Identification and genetic characterization of a novel picornavirus from chickens.

A novel picornavirus from commercial broiler chickens (Gallus gallus domesticus) has been identified and genetically characterized. The viral genome consists of a single-stranded, positive-sense RNA genome of >9243 nt excluding the poly(A) tail and as such represents one of the largest picornavirus genomes reported to date. The virus genome is GC-rich with a G+C content of 54.5 %. The genomic organization is similar to other picornaviruses: 5' UTR-L-VP0-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3' UTR. The partially characterized 5' UTR of >373 nt appears to possess a type II internal ribosomal entry site (IRES), which is also found in members of the genera Aphthovirus and Cardiovirus. This IRES exhibits significant sequence similarity to turkey 'gallivirus A'. The 3' UTR of 278 nt contains the conserved 48 nt 'barbell-like' structure identified in 'passerivirus', 'gallivirus', Avihepatovirus and some Kobuvirus genus members. A predicted large open reading frame (ORF) of 8592 nt encodes a potential polyprotein precursor of 2864 amino acids. In addition, the virus contains a predicted large L protein of 462 amino acids. Pairwise sequence comparisons, along with phylogenetic analysis revealed the highest percentage identity to 'Passerivirus A' (formerly called turdivirus 1), forming a monophyletic group across the P1, P2 and P3 regions, with <40, <40 and <50 % amino acid identity respectively. Reduced identity was observed against 'gallivirus A' and members of the Kobuvirus genus. Quantitative PCR analysis estimated a range of 4×10(5) to 5×10(8) viral genome copies g(-1) in 22 (73 %) of 30 PCR-positive faeces. Based on sequence and phylogenetic analysis, we propose that this virus is the first member of a potential novel genus within the family Picornaviridae. Further studies are required to investigate the pathogenic potential of this virus within the avian host.

Development of a real-time RT-PCR and Reverse Line probe Hybridisation assay for the routine detection and genotyping of Noroviruses in Ireland.

BACKGROUND: Noroviruses are the most common cause of non-bacterial gastroenteritis. Improved detection methods have seen a large increase in the number of human NoV genotypes in the last ten years. The objective of this study was to develop a fast method to detect, quantify and genotype positive NoV samples from Irish hospitals. RESULTS: A real-time RT-PCR assay and a Reverse Line Blot Hybridisation assay were developed based on the ORF1-ORF2 region. The sensitivity and reactivity of the two assays used was validated using a reference stool panel containing 14 NoV genotypes. The assays were then used to investigate two outbreaks of gastroenteritis in two Irish hospitals. 56 samples were screened for NoV using a real-time RT-PCR assay and 26 samples were found to be positive. Genotyping of these positive samples found that all positives belonged to the GII/4 variant of NoV. CONCLUSION: The combination of the Real-time assay and the reverse line blot hybridisation assay provided a fast and accurate method to investigate a NoV associated outbreak. It was concluded that the predominant genotype circulating in these Irish hospitals was GII/4 which has been associated with the majority of NoV outbreaks worldwide. The assays developed in this study are useful tools for investigating NoV infection.

Carlow virus, a 2002 GII.4 variant Norovirus strain from Ireland.

BACKGROUND: Noroviruses are the leading cause of infectious non-bacterial gastroenteritis in Ireland (population 4 million). Due to the number of outbreaks, its massive impact on the Irish health service and its seasonality, Norovirus has gained public notoriety as The Winter Vomiting Bug. The increase in cases in Ireland in the 2002-2003 season coincided with the emergence of two new Genogroup II genotype 4 variant clusters of Norovirus worldwide. RESULTS: Little research has been done on the epidemiology or molecular biology of Norovirus strains in Ireland. In an effort to combat this discrepancy, we cloned a full length human norovirus genome as a cDNA clone (J3) which can produce full length transcripts in vitro. A polymerase mutant cDNA clone (X1), in addition to a sub genomic cDNA clone (1A) were produced for use in future work. Carlow virus (Hu/NoV/GII/Carlow/2002/Ire) genome is 7559 nts in length, excluding the 3-end poly A tail and represents the first Norovirus strain from Ireland to be sequenced. CONCLUSION: Carlow virus is a member of the Farmington Hills variant cluster of Genogroup II genotype 4 noroviruses.

Cell-line-induced mutation of the rotavirus genome alters expression of an IRF3-interacting protein.

Rotavirus, a cause of severe gastroenteritis, contains a segmented double-stranded (ds)RNA genome that replicates using viral mRNAs as templates. The highly conserved 3'-consensus sequence (3'CS), UGUGACC, of the mRNAs promotes dsRNA synthesis and enhances translation. We have found that the 3'CS of the gene (g5) encoding NSP1, an antagonist of interferon signaling, undergoes rapid mutation when rhesus rotavirus (RRV) is serially passaged at high multiplicity of infection (MOI) in cells permitting high titer growth. These mutations increase the promoter activity of the g5 3'-sequence, but decrease its activity as a translation enhancer. The location of the mutations defines the minimal essential promoter for dsRNA synthesis as URN0-5CC. Under passage conditions where cell-to-cell spread of the virus is required to complete infection (low MOI), the 3'CS is retained due to the need for NSP1 to be expressed at levels sufficient to prevent establishment of the antiviral state. These data demonstrate that host cell type and propagation conditions affect the capacity of RRV to produce the virulence gene product NSP1, an important consideration in producing RRV-based vaccines.