Characterization of a peribunyavirus isolated from largemouth bass (Micropterus salmoides).

We report the complete genome sequencing of the first fish peribunyavirus determined using a next-generation sequencing approach. The virus was isolated during a routine health assessment of wild largemouth bass (Micropterus salmoides) in Wisconsin in April of 2009. Further research is needed to determine the epidemiology and pathogenicity of the largemouth bass bunyavirus.

Viral protein requirements for assembly and release of human parainfluenza virus type 3 virus-like particles.

To understand the roles of human parainfluenza virus 3 (HPIV3) proteins in assembly and release, viral proteins were expressed individually and in combination in 293T cells. Expression of the matrix (M) protein triggered release of enveloped, matrix-containing virus-like particles (VLPs) from cells. When M was co-expressed with the nucleocapsid (N), fusion (F) or haemagglutinin-neuraminidase (HN) proteins, VLPs that contained M+N, M+F and M+HN, respectively, were generated, suggesting that M can independently interact with each protein to facilitate assembly and release. Additionally, expression of N protein enabled incorporation of the phosphoprotein (P) into VLPs, likely due to known N-P interactions. Finally, the HPIV3 C protein did not enhance VLP release, in contrast to observations with the related Sendai virus. These findings reinforce the central importance of the M protein in virus assembly and release, but also illustrate the variable roles of other paramyxovirus proteins during these processes.

A multiplex RT-PCR assay for the detection of fish picornaviruses.

With the emergence of high profile fish diseases in the Great Lakes region, surveillance and regulatory inspections of fish populations have increased. This has resulted in a better understanding of known pathogens and isolation of many new pathogens of fish. In this study, a multiplex RT-PCR assay was developed for the detection of three newly discovered fish picornaviruses: bluegill picornavirus-1 (BGPV-1), fathead minnow picornavirus (FHMPV), and eel picornavirus-1 (EPV-1). This assay was found to be very sensitive with a detection limit of 81.9pg/µl of extracted RNA from a pool of FHMPV and BGPV-1 and was able to detect 501 and 224 gene copies/µl of BGPV-1 and FHMPV, respectively. The assay was highly reproducible and did not cross react with other closely related pathogens. We believe that this new assay provides a rapid and cost effective tool for confirming cell culture isolates and conducting prevalence studies of these newly detected fish picornaviruses.

Characterization of a new picornavirus isolated from the freshwater fish Lepomis macrochirus.

The freshwater fish Lepomis macrochirus (bluegill) is common to North American waters, and important both ecologically and as a sport fish. In 2001 an unknown virus was isolated from bluegills following a bluegill fish kill. This virus was identified as a picornavirus [termed bluegill picornavirus (BGPV)] and a diagnostic reverse transcriptase PCR was developed. A survey of bluegills in Wisconsin waters showed the presence of BGPV in 5 of 17 waters sampled, suggesting the virus is widespread in bluegill populations. Experimental infections of bluegills confirmed that BGPV can cause morbidity and mortality in bluegills. Molecular characterization of BGPV revealed several distinct genome characteristics, the most unusual of which is the presence of a short poly(C) tract in the 3' UTR. Additionally, the genome encodes a polyprotein lacking a leader peptide and a VP0 maturation cleavage site, and is predicted to encode two distinct 2A proteins. Sequence comparison showed that the virus is most closely related to a phylogenetic cluster of picornaviruses that includes the genera Aquamavirus, Avihepatovirus and Parechovirus. However, it is distinct enough, for example sharing only about 38% sequence identity to the parechoviruses in the 3D region, that it may represent a new genus in the family Picornaviridae.

Analysis of nucleotides 13-96 of the human parainfluenza virus type 3 antigenomic promoter reveals positive- and negative-acting replication elements.

During replication of human parainfluenza virus type 3 (HPIV3), the 96-nucleotide antigenomic promoter (AGP) of HPIV3 directs the synthesis of genomic RNA. Previous work showed that nucleotides 1-12 were critical in promoting replication of an HPIV3 minireplicon, but nucleotides 13-96 were not investigated. In this study, the role of nucleotides 13-96 in AGP function was analyzed by creating and assaying mutations in an HPIV3 minireplicon. A replication promoting element known as promoter element II (nt 79-96) was confirmed in the HPIV3 AGP. Additionally, nucleotides 13-39 were found to constitute an additional positive-acting cis-element. However, detailed analysis of the 13-39 element revealed a complicated control element with both stimulatory and repressing elements. Specifically, nucleotides 21-28 were shown to repress RNA replication, while flanking sequences had a stimulatory effect.

Significant pathogens isolated from surgical site infections at a community hospital in the Midwest.

BACKGROUND: Studies examining the incidence of microorganisms isolated from surgical site infections (SSIs) have been conducted primarily at large academic health care centers. Results from these studies have revealed that methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a significant pathogen in SSIs. Minimal data are available from smaller, community hospitals on the incidence of microorganisms associated with SSIs, particularly the incidence of MRSA in SSIs. METHODS: A retrospective study was performed to identify the microorganisms associated with SSIs in patients who underwent class I and II surgeries at a small urban to rural community hospital from January 2003 through December 2004. RESULTS: A total of 10,672 surgeries was performed, and 89 SSIs were identified. Staphylococcus aureus was the most common pathogen (25.8%). Enterobacteriaceae were the second most frequently isolated organisms (12.4%), followed by streptococci species (11.2%), coagulase-negative staphylococci (10.1%), enterococci species (7.9%), and Pseudomonas aeruginosa (6.7%). MRSA was isolated from 4.5% of the SSIs. CONCLUSION: We have demonstrated that the spectrum of microorganisms isolated in SSIs at a community hospital is comparable with that reported in studies conducted at large academic health care centers, including the emergence of MRSA as a pathogen in SSIs. This information will guide future infection control initiatives to reduce SSIs.

Roles of human parainfluenza virus type 3 bases 13 to 78 in replication and transcription: identification of an additional replication promoter element and evidence for internal transcription initiation.

The genomic promoter of human parainfluenza virus type 3 (HPIV3) contains multiple cis-elements controlling transcription and replication. Previous work showed that regions 1 to 12 and 79 to 96 were critical in promoting replication of an HPIV3 minireplicon, while the intergenic sequence and N gene start signal (IS/Ngs, bases 49 to 61) were important for transcription. Because these data were collected primarily using point mutations, not every base from position 1 to 96 was analyzed, and some important control elements may have been missed. To clarify the role of bases 13 to 78 in transcription and replication, a series of mutations were made which collectively scanned this entire region. Mutation of bases 13 to 28 resulted in markedly decreased HPIV3 minireplicon replication, indicating these bases constitute an additional cis-element involved in the synthesis of the HPIV3 antigenomic RNA. The position dependence of the IS/Ngs was also examined. Analysis of mutants in which the IS/Ngs was shifted 5' or 3' showed that this segment could be moved without significantly disrupting transcription initiation. Additional mutants which contained two successive IS/Ngs segments were created to test whether the polymerase accessed the gene start signal by proceeding along the template 3' to 5' or by binding internally at the gene start signal. Based on analysis of the double gene start mutants, we propose a model of internal transcription initiation in which the polymerase enters the template at approximately the location of the natural N gene start but then scans the template bidirectionally to find a gene start signal and initiate transcription.

The human parainfluenza virus type 3 (HPIV 3) C protein inhibits viral transcription.

The C protein of human parainfluenza virus type 3 (HPIV 3), like other paramyxovirus C proteins, is synthesized from an alternate open reading frame (ORF) encoded within the phosphoprotein (P) mRNA, in addition, to two other proteins, namely D and V, which arise from the same mRNA by a process of transcriptional editing. The precise role of the C, D, and V proteins in viral transcription and replication, and their interaction, if any, with other viral proteins remains unknown. To ascertain the role of the C protein, we have examined its effect on transcription using an HPIV 3 minigenome construct and monitoring the luciferase reporter gene expression. Our results demonstrate that the HPIV 3 C protein effectively inhibits minigenome transcription in a dose-dependent manner. Interestingly, the Sendai virus (Se-V) C protein was also capable of inducing an inhibitory effect on the HPIV 3 minigenome transcription, thus demonstrating a heterologous interaction. A coiled-coil motif within the C protein has been identified, and a deletion mutant within this motif abrogated the inhibitory effect significantly thereby implying that oligomerization of the C protein may be involved in inhibition of transcription.