Pestiviruses are some of the most significant pathogens affecting ruminants and swine. Here, we assembled a 11 276 bp contig encoding a predicted 3635 aa polyprotein from porcine serum with 68 % pairwise identity to that of a recently partially characterized Rhinolophus affinis pestivirus (RaPV) and approximately 25-28 % pairwise identity to those of other pestiviruses. The virus was provisionally named atypical porcine pestivirus (APPV). Metagenomic sequencing of 182 serum samples identified four additional APPV-positive samples. Positive samples originated from five states and ELISAs using recombinant APPV Erns found cross-reactive antibodies in 94 % of a collection of porcine serum samples, suggesting widespread distribution of APPV in the US swine herd. The molecular and serological results suggest that APPV is a novel, highly divergent porcine pestivirus widely distributed in US pigs.

Porcine reproductive and respiratory syndrome (PRRS) virus nonstructural protein 2 (nsp2) contains a cysteine protease domain at its N terminus, which belongs to the ovarian tumor (OTU) protease family. In this study, we demonstrated that the PRRSV nsp2 OTU domain antagonizes the type I interferon induction by interfering with the NF-B signaling pathway. Further analysis revealed that the nsp2 OTU domain possesses ubiquitin-deconjugating activity. This domain has the ability to inhibit NF-B activation by interfering with the polyubiquitination process of IB␣, which subsequently prevents IB␣ degradation. To determine whether the nsp2 protein antagonist function can be ablated from the virus, we introduced point mutations into the OTU domain region by use of reverse genetics. The D458A, S462A, and D465A mutations targeting on a B-cell epitope in the OTU domain region generated the viable recombinant viruses, and the S462A and D465A mutants were attenuated for growth in cell culture. The OTU domain mutants were examined to determine whether mutations in the nsp2 OTU domain region altered virus ability to inhibit NF-B activation. The result showed that certain mutations lethal to virus replication impaired the ability of nsp2 to inhibit NF-B activation but that the viable recombinant viruses, vSD-S462A and vSD-D465A, were unable to inhibit NF-B activation as effectively as the wild-type virus. This study represents a fundamental step in elucidating the role of nsp2 in PRRS pathogenesis and provides an important insight in future modified live-virus vaccine development.

Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a paramyxovirus often referred to as canine parainfluenza virus (CPI) in the veterinary field. PIV5 is thought to be a contributing factor to kennel cough. Kennel cough vaccines containing live PIV5 have been used in dogs for many decades. PIV5 is not known to cause any diseases in humans or other animals. PIV5 has been used as a vector for vaccine development for humans and animals. One critical question concerning the use of PIV5 as a vector is whether prior exposure to PIV5 would prevent the use of PIV5-based vaccines. In this work, we have examined immunogenicity of a recombinant PIV5 expressing hemagglutinin (HA) of influenza A virus subtype 3 (rPIV5-H3) in dogs that were immunized against PIV5. We found that vaccination of the dogs containing neutralizing antibodies against PIV5 with rPIV5-H3 generated immunity against influenza A virus, indicting that PIV5-based vaccine is immunogenic in dogs with prior exposure. Furthermore, we have examined exposure of PIV5 in human populations. We have detected neutralizing antibody (nAb) against PIV5 in 13 out of 45 human serum samples (about 29 percent). The nAb titers in humans were lower than that in vaccinated dogs, suggesting that nAb in humans is unlikely to prevent PIV5 from being an efficacious vector in humans.

Non-structural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the largest protein of this virus. In addition to its crucial role in virus replication, recent studies have indicated its involvement in modulating host immunity. In this study, each of the six identified immunodominant nsp2 B-cell epitopes (ES2-ES7) was deleted using a type I PRRSV cDNA infectious clone. Deletion of ES3, ES4 or ES7 allowed the generation of viable virus. In comparison with the parental virus, the DES3 mutant showed increased cytolytic activity and more vigorous growth kinetics, whilst the DES4 and DES7 mutants displayed decreased cytolytic activity and slower growth kinetics in MARC-145 cells. These nsp2 mutants were characterized further in a nursery pig disease model. The results showed that the DES4 and DES7 mutants exhibited attenuated phenotypes, whereas the DES3 mutant produced a higher peak viral load in pigs. The antibody response reached similar levels, as measured by IDEXX ELISA at 21 days post-infection, and slightly higher levels of mean virus neutralizing titres were observed from pigs infected by the DES4 and DES7 mutants. The expression of innate and T-helper 1 cytokines was measured in peripheral blood mononuclear cells or virus-infected macrophages. The results consistently showed that interleukin-1b and tumour necrosis factor alpha expression levels were downregulated in cells that were stimulated (or infected) with the DES3 mutant compared with parental virus and the other nsp2 deletion mutants. These results suggest that certain regions in nsp2 are non-essential for PRRSV replication but may play an important role in modulation of host immunity in vivo. INTRODUCTIONPorcine reproductive and respiratory syndrome virus (PRRSV) causes late-term reproductive failure in sows and severe pneumonia in neonatal pigs. Since its emergence in the 1980s, PRRSV has been estimated to cost at least US$600 million annually in the USA alone (Neumann et al., 2005). The recent outbreak of porcine high-fever disease in China, caused by a highly pathogenic PRRSV (HP-PRRSV), has increased the threat to the swine industry worldwide (Tian et al., 2007).PRRSV is a small, enveloped virus containing a single, positive-sense RNA genome. Genomic sequence comparisons have shown that PRRSV consists of two genotypes: European (type I) and North American (type II). These two genotypes share only about 60 % sequence identity (Allende et al., 1999;Nelsen et al., 1999). The PRRSV genome is about 15 kb in length and contains nine open reading frames (ORFs). The replicase-associated genes, ORF1a and ORF1b, are situated at the 59 end of the genome and represent nearly 75 % of the viral RNA. From studies of the related equine arteritis virus, PRRSV ORF1a and ORF1ab encode replicase polyproteins, pp1a and pp1ab, which are predicted to be cleaved into 13 nonstructural protein (nsp) products: nsp1a, nsp1b and nsp2-nsp12 (Snijder et al., 1994;den Boon et al., 1995; van Dinten et al., 1996;Ziebuhr et al., 2000). The nsp2 i...

The aim of this study was to evaluate the functional efficacy of retinal progenitor cell (RPC) containing sheets with BDNF microspheres following subretinal transplantation in a rat model of retinal degeneration. Sheets of E19 RPCs derived from human placental alkaline phosphatase (hPAP) expressing transgenic rats were coated with poly-lactide-co-glycolide (PLGA) microspheres containing brain-derived neurotrophic factor (BDNF) and transplanted into the subretinal space of S334ter line 3 rhodopsin retinal degenerate rats. Controls received transplants without BDNF or BDNF microspheres alone. Visual function was monitored using optokinetic head-tracking behavior. Visually evoked responses to varying light intensities were recorded from the superior colliculus (SC) by electrophysiology at 60 days after surgery. Frozen sections were studied by immunohistochemistry for photoreceptor and synaptic markers. Visual head tracking was significantly improved in rats that received BDNF-coated RPC sheets. Relatively more BDNF-treated transplanted rats (80%) compared to non-BDNF transplants (57%) responded to a ''low light'' intensity of 1 cd/m 2 in a confined SC area. With bright light, the onset latency of SC responses was restored to a nearly normal level in BDNF-treated transplants. No significant improvement was observed in the BDNF-only and no surgery transgenic control rats. The bipolar synaptic markers mGluR6 and PSD-95 showed normal distribution in transplants and abnormal distribution of the host retina, both with or without BDNF treatment. Red-green cones were significantly reduced in the host retina overlying the transplant in the BDNF-treated group. In summary, BDNF coating improved the functional efficacy of RPC grafts. The mechanism of the BDNF effectsdeither promoting functional integration between the transplant and the host retina and/or synergistic action with other putative humoral factors released by the RPCsdstill needs to be elucidated. Ó 2007 Elsevier Ltd. All rights reserved.Keywords: retinal transplantation; superior colliculus; head-tracking behavior; BDNF microsphere; retinal progenitor cells; S334terAbbreviations: BDNF, brain-derived neurotrophic factor; DAPI, 4 0 6 0 -diamidino-2-phenylindole hydrochloride; CNS, central nervous system; GC, ganglion cell layer; H, host retina; hPAP, human placental alkaline phosphatase; IN, inner nuclear layer; IP, inner plexiform layer; mGluR6, metabotropic glutamate receptor 6 (synaptic receptor on bipolar cells); ms, milliseconds; ON, outer nuclear layer; OP, outer plexiform layer; OS, outer segments; PBS, phosphate-buffered saline; PKC, protein kinase C (marker for rod bipolar cells); PLGA, poly-lactide-co-glycolide; PSD-95, post-synaptic density protein 95 (post-synaptic marker on bipolar cell dendrites); RG-opsin, red-green opsin; RPC, retinal progenitor cells; SC, superior colliculus; RCS, Royal College of Surgeons; RPE, retinal pigment epithelium; T, transplant.

This study aimed to test the hypothesis that visual responses in the superior colliculus (SC) originate from synaptic connections between fetal retinal transplants and degenerating host retinas. Sheets of embryonic day 19 rat retina expressing human placental alkaline phosphatase were transplanted to the subretinal space of 3-to 4-week-old S334ter-line-3 rats with fast retinal degeneration. Several months later, visual responses were recorded from the SC. Attenuated pseudorabies virus that is specifically transferred between neurons at synapses (strains PRV-152, expressing green fluorescent protein (GFP) or BaBlu, expressing Escherichia coli bgalactosidase) was injected into the visually responsive site of the SC. After survival times of 1-2 days, the virus was detected in the retina by immunohistochemistry in combination with different retinal cell markers, such as protein kinase C, recoverin, calciumcalmodulin-dependent protein kinase II and glutamine synthetase. Transplanted rats had a mean response threshold of )3.1 log cd ⁄ m 2 in a small area of the SC corresponding to the location of the graft in the retina. By 30 h after injection into this SC area, the virus traced back to host ganglion cells overlying the transplant and in close proximity to the transplant. By 2 days after injection, extensive virus label was found in the host retina and many cells in the transplant were also labeled. Virus-labeled cells in the transplant were double labeled for neuronal and glial cell markers. This study provides anatomical evidence that synaptic connections between fetal retinal transplants and host retinas contribute to the visual responses in the SC.

A full-length cDNA infectious clone, pKS15-01-Clone, was constructed from an emerging Senecavirus A (SVA; strain KS15-01). To explore the potential use as a viral backbone for expressing marker genes, the enhanced green fluorescent protein (EGFP)-tagged reporter virus (vKS15-01-EGFP) was generated using reverse genetics. Compared to the parental virus, the pKS15-01-Clone derived virus (vKS15-01-Clone) replicated efficiently in vitro and in vivo, and induced similar levels of neutralizing antibody and cytokine responses in infected animals. In contrast, the vKS15-01-EGFP virus showed impaired growth ability and induced lower level of immune response in infected animals. Lesions on the dorsal snout and coronary bands were observed in all pigs infected by parental virus KS15-01, but not in pigs infected with vKS15-01-Clone or vKS15-01-EGFP viruses. These results demonstrated that the infectious clone and EGFP reporter virus could be used as important tools in further elucidating the SVA pathogenesis and development of control measures.

8 PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines.