Epizootic Hemorrhagic Disease Agar Gel Immunodiffusion Assay.

Agar gel immunodiffusion assay (AGID) is a laboratory test which detects specific antigen-antibody interactions by the development of visible precipitation lines in a semisolid matrix. Here we describe the preparation of agar gel plates, the method to test serum samples by AGID for the presence of EHDV antibodies, and the interpretation of test results. This test has known cross-reactivity to bluetongue antibodies; therefore positive samples by this assay require additional confirmatory testing; generally, its use should be limited to healthy animal attestations where required.

Outbreak of epizootic hemorrhagic disease in captive reindeer (Rangifer tarandus).

In September 2020, an outbreak of epizootic hemorrhagic disease occurred in captive reindeer (Rangifer tarandus) and was associated with neurological signs and mortality. Four reindeer died or were euthanized after acute illness over a 12-day period. Affected reindeer displayed abnormal behavior, neurologic signs, lethargy, and/or lameness. The most consistent gross finding was dark red streaks throughout the adrenal gland cortices (4/4). One animal had acute hemorrhage involving the subcutis and skeletal muscles over the ventrolateral body wall and back, and abomasal serosa. Histologically, the most common lesions were adrenal gland cortical hemorrhage (4/4) with necrosis (3/4) and lymphoplasmacytic meningoencephalitis with gliosis, glial nodules, satellitosis, and nonsuppurative perivascular cuffing (4/4). The brain lesions were most frequent in the gray matter of the cerebrum, hippocampus, and thalamus but also involved the cerebellum and brainstem. Epizootic hemorrhagic disease virus serotype 6 was detected through PCR and sequencing of the spleen in all cases.

Transcriptomic Analysis of Liver Indicates Novel Vaccine to Porcine Reproductive and Respiratory Virus Promotes Homeostasis in T-Cell and Inflammatory Immune Responses Compared to a Commercial Vaccine in Pigs.

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.

Detection of pseudorabies virus antibody in swine serum and oral fluid specimens using a recombinant gE glycoprotein dual-matrix indirect ELISA.

Pseudorabies (Aujeszky disease) virus (PRV) was eliminated from domestic swine in many countries using glycoprotein E (gE)-deleted vaccines and serum antibody gE ELISAs, but PRV continues to circulate in some regions and in most feral swine populations in the world. We created a dual-matrix (serum and oral fluid) indirect IgG gE ELISA (iELISA) and evaluated its performance using samples from 4 groups of 10 pigs each: negative control (NC), vaccination (MLV), PRV inoculation (PRV), and vaccination followed by challenge (MLV-PRV). All serum and oral fluid samples collected before PRV challenge and all NC samples throughout the study were negative for gE antibodies by commercial blocking ELISA (bELISA) and our iELISA. Nasal swab samples from 9 of 10 animals in the PRV group were gB quantitative PRC (qPCR) positive at 2 days post-inoculation (dpi). The oral fluid iELISA detected a significant S/P response in the PRV (p = 0.03) and MLV-PRV (p = 0.01) groups by 6 dpi. ROC analyses of serum bELISA (n = 428), serum iELISA (n = 426), and oral fluid iELISA (n = 247) showed no significant differences in performance (p > 0.05). Our data support the concept of PRV surveillance based on oral fluid samples tested by an indirect gE ELISA.

Pseudorabies (Aujeszky's disease) virus DNA detection in swine nasal swab and oral fluid specimens using a gB-based real-time quantitative PCR.

In this study, the detection of PRV DNA in nasal swab (n = 440) and oral fluid (n = 1,545) samples collected over time from experimentally PRV vaccinated and/or PRV inoculated pigs (n = 40) was comparatively evaluated by real-time PCR. Serum samples (n = 440) were tested by PRV gB/gE blocking ELISAs (Pseudorabies Virus gB Antibody Test Kit and Pseudorabies Virus gpI Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME) to monitor PRV status over time. Following exposure to a gE-deleted modified live vaccine (Ingelvac® Aujeszky MLV, Boehringer Ingelheim, Ridgefield, CT) and/or a wild-type virus (3 CR Ossabaw), PRV gB DNA was detected in oral fluid specimens in a pattern similar to that of nasal swabs. For quantitative analyses, PRV PCR quantification cycle (Cq) results were re-expressed as "efficiency standardized Cqs (ECqs)" as a function of PCR efficiency using plate-specific positive amplification controls. ROC analyses of the PRV gB PCR ECqs results showed a similar performance of the PRV gB PCR for nasal swab and oral fluid specimens (area under the ROC curve = 85 % vs 83 %) and, based on an ECq cutoff of 0.01 a diagnostic specificity of 100 % and diagnostic sensitivities for oral fluid and nasal swab specimens of 53 % (95 % CI: 43 %, 62 %) and 70 % (95 % CI: 55 %, 83 %), respectively. Thus, the results described herein demonstrated the detection of PRV gB DNA in swine oral fluid and supported the use of this specimen in PRV diagnosis and surveillance.

Detection of pseudorabies virus antibody in swine oral fluid using a serum whole-virus indirect ELISA.

We evaluated the detection of pseudorabies virus (PRV) antibodies in swine oral fluid. Oral fluid and serum samples were obtained from 40 pigs allocated to 4 treatment groups (10 pigs/group): negative control (NC); wild-type PRV inoculation (PRV 3CR Ossabaw; hereafter PRV); PRV vaccination (Ingelvac Aujeszky MLV; Boehringer Ingelheim; hereafter MLV); and PRV vaccination followed by PRV inoculation at 21 d post-vaccination (MLV-PRV). Using a serum PRV whole-virus indirect IgG ELISA (Idexx Laboratories) adapted to the oral fluid matrix, PRV antibody was detected in oral fluid samples from treatment groups PRV, MLV, and MLV-PRV in a pattern similar to serum. Vaccination alone produced a low oral fluid antibody response (groups MLV and MLV-PRV), but a strong anamnestic response was observed following challenge with wild-type virus (group PRV). Analyses of the oral fluid PRV indirect IgG ELISA results showed good binary diagnostic performance (area under ROC curve = 93%) and excellent assay repeatability (intra-class correlation coefficient = 99.3%). The demonstrable presence of PRV antibodies in swine oral fluids suggests the possible use of oral fluids in pseudorabies surveillance.

Stage of Gestation at Porcine Epidemic Diarrhea Virus Infection of Pregnant Swine Impacts Maternal Immunity and Lactogenic Immune Protection of Neonatal Suckling Piglets.

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.

Experimental Seneca Valley virus infection in market-weight gilts.

Seneca Valley virus (SVV) is a picornavirus that causes vesicular disease in swine. Since it is clinically indistinguishable from vesicular disease caused by food-and-mouth disease virus (FMDV), investigations must be performed to rule out this high consequence pathogen. A large portion of these investigations have involved market-weight swine at slaughter plants. The objective of this study was to describe acute infection dynamics of market-weight gilts (8 months of age) experimentally infected with SVV. At 0 days post inoculation (dpi) all gilts (n=15) were given an intranasal SVV inoculation. Vesicular lesions on the coronary band were first observed on one or more feet by 2 dpi in 4 of the 15 gilts and in all by 5 dpi. Vesicles on the snout were observed in 6 of the 15 gilts beginning at 4 dpi. All gilts became viremic post challenge for about 7 days and developed anti-SVV neutralizing antibodies by 7 dpi. Most vesicular lesions were resolved by 14 dpi. Understanding the pathogenesis of SVV is critical in order to inform decisions that veterinarians and producers must make at the farm level to control this disease.

Coronavirus Endoribonuclease Activity in Porcine Epidemic Diarrhea Virus Suppresses Type I and Type III Interferon Responses.

Identifying viral antagonists of innate immunity and determining if they contribute to pathogenesis are critical for developing effective strategies to control emerging viruses. Previously, we reported that an endoribonuclease (EndoU) encoded by murine coronavirus plays a pivotal role in evasion of host innate immune defenses in macrophages. Here, we asked if the EndoU activity of porcine epidemic diarrhea coronavirus (PEDV), which causes acute diarrhea in swine, plays a role in antagonizing the innate response in porcine epithelial cells and macrophages, the sites of viral replication. We constructed an infectious clone of PEDV-Colorado strain (icPEDV-wt) and an EndoU-mutant PEDV (icPEDV-EnUmt) by changing the codon for a catalytic histidine residue of EndoU to alanine (His226Ala). We found that both icPEDV-wt and icPEDV-EnUmt propagated efficiently in interferon (IFN)-deficient Vero cells. In contrast, the propagation of icPEDV-EnUmt was impaired in porcine epithelial cells (LLC-PK1), where we detected an early and robust transcriptional activation of type I and type III IFNs. Infection of piglets with the parental Colorado strain, icPEDV-wt, or icPEDV-EnUmt revealed that all viruses replicated in the gut and induced diarrhea; however, there was reduced viral shedding and mortality in the icPEDV-EnUmt-infected animals. These results demonstrate that EndoU activity is not required for PEDV replication in immortalized, IFN-deficient Vero cells, but is important for suppressing the IFN response in epithelial cells and macrophages, which facilitates replication, shedding, and pathogenesis in vivo We conclude that PEDV EndoU activity is a key virulence factor that suppresses both type I and type III IFN responses.IMPORTANCE Coronaviruses (CoVs) can emerge from an animal reservoir into a naive host species to cause pandemic respiratory or gastrointestinal diseases with significant mortality in humans or domestic animals. Porcine epidemic diarrhea virus (PEDV), an alphacoronavirus (alpha-CoV), infects gut epithelial cells and macrophages, inducing diarrhea and resulting in high mortality in piglets. How PEDV suppresses the innate immune response was unknown. We found that mutating a viral endoribonuclease, EndoU, results in a virus that activates both the type I interferon response and the type III interferon response in macrophages and epithelial cells. This activation of interferon resulted in limited viral replication in epithelial cell cultures and was associated with reduced virus shedding and mortality in piglets. This study reveals a role for EndoU activity as a virulence factor in PEDV infection and provides an approach for generating live-attenuated vaccine candidates for emerging coronaviruses.

Dexamethasone treatment did not exacerbate Seneca Valley virus infection in nursery-age pigs.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Delivery of ALX-0171 by inhalation greatly reduces respiratory syncytial virus disease in newborn lambs.

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory disease in infants and young children worldwide. Currently, treatment is supportive and no vaccines are available. The use of newborn lambs to model hRSV infection in human infants may provide a valuable tool to assess safety and efficacy of new antiviral drugs and vaccines. ALX-0171 is a trivalent Nanobody targeting the hRSV fusion (F) protein and its therapeutic potential was evaluated in newborn lambs infected with a human strain of RSV followed by daily ALX-0171 nebulization for 3 or 5 consecutive days. Colostrum-deprived newborn lambs were infected with hRSV-M37 before being treated by daily nebulization with either ALX-0171 or placebo. Two different treatment regimens were examined: day 1-5 or day 3-5 post-infection. Lambs were monitored daily for general well-being and clinical parameters. Respiratory tissues and bronchoalveolar lavage fluid were collected at day 6 post-inoculation for the quantification of viral lesions, lung viral titers, viral antigen and lung histopathology. Administration by inhalation of ALX-0171 was well-tolerated in these hRSV-infected newborn lambs. Robust antiviral effects and positive effects on hRSV-induced lung lesions and reduction in symptoms of illness were noted. These effects were still apparent when treatment start was delayed and coincided with peak viral loads (day 3 post-infection) and at a time point when signs of RSV disease were apparent. The latter design is expected to have high translational value for planned clinical trials. These results are indicative of the therapeutic potential of ALX-0171 in infants.

Porcine reproductive and respiratory disease virus: Evolution and recombination yields distinct ORF5 RFLP 1-7-4 viruses with individual pathogenicity.

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.

Interferon alpha inhibits replication of a live-attenuated porcine reproductive and respiratory syndrome virus vaccine preventing development of an adaptive immune response in swine.

Type I interferons, such as interferon alpha (IFN-α), contribute to innate antiviral immunity by promoting production of antiviral mediators and are also involved in promoting an adaptive immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating and costly viruses to the swine industry world-wide and has been shown to induce a meager IFN-α response. Previously we administered porcine IFN-α using a replication-defective adenovirus vector (Ad5-IFN-α) at the time of challenge with virulent PRRSV and demonstrated an increase in the number of virus-specific IFNγ secreting cells, indicating that the presence of IFN-α at the time of infection can alter the adaptive immune responses to PRRSV. In the current experiment, we explored the use of IFN-α as an adjuvant administered with live-attenuated PRRSV vaccine as a method to enhance immune response to the vaccine. Unlike the previous studies with fully virulent virus, one injection of the Ad5-IFN-α abolished replication of the vaccine virus and as a result there was no detectible adaptive immune response. Although IFN-α did not have the desired adjuvant effect, the results further highlight the use of IFN-α as a treatment for PRRSV infection.

Kinetics of Respiratory Syncytial Virus (RSV) Memphis Strain 37 (M37) Infection in the Respiratory Tract of Newborn Lambs as an RSV Infection Model for Human Infants.

RATIONALE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Human respiratory syncytial virus memphis 37 causes acute respiratory disease in perinatal lamb lung.

Respiratory syncytial virus (RSV) is the leading cause of hospitalization due to respiratory illness among infants and young children of industrialized countries. There is a lack of understanding of the severe disease mechanisms as well as limited treatment options, none of which are fully satisfactory. This is partly due to lack of a relevant animal model of perinatal RSV infection that mimics moderate to severe disease in infants. We and others have shown mild disease in perinatal lambs with either a bovine or a human A2 strain of RSV. The Memphis 37 clinical strain of human RSV has been used to produce mild to moderate upper respiratory disease in healthy adult volunteers. We hypothesized that the Memphis 37 strain of RSV would infect perinatal lambs and produce clinical disease similar to that in human infants. Perinatal (3- to 5-day-old) lambs were inoculated intranasally with 2 mL/nostril of 1×10(5) focus-forming units (FFU)/mL (n=2) or 2.1×10(8) FFU/mL (n=3) of RSV Memphis 37. Clinical signs, gross and histological lesions, and immune and inflammatory responses were assessed. Memphis 37 caused moderate to severe gross and histologic lesions along with increased mRNA expression of macrophage inflammatory protein. Clinically, four of the five infected lambs had a mild to severe increase in expiratory effort. Intranasally administered RSV strain Memphis 37 infects neonatal lambs with gross, histologic, and immune responses similar to those observed in human infants.

Sucrose stabilization of Respiratory Syncytial Virus (RSV) during nebulization and experimental infection.

BACKGROUND: Respiratory syncytial virus (RSV) is a common respiratory pathogen that can cause severe pneumonia. In vivo studies of RSV can be difficult due to variation in viral infection and disease severity in some animal models. Factors that may contribute to the variation are decreases in viral titer due to preparation and storage and method of virus administration. Nebulization is one method of RSV administration that provides even distribution of virus to all lung lobes; however, the exact quantity of the virus killed by nebulization is not defined. To test the hypothesis that sucrose enhances RSV stability and infectivity, a series of in vitro experiments were conducted with RSV strain Memphis 37 stored at varying concentrations (0%, 3%, 5%, 8%, 10%, 15%, and 20%) of sucrose as a possible cryo- and nebulization protectant. The optimal in vitro concentration was then assessed in vivo in a lamb model. METHODS: Prior to titering the virus on HEp-2 cells, the various virus solutions were subjected to one freeze-thaw cycle and one nebulization cycle. Forty-eight hours after viral plating, infectious foci were detected and counted using immunofluorescent imaging. Titers were determined after freeze-thaw and after freeze-thaw followed by nebulization, then compared to the stock titers (before freezing) as well as to one another to determine the loss of infectivity. To further test this in vivo, lambs 2 to 3-days-old were infected via nebulization with RSV using inoculate containing either 20% sucrose or no sucrose followed by assessments of infection severity. RESULTS: Nebulization of virus in 0% sucrose resulted in a 0.580 log reduction in infectivity while virus in 20% sucrose exhibited a 0.297 log reduction. In vivo studies demonstrated that 20% sucrose enhanced RSV lesions and antigen distribution. CONCLUSIONS: The data suggests that both nebulization and freeze-thawing of RSV in the absence of sucrose cause unacceptable losses in viral infectivity and that sucrose acts as a RSV protectant in both regards.

Increased concentration of iodide in airway secretions is associated with reduced respiratory syncytial virus disease severity.

Recent studies have revealed that the human and nonrodent mammalian airway mucosa contains an oxidative host defense system. This three-component system consists of the hydrogen peroxide (H2O2)-producing enzymes dual oxidase (Duox)1 and Duox2, thiocyanate (SCN(-)), and secreted lactoperoxidase (LPO). The LPO-catalyzed reaction between H2O2 and SCN(-) yields the bactericidal hypothiocyanite (OSCN(-)) in airway surface liquid (ASL). Although SCN(-) is the physiological substrate of LPO, the Duox/LPO/halide system can generate hypoiodous acid when the iodide (I(-)) concentration is elevated in ASL. Because hypoiodous acid, but not OSCN(-), inactivates respiratory syncytial virus (RSV) in cell culture, we used a lamb model of RSV to test whether potassium iodide (KI) could enhance this system in vivo. Newborn lambs received KI by intragastric gavage or were left untreated before intratracheal inoculation of RSV. KI treatment led to a 10-fold increase in ASL I(-) concentration, and this I(-) concentration was approximately 30-fold higher than that measured in the serum. Also, expiratory effort, gross lung lesions, and pulmonary expression of an RSV antigen and IL-8 were reduced in the KI-treated lambs as compared with nontreated control lambs. Inhibition of LPO activity significantly increased lesions, RSV mRNA, and antigen. Similar experiments in 3-week-old lambs demonstrated that KI administration was associated with reduced gross lesions, decreased RSV titers in bronchoalveolar lavage fluid, and reduced RSV antigen expression. Overall, these data indicate that high-dose KI supplementation can be used in vivo to lessen the severity of RSV infections, potentially through the augmentation of mucosal oxidative defenses.

Effects of formalin-inactivated respiratory syncytial virus (FI-RSV) in the perinatal lamb model of RSV.

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.

Human respiratory syncytial virus Memphis 37 grown in HEp-2 cells causes more severe disease in lambs than virus grown in Vero cells.

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants and young children. A small percentage of these individuals develop severe and even fatal disease. To better understand the pathogenesis of severe disease and develop therapies unique to the less-developed infant immune system, a model of infant disease is needed. The neonatal lamb pulmonary development and physiology is similar to that of infants, and sheep are susceptible to ovine, bovine, or human strains of RSV. RSV grown in Vero (African green monkey) cells has a truncated attachment G glycoprotein as compared to that grown in HEp-2 cells. We hypothesized that the virus grown in HEp-2 cells would cause more severe clinical symptoms and cause more severe pathology. To confirm the hypothesis, lambs were inoculated simultaneously by two different delivery methods (intranasal and nebulized inoculation) with either Vero-grown or HEp-2-grown RSV Memphis 37 (M37) strain of virus to compare viral infection and disease symptoms. Lambs infected with HEp-2 cell-derived virus by either intranasal or nebulization inoculation had significantly higher levels of viral RNA in lungs as well as greater clinical disease including both gross and histopathologic lesions compared to lambs similarly inoculated with Vero-grown virus. Thus, our results provide convincing in vivo evidence for differences in viral infectivity that corroborate previous in vitro mechanistic studies demonstrating differences in the G glycoprotein expression by RSV grown in Vero cells.

Exogenous administration of vascular endothelial growth factor prior to human respiratory syncytial virus a2 infection reduces pulmonary pathology in neonatal lambs and alters epithelial innate immune responses.

Human respiratory syncytial virus (RSV) affects thousands of children every year. Vascular endothelial growth factor (VEGF) is a regulator of vasculogenesis, pulmonary maturation, and immunity. In order to test the extent to which VEGF may alter RSV infection, 4 groups of lambs received either human recombinant VEGF (rhVEGF) or phosphate-buffered saline (PBS) pretreatment followed by inoculation with human RSV strain A2 or sterile medium. Lambs in each group were sacrificed at 2, 4, and 6 days post infection. Expression of surfactant protein-A (SP-A), surfactant protein-D (SP-D), sheep ß-defensin-1 (SBD-1), tumor necrosis factor α (TNFα), interleukin (IL)-6, IL-8, interferon ß, and endogenous VEGF were measured to determine effect of rhVEGF pretreatment. RSV lambs pretreated with rhVEGF had reduced viral mRNA and decreased pulmonary pathology at day 6. Pretreatment with rhVEGF increased mRNA expression of SP-A, SBD-1, and TNFα, with alteration of expression in RSV lambs. Endogenous VEGF mRNA levels were increased at day 2 regardless of pretreatment. Pretreatment with rhVEGF increased pulmonary cellular proliferation in RSV lambs at day 4 post infection. Overall, these results suggest that pretreatment with rhVEGF protein may have therapeutic potential to decrease RSV viral load, decrease pulmonary lesion severity, and alter both epithelial innate immune responses and epithelial cell proliferation.