Abstract

This is a pioneering molecular report investigating the genetic diversity and phylogenic analysis of BVDV in cattle of Nineveh province, Iraq. A total of 494 ear notch samples was collected from cattle in five different regions of Nineveh province and tested by multiplex PCR assay. The overall prevalence of BVDV genotypes was 13.96% (69 out of 494), for BVDV1 it was 12.95% and for BVDV2 it was 1.01%. Bovine viral diarrhea virus 1 was significantly more detected than BVDV2 (P< 0.05). The individual sequencing analysis (BLASTn) of 69 sequences of 5’UTR gene, revealed for the first time the existence of three sub genotypes of BVDV1 in Nineveh province: sub genotype A, B and E were 3.44%, 7.89%, and 1.61% respectively. BVDV 1 sub genotype B was the most commonly detected in cattle (P< 0.05). In addition, for the first time, two sub genotypes of BVDV2 were found in Nineveh province (sub genotype A 0.60% and sub genotype B 0.40%). Bovine viral diarrhea virus 2 sub genotype A was commonly detected in cattle. It has been concluded that the current study found the genetic diversity of BVDV which circulating among the cattle population in Nineveh province and this result could have a significant implications for the epidemiology, diagnosis and control of the disease in Iraq.

Key words: BVDV, Genetic Diversity, Phylogenetic Analysis, Nineveh, Iraq.

Introduction

Bovine Viral Diarrhea virus BVDV affects cattle, regardless of age and also pregnant dam and this can lead to substantial economic losses because of the consequential reproductive failures, calf mortality, and also enteric and respiratory disease [1]. The BVD is a significant pathogen of cattle and globally distributed. In cattle, infections of BVDV may be as persistent infection (PI) or transient infections (TI) disease [2].Bovine Viral Diarrhea virus, classical swine fever virus (CSFV) and sheep Border disease virus (BDV), are classified in genus Pestivirus in the Flaviviridae family [3,4]. The genome of BVDV 12.5 kb length is a positive single strand RNA, which encodes a long open reading frame (ORF) that is flanked by 5 and 3 untranslated regions (UTRs). The translated polyprotein is cleaved by cellular and viral proteases to provide the viral structural and non-structural (NS) proteins [5]. Bovine viral diarrhea transmission occurs either horizontally or vertically from a pregnant cow to its fetus [6]. When infection occurs in the first trimester of gestation the consequence could be immune tolerance, and fetuses that survive are born as PI (persistently infected) calves. PI animals have no detectable antibodies against BVDV, but continuously shed the virus in their excretions and secretions, and are thus primary sources of BVDV infection [7]. Bovine viral diarrhea virus has been categorized into two genotypes, BVDV1 and BVDV2 based on the antigenic variation and sequence differences in the 5' UTR region [8]. BVDV strains have two biotypes, cytopathic (cp) and non-cytopathic (ncp), which are distinguishable based on their impact on cultured bovine cells [9]. Both biotypes of the virus can cause disease in cattle, however, high incidence of the disease is caused by the non-cytopathic biotype [10].The 5'UTR region is a highly-conserved component of the viral genome and consists of an internal ribosome entry site involved in translating the viral polyprotein [11].The features and the biological roles of the Pestivirus proteins, properties of the 5 and 3'UTR, as well as molecular aspects of the replication and cytopathogenicity of the virus were reviewed recently [12,13]. Three different mechanisms result genetic changes in Pestivirus genomes first accumulation of point mutations, second non-homologous RNA recombination; and third homologous RNA recombination [14]. Different genomic regions 5' UTR, Npro, E2, NS2-3, and NS5B-3'UTR have often been employed to genotype and classify BVDV and other pestiviruses. Partial 5'UTR sequences are being commonly utilized phylogenetic ally analyzer and genetically type BVDV isolates [15]. Subtypes of the two genotypes have also been defined [16]. In recent times, reverse transcription-PCR (RT-PCR) approaches have been used in facilitating the typing of BVDV at the genotype level directly from blood samples of PI cattle [17]. Several of these sequences of BVDV which have normally been carried out utilizing genetic typing sequences from the mention regions are available in the Gen Bank [18]. Phylogenetic analysis shows the presence of sub genotypes BVDV-1 and BVDV- 2, 12 for genotype BVDV-1 (1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i, 1j, 1k, 1l), five further sub genotypes of BVDV1 have been suggested (1l, 1m, 1n, 1o and 1p) and two for genotype BVDV-2 (2a, 2b) sub genotypes [19, 20]. No less than 21 BVDV-1 sub genotypes (BVDV-1a to-1u) and four BVDV-2 sub genotypes (BVDV-2a to-2d) have been defined to date [21].Serological and molecular approaches have been employed to detect and identify genotypes and sub-genotypes of BVDV [15]. The use of molecular techniques has increased because of their speed and accuracy. The nucleic acid-based techniques are useful tools for detecting and simultaneous genotyping of BVDV without isolation and propagation in cell cultures [21]. Several researchers have used a different type of PCR technique, including reverse transcriptase polymerase chain reaction (RT-PCR) [22], multiplex RT-PCR [21]. And nested RT-PCR [23] to detect and type BVDV. The aim of the present study was to explore the genetic diversity and phylogenetic analysis of BVDV circulating in cattle populations in Nineveh region north of Iraq.

Materials and Methods

Animal and sample collection
From Jan 2017 to Aug 2017, a total of 494 ear notch samples (1 centimeter square) were obtained from the external ear pinna by utilizing sterile disposable surgical blades. The samples were placed in a sterile test tube, and kept in an ice bag and taken to the laboratory to be stored at-20 °C until testing.

RNA extraction and amplification from cattle ear notches:
The RNA of BVDV was extracted from 494 ear notches employing the QIAamp® Viral RNA kit (RNA extraction from ear notches without purification) (ADIAGENE, BIOX DIGNOSTIC. FRANCE). The procedure was adapted from the literature that came with the extraction kit. Amplifying the highly conserved region 5’ UTR gene of BVDV from ear notch samples (n=494) was a target in multiplex PCR technique utilizing QIAGEN One-step RT-PCR Kit (QIAGEN, GERMANY) for BVDV detection and determination of genotypes. The oligonucleotides of specific primers were designed by [17] and provided by FIRST BASE LABORATORIES SDN. BHD. MALAYSIA (Table 1).

Table 1: The Oligonucleotide primers used to amplify the 5’UTR gene.

In this PCR process, two positive cDNA derived from persistently infected calve were used as positive control. The GenBank accession numbers for cDNA positive controls were (MF347399, MF491394) for BVDV1 and BVDV2 respectively. Further, cDNA extracted from non-infected calve were used as negative control for each PCR amplification. In this study, all the animals tested positive were re-sampled 30 days following the first round of testing to differentiate PI animals. Multiplex PCR reactions were conducted in a total volume of 50 μl, composed of 10μl 5XQaigen RT PCR buffer, 3μl of each primer, 2μl of dNTP mix, 2μl of RT-PCR enzyme, 10μl of template (RNA sample) and 17μl dH2o. The mixture was centrifuged briefly and reverse transcription was carried out at 55 ºC for 30 min in the thermo-cycler machine, then subjected to initial denaturation at 95 ºC for 15 min, followed by 40 cycles of denaturation at 94 ºC for 20 s, annealing at 50 ºC for 30 s, extension at 72 ºC for 30 s and final extension at -72 ºC for 15 min, The samples were removed at the end of the program when the temperature on the screen showed 4ºC

cDNA sequencing
A total of 69 PCR amplicons from cattle ear notches that were positive for BVDV1 and BVDV2 by multiplex PCR, were sent to a commercial company for purification and sequencing (My TACG Bioscience Enterprise, Kuala Lumpur, Malaysia). Sequences of the cDNA were subjected to analysis using Bio edit program version 7.2.5 [24] and blasted against other published BVDV1 and BVDV2 sequences from the GenBank using NCBI BLAST (BLASTn) from NCBI (available at http://www.ncbi.nlm.nih.gov)
Statistical analysis
The Statistical analysis in this study was done by using computed 2 by 2 tables in Epi-InfoTM 7 software (version 7).

Results

In the current study, a total of 494 ear notch samples were tested by Multiplex PCR technique. The overall prevalence of BVD in Nineveh province was 13.96% (69 out of 494), for BVDV1 it was 12.95% and for BVDV2 it was 1.01%. BVDV 1 was significantly more detected than BVDV2 (P< 0.05) (Table 2). For the first time, three sub genotypes of BVDV1 were detected in Nineveh province: sub genotype A 3.44%, sub genotype B 7.89%, and sub genotype E 1.61%. BVDV 1 sub genotype B was the most commonly detected in cattle (P< 0.05) (Table 3) In addition, for the first time two sub genotypes of BVDV2 were detected in Nineveh province: sub genotype A 0.60% and sub genotype B 0.40%. Bovine viral diarrhea virus 2 sub genotype A was commonly detected in cattle (Table 3). In this study, a total of 69 amplicons obtained from the extracted cattle ear notch using multiplex PCR was sequenced. Nine variable sequences of BVDV1 from cattle were deposited in the GenBank under accession numbers MF347398-MF347406 (Table 4). Moreover, four variable sequences of BVDV2 obtained from cattle were also deposited in the GenBank under accession numbers MF491394- MF491397 (Table 4). In this study, phylogenetic tree analysis using the neighbor-joining BVDV1 and BVDV2 yielded a tree with almost identical topologies and high bootstrap or nodal support values.

Table 2: Value significantly different (P < 0.05) are labeled with different letters (a, b or c). Detection rate of BVDV genotypes in ear notch cattle samples using multiplex PCR techniques (n= 494)

Table 3: Value significantly different (P < 0.05) are labeled with different letters (a, b or c). Detection rate of BVDV1 and BVDV2 sub genotypes in cattle base on individual BLASTn analysis of positive samples.

Table 4: GenBank accession numbers of Nineveh BVDV1 and BVDV2 sub genotypes.

Figure 1: Phylogenetic tree of BVDV1 and BVDV2 obtained with partial sequences of the 5’UTR gene. The numbers on the branches indicate bootstrap supports (100 replications). Sequences in bold indicate those obtained sequences in this study. Cattle and locations are in between brackets. V1-A, V1-B and V1-E indicate to Sub genotypes of BVDV1, V2-A, V2-B indicate Sub genotypes of BVDV2 and used as out group.

Nineveh BVDV1 of 5’UTR gene sequences exhibited three major classes comprising the previously published sub genotypes of BVDV1 [49], were present in the current study (Figure 1). BVDV 1 tree demonstrated three genetically distinguishing sub genotypes represented by cladding (sub genotype A, sub genotypes B and sub genotype E). Nineveh BVDV2 of 5’UTR gene sequences showed two major clads comprising the previously published sub genotypes of BVDV2, were present in the current study (Figure 1). Bovine viral diarrhea virus 1 tree demonstrated three genetically distinguishing sub genotypes represented by cladding (sub genotype A and sub genotypes B). The tree was rooted with BVDV2 (U949142A, AY443026, MF491394 and MF491396) as an out group. In the phylogenetic tree of BVDV1 and BVDV2, the samples were obtained from cattle ear notch, from different regions in Nineveh province.

Discussion

BVDV is a widely spread virus that causes various clinical syndromes leading to very substantial economic losses, Furthermore, the two species, BVDV-1 and BVDV-2, are dissimilar and differentiated based on genetic and antigenic variances, and are categorized in the genus Pesti virus within the Flaviviridae family and found on all continents of the world [11]. The results illustrated that the overall prevalence of BVDV in Nineveh was 13.96% compared to previous studies in Iraq and other countries that used the PCR technique to diagnose this disease. The prevalence was 6% in cattle in regions around Baghdad [15]. In Basrah and Nasiriya cities in Iraq, the prevalence of BVDV was 10 % [26]. In Tunisia, it was 2.65% [27]. In Egypt it was 10.4% [28]. Regional differences within the same country could be attributed to the different management practices, and how sensitive the diagnostics methods used are, incidence of competent vectors, cattle activity, presence and efficacy of control programs, the climatic variations, extensive cattle trade and uncontrolled animal movement as well as variations in the cattle population size, Biosecurity, and persistence of BVDV [29]. In the current study, based on conserved untranslated regions 5’UTR, multiplex polymerase chain reaction (PCR) allow for the detection and differentiation between BVDV genotype 1, 2. These results suggest that multiplex PCR can be used to amplify two or more DNA fragments in a single reaction. In comparison with single template PCR, multiplex PCR can lower the time, number of tubes, the cost of the reagents, and possible contamination. This finding was consistent with [21, 30, 31], who showed that the BVDV could be typed directly from blood samples by using multiplex polymerase chain reaction (PCR).
This study has demonstrated that the 5’-UTR region of the genome is very conservative and can be utilized as a target sequence for diagnostic RT-PCR. This finding agrees with [32, 33]. Moreover, the 5’UTR gene is highly important in the epidemiological genetic diversity and phylogenetic studies of pestiviruses [34, 35]. The results in this study revealed that the overall detection of both genotypes was 13.96%, with a detection rate of 12.95% and 1.01% for BVDV1 and BVDV2 respectively in Nineveh province. This may be due to the fact that BVDV1and BVDV2 is globally distributed. This finding corresponds with other studies in different countries such as in Iran [36], Turkey [36], Tunisia [27], Spain [38] and Italy [39]. It should be noted though that these results differ from those documented for another province of Iraq by [27], who detected BVDV genotype1 only in Basrah and Nasiriya cities in Iraq.
The results of the current study indicated that that BVDV1was significantly more detected than BVDV2 in Nineveh province. This result is consistent with previous studies in other countries [38, 40, 41]. In addition, Yesilbag [27] mentioned that the global distribution rate of BVDV-1 was 88.2%, considerably higher than for BVDV-2, which was 11.8% based on phylogenetic of BVDV in different countries. In the current study regarding sequencing BLASTn of PCR amplicons of the 5’UTR gene for BVDV1 and BVDV2 were obtained from cattle ear notch samples. These findings suggest that there is extensive genetic diversity found within BVDV type 1. Three sub genotypes of BVDV1 were detected for the first time in Nineveh province, including sub genotypes A, B and E. This is almost similar to previously known genotypes in other parts of the world such as reported by [42, 43]. The absence of possible BVDV1 sub genotypes C, F, G, H and U, in this current study reflects the findings of other studies by Factor et al. [44, 45]. In addition, two sub genotypes of BVDV2 were detected for the first time in Nineveh province including sub genotypes A and B. This result is similar to previously reported sub genotypes in other countries such as China, Brazil, Argentina, Italy and USA [39, 46-49].
BVDV2 sub genotype A was more commonly detected in cattle in Nineveh province compared to sub genotypes B. This is similar to previous studies in other parts of the world as reported by [25]. These variations in the distribution of BVDV sub genotypes can be interpreted based on the insertion or deletion in the 5’UTR gene sequences, which can be valuable to the virus by way of modifying the gene expression, which supports their adaptation to a new environment such as re-occurring PI, a different host (sheep, goats and cattle) and climatic changes [50].

Acknowledgements

This work was supported by the Faculty Veterinary Medicine, University of Mosul, Iraq.