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Group A rotavirus is one of the most common causes of severe diarrhea in human infants and newborn animals. Rotavirus virions are triple-layered particles. The outer capsid proteins VP4 and VP7 are highly variable and represent the major neutralizing antigens. The inner capsid protein VP6 is conserved among group A rotaviruses, is highly immunogenic, and is the target antigen of most immunodiagnosis tests. Llama-derived single-chain antibody fragments (VHH) are the smallest molecules with antigen-binding capacity and can therefore be expected to have properties different from conventional antibodies. In this study a library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated. Binders directed to VP6, in its native conformation within the viral particle, were selected and characterized. Four selected VHH directed to conformational epitopes of VP6 recognized all human and animal rotavirus strains tested and could be engineered for their use in immunodiagnostic tests for group A rotavirus detection. Three of the four VHH neutralized rotavirus in vivo independently of the strain serotype. Furthermore, this result was confirmed by in vivo partial protection against rotavirus challenge in a neonatal mouse model. The present study demonstrates for the first time a broad neutralization activity of VP6 specific VHH in vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool for the prevention and treatment of rotavirus diarrhea.Group A rotavirus (RV) is the leading cause of acute gastroenteritis in human infants less than 5 years old, causing 611,000 deaths per year (41). It is also the main cause of severe diarrhea in the neonates of many animal species of economic interest (43,47). RV virions are triple-layered particles composed by a core (protein VP2), an inner capsid (protein VP6), and an outer capsid (proteins VP7 and VP4) (16,29). The inner capsid protein, VP6, is a trimer representing 51% of the virion mass. According to the antigenic variation of VP6, RVs are classified into seven groups (A to G) (16). Depending on the presence or absence of two different epitopes in the VP6 protein, group A RV strains are further divided into subgroups (Sb) I, II, IϩII, and no I no II. Despite the different subgroups mentioned, VP6 is a strongly conserved protein among all group A RVs (Ͼ90% amino acid homology). It is highly immunogenic and constitutes the target antigen of most immunodiagnosis tests for group A RV detection. In contrast, the outer capsid proteins VP7 (glycoprotein) and VP4 (protease sensitive) are highly variable and constitute the major neutralizing antigens. Based on the variation of VP7 and VP4, group A RVs are further classified into G and P types, respectively.

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In this study, the complete genome sequences of seven equine group A rotavirus (RVA) strains (RVA/Horse-tc/GBR/L338/1991/G13P [18] [12] from South Africa) were determined. Multiple novel genotypes were identified and genotype numbers were assigned by the Rotavirus Classification Working Group: R9 (VP1), C9 (VP2), N9 (NSP2), T12 (NSP3), E14 (NSP4), and H7 and H11 (NSP5). The genotype constellation of L338 was unique: G13-P[18]-I6-R9-C9-M6-A6-N9-T12-E14-H11. The six remaining equine RVA strains showed a largely conserved genotype constellation: G3/G14-P[12]-I2/I6-R2-C2-M3-A10-N2-T3-E2/E12-H7, which is highly divergent from other known non-equine RVA genotype constellations. Phylogenetic analyses revealed that the sequences of these equine RVA strains are related distantly to nonequine RVA strains, and that at least three lineages exist within equine RVA strains. A small number of reassortment events were observed. Interestingly, the three RVA strains from Argentina possessed the E12 genotype, whereas the three RVA strains from Ireland and South Africa possessed the E2 genotype. The unusual E12 genotype has until now only been described in Argentina among RVA strains collected from guanaco, cattle and horses, suggesting geographical isolation of this NSP4 genotype. This conserved genetic configuration of equine RVA strains could be useful for future vaccine development or improvement of currently used equine RVA vaccines. INTRODUCTIONEquine group A rotavirus (RVA) strains were first detected in diarrhoeic foals in England in 1975(Flewett et al., 1975 and are a major cause of dehydrating diarrhoea in young 3Present address: Deltamune (Pty) Ltd, 248 Jean Avenue, Lyttelton, Centurion, 0140, South Africa.The GenBank/EMBL/DDBJ accession numbers for the equine rotavirus strains described in this study are JF712555-JF712565, JF712566-JF712576, JF712577-JF712587, JN872865-JN872875, JQ345489-JQ345499 and JN903507-JN903528. foals (Browning & Begg, 1996;Frederick et al., 2009; Imagawa et al., 1991;Saif et al., 1994). Serological data from Japan, the USA and France suggest that RVA is a ubiquitous pathogen in horse populations (Conner & Darlington, 1980;Imagawa et al., 1982;Pearson et al., 1982;Takahashi et al., 1979).RVA strains are icosahedral, non-enveloped viruses possessing a genome of 11 segments of dsRNA. The two outer capsid proteins, VP7 and VP4, elicit neutralizing antibodies independently and are used to differentiate RVA strains into G-types (glycoprotein) and P-types (proteasesensitive), respectively (Ciarlet & Estes, 2002 (Browning et al., 1991a). L338 was shown to possess the unique G13 and P[18] genotypes and a highly divergent NSP1 gene sequence, and to be distinct from other human and animal RVA strains by using RNA-RNA hybridization assays (Browning et al., 1991a; Iša & Snodgrass, 1994;Kojima et al., 1996;Taniguchi et al., 1994;Wu et al., 1993). In addition, a limited number of porcine-, bovineand feline-like RVA strains have been detected in horses. Examples include the G5P [7] RVA strain RVA/Horse-tc/ ...

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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.