Reverse Transcriptase-polymerase Chain Reaction Products Research Articles

Molecular and pathological investigation of avian reovirus (ARV) in Egypt with the assessment of the genetic variability of field strains compared to vaccine strains.

Avian orthoreovirus (ARV) is among the important viruses that cause drastic economic losses in the Egyptian poultry industry. Despite regular vaccination of breeder birds, a high prevalence of ARV infection in broilers has been noted in recent years. However, no reports have revealed the genetic and antigenic characteristics of Egyptian field ARV and vaccines used against it. Thus, this study was conducted to detect the molecular nature of emerging ARV strains in broiler chickens suffering from arthritis and tenosynovitis in comparison to vaccine strains. Synovial fluid samples (n = 400) were collected from 40 commercial broiler flocks in the Gharbia governorate, Egypt, and then pooled to obtain 40 samples, which were then used to screen ARV using reverse transcriptase polymerase chain reaction (RT-PCR) with the partial amplification of ARV sigma C gene. The obtained RT-PCR products were then sequenced, and their nucleotide and deduced amino acid sequences were analyzed together with other ARV field and vaccine strains from GenBank. RT-PCR successfully amplified the predicted 940 bp PCR products from all tested samples. The phylogenetic tree revealed that the analyzed ARV strains were clustered into six genotypic clusters and six protein clusters, with high antigenic diversity between the genotypic clusters. Surprisingly, our isolates were genetically different from vaccine strains, which aligned in genotypic cluster I/protein cluster I, while our strains were aligned in genotypic cluster V/protein cluster V. More importantly, our strains were highly divergent from vaccine strains used in Egypt, with 55.09-56.23% diversity. Sequence analysis using BioEdit software revealed high genetic and protein diversity between our isolates and vaccine strains (397/797 nucleotide substitutions and 148-149/265 amino acid substitutions). This high genetic diversity explains the vaccination failure and recurrent circulation of ARV in Egypt. The present data highlight the need to formulate a new effective vaccine from locally isolated ARV strains after a thorough screening of the molecular nature of circulating ARV in Egypt.

Recent detection and Genotypic characterization of duck hepatitis virus A type 1and H5 avian influenza virus from ducks.

Objective: To identify some viruses affecting ducks by Serological and molecular studies.Design: Diagnostic study.Procedures: Twenty pooled liver samples were collected from ducklings less than 2 weeks showed nervous signs and high mortality suspected to be infected by duck hepatitis virus (DHV). All samples were inoculated in allantoic cavity of SPF embryonated chicken eggs (ECGs) for three successive passages. Isolates were serologically identified by agar gel precipitation (AGPT) and fluorescent antibody technique (FAT) by using DHAV-1 antibodies as well. Also, other 20 pooled lung and pancreas samples from ducks more than one month suspected to be infected by avian influenza virus (AIV) showed nervous signs; ataxia and torticolus. Suspected AIV samples were tested by reverse transcriptase polymerase chain reaction (RT-PCR) of H5 gene. Finally, sequencing and sequencing analysis of our RT-PCR products of DHAV-1 3D gene and AIV H5 gene with that published in GenBank.Results: Twelve out of 20 local isolates of DHAV-1 were positive in AGPT and 15 gave positive result in FAT. Twelve samples suspected to be AIV gave positive HA reaction. By RT-PCR 5 selected isolates of DHAV-1 and 5 out of 10 tested samples of AIV were positive and gave the expected bands. Our DHAV-1 isolates showed 100% identity to other Egyptian sequences from GenBank and H5 AIV showed 96-97% identity.Conclusion and clinical relevance: Both DHAV-1 and H5 AIV still in ducks could be identified by serological and molecular methods.

Isolation and Cloning of Tropomyosin and Arginine Kinase from Tiger Prawn Penaeus monodon and Blue Swimming Crab Portunus trituberculatus

Shellfish is an important source of food and plays a significant role in human nutrition and health. However, shellfish allergy is a long-lasting disorder which mostly persists throughout life and is often associated with severe reactions [1]. Among the various consumed shellfish, prawns and crabs are the most widely consumed and can lead to the most severe reactions. At present, allergies to shellfish are diagnosed similarly to other food allergies. The diagnosis relies upon careful evaluation of history, the presence of appropriate clinical signs and confirmation with in vivo or in vitro tests to demonstrate the presence of allergen-specific immunoglobulin E (IgE) [2]. However, both in vivo or in vitro diagnostic approaches are mainly based on the use of crude allergen extracts. Crude allergen extracts are obtained from biological sources and consist of mixture of allergenic components with high amounts of undesirable products that can interfere with diagnosis. In many cases, only few of the several proteins found in crude allergen extracts act as the essential allergens in the majority of patients that are allergic to the substance. The most important ones are called major allergens. Problems associated with using crude allergen extracts for allergy diagnosis may be overcome with recombinant allergens. Recombinant allergens with high purity can be produced by using controlled production procedures that yield defined molecules with known molecular, immunologic and biological characteristics [1]. Tiger prawn Penaeus monodon and blue swimming crab Portunus trituberculatus, are among the widely consumed shellfish in Malaysia. Our earlier study involving 131 atopic patients in Allergy Clinic, Kuala Lumpur Hospital demonstrated that patients in Malaysia suffering from allergic responses to shellfish including tiger prawn Penaeus monodon and blue swimming crab Portunus trituberculatus. Amongst the shellfish extracts tested, prawn elicited the highest frequency of positive reactivity in 39% of the patients. Further, crab was the second most common shellfish to elicit a positive reaction in 24% of the patients [3]. Our first phase study has successfully identified tropomyosin and arginine kinase as the major allergens in both species of shellfish. However, more information about the individual allergenic species-specific components is needed. Therefore, we continued our study to isolate and clone the tropomyosin and arginine kinase from these two species of shellfish, tiger prawn Penaeus monodon and blue swimming crab Portunus trituberculatus. Tropomyosin and arginine kinase were isolated from the total RNA (Ribonucleic Acid) obtained from both prawn and crab muscles followed by RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). The RT-PCR products were then cloned into the cloning vector, pJET 1.2 and transformed into Escherichia coli host. Transformants were screened for positive clones by PCR (Polymerase Chain Reaction) colony and sequenced. The 855 bp tropomyosins have been isolated and sequenced from both prawn and crab. Arginine kinases isolated and sequenced from prawn and crab were 1071 bp and 1074 bp, respectively (Figure 1). The GenBank BLAST search for the sequences showed high homology to the targeted proteins as shown in Table 1. Tropomyosin is a 34 to 38 kDa heat-stable protein that belongs to a highly conserved family of actin filament binding proteins, which plays a functional role in contractile activities in muscle cells [4]. Arginine kinase is a 40 to 42 kDa heat-labile protein that plays an important role in regenerating adenosine triphosphate (ATP) during bursts of cellular activity [5]. Tropomyosin and arginine kinase from the prawn and crab have been isolated and the full-length sequences were obtained. Current ongoing study focuses on sub-cloning and full-length expression of tropomyosin and arginine kinase in order to produce respective recombinant proteins, and subsequently investigate their physicochemical and allergenic characteristics.

Feasibility of Qualitative Testing of BCR-ABL and JAK2 V617F in Suspected Myeloproliperative Neoplasm (MPN) Using RT-PCR Reversed Dot Blot Hybridization (RT-PCR RDB)

BackgroundDefining the presence of BCR-ABL transcript in suspected myeloproliferative neoplasm is essential in establishing chronic myeloid leukemia. In the absence of BCR-ABL, the conventional diagnostic algorithm recommends JAK2 V617F mutation testing to support diagnosis of other MPN diseases such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis. In certain cases of thrombocythemia, simultaneous upfront testing of both BCR-ABL and JAK2 may be desirable. We wanted to test the feasibility of multiplex detection of BCR-ABL transcript variants and JAK2 V617F mutation simultaneously using the reverse transcriptase polymerase chain reaction (RT-PCR)-based reverse dot-blot hybridization (RDB) method. Material and MethodsSeparate biotinylated RT-PCR primers were designed to amplify specific BCR-ABL transcripts and JAK2 V617F mutant alleles. Specific hybridization of RT-PCR products with arrays of membrane-bound probes followed by colorimetric development would allow simultaneous visualization of BCR-ABL and/or JAK2 mutant transcripts in a given specimen. To validate the RDB method, we used cDNA specimens previously referred to our laboratory for routine clinical testing of BCR-ABL and/or JAK2. ResultsThe limit of detection or analytical sensitivity of the RDB method using cDNA specimens was 0.5% and 6.25% in detecting BCR-ABL and JAK2 mutant transcripts, respectively. The diagnostic specificity and sensitivity to detect BCR-ABL and JAK2 were 100% and 92.3% (N = 38); and 100% and 100% (N = 27), respectively. RDB also detected BCR-ABL transcripts in 22% of JAK2 V617F mutation-positive samples (N = 14). ConclusionsRT-PCR RDB is a promising qualitative multiplex method to detect BCR-ABL and JAK2 mutant transcripts simultaneously.

Clinical, molecular detection and phylogenetic analysis study of local foot-and-mouth disease virus in Al-Qadisiyah province of Iraq.

Aim:This study was directed during an outbreak of suspected foot-and-mouth disease (FMD) in cattle in Al-Qadisiyah province, Iraq 2016. The disease has made a huge economic loss in livestock. It was suspected that the vaccination has failed to protect the animals from the infection because of the difference in the strains. Consequently, we designed the study to make the diagnosis and detect the strain of the causative virus.Materials and Methods:The extraction of the DNA was done on 73 samples and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was used in the detection of FMD virus (FMDV) for primary diagnosis, and serotype-specific diagnosis was done with universal primer sets 1F/1R, A-1C612, and O-ARS4 with the expected band of 329, 865, and 1301 bp, respectively.Results:Universal primer pair 1F/1R detected FMD in 55 of 73 (75.3%); of these, 37 (67.3%) were females and 18 (32.7%) were males, with high significance (p<0.01) between males and females in the PCR positivity ratio. The tested samples with positive universal primer were amplified with specific primers A-IC612 with no reaction for serotype O-ARS4.Conclusion:The products of RT-PCR were sent for RNA sequencing, and the results were 100% positive to serotype A which means that it is the predominant type in Iraq. It may help in the importing or production of the vaccine to make a preventive plan for the disease. The virus of FMD is contagious and dangerous due to its role in the huge economic loses. The detection of this virus is widely explained in lots of articles, but it is more specific and sensitive in RT-PCR and sequencing. Consequently, the authorities responsible for importing and/or production vaccines have to avoid the importing of other serotypes because it will be losing money and more outbreaks will explode.

Detection and localization of the thyroid hormone receptor beta mRNA in the immature olfactory receptor neurons of chum salmon

Thyroid hormone (TH) plays an important role in regulating multiple cellular and metabolic processes, including cell proliferation, cell death, and energy metabolism, in various organs and tissues of vertebrates. It is generally accepted that anadromous Pacific salmon (Oncorhynchus spp.) imprint odorants from their natal stream during their seaward migration, and they then use olfaction to discriminate their natal stream during the spawning migration. Both serum TH levels and the specific binding values of TH in the salmon olfactory epithelium were markedly increased during the seaward migration. However, thyroid hormone receptor (TR) expression in the olfactory epithelium has not been confirmed in vertebrates. We investigated gene expression of TR isoforms in chum salmon (O. keta) by both molecular biological and histochemical techniques. Expression of TRβ mRNA was detected in the olfactory epithelium by reverse transcriptase polymerase chain reaction (RT-PCR). Nucleotide sequencing demonstrated the existence of a remarkable homology between the RT-PCR product and part of the ligand-binding domain of other teleost TRβ isoforms. By in situ hybridization using a digoxygenin-labeled salmon olfactory TRβ cRNA probe, signals for salmon olfactory TRβ mRNA were observed preferentially in the perinuclear regions of immature olfactory receptor neurons (ORNs), as protein gene product 9.5 (PGP9.5)-immunopositive ORNs. Our results provide the first detection of TRβ gene expression in the olfactory epithelium, and suggested the possibility that TRβ may be involved in cell maturation and/or cell differentiation of the ORNs in Pacific salmon.

Molecular detection of influenza A(H1N1)pdm09 viruses with M genes from human pandemic strains among Nigerian pigs, 2013-2015: implications and associated risk factors.

In the post-pandemic period, influenza A(H1N1)pdm09 virus has been detected in swine populations in different parts of the world. This study was conducted to determine the presence and spatial patterns of this human pandemic virus among Nigerian pigs and identify associated risk factors. Using a two-stage stratified random sampling method, nasal swab specimens were obtained from pigs in Ibadan, Nigeria during the 2013-2014 and 2014-2015 influenza seasons, and the virus was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Purified RT-PCR products were sequenced in both directions, and sequences were aligned using MUSCLE. Phylogenetic analysis was conducted in MEGA6. Purely spatial scan statistics and a spatial lag regression model were used to identify spatial clusters and associated risk factors. The virus was detected in both seasons, with an overall prevalence of 8·7%. Phylogenetic analyses revealed that the M genes were similar to those of pandemic strains which circulated in humans prior to and during the study. Cluster analysis revealed a significant primary spatial cluster (RR = 4·71, LLR = 5·66, P = 0·0046), while 'hours spent with pigs (R 2 = 0·90, P = 0·0018)' and 'hours spent with pigs from different farms (R 2 = 0·91, P = 0·0001)' were identified as significant risk factors (P < 0·05). These findings reveal that there is considerable risk of transmission of the pandemic virus, either directly from pig handlers or through fomites, to swine herds in Ibadan, Nigeria. Active circulation of the virus among Nigerian pigs could enhance its reassortment with endemic swine influenza viruses. Campaigns for adoption of biosecurity measures in West African piggeries and abattoirs should be introduced and sustained in order to prevent the emergence of a new influenza epicentre in the sub-region.

Biological microchip for establishing the structure of fusion transcripts involving MLL in children with acute leukemia

MLL is involved in fusion genes with more than 100 partner genes, approximately 80 of which have been characterized at the molecular level. MLL fusion genes are often found in infants (60-80% of acute lymphoblastic leukemia (ALL) cases and 40-50% of acute myeloblastic leukemia (AML) cases) and are appreciably rarer (8-10%) in children older than 1 year of age. MLL rearrangements are important markers in diagnosis and treatment choice. To identify the partner gene is of primary importance for prognosis and minimal residual disease monitoring. The structure of the fusion gene, including localization of the MLL breakpoints, is also informative. A method was developed to examine the fusion transcripts in order to identify the partner gene among the six most common ones and to establish the exon structure of the rearranged MLL. The method includes a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify and to fluorescently label a fusion transcript fragment and subsequent hybridization of the product on a biological microchip with immobilized oligonucleotides complementary to exons of MLL and its partner genes AFF1, MLLT1, MLLT3, MLLT4, MLLT10, and ELL. Hybridization results were verified by sequencing the RT-PCR products and, in some cases, performing long-distance inverse PCR (LDI-PCR). The study involved 38 bone marrow samples from ALL patients (including 33 children younger than 1 year of age) and 15 samples from AML patients (including 10 from children younger than 1 year of age). The main partner genes were AFF1 (49%), MLLT1 (27%), MLLT3 (12%), and MLLT10 (12%) in ALL and MLLT3 (80%), MLLT10 (10%), and MLLT4 (10%) in AML. Fusion gene transcripts most commonly included MLL exon 11 (58% of ALL cases and 50% of AML cases), suggesting a breakpoint in MLL intron 11.

Identification of avian bornavirus in a Himalayan monal (Lophophorus impejanus) with neurological disease

A one-year-old male Himalayan monal (Lophophorus impejanus) was presented for veterinary attention with a history of chronic wasting, weakness and ataxia. The bird died, and post-mortem findings included mild non-suppurative encephalitis and degenerative encephalopathy, lymphoplasmacytic myenteric ganglioneuritis (particularly of the proventriculus), and Wallerian degeneration of the sciatic nerves. Avian bornavirus (ABV) was identified in the brain by immunohistochemistry and reverse-transcriptase polymerase chain reaction. Sequencing of the reverse-transcriptase polymerase chain reaction product indicated the presence of ABV genotype 4, which is generally associated with disease in psittacine birds. Subsequent to the death of the pheasant, ABV genotype 4 was identified at autopsy from a juvenile white-bellied caique (Pionites leucogaster) in the same collection. We hypothesize that the pheasant became infected through contact with psittacine birds with which it shared an aviary. We believe this to be the first reported case of natural ABV infection in a bird in the Order Galliformes.

A case of pediatric ALL with t(16;21)(p11.2;q22) and FUS-ERG rearrangement.

A case of pediatric ALL with t(16;21)(p11.2;q22) and FUS-ERG rearrangement.

Expression of Drosophila forkhead transcription factors during kidney development

The Drosophila forkhead (Dfkh) family of transcription factors has over 40 family members. One Dfkh family member, BF2 (aka FoxD1), has been shown, by targeted disruption, to be essential for kidney development. In order to determine if other Dfkh family members were involved in kidney development and to search for new members of this family, reverse transcriptase polymerase chain reaction (RT-PCR) was performed using degenerate primers of the consensus sequence of the DNA binding domain of this family and developing rat kidney RNA. The RT-PCR product was used to probe RNA from a developing rat kidney (neonatal), from a 20-day old kidney, and from an adult kidney. The RT-PCR product hybridized only to a developing kidney RNA transcript of ∼2.3kb (the size of BF2). A lambda gt10 mouse neonatal kidney library was then screened, using the above-described RT-PCR product as a probe. Three lambda phage clones were isolated that strongly hybridized to the RT-PCR probe. Sequencing of the RT-PCR product and the lambda phage clones isolated from the developing kidney library revealed Dfkh BF2. In summary, only Dfkh family member BF2, which has already been shown to be essential for nephrogenesis, was identified in our screen and no other candidate Dfkh family members were identified.

High genetic diversity of hepatitis delta virus in eastern Turkey

Hepatitis delta virus (HDV) is a serious cause of liver-related mortality in patients infected with hepatitis B virus (HBV). Determination of genotypes of HDV and phylogenetic analysis are important for better understanding the pathogenesis of the liver diseases associated with HBV infection. The aim of this study was to determine the genotype or genotypes of HDV among chronically infected patients with HBV in eastern Turkey. A group of 113 patients infected with HBV and HDV were included in this study. The samples taken from the patients were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) and restriction enzyme cleavage. According to the results of the restriction enzyme analysis, all of the RT-PCR products were determined to be HDV genotype I. Furthermore, for phylogenetic analysis and genotyping, 40 of HDV RT-PCR positive products were sequenced. Phylogenetic analysis of the sequences showed that all of the samples were infected with HDV genotype I. In addition, the results of the alignment analysis showed that the sequences of clinical samples were 82%-95% similar. These results indicate that high genetic diversity of the virus is possible in endemic areas such as Turkey.

EWSR1-CREB3L1 Gene Fusion

Low-grade fibromyxoid sarcoma (LGFMS) is an uncommon sarcoma with a deceptively bland-looking morphology that disguises its malignant clinical behavior. It shows distinctive chromosomal translocations resulting in fusion of FUS with the CREB3L2 gene in most cases and CREB3L1 in rare cases. Thus molecular studies are particularly helpful in the diagnosis of this bland-looking sarcoma. We report 2 cases of LGFMS serendipitously found to harbor a novel alternative EWSR1-CREB3L1 gene fusion, as confirmed by DNA sequencing of reverse transcriptase-polymerase chain reaction products and fluorescence in situ hybridization. One patient was a child who presented with a subcutaneous nodule on the lower leg, and the other was a middle-aged woman who had a mass lesion over the proximal thigh. Morphologically, one case showed a spindle cell tumor with hyalinization and giant rosettes, whereas the other showed classical histology of LGFMS with focal metaplastic bone formation. Immunostaining for MUC4 showed extensive positive staining. Our findings therefore expand the spectrum of gene fusions that characterize LGFMS and suggest that the EWSR1 gene may substitute for the function of FUS in gene fusions of sarcoma.

Impact of probiotic-supplemented diet on the expression level of lactate dehydrogenase in the leukocytes of rabbits

Probiotics are known as living, nonpathogenic microorganisms that colonize the intestine and provide benefit to the host. The present study aims to measure one important energy metabolism-related enzyme activity in blood of rabbits fed on probiotics of recommended concentration. In addition, it also aims for the evaluation of the expression level of lactate dehydrogenase (LDH) enzyme using reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Two groups of rabbits are used: control group receiving normal standardized diet and the other probiotic-supplemented group receiving the same diet containing probiotic, namely, Mega acidophilus (200millioncfu/kg body weight/day) for 4weeks. The obtained results revealed that the rabbits supplemented with probiotics showed a significant decrease in the levels of serum total cholesterol (TC), triacylglycerol, high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) when compared with control group. Risk factors detected by measuring TC/HDL-c and LDL-c/HDL-c ratios showed statistically significant decrease in probiotic-supplemented rabbits when compared with control group. In addition, blood glucose and total LDH activity were elevated in probiotic-supplemented rabbits when compared with control group. RT-PCR products of LDH-M gene produced two specific amplicons. One amplicon has the expected size of 243bp from all samples of rabbits as revealed by GelPro software. The level of LDH-M expression was found to be increased in the probiotic-supplemented group. However, unexpected amplicons are produced at 586bp in all the samples, which may be a dimeric form of the amplified region. It was concluded that this probiotic blend is beneficiary for the metabolic reactions of lipids in the body. Moreover, LDH expression level can be considered as a biomarker for the effect of probiotic and hence monitoring the metabolic changes as reflected from its administration.

PCR–ELISA detection of estrogen receptor β mRNA expression and plasma vitellogenin induction in juvenile sole (Solea solea) exposed to waterborne 4-nonylphenol

In this study, the effect of 4-nonylphenol (4NP) on reproductive axis of sole (Solea solea) has been investigated by using selected biomarkers of estrogenic effects: vitellogenin (VTG) and estrogen receptor β (ERβ) mRNA. Furthermore, an enzyme linked immunosorbent assay (ELISA) detection system of reverse transcriptase-polymerase chain reaction (RT-PCR) products for the analysis of sole ERβ mRNA expression was developed and validated. The proposed method allows large-scale analyses of different mRNAs in fast and not expensive way. Our results have demonstrated that the PCR–ELISA method developed shows high sensitivity, good reproducibility and also the potential for semi-quantitative analysis of hepatic ERβ mRNAs. Both plasma VTG level and ERβ mRNA expression were increased in tested animals following a short exposure to environmental relevant concentrations (10−6M) of 4NP, confirming the functional role of ERβ in the regulation of xenoestrogens-induced production of VTG. The methodology provided in the present study together with the preliminary results on the hepatic expression of ERβ may be useful in environmental xenoestrogens monitoring studies, using flatfish as “sentinel” species.

Sequence and expression of the chicken membrane-associated phospholipases A1 alpha (LIPH) and beta (LIPI)

Cancer/testis antigens (CTA) are a heterogeneous group of antigens that are expressed preferentially in tumor cells and testis. Based on this definition the human membrane-associated phospholipase A1 beta (lipase family member I, LIPI) has been identified as CTA. The high homology of LIPI and the membrane-associated phospholipase A1 alpha (lipase family member H, LIPH) suggests that both genes are derived from a common ancestor by gene duplication. In contrast to human LIPI, human LIPH is expressed in several tissues. LIPI sequences have only been identified in mammals. Here, we describe the identification of LIPI in non-mammalian vertebrates. Based on the conserved genomic organization of LIPI and LIPH we identified sequences for both lipases in birds and fishes. In all vertebrates the LIPI locus is neighbored by a member of the RNA binding motif (RBM) family, RBM11. By sequencing of reverse transcriptase-polymerase chain reaction products we determined the sequences of LIPI and LIPH messenger RNA from broilers. We found that the sequence homology between LIPI and LIPH is much higher in non-mammalian species than in mammals. In addition, we found broad expression of LIPI in broilers, resembling the expression profile of LIPH. Our data suggest that LIPI is a CTA only in mammalian species and that the unique sequence features of the mammalian LIPI/RBM11 locus have evolved together with the CTA-like expression pattern of LIPI.

First isolation and genotyping of viruses from recent outbreaks of viral haemorrhagic septicaemia (VHS) in Slovenia

In November and December 2007, the virus causing viral haemorrhagic septicaemia (VHS) was detected in rainbow trout Oncorhynchus mykiss from 2 fish farms in Slovenia. During 2008 and 2009 the infection spread only among rainbow trout farms and 4 new outbreaks were confirmed. High mortality and clinical signs of VHS were observed among the diseased fish. VHSV was confirmed by virus isolation, immunoperoxidase test, reverse transcriptase polymerase chain reaction (RT-PCR) and phylogenetic analysis. Based on 1 complete (1524 nucleotides [nt]) and 9 partial (600 nt) glycoprotein gene nucleotide sequences, 9 VHSV isolates from the 6 VHS outbreaks were genetically closely related (99 to 100% identity), and were classified into the Subgroup I-a of Genotype I, most closely related to the German isolates Dstg21-07, Dstg36-06, and Dstg54-1-07 (99 to 100% identity). Phylogenetic analysis and epidemiological investigations confirmed that the VHS virus had been (re)introduced with imported live fish, and that subsequent outbreaks were linked to the initial infection. Our study shows that direct nucleotide sequencing of RT-PCR products, amplified from the tissue of VHSV-infected fish, represents a reliable tool for fast routine genotyping in diagnostic laboratories. This is the first report of a natural epidemic associated with VHSV infection in Slovenia since the eradication of the disease in 1977.

Overexpression of aldo‐keto reductase 1C2 is associated with disease progression in patients with prostatic cancer

Prostatic cancer is resistant to chemotherapy. Expression of aldo-keto reductase 1C (AKR1C) has been associated with drug resistance and disease progression in several cancers. The aim was to investigate the relationship between AKR1C expression and disease progression in prostatic cancer. From January 1996 to December 2005, 86 pathological samples were collected from patients with prostatic cancer. A tissue microarray containing 31 prostatic cancers from American patients was used for comparison between Chinese and American patients. Using immunohistochemistry, aldo-keto reductase family 1, member C2 (AKR1C2) expression was assessed in tissue sections. The AKR1C2 was determined by two-dimensional immunoblotting and DNA sequencing of reverse transcriptase-polymerase chain reaction products. The relationship between AKR1C2 expression and clinicopathological variables was statistically analysed. In vitro, the association between AKR1C2 expression and drug resistance was investigated in androgen-sensitive and androgen-insensitive prostatic cancer cells. DNA sequencing and two-dimensional immunoblotting showed that prostatic cancer expressed AKR1C2. It was overexpressed in 77 of 86 (89.5%) Chinese and in 28 of 31 (90.3%) American samples. No difference was found in AKR1C2 expression between Chinese and American prostatic cancer patients. In vitro, increased expression of AKR1C2 and prostaglandin F2α correlated with cytoprotection against anticancer drugs and lycopene. Overexpression of AKR1C2 is associated with disease progression in prostatic cancer.

Molecular cloning of gene fragment encoding 4-coumarate: Coenzyme A ligase of Sengon (Paraserianthes falcataria)

4-coumarate:Coenzyme A ligase (4CL) plays an important role in lignin biosynthetic pathway that catalyzed the activation of coumaric acid, caffeic acid or ferulic acid to be a syringil monomer. Lignin biosynthesis control through 4CL down regulating would support lower lignin wood production. The objective of this study was to clone conserved region cDNA of gene encoding 4CL. Gene fragment isolation was conducted by means of reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate heterologous primer. The RT-PCR products were purified, sequenced and analyzed to select the highly homologous fragment to 4CL. BLASTanalysis result showed that deduction of amino acid sequences from one of two RT-PCR product nucleotide was highly homologous with the 4CL conserved region from Rubbus ideaus, Oryza sativa, Populus tomentosa, Populus balsamifera, Betulla platyphilla, Nicotiana tabacum, and Arabidopsis thaliana with identity ranging from 78-90%. Key words: 4-coumarate: Coenzyme A ligase, lignin, sengon

Cryptic EWSR1- FLI1 fusions in Ewing sarcoma: potential pitfalls in the diagnostic use of fluorescence in situ hybridization probes

Detection of EWSR1 translocations — particularly t(11;22)(q24;q12) — is of great value in the differential diagnosis of the Ewing family of tumors. We report two cases that highlight the problems and pitfalls of identifying Ewing tumors using conventional chromosome analysis and a commercial EWSR1 fluorescence in situ hybridization (FISH) probe. In both cases, the tumor karyotype was abnormal, but a visible t(11;22)(q24;q12) was not present. The commercial EWSR1 “break-apart” probe was not split in either case. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, however, identified EWSR1-FLI1 fusion transcripts in both tumors, and the gene fusions were corroborated by FISH analysis with “in house” probes and confirmed by sequencing RT-PCR products. The occurrence of cryptic EWSR1-FLI1 fusions mandates that RT-PCR should be performed, particularly in those cases in which the genetic findings are not in agreement with the histologic picture.