Antibody response in vaccinated pregnant mares to recent G3BP[12] and G14P[12] equine rotaviruses

Background Both the G3P[12] and the G14P[12] type of equine group A rotavirus (RVA) have recently become predominant in many countries, including Japan. G3 types are classified further into G3A and G3B. The G3A viruses have been circulating in Europe, Australia, and Argentina, and the G3B viruses have been circulating in Japan. However, only an inactivated vaccine containing a single G3BP[12] strain is commercially available in Japan. To assess the efficacy of the current vaccine against recently circulating equine RVA strains, we examined antibody responses in pregnant mares to recent G3BP[12] and G14P[12] strains by virus neutralization test. Findings After vaccination in five pregnant mares, the geometric mean serum titers of virus-neutralizing antibody to recent G3BP[12] strains increased 5.3- to 7.0-fold and were similar to that against homologous vaccine strain. Moreover, antibody titers to recent G14P[12] strains were also increased 3.0- to 3.5-fold. Conclusions These results suggest that inoculation of mares with the current vaccine should provide foals with virus-neutralizing antibodies against not only the G3BP[12] but also the G14P[12] RVA strain via the colostrum.


Findings
Group A rotavirus (RVA) is a non-enveloped virus belonging to the genus Rotavirus in the family Reoviridae. RVA has 11 double-stranded RNA genome segments [1]. Equine RVA infection is a major cause of diarrhea in foals up to 3 months old [2]. RVA has two outer capsid proteins, VP7 and VP4, which independently elicit the formation of neutralizing antibodies and induce protective immunity. These proteins are used to classify RVAs into G (for glycoprotein) and P (for protease-sensitive) types [1]. In addition, a whole-genome classification system based on nucleotide sequences has been proposed by using the following formula: Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx [3].
Although G3BP [12] and G14P [12] viruses have become predominant in Japan, the effectiveness of the existing vaccine against these recently circulating viruses is unclear. In this study, we used virus neutralization testing to examine the antibody responses of vaccinated pregnant mares against recently circulating equine RVA strains in Japan to evaluate the efficacy of the current vaccine.
In 2010, fecal samples were obtained from four diarrhea-affected foals in the Hidaka district of Hokkaido, Japan. All of the foals had been reared on different farms. Virus isolation was performed by using MA-104 [18] and Caco-2 [19] cells, as described previously, with a slight modification. The G type of isolated viruses was determined by semi-nested RT-PCR specific for G3 or G14 types [11], and the P type was determined by RT-LAMP specific for the P [12] type [20]. The RT-PCR products of isolated viruses were amplified by using the primers Beg9/End9 [21] and Con3/Con2 [22] and sequenced commercially at the Dragon Genomics Center (Takara Bio Inc, Mie, Japan). Phylogenetic analysis was conducted with MEGA software Version 4.0 [23]. Phylogenetic trees, based on the VP7 and VP4 gene sequences, were constructed by using the neighborjoining method. Statistical analyses of the trees were performed by employing the bootstrap test (1000 replicates) for multiple alignments.
Four equine RVA strains were isolated from the fecal samples of foals affected with diarrhea. Two G3P [12] strains isolated in MA-104 cells were designated as strains RVA/Horse-tc/JPN/No.1/2010/G3BP [12]  In the phylogenetic tree of VP7 (Figure 1) In the phylogenetic tree of VP4, all four strains belonged to the P [12] type and were located in the same cluster as the HO-5 strain and the equine RVA strains circulating from 2003 to 2008, irrespective of their G types ( Figure 2). These results are in accord with the Figure 1 Phylogenetic analysis of the VP7 gene of RVA strains G3BP [12] and G14P [12]. Rotavirus strains were denominated as reported previously [24]. The nucleotide sequences (nucleotides 104 to 906) of the VP7 gene were used to conduct a phylogenetic analysis. Black dots indicate the VP7 genes of equine RVA strains used in the virus-neutralizing test. Genome positions correspond to that of RVA/Horse-tc/JPN/HO-5/1982/G3P [12] (GenBank accession number AB046464). RVA/Horse-wt/GBR/L338/1991/G13P [18] is included as an outgroup. Percentage bootstrap support is indicated by the value at each node; values of <70 have been omitted. data that we have investigated using fecal samples collected from 2003 to 2008 [10].
In 2010 to 2011, five pregnant mares (4 to 11 years old, average age 8.2 years) that had no history of vaccination against equine RVA and had been reared at the Hidaka Training Farm of the Japan Racing Association, in the Hidaka district of Hokkaido, were twice inoculated with 2 ml of inactivated equine RVA vaccine (Nisseiken Co., Ltd., Tokyo, Japan.) at an interval of 4 to 8 weeks. These mares delivered within 3 to 6 weeks after the second vaccination. Paired sera were collected from the mares 0 to 7 days before the first vaccination and at parturition.
Virus neutralization tests were performed by using the fluorescent focus neutralization test and MA-104 cells, as described previously [25]. Virus-neutralizing antibody titers were expressed as the reciprocal of the highest serum dilution that resulted in an 80% or greater reduction in fluorescent foci. RVA strains used as reference viruses were HO-5 and RVA/Horse-tc/JPN/JE77/1997/ G14P [12] (JE77).
The virus-neutralizing antibody titers of the horse sera are shown in Table 1. After vaccination, the geometric mean titers against the homologous strain HO-5 (G3BP [12]) and against No.  (Figure 1), we expected that the antigenicity of these strains was conserved. In contrast, the geometric mean antibody titers to the G14P [12] viruses were lower than that to the homologous virus, although they were 3.0-to 3.5-fold increased after vaccination. Browning et al. have reported that mares immunized with a single-type (G6) inactivated bovine rotavirus vaccine develop antibodies in the serum and milk not only to the G6 type but also to the G2 and the G3 type [26]. Such a heterotypic response has been also reported in adult cows [27,28]. Our present result indicates that the G3BP [12] vaccine would also induce virus-neutralizing antibody against heterologous G14P [12] viruses. In addition to the immunogenicity of the G3BP [12] vaccine, investigations into that of the G3AP [12] vaccine is needed to understand vaccine efficacy and future vaccine design.
Unfortunately, we could not obtain colostrum from the vaccinated mares or sera from their foals. However, because antibody titers against equine RVA in the sera of pregnant mares are closely related to those of their colostrum and their foals' sera [17], it should be possible Figure 2 Phylogenetic analysis of the VP4 gene of RVA strains G3BP [12] and G14P [12]. The nucleotide sequences (nucleotides 114 to 755) of the VP4 gene were used to conduct a phylogenetic analysis. Black dots indicate the VP4 genes of the equine RVA strains used in the virus-neutralizing test. Genome positions correspond to that of RVA/Horse-tc/JPN/HO-5/1982/G3P [12] (Genbank accession number AB046471). RVA/Horse-wt/GBR/L338/1991/G13P [18] is included as an outgroup. Percentage bootstrap support is indicated by the value at each node; values of <70 have been omitted. to deduce the immune status of the foals from the serum antibody titers of their mares. Therefore, this study shows that the current vaccine is likely to provide foals with antibodies against recent G3BP [12] RVA strains at the same level as those against the vaccine strain. Of note, in the late 1990s it was reported that the current vaccine reduced the duration and clinical signs of diarrhea caused by G14 viruses [16]. Mares immunized with the current vaccine neutralized strains No. 24/2010 and No. 50/2010 at titers similar to those at which they neutralized strain JE77 isolated in the late 1990s (Table 1). These results suggest that inoculation with the current vaccine also appears to provide foals with virus-neutralizing antibodies against recent G14P [12] RVA strains via the colostrum. We used only a small number of samples in this study, and the results need to be confirmed with a large number of samples. Virus challenge studies in foals might be needed to clarify the protective efficacy of the vaccine against recent G14P [12] strains.