Effects of cortisol and lipopolysaccharide on expression of select growth-, stress- and immune-related genes in rainbow trout liver.

Many studies have shown that stress-induced cortisol levels negatively influence growth and immunity in finfish. Despite this knowledge, few studies have assessed the direct effects of cortisol on liver immune function. Using real-time PCR, the expression of three cortisol-responsive genes (GR: glucocorticoid receptor, IGF-1: insulin-like growth factor-I and SOCS-1: suppressor of cytokine signaling-I), genes involved with innate and adaptive immunity (IL-1ß: interleukin-1 beta, IgM: immunoglobin-M and Lyz: lysozyme), and liver-specific antimicrobial peptides (hepcidin and LEAP-2A: liver-expressed antimicrobial peptide-2A) was studied in vitro using rainbow trout liver slices. The abundances of GR, SOCS-1 and IGF-1 mRNAs were suppressed by cortisol treatment. Abundance of IL-1ß mRNA was upregulated by LPS and suppressed by cortisol treatment in a time-dependent manner. While abundance of IgM mRNA was suppressed by cortisol treatment and stimulated by LPS, there were no effects of cortisol or LPS on abundance of Lyz mRNA. Abundance of hepcidin and LEAP-2A mRNA levels were suppressed by cortisol treatment and stimulated by LPS. These results demonstrate that cortisol directly suppresses abundance of GR, IGF-1, IL-1ß, IgM, hepcidin, LEAP-2A and SOCS-1 mRNA transcripts in the rainbow trout liver. We report for the first time, a suppressive effect of cortisol (within 8 h of treatment) on hepcidin and LEAP-2A mRNAs in rainbow trout liver, which suggests that acute stress may negatively affect liver immune function in rainbow trout.

Progress toward an enhanced vaccine: Eight marked attenuated viruses to porcine reproductive and respiratory disease virus.

Recombinant viruses of strain Ingelvac® PRRS porcine reproductive and respiratory syndrome virus (PRRSV) modified live virus vaccine were produced with two individual small in-frame deletions in nonstructural protein 2 (nsp2; Δ23 and Δ87) and also the same deletions supplanted with foreign tags (Δ23-V5, Δ23-FLAG, Δ23-S, Δ87-V5, Δ87-FLAG, Δ87-S). The viruses, but one (Δ87-FLAG), were stable for 10 passages and showed minimal effects on in vitro growth. Northern hybridization showed that the Δ23-tagged probe detected intracellular viral genome RNA as well as shorter RNAs that may represent heteroclite species, while the Δ87-tagged probe detected predominantly only genome length RNAs. When the tagged viruses were used to probe nsp2 protein in infected cells, perinuclear localization similar to native nsp2 was seen. Dual infection of Δ23-S and Δ87-S viruses allowed some discrimination of individual tagged nsp2 protein, facilitating future research. The mutants could potentially also be used to differentiate infected from vaccinated animals.

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription.

Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression.IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.

Comparison of Asian porcine high fever disease isolates of porcine reproductive and respiratory syndrome virus to United States isolates for their ability to cause disease and secondary bacterial infection in swine.

Epidemiologic data from Asian outbreaks of highly-pathogenic (HP) porcine reproductive and respiratory syndrome virus (PRRSV) suggest that disease severity was associated with both the virulence of the PRRSV isolates and secondary bacterial infections. Previous reports have indicated that U.S. isolates of PRRSV predispose to secondary bacterial infections as well, but the severity of disease that occurred in Asia in pigs infected with these HP-PRRSV strains has not been reported in the U.S. The objectives of this research were to compare the pathogenesis of Asian and U.S. PRRSV isolates with regard to their ability to cause disease and predispose to secondary bacterial infections in swine. To address these objectives groups of pigs were infected with 1 of 2 Asian HP-PRRSV strains (rJXwn06 or rSRV07) or 1 of 2 U.S. PRRSV strains (SDSU73 or VR-2332) alone or in combination with Streptococcus suis, Haemophilus parasuis, and Actinobacillus suis. Pigs infected with rJXwn06 exhibited the most severe clinical disease while the pigs infected with rSRV07 and SDSU73 exhibited moderate clinical disease, and pigs infected with VR-2332 exhibited minimal clinical signs. The frequency of secondary bacterial pneumonia was associated with the clinical severity induced by the PRRSV strains evaluated. The levels of proinflammatory cytokines in the serum were often lower for pigs coinfected with virus and bacteria compared to pigs infected with PRRSV alone indicating an alteration in the immune response in coinfected pigs. Combined our results demonstrate that severity of disease appears to be dependent on virulence of the PRRSV strain, and development of secondary bacterial infection.

Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex.

The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showed that 3D entered the replication complex in the form of its precursor, P3 (or 3CD), and was cleaved to release active 3D polymerase. Furthermore, our results showed that P3 is the preferred precursor that binds to the 5'CL. Using reciprocal complementation assays, we showed that one molecule of P3 binds the 5'CL and that a second molecule of P3 provides 3D. In addition, we showed that a second molecule of P3 served as the VPg provider. These results support a model in which P3 binds to the 5'CL and recruits additional molecules of P3, which are cleaved to release either 3D or VPg to initiate RNA replication.

Development of a genome copy specific RT-qPCR assay for divergent strains of type 2 porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) became a significant pathogen of swine upon its emergence in the late 1980s and since then has exemplified a rapidly evolving, constantly re-emerging pathogen. In addition to the challenges faced in development of vaccines and diagnostics, research on the basic molecular pathogenesis of PRRSV is also restrained by the ability to accurately and comparatively quantitate levels of replication in different tissues and between strains. This is further complicated by the presence of non-genomic RNA within infected tissues which are generally detected with equivalent efficiency by RT-qPCR based techniques, thereby introducing inherent error in these measurements that may differ significantly by tissue and strain. To address this, an RT-qPCR based technique was developed which targets the viral RNA-dependent RNA polymerase gene (nsp9) which is unique to genomic RNA, being absent from all subgenomic and heteroclite RNAs. This assay targets a region of considerable sequence conservation, and based on sequence only, should be quantitative for approximately 40% of all Type 2 PRRSV strains in GenBank for which nsp9 sequence is available. The assay was demonstrated to be linear over nine orders of magnitude (10(10)-10(2) copies) and can be readily adapted for multiplex detection of additional divergent PRRSV strains. This assay will add significantly to the ability to assess and compare PRRSV replication in a variety of tissues and between divergent strains, including highly pathogenic strains of considerable concern to the global pork industry.

The vOTU domain of highly-pathogenic porcine reproductive and respiratory syndrome virus displays a differential substrate preference.

Arterivirus genus member Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically devastating disease, recently exacerbated by the emergence of highly pathogenic strains (HP-PRRSV). Within the nonstructural protein 2 of PRRSV is a deubiquitinating enzyme domain belonging to the viral ovarian tumor (vOTU) protease superfamily. vOTUs, which can greatly vary in their preference for their host ubiquitin (Ub) and Ub-like substrates such as interferon stimulated gene 15 (ISG15), have been implicated as a potential virulence factor. Since various strains of PRRSV have large variations in virulence, the specificity of vOTUs from two PRRSV strains of varying virulence were determined. While both vOTUs showed de-ubiquitinating activity and markedly low deISGylating activity, HP-PRRSV demonstrated a strong preference for lysine 63-linked poly-Ubiquitin, tied to innate immune response regulation. This represents the first report of biochemical activity unique to HP-PRRSV that has implications for a potential increase in immunosuppression and virulence.

Occurrence, sequence polymorphism and population structure of Circulifer tenellus virus 1 in a field population of the beet leafhopper.

Circulifer tenellus virus 1 (CiTV1) is the prototypical example of an unusual group of dsRNA viruses associated with insects and for which ecological data are lacking. A San Joaquin Valley (SJV), California population of the beet leafhopper (BLH; Circulifer tenellus [Baker]) was sampled for CiTV1 in 2010. Among 365 BLH sampled, 119 (32.6%) were positive for CiTV1, with at least one CiTV1-positive BLH collected from each of 35 locations. Chi-square tests indicated that observed CiTV1 incidence differed from expected values based on collection season but not geography within the SJV. Sequence comparisons identified three CiTV1 strains, designated A, B, and C. Strain A predominated (82.4%), strain B was less common (16.8%), and only one (0.8%) strain C isolate was encountered. Chi-square tests demonstrated that observed frequencies of strains A and B did not differ from expected values in space or time, indicating that the SJV population of CiTV1 was unstructured.

Reovirus genomes from plant-feeding insects represent a newly discovered lineage within the family Reoviridae.

A complex set of double-stranded RNAs (dsRNAs) was isolated from threecornered alfalfa hopper (Spissistilus festinus), a plant-feeding hemipteran pest. A subset of these dsRNAs constitute the genome of a new reovirus, provisionally designated Spissistilus festinus reovirus (SpFRV). SpFRV was present in threecornered alfalfa hopper populations in the San Joaquin Valley of California, with incidence ranging from 10% to 60% in 24 of 25 sample sets analyzed. The 10 dsRNA segments of SpFRV were completely sequenced and shown to share conserved terminal sequences (5'-AGAGA and CGAUGUUGU-3') of the positive-sense strand that are distinct from known species of the family Reoviridae. Comparisons of the RNA directed RNA polymerase (RdRp) indicated SpFRV is most closely related (39.1% amino acid identity) to another new reovirus infecting the angulate leafhopper (Acinopterus angulatus) and provisionally designated Acinopterus angulatus reovirus (AcARV). The RdRp of both viruses was distantly related to Raspberry latent virus RdRp at 27.0% (SpFRV) and 30.0% (AcARV) or Rice ragged stunt virus RdRp at 26.2% (SpFRV) and 29.0% (AcARV) amino acid identity. RdRp phylogeny confirmed that SpFRV and AcARV are sister taxa sharing a most recent common ancestor. SpFRV segment 6 encodes a protein containing two NTP binding motifs that are conserved in homologs of reoviruses in the subfamily Spinareovirinae. The protein encoded by SpFRV segment 4 was identified as a guanylyltransferase homolog. SpFRV segments 1, 3, and 10 encode homologs of reovirus structural proteins. No homologs were identified for proteins encoded by SpFRV segments 5, 7, 8, and 9. Collectively, the low level of sequence identity with other reoviruses, similar segment terminal sequences, RdRp phylogeny, and host taxa indicate that SpFRV and AcARV may be considered members of a proposed new genus of the family Reoviridae (subfamily Spinareovirinae), with SpFRV assigned as the type species.

Plant-feeding insects harbor double-stranded RNA viruses encoding a novel proline-alanine rich protein and a polymerase distantly related to that of fungal viruses.

Novel double-stranded RNAs (approximately 8 kbp) were isolated from threecornered alfalfa hopper (Spissistilus festinus) and beet leafhopper (Circulifer tenellus), two plant-feeding hemipteran insect pests. The two new viruses, designated Spissistilus festinus virus 1 (SpFV1) and Circulifer tenellus virus 1 (CiTV1), do not appear to be encapsidated in conventional virions and shared a genome organization similar to that of several unclassified fungal viruses. SpFV1 and CiTVl encode a proline-alanine rich protein (PArp) and an RNA-directed RNA polymerase (RdRp). Expression of the 3'-proximal RdRp ORF appears to result from -1 translational frameshifting of the PArp ORF. Phylogenetic analysis of the RdRp indicated that SpFV1 and CiTV1 were most closely related to each other and the unclassified plant virus Cucurbit yellows associated virus, and more distantly related to the unclassified fungal dsRNA viruses Phlebiopsis gigantea virus 2 and Fusarium graminearum virus 3.

The 5'CL-PCBP RNP complex, 3' poly(A) tail and 2A(pro) are required for optimal translation of poliovirus RNA.

In this study, we showed that the 5'CL-PCBP complex, 3' poly(A) tail and viral protein 2A(pro) are all required for optimal translation of PV RNA. The 2A(pro)-mediated stimulation of translation was observed in the presence or absence of both the 5'CL and the 3' poly(A) tail. Using protein-RNA tethering, we established that the 5'CL-PCBP complex is required for optimal viral RNA translation and identified the KH3 domain of PCBP2 as the functional region. We also showed that the 5'CL-PCBP complex and the 3' poly(A) tail stimulate translation independent of each other. In addition to the independent function of each element, the 5'CL and the 3' poly(A) tail function synergistically to stimulate and prolong translation. These results are consistent with a model in which the 5'CL-PCBP complex interacts with the 3' poly(A)-PABP complex to form a 5'-3' circular complex that facilitates ribosome reloading and stimulates PV RNA translation.

Functional role of the 5' terminal cloverleaf in Coxsackievirus RNA replication.

Using cell-free reactions, we investigated the role of the 5' cloverleaf (5'CL) and associated C-rich sequence in Coxsackievirus B3 RNA replication. We showed that the binding of poly(C) binding protein (PCBP) to the C-rich sequence was the primary determinant of RNA stability. In addition, inhibition of negative-strand synthesis was only observed when PCBP binding to both stem-loop 'b' and the C-rich sequence was inhibited. Taken together, these findings suggest that PCBP binding to the C-rich sequence was sufficient to support RNA stability and negative-strand synthesis. Mutational analysis of the three conserved structural elements in stem-loop 'd' showed that they were required for efficient negative- and positive-strand synthesis. Finally, we showed an RNA with a 5' terminal deletion (Delta49TD RNA), which was previously isolated from persistently infected cells, replicated at low but detectable levels in these reactions. Importantly, the critical replication elements identified in this study are still present in the Delta49TD RNA.

Protein-RNA tethering: the role of poly(C) binding protein 2 in poliovirus RNA replication.

The exploitation of cellular functions and host proteins is an essential part of viral replication. The study of this interplay has provided significant insight into host cell processes in addition to advancing the understanding of the viral life-cycle. Poliovirus utilizes a multifunctional cellular protein, poly(C) binding protein 2 (PCBP2), for RNA stability, translation and RNA replication. In its cellular capacity, PCBP2 is involved in many functions, including transcriptional activation, mRNA stability and translational silencing. Using a novel protein-RNA tethering system, we establish PCBP2 as an essential co-factor in the initiation of poliovirus negative-strand synthesis. Furthermore, we identified the conserved KH domains in PCBP2 that are required for the initiation of poliovirus negative-strand synthesis, and showed that this required neither direct RNA binding or dimerization of PCBP2. This study demonstrates the novel application of a protein-RNA tethering system for the molecular characterization of cellular protein involvement in viral RNA replication.