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1.  Determination of the infectious titer and virulence of an original US porcine epidemic diarrhea virus PC22A strain 
Veterinary Research  2015;46:109.
The infectious dose of a virus pool of original US PEDV strain PC22A was determined in 4-day-old, cesarean-derived, colostrum-deprived (CDCD) piglets. The median pig diarrhea dose (PDD50) of the virus pool was determined as 7.35 log10 PDD50/mL, similar to the cell culture infectious titer, 7.75 log10 plaque-forming units (PFU)/mL. 100 PDD50 caused watery diarrhea in all conventional suckling piglets (n = 12) derived from a PEDV-naive sow, whereas 1000 and 10 000 PDD50 did not cause diarrhea in piglets derived from two PEDV-field exposed-recovered sows. This information is important for future PEDV challenge studies and validation of PEDV vaccines.
PMCID: PMC4582625  PMID: 26408019
2.  Antigenic Relationships among Porcine Epidemic Diarrhea Virus and Transmissible Gastroenteritis Virus Strains 
Journal of Virology  2015;89(6):3332-3342.
Porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are economically important swine enteropathogenic coronaviruses. These two viruses belong to two distinct species of the Alphacoronavirus genus within Coronaviridae and induce similar clinical signs and pathological lesions in newborn piglets, but they are presumed to be antigenically distinct. In the present study, two-way antigenic cross-reactivity examinations between the prototype PEDV CV777 strain, three distinct U.S. PEDV strains (the original highly virulent PC22A, S indel Iowa106, and S 197del PC177), and two representative U.S. TGEV strains (Miller and Purdue) were conducted by cell culture immunofluorescent (CCIF) and viral neutralization (VN) assays. None of the pig TGEV antisera neutralized PEDV and vice versa. One-way cross-reactions were observed by CCIF between TGEV Miller hyperimmune pig antisera and all PEDV strains. Enzyme-linked immunosorbent assays, immunoblotting using monoclonal antibodies and Escherichia coli-expressed recombinant PEDV and TGEV nucleocapsid (N) proteins, and sequence analysis suggested at least one epitope on the N-terminal region of PEDV/TGEV N protein that contributed to this cross-reactivity. Biologically, PEDV strain CV777 induced greater cell fusion in Vero cells than did U.S. PEDV strains. Consistent with the reported genetic differences, the results of CCIF and VN assays also revealed higher antigenic variation between PEDV CV777 and U.S. strains.
IMPORTANCE Evidence of antigenic cross-reactivity between porcine enteric coronaviruses, PEDV and TGEV, in CCIF assays supports the idea that these two species are evolutionarily related, but they are distinct species defined by VN assays. Identification of PEDV- or TGEV-specific antigenic regions allows the development of more specific immunoassays for each virus. Antigenic and biologic variations between the prototype and current PEDV strains could explain, at least partially, the recurrence of PEDV epidemics. Information on the conserved antigenicity among PEDV strains is important for the development of PEDV vaccines to protect swine from current highly virulent PEDV infections.
PMCID: PMC4337547  PMID: 25589635
3.  Detection and Full-Length Genome Characterization of Novel Canine Vesiviruses 
Emerging Infectious Diseases  2015;21(8):1433-1436.
Vesiviruses have been detected in several animal species and as accidental contaminants of cells. We detected vesiviruses in asymptomatic kennel dogs (64.8%) and symptomatic (1.1%) and asymptomatic (3.5%) household dogs in Italy. The full-length genome of 1 strain, Bari/212/07/ITA, shared 89%–90% nt identity with vesiviruses previously detected in contaminated cells.
PMCID: PMC4517720  PMID: 26196075
calicivirus; vesivirus; dogs; genome; canine; detection; characterization; novel; viruses; Italy
4.  Pathogenicity of 2 Porcine Deltacoronavirus Strains in Gnotobiotic Pigs 
Emerging Infectious Diseases  2015;21(4):650-654.
To verify whether porcine deltacoronavirus infection induces disease, we inoculated gnotobiotic pigs with 2 virus strains (OH-FD22 and OH-FD100) identified by 2 specific reverse transcription PCRs. At 21–120 h postinoculation, pigs exhibited severe diarrhea, vomiting, fecal shedding of virus, and severe atrophic enteritis. These findings confirm that these 2 strains are enteropathogenic in pigs.
PMCID: PMC4378491  PMID: 25811229
porcine deltacoronavirus; PDCoV; coronavirus; viruses; strains; strain OH-FD22; strain OH-FD100; pathogenicity; pigs; gnotobiotic pigs; PCR
5.  In vivo gut transcriptome responses to Lactobacillus rhamnosus GG and Lactobacillus acidophilus in neonatal gnotobiotic piglets 
Gut Microbes  2014;5(2):152-164.
Probiotics facilitate mucosal repair and maintain gut homeostasis. They are often used in adjunct with rehydration or antibiotic therapy in enteric infections. Lactobacillus spp have been tested in infants for the prevention or treatment of various enteric conditions. However, to aid in rational strain selection for specific treatments, comprehensive studies are required to delineate and compare the specific molecules and pathways involved in a less complex but biologically relevant model (gnotobiotic pigs). Here we elucidated Lactobacillus rhamnosus (LGG) and L. acidophilus (LA) specific effects on gut transcriptome responses in a neonatal gnotobiotic (Gn) pig model to simulate responses in newly colonized infants. Whole genome microarray, followed by biological pathway reconstruction, was used to investigate the host-microbe interactions in duodenum and ileum at early (day 1) and later stages (day 7) of colonization. Both LA and LGG modulated common responses related to host metabolism, gut integrity, and immunity, as well as responses unique to each strain in Gn pigs. Our data indicated that probiotic establishment and beneficial effects in the host are guided by: (1) down-regulation or upregulation of immune function-related genes in the early and later stages of colonization, respectively, and (2) alternations in metabolism of small molecules (vitamins and/or minerals) and macromolecules (carbohydrates, proteins, and lipids). Pathways related to immune modulation and carbohydrate metabolism were more affected by LGG, whereas energy and lipid metabolism-related transcriptome responses were prominently modulated by LA. These findings imply that identification of probiotic strain-specific gut responses could facilitate the rational design of probiotic-based interventions to moderate specific enteric conditions.
PMCID: PMC4063840  PMID: 24637605
lactobacilli; probiotics; transcriptome; gnotobiotic pigs; gut
6.  Pathogenesis of GIII.2 bovine norovirus, CV186-OH/00/US strain in gnotobiotic calves 
Veterinary microbiology  2013;168(1):202-207.
The pathogenesis of GIII.2 bovine norovirus (BoNoV) is not well understood. Our study demonstrated persisting diarrhea and prolonged fecal shedding, but with a lack of significant intestinal lesions in gnotobiotic (Gn) calves infected with GIII.2 BoNoV, CV186-OH/00/US strain. Nine 4 to 7-day-old Angus/Jersey crossbred Gn calves were orally inoculated with 10.0-11.9 log10 genomic equivalents (GE)/calf of CV186-OH (n=7) or mock (n=2). Calves were euthanized at post-inoculation day (PID) 1 (n=1) when moderate to severe lethargy was observed and at PIDs 2-6 (n=4) after lethargy had subsided. Two calves were kept longer term (until PID 30) for monitoring fecal shedding patterns by TaqMan real-time RT-PCR (qRT-PCR). Most infected calves exhibited two clinical signs: i) acute but persisting diarrhea and ii) acute moderate to severe lethargy. The two infected calves, followed longer-term, had prolonged fecal viral RNA shedding [peak average titer of 11.8 (± 0.2) log10 GE/ml] at least until PID 20. By qRT-PCR, 5 infected calves had low viral RNA titers in serum, ranging from 4.0 to 5.8 log10 GE/ml, at PIDs 1-5, but not (<2.7 log10 GE/ml) at PIDs 6-30. The latter observation coincided with the presence of serum IgG antibody to BoNoV at PIDs 8-30. Collectively, the GIII.2 BoNoV strain CV186-OH induced only mild enteropathogenicity, evident by the lack of significant intestinal lesions, but it led to persisting mild diarrhea and prolonged fecal virus shedding in Gn calves. The prolonged fecal shedding of GIII.2 BoNoV might partially explain how this virus is maintained as endemic infections in cattle.
PMCID: PMC3905316  PMID: 24309403
Norovirus; Pathogenesis; Cattle; Prolonged virus shedding
7.  Integrating Bacterial and Viral Water Quality Assessment to Predict Swimming-Associated Illness at a Freshwater Beach: A Cohort Study 
PLoS ONE  2014;9(11):e112029.
Background & Objective
Recreational waters impacted by fecal contamination have been linked to gastrointestinal illness in swimmer populations. To date, few epidemiologic studies examine the risk for swimming-related illnesses based upon simultaneous exposure to more than one microbial surrogate (e.g. culturable E. coli densities, genetic markers). We addressed this research gap by investigating the association between swimming-related illness frequency and water quality determined from multiple bacterial and viral genetic markers.
Viral and bacterial genetic marker densities were determined from beach water samples collected over 23 weekend days and were quantified using quantitative polymerase chain reaction (qPCR). These genetic marker data were paired with previously determined human exposure data gathered as part of a cohort study carried out among beach users at East Fork Lake in Ohio, USA in 2009. Using previously unavailable genetic marker data in logistic regression models, single- and multi-marker/multi-water quality indicator approaches for predicting swimming-related illness were evaluated for associations with swimming-associated gastrointestinal illness.
Data pertaining to genetic marker exposure and 8- or 9-day health outcomes were available for a total of 600 healthy susceptible swimmers, and with this population we observed a significant positive association between human adenovirus (HAdV) exposure and diarrhea (odds ratio  = 1.6; 95% confidence interval: 1.1–2.3) as well as gastrointestinal illness (OR  = 1.5; 95% CI: 1.0–2.2) upon adjusting for culturable E. coli densities in multivariable models. No significant associations between bacterial genetic markers and swimming-associated illness were observed.
This study provides evidence that a combined measure of recreational water quality that simultaneously considers both bacterial and viral densities, particularly HAdV, may improve prediction of disease risk than a measure of a single agent in a beach environment likely influenced by nonpoint source human fecal contamination.
PMCID: PMC4237328  PMID: 25409012
8.  The Global One Health Paradigm: Challenges and Opportunities for Tackling Infectious Diseases at the Human, Animal, and Environment Interface in Low-Resource Settings 
Zoonotic infectious diseases have been an important concern to humankind for more than 10,000 years. Today, approximately 75% of newly emerging infectious diseases (EIDs) are zoonoses that result from various anthropogenic, genetic, ecologic, socioeconomic, and climatic factors. These interrelated driving forces make it difficult to predict and to prevent zoonotic EIDs. Although significant improvements in environmental and medical surveillance, clinical diagnostic methods, and medical practices have been achieved in the recent years, zoonotic EIDs remain a major global concern, and such threats are expanding, especially in less developed regions. The current Ebola epidemic in West Africa is an extreme stark reminder of the role animal reservoirs play in public health and reinforces the urgent need for globally operationalizing a One Health approach. The complex nature of zoonotic diseases and the limited resources in developing countries are a reminder that the need for implementation of Global One Health in low-resource settings is crucial. The Veterinary Public Health and Biotechnology (VPH-Biotec) Global Consortium launched the International Congress on Pathogens at the Human-Animal Interface (ICOPHAI) in order to address important challenges and needs for capacity building. The inaugural ICOPHAI (Addis Ababa, Ethiopia, 2011) and the second congress (Porto de Galinhas, Brazil, 2013) were unique opportunities to share and discuss issues related to zoonotic infectious diseases worldwide. In addition to strong scientific reports in eight thematic areas that necessitate One Health implementation, the congress identified four key capacity-building needs: (1) development of adequate science-based risk management policies, (2) skilled-personnel capacity building, (3) accredited veterinary and public health diagnostic laboratories with a shared database, and (4) improved use of existing natural resources and implementation. The aim of this review is to highlight advances in key zoonotic disease areas and the One Health capacity needs.
PMCID: PMC4230840  PMID: 25393303
9.  Distinct Characteristics and Complex Evolution of PEDV Strains, North America, May 2013–February 2014 
Emerging Infectious Diseases  2014;20(10):1620-1628.
Sequence analysis showed heterogeneity among 74 strains and distinct molecular characteristics of highly virulent strains and variants.
Porcine epidemic diarrhea virus (PEDV), which emerged in the United States in 2013, has spread throughout North America. Limited availability of PEDV complete genomes worldwide has impeded our understanding of PEDV introduction into the United States. To determine the relationship between the North American strains and global emerging and historic PEDV strains, we sequenced and analyzed complete genomes of 74 strains from North America; the strains clustered into 2 distinct clades. Compared with the initially reported virulent US PEDV strains, 7 (9.7%) strains from 4 states contained insertions and deletions in the spike gene (S INDELs). These S INDEL strains share 99.8%–100% nt identity with each other and 96.2%–96.7% nt identity with the initial US strains. Furthermore, the S INDEL strains form a distinct cluster within North American clade II, sharing 98.6%–100% nt identity overall. In the United States, the S INDEL and original PEDV strains are co-circulating and could have been introduced simultaneously.
PMCID: PMC4193278  PMID: 25279722
porcine epidemic diarrhea virus; virulent strains; S INDEL strains; complete genome analysis; phylogenetic analysis; recombination identification analysis; spike gene; PEDV variant; US strains; pigs; single nucleotide polymorphisms; open reading frame 1; ORF 1; viruses
10.  Divergent Immunomodulating Effects of Probiotics on T Cell Responses to Oral Attenuated Human Rotavirus Vaccine and Virulent Human Rotavirus Infection in a Neonatal Gnotobiotic Piglet Disease Model 
Rotaviruses (RVs) are a leading cause of childhood diarrhea. Current oral vaccines are not effective in impoverished countries where the vaccine is needed most. Therefore, alternative affordable strategies are urgently needed. Probiotics can alleviate diarrhea in children and enhance specific systemic and mucosal Ab responses, but the T cell responses are undefined. In this study, we elucidated the T cell and cytokine responses to attenuated human RV (AttHRV) and virulent human RV (HRV) in gnotobiotic pigs colonized with probiotics (Lactobacillus rhamnosus strain GG [LGG] and Bifidobacterium lactis Bb12 [Bb12]), mimicking gut commensals in breastfed infants. Neonatal gnotobiotic pigs are the only animal model susceptible to HRV diarrhea. Probiotic colonized and nonvaccinated (Probiotic) pigs had lower diarrhea and reduced virus shedding postchallenge compared with noncolonized and nonvaccinated pigs (Control). Higher protection in the Probiotic group coincided with higher ileal T regulatory cells (Tregs) before and after challenge, and higher serum TGF-β and lower serum and biliary proinflammatory cytokines postchallenge. Probiotic colonization in vaccinated pigs enhanced innate serum IFN-α, splenic and circulatory IFN-γ−producing T cells, and serum Th1 cytokines, but reduced serum Th2 cytokines compared with noncolonized vaccinated pigs (Vac). Thus, LGG+Bb12 induced systemic Th1 immunostimulatory effects on oral AttHRV vaccine that coincided with lower diarrhea severity and reduced virus shedding postchallenge in Vac+Pro compared with Vac pigs. Previously unreported intestinal CD8 Tregs were induced in vaccinated groups postchallenge. Thus, probiotics LGG+Bb12 exert divergent immunomodulating effects, with enhanced Th1 responses to oral AttHRV vaccine, whereas inducing Treg responses to virulent HRV.
PMCID: PMC4136549  PMID: 23918983
11.  Bovine Respiratory Coronavirus 
PMCID: PMC4094360  PMID: 20619189
Bovine respiratory coronavirus; shipping fever; wild ruminant coronaviruses
12.  Prevalence and molecular characterization of porcine enteric caliciviruses and first detection of porcine kobuviruses in US swine 
Archives of virology  2013;158(7):1583-1588.
The prevalence of porcine sapoviruses (SaVs) and noroviruses (NoVs) in nursing piglets on three pig farms in Ohio was studied. Fecal samples (n = 139) were collected from individual pigs and screened for caliciviruses by RT-PCR. Phylogenetic analysis was conducted using partial sequences of the RNA polymerase region. Three different SaV genogroups, including a newly emerging one (DO19 Korea-like) were detected. No NoVs were detected. Kobuviruses, emerging members of the family Picornaviridae, were detected by primers designed for SaV. To our knowledge, this is the first report of porcine DO19 Korea-like SaV and kobuvirus in the United States.
PMCID: PMC4094368  PMID: 23456421
13.  Molecular detection and genetic characterization of kobuviruses and astroviruses in asymptomatic local pigs in East Africa 
Archives of virology  2013;159(6):1313-1319.
In this study, swine fecal specimens (n = 251) collected from nursing and weaned piglets raised under smallholder production systems were screened for the presence of kobuviruses by RT-PCR. Porcine kobuviruses were detected in 13.1 % (33/251) of the samples. We demonstrated that porcine kobuvirus infections exist in indigenous pigs in Kenya and Uganda and that the prevalence was higher in young piglets than older pigs: nursing piglets (15 %), post-weaning (3-month-old) pigs (17 %), 4-month-old pigs (10 %). Genetic analysis of the partial RNA-dependent RNA polymerase (RdRp) region (690 nt) revealed that kobuviruses circulating in East Africa are diverse, sharing nucleotide sequence identities ranging from 89.7 to 99.1 % and 88 to 92.3 % among them and with known porcine kobuviruses, respectively. The nucleotide sequence identities between our kobuvirus strains and those of human, bovine and canine kobuviruses were 69.4-70.7 %, 73.1-74.4 % and 67-70.7 %, respectively. Additionally, upon sequencing selected samples that showed consistent 720-bp RT-PCR bands while using the same primer set, we detected porcine astroviruses in our samples belonging to type 2 and type 3 mamastroviruses. To our knowledge, this study reports the first detection and molecular analysis of both porcine kobuviruses and astroviruses in an African region. Further studies are required to determine the role of these viruses in gastrointestinal infections of pigs in this region and to determine the genetic diversity of the circulating strains to develop accurate diagnostic tools and implement appropriate control strategies.
PMCID: PMC4094370  PMID: 24327095
14.  A human norovirus-like particle vaccine adjuvanted with ISCOM or mLT induces cytokine and antibody responses and protection to the homologous GII.4 human norovirus in a gnotobiotic pig disease model 
Vaccine  2007;25(50):8448-8459.
We inoculated gnotobiotic pigs oraly/intranasally with human norovirus GII.4 HS66 strain virus-like particles (VLP) and immunostimulating complexes (ISCOM) or mutant E. coli LT toxin (mLT, R192G) as mucosal adjuvants, then assessed intestinal and systemic antibody and cytokine responses and homologous protection. Both vaccines induced high rates of seroconversion (100%) and coproconversion (75–100%). The VLP+mLT vaccine induced Th1/Th2 serum cytokines and cytokine secreting cells, whereas the VLP+ISCOM vaccine induced Th2 biased responses with significantly elevated IgM, IgA and IgG antibody-secreting cells in intestine. Nevertheless, both vaccines induced increased protection rates against viral shedding and diarrhea (75–100%) compared to controls; however, only 57% of controls shed virus.
PMCID: PMC4094375  PMID: 18022293
Norovirus; immunity; VLP; calicivirus; vaccine; gnotobiotic pig model
15.  Transfer of maternal cytokines to suckling piglets: in vivo and in vitro models with implications for immunomodulation of neonatal immunity 
Maternal cytokines may play instructive roles in development of the neonatal immune system. However, cytokines in colostrum and milk and their transfer from mothers to neonates have not been well documented, except for TGF-β. Swine provide a unique model to study lactogenic cytokines because the sow's impermeable placenta prohibits transplacental passage. We investigated IL-6 and TNF-α (pro-inflammatory), IFN-γ and IL-12, (Th1), IL-10 and IL-4 (Th2) and TGF-β1 (Th3) concentrations in sow serum and colostrum/milk and serum of their suckling and weaned piglets and in age-matched colostrum-deprived gnotobiotic piglets. All cytokines were detected in colostrum/milk and correlated with concentrations in sow serum except for mammary-derived TNF-α and TGF-β1. Detection of IL-12 and TGF-β1 in pre-suckling and colostrum-deprived gnotobiotic piglet serum suggests constitutive production: other cytokines were undetectable confirming absence of transplacental transfer. Peak median cytokine concentrations in suckling piglet serum occurred at post-partum days 1-2 (IL-4>IL-6>IFN-γ>IL-10). The effects in vitro of physiologically relevant concentrations of the two predominant lactogenic cytokines (TGF-β1 and IL-4) on porcine naive B cell responses to lipopolysaccharide (LPS) and rotavirus (RV) were investigated. High (10ng/ml) TGF-β1 suppressed immunoglobulin secreting cell responses to LPS and rotavirus; low concentrations (0.1ng/ml) promoted isotype switching to IgA antibody. Interleukin-4 induced inverse dose-dependent (0.1>10ng/ml) isotype switching to IgA and enhanced IgM secreting cell responses to LPS and rotavirus. In summary, we documented the transfer and persistence of maternal cytokines from colostrum/milk to neonates and their potential role in Th-2 biased IgA responses and reduced immunologic responsiveness of neonates.
PMCID: PMC4094377  PMID: 17403542
Cytokines; Milk; Neonate; Swine; Th2 bias
16.  Prenatally acquired vitamin A deficiency alters innate immune responses to human rotavirus in a gnotobiotic pig model§ 
We examined how prenatally acquired vitamin A deficiency (VAD) modulates innate immune responses and human rotavirus (HRV) vaccine efficacy in a gnotobiotic (Gn) piglet model of HRV diarrhea. The VAD and vitamin A sufficient (VAS) Gn pigs were vaccinated with attenuated HRV (AttHRV) with or without concurrent oral vitamin A supplementation (100,000IU) and challenged with virulent HRV (VirHRV).
Regardless of vaccination status, the numbers of conventional and plasmacytoid dendritic cells (cDCs and pDCs) were higher in VAD piglets pre-challenge, but decreased substantially post-challenge as compared to VAS pigs. We observed significantly higher frequency of CD103 (integrin αEβ7) expressing DCs in VAS vs. VAD piglets post-challenge, indicating that VAD may interfere with homing (including intestinal) phenotype acquisition. Post VirHRV challenge, we observed longer and more pronounced diarrhea and higher VirHRV fecal titers in non-vaccinated VAD piglets. Consistent with higher VirHRV shedding titers, higher IFNα levels were induced in control VAD vs. VAS piglet sera at post-challenge day (PCD)2. Ex vivo HRV-stimulated mononuclear cells (MNCs) isolated from spleen and blood of VAD pigs pre-challenge also produced more IFNα. In contrast at PCD10, we observed reduced IFNα levels in VAD pigs that coincided with decreased TLR3+ MNC frequencies. Numbers of necrotic MNCs were higher in VAD pigs in spleen (coincident with splenomegaly in other VAD animals) pre-challenge and intestinal tissues (coincident with higher VirHRV induced intestinal damage) post-challenge. Thus, prenatal VAD caused an imbalance in innate immune responses and exacerbated VirHRV infection, whereas vitamin A supplementation failed to compensate for these VAD effects.
PMCID: PMC3633669  PMID: 23536630
17.  Probiotics and colostrum/milk differentially affect neonatal humoral immune responses to oral rotavirus vaccine 
Vaccine  2013;31(15):1916-1923.
Breast milk (colostrum [col]/milk) components and gut commensals play important roles in neonatal immune maturation, establishment of gut homeostasis and immune responses to enteric pathogens and oral vaccines. We investigated the impact of colonization by probiotics, Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) with/without col/milk (mimicking breast/formula fed infants) on B lymphocyte responses to an attenuated (Att) human rotavirus (HRV) Wa strain vaccine in a neonatal gnotobiotic pig model. Col/milk did not affect probiotic colonization in AttHRV vaccinated pigs. However, unvaccinated pigs fed col/milk shed higher numbers of probiotic bacteria in feces than non-col/milk fed colonized controls. In AttHRV vaccinated pigs, col/milk feeding with probiotic treatment resulted in higher mean serum IgA HRV antibody titers and intestinal IgA antibody secreting cell (ASC) numbers compared to col/milk fed, non-colonized vaccinated pigs. In vaccinated pigs without col/milk, probiotic colonization did not affect IgA HRV antibody titers, but serum IgG HRV antibody titers and gut IgG ASC numbers were lower, suggesting that certain probiotics differentially impact HRV vaccine responses. Our findings suggest that col/milk components (soluble mediators) affect initial probiotic colonization, and together, they modulate neonatal antibody responses to oral AttHRV vaccine in complex ways.
PMCID: PMC3606660  PMID: 23453730
Probiotics (LGG+Bb12); Colonization; Antibody responses; Rotavirus vaccine; Colostrum/milk; TGFβ
18.  First Complete Genome Sequences of Genogroup VI Porcine Sapoviruses 
Genome Announcements  2014;2(2):e00275-14.
Sapoviruses, members of the family Caliciviridae, are genetically diverse and divided into multiple genogroups. Only a few complete genome sequences of animal strains are available. We report the first complete genome sequences of genogroup VI sapoviruses, those of strains JJ674 and JJ681, isolated from fecal samples from diarrheic pigs.
PMCID: PMC3974945  PMID: 24699963
19.  Pathology of US Porcine Epidemic Diarrhea Virus Strain PC21A in Gnotobiotic Pigs 
Emerging Infectious Diseases  2014;20(4):662-665.
To understand the progression of porcine epidemic diarrhea virus infection, we inoculated gnotobiotic pigs with a newly emerged US strain, PC21A, of the virus. At 24–48 hours postinoculation, the pigs exhibited severe diarrhea and vomiting, fecal shedding, viremia, and severe atrophic enteritis. These findings confirm that strain PC21A is highly enteropathogenic.
PMCID: PMC3966387  PMID: 24795932
porcine epidemic diarrhea virus; porcine epidemic diarrhea, pathogenesis, outbreak, USA, gnotobiotic pigs, US strain, pigs, strain PC21A, viruses, enteropathogenic
21.  Prevalence of Porcine Noroviruses, Molecular Characterization of Emerging Porcine Sapoviruses from Finisher Swine in the United States, and Unified Classification Scheme for Sapoviruses 
Journal of Clinical Microbiology  2013;51(7):2344-2353.
Noroviruses (NoVs) and sapoviruses (SaVs) are important human pathogens. Although the involvement of porcine NoVs in disease in pigs is unclear, they are genetically and antigenically closely related to human NoVs. Human NoV-like strains have been detected in pigs, raising public health concerns of potential interspecies transmission. Porcine SaVs are highly diverse and emerging in swine populations. Recently, at least three new genogroups of porcine SaVs have been proposed. In this study, we tested 413 pooled fecal samples collected from apparently healthy finisher pigs in North Carolina swine farms during 2009. Reverse transcription (RT)-PCR coupled hybridization assays were performed to detect known porcine NoVs. The overall prevalence of porcine NoVs determined was 18.9% based on this method. Samples were then tested by RT-PCR targeting the 5′ end of the capsid region for genogroup II (GII) NoVs, a group which includes human NoVs, followed by sequence analysis. All NoVs identified belonged to typical porcine NoV genotypes, and no human NoV-like strains were detected in specimens from these pigs. Porcine NoV-negative samples (n = 335) were subsequently screened using universal calicivirus primers, and 17 SaV strains were confirmed by sequencing. Based on the partial RNA-dependent RNA polymerase (RdRp) region, they clustered with GIII, GVII, and GVIII and with currently unclassified SaVs. According to analysis of the complete capsid sequences, 7 representative strains clustered with GVII, GVIII, and GIX? SaVs. We tentatively classified SaVs into 14 genogroups based on the complete capsid protein VP1. In summary, porcine NoVs and highly divergent SaVs were present in North Carolina finisher pigs.
PMCID: PMC3697660  PMID: 23678065
22.  Vitamin A Deficiency Impairs Adaptive B and T Cell Responses to a Prototype Monovalent Attenuated Human Rotavirus Vaccine and Virulent Human Rotavirus Challenge in a Gnotobiotic Piglet Model 
PLoS ONE  2013;8(12):e82966.
Rotaviruses (RV) are a major cause of gastroenteritis in children. Widespread vitamin A deficiency is associated with reduced efficacy of vaccines and higher incidence of diarrheal infections in children in developing countries. We established a vitamin A deficient (VAD) gnotobiotic piglet model that mimics subclinical vitamin A deficiency in children to study its effects on an oral human rotavirus (HRV) vaccine and virulent HRV challenge. Piglets derived from VAD and vitamin A sufficient (VAS) sows were orally vaccinated with attenuated HRV or mock, with/without supplemental vitamin A and challenged with virulent HRV. Unvaccinated VAD control piglets had significantly lower hepatic vitamin A, higher severity and duration of diarrhea and HRV fecal shedding post-challenge as compared to VAS control pigs. Reduced protection coincided with significantly higher innate (IFNα) cytokine and CD8 T cell frequencies in the blood and intestinal tissues, higher pro-inflammatory (IL12) and 2-3 fold lower anti-inflammatory (IL10) cytokines, in VAD compared to VAS control pigs. Vaccinated VAD pigs had higher diarrhea severity scores compared to vaccinated VAS pigs, which coincided with lower serum IgA HRV antibody titers and significantly lower intestinal IgA antibody secreting cells post-challenge in the former groups suggesting lower anamnestic responses. A trend for higher serum HRV IgG antibodies was observed in VAD vs VAS vaccinated groups post-challenge. The vaccinated VAD (non-vitamin A supplemented) pigs had significantly higher serum IL12 (PID2) and IFNγ (PID6) compared to vaccinated VAS groups suggesting higher Th1 responses in VAD conditions. Furthermore, regulatory T-cell responses were compromised in VAD pigs. Supplemental vitamin A in VAD pigs did not fully restore the dysregulated immune responses to AttHRV vaccine or moderate virulent HRV diarrhea. Our findings suggest that that VAD in children in developing countries may partially contribute to more severe rotavirus infection and lower HRV vaccine efficacy.
PMCID: PMC3846786  PMID: 24312675
23.  Lactobacilli and Bifidobacteria Promote Immune Homeostasis by Modulating Innate Immune Responses to Human Rotavirus in Neonatal Gnotobiotic Pigs 
PLoS ONE  2013;8(10):e76962.
The effects of co-colonization with Lactobacillus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) on 3-dose vaccination with attenuated HRV and challenge with virulent human rotavirus (VirHRV) were assessed in 4 groups of gnotobiotic (Gn) pigs: Pro+Vac (probiotic-colonized/vaccinated), Vac (vaccinated), Pro (probiotic-colonized, non-vaccinated) and Control (non-colonized, non-vaccinated). Subsets of pigs were euthanized pre- [post-challenge day (PCD) 0] and post (PCD7)-VirHRV challenge to assess diarrhea, fecal HRV shedding and dendritic cell/innate immune responses. Post-challenge, Pro+Vac and Vac groups were completely protected from diarrhea; protection rates against HRV shedding were 100% and 83%, respectively. Diarrhea and HRV shedding were reduced in Pro compared to Control pigs following VirHRV challenge. Diarrhea scores and virus shedding were significantly higher in Controls, compared to all other groups, coincident with significantly higher serum interferon-alpha levels post-challenge. LGG+Bb12 colonization ±vaccine promoted immunomaturation as reflected by increased frequencies of CD4, SWC3a, CD11R1, MHCII expressing mononuclear cells (MNCs) and conventional dendritic cells in intestinal tissues and blood post-challenge. Colonization decreased frequencies of toll-like receptors (TLR) 2 and TLR4 expressing MNCs from vaccinated pigs (Pro+Vac) pre-challenge and increased frequencies of TLR3 expressing MNCs from Pro pigs post-challenge, suggesting that probiotics likely exert anti-inflammatory (TLR2 and 4 down-regulation) and antiviral (TLR3 up-regulation by HRV dsRNA) actions via TLR signaling. Probiotic colonization alone (Pro) increased frequencies of intestinal and systemic apoptotic MNCs pre-challenge, thereby regulating immune hyperreactivity and tolerance. However, these frequencies were decreased in intestinal and systemic tissues post-challenge, moderating HRV-induced apoptosis. Additionally, post-challenge, Pro+Vac and Pro groups had significantly decreased MNC proliferation, suggesting that probiotics control excessive lymphoproliferative reactions upon VirHRV challenge. We conclude that in the neonatal Gn pig disease model, selected probiotics contribute to immunomaturation, regulate immune homeostasis and modulate vaccine and virulent HRV effects, thereby moderating HRV diarrhea.
PMCID: PMC3788735  PMID: 24098572
24.  Complete Genome Sequence of the Feline Calicivirus 2280 Strain from the American Tissue Culture Collection 
Genome Announcements  2013;1(3):e00349-13.
Feline calicivirus (FCV) is a highly contagious pathogen of cats that can be grown in cultured cells. FCV is used as a model to study nonculturable caliciviruses, such as noroviruses. We determined the complete genome sequence of the FCV 2280 strain from the American Tissue Culture Collection.
PMCID: PMC3707591  PMID: 23788542
25.  First Complete Genome Sequence of a Genogroup II Genotype 18 Porcine Norovirus, Strain QW125 
Genome Announcements  2013;1(3):e00344-13.
Noroviruses, members of the family Caliciviridae, are genetically diverse. We report the first complete genome sequence of a genogroup II genotype 18 porcine norovirus, strain QW125. A protein BLAST search revealed that identity scores of this strain compared to other norovirus strains were highest in the predicted protease region.
PMCID: PMC3707576  PMID: 23766405

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