Rapid detection and characterization from field cases of infectious laryngotracheitis virus by real-time polymerase chain reaction and restriction fragment length polymorphism.

A real-time polymerase chain reaction (PCR) assay was developed to specifically amplify infectious laryngotracheitis virus (ILTV) DNA from field samples. The 222-base-pair PCR fragment was amplified using primers located in a conserved region of the infected cell protein 4 gene that was demonstrated in this work to encompass a single nucleotide polymorphism. Subsequent restriction fragment length polymorphism (RFLP) analysis of real-time PCR amplified fragments from a range of ILTV isolates using the restriction endonuclease MspI enabled differentiation between older ILTV isolates that were prevalent in the 1960s prior to the availability of vaccine strains and more recent isolates that predominantly are identical to vaccine strains. The assay, using real-time PCR, RFLP and sequence analysis, was used to characterize two recent field cases of infectious laryngotracheitis from Northern Ireland. One of the field cases was demonstrated to be similar to older "wild-type" isolates, while the other field case was identified to have a concurrent ILTV infection of both "wild-type" and vaccinal type origin. The assay described here using real-time PCR and RFLP provides a rapid, specific method that enables detection and characterization of ILTV directly from field cases.     

Genetic characterization of E2 gene of classical swine fever virus by restriction fragment length polymorphism and phylogenetic analysis

An RT-nested PCR (RT-nPCR)-based restriction fragment length polymorphism (RFLP) analyses of the E2 gene were developed for genetic subtyping and differentiation of vaccinated and infected classical swine fever virus (CSFV) strains. RT-nPCR identified 96 CSFV-positive samples from 321 clinical specimens from southeastern China during 2003–2008. The PCR products of positive samples were further differentiated using MspI digestion, 23 were identified as the C-strain, 62 as field strains, and 11 as mixture of the vaccine strain and field ones. RFLP with BglI, DdeI, DraI, and PstI were then used for subtyping of the field CSFV isolates. Thirty-eight field isolates phylogenetically classified as subgroup 2.1 based on E2 were divided into 11 subtypes by this RFLP scheme. Both RFLP profiling and sequence-based phylogenetic analysis revealed genetic diversity of CSFV in the field. Three novel substitutions at amino acid positions 17, 93, and 286 were identified in the predominant subtype VI strains isolated in 2008 as compared to other strains including historical subtype VI strains. These results suggest that CSFV in China experienced gradual variations and evolutionary accumulation progress. Thus, the RFLP methods targeting on the CSFV E2 gene are suitable for epidemiological survey in endemic area where the C-strain is applied for vaccination. Combination of the RFLP schemes with sequence-based phylogenetic analysis could provide more detailed information on transmission of CSFV in the region or even its evolution.     

Genetic grouping for the isolates of avian infectious bronchitis virus in Taiwan

Summary In order to differentiate recent isolates of avian infectious bronchitis virus (IBV) in Taiwan, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and direct sequencing methods were used to type 25 IBV Taiwan isolates. Two conserved sequences that flank the hypervariable region I (HVR I) in the N-terminus of S1 protein gene were chosen as primers. Sequences of 228–231 base pairs (bp) were amplified by PCR from 25 Taiwan isolates and 4 reference strains (H120, Conn, JMK, Holte). PCR products were digested with 5 restriction endonucleases,BsoFI,DdeI,MboII,AluI,RsaI, and different IBV isolates were grouped according to their RFLP patterns. The RFLP patterns of the 4 reference strains in this study matched the published sequences in GenBank. Except 1 vaccine strain, the other 24 Taiwan isolates were different from these 4 and 18 other IBV strains whose sequences were published. The data from PCR-RFLP and sequencing of IBV genomes showed that the 24 Taiwan isolates can be divided into 2 distinct groups, I and II. Seven RFLP patterns are identified in group I and only 1 in group II.     

Restriction fragment length polymorphism analysis of open reading frame 5 gene of porcine reproductive and respiratory syndrome virus isolates in Korea

Summary.  The genetic variability of porcine reproductive and respiratory syndrome virus (PRRSV) was studied by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified fragments among 50 Korean isolates from open reading frame 5. All Korean PRRSVs were isolated from the field cases after the marketing of an U.S. ATCC VR2332-derived modified live PRRSV vaccine. Combining the restriction enzyme digestion patterns obtained with MluI, HincII, SacII, and HaeIII, we observed 19 distinct RFLP patterns. Seventeen out of 50 PRRSV isolates (34%) exhibited the modified live PRRSV vaccine RFLP pattern. The genomic variations that have been identified in the present study seemed to represent characteristic features of the Korean PRRSV isolates. PCR-based RFLP analysis using several restriction enzymes provides a good genetic estimate for isolate differentiation. Received December 20, 1999 Accepted January 28, 2000     

Restriction endonuclease analysis of Aujeszky's disease (pseudorabies) virus DNA: Comparison of Northern Ireland isolates and isolates from other countries

Summary Aujeszky's disease (AD; pseudorabies) viruses isolated in Northern Ireland over a 20 year period were compared with isolates from other parts of the world using restriction endonuclease analysis of virus DNA. When the numbers of Bam H1, Kpn 1 and Sal 1 restriction sites were considered, pathogenic Northern Ireland isolates resembled viruses isolated in England, Hungary and the U.S.A. and could be differentiated from viruses isolated in Denmark, Belgium and the Netherlands. The avirulent Northern Ireland isolate NIA4 and the Bartha vaccine strain were very similar to each other and could be distinguished from pathogenic isolates.While almost all the pathogenic viruses isolated in Northern Ireland from 1963 to 1983 appeared to possess the same number of restriction sites none of the viruses, even those made at the same farm during one outbreak of infection, were identical. The differences were confined to variation in the sizes of certain fragments which map in variable regions of the genome.With 4 Figures     

Molecular characterization of Blastocystis isolates in the Philippines by riboprinting

Extensive genomic polymorphism has been demonstrated among morphologically identical Blastocystis isolates. To this end, 32 Blastocystis isolates from the Philippines (12 from humans, 12 from pigs and 8 from chickens) were analyzed genetically by riboprinting or restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified small subunit rDNA. Three distinct riboprint patterns were observed from the HinfI digestion, while four patterns resulted from the RsaI digestion of Blastocystis SSU rDNA. Restriction fragment profiles between Blastocystis isolates from different hosts were generally different from each other. However, Blastocystis isolates within each host group were practically the same. Cluster analysis of the riboprint patterns revealed seven distinct groups of the Blastocystis isolates, including a zoonotic strain. These results demonstrate the genetic heterogeneity of Blastocystis in the Philippines and a support to the idea of the organisms zoonotic potential.Declaration: The experiments conducted in this study comply with the current laws of the Philippines.     

Genotype identification of human cystic echinococcosis in Isfahan, central Iran

Echinococcosis/hydatidosis is one of the most important zoonotic diseases commonly found in different regions of Iran with a major economic and public health importance. In the current study, Echinococcus granulosus isolates were collected from hospitalized patients in Isfahan, central Iran. The genotypes of 30 samples were determined by polymerase chain reaction amplification of internal transcribed spacer-1 region of ribosomal DNA, followed by restriction fragment length polymorphism (RFLP) with two restriction enzymes namely AluI and MspI. As expected, each isolate yielded an approximately 1-kbp DNA fragment on the electrophoresis gel. According to RFLP results for both enzymes, all isolates had an equal pattern indicating the G1 genotype. Our findings confirmed that G1 is the dominant genotype of cystic echinococcosis in human in central Iran, with predilection to different organs including liver, lung, and brain, and warrants the importance of sheep dog cycle in public health.     

Length heterogeneity in ITS 2 and the methylation status of CCGG and GCGC sites in the rRNA genes of the genus Peronosclerospora

Summary The polymerase chain reaction (PCR) was used with primers complementary to conserved flanking sequences to amplify the internal transcribed spacer 2 (ITS 2) of the rDNA repeat units of five Peronoscleropora isolates, one each of P. sorghi, P. maydis, P. sacchari and tow of P. zeae. In contrast to the situation found in mostfungi that have been examined, length heterogeneity was evident in each sample. The rDNA composition of the amplified bands was confirmed by Southern hybridizations using an ITS 2 amplified from P. sorghi and cloned rDNA from Neurospora crassa as probes. Length heterogeneity was also detected in genomic DNA digests using the same probes. In addition to one dominant fragment for each isolate, there were several less frequent fragments of different sizes, and the isolate(s) for each species had a unique banding pattern for ITS 2. The absence of 5-methylcytosine residues in CCGG and GCGC sequences in the ribosomal genes of these four Peronosclerospora species was demonstrated by the production of identical banding patterns with ribosomal DNA probes following digestion of genomic DNA with MspI and HpaII, and by complete digestion with CfoI.     

A molecular taxonomy for cricket paralysis virus including two new isolates from Australian populations ofDrosophila (Diptera: Drosophilidae)

Summary Two new isolates of cricket paralysis virus, TAR and SIM, are described that were originally isolated from laboratory colonies ofDrosophila melanogaster andDrosophila simulans respectively. Using a combination of biological, serological and molecular characters it was possible to distinguish the SIM isolate from all other isolates and it is thus described as a new strain; CrPV_SIM. The TAR isolate however, was indistinguishable from the CrPV reference isolate CrPV_VIC/Gm/D2²/Gm/D2² (Teleogryllus commodus, Victoria, Australia, 1968). The molecular characters used in the present study were obtained by combining PCR and restriction endonuclease digestion of the amplified fragments. This work demonstrates that such molecular characters, when used in combination with others, provide a powerful set of taxonomic characters for classifying CrPV isolates and strains and assessing their genetic relatedness.     

Genetic variability in the coat protein genes of lettuce big-vein associated virus and Mirafiori lettuce big-vein virus

Summary. Available data suggests that lettuce big-vein disease is caused by the ophiovirus Mirafiori lettuce big-vein virus (MLBVV) but not by the varicosavirus Lettuce big-vein-associated virus (LBVaV), although the latter is frequently associated with the disease. Since the disease occurs worldwide, the putative coat protein (CP) open reading frames of geographically distinct isolates of MLBVV and LBVaV were sequenced. Comparison of both nucleotide and amino acid sequences showed a high level of sequence similarity among LBVaV isolates. Phylogenetic analysis of LBVaV CP nucleotide sequences showed that most of the Spanish isolates clustered in a phylogenetic group whereas English isolates were more similar to the USA isolate. An Australian isolate was closely related to the Dutch isolate. Genetic diversity among MLBVV CP nucleotide sequences was higher ranging from 0.2% to 12%. Phylogenetic analysis of MLBVV CP nucleotide sequences revealed two distinct subgroups. However, this grouping was not correlated with symptom development on lettuce or the geographic origin of the MLBVV isolates. Finally, a quick method based on RFLP analysis of RT-PCR amplicons was developed for assigning MLBVV isolates to the two subgroups.     

MinION sequencing to genotype US strains of infectious laryngotracheitis virus

Over the last decade the US broiler industry has fought long-lasting outbreaks of infectious laryngotracheitis (ILTV). Previously, nine genotypes (I-IX) of ILTVs have been recognized using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method with three viral alleles (gB, gM and UL47/gG). In this study, the genotyping system was simplified to six genotypes by amplicon sequencing and examining discriminating single nucleotide polymorphisms (SNPs) within these open reading frames. Using phylogenomic analysis of 27 full genomes of ILTV, a single allele (ORF A/ORF B) was identified containing SNPs that could differentiate ILTVs into genotypes congruent with the phylogenetic partitioning. The allelic variations allowed for the cataloging of the 27 strains into 5 genotypes: vaccinal TCO, vaccinal CEO, virulent CEO-like, virulent US and virulent US backyard flocks from 1980 to 1990, correlating with the PCR-RFLP genotypes I/ II/ III (TCO), IV (CEO), V (virulent CEO-like), VI (virulent US) and VII/VIII/IX (virulent US backyard flock isolates). With the unique capabilities of third generation sequencing, we investigated the application of Oxford Nanopore MinION technology for rapid sequencing of the amplicons generated in the single-allele assay. This technology was an improvement over Sanger-based sequencing of the single allele amplicons due to a booster amplification step in the MinION sequencing protocol. Overall, there was a 90% correlation between the genotyping results of the single-allele assay and the multi-allele assay. Surveillance of emerging ILTV strains could greatly benefit from real-time amplicon sequencing using the single-allele assay and MinION sequencing.

RESEARCH HIGHLIGHTS

• A multi-allelic assay identified nine ILTV genotypes circulating in the US

• Single-allele genotyping is congruent with whole genome phylogenetic partitioning

• US ILTV strains can be grouped into five genotypes using the single-allele assay

• The single-allele assay can be done using MinION sequencing of barcoded amplicons

     

Use of pulsed-field gel electrophoresis for comparison of similar but distinguishable isolates of Shigella sonnei collected in Ireland and Italy

Comparison of pulsed-field gel electrophoresis patterns (generated with XbaI and BlnI) of Shigella sonnei isolates from Ireland and Italy suggests that two possibly distantly related lineages are present in both countries. Smaller, more closely related groups, including isolates from Ireland and Italy, were also noted. These groups raise the possibility that the dissemination of clonal groups of S. sonnei may have occurred in recent years.     

Molecular,serological and biological variation among chickpea chlorotic stunt virus isolates from five countries of North Africa and West Asia

Chickpea chlorotic stunt virus (CpCSV), a proposed new member of the genus Polerovirus (family Luteoviridae), has been reported only from Ethiopia. In attempts to determine the geographical distribution and variability of CpCSV, a pair of degenerate primers derived from conserved domains of the luteovirus coat protein (CP) gene was used for RT-PCR analysis of various legume samples originating from five countries and containing unidentified luteoviruses. Sequencing of the amplicons provided evidence for the occurrence of CpCSV also in Egypt, Morocco, Sudan, and Syria. Phylogenetic analysis of the CP nucleotide sequences of 18 samples from the five countries revealed the existence of two geographic groups of CpCSV isolates differing in CP sequences by 8–10%. Group I included isolates from Ethiopia and Sudan, while group II comprised those from Egypt, Morocco and Syria. For distinguishing these two groups, a simple RFLP test using HindIII and/or PvuII for cleavage of CP-gene-derived PCR products was developed. In ELISA and immunoelectron microscopy, however, isolates from these two groups could not be distinguished with rabbit antisera raised against a group-I isolate from Ethiopia (CpCSV-Eth) and a group-II isolate from Syria (CpCSV-Sy). Since none of the ten monoclonal antibodies (MAbs) that had been produced earlier against CpCSV-Eth reacted with group-II isolates, further MAbs were produced. Of the seven MAbs raised against CpCSV-Sy, two reacted only with CpCSV-Sy and two others with both CpCSV-Sy and -Eth. This indicated that there are group I- and II-specific and common (species-specific) epitopes on the CpCSV CP and that the corresponding MAbs are suitable for specific detection and discrimination of CpCSV isolates. Moreover, CpCSV-Sy (group II) caused more severe stunting and yellowing in faba bean than CpCSV-Eth (group I). In conclusion, our data indicate the existence of a geographically associated variation in the molecular, serological and presumably biological properties of CpCSV.     

Analysis of fliC variation among clinical isolates of Burkholderia cepacia.

PCR and restriction fragment length polymorphism (RFLP) typing of flagellin genes (fliC) from 57 clinical isolates of Burkholderia cepacia indicated that only type 11 flagellins were present. Twenty-two isolates previously identified as the epidemic UK cystic fibrosis strain were indistinguishable by this method, as were 11 isolates from a pseudo-outbreak in Senegal. Other clinical isolates, including 19 from disparate sources in Malaysia, were separated into nine fliC RFLP groups, exhibiting a large degree of divergence. When isolates were indistinguishable by fliC genotyping, their similarity was confirmed by whole genome macro-restriction analysis with pulsed-field gel electrophoresis following XbaI digestion. The variation in fliC sequences of B. cepacia was far greater than that with B. pseudomallei, supporting the view that 'B. cepacia', as currently defined, may comprise several different genomic species.     

Increased Population Risk of AIP‐Related Acromegaly and Gigantism in Ireland

The aryl hydrocarbon receptor interacting protein (AIP) founder mutation R304^* (or p.R304^*; NM_003977.3:c.910C>T, p.Arg304Ter) identified in Northern Ireland (NI) predisposes to acromegaly/gigantism; its population health impact remains unexplored. We measured R304^* carrier frequency in 936 Mid Ulster, 1,000 Greater Belfast (both in NI) and 2,094 Republic of Ireland (ROI) volunteers and in 116 NI or ROI acromegaly/gigantism patients. Carrier frequencies were 0.0064 in Mid Ulster (95%CI = 0.0027–0.013; P = 0.0005 vs. ROI), 0.001 in Greater Belfast (0.00011–0.0047) and zero in ROI (0–0.0014). R304^* prevalence was elevated in acromegaly/gigantism patients in NI (11/87, 12.6%, P < 0.05), but not in ROI (2/29, 6.8%) versus non‐Irish patients (0–2.41%). Haploblock conservation supported a common ancestor for all the 18 identified Irish pedigrees (81 carriers, 30 affected). Time to most recent common ancestor (tMRCA) was 2550 (1,275–5,000) years. tMRCA‐based simulations predicted 432 (90–5,175) current carriers, including 86 affected (18–1,035) for 20% penetrance. In conclusion, R304^* is frequent in Mid Ulster, resulting in numerous acromegaly/gigantism cases. tMRCA is consistent with historical/folklore accounts of Irish giants. Forward simulations predict many undetected carriers; geographically targeted population screening improves asymptomatic carrier identification, complementing clinical testing of patients/relatives. We generated disease awareness locally, necessary for early diagnosis and improved outcomes of AIP‐related disease.     

Restriction fragment length polymorphism analysis of clinical isolates of Mycobacterium haemophilum.

Mycobacterium haemophilum is an emerging opportunistic pathogen, and since 1989, infections caused by this organism have been identified more frequently in the New York City area than in any other region of the United States. A DNA fingerprinting method, based on restriction fragment length polymorphisms (RFLPs) was developed. A genomic library of M. haemophilum isolate 1A was constructed; screening the library yielded a recombinant strain that incorporated a genetic element present in multiple copies in the M. haemophilum genome. This clone was used to produce a probe for RFLP analyses of PvuII digests of genomic DNA. We used this probe to determine the RFLP patterns of 43 clinical isolates of M. haemophilum from 28 patients. A total of six distinct patterns were observed. Two patterns, designated types 1 and 2, accounted for 91% of the infections in patients from the New York City area. Two isolates from Arizona had identical patterns but were distinct from those of New York isolates, and an isolate from Israel, the type strain, had another distinct pattern (type 6). The type 6 pattern was also seen in a recent isolate from Norway. All of the type 1 isolates and 60% of the type 2 isolates were recovered from patients with AIDS in the New York City area. This molecular subtyping method should provide a useful tool for epidemiological studies and may help identify the associated risk factors, vehicles, and possible reservoirs of this newly emerging pathogen.     

Molecular diversity of native rhizobia trapped by five field pea genotypes in Indian soils

Five pea cultivars; HFP 4, HVP 3–5, HFP 9426, Jayanti and Hariyal, being grown in CCS Haryana Agricultural University farm were used to isolate native rhizobia. Selected 54 rhizobia, from all cultivars, were authenticated as rhizobia by plant infectivity test. Along with nodulation, symbiotic effectiveness in terms of symbiotic ratios showed wide range of effectiveness of pea rhizobia from 1.11 to 5.0. DNA of all the 54 rhizobia was extracted and amplified by PCR, using ERIC and 16S rDNA primers. Dendrogram based on ERIC profiles of these 54 rhizobia showed the formation of 13 subclusters at 80% level of similarity. Dendrogram based on RFLP of 16S rDNA by three restriction endonucleases; Msp I, Csp 6I and Rsa I; also formed 13 subclusters at 80% level of similarity. However, positioning of subclusters was different from that of ERIC based dendrogram. Majority of the isolates i.e. 64.8% by ERIC profiles and 44.4% by RFLP of 16S rDNA formed one cluster. Isolates from same nodule were not 100% similar. Considering each cluster representing a rhizobial genotype, both techniques used to assess molecular diversity indicated the presence of 13 genotypes of field pea rhizobia in CCS Haryana Agricultural University farm soil. Two pea rhizobial genotypes were able to nodulate all the five pea cultivars. Furthermore, high strain richness index (0.43–0.5) of field pea rhizobia was observed by both the techniques. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Use of monoclonal antibodies in the characterisation of avian paramyxovirus type 1 (Newcastle disease virus) isolates submitted to an international reference laboratory

A total of 106 Newcastle disease viruses submitted to the International Reference Laboratory at the Central Veterinary Laboratory, Weybridge from field investigations in 15 different countries was characterised using pathogenicity index tests in chickens and mouse monoclonal antibodies raised against NDV-Ulster 2C (Russell and Alexander, Archives of Virology, 75: 243, 1983) and pigeon isolate 617/83. These isolates could be placed into six distinct groups on the basis of their reaction with the monoclonal antibodies although four isolates gave ambiguous results and remained untyped. Forty isolates, obtained from chickens (21), pigeons (16), a duck (1), a sparrow (1) and a kestrel (1), were indistinguishable from isolates which were responsible for the recent panzootic in pigeons. Twenty-one isolates from domestic poultry, one isolate from a pheasant and one from a chicken in quarantine were identified as vaccinal virus of Bi or La Sota type. Thirty-five isolates placed in the same monoclonal antibody group were velogenic viruses. These had been obtained from domestic poultry in Italy, Austria, Mauritius and Saudi Arabia during 1983-1985, commercial pigeons in Hong Kong in 1986 and exotic birds in Italy, Great Britain and the Federal Republic of Germany during 1981-1985. This group was distinguishable from velogenic viruses responsible for disease outbreaks in poultry during the 1970s. Two lentogenic isolates from commercial ducks in England showed different monoclonal antibody binding patterns both of which have been associated with feral ducks. An isolate from chickens in Italy was also placed in one of these groups. A single isolate from a loon (Gavia sp) in the USA showed a monoclonal antibody binding pattern which had not been seen previously. In addition, 11 vaccinal or laboratory viruses were received for confirmatory characterisation which was greatly aided by the use of the monoclonal antibodies.