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.
•Next generation sequencing of 17 PRRSV isolates with an ORF5 RFLP of 1-7-4 was completed.•The viruses were diverse and showed evidence for recombination events.•Swine were challenged with four such viruses to examine degree of pathogenic severity.•Isolate NADC34 was the most pathogenic and the least virulent isolate was ISU-2.
Neonatal mortality has been increasingly reported on swine breeding farms experiencing swine idiopathic vesicular disease (SIVD) outbreaks, which can be accompanied by lethargy, diarrhea, and ...neurologic signs in neonates. Seneca Valley Virus (SVV), or Senecavirus A, has been detected in clinical samples taken from pigs with SIVD. Experimental SVV inoculation has caused vesicular disease in pigs, particularly during the stages from weaning to finishing. However, it remains crucial to investigate whether SVV directly contributes to the increase in neonatal mortality rates. The following study was conducted to chronicle the pathogenesis of SVV infection in sows and their offspring. Ten sows were intranasally inoculated with 4.75 × 10
plaque-forming units of the virus per sow either late in gestation (n = 5) or within fourteen days of farrowing (n = 5). Each sow replicated SVV following intranasal inoculation, but only one out of ten sows developed a vesicular lesion on the snout. Evidence of transplacental infection was observed in two litters, and an additional two litters became infected following parturition out of five litters from sows inoculated in late gestation. No clinical signs were observed in the infected neonates. Likewise, no clinical signs were observed in the other five litters inoculated after farrowing, although each piglet did replicate the challenge virus. In this study, the experimental challenge of SVV did not result in neonatal mortality in contrast to observations in the field; however, it has shed light on the pathogenesis of the virus, the transmission of SVV between sows and their offspring, and host immune response that can help shape control measures in the field.
During pregnancy, the maternal immune response changes dramatically over the course of gestation. This has implications for generation of lactogenic immunity and subsequent protection in suckling ...neonates against enteric viral infections. For example, porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus that causes acute diarrhea in neonatal piglets. Due to the high virulence of PEDV and the naïve, immature immune system of neonatal suckling piglets, passive lactogenic immunity to PEDV induced during pregnancy, via the gut-mammary gland (MG)-secretory IgA (sIgA) axis, is critical for piglet protection. However, the anti-PEDV immune response during pregnancy and stage of gestation required to optimally stimulate the gut-MG-sIgA axis is undefined. We hypothesize that there is a gestational window in which non-lethal PEDV infection of pregnant gilts influences maximum lymphocyte mucosal trafficking to the MG, resulting in optimal passive lactogenic protection in suckling piglets. To understand how the stages of gestation affect maternal immune responses to PEDV, three groups of gilts were orally infected with PEDV in the first, second or third trimester. Control (mock) gilts were inoculated with medium in the third trimester. To determine if lactogenic immunity correlated with protection, all piglets were PEDV-challenged at 3-5 days postpartum. PEDV infection of gilts at different stages of gestation significantly affected multiple maternal systemic immune parameters prepartum, including cytokines, B cells, PEDV antibodies (Abs), and PEDV antibody secreting cells (ASCs). Pregnant second trimester gilts had significantly higher levels of circulating PEDV IgA and IgG Abs and ASCs and PEDV virus neutralizing (VN) Abs post PEDV infection. Coinciding with the significantly higher PEDV Ab responses in second trimester gilts, the survival rate of their PEDV-challenged piglets was 100%, compared with 87.2, 55.9, and 5.7% for first, third, and mock litters, respectively. Additionally, piglet survival positively correlated with PEDV IgA Abs and ASCs and VN Abs in milk and PEDV IgA and IgG Abs in piglet serum. Our findings have implications for gestational timing of oral attenuated PEDV maternal vaccines, whereby PEDV intestinal infection in the second trimester optimally stimulated the gut-MG-sIgA axis resulting in 100% lactogenic immune protection in suckling piglets.
Respiratory syncytial virus (RSV) infection in preterm and newborn infants can result in severe bronchiolitis and hospitalization. The lamb lung has several key features conducive to modeling RSV ...infection in human infants, including susceptibility to human strains of RSV such as the A2, Long, and Memphis Strain 37 (M37). In this study, the kinetics of M37 infection was investigated in newborn lambs in order to better define clinical, viral, physiological, and immunological parameters as well as the pathology and lesions.
Newborn lambs were nebulized with M37 hRSV (6 mL of 1.27 x 10(7) FFU/mL), monitored daily for clinical responses, and respiratory tissues were collected from groups of lambs at days 1, 3, 4, 6, and 8 post-inoculation for the assessment of viral replication parameters, lesions and also cellular, immunologic and inflammatory responses.
Lambs had increased expiratory effort (forced expiration) at days 4, 6, and 8 post-inoculation. Nasal wash lacked RSV titers at day 1, but titers were present at low levels at days 3 (peak), 4, and 8. Viral titers in bronchoalveolar lavage fluid (BALF) reached a plateau at day 3 (4.6 Log10 FFU/mL), which was maintained until day 6 (4.83 Log10 FFU/mL), and were markedly reduced or absent at day 8. Viral RNA levels (detected by RT-qPCR) in BALF were indistinguishable at days 3 (6.22 ± 0.08 Log10 M37 RNA copies/mL; mean ± se) and 4 (6.20 ± 0.16 Log10 M37 RNA copies/mL; mean ± se) and increased slightly on day 6 (7.15 ± 0.2 Log10 M37 RNA copies/mL; mean ± se). Viral antigen in lung tissue as detected by immunohistochemistry was not seen at day 1, was present at days 3 and 4 before reaching a peak by day 6, and was markedly reduced by day 8. Viral antigen was mainly present in airways (bronchi, bronchioles) at day 3 and was increasingly present in alveolar cells at days 4 and 6, with reduction at day 8. Histopathologic lesions such as bronchitis/bronchiolitis, epithelial necrosis and hyperplasia, peribronchial lymphocyte infiltration, and syncytial cells, were consistent with those described previously for lambs and infants.
This work demonstrates that M37 hRSV replication in the lower airways of newborn lambs is robust with peak replication on day 3 and sustained until day 6. These findings, along with the similarities of lamb lung to those of infants in terms of alveolar development, airway branching and epithelium, susceptibility to human RSV strains, lesion characteristics (bronchiolitis), lung size, clinical parameters, and immunity, further establish the neonatal lamb as a model with key features that mimic RSV infection in infants.
Kaposi's sarcoma-associated herpesvirus (KSHV) has been linked to the development of Kaposi's sarcoma, a major AIDS-associated malignancy, and to hematologic malignancies, including primary effusion ...lymphoma and multicentric Castleman's disease. Like other herpesviruses, KSHV is capable of both latent and lytic replication. Understanding the molecular details associated with this transition from latency to lytic replication is key to controlling virus spread and can affect the development of intervention strategies. Here, we report that Kruppel-associated box domain-associated protein-1 (KAP-1)/transcriptional intermediary factor 1beta, a cellular transcriptional repressor that controls chromosomal remodeling, participates in the process of switching viral latency to lytic replication. Knockdown of KAP-1 by small interfering RNA leads to KSHV reactivation mediated by K-Rta, a key transcriptional regulator. In cells harboring latent KSHV, KAP-1 was associated with the majority of viral lytic-gene promoters. K-Rta overexpression induced the viral lytic cycle with concomitant reduction of KAP-1 binding to viral promoters. Association of KAP-1 with heterochromatin was modulated by both sumoylation and phosphorylation. During lytic replication of KSHV, KAP-1 was phosphorylated at Ser(824). Several lines of evidence directly linked the viral protein kinase to this post-translational modification. Additional studies showed that this phosphorylation of KAP-1 produced a decrease in its sumoylation, consequently decreasing the ability of KAP-1 to condense chromatin on viral promoters. In summary, the cellular transcriptional repressor KAP-1 plays a role in regulating KSHV latency, and viral protein kinase modulates the chromatin remodeling function of this repressor.
One of the largest impediments for commercial swine production is the presence of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a devastating RNA viral infection that is responsible ...for over $1 billion in loss in the U.S. annually. The challenge with combating PRRSV is a combination of the effect of an extraordinary rate of mutation, the ability to infect macrophages, and subversion of host immune response through a series of actions leading to both immunomodulation and immune evasion. Currently there are a handful of commercial vaccines on the market that have been shown to be effective against homologous infections, but struggle against heterologous or mixed strain infections. However, vaccination is the current best strategy for combating PRRSV, making research into new vaccine technology key. To address these issues with PRRSV and host antiviral functions a novel modified-live vaccine (MLV) able to stimulate known antiviral interferons was created and examined for its ability to potentiate effective immunity and better protection. Here, we examine gene expression in the liver of pigs vaccinated with our novel vaccine, given the liver's large role in antiviral responses and vaccine metabolism. Our study indicated that pigs administered the novel vaccine experience homeostatic gene expression consistent with less inflammation and T-cell depletion risk than pigs administered the commercial vaccine.
Previously, we have shown that CMV isolated from deer mouse could be used in vivo and in vitro to express Sin Nombre virus (SNV) glycoprotein G1 in deer mice Rizvanov AA, van Geelen AG, Morzunov S, ...et al. Generation of a recombinant cytomegalovirus for expression of a hantavirus glycoprotein. J.Virol. 2003;77(22):12203–10. In this report, we further characterize replication of wild-type (wt) and recombinant
Peromyscus CMV (PCMV) in vivo and in vitro using realtime PCR, and infectious center assays. Our findings indicate that both wt PCMV and recombinant PCMV establish persistent infections in
P. maniculatus. In addition, we demonstrated that gamma irradiation of PCMV infected mice resulted in reactivation of recombinant PCMV in persistently infected mice.
Senecavirus 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.
Vesicular 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.
Treatment 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.
Abstract The effects of ethanol exposure on fetal lungs remain under investigation. Previously, we demonstrated that lambs exposed to ethanol during gestation had impaired expression of pulmonary ...surfactant protein A, a crucial component of lung immunity. In this study, we investigated the effects of in utero exposure to ethanol on maturation and immunity of the fetal lung. Pregnant ewes were surgically implanted with an abomasal cannula and administered 1 g ethanol/kg ( n = 8) or water ( n = 8) during the last trimester of pregnancy. Lambs were delivered prematurely or naturally. Neonatal lungs were assessed for maturation markers (hypoxia-inducible factor-1α HIF-1α, HIF-2α, HIF-3α, vascular endothelial growth factor-A VEGF-A, VEGFR-1, VEGFR-2, glycogen, and lung protein levels) and immunity (cytokines and chemokines). Preterm animals exposed to ethanol had significantly reduced VEGF-A mRNA ( P = .066) and protein levels, HIF-1α ( P = .055), HIF-2α ( P = .019), VEGFR-1 ( P = .088), and VEGFR-2 ( P = .067) mRNA levels but no changes in HIF-3α mRNA. No significant changes occurred in full-term animals exposed to ethanol. Glycogen levels were significantly higher in preterm animals exposed to ethanol ( P = .006) but not in full-term animals. Ethanol exposure was associated with significantly lower lung protein levels in preterm ( P = .03) but not full-term animals. Preterm animals exposed to ethanol had significantly reduced TNF-α ( P = .05), IL-10 ( P = .03), chemokine (C-C motif) ligand 5 (CCL5) ( P = .017), and monocyte chemotactic protein-1 (MCP-1) ( P = .0004) mRNA. In full-term animals exposed to ethanol, the immune alterations were either sustained (TNF-α, P = .009; IL-10, P = .03) or returned to near baseline levels (CCL5 and MCP-1). The ethanol-mediated alterations in fetal lung maturation and immunity may explain the increased incidence of respiratory infections in neonates exposed to ethanol in utero.
Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in infants and children worldwide. There are currently no licensed vaccines or effective antivirals. The lack of a ...vaccine is partly due to increased caution following the aftermath of a failed clinical trial of a formalin-inactivated RSV vaccine (FI-RSV) conducted in the 1960's that led to enhanced disease, necessitating hospitalization of 80% of vaccine recipients and resulting in two fatalities. Perinatal lamb lungs are similar in size, structure and physiology to those of human infants and are susceptible to human strains of RSV that induce similar lesions as those observed in infected human infants. We sought to determine if perinatal lambs immunized with FI-RSV would develop key features of vaccine-enhanced disease. This was tested in colostrum-deprived lambs immunized at 3-5 days of age with FI-RSV followed two weeks later by RSV infection. The FI-RSV-vaccinated lambs exhibited several key features of RSV vaccine-enhanced disease, including reduced RSV titers in bronchoalveolar lavage fluid and lung, and increased infiltration of peribronchiolar and perivascular lymphocytes compared to lambs either undergoing an acute RSV infection or naïve controls; all features of RSV vaccine-enhanced disease. These results represent a first step proof-of-principle demonstration that the lamb can develop altered responses to RSV following FI-RSV vaccination. The lamb model may be useful for future mechanistic studies as well as the assessment of RSV vaccines designed for infants.
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