Linda J Saif

Publications (87)251.77 Total impact

• Article: Prenatally Acquired Vitamin A Deficiency Alters Innate Immune Responses to Human Rotavirus in a Gnotobiotic Pig Model.

  Anastasia N Vlasova, Kuldeep S Chattha, Sukumar Kandasamy, Christine S Siegismund, Linda J Saif
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  ABSTRACT: 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 (100,000 IU) 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 prechallenge, but decreased substantially postchallenge as compared with VAS pigs. We observed significantly higher frequency of CD103 (integrin αEβ7) expressing DCs in VAS versus VAD piglets postchallenge, 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 nonvaccinated VAD piglets. Consistent with higher VirHRV shedding titers, higher IFN-α levels were induced in control VAD versus VAS piglet sera at postchallenge day 2. Ex vivo HRV-stimulated mononuclear cells (MNCs) isolated from spleen and blood of VAD pigs prechallenge also produced more IFN-α. In contrast, at postchallenge day 10, we observed reduced IFN-α 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) prechallenge and intestinal tissues (coincident with higher VirHRV induced intestinal damage) postchallenge. 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.
  The Journal of Immunology 03/2013; · 5.79 Impact Factor
• Article: Probiotics and colostrum/milk differentially affect neonatal humoral immune responses to oral rotavirus vaccine.

  Kuldeep S Chattha, Anastasia N Vlasova, Sukumar Kandasamy, Malak A Esseili, Christine Siegismund, Gireesh Rajashekara, Linda J Saif
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  ABSTRACT: 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.
  Vaccine 02/2013; · 3.77 Impact Factor
• Article: Human rotavirus virus-like particle vaccines evaluated in a neonatal gnotobiotic pig model of human rotavirus disease.

  Marli P Azevedo, Anastasia N Vlasova, Linda J Saif
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  ABSTRACT: The authors discuss here rotavirus-like particle vaccines as an alternative approach to oral live-attenuated rotavirus vaccine and their efficacy in a gnotobiotic pig model. Rotavirus virus-like particles (VLPs) were evaluated in different doses, and routes of administration, and combined with live-attenuated virus and adjuvants. A VLP vaccine composed of rotavirus VP2 and VP6 was immunogenic in gnotobiotic pigs when inoculated intranasally; however, this vaccine failed to confer protection. A combination of oral attenuated human rotavirus and intranasal 2/6VLP vaccines conferred immunogenicity, partial protection against a human rotavirus challenge and induced IFN-γ-producing T cells in the ileum of pigs with similar frequencies to human rotavirus infection. Vaccination through a combination of mucosal inductive sites and live-attenuated vaccine combined with VLP vaccines was the most effective regimen, compared with the use of a single route or a single vaccine alone. However, if formulated with neutralizing antigens, VLP vaccines may constitute a better approach in a high maternal antibody scenario.
  Expert Review of Vaccines 02/2013; 12(2):169-81. · 4.25 Impact Factor
• Article: Complete Genome Sequence of GII.4 Human Norovirus HS191.

  Stanislav V Sosnovtsev, Karin Bok, Qiuhong Wang, Linda J Saif, Kim Y Green
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  ABSTRACT: Noroviruses are a common cause of gastrointestinal disease in humans worldwide. Here, we report the full-length genomic characterization of GII.4 norovirus strain HS191, which was associated with gastroenteritis in a laboratory worker in 2004.
  Genome announcements. 01/2013; 1(1).
• Article: Internalization of sapovirus, a surrogate for norovirus, in romaine lettuce and the effect of lettuce latex on virus infectivity.

  Malak A Esseili, Qiuhong Wang, Zhenwen Zhang, Linda J Saif
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  ABSTRACT: Noroviruses are the leading cause of food-borne outbreaks, including those that involve lettuce. The culturable porcine sapovirus (SaV) was used as a norovirus surrogate to study the persistence and the potential transfer of the virus from roots to leaves and from outer to inner leaves of lettuce plants. Treatment of lettuce with SaV was done through the roots of young plants, the soil, or the outer leaves of mature plants. Sampling of roots, xylem sap, and inner and outer leaves followed by RNA extraction and SaV-specific real-time reverse transcription (RT)-PCR was performed at 2 h and on postinoculation days (PID) 2, 5, 7, 14, and/or 28. When SaV was inoculated through the roots, viral RNA persisted on the roots and in the leaves until PID 28. When the virus was inoculated through the soil, viral RNA was detected on the roots and in the xylem sap until PID 14; viral RNA was detected in the leaves only until PID 2. No infectious virus was detected inside the leaves for either treatment. When SaV was inoculated through the outer leaves, viral RNA persisted on the leaves until PID 14; however, the virus did not transfer to inner leaves. Infectious viral particles on leaves were detected only at 2 h postinoculation. The milky sap (latex) of leaves, but not the roots' xylem sap, significantly decreased virus infectivity when tested in vitro. Collectively, our results showed the transfer of SaV from roots to leaves through the xylem system and the capacity of the sap of lettuce leaves to decrease virus infectivity in leaves.
  Applied and environmental microbiology 06/2012; 78(17):6271-9. · 3.69 Impact Factor
• Article: Stability of bovine coronavirus on lettuce surfaces under household refrigeration conditions.

  Lisa Mullis, Linda J Saif, Yongbin Zhang, Xuming Zhang, Marli S P Azevedo
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  ABSTRACT: Fecal suspensions with an aerosol route of transmission were responsible for a cluster of severe acute respiratory syndrome (SARS) cases in 2003 in Hong Kong. Based on that event, the World Health Organization recommended that research be implemented to define modes of transmission of SARS coronavirus through sewage, feces, food and water. Environmental studies have shown that animal coronaviruses remain infectious in water and sewage for up to a year depending on the temperature and humidity. In this study, we examined coronavirus stability on lettuce surfaces. A cell culture adapted bovine coronavirus, diluted in growth media or in bovine fecal suspensions to simulate fecal contamination was used to spike romaine lettuce. qRT-PCR detected viral RNA copy number ranging from 6.6 × 10⁴ to 1.7 × 10⁶ throughout the experimental period of 30 days. Whereas infectious viruses were detected for at least 14 days, the amount of infectious virus varied, depending upon the diluent used for spiking the lettuce. UV and confocal microscopic observation indicated attachment of residual labeled virions to the lettuce surface after the elution procedure, suggesting that rates of inactivation or detection of the virus may be underestimated. Thus, it is possible that contaminated vegetables may be potential vehicles for coronavirus zoonotic transmission to humans.
  Food Microbiology 05/2012; 30(1):180-6. · 3.28 Impact Factor
• Article: Stability of and attachment to lettuce by a culturable porcine sapovirus surrogate for human caliciviruses.

  Qiuhong Wang, Zhenwen Zhang, Linda J Saif
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  ABSTRACT: Human noroviruses (HuNoVs) are the leading cause of food-borne illness, accounting for 58% of U.S. cases. Because HuNoVs are unculturable, surrogates are needed to investigate transmission routes and evaluate disinfection methods. However, the current surrogates, feline calicivirus (FCV) and murine NoV (MNV), are less tolerant than HuNoVs to acid and chlorine, respectively. Porcine sapovirus (SaV) is the only culturable enteropathogenic calicivirus. In this study, the resistance of SaV to physicochemical treatments was compared to that of HuNoVs (by reverse transcription-PCR), FCV, and MNV (by infectivity assays). Sapovirus and HuNoV (viral RNA) showed similar resistances to heat (56°C) and to different concentrations of chlorine. However, SaV was more resistant than HuNoVs to ethanol treatment (60% and 70%). Like HuNoVs, SaV was stable at pH 3.0 to 8.0, with a <1.0 log(10) 50% tissue culture infective dose (TCID(50)) reduction at pH 3.0 compared to the value for pH 4.0 to 8.0. SaV and MNV showed similar resistances, and both were more resistant than FCV to heat inactivation (56°C). FCV was more resistant than MNV and SaV to ethanol, and all three viruses showed similar resistances to treatment with low concentrations of chlorine for 1 min. Those results indicate that SaV is a promising surrogate for HuNoVs. Next, we used SaV as a surrogate to examine virus attachment to lettuce at different pHs. Sapovirus attached to lettuce leaves significantly at its capsid isoelectric point (pH 5.0), and the attached viral particles remained infectious on lettuce after 1 week of storage at 4°C. The culturable SaV is a good surrogate for studying HuNoV contamination and transmission in leafy greens and potential disinfectants.
  Applied and environmental microbiology 03/2012; 78(11):3932-40. · 3.69 Impact Factor
• Article: The effects of simvastatin or interferon-α on infectivity of human norovirus using a gnotobiotic pig model for the study of antivirals.

  Kwonil Jung, Qiuhong Wang, Yunjeong Kim, Kelly Scheuer, Zhenwen Zhang, Quan Shen, Kyeong-Ok Chang, Linda J Saif
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  ABSTRACT: The lack of an animal model for human norovirus (HuNoV) has hindered the development of therapeutic strategies. This study demonstrated that a commonly used cholesterol-lowering statin medication, simvastatin, which increases HuNoV replication in an in vitro replicon system, also enhances HuNoV infectivity in the gnotobiotic (Gn) pig model. In contrast, oral treatment with interferon (IFN)-α reduces HuNoV infectivity. Young piglets, all with A or H1 histo-blood group antigens on enterocytes, were treated orally with 8 mg/kg/day of simvastatin; 5 days later, the pigs were inoculated orally with a GII.4 HuNoV (HS194/2009/US strain) and then treated with simvastatin for 5 more days. Simvastatin induced significantly earlier onset and longer duration of HuNoV fecal shedding in treated pigs, frequently with higher fecal viral titers. Simvastatin impaired poly (I:C)-induced IFN-α expression in macrophages or dendritic cells, possibly due to lowered toll-like receptor (TLR) 3 expression; however, the mechanisms were not related to interferon regulatory factor 3 or nuclear factor kappa B signaling pathway. Thus, the enhanced, earlier infectivity of HuNoV in simvastatin-treated pigs coincided with the inhibitory effect of simvastatin on innate immunity. In contrast to the increased HuNoV shedding that simvastatin induced, viral shedding during the treatment period was reduced or curtailed in the HuNoV-inoculated pigs pre-treated/treated with human IFN-α. Our findings are the first to indicate that IFN-α has potential as antiviral therapy against HuNoV. Based on these intriguing and novel findings using the Gn pig model, we confirmed that HuNoV infectivity is altered by treatment with simvastatin or IFN-α. Collectively, these findings indicate that Gn pigs are a useful model to test immunomodulators or efficacy of antivirals against HuNoV.
  PLoS ONE 01/2012; 7(7):e41619. · 4.09 Impact Factor
• Source

  Available from: Vito Martella

  Article: Discovery and genomic characterization of noroviruses from a gastroenteritis outbreak in domestic cats in the US.

  Pierfrancesco Pinto, Qiuhong Wang, Ning Chen, Edward J Dubovi, Joshua B Daniels, Laurie M Millward, Canio Buonavoglia, Vito Martella, Linda J Saif
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  ABSTRACT: Norovirus (NoV) RNA was detected in the stools of 6 out 14 (42.8%) 8-12-week-old cats with enteritis from a feline shelter, in New York State. Upon sequence analysis of the complete capsid, the six NoVs were found to be identical, suggesting the spread of a unique NoV strain in the shelter. The full-length genomic sequence (7839 nt) of one feline NoV, CU081210E/2010/US, was determined. In the capsid protein VP1 region, the virus displayed the highest amino acid identity to animal genogroup IV genotype 2 (GIV.2) NoVs: lion/Pistoia-387/06/IT (97.9%) and dog/Bari-170/07/IT (90.4%). These findings document the discovery of a novel feline calicivirus, different from vesiviruses, and extend the spectrum of NoV host range. Epidemiological studies using feline NoV-specific diagnostic tools and experimental infection of cats are required to understand whether NoVs have a pathogenic role in this species.
  PLoS ONE 01/2012; 7(2):e32739. · 4.09 Impact Factor
• Article: Binding of human GII.4 norovirus virus-like particles to carbohydrates of romaine lettuce leaf cell wall materials.

  Malak A Esseili, Qiuhong Wang, Linda J Saif
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  ABSTRACT: Norovirus (NoV) genogroup II genotype 4 (GII.4) strains are the dominant cause of the majority of food-borne outbreaks, including those that involve leafy greens, such as lettuce. Since human NoVs use carbohydrates of histo-blood group antigens as receptors/coreceptors, we examined the role of carbohydrates in the attachment of NoV to lettuce leaves by using virus-like particles (VLPs) of a human NoV/GII.4 strain. Immunofluorescence analysis showed that the VLPs attached to the leaf surface, especially to cut edges, stomata, and along minor veins. Binding was quantified using enzyme-linked immunosorbent assay (ELISA) performed on cell wall materials (CWM) from innermost younger leaves and outermost lamina of older leaves. The binding to CWM of older leaves was significantly (P < 0.05) higher (1.5- to 2-fold) than that to CWM of younger leaves. Disrupting the carbohydrates of CWM or porcine gastric mucin (PGM) (a carbohydrate control) using 100 mM sodium periodate (NaIO(4)) significantly decreased the binding an average of 17% in younger leaves, 43% in older leaves, and 92% for PGM. In addition, lectins recognizing GalNAc, GlcNAc, and sialic acid at 100 μg/ml significantly decreased the binding an average of 41%, 33%, and 20% on CWM of older leaves but had no effect on younger leaves. Lectins recognizing α-D-Gal, α-D-Man/α-D-Glc, and α-L-Fuc showed significant inhibition on CWM of older leaves as well as that of younger leaves. All lectins, except for the lectin recognizing α-D-Gal, significantly inhibited NoV VLP binding to PGM. Collectively, our results indicate that NoV VLPs bind to lettuce CWM by utilizing multiple carbohydrate moieties. This binding may enhance virus persistence on the leaf surface and prevent effective decontamination.
  Applied and environmental microbiology 12/2011; 78(3):786-94. · 3.69 Impact Factor
• Article: Characterization of emerging GII.g/GII.12 noroviruses from a gastroenteritis outbreak in the United States in 2010.

  Sayaka Takanashi, Qiuhong Wang, Ning Chen, Quan Shen, Kwonil Jung, Zhenwen Zhang, Masaru Yokoyama, Lisa C Lindesmith, Ralph S Baric, Linda J Saif
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  ABSTRACT: Norovirus is a major cause of acute gastroenteritis in humans. A norovirus outbreak occurred in Ohio in January 2010. Stool and saliva samples were obtained from six infected individuals. The full-length genomes of two representative strains (HS206 and HS210) were characterized. They belonged to GII.12 in the capsid but GII.g in the RNA polymerase region. Interestingly, an immunocompetent 2-year-old male shed virus for up to 30 days, as detected by real-time reverse transcription (RT)-PCR. Histo-blood group antigen (HBGA) typing of saliva showed that the norovirus strains infected various types of secretor-positive individuals (types A, B, and O). The viruslike particles of strain HS206 did not bind substantially to A/B/O antigens by synthetic HBGA binding, hemagglutination, or saliva binding assays. These results suggest that infection by this strain may not be A/B/O antigen dependent or that in vitro binding patterns do not always accurately reflect in vivo HBGA usage. This is different from the HBGA binding pattern of the previously reported GII.12/Aichi76 strain. Structural analysis of the predicted capsid of these GII.12 strains revealed two amino acid mismatches located near the HBGA binding sites. Four gnotobiotic pigs were inoculated orally with HS206 (6 × 10(10) genomic equivalents [GE]/pig). Virus shedding began at postinoculation days (PID) 1 to 3 and continued up to PID 16 (1 × 10(5) to 2 × 10(7) GE/ml). Gastroenteritis cases caused by GII.12 noroviruses have been recently reported worldwide. We observed that this emerging GII.12 norovirus infected humans regardless of A/B/O blood type. The infection of pigs by strain HS206 suggests that interspecies transmission of this strain is possible under experimental conditions.
  Journal of clinical microbiology 07/2011; 49(9):3234-44. · 4.16 Impact Factor
• Article: Development of γδ T cell subset responses in gnotobiotic pigs infected with human rotaviruses and colonized with probiotic lactobacilli.

  Ke Wen, Guohua Li, Wei Zhang, Marli S P Azevedo, Linda J Saif, Fangning Liu, Tammy Bui, Ahmed Yousef, Lijuan Yuan
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  ABSTRACT: γδ T cell responses are induced by various viral and bacterial infections. Different γδ T cells contribute to activation and regulation of the inflammatory response and to epithelial repair. How γδ T cells respond to rotavirus infection and how the colonization of probiotics influences the γδ T cell response were unknown. In this study, we evaluated by multicolor flow cytometry the frequencies and distribution of total γδ T cells and three major subsets (CD2-CD8-, CD2+CD8- and CD2+CD8+) in ileum, spleen and blood of gnotobiotic (Gn) pigs at early (3-5 days) and late phases (28 days) after rotavirus infection. The Gn pigs were inoculated with the virulent human rotavirus Wa strain and colonized with a mixture of two strains of probiotics Lactobacillus acidophilus and Lactobacillus reuteri. In naïve pigs, the highest frequency of total γδ T cells was found in blood, followed by spleen and ileum at the early age (8-10 days old) whereas in older pigs (32 days of age) the highest frequency of total γδ T cells was found in ileum and spleen followed by blood. Rotavirus infection significantly increased frequencies of intestinal total γδ T cells and the putatively regulatory CD2+CD8+ γδ T cell subset and decreased frequencies of the putatively proinflammatory CD8- subsets in ileum, spleen and blood at post-infection days (PID) 3 or 5. The three γδ T cell subsets distributed and responded differently after rotavirus infection and/or lactobacilli colonization. The CD2+CD8+ subset contributed the most to the expansion of total γδ T cells after rotavirus infection in ileum because more than 77% of the total γδ T cells there were CD2+CD8+ cells. There was an additive effect between lactobacilli and rotavirus in inducing total γδ T cell expansion in ileum at PID 5. The overall effect of lactobacilli colonization versus rotavirus infection on frequencies of the CD2+CD8+ γδ T cell subset in ileum was similar; however, rotavirus-infected pigs maintained significantly higher frequencies of CD8- subsets in ileum than lactobacilli-colonized pigs. The dynamic γδ T cell responses suggest that γδ T cell subsets may play important roles in different stages of immune responses after rotavirus infection and probiotic colonization. The knowledge on the kinetics and distribution patterns of γδ T cell subsets in naïve pigs and after rotavirus infection or lactobacilli colonization provides the foundation for further mechanistic studies of their functions.
  Veterinary Immunology and Immunopathology 06/2011; 141(3-4):267-75. · 2.08 Impact Factor
• Source

  Available from: Kelly Scheuer

  Article: Characterization and prevalence of a new porcine Calicivirus in Swine, United States.

  Qiuhong Wang, Kelly Scheuer, Zhenwen Ahang, Wondwoosen A Gebreyes, Bayleyegn Z Molla, Armando E Hoet, Linda J Saif
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  ABSTRACT: Real-time reverse transcription PCR revealed that new St-Valerien-ike porcine caliciviruses are prevalent (2.6%-80%; 23.8% overall) in finisher pigs in North Carolina. One strain, NC-WGP93C, shares 89.3%-89.7% genomic nucleotide identity with Canadian strains. Whether these viruses cause disease in pigs or humans or are of food safety concern requires further investigation.
  Emerging Infectious Diseases 06/2011; 17(6):1103-6. · 6.79 Impact Factor
• Source

  Available from: Norma Santos

  Article: Uniformity of rotavirus strain nomenclature proposed by the Rotavirus Classification Working Group (RCWG).

  Jelle Matthijnssens, Max Ciarlet, Sarah M McDonald, Houssam Attoui, Krisztián Bányai, J Rodney Brister, Javier Buesa, Mathew D Esona, Mary K Estes, Jon R Gentsch, [......], Viviana Parreño, Mustafizur Rahman, Franco M Ruggeri, Linda J Saif, Norma Santos, Andrej Steyer, Koki Taniguchi, John T Patton, Ulrich Desselberger, Marc Van Ranst
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  ABSTRACT: In April 2008, a nucleotide-sequence-based, complete genome classification system was developed for group A rotaviruses (RVs). This system assigns a specific genotype to each of the 11 genome segments of a particular RV strain according to established nucleotide percent cutoff values. Using this approach, the genome of individual RV strains are given the complete descriptor of Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx. The Rotavirus Classification Working Group (RCWG) was formed by scientists in the field to maintain, evaluate and develop the RV genotype classification system, in particular to aid in the designation of new genotypes. Since its conception, the group has ratified 51 new genotypes: as of April 2011, new genotypes for VP7 (G20-G27), VP4 (P[28]-P[35]), VP6 (I12-I16), VP1 (R5-R9), VP2 (C6-C9), VP3 (M7-M8), NSP1 (A15-A16), NSP2 (N6-N9), NSP3 (T8-T12), NSP4 (E12-E14) and NSP5/6 (H7-H11) have been defined for RV strains recovered from humans, cows, pigs, horses, mice, South American camelids (guanaco), chickens, turkeys, pheasants, bats and a sugar glider. With increasing numbers of complete RV genome sequences becoming available, a standardized RV strain nomenclature system is needed, and the RCWG proposes that individual RV strains are named as follows: RV group/species of origin/country of identification/common name/year of identification/G- and P-type. In collaboration with the National Center for Biotechnology Information (NCBI), the RCWG is also working on developing a RV-specific resource for the deposition of nucleotide sequences. This resource will provide useful information regarding RV strains, including, but not limited to, the individual gene genotypes and epidemiological and clinical information. Together, the proposed nomenclature system and the NCBI RV resource will offer highly useful tools for investigators to search for, retrieve, and analyze the ever-growing volume of RV genomic data.
  Archives of Virology 05/2011; 156(8):1397-413. · 2.11 Impact Factor
• Article: Reassortment among bovine, porcine and human rotavirus strains results in G8P[7] and G6P[7] strains isolated from cattle in South Korea.

  Sang-Ik Park, Jelle Matthijnssens, Linda J Saif, Hyun-Jeong Kim, Jun-Gyu Park, Mia Madel Alfajaro, Deok-Song Kim, Kyu-Yeol Son, Dong-Kun Yang, Bang-Hun Hyun, Mun-Il Kang, Kyoung-Oh Cho
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  ABSTRACT: Group A rotaviruses (GARVs) cause severe acute gastroenteritis in children and young animals. Although zoonotic infections with bovine-like G6 and G8 GARVs have been reported in many countries, there is little evidence for reassortment between bovine GARVs and GARVs from heterologous species. The finding of bovine GARVs with the G6 and G8 genotypes in combination with the typical porcine P[7] prompted us to characterize all 11 genes of 30 bovine GARVs isolated from clinically infected calves. By the comparison of the full-length ORF of VP7 and NSP1-5, and the partial VP1-4 and VP6 nucleotide sequences between the 30 Korean and other known strains, three different genome constellations were found. Twenty seven strains showed the G8-P[7]-I5-R1-C1-M2-A1-N1-T1-E1-H1 genotypes, a single strain possessed the G6-P[7]-I2-R2-C1-M2-A1-N2-T1-E2-H1 genotype constellation and 2 strains the G6-P[7]-I2-R2-C2-M2-A3-N2-T6-E2-H3 genotype constellation. The complete genome of a single reference strains for each of these three genotype constellations (KJ25, KJ9-1 and KJ19-2) was determined and analyzed. A detailed phylogenetic analysis revealed a complicated picture, with several reassortments among bovine-like, porcine-like and human-like GARV strains, resulting in several different reassortant strains successfully infecting cattle.
  Veterinary Microbiology 04/2011; 152(1-2):55-66. · 3.33 Impact Factor
• Article: Molecular characterization of a new species in the genus Alphacoronavirus associated with mink epizootic catarrhal gastroenteritis.

  Anastasia N Vlasova, Rebecca Halpin, Shiliang Wang, Elodie Ghedin, David J Spiro, Linda J Saif
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  ABSTRACT: A coronavirus (CoV) previously shown to be associated with catarrhal gastroenteritis in mink (Mustela vison) was identified by electron microscopy in mink faeces from two fur farms in Wisconsin and Minnesota in 1998. A pan-coronavirus and a genus-specific RT-PCR assay were used initially to demonstrate that the newly discovered mink CoVs (MCoVs) were members of the genus Alphacoronavirus. Subsequently, using a random RT-PCR approach, full-genomic sequences were generated that further confirmed that, phylogenetically, the MCoVs belonged to the genus Alphacoronavirus, with closest relatedness to the recently identified but only partially sequenced (fragments of the polymerase, and full-length spike, 3c, envelope, nucleoprotein, membrane, 3x and 7b genes) ferret enteric coronavirus (FRECV) and ferret systemic coronavirus (FRSCV). The molecular data presented in this study provide the first genetic evidence for a new coronavirus associated with epizootic catarrhal gastroenteritis outbreaks in mink and demonstrate that MCoVs possess high genomic variability and relatively low overall nucleotide sequence identities (91.7 %) between contemporary strains. Additionally, the new MCoVs appeared to be phylogenetically distant from human (229E and NL63) and other alphacoronaviruses and did not belong to the species Alphacoronavirus 1. It is proposed that, together with the partially sequenced FRECV and FRSCV, they comprise a new species within the genus Alphacoronavirus.
  Journal of General Virology 02/2011; 92(Pt 6):1369-79. · 3.36 Impact Factor
• Article: Porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs.

  Gourapura J Renukaradhya, Konstantin Alekseev, Kwonil Jung, Ying Fang, Linda J Saif
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  ABSTRACT: We performed a comprehensive analysis of innate and adaptive immune responses in dual-virus infected pigs to understand whether a pre-existing immunomodulatory respiratory viral infection affects the overall immunity to a subsequent porcine respiratory coronavirus (PRCV) infection in pigs. Pigs were either mock-infected or infected with porcine reproductive and respiratory syndrome virus (PRRSV), a virus known to cause immunosuppressive respiratory disease, and then pigs were co-infected with PRCV, which normally causes subclinical respiratory infection. We collected samples for six independent experiments from 178 pigs that were also used for pathological studies. We detected a significant reduction in innate NK-cell-mediated cytotoxic function in PRRSV-infected pigs, which was synergistically further decreased in pigs co-infected with PRCV. Subsequently, in association with clinical signs we observed elevated levels of proinflammatory (IL-6), Th-1 (IL-12), and regulatory (IL-10 and TGF-β) cytokines. Increased frequencies of CD4CD8 double-positive T lymphocytes and myeloid cells, in addition to the elevated Th-1 and proinflammatory cytokines in dual-infected pigs, contributed to the severity of lung disease in pigs. The results of our study clarify how each virus modulates the host innate and adaptive immune responses, leading to inflammatory reactions and lung pathology. Thus measurements of cytokines and frequencies of immune cells may serve as indicators of the progression of respiratory viral co-infections, and provide more definitive approaches for treatment.
  Viral immunology 10/2010; 23(5):457-66. · 1.78 Impact Factor
• Article: Nitric oxide is elicited and inhibits viral replication in pigs infected with porcine respiratory coronavirus but not porcine reproductive and respiratory syndrome virus.

  Kwonil Jung, Ashita Gurnani, Gourapura J Renukaradhya, Linda J Saif
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  ABSTRACT: There is little information on the role of nitric oxide (NO) in innate immunity to respiratory coronavirus (CoV) infections. We examined NO levels by Greiss assay in bronchoalveolar lavage (BAL) of pigs infected with either porcine respiratory coronavirus (PRCV) or porcine reproductive and respiratory syndrome virus (PRRSV), a member of Nidovirales, like CoV. The antiviral effects of NO on these two viruses were tested in an in vitro system using a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). We detected a large increase in NO levels in BAL fluids of PRCV-infected pigs, but not in PRRSV-infected pigs. Pulmonary epithelial cell necrosis induced by PRCV coincided with increased NO. Moreover, NO levels in cell culture medium of PRRSV-infected alveolar macrophages (AMs) did not differ from that of mock-infected AMs. Antiviral assays showed that NO significantly inhibited PRCV replication in swine testicular (ST) cells, whereas PRRSV was not susceptible to NO based on the conditions tested. Our study suggests that unlike PRRSV which induces apoptosis in AMs, respiratory CoVs such as PRCV that infect pulmonary epithelial cells and cause cytolysis, induce NO production in the respiratory tract. Thus, NO may play a role in innate immunity to respiratory CoV infections by inhibiting viral replication.
  Veterinary Immunology and Immunopathology 08/2010; 136(3-4):335-9. · 2.08 Impact Factor
• Article: Inactivated rotavirus vaccine induces protective immunity in gnotobiotic piglets.

  Yuhuan Wang, Marli Azevedo, Linda J Saif, Jon R Gentsch, Roger I Glass, Baoming Jiang
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  ABSTRACT: Live oral rotavirus vaccines that are effective in middle and high income countries have been much less immunogenic and effective among infants in resource-limited settings. Several hypotheses might explain this difference, including neutralization of the vaccine by high levels of maternal antibody in serum and breast milk, severe malnutrition, and interference by other flora and viruses in the gut. We have pursued development of an alternative parenteral rotavirus vaccine with the goal of inducing comparable levels of immunogenicity and efficacy in populations throughout the world regardless of their income levels. In the present study, we assessed the immunogenicity and protection of a candidate inactivated rotavirus vaccine (IRV), the human strain CDC-9 (G1P[8]) formulated with aluminum phosphate, against rotavirus infection in gnotobiotic piglets. Three doses of IRV induced high titers of rotavirus-specific IgG and neutralizing activity in the sera of gnotobiotic piglets and protection against shedding of rotavirus antigen following oral challenge with a homologous virulent human strain Wa (G1P[8]). Our findings demonstrate the proof of concept for an IRV in a large animal model and provide evidence and justification for further clinical development as an alternative candidate vaccine.
  Vaccine 07/2010; 28(33):5432-6. · 3.77 Impact Factor
• Article: Bovine respiratory coronavirus.

  Linda J Saif
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  ABSTRACT: Bovine coronaviruses (BCoVs) cause respiratory and enteric infections in cattle and wild ruminants. BCoV is a pneumoenteric virus that infects the upper and lower respiratory tract and intestine. It is shed in feces and nasal secretions and also infects the lung. BCoV is the cause of 3 distinct clinical syndromes in cattle: (1) calf diarrhea, (2) winter dysentery with hemorrhagic diarrhea in adults, and (3) respiratory infections in cattle of various ages including the bovine respiratory disease complex or shipping fever of feedlot cattle. No consistent antigenic or genetic markers have been identified to discriminate BCoVs from the different clinical syndromes. At present, there are no BCoV vaccines to prevent respiratory BCoV infections in cattle, and the correlates of immunity to respiratory BCoV infections are unknown. This article focuses on respiratory BCoV infections including viral characteristics; epidemiology and interspecies transmission; diagnosis, pathogenesis, and clinical signs; and immunity and vaccines.
  Veterinary Clinics of North America Food Animal Practice 07/2010; 26(2):349-64. · 1.47 Impact Factor