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PubMed2004

[Antigenic and genetic characterizations of group A influenza viruses H3N2 circulated in men in China during 2000-2002].

OBJECTIVE: To understand the antigenic and genetic characteristics of influenza A H3N2 viruses circulated in man in China from 2000 to 2002. METHODS: Embryonated chicken eggs inoculated with virus for amplification of viral yield. The harvesting egg allantoic fluids with influenza viruses were provided for testing antigen and RNA extraction. Virion RNA was transcribed into cDNA by reverse transcriptase, cDNA amplified by PCR, and the product of PCR was purified. Afterward RNA sequence analysis was performed by the dideoxynucleotide chain termination method using synthetic oligodeoxynucleotide primers. Finally the phylogenetic tree was analyzed with MegAlign software. RESULTS: The H3N2 viruses isolated during 2000-2002 were different in amino acid sequences on HA1 domain protein molecule from those of A/Wuhan359/1995 H3N2 as well as those of A/Sydney/7/1997 H3N2 strains. There were four different positions of amino acid sequence on HA1 domain protein molecule among the H3N2 viruses isolated in 2000 and during 2001-2002. They located at 83, 186, 202, 222 and 225 position, respectively. Of them 83 and 186 were in antigenic determinant E and B, respectively. The others located at left wall of the receptor binding site (RBS). CONCLUSION: From the end of 2001 to the beginning of 2002, the influenza epidemic in Northern China caused by H3N2 virus was due to occurrence of antigenic and genetic changes of influenza A(H3N2) virus.

Amino Acid Sequence↗
PubMed2002

Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States.

Hepatitis E virus (HEV) is an important public health concern in many developing countries. HEV is also endemic in some industrialized counties, including the United States. With our recent discovery of swine HEV in pigs that is genetically closely related to human HEV, hepatitis E is now considered a zoonotic disease. Human strains of HEV are genetically heterogenic. So far in the United States, only one strain of swine HEV has been identified and characterized from a pig. To determine the extent of genetic variations and the nature of swine HEV infections in U.S. pigs, we developed a universal reverse transcription-PCR (RT-PCR) assay that is capable of detecting genetically divergent strains of HEV. By using this universal RT-PCR assay, we tested fecal and serum samples of pigs of 2 to 4 months of age from 37 different U.S. swine farms for the presence of swine HEV RNA. Thirty-four of the 96 pigs (35%) and 20 of the 37 swine herds (54%) tested were positive for swine HEV RNA. The sequences of a 348-bp region within the ORF2 gene of 27 swine HEV isolates from different geographic regions were determined. Sequence analyses revealed that the 27 U.S. swine HEV isolates shared 88 to 100% nucleotide sequence identities with each other and 89 to 98% identities with the prototype U.S. strain of swine HEV. These U.S. swine HEV isolates are only distantly related to the Taiwanese strains of swine HEV, with about 74 to 78% nucleotide sequence identities; to most known human strains of HEV worldwide, with <79% sequence identities; and to avian HEV, with 54 to 56% sequence identities. Phylogenetic analysis showed that all the U.S. swine HEV isolates identified in this study clustered in the same genotype with the prototype U.S. swine HEV and the two U.S. strains of human HEV. The data from this study indicated that swine HEV is widespread and enzoonotic in U.S. swine herds and that, as is with human HEV, swine HEV isolates from different geographic regions of the world are also genetically heterogenic. These data further raise potential concerns for zoonosis, xenozoonosis, and food safety.

Animals↗
PubMedDate not supplied

Genetic characterization of poliovirus isolates in Canada, 1962-1981.

From 1962 to 1981, 629 poliovirus strains of human and sewage origin were assayed for genetic markers in the kinetic serum neutralization ( KSN ) and temperature marker (rct) tests. The KSN test proved to be most dependable: 97% of strains tested could be classified as either vaccine-like (VL) or non-vaccine-like ( NVL ). All but two of the 18 virus strains whose classification was inconclusive in the KSN test were further characterized by micro serum neutralization assay. The correlation between the results of the rct and KSN marker tests was not satisfactory for the characterization of poliovirus types 1 and 3. Numerous type 1 virus strains were analyzed with strain-specific monoclonal antibodies for antigenic markers and were examined in polyacrylamide gel electrophoreses for viral proteins. Whereas greater than 90% of poliovirus types 2 and 3 were found to be VL in the KSN test, 40% of type 1 isolates were classified as NVL . The latter were detected not only in the epidemics of 1962 and 1978-1979 caused by type 1 poliovirus but also were present in endemic form in the late 1960s and 1970s in various provinces. In contrast, no NVL type 2 strains were detected in any specimen, and the last type 3 NVL strains were demonstrated in 1963-1964. Thus only type 1 poliovirus appears to be endemic in Canada.

Adolescent↗
PubMed1989

Genetic characterization of FLA, the cat major histocompatibility complex.

The major histocompatibility complex (MHC) of the domestic cat (termed FLA) has been refractile to genetic and serological definition largely because of repeated failure to detect cytotoxic antibodies in multiparous cats or to elicit antibody following allogeneic lymphocyte immunization. We have developed a protocol for producing cytotoxic alloantisera in the cat following rejection of multiple surgical skin grafts. Of 59 cats subjected to grafting, 13 produced lymphocytotoxic antisera which had varying specificities among a panel of outbred cat cells. A population cluster analysis of the 13 alloantisera permitted the identification of six clusters of overlapping FLA specificities. Serological analysis of cells from 12 cat kindreds led to the definition of 24 allogeneic haplotypes, which segregate as a single Mendelian complex. Feline FLA antisera were characterized as class I or class II specific by immunoprecipitation of FLA gene products on lymphocyte cell surfaces. Abundant antigenic polymorphisms for both class I and class II MHC determinants were discovered, a result consistent with precedence in other species and the common expectation of the adaptive value of MHC variation. Development of feline MHC typing reagents and the definition of haplotypes for the cat hold promise for experimental analysis of valuable feline models for virus-induced immune deficiencies.

Animals↗
PubMed1995

Genetic characterization of geographic populations using morphometrical traits in Drosophila melanogaster: isogroups versus isofemale lines.

Studies of short or medium range geographic variations play an increasing role in ecological genetics, and sensitive techniques are required to detect them. In this respect, two sampling techniques were compared in D. melanogaster. The biological data were provided by the analysis of four natural populations from the same geographic area, Spain (one) and Southern France (three), for four morphometrical traits: abdomen and thoracic pigmentation, and wing and thorax lengths. Traits were measured on wild living females and on their progeny reared in the laboratory at 25 degrees C. For progeny analyses, two techniques were compared: the usual isofemale line technique, sib families issued from a single female, and a new isogroup technique, the progeny produced by a group of 20 wild-collected parents. Large phenotypic variations were observed in wild living flies, corresponding to the unstability of natural environmental conditions during their development. Among laboratory grown flies, variations were much smaller. Between isogroups, differences were small, due to sampling error and some common environment effects. Variations between lines were much greater, thus demonstrating a strong genetic component. When different populations have to be compared, the isogroup technique should be preferred since, for the same amount of work, the lesser variability between groups provides a more precise characterization of the population means.

Animals↗
PubMed1991

Phenotypic and genetic characterization of a unique B lymphocyte deficiency in strain A/WySnJ mice.

The elucidation of B lymphocyte development has been partially achieved through genetic models such as the X-linked immunodeficiency (xid) mutation. We discovered a unique B lymphocyte developmental defect in strain A/WySnJ mice. We used single- and two-color flow cytometry to analyze lymphocytes from A/J and A/WySnJ mice. Adult A/WySnJ mice had a severe B cell deficiency, which was apparent in the spleen, lymph nodes, peritoneum and peripheral blood, compared to adult A/J mice. An ontogeny study revealed that a developmental defect, inhibiting B lymphocyte maturation or differentiation but not B lymphopoiesis, was responsible for the deficiency. This maturational defect blocked the production of B220hi/Iahi/surface IgMlo B cells, and was manifested in the adult bone marrow and neonatal spleen, but not the adult spleen. Neither Ly-1 B cells nor peripheral T cells were apparently affected by the A/WySnJ defect. The B cell immunodeficiency segregated as an autosomal co-dominant trait in F1 and F2 mice. We propose that A/WySnJ mice have a novel genetic defect arresting B cell differentiation in adult bone marrow and neonatal spleen.

Animals↗
PubMed2006

Genetic characterization of a diverse Escherichia coli O157:H7 population from a variety of farm environments.

Many of the current studies on the genetic diversity of Escherichia coli O157:H7 have focused on pathogenic clinical, veterinary, or food isolates. These studies did not explore the diversity of the larger population in the farm environment. Research on selected farm isolates address this wider diversity but have typically been limited to a specific geographic locale or farm type, thus giving limited insight into the greater diversity across geographic regions and varied environments. The objective of this study was to evaluate a diverse population of E. coli O157:H7 collected from a variety of locations and farm environments. Eighty-eight isolates were collected from four farm types (swine, dairy, beef, and poultry) across the southeastern and western United States. Eighteen farms were sampled every 3 months over a period of 24 months. Isolates were analyzed by ribotyping and pulsed field gel electrophoresis (PFGE). Real-time PCR was used to determine the presence or absence of key pathogenic genes (stx1, stx2, and eae). The data indicate a significant amount of genetic diversity, however, ribotype analysis revealed meaningful clusters within the larger population. These groupings were consistent with PFGE analysis. Most of these isolates were clustered by location (i.e. from the same state or region) or farm type. Of the isolates in these clusters, most did not contain pathogenic genes. Of notable interest is a single group in which the majority of isolates, collected from four of the five states sampled, contained at least one stx gene and the eae gene suggesting the existence of a specific pathogenic cluster. These data suggest that, while there is notable diversity within the broader E. coli O157:H7 population, pathogenic isolates may be limited to a subset of strains within the population.

Animal Husbandry↗
PubMed1985

Genetic characterization and partial sequence determination of a Treponema pallidum operon expressing two immunogenic membrane proteins in Escherichia coli.

A detailed physical and genetic map of a previously cloned 5.5-kilobase segment of Treponema pallidum DNA is described. This segment expressed two proteins that are cell membrane associated in Escherichia coli. The structural genes of these treponemal membrane proteins, tmpA and tmpB, are coordinately expressed, and transcription in E. coli can start from at least two different treponemal promoters. The tmpA and tmpB proteins are the products of in vivo proteolytic cleavage from precursor proteins which are 2 and 4 kilodaltons larger, respectively, than the mature proteins. Because the sizes of the corresponding proteins produced in T. pallidum were identical to those of the mature membrane proteins in E. coli, we concluded that a similar proteolytic processing takes place in both E. coli and T. pallidum. Although tmpA and tmpB were controlled by the same transcription signals, tmpB was expressed to a higher extent than tmpA, and only the tmpB product could be overproduced by placing the left lambda promoter in front of the structural genes. The nucleotide sequence of the T. pallidum tmpA gene was established. This is the first T. pallidum gene sequenced. Codon usage and the nature of transcriptional and translational signals are discussed. The deduced amino acid sequence indicated the presence of a sequence that was characteristic for a signal peptide. This sequence information allowed the construction of hybrid genes coding for proteins having beta-galactosidase enzyme activity as well as TmpA epitopes. The enzyme-linked antigen was expressed at a high level in E. coli when transcriptional and translational signals from coliphage lambda were used. In this case the protein produced was a sandwich protein consisting of 21 amino acids of the lambda cro protein, 204 amino acids of the T. pallidum TmpA protein, and 1,020 amino acids of the E. coli lambda-galactosidase. The potential use of this enzyme-linked antigen for the serodiagnosis of syphilis is discussed.

Amino Acid Sequence↗
PubMed2000

Phenotypic and genetic characterization of Lactococcus garvieae isolated in Spain from lactococcosis outbreaks and comparison with isolates of other countries and sources.

The phenotypic and genetic analysis results for 84 isolates of Lactococcus garvieae (including 62 strains from trout with lactococcosis from four different countries, 7 strains from cows and water buffalos with subclinical mastitis, 3 from water, and 10 from human clinical samples) are presented. There was great phenotypic heterogeneity (13 different biotypes) based on the acidification of saccharose, tagatose, mannitol, and cyclodextrin and the presence of the enzymes pyroglutamic acid arylamidase and N-acetyl-beta-glucosaminidase. L. garvieae also exhibited high genetic diversity by pulsed-field gel electrophoresis (PFGE), with 19 different pulsotypes among the isolates of L. garvieae studied. Only epidemiologically related strains, like the Spanish and Italian fish isolates and the cow and water buffalo isolates, displayed a close genetic relationship by PFGE, while the strains isolated from sporadic clinical cases, like the human isolates, were genetically unrelated. Overall, a general correlation between phenotypic and genetic data was observed. Epidemiological analysis of biotype and PFGE results indicated that the trout lactococcosis outbreaks in Spain and Portugal and those in France and Italy were produced by genetically unrelated clones. In Spain, two different clones were detected; the outbreaks diagnosed from 1995 onward were produced by a clone (biotype 2, pulsotype A1) which, although genetically related, was different from the one that was responsible for the outbreaks studied between 1991 and 1994 (biotype 1, pulsotype B). The Portuguese isolate had a biochemical profile identical to that of the Spanish strain isolated from 1995 onward and is also genetically closely related to this strain (pulsotype A2). There was a close relationship between the two pulsotypes (E and F) found in the Italian isolates. The French isolate (biotype 3, pulsotype D) was not genetically related to any other L. garvieae fish isolate. These results suggest the existence of diverse infection sources for the different lactococcosis outbreaks.

Animals↗
PubMed1977

Physical and genetic characterization of deletion mutants of simian virus 40 constructed in vitro.

Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting CV-1P cells with linear SV40 DNA and DNA of an appropriate helper virus. The linear DNA was obtained by complete cleavage of closed circular DNA with Hae II or Bam HI endonuclease or partial cleavage with either Hae III endonuclease or nuclease S1, followed, in some cases, by mild digestion with phage lambda 5' -exonuclease. The following mutants with deletions in the late region of the SV40 genome were obtained and characterized. Ten, containing deletions at the Hae II endonuclease site (map location 0.83), define a new genetic complementation group, E, grow extremely slowly without helper virus, and cause alterations only in VP2. Two mutants with deletions in the region 0.92 to 0.945 affect both VP2 and VP3, demonstrating that VP3 shares sequences with the C-terminal portion of VP2. The mutant with a deletion at 0.93 is the first deletion mutant in the D complementation group and is also temperature sensitive; the mutant with a deletion at 0.94 is viable and grows normally. Three mutants with deletions at the EcoRI endonuclease site (0/1.0) and eleven with deletions at the BamHI endonuclease site (0.15) fall into the B/C complementation group. Six additional mutants with deletions at the BamHI endonuclease site are viable, growing more slowly than wild type. VP1 is the only polypeptide affected by mutants in the B/C group. A mutant with a deletion of the region 0.72 to 0.80 has a polar effect, failing to express the E, D, and B/C genes. Mutants with deletions in the early region (0.67 counterclockwise to 0.17) at 0.66 to 0.59, 0.48, 0.47, 0.33, and 0.285 to 0.205 are all members of the A complementation group. Thus, the A gene is the only viral gene in the early region whose expression is necessary for productive infection of permissive cells. Since mutants with deletions in the region 0.59 to 0.54 are viable, two separate regions are essential for expression of the gene A function: 0.66 to 0.59 and 0.54 to 0.21. Mutants with deletions at 0.21 and 0.18 are viable. Approximate map locations of SV40 genes and possible models for their regulation are discussed.

Cell Line↗
PubMed1997

Correlation of patient immune responses with genetically characterized small round-structured viruses involved in outbreaks of nonbacterial acute gastroenteritis in the United States, 1990 to 1995.

Small round-structured viruses (SRSVs) are a genetically and antigenically diverse group of caliciviruses that are the most common cause of outbreaks of acute nonbacterial gastroenteritis. We have applied both molecular techniques to characterize SRSVs in fecal specimens and serologic assays using four different expressed SRSV antigens to examine the distribution of outbreak strains in the United States and determine if the immune responses of patients were strain specific. Strains from 23 outbreaks of SRSV gastroenteritis were characterized by reverse transcription-PCR and nucleotide sequencing of a 277-base region of the capsid gene. These strains segregated into two distinct genogroups, I and II, comprising four and six clusters of strains respectively, each representing a distinct phylogenetic lineage. Serum IgG responses in patients were measured by enzyme immunoassay using expressed capsid antigens of Norwalk virus (NV), Toronto virus (TV), Hawaii virus (HV), and Lordsdale virus (LV), representing four of the 10 clusters. While strains in genogroups I and II were antigenically distinct, within genogroups, the specificity of the immune response varied greatly. Patients infected with genogroup I strains which had as much as 38.5% aa divergence from NV demonstrated relatively homologous seroresponses to the single NV antigen. In contrast, in genogroup II, homologous seroresponses to TV and HV were only present when the infecting strains showed less than 6.5% aa divergence from these antigens. These results suggest that TV and HV represent not only separate genetic clusters in genogroup II but also separate antigenic groups, each of which is related but distinguishable. In addition, two genetically distinct SRSV strains were identified for which we have no homologous antigen. This study suggests that while current molecular diagnostics are capable of detecting the full range of SRSVs, additional expressed antigens will be required to detect an immune response to SRSV infection caused by all the antigenically diverse strains.

Acute Disease↗
PubMed2002

Social system of the Alaotran gentle lemur (Hapalemur griseus alaotrensis): genetic characterization of group composition and mating system.

Group composition and mating system were investigated in wild Alaotran gentle lemurs (Hapalemur griseus alaotrensis) using genetic markers. These small-bodied herbivorous and cathemeral primates are endemic to the reed and papyrus beds around Lac Alaotra in Madagascar. They live in small groups in small, defended territories. Data were collected during the rainy seasons in 1996, 1997, and 1999, and include 99 individuals from 22 neighboring social groups and an additional 30 animals from other areas representing most of the geographic range. Animals were located by researchers canoeing in the marshes bordering the lake. After a group's size was determined by direct observation, all individuals were captured, marked, and released. During handling each individual was weighed and sexed, and hair samples for DNA extraction were collected. A 342 bp mtDNA control region sequence and 10 nuclear microsatellite loci provided multilocus genotypes that were used to assess pedigree relations and population structure. Alaotran gentle lemurs were found in groups of two to nine individuals (mean: 4.3), comprising one or two breeding females, their offspring, and one reproducing male. Solitary individuals of both sexes were encountered rarely. Breeding females were the permanent core of the social groups, whereas intergroup transfer of reproductive males was relatively frequent. Forty percent of all reproducing groups contained two breeding females, which were related to each other as closely as mother-daughter or full sisters. Parentage assessment revealed a variable mating system ranging from serial monogamy to polygyny within social groups. At least 8% of paternities involved extragroup males. Additional data on life history and reproduction are presented, and the social system of the Alaotran gentle lemur is discussed in the light of the new genetic findings.

Animals↗
PubMed2000

PCR-RFLP and sequence analysis of a non-ribosomal fragment for genetic characterization of European stone fruit yellows phytoplasmas infecting various Prunus species.

A 927 bp non-ribosomal fragment was used to assess the genetic variability of the European stone fruit yellows (ESFY) phytoplasma infecting 14 different Prunus species. For this, 175 isolates originating from four different Mediterranean countries were tested by PCR-RFLP analysis with seven restriction enzymes. No polymorphism among the ESFY phytoplasma could be observed but 12 out of 18 restriction sites differed between the homologous fragments of ESFY and apple proliferation (AP) phytoplasmas. An 846 bp fragment of a French ESFY isolate was sequenced, it included the 3'-end of a putative nitroreductase gene, an intergenic region and a truncated open reading frame. This ESFY phytoplasma sequence showed 89.7% identity with the equivalent AP phytoplasma nucleotide sequence (83. 9% identity at the amino acid level). The G+C content of the entire sequence was extremely low (15.4%) and A+T-rich codons were highly preferred in codon usage. In this paper, we report the presence of the ESFY phytoplasma for the first time in Turkey and in five Prunus hosts never reported previously. Our results also indicate that the ESFY phytoplasma isolates affecting various Prunus species are genetically homogenous but can be distinguished from the AP phytoplasma. Therefore, they are likely to represent different taxons.

Amino Acid Sequence↗
PubMed2000

Genetic characterization of the 44D-45B region of the Drosophila melanogaster genome based on an F2 lethal screen.

We have performed an F2 genetic screen to identify lethal mutations that map to the 44D-45B region of the Drosophila melanogaster genome. By screening 8500 mutagenized chromosomes for lethality over Df(2R)Np3, a deficiency which encompasses nearly 1% of the D. melanogaster euchromatic genome, we recovered 125 lines with lethal mutations that represent 38 complementation groups. The lethal mutations have been mapped to deficiencies that span the 44D-45B region, producing an approximate map position for each complementation group. Lethal mutations were analyzed to determine the phase of development at which lethality occurred. In addition, we have linked some of the complementation groups to P element-induced lethals that map to 44D-45B, thus possibly providing new alleles of a previously tagged gene. Some of the complementation groups represent potentially novel alleles of previously identified genes that map to the region. Several genes have been mapped by molecular means to the 44D-45B region, but do not have any reported mutant alleles. This screen may have uncovered mutant alleles of these genes. The results of complementation tests with previously identified genes in 44D-45B suggests that over half of the complementation groups identified in this screen may be novel.

Alleles↗
PubMed1992

Immunologic and genetic characterization of S180, a cell line of murine origin capable of growing in different inbred strains of mice.

Some of the immunologic and genetic properties of the cell line S180 have been examined. These cells grew without restrictions in the peritoneal cavity of different inbred strains of mice and invariably killed the animals. With Northern blots it was demonstrated that S180 cells contained class I mRNAs but failed to transcribe B2m genes. However, under experimental conditions, a protective humoral immune response mediated by cytotoxic antibodies and complement against S180 cells was obtained through non-H-2 antigens in C57BL/6J mice.

Animals↗
PubMed2024

Genetic characterization and selection of litter size traits of Guizhou Black goat and Meigu goat.

The aim of this study is to explore the genetic characteristics of Guizhou Black goats and Meigu goats and their relationship to reproductive performance through population structure analysis, genetic diversity assessment, and selection signal analysis. Blood samples of 19 Guizhou Black goats and 11 Meigu goats were collected for whole-genome high-throughput sequencing. Using PCA and ADMIXTURE analyses, their population structure and genetic relationships were revealed. Further genetic diversity analysis showed that although there is significant population differentiation, the levels of genetic diversity are similar. Subsequently, these goats were categorized into high-yield and low-yield groups based on their litter sizes, with 15 goats in each group. Then, a selection signal analysis was performed using FST and &#x3c0; ratios for 33,563 SNP loci. The results identified six candidate genes, including KCNIP4, GFRA2, and DGKH, which are significantly associated with high litter performance. These findings enhanced our understanding of the genetic characteristics and population structure of Guizhou Black goats and Meigu goats. Moreover, they provide an important theoretical foundation and scientific basis for further breeding improvements.

Animals↗