Reverse Transcription-polymerase Chain Reaction Primers Research Articles

Development of multiplex reverse transcription-polymerase chain reaction for differentiation of strains of infectious bursal disease virus and primary screening of the virus in Thailand.

Background and Aim:A new set of primers (400 base pairs partial of VP2) was designed and used for the infectious bursal disease virus (IBDV) screening test. Using this new primer set, the enzymes MboI and BstNI were unable to differentiate the field and vaccine strains. As a result, a new simple, cheap, and appropriate tool for strain differentiation is required. The objective of this study was to develop the appropriate restriction fragment length polymorphism (RFLP) and multiplex reverse transcription-polymerase chain reaction (RT-PCR) for the differentiation of classic IBDV (cIBDV) strains and very virulent IBDV (vvIBDV) strains in Thailand.Materials and Methods:Ninety seven bursa of Fabricius from 16 farms were collected from farms in the eastern and central regions of Thailand. RT-PCR screening showed that 82 samples were positive for IBDV and 15 samples were negative. Then, selected samples were sequenced from each farm with a positive test.Results:The sequencing results showed that samples from six of the farms were vvIBDV and samples from the other six farms were cIBDV. Although the whole genome sequencing was incomplete, both the sequencing results of segment A and segment B showed high similarity between cIBDV and vvIBDV. Restriction enzyme cutting site and primers for multiplex RT-PCR were hard to design. An RT-PCR-RFLP method was developed, but it failed to differentiate IBDV strains. However, the multiplex RT-PCR was able to differentiate cIBDV from vvIBDV. Four primers were used in the multiplex RT-PCR.Conclusion:These four primers were used together in one reaction at an annealing temperature of 45°C. Therefore, multiplex RT-PCR is a less complicated, cheaper, and less time-consuming method for the differentiation of cIBDV and vvIBDV strains.

Development of diagnostic systems for wide range and highly sensitive detection of two waterborne hepatitis viruses from groundwater using the conventional reverse transcription nested PCR assay

Waterborne epidemics of human hepatitis virus A and E (HAV and HEV) have been reported worldwide. Molecular biology techniques, such as reverse transcription polymerase chain reaction (RT-PCR), have been widely used to detect the two hepatitis viruses. However, comparative studies of various types of samples are needed, and different environmental factors, including the low copy pathogens, presence of PCR inhibitors in the sample, unknown non-specific reaction with template, and sequence diversity leading to new variants in viruses, should be considered. In addition, standard positive material is required to determine the accuracy of the PCR and should be able to distinguish between false and real positives. In this study, we developed RT-PCR primer sets and optimised standard templates for HAV and HEV detection to address the above concerns associated with test sensitivity and possible PCR inhibition. Finally, previously reported diagnostic methods of HAV and HEV were compared and an applicability test using groundwater was performed. The nested RT-PCR developed in this study is expected to contribute to assess water safety by monitoring HAV and HEV in non-disinfected water, like groundwater.

Analysis and forecasting of global real time RT-PCR primers and probes for SARS-CoV-2

Rapid tests for active SARS-CoV-2 infections rely on reverse transcription polymerase chain reaction (RT-PCR). RT-PCR uses reverse transcription of RNA into complementary DNA (cDNA) and amplification of specific DNA (primer and probe) targets using polymerase chain reaction (PCR). The technology makes rapid and specific identification of the virus possible based on sequence homology of nucleic acid sequence and is much faster than tissue culture or animal cell models. However the technique can lose sensitivity over time as the virus evolves and the target sequences diverge from the selective primer sequences. Different primer sequences have been adopted in different geographic regions. As we rely on these existing RT-PCR primers to track and manage the spread of the Coronavirus, it is imperative to understand how SARS-CoV-2 mutations, over time and geographically, diverge from existing primers used today. In this study, we analyze the performance of the SARS-CoV-2 primers in use today by measuring the number of mismatches between primer sequence and genome targets over time and spatially. We find that there is a growing number of mismatches, an increase by 2% per month, as well as a high specificity of virus based on geographic location.

Molecular epidemiology and phylogenetic analysis of bovine picobirnaviruses causing calf diarrhea, in Iran.

Picobirnavirus (PBV) is an enteropathogen virus causing diarrhea as an opportunistic virus in its vertebrate host. There is no information about human or animal PBVs in Iran. The aim of the present study was the investigation of the epidemiology of bovine PBV in the broad geographical area of Iran. Four hundred and eighty-five stool samples of up to 1 month old diarrheic calves were collected from 14 provinces and were tested with polyacrylamide gel electrophoresis (PAGE), and reverse transcription polymerase chain reaction (RT-PCR). Five samples were positive in PAGE assay (1.00%) and all of them were amplified using GI specific primers in RT-PCR. Phylogenetic analysis of one of the amplicons (strain Nazaktabar-14) revealed a low relationship to bovine PBV sequences and more identity to PBV isolates from other hosts. The structural alignment of the deduced amino acids of the partially sequenced RdRp gene of the Nazaktabar-14 strain showed high conservation. Sequences obtained from other amplicons showed a high mutation rate and further analysis of one of them showed that, despite the potential of forming deleterious mutations, most of the point mutations occurred in the RdRp gene of PBVs may be a silent mutation. There is little information about the molecular epidemiology of bovine PBVs. This study was the first report on the occurrence of PBVs in Iran and the first study on the molecular epidemiology of bovine PBV in the Middle East, revealing its low frequency as a diarrhea causative agent.

English

Most patients presenting with febrile conditions are often treated for malaria, especially in the developing world, whereas some of them may be of arboviral origin as they also present with similar symptoms. Reverse transcription-polymerase chain reaction (RT-PCR) has been found to be the method of choice for the early detection and confirmation of virus in clinical samples, especially where there is an overlap of symptoms. The objective of this study was to detect the involvement of some arboviruses in febrile conditions in humans visiting two health institutions in Ile-Ife, Nigeria. Consenting febrile patients numbering one hundred and sixty five that were to be screened for malaria parasites at the hospitals were recruited for the study. From each patient, 2 ml venous blood was collected and processed for RNA extraction using QiAmp RNA Extraction kit (Qiagen, Hilden, Germany) and amplified using one step RT-PCR and appropriate primers for West Nile, Chikungunya, Dengue and Rift valley fever viruses. The detection was done in 2% agarose gel electrophoresis and viewed using a gel imager. The study reports the detection of West Nile RNA in 6 (3.6%) patients and Chikungunya RNA in 3 (1.8%) out of the 165 serum samples which have been pre-screened for malaria parasite by the hospital where the samples were collected from. Rift valley fever virus RNA and Dengue virus RNA were not detected in any of the samples. Out of the malaria parasite negative patients, 3 tested positive for the West Nile RNA and 1 showed detectable Chikungunya virus RNA, thus suggesting the role of these arboviruses in febrile conditions, first to be reported in Osun state, Nigeria. The involvement of viruses in febrile conditions as shown by this study has buttressed the need to extend laboratory examination of febrile conditions beyond malaria parasite to some of these arboviruses whose vectors are abundant and extending geographical coverage. Key words: Febrile, malaria, Chikungunya virus, West Nile virus, Dengue virus, reverse transcription-polymerase chain reaction (RT-PCR).

Serosurveillance and establishment of a reverse transcription-polymerase chain reaction assay for bovine parainfluenza virus type 5

Abstract : Bovine parainfluenza virus type 5 (bPIV5) was isolated from cattle with downer cow syndrome in 2012,and included both respiratory and neurotropic pathogens from a variety of animals. In the current study, we conductedserosurveillance using sera obtained from seven Korean farms and optimized a reverse transcription-polymerase chainreaction (RT-PCR) assay to detect bPIV5. The overall seropositive rate for Korean cattle was 21.4% (163/760). Afarm located near the city of Milyang in Gyeoungnam province had a markedly elevated seropositive rate for bPIV5compared to that of the other six farms. The regional seropositive rates were 4.2% (8/192) for Haman, 19.5% (18/55) for Hwasung, 73.9% (65/88) for Milyang, 26.0% (50/192) for Namwon, 1.0% (1/96) for Uljin, 13.5% (13/96)for Yeongju, and 32.7% (8/41) for Yongin. The sensitivity and specificity of three RT-PCR primer sets used to amplifythe conserved fusion gene of bPIV5 were also evaluated. An RT-PCR assay using the bPIVFR3 primer set was 10-fold more sensitive than the assays using the two other primer sets and did not result in non-specific amplification.These results demonstrated that the bPIFR3 primer set can be used to detect bPIV5.Keywords : bovine, parainfluenza virus 5, reverse transcription-polymerase chain reaction, serosurveillance

Identification and Molecular Characterization of Nuclear Citrus leprosis virus, a Member of the Proposed Dichorhavirus Genus Infecting Multiple Citrus Species in Mexico.

Citrus leprosis is one of the most destructive diseases of Citrus spp. and is associated with two unrelated virus groups that produce particles primarily in either the cytoplasm or nucleus of infected plant cells. Symptoms of leprosis, including chlorotic spots surrounded by yellow haloes on leaves and necrotic spots on twigs and fruit, were observed on leprosis-affected mandarin and navel sweet orange trees in the state of Querétaro, Mexico. Serological and molecular assays showed that the cytoplasmic types of Citrus leprosis virus (CiLV-C) often associated with leprosis symptomatic tissues were absent. However, using transmission electron microscopy, bullet-shaped rhabdovirus-like virions were observed in the nuclei and cytoplasm of the citrus leprosis-infected leaf tissues. An analysis of small RNA populations from symptomatic tissue was carried out to determine the genome sequence of the rhabdovirus-like particles observed in the citrus leprosis samples. The complete genome sequence showed that the nuclear type of CiLV (CiLV-N) present in the samples consisted of two negative-sense RNAs: 6,268-nucleotide (nt)-long RNA1 and 5,847-nt-long RNA2, excluding the poly(A) tails. CiLV-N had a genome organization identical to that of Orchid fleck virus (OFV), with the exception of shorter 5' untranslated regions in RNA1 (53 versus 205 nt) and RNA2 (34 versus 182 nt). Phylogenetic trees constructed with the amino acid sequences of the nucleocapsid (N) and glycoproteins (G) and the RNA polymerase (L protein) showed that CiLV-N clusters with OFV. Furthermore, phylogenetic analyses of N protein established CiLV-N as a member of the proposed genus Dichorhavirus. Reverse-transcription polymerase chain reaction primers for the detection of CiLV-N were designed based on the sequence of the N gene and the assay was optimized and tested to detect the presence of CiLV-N in both diseased and symptom-free plants.

Establishment and Application of a Reverse Transcription Loop-mediated Isothermal Amplification Assay for Detection of Grapevine Fanleaf Virus

Grapevine degeneration in grapevines caused by Grapevine Fanleaf Virus (GFLV) has been documented in many viticulture regions worldwide. It is one of the major economically important virus diseases affecting the longevity of grapevines and reducing the fruit yield and fruit quality. To diminish the time required for some diagnostic assays, including reverse transcription polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP) and also double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) into a minimum level, an innovative immunocapture RT-LAMP (IC-RT-LAMP) and immunocapture RT-PCR (IC-RT-PCR) protocol on the basis of GFLV genome were used and the reaction conditions were optimized for rapid detection of GFLV. In this regard, DAS-ELISA was employed first to validate the existence of the virus. All six RT-LAMP primers (i.e., F3, B3, FIP, BIP, LF and LB) together with RT-PCR primers (F and B) were designed on the basis of coat protein (CP) gene of GFLV. LAMP method, on the whole, had the following advantages over the other mentioned procedures: (I) fascinatingly, no need of RNA extraction; (II) no requirement of expensive and sophisticated tools for amplification and detection; (III) no post-amplification treatment of the amplicons; and (IV) a flexible and easy detection approach, which is visually detected by naked eyes using diverse visual dyes.

A Novel Virus of the Genus Cilevirus Causing Symptoms Similar to Citrus Leprosis

Citrus leprosis in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR)-based diagnostic methods failed to identify CiLV-C from citrus samples with symptoms similar to citrus leprosis; however, virions similar to CiLV-C were observed in the cytoplasm of the symptomatic leaves by transmission electron microscopy. Furthermore, the causal organism was transmitted by the false spider mite, Brevipalpus phoenicis, to healthy citrus seedlings. A library of small RNAs was constructed from symptomatic leaves and used as the template for Illumina high-throughput parallel sequencing. The complete genome sequence and structure of a new bipartite RNA virus was determined. RNA1 (8,717 nucleotides [nt]) contained two open reading frames (ORFs). ORF1 encoded the replication module, consisting of five domains: namely, methyltransferase (MTR), cysteine protease-like, FtsJ-MTR, helicase (Hel), and RNA-dependent RNA polymerase (RdRp); whereas ORF2 encoded the putative coat protein. RNA2 (4,989 nt) contained five ORFs that encode the movement protein (MP) and four hypothetical proteins (p7, p15, p24, and p61). The structure of this virus genome resembled that of CiLV-C except that it contained a long 3' untranslated terminal region and an extra ORF (p7) in RNA2. Both the RNA1 and RNA2 of the new virus had only 58 and 50% nucleotide identities, respectively, with known CiLV-C sequences and, thus, it appears to be a novel virus infecting citrus. Phylogenetic analyses of the MTR, Hel, RdRp, and MP domains also indicated that the new virus was closely related to CiLV-C. We suggest that the virus be called Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) and it should be unambiguously classified as a definitive member of the genus Cilevirus. A pair of CiLV-C2 genome-specific RT-PCR primers was designed and validated to detect its presence in citrus leprosis samples collected from the Casanare and Meta states in Colombia.

Development of a sensitive RT-PCR method for amplifying and sequencing near full-length HCV genotype 1 RNA from patient samples

BackgroundDirect-acting antiviral (DAAs) agents for hepatitis C virus (HCV) span a variety of targets, including proteins encoded by the NS3/4A, NS4B, NS5A, and NS5B genes. Treatment with DAAs has been shown to select variants with sequence changes in the HCV genome encoding amino acids that may confer resistance to the treatment. In order to assess these effects in patients, a Reverse Transcription Polymerase Chain Reaction (RT-PCR) method was developed to sequence these regions of HCV from patient plasma.MethodsA method was developed to amplify and sequence genotype 1 HCV RNA from patient plasma. Optimization of HCV RNA isolation, cDNA synthesis, and nested PCR steps were performed. The optimization of HCV RNA isolation, design of RT-PCR primers, optimization of RT-PCR amplification conditions and reagents, and the evaluation of the RT-PCR method performance is described.ResultsThe optimized method is able to successfully, accurately, and reproducibly amplify near full-length genotype 1 HCV RNA containing a wide range of concentrations (103 to 108 IU/mL) with a success rate of 97%. The lower limit of detection was determined to be 1000 IU/mL HCV RNA.ConclusionsThis assay allows viral sequencing of all regions targeted by the most common DAAs currently in development, as well as the possibility to determine linkage between variants conferring resistance to multiple DAAs used in combination therapy.

Isolating of a putative glyceraldehyde-3 phosphate dehydrogenase (GAPDH) from Calophyllum brasiliense, an important tropical forest tree

Abstract Calophyllum brasiliense Cambess. has characteristics that made it an excellent candidate model for ecogenomics in rain forest trees such as widespread natural occurrence and geographical patterns of adaptive genetic variation. Besides, it is also becoming a popular species for reforestation efforts in Brazil. Although, very little is known about its genetic diversity and the molecular mechanisms involved genetic adaptation traits. The first difficulty in launching genetic studies in a wild wood species is the lack of an optimized protocol for RNA and DNA isolation. In this work we built the essential framework for molecular genetics research with C. brasiliense comparing four distinct methods of RNA extraction from of three different tissues: leaves, stems and roots. We also were successful in the isolation of genomic DNA by an optimized CTAB method. Finally, degenerated oligonucleotide primers were designed for isolating of the glyceraldehyde-3 phosphate dehydrogenase (GAPDH) gene of C. brasiliense from the corresponding gene in closely related species. This gene is commonly used in plants as reference in expression gene analysis by Reverse Transcription Polymerase Chain Reaction (RT-PCR). Primers for RT-PCR were designed based on partial sequence obtained using degenerate primers designed. The optimized methods of RNA and DNA extraction combined with the identification, isolation and specific primer design for RT-PCR of a traditional Reference Gene provide the essential framework for molecular genetics research with C. brasiliense.

Gene Expression and Metabolite Profiling of Developing Highbush Blueberry Fruit Indicates Transcriptional Regulation of Flavonoid Metabolism and Activation of Abscisic Acid Metabolism

Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3'-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3'5'-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of blueberry flavonoid biosynthesis.

RT-PCR Detection of Five Quarantine Plant RNA Viruses Belonging to Potyand Tospoviruses

In order to detect quarantine plant viruses, we developed reverse transcription-polymerase chain reaction (RT-PCR) primer pairs for five single-stranded (ss) plant RNA viruses that are not currently reported in Korea but could be potential harmful plant viral pathogens. Three viruses such as Chilli veinal mottle virus (ChiVMV), Colombian datura virus (CDV), and Tobacco etch virus (TEV) belong to the genus Potyvirus while Chrysanthemum stem necrosis virus (CSNV) and Iris yellow spot virus (IYSV) are members of the genus Tospovirus. To design RT-PCR primers, we used reported gene sequences corresponding to the capsid protein and polyprotein for ChiVMV, CDV, and TEV while using nucleocapsid protein regions for CSNV and IYSV. At least two different primer pairs were designed for each virus. Fifteen out of 16 primer pairs were successfully applied in detection of individual quarantine virus with high specificity and efficiency. Taken together, this study provides a rapid and useful protocol for detection of five quarantine viruses.

Snake melon asteroid mosaic virus, a Tentative New Member of the Genus Sobemovirus Infecting Cucurbits.

A virus isolate (Su-95-67) was obtained from a snake melon (Cucumis melo var. flexuosus) plant presenting severe chlorotic spots, mosaic, stunting, and leaf deformations collected in Eastern Sudan in 1995. Su-95-67 was easily mechanically transmissible and had a host range limited to a few cucurbit species. Isometric virus particles approximately 30 nm in diameter were observed in leaf dip preparations. A cytopathological study did not reveal alterations specific for a virus genus or family. A polyclonal antiserum was obtained and used in double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Su-95-67 was transmitted by seed at a low rate, by the red melon beetle (Aulacophora foveicollis), but not by the melon aphid (Aphis gossypii). Because Su-95-67 shared several properties with sobemoviruses, generic Sobemovirus reverse-transcription polymerase chain reaction primers were developed. They allowed amplification of a 384-bp fragment from extracts of plants infected by two sobemoviruses or by Su-95-67 but not from healthy plants extracts. Sequence comparison confirmed that Su-95-67 belongs to a new tentative Sobemovirus species for which we propose the name Snake melon asteroid mosaic virus (SMAMV). DAS-ELISA tests conducted on extracts of virus-infected cucurbit plants collected from 1992 to 2003 revealed the presence of SMAMV in 10.2% of 600 samples originating from different regions of Sudan.

Large-Scale Analysis of Putative Soybean Regulatory Gene Expression Identifies a Myb Gene Involved in Soybean Nodule Development

Nodulation is the result of a symbiosis between legumes and rhizobial bacteria in soil. This symbiosis is mutually beneficial, with the bacteria providing a source of nitrogen to the host while the plant supplies carbon to the symbiont. Nodule development is a complex process that is tightly regulated in the host plant cell through networks of gene expression. In order to examine this regulation in detail, a library of quantitative reverse transcription-polymerase chain reaction primer sets was developed for a large number of soybean (Glycine max) putative regulatory genes available in the current expressed sequence tag collection. This library contained primers specific to soybean transcription factor genes as well as genes involved in chromatin modification and translational regulation. Using this library, we analyzed the expression of this gene set during nodule development. A large number of genes were found to be differentially expressed, especially at the later stages of nodule development when active nitrogen fixation was occurring. Expression of these putative regulatory genes was also analyzed in response to the addition of nitrate as a nitrogen source. This comparative analysis identified genes that may be specifically involved in nitrogen assimilation, metabolism, and the maintenance of active nodules. To address this possibility, the expression of one such candidate was studied in more detail by expressing in soybean roots promoter beta-glucuronidase and green fluorescent protein fusions. This gene, named Control of Nodule Development (CND), encoded a Myb transcription factor gene. When the CND gene was silenced, nodulation was reduced. These results, associated with a strong expression of the CND gene in the vascular tissues, suggest a role for CND in controlling soybean nodulation.

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

BackgroundThe development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp.ResultsBiologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp.ConclusionAll protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

Geographical Distribution of Four Sugarcane yellow leaf virus Genotypes.

Specific primer pairs were designed to distinguish four genotypes (BRA for Brazil, CUB for Cuba, PER for Peru, and REU for Réunion Island) of Sugarcane yellow leaf virus (SCYLV) by reverse transcription-polymerase chain reaction (RT-PCR). A unique genome fragment was amplified from each genotype, with the exception of genotypes BRA and PER that are phylogenetically relatively close and were designated genotype BRA-PER. These RT-PCR primers were then used to identify the SCYLV genotype(s) present in 18 different sugarcane growing locations in the world, and 245 leaf samples infected by the virus were analyzed. Most samples were infected by only one of the three genotypes, but mixed infections occurred. Genotype BRA-PER was found in all sugarcane growing locations, whereas genotypes CUB and REU were each found in four geographical locations only. Genotypes BRA-PER, CUB, and REU were all three detected in locally bred sugarcane cultivars in Guadeloupe, indicating local transmission of these genotypes. In contrast, only genotypes BRA-PER and CUB were found in locally bred cultivars in Brazil, whereas genotype REU was detected in this country in cultivar R570 imported from Réunion. Similarly, genotypes BRA-PER and REU are both present in Réunion, but genotype BRA-PER has not, as of yet, spread on this island. Presence of several SCYLV genotypes in Brazil, Colombia, Guadeloupe, Mauritius, and Réunion suggests different virus introductions and/or different evolution histories of the virus after its introduction into a new environment.

An RT-PCR primer pair for the detection of Pospiviroid and its application in surveying ornamental plants for viroids

A primer pair for reverse transcription-polymerase chain reaction (RT-PCR), based on the conserved sequences of the members of genus Pospiviroid was designed to yield a fragment of about 200 base pairs (bp). Since pospiviroids infect a large number of plants species and a few members of the genus Pospiviroid have been already detected in some ornamental plants, the primer pair was evaluated for its efficacy using ornamental plants. The method of return-polyacrylamide gel electrophoresis (R-PAGE) was used to determine the general presence of viroids in the test samples. Efficacy of the primer pair for members of genus Pospiviroid was demonstrated by the detection of Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (TCDVd) in potato, Chrysanthemum stunt viroid and Iresine viroid in Verbena and Vinca species, and Citrus exocortis viroid in Impatiens species. Specificity of the primer pair became evident, where additional viroids were detected by R-PAGE in Coleus and Magilla species, but they were not amplified by the Pospiviroid primer. This primer pair would be of benefit in indexing ornamental plants in quarantine samples or in viroid-free certification schemes, irrespective of their actual identity.

A novel coronavirus associated with severe acute respiratory syndrome.

A worldwide outbreak of severe acute respiratory syndrome (SARS) has been associated with exposures originating from a single ill health care worker from Guangdong Province, China. We conducted studies to identify the etiologic agent of this outbreak. We received clinical specimens from patients in seven countries and tested them, using virus-isolation techniques, electron-microscopical and histologic studies, and molecular and serologic assays, in an attempt to identify a wide range of potential pathogens. None of the previously described respiratory pathogens were consistently identified. However, a novel coronavirus was isolated from patients who met the case definition of SARS. Cytopathological features were noted in Vero E6 cells inoculated with a throat-swab specimen. Electron-microscopical examination revealed ultrastructural features characteristic of coronaviruses. Immunohistochemical and immunofluorescence staining revealed reactivity with group I coronavirus polyclonal antibodies. Consensus coronavirus primers designed to amplify a fragment of the polymerase gene by reverse transcription-polymerase chain reaction (RT-PCR) were used to obtain a sequence that clearly identified the isolate as a unique coronavirus only distantly related to previously sequenced coronaviruses. With specific diagnostic RT-PCR primers we identified several identical nucleotide sequences in 12 patients from several locations, a finding consistent with a point-source outbreak. Indirect fluorescence antibody tests and enzyme-linked immunosorbent assays made with the new isolate have been used to demonstrate a virus-specific serologic response. This virus may never before have circulated in the U.S. population. A novel coronavirus is associated with this outbreak, and the evidence indicates that this virus has an etiologic role in SARS. Because of the death of Dr. Carlo Urbani, we propose that our first isolate be named the Urbani strain of SARS-associated coronavirus.

A reassortant bunyavirus isolated from acute hemorrhagic fever cases in Kenya and Somalia.

In late 1997 and early 1998, a large outbreak of hemorrhagic fever occurred in East Africa. Clinical samples were collected in Kenya and southern Somalia, and 27 of 115 (23%) hemorrhagic fever patients tested showed evidence of acute infection with Rift Valley fever (RVF) virus as determined by IgM detection, virus isolation, detection of virus RNA by reverse transcription-polymerase chain reaction (RT-PCR), or immunohistochemistry. However, two patients (one from Kenya and the other from Somalia) whose illness met the hemorrhagic fever case definition yielded virus isolates that were not RVF. Electron microscopy suggested these two virus isolates were members of the family Bunyaviridae. RT-PCR primers were designed to detect bunyavirus RNA in these samples. Regions of the S and L segments of the two isolates were successfully amplified, and their nucleotide sequences exhibited nearly complete identity with Bunyamwera virus, a mosquito-borne virus not previously associated with severe human disease. Unexpectedly, the virus M segment appeared to be reassorted, as the sequences detected exhibited 32-33% nucleotide and 28% amino acid differences relative to the corresponding M segment sequence of Bunyamwera virus. The association of this reassortant bunyavirus, proposed name Garissa virus, with severe disease is supported by the detection of the virus RNA in acute-phase sera taken from 12 additional hemorrhagic fever cases in the region.