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Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Tunisian calves with diarrhoea

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Page 1: Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Tunisian calves with diarrhoea

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

Page 2: Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Tunisian calves with diarrhoea

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.

Page 3: Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Tunisian calves with diarrhoea

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

Page 4: Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Tunisian calves with diarrhoea

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.

Page 5: Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Tunisian calves with diarrhoea

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

Page 6: Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Tunisian calves with diarrhoea

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.

Page 7: Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Tunisian calves with diarrhoea

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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