Recent cases of porcine reproductive and respiratory syndrome virus (PRRSV) infection in United States swine-herds have been associated with high mortality in piglets and severe morbidity in sows. Analysis of the ORF5 gene from such clinical cases revealed a unique restriction fragment polymorphism (RFLP) of 1-7-4. The genome diversity of seventeen of these viruses (81.4% to 99.8% identical; collected 2013-2015) and the pathogenicity of 4 representative viruses were compared to that of SDSU73, a known moderately virulent strain. Recombination analyses revealed genomic breakpoints in structural and nonstructural regions of the genomes with evidence for recombination events between lineages. Pathogenicity varied between the isolates and the patterns were not consistent. IA/2014/NADC34, IA/2013/ISU-1 and IN/2014/ISU-5 caused more severe disease, and IA/2014/ISU-2 did not cause pyrexia and had little effect on pig growth. ORF5 RFLP genotyping was ineffectual in providing insight into isolate pathogenicity and that other parameters of virulence remain to be identified.

Senecavirus A has been infrequently associated with vesicular disease in swine since 1988. However, clinical disease has not been reproduced after experimental infection with this virus. We report vesicular disease in 9-week-old pigs after Sencavirus A infection by the intranasal route under experimental conditions.

In 2013, porcine epidemic diarrhea virus (PEDV) emerged in the United States as a rapidly spreading epidemic causing dramatic death losses in suckling piglets. Neonatal piglets are most vulnerable to clinical disease and their only protection is passive immunity from their dam. At the end of the third year of the PEDV outbreak, most US sow herds have been infected and many are entering into an endemic disease with much less, but still chronic losses. This endemic state and the occasional naïve herd that breaks with PEDV demonstrate a need to immunize sows to protect piglets. Stimulating PEDV immunity in the sow using safe and efficacious vaccines is the best course of action; however, conducting such studies to develop sow vaccines is very costly and logistically difficult. This manuscript reviews the status of PEDV vaccines available in the United States and Canada, and describes an experiment evaluating the potential use of young pigs as a surrogate model to evaluate potential sow vaccines.

The L6 region of bovine adenovirus type (BAdV)-3 encodes a nonstructural protein named 33K. To identify and characterize the 33K protein, rabbit polyclonal antiserum was raised against a 33K-GST fusion protein expressed in bacteria. Anti-33K serum immunoprecipitated a protein of 42 kDa in in vitro translated and transcribed mRNA of 33K. However, three proteins of 42, 38, and 33 kDa were detected in BAdV-3 infected cells. To determine the role of this protein in virus replication, a recombinant BAV-33S1 containing insertional inactivation of 33K (a stop codon created at the seventh amino acid of 33K ORF) was constructed. Although BAV-33S1 could be isolated, the mutant showed a severe defect in the production of progeny virus. Inactivation of the 33K gene showed no effect on early and late viral gene expression in cells infected with BAV-33S1. However, formation of mature virions was significantly reduced in cells infected with BAV-33S1. Surprisingly, insertional inactivation of 33K at amino acid 97 (pFBAV-33.KS2) proved lethal for virus production. Although expression of early or late genes was not affected, no capsid formation could be observed in mutant DNA-transfected cells. These results suggest that 33K is required for capsid assembly and efficient DNA capsid interaction.

Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs and spreads rapidly after entering naïve pig populations. The objectives were to (1) compare the disease course following inoculation with PEDV isolate US/Colorado/2013 in naïve 10 day and 8 week-old pigs, and (2) contrast the naïve response to homologous challenge in 8 week-old pigs. Pigs were randomly assigned into group 1 (n = 40, no PEDV exposure), group 2 (n = 43, PEDV inoculation at 10 days of age) and group 3 (n = 48, PEDV inoculation at 8 weeks of age). Thirty-three group 2 pigs received a homologous challenge at 8 weeks of age. Following primary or secondary inoculation, 3–10 pigs were euthanized at days post-inoculation (dpi) 1, 2, 3, 7 or 14. Clinical signs were more pronounced in 10 day-old pigs compared to 8 week-old pigs at dpi 2 and 3, a higher number of 10 day-old pigs shed PEDV RNA in feces compared to 8 week-old pigs. Typical severe atrophic enteritis of PEDV infection was observed at dpi 3 in both age groups, and at dpi 4 and 14 fecal shedding patterns were also similar. While both age groups had seroconverted to PEDV by dpi 14, IgG levels were higher in 8 week-old pigs. PEDV IgA antibodies were detected in feces of approximately 50% of the pigs at dpi 44. In homologous challenged pigs, no clinical signs or lesions were found, and PEDV fecal shedding was restricted to less than 10% of the pigs indicating the existence of homologous protection 44 days after initial PEDV exposure.

The adenovirus 33K protein appears to be a multifunctional protein performing different roles in viral infection. The involvement of 33K protein in different steps of adenovirus replication may require protein-protein interaction. Using 33K protein as a bait in the yeast two-hybrid system, we screened open reading frames (ORFs) of bovine adenovirus (BAdV)-3 for potential interactions with 33K protein. Interestingly, 33K protein showed specific interaction with 100K and pV proteins. The yeast two-hybrid findings were validated by in vitro binding using in vitro synthesized transcription-translation products, GST pull down assay and in vitro co-immunoprecipitation assay using protein-specific antibodies. We demonstrated, that the interaction of 33K protein with 100K and pV proteins takes place during BAdV-3 infection. Finally, our data suggests that the stretch of amino acids 81-120 and 161-200 in 33K protein are critical for the interaction with pV and 100K proteins, respectively.

BackgroundInfectious salmon anemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon (Salmo salar); a disease first diagnosed in Norway in 1984. This virus, which was first characterized following its isolation in cell culture in 1995, belongs to the family Orthomyxoviridae, genus, Isavirus. The Isavirus genome consists of eight single-stranded RNA segments of negative sense, each with one to three open reading frames flanked by 3' and 5' non-coding regions (NCRs). Although the terminal sequences of other members of the family Orthomyxoviridae such as Influenzavirus A have been extensively analyzed, those of Isavirus remain largely unknown, and the few reported are from different ISAV strains and on different ends of the different RNA segments. This paper describes a comprehensive analysis of the 3' and 5' end sequences of the eight RNA segments of ISAV of both European and North American genotypes, and evidence of quasispecies of ISAV based on sequence variation in the untranslated regions (UTRs) of transcripts.ResultsTwo different ISAV strains and two different RNA preparations were used in this study. ISAV strain ADL-PM 3205 ISAV-07 (ADL-ISAV-07) of European genotype was the source of total RNA extracted from ISAV-infected TO cells, which contained both viral mRNA and cRNA. ISAV strain NBISA01 of North American genotype was the source of vRNA extracted from purified virus. The NCRs of each segment were identified by sequencing cDNA prepared by three different methods, 5' RACE (Rapid amplification of cDNA ends), 3' RACE, and RNA ligation mediated PCR. Sequence analysis of five clones each derived from one RT-PCR product from each NCR of ISAV transcripts of segments 1 to 8 revealed significant heterogeneity among the clones of the same segment end, providing unequivocal evidence for presence of intra-segment ISAV quasispecies. Both RNA preparations (mRNA/cRNA and vRNA) yielded complementary sequence information, allowing the simultaneous identification and confirmation of the 3' and 5' NCR sequences of the 8 RNA genome segments of both genotypes of ISAV. The 3' sequences of the mRNA transcripts of ADL-ISAV-07 terminated 13-18 nucleotides from the full 3' terminus of cRNA, continuing as a poly(A) tail, which corresponded with the location of the polyadenylation signal. The lengths of the 3' and 5' NCRs of the vRNA were variable in the different genome segments, but the terminal 7 and 11 nucleotides of the 3' and 5' ends, respectively, were highly conserved among the eight genomic segments of ISAV. The first three nucleotides at the 3' end are GCU-3' (except in segment 5 with ACU-3'), whereas at the 5' end are 5'-AGU with the polyadenylation signal of 3-5 uridines 13-15 nucleotides downstream of the 5' end terminus of the vRNA. Exactly the same features were found in the respective complementary 5' and 3' end NCR sequences of the cRNA transcripts of ADL-ISAV-07, indicating that the terminal sequences of the 8 RNA genome segments are highly conserved among the two ISAV genotypes. The 5' NCR sequence...

BackgroundSenecavirus A, commonly known as Seneca Valley virus (SVV), is a picornavirus that has been infrequently associated with porcine idiopathic vesicular disease (PIVD). In late 2014 there were multiple PIVD outbreaks in several states in Brazil and samples from those cases tested positive for SVV. Beginning in July of 2015, multiple cases of PIVD were reported in the United States in which a genetically similar SVV was also detected. These events suggested SVV could induce vesicular disease, which was recently demonstrated with contemporary US isolates that produced mild disease in pigs. It was hypothesized that stressful conditions may exacerbate the expression of clinical disease and the following experiment was performed. Two groups of 9-week-old pigs were given an intranasal SVV challenge with one group receiving an immunosuppressive dose of dexamethasone prior to challenge. After challenge animals were observed for the development of clinical signs and serum and swabs were collected to study viral shedding and antibody production. In addition, pigs were euthanized 2, 4, 6, 8, and 12 days post inoculation (dpi) to demonstrate tissue distribution of virus during acute infection.ResultsVesicular disease was experimentally induced in both groups with the duration and magnitude of clinical signs similar between groups. During acute infection [0–14 days post infection (dpi)], SVV was detected by PCR in serum, nasal swabs, rectal swabs, various tissues, and in swabs from ruptured vesicles. From 15 to 30 dpi, virus was less consistently detected in nasal and rectal swabs, and absent from most serum samples. Virus neutralizing antibody was detected by 5 dpi and lasted until the end of the study.ConclusionTreatment with an immunosuppressive dose of dexamethasone did not drastically alter the clinical disease course of SVV in experimentally infected nursery aged swine. A greater understanding of SVV pathogenesis and factors that could exacerbate disease can help the swine industry with control and prevention strategies directed against this virus.