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"D�5N#R$�D D D 7705XD D D �7&&&&���$�������������������DETECTION OF ROTAVIRUS G3P[22] GENOTYPE IN GNOTOBIOTIC RABBIT MODEL
Audrey Cillia*, Claudia M. Koikea,b, Neuza M. Frazatti-Gallinab, Maria do Carmo Sampaio Tavares Timenetskya

aAdolfo Lutz Institute, Virology Center, Enteric Diseases Laboratory, Av Dr Arnaldo, n� 355, CEP 01246-902, S�o Paulo, SP, Brazil
bButantan Institute, Av Vital Brasil, 1500, CEP S�o Paulo, SP, Brazil

*Corresponding Author: Audrey Cilli and Maria do Carmo Sampaio Tavares Timenetsky. Instituto Adolfo Lutz, Centro de Virologia, N�cleo de Doen�as Ent�ricas, Av. Dr. Arnaldo, 355 CEP 01246-902, S�o Paulo, Brasil. Tel.: +55 11 30682909; fax: +55 11 30853505. E-mail adress:  HYPERLINK "mailto:audreycilli@gmail.com" audreycilli@gmail.com (Cilli A)












1. Introduction

Rotaviruses have been recognized as important agents of acute diarrhea in both humans and animals [1]. Rotaviruses belong to the Reoviridae family and are characterized by a genome consisting of 11 segments of double-stranded RNA (dsRNA), enclosed in a triple-layered protein capsid. Serotype designations are based on independent neutralization determinants of the two outer capsid proteins VP4 (P type) and VP7 (G type) [2].  Based on molecular differences, at least 27 different G-types and 35 P-types have been described so far [3].

	The use of animal models, including the gnotobiotic rabbit, mouse, piglets, lambs and calves models, has been essential to the understanding of rotavirus infection, pathology, disease, immunity, and testing vaccines in children [4,5]. Only two animal models of rotavirus infection (rabbits and piglets) closely mimic rotavirus infection in children and allow the study of active immune response and protection [6].

	Rabbit rotaviruses have been isolated by several groups of investigators in Great Britain, Europe, Japan, and the United States [5,7,8]. The epidemiology of rotaviruses infections in rabbits is similar to that observed in other species. In the major of reports, rabbits of 4 to 6 weeks old are affected. Both clinical and subclinical infections occur, in endemically infected colonies, rotavirus infection caused low morbidity in wealing rabbits. High morbidity and mortality occurred in 1 to 3 weeks-old rabbits could be a result of recently rotavirus introduction in colonies [9]. By 2 to 3 months of age most rabbits (>90%) possessed antibody to rotavirus, suggesting that rotavirus is endemic in rabbit population [10].  
	 Rabbits are productively infected with homologous lapine rotavirus strains up to at least 5 years age, which allows examine of active and long-term immunity for vaccine studies [10]. Group A lapine rotavirus strains have been isolated in Canada, Japan, Italy, and the United States, and revealed an antigen/genetic homogeneity of LRVs, as all the viruses characterized to the VP7 genotype G3 and to the VP4 genotype P[14] and P[22] [8,10,11]. 

	The significant impact of rotavirus infections in children of developing countries has directed research efforts to the development of an effective vaccine. Currently, attenuated and reassortant vaccines are being evaluated in all world [12]. To develop the rotavirus vaccine clinical trial, rabbits and mice are used in the tests. The advantage of studying the immune response to infection in an animal model is that the viral exposure history is known and can be controlled. 

The aim of this study was to describe the first detection of rotavirus G3P[22] in rabbits in Brazil and to demonstrate the importance of monitoring gnotobiotic animals used in vaccine clinical tests. 

2. Material and Methods
2.1. Virus. Pentavalent rotavirus vaccine prototype BRV-UK, produced by Butantan Institute, from reassortants of bovine UK virus and genes of rotavirus VP7 protein of human genotypes G1-G4 and G9 were used for inoculation of the animals.

2.2. Animals. The animals used in this study were rabbits and mice acquired from specific-pathogen-free colonies, being health certified. Fecal samples of these animals were sent to Adolfo Lutz Institute to be tested for viral excretion. Thirty New Zealand White rabbits (males and females), 1 to 3 months old and Swiss White mice, 6 to 8 weeks old, were used in viral excretion tests. 

2.3. Inoculation of animals. Thirty animals were divided into 6 groups, of 5 animals per group and maintained in separate isolator units for each treatment group. The animals were inoculated 1.0 ml each of vaccine prototype or placebo, intraperitoneal or orally, at 0, 7 and 14 days. Animals groups 1, 2, 3 and 4 were inoculated with vaccine prototype diluted in a citrate-phosphate buffer and groups 5 and 6 with placebo (Eagle medium, sucrose and citrate-phosphate buffer). Groups 1 and 2 received 1 dose of vaccine at concentration of 2.6 x 106 PFU/ml, and groups 3 and 4, 3 doses, 1 x 106 PFU/ml. Animal groups 5 and 6 were inoculated with 3 doses of placebo. Fecal samples were collected at days 0, 1, 2, 8, 9, 15 and 16, to monitor virus shedding. 

2.4. Detection of virus shedding by ELISA. Fecal samples were analyzed by antigen capture enzyme-linked immunosorbent assay (ELISA) to detect antigen rotavirus utilizing a commercial test (PremierTM Rotaclone� Meridian R-Biopharm Inc., USA) following the manufactures instructions. 

2.5. Reverse transcription-PCR (RT-PCR). Rotavirus strains detected by ELISA were confirmed by RT-PCR and genotyped by PCR using the primers set and methods previously described [13,14]. The primer sequence for P[22] genotyping was freely disposable by J. Matthinjnssens.

2.6. Sequence analyses. The nucleotide sequence of G3 rotavirus was determined using the Kit ABI Prism� Big Dye"! Terminator Cycle Sequencing, Ready Reaction (Applied Biosystems, Foster City, EUA) according to the manufacture instructions. The primers RVG9 and anti-A1 (gene 9) were utilized in the reactions. The extension products were purified by Centri-cep columns (Princeton Adelphia, Ni) and the nucleotide sequences determined in an automated 3130 ABI sequencer (PE Applied Biosystems, Inc., USA). The sequences generated were compared to sequences available at GenBank database (AF52801 and AF528204) and with the standard vaccines virus UK-G3, G1, G4 and G9 using BioEdit software (Ibis Therapeutics, USA). Megalign (DNAstar, Inc., Madison, Wis. USA) was used to calculate the identity between the analyzed sequences. Sequencing chromatograms were edited using Sequencher 4.7 software. Neighbor joining tree were constructed based on Maximum Composite Likelihood model determined by MEGA software version 4.0.

 2.7. Nucleotide sequence accession numbers. The GenBank accession numbers for the nucleotide and/or aminoacid sequences of the rabbit strains are KC659997, KC659998, KC659999, KC660000, KC660001 and KC660002.

3. Results
The mice not excreted virus pre- and post inoculation. In 7 samples of rabbits pre and post-immunization (0 and 8 days) was identified rotavirus group A by ELISA. By RT-PCR genotyping of the VP7 and VP4 genes with panels of primers specific for various G and P types, the strains were characterized as G3P[22] genotype (Table 1, Fig. 1, 2). These rabbits belong to placebo group and this found were not expected. The PCR product of G3 strains was sequenced and the sequences obtained were compared to standard vaccine sample UK-G3 and to animal G3 samples available at GenBank (Fig. 3). All sequences have 100% of identity each other. Identity of 80.5% was observed between rabbit sequences and standard vaccine virus UK-G3. Comparison of the nucleotide sequence analysis with rabbit sequences available at GenBank: AF52801 e AF528204 showed 84.3 to 84.5% of nucleotide identity and 94.1 to 94.5% of aminoacid identity. As expected, the sequences compared to the standard vaccines virus UK-G1, G4 and G9 showed 73.2, 74.3 and 78.5% of nucleotide identity, respectively.

4. Discussion
Rabbits have been explored as a model of homologous and heterologous rotavirus infection, transmission, and protection studies [6,15]. Immunity against a homologous rotavirus strain was demonstrated after parenteral administration of inactivated virus [16]. However, the mechanisms of protection have not been studied further. Desselberger and Huppertz (2011) affirm that a broad heterotypic protection against rotavirus challenge was induced in rabbits by parenterally administration of inactivated rotavirus particles or VLPs [17]. 
Successful use of the rabbit as an animal model for the study of rotavirus infections is contingent on the inoculation of seronegative rabbits, which requires maintain animals under strict isolation. It is necessary to routinely monitor animals to ensure that no breaks in isolation occurred [10]. 

Rabbits seem to be animals that better mimic humans in many immunologic parameters, being this animal model useful at exam how pre-existing antibodies can affect the primary viral infection and the response of primary antibodies to infection.  Beside, the neutralization results in vitro not always reflect the response of protection in vivo [10].  

Rabbit rotavirus is not commonly monitored in laboratory animal facilities, and kits for detecting rabbit rotavirus infection have not been commercialized. Commercial kits utilizing immunochromatography are available for detecting human rotavirus antigen. These kits are expected to be adaptable to lapine rotavirus, because the antibody cross-reacts among group A rotavirus antigens. Fushuku and Fukuda (2006) study cases of epizootic diarrhea in conventional rabbits at the Center for Laboratory Animal Science of the National Defense Medical College of Japan and the applicability of a commercial human rotavirus detection kit for the detection of lapine rotavirus in these cases [18]. The rotavirus antigen detection kit has showed to be useful for detecting lapine rotavirus infection in clinical health rabbits, rabbits with diarrhea symptom and in rabbits with other symptoms than diarrhea. Additional tests of RT-PCR and restriction endonuclease analysis (REA) were performed to confirm the result and the G3 type was detected. 

Utilizing ELISA commercial kits was detected viral excretion in 7 fecal samples of non-immunized rabbits, which received only placebo. The detection of rotavirus in these animals at day 0 leads to the suspicion that could be an endemic infected population, even these animals certified free.   

The excretion of vaccine virus (reassortant human/bovine) was not expected. This reassortant vaccine virus is capable to induce immune response, but present low replication capacity at animals. The anatomic-pathological tests were unable to detected infections virus damage in organs of inoculated animals (data not show). This infectious pattern was observed by Ward et al. (1996), villous atrophy is induced by virulent RV but not by attenuated RV [19]. The explanation was that the attenuated RV after oral inoculation replicated in the tonsils and pharyngeal tissues, followed by swallorwing of virus and introduction into the stomach with limited viral replication seen in the epithelial cells of the gut.

Lower concentrations of infectious heterologous rabbit viruses are excreted than homologous viruses, although rotavirus antigen is detectable by immunoassay methods [10]. Mice and children infected with SA11 and RRV had similar restriction of heterologous virus transmission, consisting of a reason to test heterologous strains of virus for candidate human vaccines. To study the molecular determinants of pathogenesis of infection and virus transmission, the heterogeneity in the response to infection in rabbits by these different virus strains will be useful.

DiGiacomo and Thouless (1986) observed that rabbits� transplacental antibodies decline promptly, however, by 2 to 3 months of age most rabbits (>90%) possessed antibody to rotavirus, suggesting that rotavirus is endemic in rabbit population [20]. Conner et al. (1988) concludes that successful use of the rabbit as an animal model for the study of rotavirus infections is uncertain, because needs that these animals to be maintaining in total isolation [10]. These authors also show the necessity of routinely monitor animals to ensure that no breaks in isolation occurred. However, continue to be an animal model which overcomes some of the restrictions and limitations of other animal models for the study of rotavirus infections. The advantages of the rabbit model include: increased age susceptibility, transplacental transfer of antibody, modest cost compared to large animal models, animal size allows for easy sample collection and manipulation several cultivatable virus strains can easily infect rabbits.  

The strains were identified as G3P[22] genotype and did not present high genomic identify with the vaccine virus UK-G3. When comparing the sequences obtained, major identity with G3 rabbit sequences from GenBank are found. The analysis of deduced aminoacid sequences also revel high identity (94.1 to 94.5%) comparing to other G3 sequences at GenBank. This fact is corroborated by studies of Ciarlet et al. (1997) which affirm the percentage of aminoacid identity (98.5 to 94.2) of G3 rabbit strains is high and can confirm the virus serological classification [11]. Martella et al. (2003) also reffer to the high aminoacid percentage (95 to 96%) found in their rabbit strains [21].

The G3 genotype have been described in many species of animals, like piglets, birds, dogs, cats, monkeys, horses, mice, cows and lambs, being the only genotype identify in rabbits [8,22]. This is the only rotavirus genotype which such diversity of hosts has been described [23]. Contrary, P[22] rotaviruses have not yet been identified in hosts other than rabbits [21]. The appointment of rotavirus in gnotobiotic rabbits show the importance of monitoring viral excretion in animals utilized in experimentation tests. It is important to emphasize the necessity of neutralizing antibody dose and the viral excretion in animals, before the beginning of challenge. 


Acknowledgments. We thank J. Matthinjnssens for the P[22] primer sequence. Our study was supported by Butantan Foundation.

Conflicts of interest: None declared.

Ethical Approval: Previous Ethics Committee approval was granted by Butantan Institute � S�o Paulo � Brazil. 


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Table 1. Summary of fecal rotavirus shedding in rabbits after inoculation with pentavalent rotavirus vaccine prototype BRV-UK by oral and intraperitoneal routes. Fecal samples were collected at days DO, D1, D2, D8, D9, D15 and D16.


Figure. 1. Agarose gel (1.2%) eletrophoresis showing rotavirus genotype G3 amplification (374 bp) of VP7 gene product from rabbit strains. Lanes 1 and 10 100 bp DNA ladder; lanes 2 to 8 G3 rabbit DNA products; lane 9 negative control. 


Figure. 2. Agarose gel (1.2%) eletrophoresis showing rotavirus genotype P[22] amplification of VP4 gene product from rabbit strains. Lanes 1 and 10 100 bp DNA ladder; lanes 2 to 8 P[22] rabbit DNA products; lane 9 negative control. 


Figure 3. Neighbor joining phylogenetic tree of the VP7 nucleotide sequence generate with MEGA 4.0 software of rabbit rotavirus G3 strains. Sequences of group A rotavirus strains representing the G3 genotypes from GenBank (AF52801 and AF528204) and the vaccine prototype G3, G1, G4 and G9 virus.
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