Nt Deletion Research Articles

Gone over to the dark side

Gone over to the dark side

Genetic Analysis of West Nile Virus Isolates from an Outbreak in Idaho, United States, 2006–2007

West Nile virus (WNV) appeared in the U.S. in 1999 and has since become endemic, with yearly summer epidemics causing tens of thousands of cases of serious disease over the past 14 years. Analysis of WNV strains isolated during the 2006–2007 epidemic seasons demonstrates that a new genetic variant had emerged coincidentally with an intense outbreak in Idaho during 2006. The isolates belonging to the new variant carry a 13 nt deletion, termed ID-Δ13, located at the variable region of the 3′UTR, and are genetically related. The analysis of deletions and insertions in the 3′UTR of two major lineages of WNV revealed the presence of conserved repeats and two indel motifs in the variable region of the 3′UTR. One human and two bird isolates from the Idaho 2006–2007 outbreaks were sequenced using Illumina technology and within-host variability was analyzed. Continued monitoring of new genetic variants is important for public health as WNV continues to evolve.

DNA polymerase β mutations and survival of patients with esophageal squamous cell carcinoma in Linzhou City, China

Linzhou City in northern China has a high incidence of esophageal squamous cell carcinoma (ESCC). This study retrospectively analyzed the data of 231 cases with ESCC collected from 1998 to 2012. Mutations of DNA polymerase β (polβ) gene in the ESCC samples from patients in Linzhou City were examined by amplifying polβ cDNA by RT-PCR followed by cloning and sequencing. Mutations in polβ were found in 105 cases (45.9%). Nine types of mutations were identified in the polβ cDNA; the most common were 177–234 nt deletion (11.3%), 462 nt G → T (9.1%), and 648 nt G → C (6.9%). Mutations in polβ appeared to be associated with TNM status (P = 0.048). Follow-up data was used for survival analysis. The overall 5-year survival rate of the 231 patients was 37.4%; the rate for patients with wild-type (WT) polβ was 41.8%. Compared with the WT polβ group, the median survival for patients with specific mutations (177–234 nt deletion, 462 nt G → T, or 613 nt A → T) was significantly shorter (all P = 0.000), and the 5-year survival rate decreased to 0%. Patients with the 648 nt G → C mutation had improved survival (P = 0.000) with a 5-year survival rate of 100%. Our results identified nine types of mutations within polβ cDNA in ESCC patients with four mutations related to patient survival.

120 : Loss of IFN-gamma 3′untranslated region AU-rich element affects B220+ cell populations in novel murine lupus model

Interferon-gamma (IFN-g) is a key player in immunoregulation, inflammation and autoimmunity. We created a mouse with a 162 nt deletion of an AU-rich element (ARE) region in the 3’UTR of the IFN-g gene. The ARE-deleted (ARE-Del) mice have chronic circulating serum IFN-g levels and develop a lupus-like disease characterized by nuclear antigen-specific autoantibodies and glomerulonephritis. B cells and plasmacytoid dendritic cells (pDCs) play a prominent role is systemic lupus erythematosus (SLE) and we find alterations in both B220+ cell types in these mice. The percentage of CD11c+, pDCA1+, B220+, Siglec H+ pDCs increases in bone marrow and spleens from ARE-Del mice. Furthermore, addition of IFN-g to flt-3 driven in vitro bone marrow cultures results in a dramatic increase in the pDC population with increases in IRF8, but not E2-2, mRNA expression that coincides with this expansion. In contrast, the total percentage of B220+, CD19+ B cells is reduced in spleens from ARE-Del mice. There is little impact on the CD23+ follicular B cell subset; however, CD21+ marginal zone (MZ) B cells are greatly reduced or absent. Crossing the ARE-Del mice with TLR7 or IFN-alpha receptor null mice restore MZ B cells to levels seen in WT mice indicating that interplay between type I and type II interferons plays a role in splenic B cell development. Current studies are underway to understand the molecular basis for how these interferons influence B cell maturation.

A Deletion in the Glycoprotein L (gL) Gene of U.S. Marek's Disease Virus (MDV) Field Strains Is Insufficient to Confer Increased Pathogenicity to the Bacterial Artificial Chromosome (BAC)–Based Strain, RB-1B

Marek's disease (MD) is a highly transmissible, herpesvirus-associated malignancy of chickens and turkeys caused by Marek's disease virus (MDV). MD is currently controlled through the use of nonsterilizing vaccines composed of antigenically related, apathogenic herpesviruses Mardivirus 2 (MDV-2), Meleagrid herpesvirus 1 (herpesvirus of turkeys, HVT), or attenuated MDV-1 strain CVI988 (Rispens). Since the mid-1960s, field strains of MDV have increased in virulence, due, in part, to the widespread use of vaccines since the early 1970s. One mutation that we have identified common to very virulent field strains (vv and vv+MDVs) since the 1990s has been a mutation in the UL1 gene, encoding glycoprotein L (gL). This mutation, a 12-nucleotide (nt) deletion in the signal peptide of gL, has been associated with increased virulence and decreased vaccine protection in the context of challenge with a vv+MDV, strain TK. To determine whether this mutation alone was sufficient to confer increased virulence, we introduced this mutation into the transmission-competent pRB-1B bacterial artificial chromosome (BAC) using two-step, Red-mediated recombination. The resulting mutant, pRB-1BgLdelta, was tested for changes in replication in cell culture using multistep growth curves, plaque size analysis, viral burst analysis, and the ability to compete with the parental virus when co-transfected at different ratios and sequentially passaged. In addition, we examined this mutant for changes in pathogenicity in inoculated and contact-exposed unvaccinated and vaccinated chickens. Our data show minor differences in plaque sizes in cell culture, but no discernible changes in the infection of specific-pathogen-free (SPF) leghorn chickens. We therefore conclude that although this mutation is indeed common to MDV field strains isolated in the eastern United States, it is insufficient to confer increased virulence or loss of vaccine protection previously observed for a vv+MDV strain having this mutation.

Novel Mutations Associated with Resistance to Bacillus sphaericus in a Polymorphic Region of the Culex quinquefasciatus cqm1 Gene

Bin toxin from Bacillus sphaericus acts on Culex quinquefasciatus larvae by binding to Cqm1 midgut-bound receptors, and disruption of the cqm1 gene is the major cause of resistance. The goal of this work was to screen for a laboratory-selected resistance cqm1(REC) allele in field populations in the city of Recife, Brazil, and to describe other resistance-associated polymorphisms in the cqm1 gene. The cqm1(REC) allele was detected in the four nontreated populations surveyed at frequencies from 0.001 to 0.017, and sequence analysis from these samples revealed a novel resistant allele (cqm1(REC-D16)) displaying a 16-nucletotide (nt) deletion which is distinct from the 19-nt deletion associated with cqm1(REC). Yet a third resistant allele (cqm1(REC-D25)), displaying a 25-nt deletion, was identified in samples from a treated area exposed to B. sphaericus. A comparison of the three deletion events revealed that all are located within the same 208-nt region amplified during the screening procedure. They also introduce equivalent frameshifts in the sequence and generate the same premature stop codon, leading to putative transcripts encoding truncated proteins which are unable to locate to the midgut epithelium. The populations analyzed in this study contained a variety of alleles with mutations disrupting the function of the corresponding Bin toxin receptor. Their locations reveal a hot spot that can be exploited to assess the resistance risk through DNA screening.

Comparing PreXMRV-2 gag sequence diversity in laboratory and wild mice using deep sequencing

It has recently been reported that the xenotropic murine leukemia virus-related virus (XMRV) derives from a laboratory recombinant. However, sequences with characteristics of the 5′ half of XMRV (termed PreXMRV-2) have been identified in several laboratory mouse genomes and cell lines suggesting parts of the XMRV genome exist as naturally occurring retroviruses in mice. We compare here PreXMRV-2 gag sequence diversity in mice to that of reported XMRV-like sequences by testing a panel of wild mouse and common inbred laboratory mouse strain genomic DNAs and by using high throughput amplicon sequencing. Sequences with features typical of previously reported PreXMRV-2 sequences, among them a 24 nt deletion, were repeatedly identified in different wild mice and inbred mouse strains within a high background of non-XMRV-like sequences. However, Sanger sequencing of clones from amplicons failed to retrieve such sequences effectively. Phylogenetic analysis suggests that PreXMRV-2 gag sequences from mice, cell lines and patient samples belong to the same evolutionarily young clade and that such sequences are diverse and widespread within Mus musculus domesticus and laboratory mice derived from this species. No evidence of PreXMRV-2 like gag sequences could be obtained outside of the M. musculus lineage. The results suggest that accurate determination of presence, absence and relationships of specific murine retroviral strains benefit greatly from deep sequencing analysis.

Complete Genome Sequence of an Overattenuated Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

JXA1-P170 is an overattenuated highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) that has been passaged in vitro 170 times. Vaccination with JXA1-P170 cannot protect pigs against JXA1 challenge. Compared with the parental virus JXA1, JXA1-P170 contains 1 nucleotide (nt) deletion and 113 nt mutations leading to 59 amino acid substitutions. Here we announce the first complete genome sequence of the overattenuated HP-PRRSV.

New insights into the genetics of EBLV-1 from Germany.

Previous epidemiological studies on EBLVs indicated a distinct geographical distribution of EBLV-1 in Germany. In this study, 48 isolates were selected to further investigate the spatial and temporal distribution of EBLV-1 variants in Germany. The nucleoprotein-gene (N), the nucleoprotein-phosphoprotein spanning untranslated region (NP-UTR) and the UTR between G- and L-gene of each isolate were sequenced using direct cycle sequencing. Results of the subsequent phylogenetic analysis of the N-gene confirmed previous studies on EBLVs, showing a high sequence identity among German EBLV-1a isolates, and a correlation between genetic and temporal and spatial distance, respectively, was shown. Our results indicate that the GL-UTR is not suitable for phylogenetic analyses. Interestingly, 6 nt insertions in two isolates as well as a single nucleotide insertion in a different isolate were detected in the N-P UTR. Within the UTR between G- and L-gene one isolate showed a 35 nt deletion. The effect of those changes on viral properties remains elusive as such mutations have not been described for lyssaviruses before.

Loss of IFN-gamma 3’untranslated region AU-rich element affects B220+ B cell and plasmacytoid dendritic cell populations in novel murine lupus model (161.3)

Abstract Interferon-gamma (IFN-g) is a key player in immunoregulation, inflammation and autoimmunity. We created a mouse with a162 nt deletion of an AU-rich element (ARE) region in the 3’UTR of the IFN-g gene. The ARE-deleted (ARE-Del) mice have chronic circulating serum IFN-g levels and develop a lupus-like disease characterized by DNA and nuclear antigen-specific autoantibodies and glomerulonephritis. B220+ B cells and plasmacytoid dendritic cells (pDCs) play a prominent role in systemic lupus erythematosus (SLE). We find alterations in both cell types. Greater B cell percentages are found in lymph nodes and thymus with a concomitant reduction in spleen. The B cells have an activated phenotype with increased CD69 and/or CD86 expression. The ARE-Del thymic B cell population is partially composed of B1a and B1b B cells and is correlated with increased thymic mRNA expression of the B cell chemoattractant, CXCL13. Splenic B cells down-regulate CD23 and CD21 with no change in surface markers CD19, B220, IgM, and IgD. The percentage of CD11c+, pDCA1+, B220+ Siglec H+ pDCs is increased in bone marrow and spleens from AU-deleted mice. Current studies are underway to further characterize the pDC population and to investigate expression of bone marrow-specific factors important for pDC development. Overall, these data demonstrate that low chronic IFN-g levels are sufficient to alter the phenotype and potentially the development of cells important for SLE disease onset and progression.

Revertant Somatic Mosaicism by Mitotic Recombination in Dyskeratosis Congenita

Revertant mosaicism is an infrequently observed phenomenon caused by spontaneous correction of a pathogenic allele. We have observed such reversions caused by mitotic recombination of mutant TERC (telomerase RNA component) alleles in six patients from four families affected by dyskeratosis congenita (DC). DC is a multisystem disorder characterized by mucocutaneous abnormalities, dystrophic nails, bone-marrow failure, lung fibrosis, liver cirrhosis, and cancer. We identified a 4 nt deletion in TERC in a family with an autosomal-dominant form of DC. In two affected brothers without bone-marrow failure, sequence analysis revealed pronounced overrepresentation of the wild-type allele in blood cells, whereas no such skewing was observed in the other tissues tested. These observations suggest that this mosaic pattern might have resulted from somatic reversion of the mutated allele to the normal allele in blood-forming cells. SNP-microarray analysis on blood DNA from the two brothers indeed showed independent events of acquired segmental isodisomy of chromosome 3q, including TERC, indicating that the reversions must have resulted from mitotic recombination events. Subsequently, after developing a highly sensitive method of detecting mosaic homozygosity, we have found four additional cases with a mosaic-reversion pattern in blood cells; these four cases are part of a cohort of 17 individuals with germline TERC mutations. This shows that revertant mosaicism is a recurrent event in DC. This finding has important implications for improving diagnostic testing and understanding the variable phenotype of DC.

Phylogenetic and molecular characterization of coxsackievirus A24 variant isolates from a 2010 acute hemorrhagic conjunctivitis outbreak in Guangdong, China.

BackgroundAcute hemorrhagic conjunctivitis is a common disease in China. As a notifiable disease, cases are registered by ophthalmologists on the AHC surveillance system. An AHC outbreak caused by CA24v was observed in Guangdong Province in 2007 by the National Disease Supervision Information Management System. Three years later, a larger outbreak occurred in Guangdong during the August-October period (2010). To characterize the outbreak and compare the genetic diversity of CA24v, which was determined to be the cause of the outbreak, the epidemiology and the molecular characterization of CA24v were analyzed in this study.ResultsA total of 69,635 cases were reported in the outbreak. 73.5% of index cases originated from students, children in kindergarten and factory workers, with the ≦ 9 age group at the highest risk. The male to female ratio was 1.84:1 among 0-19 years. 56 conjunctival swabs were collected to identify the causative agent from five cities with the AHC outbreak. 30 virus strains were isolated, and two of the genomes had the highest identity values (95.8%) with CA24v genomes. Four CA24v genotypes were identified by phylogenetic analysis for the VP1 and 3C regions. CA24v which caused the outbreak belonged to genotype IV. Furthermore, full nucleotide sequences for four representative isolates in 2010 and 2007 were determined and compared. 20 aa mutations, two nt insertions and one nt deletion were observed in the open reading frame, with 5'- and 3'- UTR respectively between them.ConclusionsCA24v was determined to be the pathogen causing the outbreak and belongs to genotype IV. VP1 is more informative than 3CPro for describing molecular epidemiology and we hypothesize that accumulative mutations may have promoted the outbreak.

A unique nucleotide sequence variant in the coat protein region of the genome of aMaize dwarf mosaic virusisolate

During our recent four years epidemiological studies ofMaize dwarf mosaic virus (MDMV) populations in Hungary, when 86 virus isolates were collected and analysed, a unique nucleic acid sequence variant was found in the 3’end region of the viral genome. According to our sequence studies, which also included all available MDMV sequences from different databanks, the coat protein region of the viral genome prooved to be quite identical. However, in several cases an insertion was located in the same position of the coat protein region. In the unique sequence variant we also found a 27 nt deletion in addition to the insertion. According to the extensive sequence search this deletion is unique and have been located only in two other potyvirus coat protein regions, while never in the case of MDMV. The potential role of this deletion in the virus infectivity, replication or other biological characteristics is discussed.

Chronic IFN-gamma expression alters the migration pattern of peripheral T cells (160.7)

Abstract We report here on preliminary data from an IFN-gamma(IFN-γ)ARE-deleted Balb/c mouse model developed in our laboratory.This mouse has a 162 nt deletion in the 3’untranslated region of the IFN-γ mRNA to eliminate an AU-rich sequence. The deletion has a positive impact on IFN-γ mRNA accumulation by dramatically increasing its half-life. In the absence of stimulatory factors,these mice express low level of IFN-γ constitutively. IFN-γ ARE-deletion results in a severe phenotype on Balb/c mice. The knockout (KO) and heterozygous (Het) mice are smaller in size than the littermate controls, have a rough coat and a low survival rate after weaning. Additionally, these mice have an enlarged thymus with an increased percentage of mature naïve T cells. The mature thymocytes from Het animals are able to leave the thymus and enter peripheral lymphoid tissues. However, the results from chimera experiments indicate that IFN-γ may impact the thymic environment, thus resulting in abnormal T cell development. In addition, we found the migration pattern of peripheral T cells from Het mice differs from that observed with cells from control animals, further suggesting an abnormality in the Het T cell physiology. Current experiments focus on analyzing changes in the thymic environment and T cell development in newborn mice. In addition, we are analyzing chemokine receptor expression to determine if changes in these receptors may be a mitigating factor in the T cell migration patterns.

Identification of a New α Chain Variant at Codons 22–25 (–9 nts) Using the Sebia Capillarys 2 Electrophoresis System

We have identified a new α chain hemoglobin (Hb) variant in a Chinese family. Sequencing of the amplified α2-globin gene revealed a 9 nucleotide (nt) deletion (–C GAG TAT GG) at codons 22–25, which results in a predicted α-globin chain that is missing amino acid residues 23–25 (Glu-Tyr-Gly) and the formation of Hb Zhanjiang.

Hb St. Truiden [α68(E17)Asn→His] and Hb Westeinde [α125(H8)Leu→Gln]: Two New Abnormalities of the α2-Globin Gene

We report two new abnormal hemoglobins (Hbs) caused by mutations on the α2 gene. One resulted into an Asn→His substitution at position 68, the other in a Leu→Gln substitution at position 125. The first mutation was observed in a 61-year-old North European Belgian male during Hb A1c analysis and subsequently in other members of his family. The variant was expressed at a normal level and caused no hematological abnormalities in the carriers. The second was found in a 27-year-old Turkish male living in The Hague, The Netherlands, who presented with microcytic hypochromic parameters without iron deficiency and was also carrier of the common α2 IVS-I (–5 nt) deletion.

Analysis of the gene 3 region sequences of Chinese field strains of Transmissible gastroenteritis virus

The genome of Transmissible gastroenteritis virus (TGEV) displays genetic diversity especially in gene 3 region. Sequence and comparative analysis of 3a and 3b genes of eight Chinese field strains with reference TGEV strains indicated that these strains shared 87.0-100% and 51.5%-100% identities at the nucleotide level, respectively, and 86.1%-100% and 66.2%-100% identities at the amino acid level, respectively. Moreover, in one of the strains (CH/SDQ/08), a 51 nt deletion in the gene 3 region was found. Phylogenetic analysis showed that the eight Chinese strains were more closely related to TGEV strains H165, H16, Miller M6, Miller M60, TS, and CHV than to other reference strains. In addition, this study indicated the presence of different TGEV strains within the same pig herds in China.

Intraspecies diversity of SARS-like coronaviruses in Rhinolophus sinicus and its implications for the origin of SARS coronaviruses in humans

The Chinese rufous horseshoe bat (Rhinolophus sinicus) has been suggested to carry the direct ancestor of severe acute respiratory syndrome (SARS) coronavirus (SCoV), and the diversity of SARS-like CoVs (SLCoV) within this Rhinolophus species is therefore worth investigating. Here, we demonstrate the remarkable diversity of SLCoVs in R. sinicus and identify a strain with the same pattern of phylogenetic incongruence (i.e. an indication of recombination) as reported previously in another SLCoV strain. Moreover, this strain possesses a distinctive 579 nt deletion in the nsp3 region that was also found in a human SCoV from the late-phase epidemic. Phylogenetic analysis of the Orf1 region suggested that the human SCoVs are phylogenetically closer to SLCoVs in R. sinicus than to SLCoVs in other Rhinolophus species. These findings reveal a closer evolutionary linkage between SCoV in humans and SLCoVs in R. sinicus, defining the scope of surveillance to search for the direct ancestor of human SCoVs.

Cloning of Chicken Microsomal Glutathione S-transferase 1 Gene (MGST1) and Identification of Its Different Splice Variants

Mammal microsomal glutathione transferase 1 (MOST1) can conjugate many toxic or carcinogenic substances and depress oxidative stress. In this study, Chicken MOST1 and its variants were cloned for the first time and were composed of 956 or 944 nucleotides. The 12 nt deletion in the exon 2 did not alter the GT-AG rule and the ORFs for the two MGST1 variants were the same, which both comprised 465 nucletides and encoded a peptide with 155 amino acids. It was found that the two different splice variants identified using RT-PCR expressed in all three organs investigated of Dwarf Brown Chicken, namely liver, spleen and shell gland. Moreover, the expression level of MGST1 mRNA in the liver of Dwarf Brown chickens was the highest (p<0.01), and there were no significant differences between the spleen and the shell gland. These results provide a base for studying the biological function of Chicken MOST1.

Characterization of H9N2 influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation in eastern China during a 5 year period (1998–2002)

In the current study, we characterized H9N2 influenza viruses isolated from vaccinated flocks in an integrated broiler chicken operation during a 5 year period (1998-2002). Phylogenetic analysis of the 8 genes of 11 representative viruses showed that they all shared high similarity to that of the first isolate, A/Chicken/Shanghai/F/1998 (Ck/SH/F/98), and clustered to the same lineages. Furthermore, all 11 viruses had a 9 nt deletion between positions 206 and 214 of the neuraminidase gene. These genetic characteristics strongly suggest that these viruses are descendants of the first isolate. In addition, our study also showed that the H9N2 viruses circulating in the operation during this 5 year period were evolving, as shown by antigenic variations between viruses manifested by reactivity with polyclonal antisera and monoclonal antibodies, by haemagglutination with erythrocytes from different animals, by amino acid differences in haemagglutinin and neuraminidase proteins, and by variation in their ability to replicate in the respiratory and intestinal tract and to be transmitted by aerosol. Phylogenetic analysis revealed that the internal genes from some H5N1 viruses of duck origin clustered together with those from H9N2 virus and that the RNP genes of these H5N1 viruses isolated after 2001 are more closely related to the genes of the Ck/SH/F/98-like H9N2 viruses, indicating more recent reassortment events between these two subtypes of viruses. Continuous surveillance of influenza virus in poultry and waterfowl is critical for monitoring the genesis and emergence of potentially pandemic strains in this region.