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Group A rotaviruses (RVAs) are an important cause of diarrhoeal illness in humans, as well as in mammalian and avian animal species. Previous sequence analyses indicated that avian RVAs are related only distantly to mammalian RVAs. Here, the complete genomes of RVA strain 03V0002E10 from turkey (Meleagris gallopavo) and RVA strain 10V0112H5 from pheasant (Phasianus colchicus) were analysed using a combination of 454 deep sequencing and Sanger sequencing technologies. An adenine-rich insertion similar to that found in the chicken RVA strain 02V0002G3, but considerably shorter, was found in the 39 NCR of the NSP1 gene of the pheasant strain. Most genome segments of both strains were related closely to those of avian RVAs. The novel genotype N10 was assigned to the NSP2 gene of the pheasant RVA, which is related most closely to genotype N6 found in avian RVAs. However, this virus contains a VP4 gene of the novel genotype P [37], which is related most closely to RVAs from pigs, dogs and humans. This strain either may represent an avian/mammalian rotavirus reassortant, or it carries an unusual avian rotavirus VP4 gene, thereby broadening the potential genetic and antigenic variability among RVAs. INTRODUCTIONGroup A rotaviruses (RVAs) are aetiological agents of acute gastroenteritis in humans and animals. They cause severe diarrhoea in infants and children, to which are attributed approximately 453 000 childhood deaths annually (Tate et al., 2012). Recently, two attenuated live vaccines have been introduced and are used successfully for prevention of severe rotavirus-induced disease (Yen et al., 2011).Rotaviruses are classified as a genus within the family Reoviridae (Attoui et al., 2012). They are non-enveloped particles containing a genome of 11 segments of dsRNA with monocistronic coding capacity except for segment 11, which may encode two proteins. Based on antigenic and genome sequence properties, five rotavirus groups (A-E) and two tentative groups (F, G) can be distinguished, which also represent the taxonomically defined rotavirus species and tentative species, respectively (Attoui et al., 2012). In addition, the rotavirus NADRV has been described in adults in Asia (Yang et al., 2004). Recently, a classification system into the eight rotavirus species A-H (with NADRV designated rotavirus H) has been proposed, based on genetic data of genome segment 6 (Matthijnssens et al., 2012).Among the rotavirus groups, RVAs have the highest clinical importance among humans and most mammalian species. The antigenic structures of RVAs eliciting neutralizing antibodies are the outer capsid proteins VP7 and VP4, which define the G-and P-types, respectively. Originally, Gand P-serotypes were defined based on antibody reactivity (Hoshino & Kapikian, 2000). Later on, sequence data of the VP7-and VP4-encoding genome segments were used for definition of G-and P-genotypes, leading to the present list of at least 27 different G-types and 35 different P-types in human and animal RVAs (Matthijnssens et al., 2011). Recently, a genotypi...

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The intestinal tract and intestinal contents were collected from 34 stunted, 5-to-14-day-old broiler chicks from eight flocks with runting and stunting syndrome (RSS) in Northern Germany to investigate intestinal lesions and the presence of enteric pathogens with a special focus on rotaviruses (RVs). Seven chicks from a healthy flock were used as controls. Severe villous atrophy was seen in chicks from six flocks with RSS but not in the control flock. Lesions were often "regionally" distributed in the middle-to-distal small intestine. Transmission electron microscopy (TEM), polyacrylamide-gel electrophoresis (PAGE), reverse-transcriptase polymerase chain reaction (RT-PCR), and seminested RT-PCR were used for detection and characterization of RVs. The PAGE allows discrimination of different RV groups, and the RT-PCR was used to verify the presence of group (gp) A RVs. RVs were detected (by all methods) in 32 of 34 chicks from the flocks with RSS. By TEM (negative staining), RV particles were observed in intestinal contents of 28 chicks from the flocks with RSS. PAGE analysis showed four RV groups: gpA, gpD, gpF, and gpG. Group A RVs were detected in four chicks from two flocks with RSS, without intestinal lesions. GpD RVs were detected in 12 chicks of five flocks with RSS, 10 of them with severe villous atrophy. GpF RVs were confirmed in four chicks from three flocks with RSS and in two birds in the control flock. GpG RVs were verified in two chicks from two flocks with RSS, one with, and one without, intestinal lesions. At present, PCR methods are only available for detection of gpA RVs. Using RT-PCR, gpA RVs were identified in samples from 22 chicks including samples of two chicks from the control flock. Statistical analysis revealed a positive correlation between presence of gpD RV and severe villous atrophy in flocks with RSS. The results suggest that gpD RV plays a major role in the pathogenesis of RSS.

Avian rotaviruses are broadly distributed among birds, but only scarcely characterized on the molecular level. The VP4-, VP6-, VP7- and NSP5-encoding sequences of eight group A rotaviruses from chickens and turkeys determined here indicate a low degree of sequence similarity with mammalian rotaviruses. An NSP6-encoding region was missing in all chicken isolates except for isolate Ch2. Four novel genotypes (P[30], P[31], G22 and H8) were assigned by the Rotavirus Classification Working Group. Generally, chicken and turkey isolates clustered into separate branches of phylogenetic trees. However, chicken isolate Ch2 consistently clustered together with turkey isolates. Chicken isolate 06V0661G1 has a VP4-encoding sequence of unknown origin, but possesses VP6, VP7 and NSP5 genotypes typical for chicken isolates. These results might indicate interspecies transmission and reassortment among avian group A rotaviruses under field conditions. PCR protocols enabling amplification of avian and mammalian group A rotaviruses were developed for use in further epidemiological studies.

dSeveral real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics. Because of its extraordinary infectiousness, the zoonotic pathogen Francisella tularensis causing tularemia was in the past the subject of state-run biowarfare research programs and therefore is included on the CDC category A list of biothreat agents. It causes disease in a vast range of animals, with relevant disease transmission to humans by direct contact or via vectors such as deer flies, horse flies, mosquitoes, and hard ticks. Infection due to inhalation of aerosols can occur through contact with infected hares. There are three F. tularensis subspecies, F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and F. tularensis subsp. mediasiatica, which can be found in several environments and geographical regions. The first two subspecies and Francisella novicida cause the bulk of human infections (7,19). Infection by Francisella hispaniensis has also been described, and results of phylogenetic analysis suggest that F. novicida should be regrouped as a fourth subspecies of F. tularensis (3,12).In all scenarios dealing with intentional release of biothreat agents, timely diagnosis is regarded as essential to identifying and containing outbreaks of infectious disease (4). Many efforts have been made to reduce the assay time of PCR-based nucleic acid detection. In spite of engineering constraints regarding temperature cycling needed for the PCR assays, short protocols and miniaturized cyclers or chip platforms are being developed (5, 6, 18).In recent years a variety of isothermal amplification methods have been developed which offer the possibility of developing even simpler point-of-care systems. One example is the ESEQuant Tube Scanner device (Qiagen Lake Constance GmbH, Stockach, Germany). This device contains a sophisticated fluorescence sensor which slides back and forth under a set of eight tubes, collecting fluorescence signals over time and allowing for real-time documentation of increasing fluorescence signals. A combined threshold and signal slope analysis is used for signal interpretation, which can be confirmed by second-derivative analysis (11; also ESEQuant Tube Scanner software [Qiagen]). The recombinase polymerase amplification (RPA) assay is an isothermal amplification method which can be combined with a sequencespecific fluorescent probe for real-time detection. In RPA the phage-derived recombinase UvsX, assisted by its cofactor UvsY, aggregates with oligo...

Jena virus (JV) is a noncultivatable bovine enteric calicivirus associated with diarrhea in calves and was first described in Jena, Germany. The virus was serially passaged 11 times in colostrum-deprived newborn calves and caused diarrheal disease symptoms at each passage. The complete JV genome sequence was determined by using cDNA made from partially purified virus obtained from a single stool sample. JV has a positive-sense single-stranded RNA genome which is 7,338 nucleotides in length, excluding the poly(A) tail. JV genome organization is similar to that of the human Norwalk-like viruses (NLVs), with three separate open reading frames (ORFs) and a 24-nucleotide sequence motif located at the 5′ terminus of the genome and at the start of ORF 2. The polyprotein (ORF 1) consists of 1,680 amino acids and has the characteristic 2C helicase, 3C protease, and 3D RNA polymerase motifs also found in the NLVs. However, comparison of the N-terminal 100 amino acids of the JV polyprotein with those of the group 1 and group 2 NLVs showed a considerable divergence in sequence. The capsid protein (ORF 2) at 519 amino acids is smaller than that of all other caliciviruses. JV ORF 2 was translated in vitro to produce a 55-kDa protein that reacted with postinfection serum but not preinfection serum. Phylogenetic studies based on partial RNA polymerase sequences indicate that within the Caliciviridae JV is most closely related to the group 1 NLVs.

Objective-To investigate prospectively abnormalities of brain glucose utilisation in relation to major or minor neuropsychiatric symptoms in systemic lupus erythematosus (SLE). Methods-Positron emission tomography (PET) using F-18-labelled fluorodeoxyglucose was performed in 28 patients with SLE. Patients were classified as having severe neuropsychiatric manifestations (seizures, focal neurological deficits, acute confusional states, mood disorders) (n=12), or mild neuropsychiatric manifestations (headache, reactive depression, cognitive dysfunction, anxiety disorders) (n=11) and five patients without signs of central nervous system (CNS) involvement. Ten clinically and neurologically healthy volunteers served as controls. In 26 patients magnetic resonance imaging (MRI) was performed and autoantibodies against CNS tissue, ribosomal P protein and cardiolipin were measured. In 14 patients follow up PET scans were performed after a mean (SD) period of 11.6 (9.5) months. Results-PET scans showed hypometabolism in at least one brain region in all patients with severe or mild CNS symptoms (100%) as compared with patients without cerebral symptoms (40%) (p<0.0025). Parieto-occipital regions were most commonly aVected (96%), followed by parietal regions (32%). In contrast, MRI images were abnormal in only 11 of 22 patients (50%) with neuropsychiatric symptoms and in one of four patients (25%) without symptoms. In 12 of 14 patients examined in follow up PET scans persistence, improvement or worsening of cerebral symptoms were associated with unchanged, decreased or increased brain hypometabolism, respectively. No significant correlation was found between PET or MRI findings and autoantibody profiles. Conclusions-PET imaging represents a sensitive tool to detect manifest or subclinical CNS involvement in SLE and PET findings correlate well with the clinical course of disease.