Endonuclease Cleavage Patterns Research Articles

Molecular discrimination of Hyalomma tick species serving as reservoirs and vectors for Crimean-Congo hemorrhagic fever virus in sub-Saharan Africa

The species identification of tick vectors of Crimean-Congo hemorrhagic fever virus (CCHFV), especially Hyalomma (H.) species, is a prerequisite to understand the eco-epidemiology of this disease and to reveal vector and virus reservoir species. However, the morphologic species discrimination can be difficult for damaged or blood-fed ticks and in case of species intercrosses. Therefore, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and restriction fragment length polymorphism (RFLP) analysis to distinguish the most common Hyalomma species from sub-Saharan Africa (H. truncatum, H. rufipes and H. dromedarii). Within the last years, MALDI-TOF MS analysis based on tick leg proteins has been shown to be a reliable method to distinguish several tick species. For this purpose, a reference spectral library of several European, American and African tick species was established. In this study, six different Hyalomma species were tested, all of which were all clearly distinguishable by mass spectrometric analyses. Moreover, MALDI TOF- MS was able to confirm morphologic findings where sequencing provided ambiguous results. In addition, a polymerase chain reaction (PCR) based on the CO1 gene amplification of ticks has been developed for the unequivocal species identification by amplicon sequencing and specific restriction endonuclease cleavage pattern analysis. RFLP proved to be a feasible auxiliary discrimination tool for selected Hyalomma species when access to sequencing methods is not available, as for instance during field studies.

Base‐Modified Nucleic Acids as a Powerful Tool for Synthetic Biology and Biotechnology

The ability of various nucleoside triphosphate analogues of deoxyguanosine and deoxycytidine with 7-deazadeoxyadenosine (A1 ) and 5-chlorodeoxyuridine (T1 ) to serve as substrates for Taq DNA polymerase was evaluated. The triphosphate set composed of A1 , T1 , and 7-deazadeoxyguanosine with either 5-methyldeoxycytidine or 5-fluorodeoxycytidine was successfully employed in the polymerase chain reaction (PCR) of 1.5 kb fragments as well as random oligonucleotide libraries. Another effective combination of triphosphates for the synthesis of a 1 kb PCR product was A1 , T1 , deoxyinosine, and 5-bromodeoxycytidine. In vivo experiments using an antibiotic-resistant gene containing the latter set demonstrated that the bacterial machinery accepts fully modified sequences as genetic templates. Moreover, the ability of the base-modified segments to selectively protect DNA from cleavage by restriction endonucleases was shown. This approach can be used to regulate the endonuclease cleavage pattern.

Molecular epidemiology of an outbreak of clinical mastitis in sheep caused by Mannheimia haemolytica

The aetiology and epidemiology of outbreaks of clinical mastitis in sheep under extensive pastoral conditions are incompletely understood. The objective of this study was to conduct a detailed investigation of a clinical mastitis outbreak that affected more than 10% of 230 at-risk ewes on a sheep and grain producing property in south east Australia during drought conditions in 2009. Milk samples were collected aseptically from all affected ewes and plated on sheep blood agar for bacterial identification. M. haemolytica was isolated from 80% of the samples that yielded cultivable microorganisms and thus was the main microorganism responsible for the outbreak. Analysis of the restriction endonuclease cleavage patterns of the isolates using pulsed field gel electrophoresis revealed some evidence of clonality, suggesting the possibility of horizontal transmission, but there was also considerable diversity between the clusters of closely related isolates. Multilocus sequence typing of the M. haemolytica isolates revealed most of the isolates belonged to ST1 with no association between the PFGE and MLST fingerprints of the isolates. Resistance to neomycin, streptomycin and sulphafurazole was detected in some of the isolates, but they were all susceptible to penicillin, ampicillin, ceftiofur, amoxycillin/clavulanic acid, ciprofloxacin, tetracycline, erythromycin and trimethoprim. This is the first published record of a comparison of the strains of M. haemolytica involved in a clinical mastitis outbreak in sheep and demonstrates the importance of this pathogen in sheep production systems, particularly during adverse climatic conditions and increased stocking rate.

The upper respiratory tract is a natural reservoir of haemolytic Mannheimia species associated with ovine mastitis

Lamb suckling has been suggested to be an important way of infecting a ewe's udder with different bacteria, including Mannheimia haemolytica. To test the potential role of lambs in transferring Mannheimia species to the ewe’s udder, the restriction endonuclease cleavage patterns of isolates obtained from nasopharyngeal swabs were compared with those obtained from cases of mastitis. Sterile cotton swabs were used to collect nasopharyngeal samples from 50 ewes and 36 lambs from three flocks. M. haemolytica and Mannheimia glucosida as well as haemolytic Mannheimia ruminalis-like organisms were detected in the upper respiratory tract of lambs and ewes. Comparison of the restriction endonuclease cleavage patterns of the isolates suggested that the M. haemolytica isolates obtained from different milk samples from ewes with mastitis were more clonal than those obtained from the nasal swabs. However, some nasal isolates within both Mannheimia species had restriction endonuclease cleavage patterns identical to those obtained from milk samples from ewes with mastitis, indicating that lambs may have a role in transferring these organisms to the udder. More clonality was observed between the M. glucosida isolates than between M. haemolytica isolates.

Human adenovirus type identification.

Dear editor The published paper in your journal entitling “Human adenovirus type 8 epidemic keratoconjunctivitis with large corneal epithelial full-layer detachment: an endemic outbreak with uncommon manifestations” has come into our attention.1 The article provides interesting clinical presentation of corneal epithelial layer detachment among 25% (4 out of 16 human adenovirus [HAdV]-positive cases) of patients diagnosed with epidemic keratoconjunctivitis (EKC). However, we have some opinions about the article that are stated below: First, we have noticed that the authors have used the combination of hexon gene sequence and BLASTN for the typing of HAdV. Due to the appearance of many recombinant types of HAdVs, especially the EKC causing ones, for the last few years, many isolates were wrongly diagnosed as HAdV-8 by serological method. For example, type HAdV-54 was diagnosed as HAdV-8 due to cross reaction in neutralization test.2 Even this was described as genome type HAdV-8I due to its misidentification as HAdV-8.3,4 Nowadays, HAdV-54 has become the major agent of EKC in Japan, although a few HAdV-8 is still being isolated in some places.5–9 Another EKC causing recombinant type HAdV-53 carries the fiber gene of HAdV-8, penton of HAdV-37, and the hexon of HAdV-22.9 Therefore, current recommended type identification method of HAdV is sequencing of the penton (P)-hexon (H)-fiber (F) genes (molecular typing) or whole genome sequence.10 Second, the authors used the term HAdV-8 genotype on the basis of HAdV-8 hexon sequence followed by BLASTN. Now a days, PCR amplification and sequencing of variable regions within the hexon, penton base, and fiber genes is designated as molecular typing method. The term genotype is used for the recognition of a new type by full genome sequence. A genotype number is assigned where sequence data reveal that either i) the virus isolate encodes novel hexon (loops 1 and 2), penton base (hypervariable region), and fiber (knob) sequences, or ii) is a recombinant with one or two of these regions derived from previously designated genotypes, or iii) is a recombinant that has a unique combination of these three regions derived from previously recognized genotypes.10 Third, in the discussion, the authors described HAdV-8C–H genome types as genotypes. Traditionally, genome types of HAdV are designated by the restriction endonuclease cleavage pattern analysis of viral DNA.11 We hope the authors will perform the currently recommended methods to confirm the identity of their EKC causing HAdVs.

ISOLATION AND CHARACTERIZATION OF A NOVEL HERPESVIRUS FROM A FREE-RANGING EASTERN GREY KANGAROO (MACROPUS GIGANTEUS)

We isolated a macropodid herpesvirus from a free-ranging eastern grey kangaroo (Macropus giganteous) displaying clinical signs of respiratory disease and possibly neurologic disease. Sequence analysis of the herpesvirus glycoprotein G (gG) and glycoprotein B (gB) genes revealed that the virus was an alphaherpesvirus most closely related to macropodid herpesvirus 2 (MaHV-2) with 82.7% gG and 94.6% gB amino acid sequence identity. Serologic analyses showed similar cross-neutralization patterns to those of MaHV-2. The two viruses had different growth characteristics in cell culture. Most notably, this virus formed significantly larger plaques and extensive syncytia when compared with MaHV-2. No syncytia were observed for MaHV-2. Restriction endonuclease analysis of whole viral genomes demonstrated distinct restriction endonuclease cleavage patterns for all three macropodid herpesviruses. These studies suggest that a distinct macropodid alphaherpesvirus may be capable of infecting and causing disease in eastern grey kangaroos.

Morphology and General Characteristics of Bacteriophages Infectious to Robinia pseudoacacia Mesorhizobia

Four phages infectious to Mesorhizobium strains were identified in soil samples taken from local Robinia pseudoacacia stands. Based on their polyhedral heads and short noncontractile tails, three of the phages, Mlo30, Mam12, and Mam20, were assigned to group C of Bradley's classification, the Podoviridae family, while phage Mlo1, with its elongated hexagonal head and a long flexible tail represented subgroup B2 bacteriophages, the Siphoviridae family. The phages were homogeneous in respect of their virulence, as they only lysed Mesorhizobium strains, but did not affect strains of Rhizobium or Bradyrhizobium. On the basis of one-step growth experiments, the average virus yield was calculated as approximately 10-25 phage particles for phages Mlo30, Mam12 and Mam20, and as many as 100-120 for phage Mlo1. The rate of phage adsorption to heat-treated cells showed differences in the nature of their receptors, which seemed to be thermal sensitive, thermal resistant, or a combination of the two. Only the receptor for phage Mlo30 was likely to be an LPS molecule, which was supported by a neutralization test. The smooth LPS with O-antigenic chains of the phage-sensitive M. loti strain completely reduced the bactericidal activity of virions at a concentration of 1 μg/ml. The molecular weights of phage DNAs estimated from restriction endonuclease cleavage patterns were in the range from approximately 39 kb for group C phages to approximately 80 kb for B2.

PCR-RFLP of mitochondrial DNA of swamp buffaloes in breeding station

Methods have been devised for detecting polymorphisms in the D-loop regions using the PCR followed by restriction enzyme digestion to reveal restriction fragment length polymorphism. Total DNA were extracted from the white blood cell of swamp buffaloes. Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) of local swamp buffalo from Lamphayaklang Livestock Research and Breeding Station in Thailand were analyzed using three enzymes (Ava lI, Bam H I and Hae III) which recognize 4 and 6 nucleotides. Among the 49 animals were analyzed, Ava II, BamH I and Hae III revealed different polymorphisms. Ava II showed two types of cleavage pattern. BamH I showed two type of cleavage pattern. And Hae III showed four types of cleavage pattern. The polymorphic fragments patterns for these three restriction endonuclease (Ava II- BamH I- Hae III) revealed five types of D-loop mtDNA in all the animals examined.These preliminary results indicated that polymorphism in the D-loop region of swamp buffalo mtDNA can be revealed by PCR and restriction enzyme analysis. And these enzymes would be able to show the genetic difference among the swamp buffaloes.These preliminary results indicated that polymorphism in the D-loop region of swamp buffalo mtDNA can be revealed by PCR and restriction enzyme analysis. And these enzymes would be able to show the genetic difference among the swamp buffaloes.

Comparison of virulence and restriction endonuclease cleavage patterns of infectious laryngotracheitis viruses isolated in Korea

Isolates of infectious laryngotracheitis virus (ILTV) obtained from field disease outbreaks in Korea from 1982 to 1998 were compared by virulence testing and by examining restriction endonuclease (REN) cleavage patterns of viral DNA. Based on pathogenicity tests, eight of 11 ILTV strains were classified as virulent, because these strains caused 40 to 80% mortality in specific pathogen free chickens, while three strains were classified as low virulence because these did not cause mortality. The REN cleavage patterns of the low virulence strains were identical with those of two reference vaccine strains, which were of chicken embryo origin. However, the DNA cleavage patterns of the virulent strains differed from those of both the low virulence and the vaccine strains. Furthermore, one virulent Korean strain N87278 had REN cleavage patterns that were clearly different from other virulent strains. In the present study, ILTV strains examined could be classified into two groups (virulent and low virulence strains) by pathogenicity testing, and three groups based on their REN cleavage patterns. These results suggest that most outbreaks of infectious laryngotracheitis were not likely to be associated with vaccine strains, but some were associated with viruses indistinguishable from commercial vaccine strains. At least three genetically distinct groupings of ILTV have been involved in outbreaks of infectious laryngotracheitis in Korea.

Characterization of a bovine herpesvirus type 4 isolated from the spinal cord of a cow with astasia.

A bovine herpesvirus (BHV) strain designated B11-41, was recently isolated from the spinal cord of a cow with astasia. By BHV type specific polymerase chain reaction (PCR), a BHV-4 specific fragment was amplified from the DNA of strain B11-41. The nucleotide sequence of the PCR product revealed high homologies (98.4% and 99.8%) with those of two BHV-4 strains (Movar 33/63 and 86-068). The BamHI restriction endonuclease cleavage patterns of the B11-41 were more similar to those of BHV-4 than those of other types of BHV. BHV-4 is divided into two groups, European and American. The EcoRI, and BamHI, and HindIII restriction endonuclease analysis demonstrated that strain B11-41 was closely related to the American type of BHV-4. It was concluded that B11-41 was identified as a BHV-4 that belongs to the American group. Additionally, the results of this study may indicate that the nervous system is one of the sites of viral latency in natural infection.

Molecular characterisation and differential diagnosis of a necrotic PVY isolate in tomato

SummaryA PVY isolate which causes strong necrotic lesions in tomato was isolated and characterised. It did not infect potato and did not react with antiserum specific to the N‐strain of PVY. Restriction endonuclease cleavage patterns of PCR products derived from the 5′‐end of the virus genome (Hinc II and Rsa I) clearly distinguished the tomato, pepper and potato strains. Additional sequence analysis indicated that the tomato and pepper isolates were closely related, while both markedly differed from the necrotic strains of potato. Hence, it was concluded that the necrotic tomato isolate is uniquely specific for tomato.

Partial characterization of a Brazilian strain of Aujeszky’s disease virus recovered from a pig with subclinical infection

One Brazilian strain of Aujeszky’s disease virus isolated from a piglet in which the disease had not been observed was studied as for its virulence in pigs. The genome of the virus was molecularly analysed as for their restriction endonuclease cleavage pattern. Fifty-day-old non-immune weanlings exposed to this strain showed no disease although the virus was present in their oropharyngeal area for at least three days. All animals developed moderate titers of neutralizing antibody. Based on number of bands and migration rate of restriction fragments the isolate was classified into Herrmann’s type I group. Latent infection was detected in all pigs by PCR. Some variations were detected in the cleavage pattern of the strain ASB Piau when compared to LA031 virulent Brazilian strain, that could be related to differences in the virulence.

The Mycoplasma hominis P120 membrane protein contains a 216 amino acid hypervariable domain that is recognized by the human humoral immune response.

In the antigenically heterogeneous species Mycoplasma hominis a monoclonal antibody, mAb 26.7D, was previously found to recognize a 120 kDa polypeptide from M. hominis 7488. This antibody did not react with the type strain PG21. The homologous gene from M. hominis PG21 was cloned and sequenced and found to have a sequence identity of 91% with the gene of strain 7488. One hypervariable and two semivariable regions were detected. The epitope for mAb 26.7D was mapped to the hypervariable domain by expression of various parts of this domain in Escherichia coli using expression vector systems. A polyclonal antiserum (pAb 121) generated against the hypervariable region of P120 from PG21 identified the P120 homologue in M. hominis PG21. Fusion proteins of the hypervariable and constant parts of the proteins were constructed and tested for reactivity with 21 human sera. Twelve sera reacted with the 7488 hypervariable fusion protein, but only four reacted with PG21 hypervariable fusion protein. No reactivity was seen with a fusion protein containing part of the constant region of P120. Gene fragments amplified from 18 M. hominis isolates by PCR confirmed the heterogeneity of the hypervariable domain. Based on restriction endonuclease cleavage patterns of the hypervariable domain the 18 isolates could be divided into four cases. Reactivity with both mAb 26.7D and pAb 121 confirmed these classes. The hypervariable, but not the constant, part of P120 was recognized by the human humoral immune response. Such a variable domain may be important in evasion of the host's immune response, and thus aid survival of the micro-organism.

Comparative Analysis of the Restriction Endonuclease Cleavage Patterns of Mitochondrial DNA in the Genus Gallus.

Comparative Analysis of the Restriction Endonuclease Cleavage Patterns of Mitochondrial DNA in the Genus Gallus.

Polymorphism of mitochondrial DNAs of Yunnan domestic water buffaloes, Bubalus bubalis, in China, based on restriction endonuclease cleavage patterns.

Restriction endonuclease cleavage patterns of mitochondrial DNA(mtDNA) of three local types of Yunnan native water buffalo were analyzed using 18 enzymes which recognize six nucleotides. Among the 12 animals analyzed, 3 of 18 enzymes, BamHI, EcoRI, and Scal, revealed polymorphisms. Three mtDNA types were identified. The results indicate that a relatively low level of mtDNA variation exists in Yunnan domestic water buffaloes. The origin of Chinese buffalo derived from Yunnan province of China is discussed.

Comparison of Mycoplasma agalactiae isolates by pulsed field gel electrophoresis, SDS-PAGE and immunoblotting

We have analyzed 81 isolates of Mycoplasma agalactiae from four different regions of Italy between 1990 and 1995 in order to identify antigenic differences through SDS-PAGE and Western blotting and chromosomal DNA restriction endonuclease cleavage pattern differences. Antigenic variability in M. agalactiae isolates was investigated analyzing hydrophobic membrane protein fractions by immunoblotting using pooled sheep antiserum from naturally infected sheep. Large restriction fragments obtained cleaving genomic DNAs with SmaI, NruI, SalI, XhoI, BssHII and KpnI were analyzed by pulsed field gel electrophoresis. Genetic analysis indicates that isolates are all similar without intraspecific differences. This homogeneity was confirmed by immunoblotting: 80 and 50 kDa antigens are present in all strains analyzed.

Nucleotide diversity of mitochondrial DNAs between the swamp and the river types of domestic water buffaloes,Bubalus bubalis, based on restriction endonuclease cleavage patterns

Cleavage patterns of mitochondrial DNAs (mtDNAs) by 15 restriction endonucleases were analyzed for 10 swamp and 13 river types of domestic water buffaloes. Digestions with nine enzymes exhibited polymorphisms giving two or three kinds of cleavage patterns. Five mtDNA types were identified, three types in the swamp buffaloes of the Philippines, Vietnam, and Indonesia (S-types) and two types in the river buffaloes of Bangladesh and Pakistan (R-types). Nucleotide diversities ranged from 0.2 to 0.6% within the S- and R-types and from 1.9 to 2.4% between the R-types and the S-types. These values indicated that R-type and S-type mtDNAs differentiated at the subspecific level of other mammalian species reported. The possibility of polyphyletic domestication in different places is discussed for the origin of two distinct types of domestic water buffaloes.

Characterization of Cytoplasmic and Nuclear Polyhedrosis Viruses Recovered from the Nest of Polistes hebraeus F. (Hymenoptera; Vespidae)

Viruses (CPVs and NPVs) present in nests of the paper wasp Polistes hebraeus were compared with six wild isolates obtained from some lepidopterous prey of the wasp. The electrophoresis of viral genomes was used to look for homology between isolates. The genomic size (126 kbp) and the restriction endonuclease cleavage pattern of NPV from nest were identical to those of viruses found in field populations of Catopsilia thauruma (CtMNPV) and Catopsilia florella. Two different CPV electrophoretypes were also characterized. One type was common to a CPV extracted from nests and to 4 CPVs naturally isolated from the noctuids Calixena versicolora, Microplexia costimuculalis, and Polydesma umbricola and from the pierid Catopsilia florella. This type is, moreover, identical to Bombyx mori CPV (CPV type 1), with a calculated size of 30.79 kbp. The other electrophoretype (263 CPV type) (30.53 kbp) characterizes a CPV from wasp nest multiplied in a substitution host (Eagris sabadius); the natural host of this CPV is still unknown. From these results it appears that through electrophoretic analysis of viral genomes, the nest of Vespidae can be used to study populations of insect viruses and to detect the presence of peculiar viruses in biotopes.

Outbreak of adenovirus type 4 conjunctivitis in South Australia.

An outbreak of adenovirus type 4 conjunctivitis occurred in South Australia between April and November 1992. Eye swabs were submitted by general practitioners and ophthalmologists who had seen patients with clinical conjunctivitis or keratitis. Apart from interfamilial spread, there were no other common epidemiological factors. Adenovirus was isolated from the eye swabs of 38 patients. Isolates were typed by neutralisation tests and restriction endonuclease cleavage patterns and found to be adenovirus type 4. This report serves to illustrate an infrequent cause of epidemic conjunctivitis, namely adenovirus type 4. There was no demonstrable focus of the outbreak.

Clonal relationships among classical Vibrio cholerae O1 strains isolated between 1961 and 1992 in Bangladesh.

In Bangladesh, the replacement of classical Vibrio cholerae by the E1 Tor biotype in 1968 and the reappearance of the classical biotype and its coexistence with the E1 Tor biotype after 1982 were never adequately explained. We have analyzed 23 classical V. cholerae isolates collected between 1961 and 1968, 14 classical isolates collected between 1982 and 1992 from the capital city, Dhaka, and 6 classical V. cholerae isolates collected from two southern districts of Bangladesh and studied restriction endonuclease cleavage patterns of rRNA genes (ribotypes) to investigate the clonal relationships among the isolates. Southern blots of total DNA digested with restriction enzyme BamHI, BglI, EcoRI, HindIII, or PstI were probed, using a cloned Escherichia coli rRNA operon. While restriction enzymes BamHI, EcoRI, and PstI failed to differentiate the isolates on the basis of ribotyping, BglI and HindIII produced digestion patterns that allowed differentiation. Ribotyping the isolates with BglI and HindIII revealed five different clones (ribotypes IA, IB, IIA, IC, and IIC) of classical vibrios in Bangladesh. Strains belonging to ribotypes IA and IB were isolated in Dhaka before 1968, and one ribotype (IA) was again isolated between 1982 and 1992. Ribotype IIA was isolated in 1988 and 1989, when both clones (IA and IIA) of classical vibrios coexisted with the EI Tor vibrios. Isolates belonging to ribotypes IC and IIC were collected in the southern districts of Bangladesh and were clearly different from those collected in Dhaka between 1968 and 1992 by ribotyping analysis with BglI. These results support the previous assumption that classical vibrios were never completely replaced in Bangladesh and also demonstrate the existence of more than one genetically different clone of classical V. cholerae.