Rotavirus C: prevalence in suckling piglets and development of virus-like particles to assess the influence of maternal immunity on the disease development

International audienceAbstractRotavirus C (RVC) has been detected increasingly in humans and swine in different countries, including the US. It is associated with significant economic losses due to diarrheal disease in nursing piglets. In this study we aimed: (1) to determine the prevalence of RVC in healthy and diarrheic suckling piglets on US farms; and (2) to evaluate if maternal antibody (Ab) levels were associated with protection of newborn suckling piglets against RVC. There was a significantly higher prevalence (p = 0.0002) of litters with diarrhea born to gilts compared with those born to multiparous sows. Of 113 nursing piglet fecal samples tested, 76.1% were RVC RNA positive. Fecal RVC RNA was detected in significantly (p = 0.0419) higher quantities and more frequently in piglets with diarrhea compared with healthy ones (82.5 vs. 69.9%). With the exception of the historic strain Cowden (G1 genotype), field RVC strains do not replicate in cell culture, which is a major impediment for studying RVC pathogenesis and immunity. To circumvent this, we generated RVC virus-like particles (VLPs) for Cowden (G1), RV0104 (G3) and RV0143 (G6) and used them as antigens in ELISA to detect swine RVC Abs in serum and milk from the sows. Using RVC-VLP Ab ELISA we demonstrated that sows with diarrheic litters had significantly lower RVC IgA and IgG Ab titers in milk compared to those with healthy litters. Thus, our data suggest that insufficient lactogenic protection provided by gilts plays a key role in the development of and the increased prevalence of clinical RVC disease

Oral vitamin A supplementation of porcine epidemic diarrhea virus infected gilts enhances IgA and lactogenic immune protection of nursing piglets

International audienceAbstractVitamin A (VA) has pleiotropic effects on the immune system and is critical for mucosal immune function and intestinal lymphocyte trafficking. We hypothesized that oral VA supplementation of porcine epidemic diarrhea virus (PEDV)-infected pregnant gilts would enhance the gut-mammary gland-secretory IgA axis to boost lactogenic immunity and passive protection of nursing piglets against PEDV challenge. Gilts received daily oral retinyl acetate (30 000 IU) starting at gestation day 76 throughout lactation. At 3–4 weeks pre-partum, VA-supplemented (PEDV + VA) and non-supplemented (PEDV) gilts were PEDV or mock inoculated (mock + VA and mock, respectively). PEDV + VA gilts had decreased mean PEDV RNA shedding titers and diarrhea scores. To determine if lactogenic immunity correlated with protection, all piglets were PEDV-challenged at 3–5 days post-partum. The survival rate of PEDV + VA litters was 74.2% compared with 55.9% in PEDV litters. Mock and mock + VA litter survival rates were 5.7% and 8.3%, respectively. PEDV + VA gilts had increased PEDV IgA antibody secreting cells and PEDV IgA antibodies in serum pre-partum and IgA+β7+ (gut homing) cells in milk post piglet challenge compared with PEDV gilts. Our findings suggest that oral VA supplementation may act as an adjuvant during pregnancy, enhancing maternal IgA and lactogenic immune protection in nursing piglets

Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea

Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.Fil: Vega, Celina Guadalupe. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bok, Marina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vlasova, Anastasia N.. Ohio State University; Estados UnidosFil: Chattha, Kuldeep S.. Ohio State University; Estados UnidosFil: Gómez Sebastián, Silvia. Universidad Politécnica de Madrid; EspañaFil: Nuñez, Carmen. Universidad Politécnica de Madrid; EspañaFil: Alvarado, Carmen. Universidad Politécnica de Madrid; EspañaFil: Lasa, Rodrigo. Universidad Politécnica de Madrid; EspañaFil: Escribano, José M.. Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria. Departamento Mejora Genética y Biotecnología; EspañaFil: Garaicoechea, Lorena Laura. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernández, Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Bok, Karin. National Institutes of Health; Estados UnidosFil: Wigdorovitz, Andrés. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Saif, Linda J.. Ohio State University; Estados UnidosFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

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

Characterization of Developmental Pathway of Natural Killer Cells from Embryonic Stem Cells In Vitro

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34(+) EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34(+) EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34(+) EB cells, NK and T cell potentials were detected in a CD45(−) subset, whereas CD45(+) EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34(+)CD45(−) EB cells showed that CD45(+)Mac-1(−)Ter119(−) ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45(−)CD34(+) EB cells have lymphoid potential, and they differentiate into more mature CD45(+)Lin(−) hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122(−) and they rapidly acquire CD122 as they differentiate along the NK lineage

Care of patients with inborn errors of immunity in thirty J Project countries between 2004 and 2021

IntroductionThe J Project (JP) physician education and clinical research collaboration program was started in 2004 and includes by now 32 countries mostly in Eastern and Central Europe (ECE). Until the end of 2021, 344 inborn errors of immunity (IEI)-focused meetings were organized by the JP to raise awareness and facilitate the diagnosis and treatment of patients with IEI.ResultsIn this study, meeting profiles and major diagnostic and treatment parameters were studied. JP center leaders reported patients’ data from 30 countries representing a total population of 506 567 565. Two countries reported patients from JP centers (Konya, Turkey and Cairo University, Egypt). Diagnostic criteria were based on the 2020 update of classification by the IUIS Expert Committee on IEI. The number of JP meetings increased from 6 per year in 2004 and 2005 to 44 and 63 in 2020 and 2021, respectively. The cumulative number of meetings per country varied from 1 to 59 in various countries reflecting partly but not entirely the population of the respective countries. Altogether, 24,879 patients were reported giving an average prevalence of 4.9. Most of the patients had predominantly antibody deficiency (46,32%) followed by patients with combined immunodeficiencies (14.3%). The percentages of patients with bone marrow failure and phenocopies of IEI were less than 1 each. The number of patients was remarkably higher that those reported to the ESID Registry in 13 countries. Immunoglobulin (IgG) substitution was provided to 7,572 patients (5,693 intravenously) and 1,480 patients received hematopoietic stem cell therapy (HSCT). Searching for basic diagnostic parameters revealed the availability of immunochemistry and flow cytometry in 27 and 28 countries, respectively, and targeted gene sequencing and new generation sequencing was available in 21 and 18 countries. The number of IEI centers and experts in the field were 260 and 690, respectively. We found high correlation between the number of IEI centers and patients treated with intravenous IgG (IVIG) (correlation coefficient, cc, 0,916) and with those who were treated with HSCT (cc, 0,905). Similar correlation was found when the number of experts was compared with those treated with HSCT. However, the number of patients treated with subcutaneous Ig (SCIG) only slightly correlated with the number of experts (cc, 0,489) and no correlation was found between the number of centers and patients on SCIG (cc, 0,174).Conclusions1) this is the first study describing major diagnostic and treatment parameters of IEI care in countries of the JP; 2) the data suggest that the JP had tremendous impact on the development of IEI care in ECE; 3) our data help to define major future targets of JP activity in various countries; 4) we suggest that the number of IEI centers and IEI experts closely correlate to the most important treatment parameters; 5) we propose that specialist education among medical professionals plays pivotal role in increasing levels of diagnostics and adequate care of this vulnerable and still highly neglected patient population; 6) this study also provides the basis for further analysis of more specific aspects of IEI care including genetic diagnostics, disease specific prevalence, newborn screening and professional collaboration in JP countries

Prevalence and Genetic Diversity of Rotaviruses among under-Five Children in Ethiopia: A Systematic Review and Meta-Analysis

Rotavirus infection is the major cause of acute gastroenteritis among children globally. Sub-Saharan Africa including Ethiopia is disproportionally affected by the disease. The aims of this review were to determine the pooled prevalence of rotavirus infection among children under-five and to identify the dominant rotavirus genotypes in Ethiopia. Twelve studies were included to estimate the pooled prevalence of rotavirus acute gastroenteritis and five studies were used to determine predominantly circulating genotypes of rotavirus. The pooled prevalence of rotavirus infection was 23% (95% CI = 22%–24%). G3 (27.1%) and P[8] (49%) were the dominant G and P types, respectively. The G8 G-type uncommon in humans but highly prevalent in cattle was also reported accounting for 1% of all cases. The major G/P combinations were G12P[8] (15.4%), G3P[6] (14.2%), G1P[8] (13.6%) and G3P[8] (12.9%) collectively accounting for 56.1% of rotavirus strains. Similar to other parts of the world, the dominance of G1, G3, P[6] and P[8] genotypes was noted in Ethiopia. The increased prevalence of G12P[8] strains observed in Ethiopia was similar to observations in other geographic regions in the post-vaccine introduction period. Thus, further studies are required on the vaccine effectiveness, genotype distribution and inter-species transmission potential of rotaviruses in Ethiopia

Porcine Rotaviruses: Epidemiology, Immune Responses and Control Strategies

Rotaviruses (RVs) are a major cause of acute viral gastroenteritis in young animals and children worldwide. Immunocompetent adults of different species become resistant to clinical disease due to post-infection immunity, immune system maturation and gut physiological changes. Of the 9 RV genogroups (A–I), RV A, B, and C (RVA, RVB, and RVC, respectively) are associated with diarrhea in piglets. Although discovered decades ago, porcine genogroup E RVs (RVE) are uncommon and their pathogenesis is not studied well. The presence of porcine RV H (RVH), a newly defined distinct genogroup, was recently confirmed in diarrheic pigs in Japan, Brazil, and the US. The complex epidemiology, pathogenicity and high genetic diversity of porcine RVAs are widely recognized and well-studied. More recent data show a significant genetic diversity based on the VP7 gene analysis of RVB and C strains in pigs. In this review, we will summarize previous and recent research to provide insights on historic and current prevalence and genetic diversity of porcine RVs in different geographic regions and production systems. We will also provide a brief overview of immune responses to porcine RVs, available control strategies and zoonotic potential of different RV genotypes. An improved understanding of the above parameters may lead to the development of more optimal strategies to manage RV diarrheal disease in swine and humans

Animal Rotaviruses

Rotaviruses (RVs) are ubiquitous and remain the major cause of acute viral gastroenteritis in young animals, bird species and children worldwide. The disease is acute, occurs predominantly in intensively reared animals and characterized by a short incubation period, anorexia and diarrhoea. Post-infection immunity and immune system and intestinal microbiome maturation make immunocompetent adults of different species resistant to clinical RV disease. RVs of groups A, B, C, E, H, I and J have been detected in sporadic, endemic or epidemic infections of various mammalian species, whereas RV strains of groups D, F and G are only found in poultry, such as chickens and turkeys. Recently identified novel RVs in sheltered dogs in Hungary and bats in Serbia are tentatively identified as group I and J, respectively. Historically, diagnosis of RV infections relied on conventional techniques such as isolation in cell culture, electron microscopy, electropherotyping and various serological tests. Presently, RT-PCR assays and molecular typing using sequencing or genomic hybridization techniques are used predominantly for RV diagnosis and classification. Because RVs are endemic in most animal populations and exhibit extreme genetic diversity due to frequent mutations and re-assortment events, available RV vaccines are only marginally efficient, and eradication of the pathogen remains a challenge. Thus, a better understanding of the historic and current prevalence and genetic diversity of animal RVs in different geographic regions, disease pathogenesis, available control strategies and zoonotic potential is needed. This knowledge will lead to the development of more optimal strategies to manage RV diarrhoeal disease in animals, birds and humans