotide positions are not tolerated, so that substitutions at the positions which can change are saturated, thereby obscuring the evolutionary relationships.     

Rapid and simple approach for identification of Mycobacterium tuberculosis and M. bovis by detection of regulatory gene whiB7

Identification of Mycobacterium tuberculosis and M. bovis is necessary for the application of adequate drug therapy. PCR amplification is a good tool for this purpose, but choosing proper target is of a great concern. We describe a PCR assay for fast detection of M. tuberculosis and M. bovis.As a BLAST and BLASTP search we selected regulatory gene whiB7 that encodes multi-drug resistance in this bacterium. Thirty clinical isolates of M. tuberculosis were sequenced and all the mutations in gene whiB7 were detected. The best set of several pairs of primers was selected and used in comparison by rpoB gene for differentiation of M. bovis, M. avium, M. kansasii, M. phlei, M. fortuitum, M. terrae, seven non-pathogenic Mycobacterium isolates and 30 clinical isolates of M. tuberculosis.It was proved that only clinical isolates of M. tuberculosis and M. bovis have positive bands of 667 bp whiB7. Other non-tuberculous and non-pathogenic isolates did not show any positive sign. Furthermore, 667-bp PCR products of whiB7 gene were observed for ten positive sputum samples (preliminarily approved to be positive for M. tuberculosis by commercially real-time based method), but no bands were detected in 5 negative sputum samples. RpoB gene could not differentiate non-tuberculous strains and non-pathogenic isolates from pathogenic clinical isolates. We concluded that PCR amplification of the gene coding for the WhiB7 protein could be successfully used as a good tool for rapid identification of M. tuberculosis and M. bovis. We propose application of this method as a rapid and simple approach in mycobacteriological laboratories.     

Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment.

This paper reports the establishment of a model for hantavirus host adaptation. Wild-type (wt) (bank vole-passaged) and Vero E6 cell-cultured variants of Puumala virus strain Kazan were analyzed for their virologic and genetic properties. The wt variant was well adapted for reproduction in bank voles but not in cell culture, while the Vero E6 strains replicated to much higher efficiency in cell culture but did not reproducibly infect bank voles. Comparison of the consensus sequences of the respective viral genomes revealed no differences in the coding region of the S gene. However, the noncoding regions of the S gene were found to be different at positions 26 and 1577. In one additional and independent adaptation experiment, all analyzed cDNA clones from the Vero E6-adapted variant were found to carry substitutions at position 1580 of the S segment, just 3 nucleotides downstream of the mutation observed in the first adaptation. No differences were found in the consensus sequences of the entire M segments from the wt and the Vero E6-adapted variants. The results indicated different impacts of the S and the M genomic segments for the adaptation process and selective advantages for the variants that carried altered noncoding sequences of the S segment. We conclude that the isolation in cell culture resulted in a phenotypically and genotypically altered hantavirus.     

中国汉族两个马凡综合征家系FBN1基因的一个新插入突变和一个复发点突变

目的:明确两个中国北方汉族马凡综合征(Marfan syndrome,MFS)家系的临床特点,并对其进行基因诊断。方法:对两个家系进行家系调查和系谱分析,应用聚合酶链式反应-DNA测序方法对原纤维蛋白1基因(Fibrillin-1,FBN1)的所有外显子进行测序。应用Swiss-model、Polyphen-2和SIFT软件对发现的变异位点进行功能预测。结果:两个家系均呈常染色显性遗传特点,在家系1患者中发现一个新的插入突变,即第13外显子1691位碱基处插入碱基A(1691 ins A),导致蛋白在第571位氨基酸处翻译提前终止。此外,在家系2患者中发现一个已知的点突变,即第27外显子第3463位碱基由G变为A(3463 GA),导致第1155位氨基酸由天冬氨酸变为天冬酰胺。这两个变异位点在家系的健康人及50例健康对照中均未出现。功能预测发现这两个变异位点均可能会影响FBN1蛋白的结构或功能。结论:在两个MFS家系中发现一个新插入突变位点(1691 ins A)和一个已知点突变位点(3463 GA),为扩大FBN1基因的突变谱及进一步阐明FBN1基因突变在MFS中的作用提供理论依据。