Specific Reverse Transcription Research Articles

Genogroup I and II Picobirnaviruses in Respiratory Tracts of Pigs

Sequence-independent amplification and specific reverse transcription PCRs identified genogroup I and II picobirnaviruses in respiratory tracts of pigs. These data expand knowledge of picobirnavirus diversity and tropism. Genetic relationships between porcine respiratory and human enteric picobirnaviruses suggest cross-species transmission of picobirnaviruses between pigs and humans.

Development of reverse transcriptase polymerase chain reaction enzyme-linked immunosorbent assay for the detection of hepatitis A virus in vegetables

This study’s aim was to develop a specific and sensitive reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (RT-PCR-ELISA) for hepatitis A virus (HAV). Three concentration methods were compared using samples from cabbage, lettuce, and sesame leaves that were artificially contaminated with HAV. The specificity of the assay was tested against human norovirus, hepatitis E virus, rotavirus, enterovirus, and FCV. The sensitivity and specificity of RT-PCR-ELISA that were targeted to the 5′NCR and VP1 regions of HAV were compared. Nonspecific reactions were not observed. An optimal primer and probe set for RT-PCR-ELISA was selected for each VP1 and 5′NCR. The detection limit of the RT-PCR-ELISA was enhanced by 10–100 fold more than nested RT-PCR. Our new RT-PCR-ELISA was successfully optimized to screen vegetables for HAV contamination with high sensitivity in large samples.

Development and validation of a reverse transcription quantitative PCR for universal detection of viral hemorrhagic septicemia virus

Viral hemorrhagic septicemia virus (VHSV) infects over 70 fish species inhabiting marine, brackish or freshwater environments throughout the Northern Hemisphere. Over its geographic range, 4 VHSV genotypes and multiple subtypes exist. Here, we describe the development and validation of a rapid, sensitive and specific real-time reverse transcription quantitative PCR assay (RT-qPCR) that amplifies sequence from representative isolates of all VHSV genotypes (I, II, III and IV). The pan-specific VHSV RT-qPCR assay reliably detects 100 copies of VHSV nucleoprotein RNA without cross-reacting with infectious hematopoietic necrosis virus, spring viremia of carp virus or aquatic birnavirus. Test performance characteristics evaluated on experimentally infected Atlantic salmon Salmo salar L. revealed a diagnostic sensitivity (DSe) > or = 93% and specificity (DSp) = 100%. The repeatability and reproducibility of the procedure was exceptionally high, with 93% agreement among test results within and between 2 laboratories. Furthermore, proficiency testing demonstrated the VHSV RT-qPCR assay to be easily transferred to and performed by a total of 9 technicians representing 4 laboratories in 2 countries. The assay performed equivalent to the traditional detection method of virus isolation via cell culture with the advantage of faster turnaround times and high throughput capacity, further suggesting the suitability of the use of this VHSV RT-qPCR in a diagnostic setting.

Development of duplex RT-PCR-ELISA for the simultaneous detection of hepatitis A virus and hepatitis E virus

This study aimed to develop a specific and sensitive duplex reverse transcription polymerase chain reaction enzyme-linked immunosorbent assay (duplex RT-PCR-ELISA) for hepatitis A virus (HAV) and hepatitis E virus (HEV). Duplex RT-PCR-ELISA could detect and differentiate HAV and HEV with specific probes. When ELISA technique was used to detect probe-bound RT-PCR products, duplex RT-PCR-ELISA could detect as little as 0.1ng/μL HAV and HEV from clinical samples. Human norovirus, enterovirus, poliovirus, murine norovirus and feline calicivirus were used for the specificity test; all were negative. Therefore duplex RT-PCR-ELISA can be used for the simultaneous detection of HAV and HEV in contaminated fecal samples.

DNA methylation and histone modification relate to RASSF1A gene deletion in laryngeal carcinoma tissues

To investigate the relationship between RASSF1A gene expression and DNA methylation or histone modification in laryngeal carcinoma tissues. Chromatin immunoprecipitation (ChIP), methylation specific polymerase chain reaction (MSP) and realtime quantitative reverse transcription polymerase chain reaction (realtime RT-PCR) were used to analyze RASSF1A gene promoter region histone H3 lysine 9 methylation, H3 lysine 4 methylation, H3 lysine 9 acetylation, DNA methylation, and RASSF1A gene expression in laryngeal carcinoma tissue of 50 cases. DNA methylation rate of gene RASSF1A was 62% in 50 cases of laryngeal carcinoma, but no DNA methylation was found in normal control group, with a significant difference (χ(2) = 15.381, P < 0.05). DNA methylation had no correlation with age, gender, differentiation degree, T stage, pathological type and lymph node metastasis (P > 0.05). The affection of DNA methylation group was more than unmethylation group to expression of gene RASSF1A (t = -3.108, P < 0.01). There was positive correlation between RASSF1A deletion and gene hypermethylation or between H3 lysine 9 methylation of RASSF1A gene promoter and DNA methylation in laryngeal carcinoma tissue(r = 0.816, P < 0.05), but there was negative correlation between H3 lysine 4 methylation of RASSF1A gene promoter and DNA methylation (r = -0.837, P < 0.05) and no correlation between H3 lysine 9 acetylation and DNA methylation (r = -0.383, P > 0.05). Laryngeal tumor suppressor gene RASSF1A promoter methylation is a key factor down-regulating the gene expression, and histone modifications also plays an important role in tumor development.

Survey and molecular detection of two citrus viroids affecting commercial citrus orchards in the Northern part of Sudan

Two extensive surveys and laboratory work were conducted to determine the occurrence of citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd), the causal agent of cachexia (CVd-IIb) disease in the main citrus growing areas in three states in the northern part of Sudan, viz, Northern, River Nile and Khartoum. For CEVd, all, but one of the examined citrus trees failed to show typical exocortis symptoms. The only symptomatic orange tree encountered in the survey displayed the characteristic CEVd symptoms including tree stunting and bark cracking or bark splitting of the rootstock. While for the cachexia disease, most of the commercial citrus trees were found to be symptomless carriers except mandarin. Symptomatic mandarin trees which were grafted on sour orange rootstock displayed heavy gum impregnation (bark gumming), wood staining and deep bark cracking, in addition to severe stem pitting, bark pegging, twig dieback and yellowing of leaves. Pitting and gum development were usually encountered near the budunion but could spread to other sensitive portions of the infected tree. However, using viroid specific primers and reverse transcription polymerase chain reaction (RT-PCR) approach, both viroids were shown to exist in total RNA preparations from symptomatic and asymptomatic citrus species. Bands of 370 bp and 300 bp corresponding to the full length genomes of CEVd and CVd-IIb, respectively, were detected. The test was positive in 54 for CEVd and 20 for CVd-IIb out of 200 random samples tested from orange, grapefruit and mandarin. The results indicated that a considerably high percentage of citrus species in the northern part of Sudan, particularly in Khartoum State, were symptomless carriers of both CEVd and CVd-IIb. Being rapid, sensitive and in most cases reliable, the molecular approach could be considered indispensable for viroid detection and in testing programs to produce certified viroid-free planting materials. Key wards: Citrus viroids, Exocortis, Cachexia, Molecular detection.

Nosocomial outbreak of Crimean-Congo haemorrhagic fever

We report a nosocomial outbreak of Crimean-Congo haemorrhagic fever (CCHF) that affected six patients in June 2009 in Ghaem Hospital, Mashhad, Iran, apparently related to one index case. The last four cases were healthcare workers. Infection was spread by percutaneous exposure to two cases, and probably by direct contact with blood, clothes and sheets, to three others. The diagnosis in the two fatal cases was not confirmed virologically. The diagnosis in four cases who survived was confirmed by specific reverse transcription polymerase chain reaction. The patients were treated with ribavirin. In endemic areas, every patient presenting with a febrile haemorrhagic syndrome should be considered to have a viral haemorrhagic fever until proven otherwise. Patients who meet the criteria for probable CCHF should be admitted to hospital and treated with ribavirin. Appropriate isolation precautions should be immediately initiated.

Nucleic Acid Interaction Kinetics Modulate the Chaperone Activity of Retroviral Nucleocapsid and Gag Proteins

Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are believed to be critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins’nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone. This work was funded in part by Federal Funds from NCI, NIH under contract N01-CO-12400 (RJG).

Development of Real Time RT-PCR Assays for Detection of Type A Influenza Virus and for Subtyping of Avian H5 and H7 Hemagglutinin Subtypes

Rapid detection and subtyping of H5 and H7 subtypes influenza A viruses are important for disease control in poultry and potential transmission to humans. Currently, virus isolation and subsequent HA and NA subtyping constitute the standard for avian influenza viruses detection and subtype identification. These methods are highly accurate and sensitive but are also laborious and time-consuming. Reverse transcription PCR and real time reverse transcription PCR assays, suitable tests for rapid detection, have previously been used for the specific diagnosis of H5 and H7 viruses, however, at present, no primer and probe sets are available for the identification of all H5 and H7 strains. Herein, we have developed specific and sensitive real time reverse transcription PCR assays for the detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes and we have also compared these molecular assays with viral isolation in terms of sensitivity. Our results demonstrate that the real time reverse transcription PCR assays are more sensitive, specific, less expensive compared to viral isolation. In conclusion, molecular assays could represent an useful tool for rapid detection and screening of H5 and H7 isolates during influenza A virus outbreaks alternatively to viral isolation.

Identification of Nutrient-Responsive Arabidopsis and Rapeseed MicroRNAs by Comprehensive Real-Time Polymerase Chain Reaction Profiling and Small RNA Sequencing

Comprehensive expression profiles of Arabidopsis (Arabidopsis thaliana) MIRNA genes and mature microRNAs (miRs) are currently not available. We established a quantitative real-time polymerase chain reaction platform that allows rapid and sensitive quantification of 177 Arabidopsis primary miR transcripts (pri-miRs). The platform was used to detect phosphorus (P) or nitrogen (N) status-responsive pri-miR species. Several pri-miR169 species as well as pri-miR398a were found to be repressed during N limitation, whereas during P limitation, pri-miR778, pri-miR827, and pri-miR399 species were induced and pri-miR398a was repressed. The corresponding responses of the biologically active, mature miRs were confirmed using specific stem-loop reverse transcription primer quantitative polymerase chain reaction assays and small RNA sequencing. Interestingly, the latter approach also revealed high abundance of some miR star strands. Bioinformatic analysis of small RNA sequences with a modified miRDeep algorithm led to the identification of the novel P limitation-induced miR2111, which is encoded by two loci in the Arabidopsis genome. Furthermore, miR2111, miR169, a miR827-like sequence, and the abundances of several miR star strands were found to be strongly dependent on P or N status in rapeseed (Brassica napus) phloem sap, flagging them as candidate systemic signals. Taken together, these results reveal the existence of complex small RNA-based regulatory networks mediating plant adaptation to mineral nutrient availability.

Evaluation of murine norovirus as a surrogate for human norovirus and hepatitis A virus in heat inactivation studies

Aims: To determine the suitability of murine norovirus (MNV) as a surrogate for human norovirus (HuNoV) in heat inactivation studies. Methods and Results: MNV, hepatitis A virus (HAV) and HuNoV genogroup I and II (GI and GII) specific real‐time quantitative reverse transcription (qRT)‐PCR assays were used to determine the effects of heat exposure (63 and 72°C) for up to 10 min in water and milk. Using culture assays, MNV and HAV showed similar reductions in infectivity over time. Both HuNoV GI and GII showed lower log reductions in qRT‐PCR titre following heat exposure than either MNV or HAV. No significant protective effect of milk was observed for any virus. Conclusions: MNV is as suitable a surrogate for HuNoV as HAV. In heat inactivation studies at 63 and 72°C, qRT‐PCR results indicate that HuNoV is less susceptible to heat than either HAV or MNV and so neither virus may be an appropriate surrogate for HuNoV. Significance and Impact of the Study: Caution should be used when extrapolating surrogate virus data for HuNoV. Although not conclusive, our results suggest that HuNoV may be more resistant to heat than either HAV or MNV.

Characterization of Moloney Murine Leukaemia Virus/Avian Myeloblastosis Virus Chimeric Reverse Transcriptases

Reverse transcriptases (RTs) from Moloney murine leukaemia virus (MMLV) and avian myeloblastosis virus (AMV) contain all the fingers, palm, thumb, connection and RNase H domains. The fingers, palm and thumb domains are thought to be involved in the reverse transcription activity, and the RNase H domain is in the RNase H activity. In this study, we characterized four chimeric RTs which comprise one of the fingers, palm, thumb and RNase H domains originated from AMV RT and the other three and connection domains originated from MMLV RT. Unexpectedly, all chimeric RTs exhibited the same characteristics: their specific reverse transcription activities decreased to less than 0.1% of that of MMLV RT, while their specific RNase H activities were approximately 20% of that of MMLV RT. The decreases in the two activities of the chimeric RTs were ascribed to the decreases in k(cat). Based on that the reverse transcription activity of MMLV RT was impaired by substituting its RNase H domain with that from AMV RT, we propose that in MMLV RT, there might be an interaction between the fingers/palm/thumb domain and the RNase H domain.

Detection and identification of human metapneumovirus by real time reverse transcription PCR

To develop a rapid, sensitive and specific real time reverse transcription PCR for detecting and identifying human metapneumovirus. The Hmpv-L gene of human metapneumovirus was chosen as target gene, the primers and TaqMan probe were designed, and the PCR reaction was optimized systematically. The total RNA was extracted from respiratory specimens, and reverse transcription was performed through random primer. The cDNA was detected by using real time PCR. The specificity, sensitivity and reproducibility of real time PCR were estimated. The real time PCR was applied to detect 180 clinical respiratory specimens. The human metapneumovirus can be detected using real time reverse transcription PCR accurately and quickly, and the sensitivity was 1 copy/microl. The coefficient of variation of intra-assay and inter-assay was less than 5%. Among those 180 specimens, 28 (15.56%) were positive for human metapneumovirus, the clinical diagnoses for these 28 patients were pneumonia (15.60%, 17/109) and bronchiolitis (15.49%, 11/71). 21 positive specimens were from patients under 2 years of age, and 6 positive specimens were from patients between 2 and 5 years of age, only 1 positive specimens was from patients over 5 years. It is demonstrated that real time reverse transcription PCR is a reliable, accurate and feasible assay for human metapneumovirus, which has become one of the most important pathogens induced acute respiratory infections in pediatric patients.

Human Rhinovirus Group C Infection in Children with Lower Respiratory Tract Infection

Human Rhinovirus Group C Infection in Children with Lower Respiratory Tract Infection

First Report on the Occurrence of Grapevine leafroll-associated virus 7 and 9 in Chilean Grapevines.

Grapevine is one of the oldest horticultural crops and represents a highly valuable agricultural commodity. So far, nine distinct Grapevine leafroll-associated viruses (GLRaVs) within the Closteroviridae family have been found to be associated with grapevine leafroll disease (3). Previous studies have demonstrated a high incidence of GLRaV-1, -2, and -3 in Chile (2). To determine if other GLRaVs were present, 21 dormant cane samples were screened with a comprehensive 70-mer oligonucleotide microarray designed to simultaneously detect all grapevine viruses with total or partial genomic sequence available. The array contained 570 unique probes designed against specific regions of more than 40 viral genomes (E. Engel et al., 15th ICVG [Abstr.], 2006). One sample (cv. Black Seedless) showing a microarray hybridization pattern compatible with a mixed infection of GLRaV-7 and GLRaV-1 was analyzed by ELISA using GLRaV-7 specific antibodies (Agritest, Valenzano, Italy) and reverse transcription (RT)-PCR using virus-specific primers LR7-F: 5'- TAT ATC CCA ACG GAG ATG GC -3' and LR7-R: 5'- ATG TTC CTC CAC CAA AAT CG -3' (based on GenBank Accession No. Y15987). The serological analysis confirmed the presence of GLRaV-7 with further confirmation by the RT-PCR product of 502 bp corresponding to a fragment of the HSP70h gene that was cloned and sequenced. The Chilean GLRaV-7 sequence (GenBank Accession No. EU334662) showed 94% nucleotide and 95% amino acid identity when compared with a corresponding region of another GLRaV-7 isolate from Albania (GenBank Accession No. Y15987). GLRaV-1 infection was confirmed by ELISA (Bioreba AG, Reinach, Switzerland) and RT-PCR. A second sample (cv. Tintorera) showing microarray hybridization pattern compatible with a mixed infection of GLRaV-9 and Grapevine virus A (GVA) was analyzed by RT-PCR using virus-specific primers LR9-F: 5'- CGG CAT AAG AAA AGA TGG CAC -3' and LR9-R: 5'- TCA TTC ACC ACT GCT TGA AC -3' (1). The RT-PCR product of 393 bp corresponding to a fragment of the HSP70h gene was cloned and sequenced (GenBank Accession No. EU334663), showing 94% nucleotide and 95% amino acid identity when compared with a corresponding region of another GLRaV-9 isolate from the United States (GenBank Accession No. AY297819). Since there are no commercial antibodies available for GLRaV-9 detection, a second pair of primers, LR9-F1: 5'- AAA GGT TTC TGC TGG TTA CC -3' and LR9-R1: 5'- CTT TCA GAA CAG TCC TCC TC -3' that amplified a fragment of ORF1a was also used. The 301-bp product was cloned and sequenced (GenBank Accession No. EU588989) showing 93.7% nucleotide and 98% amino acid identity when compared with a corresponding region of another GLRaV-9 isolate (GenBank Accession No. AY297819). GVA infection was confirmed by ELISA (Bioreba AG) and RT-PCR. To our knowledge, this is the first report of GLRaV-7 and GLRaV-9 in Chile. Further studies will help determine the effect and incidence of these viruses in Chilean grapevines.

Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies

BackgroundDue to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan® PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp).ResultsT-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 ± 0.33 Ct values. The coefficients for inter- (1.89 ± 0.48%) and intra-assay variation (0.85 ± 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 ± 0.33, without significant differences between self-designed and ABI inventoried Taqman® gene assays. Only two of the tested Taqman® ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 ± 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 ± 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 ± 0.74), albeit higher inter-assay CVs (5.38 ± 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts.ConclusionIn comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.

Effects of nucleic acid local structure and magnesium ions on minus-strand transfer mediated by the nucleic acid chaperone activity of HIV-1 nucleocapsid protein

HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which is required for highly specific and efficient reverse transcription. Here, we demonstrate that local structure of acceptor RNA at a potential nucleation site, rather than overall thermodynamic stability, is a critical determinant for the minus-strand transfer step (annealing of acceptor RNA to (−) strong-stop DNA followed by reverse transcriptase (RT)-catalyzed DNA extension). In our system, destabilization of a stem-loop structure at the 5′ end of the transactivation response element (TAR) in a 70-nt RNA acceptor (RNA 70) appears to be the major nucleation pathway. Using a mutational approach, we show that when the acceptor has a weak local structure, NC has little or no effect. In this case, the efficiencies of both annealing and strand transfer reactions are similar. However, when NC is required to destabilize local structure in acceptor RNA, the efficiency of annealing is significantly higher than that of strand transfer. Consistent with this result, we find that Mg2+ (required for RT activity) inhibits NC-catalyzed annealing. This suggests that Mg2+ competes with NC for binding to the nucleic acid substrates. Collectively, our findings provide new insights into the mechanism of NC-dependent and -independent minus-strand transfer.

First Report of Apple chlorotic leaf spot virus in Quince in Greece.

The sanitary status of pome fruit trees was evaluated in central and northern Greece during a survey in the autumn of 2004 and spring of 2005. Twenty-six leaf samples were collected from five symptomless and 21 symptomatic quince trees showing fruit deformation (FD) symptoms and diffuse chlorotic leaf spots. All samples were tested for the presence of Apple chlorotic leaf spot virus (ACLSV), a member of the Trichovirus genus, initially by ELISA and then by a specific reverse transcription (RT)-PCR assay reported previously (1). ACLSV has a broad host range that includes most, if not all, Prunoidae (peach, apricot, plum, and cherry) and Maloidae (apple, pear, and quince) fruit tree species. Although it has been tentatively linked with fruit, leaf, bark, and growth retardation symptoms in quince (2,3), its geographic distribution and association with specific symptoms is still poorly determined. ACLSV was initially detected by serology in two plants, one symptomless and one showing FD symptoms. ACLSV presence in these two samples and in an additional symptomless plant was confirmed by the ACLSV-specific RT-PCR assay. Sequencing of the RT-PCR amplicon from the symptomatic isolate (EMBL Accession No. AM292923), which was positive in both assays, confirmed the identification of ACLSV. The obtained sequence shows 93% nucleotide identity with an apple isolate of ACLSV (EMBL Accession No. AY677103). To our knowledge, these findings represent the first report of the presence of ACLSV in quince in Greece. They further indicate that at least for some host-cultivar/virus isolate combinations, ACLSV may be asymptomatic in quince and that the symptoms observed in the plants sampled are unrelated to ACLSV infection.

Detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in Rome, Italy

Detection of a broad number of respiratory viruses is not undertaken currently for the diagnosis of acute respiratory infection due to the large and always increasing list of pathogens involved. A 1‐year study was undertaken on children hospitalized consecutively for acute respiratory infection in a Pediatric Department in Rome to characterize the viruses involved. Two hundred twenty‐seven children were enrolled in the study with a diagnosis of asthma, bronchiolitis, bronchopneumonia, or laringo‐tracheo bronchitis. A molecular approach was adopted using specific reverse transcription (RT)‐PCR assays detecting 13 respiratory viruses including metapneumovirus (hMPV) and the novel coronaviruses NL63 and HKU1; most amplified fragments were sequenced to confirm positive results and differentiate the strain. Viral pathogens were detected in 97 samples (42.7%), with 4.8% of dual infections identified; respiratory syncytial virus (RSV) was detected in 17.2% of children, followed by rhinovirus (9.7%), parainfluenza virus type 3 (PIV3) (7.5%), and influenza type A (4.4%). Interestingly, more than half the patients (9/17) that have rhinovirus as the sole respiratory pathogen had pneumonia. HMPV infected children below 3 years in two peaks in March and June causing bronchiolitis and pneumonia. One case of NL63 infection is described, documenting NL63 circulation in central Italy. In conclusion, the use of a comprehensive number of PCR‐based tests is recommended to define the burden of viral pathogens in patients with respiratory tract infection. J. Med. Virol. 79:463–468, 2007. © 2007 Wiley‐Liss, Inc.

Mobile mRNA Makes Potatoes

Tuber formation in potato plants is promoted when plants are exposed to short-day photoperiods and inhibited in plants that experience summerlike long periods of daylight. This process requires that a signal be passed from the leaves to the growing tuber underground. Banerjee et al . report a series of experiments that implicate specific mRNAs as the molecules that carry the signal. The mRNA encodes a transcription factor called St BEL5 that acts with another transcription factor, POTH1, to control hormone concentrations in the tuber that regulate growth. The authors used laser microdissection of specific cell types and reverse transcription polymerase chain reaction amplification to pinpoint specific mRNAs in phloem and other cell types. When a leaf-specific promoter was used to drive expression of St BEL5 RNA, exposure to short days enhanced mobility of the RNA to the root region. Overexpression of the St BEL5 transcript enabled transgenic plants to undertake tuber formation even when exposed to long photoperiods. The authors propose that the mRNA transport reflects light-dependent changes in the abundance of RNA-binding proteins that facilitate transport and targeting of mRNA to specific cells. A. K. Banerjee, M. Chatterjee, Y. Yu, S.-G. Suh, W. A. Miller, D. J. Hannapel, Dynamics of a mobile RNA of potato involved in a long-distance signaling pathway. Plant Cell 18 , 3443-3457 (2006). [Abstract] [Full Text]