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ORIGINAL ARTICLE
Distribution of G (VP7) and P (VP4) genotypes of group Abovine rotaviruses from Tunisian calves with diarrhoeaM. Hassine-Zaafrane1,2, I. Ben Salem1, K. Sdiri-Loulizi1,2, J. Kaplon2, L. Bouslama1, Z. Aouni1,N. Sakly3, P. Pothier2, M. Aouni1 and K. Ambert-Balay2
1 Laboratory of Infectious Diseases and Biological Agents, Faculty of Pharmacy, University of Monastir, Monastir, Tunisia
2 National Reference Center for Enteric Viruses, Laboratory of Virology, CHU of Dijon, 2 Rue Ang�elique Ducoudray, University of Bourgogne,
Dijon, France
3 Laboratory of Immunology, University Hospital Fattouma Bourguiba, Monastir, Tunisia
Keywords
Bovine rotaviruses, diarrhoea, molecular
genotyping, Tunisia.
Correspondence
Mouna Hassine-Zaafrane, Laboratory of Infec-
tious Diseases and Biological Agents, Faculty
of Pharmacy, University of Monastir, TU-5000
Monastir, Tunisia.
E-mail: [email protected]
2013/2505: received 13 December 2013,
revised 30 January 2014 and accepted 3
February 2014
doi:10.1111/jam.12469
Abstract
Aims: To investigate the incidence, viral load and genetic diversity of bovine
rotaviruses strains in Tunisia.
Methods and Results: A total of 169 faecal specimens, collected from
diarrhoeic calves from several farms located in the central eastern regions of
Tunisia, between January 2006 and October 2010, were analysed by semi-
nested multiplex RT-PCRs for P and G genotypes identification or were
genotyped by DNA sequencing. Positive samples were tested by TaqMan real-
time RT-PCR to quantify the viral load. Group A bovine rotaviruses were
detected in 15�4% (26/169) of the total studied cases of diarrhoea. Overall, G10
was the predominant G type, detected in 12/26 samples (46�2%) and G6
accounted for 42�3% (11/26) while P[11] was the predominant P type,
detected in 12/26 samples (46�2%). Two P[5] genotypes (7�7%) were found in
the collection. Dual G or P combination and genotype G8 were not found.
The most common VP7/VP4 combinations were G6P[11] (30�8%; n = 8) and
G10P[11] (11�5%; n = 3). The combination G10P[14] was seen in one sample,
and partial typing was assessed in 53�8% (n = 14) of the cases. The viral load
determined by real-time RT-PCR showed an average of 1�68 9 109 genome
copies/g of faeces.
Conclusion: Knowledge of P and G types could help us understand the
relatedness of animal rotaviruses to viruses causing disease in humans.
Significance and Impact of the Study: This is the first time that the viral load
and P types of bovine rotaviruses have been determined in Tunisia, and this
study contributes to a better understanding of the epidemiology of such
viruses circulating in Tunisia. Nevertheless, continuous surveillance is necessary
to detect the emergence of new variants.
Introduction
Group A rotaviruses are a major cause of diarrhoea in
young children and animals including cattle (Kapikian
and Chanock 1990; Saif et al. 1994). Rotaviruses consti-
tute a genus within the Reoviridae family, characterized
by nonenveloped triple-layered viral particles with a viral
genome composed of 11 double-stranded RNA segments
(dsRNA). The inner capsid is composed of a single
protein, VP6, encoded by gene segment 6 and bearing
group and subgroup antigenic specificities. The outer
capsid is studded with VP7 and VP4 proteins, which eli-
cit neutralizing antibody responses and form the basis of
the present dual classification system of G (VP7) and P
(VP4) types (Kapikian et al. 2001). So far, 27 G geno-
types and 35 P genotypes have been characterized in
humans and animals (Matthijnssens et al. 2011). While at
least six P genotypes (P[1], P[5], P[11], P[14], P[17] and
Journal of Applied Microbiology 116, 1387--1395 © 2014 The Society for Applied Microbiology 1387
Journal of Applied Microbiology ISSN 1364-5072
P[21]) and eight G genotypes (G1, G3, G5–G8, G10 and
G15) have already been described in rotaviruses affecting
cattle, only G6, G10 and G8 combined with P[5], P[11]
and P[1] are considered epidemiologically important
(Alfieri et al. 2004; Barreiros et al. 2004; Dhama et al.
2009; Martella et al. 2010).
Several genotypes, such as G3, G6 and G8, are shared
by both humans and animals (Desselberger et al. 2001),
but direct transmissions between different animal species
and between humans and animal species have not actu-
ally been observed. However, the increasing number of
reports of new human rotavirus genotypes that are more
commonly found in animals suggests the possibility of
interspecies transmission or genetic reassortment of rota-
virus strains (Nakagomi et al. 1994; Desselberger et al.
2001).
While bovine rotavirus infections have been reported
in Tunisia on previous occasions (Libersou et al. 2004;
Fodha et al. 2005), current prevalence, viral load and the
circulating G and P genotypes are still unknown. For this
purpose, we examined diarrhoeic calves in the central
eastern regions of Tunisia for rotavirus infection during
the period extending from January 2006 to October 2010.
Furthermore, this is the first survey reporting the quanti-
fication and the P types of bovine rotaviruses circulating
in Tunisian cattle. Regarding the G types, our results add
information to previous genotyping.
Materials and methods
Faecal samples
Between January 2006 and October 2010, 169 faecal sam-
ples from mixed dairy-beef calves with diarrhoea were
collected from 17 cattle herds (designated as herds A-Q)
as part of an ongoing surveillance study of potential zoo-
notic micro-organisms associated with gastroenteritis in
humans. These cattle herds were located in the central
eastern regions of Tunisia: Mahdia, Monastir, Ouerda-
nine, Moknine and Kairouan. In these regions, 80% of
calves belong to the pure Holstein breed and are essen-
tially oriented towards milk production. Farm sizes ran-
ged from 5 to 25 animals. All calves with diarrhoea were
sampled. Sixty-six originated from Mahdia (16, 9, 14, 12, 5
and 10 from herds A, B, C, D, E and F, respectively), 34
from Monastir (12, 7 and 15 from herds G, H and I,
respectively), 13 from Ouerdanine (13 from herd J), 49
from Moknine (10, 5, 3,15, 9 and 7 from herds K, L, M, N,
O and P, respectively) and 7 from Kairouan (7 from herd
Q). The ages of the calves under experiment ranged from 3
to 90 days (mean age, 55 days; median age, 30 days). All
of the specimens were transported to the laboratory on
ice and stored at �20°C until analysis. The same faecal
samples were previously tested for the detection of bovine
caliciviruses (Hassine-Zaafrane et al. 2012).
RNA Extraction, RT-PCR, real-time RT-PCR and
genotyping of rotavirus
The viral RNA was extracted as previously described
(Hassine-Zaafrane et al. 2011) and then was analysed
using one-step RT-PCR kit (QIAGEN, Hilden, Germany)
and primers amplifying partial VP6 gene (Iturriza-
Gomara et al. 2002). The PCR conditions involved an ini-
tial reverse transcription step of 30 min at 50°C, followedby PCR activation at 95°C for 15 min, 35 cycles of ampli-
fication (1 min at 94°C, 1 min at 55°C and 1 min at
72°C), with a final extension of 10 min at 72°C. All rota-virus-positive samples were quantified by VP2 TaqMan
RT-PCR assay using primers and probe as previously
described (Guti�errez-Aguirre et al. 2008). The number of
genome copies present in each positive sample that could
be evaluated was estimated by comparing the sample Ct
value to standard curves. The detection limit of this
real-time RT-PCR assay was 100 copies of viral RNA,
indicating a good sensitivity of the assay. To obtain the
standard curves, a 531-bp fragment was amplified by
RT-PCR from the VP2 gene then cloned into the pGEM-
T Easy vector (Promega Corporation, Madison, WI). After
transformation in E. coli, and production of the clones,
the plasmid DNA was purified and quantified; then, serial
dilutions were prepared and used as standard curves. The
final concentration in the samples was adjusted based on
the volume of nucleic acids analysed and was expressed
per gram of faeces.
Bovine rotavirus G and P genotyping was performed
using semi-nested type-specific multiplex RT-PCRs that
could detect five G types and five P types. For G typing,
the first-round PCR used VP7-F and VP7-R primers to
amplify an 881-bp region of the VP7 gene (Iturriza-
Gomara et al. 2001). The nested multiplex PCR was per-
formed using a pool of internal primers (Gouvea et al.
1994b) specific for G5, G6, G8, G10 and G11 bovine
rotavirus genotypes in combination with the appropriate
forward consensus primer (VP7-F).
For P typing, primers VP4-F and VP4-R were used in
the first-round PCR to amplify a 663-bp fragment of the
VP4 gene (Simmonds et al. 2008). The second-round
PCR amplification was carried out with primer VP4-R
(antisense) and a pool of primers specific to P genotypes
P[1], P[5], P[6], P[7] and P[11] (sense) (Gentsch et al.
1993; Gouvea et al. 1994a). All PCR products were exam-
ined by gel electrophoresis in 2% agarose gels containing
0�4 lg ml�1 ethidium bromide and then visualized under
UV light. P and G genotypes were determined by the size
of the amplicons.
Journal of Applied Microbiology 116, 1387--1395 © 2014 The Society for Applied Microbiology1388
Bovine rotaviruses, molecular genotyping, diarrhoea, Tunisia M. Hassine-Zaafrane et al.
Nucleotide sequencing
All VP6 RT-PCR-positive faecal specimens that were nega-
tive in semi-nested multiplex RT-PCR were typed by
sequencing part of the VP7 (20 specimen typed) and/or
VP4 (4 specimen typed) gene. The samples were amplified
by one-step RT-PCR for VP7 and VP4 genes as previously
described (Iturriza-Gomara et al. 2001; Simmonds et al.
2008).
The amplicons of the VP7 and VP4 genes were purified
using Amicon� Ultra 0.5 30K Centrifugal Filters (Milli-
poreTM Corporation, Billerica, MA) according to the man-
ufacturer’s protocol. The purified PCR products were
used as a template for sequencing using an ABI PRISM�
Big Dye� Terminator Cycle Sequencing Ready Reaction
Kit on an automated sequencer (model 3130XL DNA
Genetic Analyzer), (Applera Corporation, Foster City,
CA) and were sequenced from both directions.
Phylogenetic analysis
Multiple alignments were carried out using Clustal W
(Thompson et al. 1994). Phylogenetic trees were designed
by imputing the aligned sequences into the MEGA pro-
gram (version 4.1) (Tamura et al. 2007) and constructed
with the neighbour-joining algorithm (Saitou and Nei
1987). Genetic distances were calculated with the
Kimura-2 parameter model (Kimura 1980) with a transi-
tion/transverse ratio of 2�0, and the reliability of the trees
was determined by bootstrap analysis with 100 pseudo-
replicates data sets.
The sequences obtained in this study have been sub-
mitted to GenBank under the following Accession Num-
bers:
VP4: B70/16-12-06/TUN [GenBank: KF724031]; B85/
02-02-07/TUN [GenBank: KF724032]; B137/22-02-09/
TUN [GenBank: KF724033]; B158/16-04-10/TUN
[GenBank: KF724034].
VP7: B21/28-05-06/TUN [GenBank: KF724035]; B31/
27-07-06/TUN [GenBank: KF724036]; B38/04-09-06/TUN
[GenBank: KF724037]; B52/23-10-06/TUN [GenBank:
KF724038]; B55/03-11-06/TUN [GenBank: KF724039];
B70/16-12-06/TUN [GenBank: KF724040]; B72/23-12-06/
TUN [GenBank: KF724041]; B85/02-02-07/TUN
[GenBank: KF724042]; B89/03-03-07/TUN [GenBank:
KF724043]; B91/05-03-07/TUN [GenBank: KF724044];
B95/05-04-07/TUN [GenBank: KF724045]; B109/27-06-
07/TUN [GenBank: KF724046]; B132/21-02-09/TUN
[GenBank: KF724047]; B135/23-02-09/TUN [GenBank:
KF724048]; B149/24-03-10/TUN [GenBank: KF724049];
B156/08-04-10/TUN [GenBank: KF724050]; B157/16-04-
10/TUN [GenBank: KF724051]; B158/16-04-10/TUN
[GenBank: KF724052]; B159/02-10-10/TUN [GenBank:
KF724053]; and B165/15-10-10/TUN [GenBank:
KF724054].
Statistical analysis
Statistical analyses were performed with SPSS� software,
version 19 as previously described (Hassine-Zaafrane
et al. 2011). P values ≤ 0�05 were considered significant.
Results
Rotaviruses in cattle
Twenty-six of 169 (15�4%) diarrhoeic animals were tested
positive to group A rotaviruses by one-step VP6 RT-PCR
assay. Five (3%) noroviruses of genogroup III were
detected as mixed infections with rotavirus.
Age and seasonal distribution
In this study, the mean age of calves found positive to
rotaviruses was 32�4 � 24�3 days and the median was
30 days.
Regarding the seasonal distribution of bovine rotavi-
ruses, despite the small number of positive samples, a sig-
nificant relationship was found between rotavirus
infection and seasonal distribution (P < 0�05). Indeed,
the prevalence of bovine rotaviruses was 25% (12/48) in
spring, 7�3% (3/41) in summer, 12�5% (5/40) in autumn
and 15% (6/40) in winter.
G and P genotyping of bovine rotavirus strains
G and P types of bovine rotavirus strains detected in the 26
positive cases are summarized in Table 1. G and P geno-
types were successfully determined for 23 (88�5%) and 15
(57�7%) samples, respectively. The VP7 gene of 3 samples
and the VP4 gene of 11 samples could not be amplified.
In the current study, G10 (46�2%) was the most pre-
dominant strain followed by G6 (42�3%). Among these
rotavirus strains, 12 were characterized as P[11] (46�2%),
2 as P[5] (7�7%) and 1 as P[14] (3�8%).
Both G and P types could be assigned to 12 (46�2%)
of 26 rotavirus-positive samples. Overall, G6 in combina-
tion with P[11] was the most prevalent strain (30�8%)
followed by G10P[11] (11�5%), both considered as the
most common G/P associations found in bovines. Alto-
gether, these data may suggest that there is a predomi-
nance in Tunisia of bovine strains with P[11] VP4 and
with either G6 or G10 VP7. One partially typed strain
G10P[?] detected in one sample was further characterized
by sequence analysis as G10P[14]. Dual G or P types
were not found.
Journal of Applied Microbiology 116, 1387--1395 © 2014 The Society for Applied Microbiology 1389
M. Hassine-Zaafrane et al. Bovine rotaviruses, molecular genotyping, diarrhoea, Tunisia
When analysing the distribution of G/P type combina-
tion through time, it was observed that G6P[11] was the
prevalent strain in 2006, 2007 and 2009, but G10P[11]
predominated in 2010. The one strain G10P[14] was
detected in February 2009.
For strains genotyped by DNA sequencing, phyloge-
netic analyses were performed by comparing the nucleo-
tide sequences obtained with strain sequences available in
the GenBank database.
Phylogenetic trees of nucleotide sequences of bovine
rotavirus isolates with representative VP7 (a) and VP4
(b) genotypes were constructed (Fig. 1).
The three strains B70/16-12-06/TUN, B85/02-02-07/
TUN and B158/16-04-10/TUN shared nucleotide identity
ranging from 99�3% to 99�8%, and they showed high nucle-
otide identity with the reference strain BO/B223 (Accession
Number D13394) ranging from 95�9% to 96�4%.
The strain B137/22-02-09/TUN showed high identity
with bovine strain BO/86 (Accession Number GU984756)
and human strain Hu/PR/1300/04 (Accession Number
EU835944) with 88�4% and 88�2% nucleotide identity,
respectively.
Therefore, B70/16-12-06/TUN, B85/02-02-07/TUN and
B158/16-04-10/TUN isolates were classified as isolates
with P[11] genotypes while B137/22-02-09/TUN as isolate
with P[14] genotype.
The G10 strains displayed nucleotide identities between
them ranging from 98�9% to 100% and clustered with
Bo/61A (Accession Number X53403).
Phylogenetic analysis revealed that 8 G6 strains were
homologous to each other (86�5–100% nucleotide iden-
tity). All these showed high nucleotide identities with the
Buff/10733 (Accession Number AY281360), Hu/Hun4
(Accession Number AJ487833), ROBVP7G (Accession
number M63266) and BO/CIT39A/02 (Accession Number
AY629556) ranging from 93�8% to 100%.
The two strains B159/02-10-10/TUN and B109/27-06-
07/TUN formed a branch separate from all the 8 estab-
lished G6 types.
Quantification of rotavirus
In this study, the quantification of rotaviruses in faecal
specimens by TaqMan RT-PCR demonstrated a mean
Table 1 G and P genotype combinations of individual bovine rotavirus isolates typed by multiplex RT-PCR or by DNA sequencing.
Strains Year Season
G typing result by P typing result by
Multiplex PCR Sequencing Multiplex PCR Sequencing
B21/28-05-06/TUN 2006 Spring ND G10 ND ND
B31/27-07-06/TUN 2006 Summer ND G10 ND ND
B38/04-09-06/TUN 2006 Autumn ND G10 ND ND
B52/23-10-06/TUN 2006 Autumn ND G10 ND ND
B55/03-11-06/TUN 2006 Autumn ND G10 ND ND
B70/16-12-06/TUN 2006 Winter ND G6 ND P[11]
B72/23-12-06/TUN 2006 Winter ND G6 P[11]
B85/02-02-07/TUN 2007 Winter ND G6 ND P[11]
B89/03-03-07/TUN 2007 Spring ND G6 ND ND
B91/05-03-07/TUN 2007 Spring ND G6 P[11]
B95/05-04-07/TUN 2007 Spring ND G6 P[11]
B97/17-04-07/TUN 2007 Spring ND ND P[11]
B109/27-06-07/TUN 2007 Summer ND G6 P[11]
B116/09-07-07/TUN 2007 Summer G6 P[11]
B132/21-02-09/TUN 2009 Winter ND G6 P[11]
B135/23-02-09/TUN 2009 Winter ND G6 ND ND
B137/23-02-09/TUN 2009 Winter G10 ND P[14]
B145/02-03-10/TUN 2010 Spring ND ND P[5]
B146/11-03-10/TUN 2010 Spring ND ND P[5]
B149/24-03-10/TUN 2010 Spring ND G10 ND ND
B155/06-04-10/TUN 2010 Spring G10 P[11]
B156/08-04-10/TUN 2010 Spring ND G10 ND ND
B157/16-04-10/TUN 2010 Spring ND G10 P[11]
B158/16-04-10/TUN 2010 Spring ND G10 ND P[11]
B159/02-10-10/TUN 2010 Autumn ND G6 ND ND
B165/15-10-10/TUN 2010 Autumn ND G10 ND ND
ND, not determined.
Shaded cells, G/P combination determined.
Journal of Applied Microbiology 116, 1387--1395 © 2014 The Society for Applied Microbiology1390
Bovine rotaviruses, molecular genotyping, diarrhoea, Tunisia M. Hassine-Zaafrane et al.
AF386920ROHVP7P
AY281360
VP7 (a)
VP4 (b)
AY816181
61
B70/16-12-06/TUNB85/02-02-07/TUNB91/05-03-07/TUNB89/03-03-07/TUNAJ487833B135/23-02-09/TUNB95/05-04-07/TUNROBVP7G
59
G6
B72/23-12-06/TUNB132/21-02-09/TUNAY629556
B159/02-10-10/TUNB109/27-06-07/TUN
B52/23-10-06/TUNB55/03-11-06/TUNB165/15-10-10/TUNB156/08-04-10/TUNB157/16-04-10/TUNX53403B38/04-09-06/TUNB31/27-07-06/TUN
64 G10
B149/24-03-10/TUNB21/28-05-06/TUNB158/16-04-10/TUN
0·01
B70/16-12-06/TUN
B85/02-02-07/TUN
94
98 P[11]
B158/16-04-10/TUN
D13394
100
B137/22-02-09/TUN
GU984756
P[14]
GU984754
EU835944
63
EF554107
AB158430
58
AY740738
EU311199
0·2
Figure 1 Phylogenetic trees based on partial
sequences of VP7 (a) and VP4 (b) genes of
bovine rotavirus strains. The numbers
adjacent to the nodes represent the
percentage of bootstrap support (of 100
replicates). Bootstrap values lower than 50%
are not shown. The strains of this study are in
bold face. For reference strains, we used
accession number in GenBank.
Journal of Applied Microbiology 116, 1387--1395 © 2014 The Society for Applied Microbiology 1391
M. Hassine-Zaafrane et al. Bovine rotaviruses, molecular genotyping, diarrhoea, Tunisia
viral load of 1�68 9 109 genome copies/g of faeces, and a
G/P combination can be determined only in samples with
viral load higher than 1�90 9 105 genome copies/g of fae-
ces (Table 2).
Discussion
Bovine group A rotaviruses play an important role in
causing gastroenteritis in young calves, and the best hope
for prevention is the development of an effective vaccine.
A rotavirus surveillance is an essential step in designing
vaccines and vaccine strategies to identify regional strain
patterns and potential emerging strains.
A previous study of the aetiological agents of calf diar-
rhoea in Tunisia has been published, which confirmed
that rotavirus was the major cause of diarrhoea in the
country (Zrelli et al. 1988). Out of this finding, we con-
ducted this study to achieve a better understanding of the
epidemiology of such viruses circulating in Tunisia.
The molecular prevalence of rotaviruses in this study is
consistent with studies conducted in Western India
(14�3%) (Chitambar et al. 2011) and France (15%)
(Midgley et al. 2012), but is lower than the prevalence of
rotavirus recorded in Denmark (46%) (Midgley et al.
2012) and in Argentina (62�5%) (Garaicoechea et al.
2006). A previous Tunisian study conducted between
December 2001 and April 2002 reported that bovine rota-
viruses were detected in 30% of dairy calves with diar-
rhoea (Fodha et al. 2005). Another study conducted in
six European countries demonstrated that lower rates
were reported when asymptomatic animals were tested
(2–16%) and higher rates when diarrhoeic animals were
assessed (12–98%) (Dhama et al. 2009).
The mean age of calves found positive to rotaviruses in
this study was higher than that found by Reynolds et al.
(1986) in Southern Britain (9�8 days) and by Garcia et al.
(2000) in Spain (12�9 days).
Regarding the seasonal distribution of bovine rotavi-
ruses, despite the small number of positive samples, a
clear detection peak was observed in spring. This distri-
bution was different from the seasonality of human rota-
viruses, which peaked in winter (Hassine-Zaafrane et al.
2011). A study conducted in Japan on healthy calves
showed that the highest detection rate of rotavirus genes
was in January followed by December (Abe et al. 2009).
Little is known about the seasonal distribution of bovine
rotavirus infection because some countries apply calving
programmes and because of the fact that these viruses
infect mostly younger calves, most faecal sampling is per-
formed at the same time.
In this study, dual G or P types were not found. How-
ever, a study conducted in India between 2007 and 2010
indicated that G3P[11] alone or in combination with
G10 or G8 was predominant among bovine populations
(Malik et al. 2012). Besides, co-infections by G8 and G6
were registered in Argentina from 2004 to 2010 (Badarac-
co et al. 2012).
The genotype P[14] was found in combination with
G10 genotype in one sample. In India, the genotype P
[14] was detected in association with G6 and G10 (Ghosh
et al. 2007), while it was found in association with G8 in
Japan (Fukai et al. 1999) and Western India (Chitambar
et al. 2011).
As in the current study, the most common G types in
cattle were G10 and G6 as recorded in Brazil (Alfieri
et al. 2004), the Netherlands (van der Heide et al. 2005),
Ireland (Cashman et al. 2010), Italy (Monini et al. 2008),
Turkey (Alkan et al. 2010), earlier in Sweden (De-Verdier
Klingenberg et al. 1999), Japan (Fukai et al. 2004) and
India (Rao et al. 2000).
The first Tunisian study has reported the isolation of a
single genotype of bovine rotaviruses: genotype G6 (Lib-
ersou et al. 2004). However, results obtained by Fodha
et al. (2005) demonstrated that genotype G8 was the
dominant strain followed by genotype G6 or that faecal
specimens contained a mixture of both.
We know that genotype G8 is considered as the third
G type of epidemiological importance in cattle. It was
previously detected in bovine in Japan (Fukai et al.
2002), Sweden (De-Verdier Klingenberg et al. 1999) and
Italy (Falcone et al. 1999), but in this study, genotype G8
was not found.
The frequency of G10P[11] (11�5%) was relatively low,
with respect to the values obtained from studies in India
(Gulati et al. 1999) (81% between 1994 and 1997) and
Italy (Falcone et al. 1999) (31�5% between 1994 and
1998), but close to values obtained in Brazil (Alfieri et al.
2004) (16% between 1996 and 1999).
Rotavirus strains bearing G10P[11] are common
pathogens of cattle in various regions (Fukai et al. 2002,
2004; Garaicoechea et al. 2006; Monini et al. 2008).
It has also been reported that G10P[11] strains are
Table 2 Distribution of mean viral load and G/P combinations in
rotavirus-positive faecal specimens from diarrhoeic calves in central
eastern Tunisia.
G/P
combinations
Number of G/P
combinations
Mean of viral load (genome
copies/g of faeces)
Mean
of Ct
G6P[11] 8 1�09 9 107 17�9G10P[11] 3 1�14 9 1010 13�1G10P[14] 1 3�61 9 108 15�4NDP[5] 2 1�21 9 104 27�1G10ND 8 1�90 9 105 25�6G6ND 3 3�81 9 103 29�4NDP[11] 1 2�48 9 102 32�8
ND, not determined.
Journal of Applied Microbiology 116, 1387--1395 © 2014 The Society for Applied Microbiology1392
Bovine rotaviruses, molecular genotyping, diarrhoea, Tunisia M. Hassine-Zaafrane et al.
associated with symptomatic and asymptomatic infections
in children in India (Iturriza-Gomara et al. 2004).
Indeed, interspecies transmissions of group A rotaviruses
have been suggested, especially between humans and cat-
tle (Das et al. 1993). Several publications reported G6
(Gerna et al. 1992; Steyer et al. 2012) and G10 (Urasawa
et al. 1992) genotype strains in humans. Also, uncommon
genotypes such as P[9], P[11] and P[14] are increasingly
detected in humans in different areas of the world (Gerna
et al. 1992; Gentsch et al. 1993; Santos and Hoshino
2005; El Sherif et al. 2011).
Concerning untypeable genotypes, in most of these
cases, there was either no amplified product after the
nested multiplex RT-PCR reaction, or the VP7 and/or
VP4 sequences of these strains could not be determined.
The large number of untypeable genotypes can be
explained by the fact that, in nature, any combination of
G and P types may occur and the untypeable genotypes
may represent other existing or new G or P types. These G
or P types may escape classification if there are no suitable
diagnostic reagents available. These samples may also be
classified as untypeable because they were tested only with
primers representing the G and P types traditionally asso-
ciated with the bovine population. Another explanation
that should be considered is that the rotavirus samples
from India and Africa are more diverse and thus less likely
to be amplified with a given set of primers (Simmonds
et al. 2008). In these cases, DNA sequencing of a part of
VP7 and VP4 genes was shown to be useful as a quick
determination of uncommon or novel strains whose geno-
typing cannot be performed by genotyping PCR. However,
the lack of amplification and the absence of sequences
could be due to inhibitors in faecal samples, conservation
problems or mismatches with the sequence of the primer.
Out of this study, it can be concluded that the usual
bovine P and G genotype rotaviruses circulate in Tunisia.
Therefore, it is suggested that the study of rotavirus G
and P genotyping of human and animal rotaviruses of
different species should be carried on and that the meth-
ods used for rotavirus typing need to be monitored and
updated regularly.
Acknowledgements
This work was supported by the AUF Project (code 2092
RR823) and the National Reference Center (NRC) for
Enteric Viruses, CHU Dijon, France. We thank Nedra
Kerkeni for her editorial assistance.
Conflict of Interest
None of the authors have a commercial or other associa-
tion that might pose a conflict of interest.
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