Isolation and Genetic Characterization of Hobi- Like Pestivirus Circulating in Egyptian Cattle during 2019-2020
Isolation and Genetic Characterization of Hobi- Like Pestivirus Circulating in Egyptian Cattle during 2019-2020
Zeinab R. Aboezz1*, Ayman S. El-Habbaa1, Rania S. El-Mohamady2, Samia A. Elnagar2, Ehab M. El-Nahas1
1Virology Department, Faculty of Veterinary Medicine, Benha University, Moshtohor 13736, Kalubia, Egypt; 2Viral Diseases Research Unit, Animal Reproduction Research Institute (ARRI), Agricultural Research Center (ARC), Giza, Egypt.
Abstract | HoBi-like Pestivirus is accused in similar clinical manifestations to those of bovine viral diarrhea virus infections. Although, HoBi-like Pestivirus infection has been recorded in multiple countries as in South America, Europe, and Asia, no clinical cases were recorded in Egypt even now. Here, for the first time, we reported natural infection of HoBi-like Pestivirus in Egyptian cattle. Serum and ovarian samples were collected from cattle during the year of 2019. RT-PCR, viral isolation, sequencing, and phylogeny revealed that the primary causative agent was HoBi-like Pestivirus. Our HoBi-like Pestivirus strain, Egypt-020-1ncp, BVDV-Egypt-020-3ncp and BVDV-Egypt-020-2ncp, shared 99.6% homology with BVD3- B1-AU, 99.2% with BVD3- B5-3-MX and BVD3-G2-BR, 98% with BVD3- Hobi-like virus SA-2016-04 and 95% with BVD3- Italy-68-13ncp isolated in Australia, Mexico, Brazil, South America, and Italy. Multiple area of mutations a long 5′ UTR amplicon, were observed by alignment of BVDV- Egypt-020-1ncp BVDV-Egypt-020-3ncp and BVDV-Egypt-020-2ncp.While Npro based detection was not succeeded to detect our HoBi-like Pestivirus in original samples or even in viral isolate despite their ability to amplify the BVDV RNA of reference strain (NDAL). Precisely, this study provides the first evidence of HoBi-like pestivirus infection in Egypt, raising prospective threat to Egyptian cattle industry.
Keywords | HoBi-like Pestivirus, Cattle, Phylogenetic analysis, 5`UTR
Received | April 06, 2022; Accepted | May 21, 2022; Published | July 14, 2022
*Correspondence | Zeinab Aboezz, Virology Department, Faculty of Veterinary Medicine, Benha University, Moshtohor 13736, Kalubia, Egypt; Email: email@example.com
Citation | Aboezz ZR, El-Habbaa AS, El-Mohamady RS, Elnagar SA, El-Nahas EM (2022). Isolation and genetic characterization of hobi- like Pestivirus circulating in Egyptian cattle during 2019-2020. Adv. Anim. Vet. Sci. 10(8):1725-1730.
ISSN (Online) | 2307-8316
Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.
Bovine pestiviruses infection might result in various clinical manifestation extending from mild to severe clinical signs, as reproductive, gastroenteritis and immune disorder and respiratory disease incriminated grave economic concerns in cattle industry. Currently joined, HoBi-like Pestivirus that known as BVDV-3 or atypical bovine Pestivirus. HoBi-like Pestivirus (HoBiPeV) causes similar clinical signs to the other BVDV in cattle ().
Bovine Pestiviruses belong to family Flaviviridae, genus Pestivirus. The official classification has been comprised Pestivirus A, Pestivirus B and Pestivirus H, that include bovine viral diarrhea virus 1 (BVDV-1), bovine viral diarrhea virus 2 (BVDV-2) and HoBi-like Pestivirus (HoBiPeV) (). Pestivirus is a small, enveloped single-stranded positive sense RNA virus. The viral genome is approximately 12.3 kb with single open reading frame (ORF) flanked by 5′UTR and 3′UTR (). ORF encodes a polyprotein (NH2-Npro-C-Erns-E1-E2-p7-NS2-3-NS4A-NS4B-NS5A-NS5B-COOH) that is cleaved by viral and cellular proteases, forming viral structural and nonstructural proteins (). The conserved region among Pestiviruses is 5′UTR, that commonly used in classification of viral strains and in phylogenetic analyses, the N-terminal protease (Npro) often used in phylogeny (; ).
HoBi-like Pestivirus was firstly identified in Germany in fetal bovine serum (FBS) batch from Brazil (). Subsequently, the HoBi-like Pestiviruses were excessively reported in commercial FBS batches and cell culture derived from several regions, as South America, Europe and Asia (; ; ; ). Natural infection was recorded firstly in aborted fetuses in Brazil in 2006 (). Subsequently, HoBi-like Pestiviruses were reported in cattle in various districts as Italy, Brazil, Thailand and Bangladesh (; ; ; ) with variation of clinical symptoms including respiratory diseases and reproductive diseases. However, natural infection of HoBi-like Pestivirus in cattle has not been identified in Egypt so far. So, the objective of this study was applied for determination the existence of HoBi-like Pestivirus in Egyptian cattle. Here, for the first time, we reported and identified a HoBi-like Pestivirus infection in cattle herd in Egypt.
Materials and Methods
Ovaries, and serum samples (n= 10) were collected from ten freshly slaughtered heifers and cows from abattoirs in different Egyptian province in the year 2019, with pervious history of reproductive problem as poor conception rate, early embryonic deaths, and abortion. Samples were collected under complete aseptic condition and were kept at -20°C for further RNA extraction and viral isolation.
Nucleic acid extraction and reverse transcriptase polymerase chain reaction (RT- PCR)
Viral genome was extracted from ovarian and serum samples by using QIAamp® Viral RNA mini-Kit (Qiagen, USA) (Cat. No. 52904) according to the manufacturer’s instructions. The extracted RNA was stored at –80°C for Reverse transcription-polymerase chain reaction (RT-PCR) using specific primer set for 5 ’UTR gene, RT-PCR was performed using One-Step RT-PCR Kit (Qiagen) (Cat. No. 210212). The oligonucleotides primer set sequence was F 5’– ATGCCCWTAGTAG GACTAGCA - 3’ (forward primer) R 5’- TCAACTCCATGT GCCATGTAC- 3’ (reverse primer) targeting 288bp sequence (). The reaction conditions were 50°C for 30 min followed by 94°C for 7 min; and 35 cycles of 94°C for 10 min 91 sec, 53°C for 30 sec, 68 °C for 30 sec; and final elongation at 68°C for 7 min. The PCR products were analyzed using 1% agarose gel. Moreover, using of Npro primer set for RT-PCR application on the clinical samples and viral isolate. The used oligonucleotides Npro primer set sequence was F 5’– TGCTACTAAAAATCTCTGCTGT - 3’ (forward primer) R 5’- CCATCTATRCAYACATARATGTGGT- 3’ (reverse primer) targeting 441bp (). The RT-PCR reactions were applied under the following conditions 50°C for 30 min followed by 94°C for 7 min; and 35 cycles of 94°C for 10 min, 53°C for 30 sec, 68 °C for 30 sec; and final elongation at 68°C for 7 min. then the PCR products were analyzed on 1% agarose gel.
Sequencing and phylogenetic analysis of 5 ’UTR amplicon of suspected viral sample and BVD Isolate RT-PCR product
The amplified PCR products were purified using a commercial purification kit (QIAGEN (QIAGEN, Valencia, USA) (Cat. No. 28706X4) according to the manufacturer’s protocol. These products were sequenced, using big dye chain terminator V3.1 cycle sequencing kit (Perkin-Elmer, Foster city, CA, USA) (Cat. No. 4337454), and analyzed, and their homology to other BVD viruses was determined based on published sequence information. This analysis was performed by using computer software as MEGA X ().
Trials for viral isolation
Madin-Darby bovine kidney (MDBK) cells were used for isolation of bovine pesti like virus. and reference NADL strain of BVD was used as a control positive. The RNA was extracted from HoBi-like virus third passage isolates and subjected to RT-PCR and sequencing as described above.
Identification of the BVD isolated virus using indirect IFA
The inoculated cells with ovarian homogenate, and serum from suspected viral samples were detected by IFT proved by immune fluorescence (IFX) staining using BVDV polyclonal antiserum that provided by the Department of virology, Animal Health Research Institute, Dokki, Giza, and Rabbit Antibovine-IgG conjugated with fluorescienisothiocyanate that supplied by Sigma (Cat. No. B8395) ().
Nucleotide accession numbers
Results and Discussion
Detection and identification of BVDV in ovary, and serum samples in cattle
In the following study 10 suspected ovarian, and serum samples were examined for existence of BVDV by one step RT-PCR assay for BVDV 5’ UTR and resulted in a positive PCR amplicon with size of 288 bp on 1% agarose gel in 7 samples out of 10 samples. While the RT-PCR assay failed to get any PCR product with clinical samples using Npro primer set in spite their efficient work with the control BVDV strain (NDAL) with product size 441bp.
BVDV was isolated from RT-PCR positive clinical samples, and they were identified by immunofluorescence, RT-PCR, and sequence analysis. As the indirect immunofluorescence assay revealed presence of a non-cytopathogenic strain of BVDV isolates. The sequence of viral isolate was designated as BVDV-Egypt-020-2ncp and deposited in gene bank under the following nucleotide sequence accession number MZ873106.
Sequence analysis of BVDV 5’ UTR amplicon
A blast search revealed that the sequence of the PCR amplified 5’ UTR fragments were related to BVDV-3. The sequences of original sample from serum and ovary were designated as BVDV-Egypt-020-1ncp and BVDV-Egypt-020-3ncp and deposited in gene bank under the following accession numbers MZ873104, MZ873105 respectively. The nucleotide sequences showed that BVDV-Egypt-020-1ncp (Accession. no, MZ873104) and BVDV-Egypt- 020-3ncp (Accession. no, MZ873105) shared identities 96.2% with each other; BVDV-Egypt-020-1ncp shared identities 99.6% with BVD3- B1-AU (Accession. no, JN967707), 99.2% with BVD3- B5-3-MX and BVD3-G2-BR (Accession. no, JN967747, JN967729); 98% with BVD3- Hobi-like virus SA-2016-04 (Accession. no, KY091655) and 95% with BVD3- Italy-68-13ncp (Accession. no, KJ627179). Meanwhile BVDV-Egypt-020-3ncp (Accession. no, MZ873105) shared nucleotide identities 99.6% with BVD3- B1-AU, 99.2% with BVD3-B5- 3-MX and BVD3-G2-BR; 98% with BVD3- Hobi-like virus SA-2016-04 (Accession. no, KY091655) and 96.2% with BVD3- Italy-68-13ncp.
The dendrogram was generated to determine the phylogenetic position of BVDV- Egypt-020-1ncp and BVDV-Egypt-020-3ncp among BVDV-1, BVDV-2 and BVDV- 3 strains (). BVDV-Egypt-020-1ncp and BVDV-Egypt-020-3ncp isolate was related to BVDV-3 strains of Australian, North and South America and Italian strains, respectively.
Rate of 5` UTR sequence diversion after isolation
BVDV-Egypt-020-1ncp and BVDV-Egypt-020-2ncp were 5 `UTR BVDV sequence from serum original sample and the third passage of serum on MDBK cells, respectively. The nucleotide analysis of 5’ UTR products of BVDV-Egypt-020-1ncp and BVDV-Egypt-020-2ncp revealed slight nucleotide variations at three sites of 5UTR products including sites 31, 32 and 96 where G nucleotide in BVDV-Egypt- 020-1ncp replaced by C, A, A in BVDV-Egypt-020-2ncp at this site, respectively .
Also, nucleotide variations were traced between BVDV-Egypt-020-1ncp (BVDV original serum sample) and BVDV-Egypt-020-3ncp (BVDV original ovary sample). The nucleotide analysis of 5` UTR products revealed nucleotide variations in the sites from 27 to 40 where ATCTGGGCTCGTGT in BVDV-Egypt-020-1ncp replaced by GATGTCAGCTAGCA in BVDV-Egypt-020-3ncp, also there is variation at the site 42 where A in BVDV-Egypt-020-1ncp replaced by T in BVDV-Egypt-020-3ncp .
HoBi-like Pestivirus was detected in cell cultures, commercial FBS, small ruminants, and cattle in several countries. It was accused in several clinical manifestations smellier to BVDV in South American, Europe and Asia. Although no available data about existence of HoBi-like Pestivirus infection in Egypt so far. We firstly recorded clinical cases with HoBi-like Pestivirus infection in cattle in Egypt. The 5′ UTR is frequently used for classification of Pestivirus strains. Interestingly, the newly isolated Pestivirus was non cytopathic strain of HoBi-like Pestivirus. It is known that Cytopathic and noncytopathic are equally able to produce severe disease in cattle, moreover, only non-cytopathic stain is produced persistent infection ().
This Egyptian HoBi-like pestivirus had a great homology in 5`UTR region with the HoBi-like pestivirus strains from Italy, Australia, Mexico, Brazil, and South America. It is possible that this HoBi-like pestivirus in Egypt was introduced from other regions through cattle imports and trading of contaminated biological products (). Further, the 5` UTR slight nucleotide variations between BVDV-3 Egyptian isolate and original BVDV-3 virus as shown in ( and ). These clarify the genetic diversity by the third passage of BVDV-3 Egyptian strain on MDBK cells as a characteristic for Pestiviruses genus (; ). Our suggestion is that the nucleotide substitutions in 5` UTR may differentiate between the passaged and un passaged virus as well as the viral tropism. Similarly, like () who distinguishing between eight isolates of low and high virulence BVDV-2. A cytosine at position 219 and an uracil at position 278 were present in the low virulence isolates, while the opposite was observed in the high virulence isolates.
Moreover; the failure of Npro primer set to amplify target sequence with our HoBi-like Pestivirus samples and isolate in spite of their work with control NDAL BVDV. This might be attributed to genetic diversity of our HoBi-like Pestivirus that might incorporate with mismatching of Npro primer sequences and viral nucleotide as described in previous studies So, HoBi-like viruses detection and identification required using HoBi-like primers.it is prospective like other pestivirus membres genetically diverse (; ).
In summary, our study described the first clinical cattle case of natural HoBi-like Pestivirus infection in Egypt. It is worthy to note that the epidemiological status and virus variation of the HoBi-like Pestivirus were still largely unclear in most countries (). In addition, there is no vaccine available to specifically control the infection of HoBi-like Pestivirus. Thus, the emerging HoBi-like Pestivirus could be a considerable threat to the cattle industry in Egypt and worldwide.
The authors would like to thank Viral Diseases Research Unit, Animal Reproduction Research Institute (ARRI), Agricultural Research Center (ARC), Giza, Egypt for providing facilities for conducting this study.
The existence of HoBi-like pestivirus was not recorded absolutely as natural infection in Egypt so far; till our study identified and reported HoBi-like pestivirus as natural infection in cattle herd.
Conceptualization of this study were performed by E.M. El-Nahas, A.S. El-Habbaa, and Z.R. Aboezz. Investigation and methodology were performed by S.A. Elnagar, E.M. El-Nahas, and R.S El-Mohamady. Data curation and phylogenetic analysis were performed by A.S. El-Habbaa and Z.R. Aboezz. The first draft of manuscript was written by Z.R. Aboezz and S.A. Elnagar. Reviewed and editing of the manuscript were performed by E.M. El-Nahas, A.S. El-Habbaa and R.S El-Mohamady. All authors have read and agreed to the published version of the manuscript.
Conflict of interest
The authors have declared no conflict of interest.
Bachofen C, Stalder H, Braun U, Hilbe M, Ehrensperger F, Peterhans E (2008). Co-existence of genetically and antigenically diverse bovine viral diarrhoea viruses in an endemic situation. Vet. Microbiol., 131(1): 93–102.
Bauermann FV, Falkenberg SM, Decaro N, Flores EF, Ridpath JF (2015). Experimental infection of calves, sheep, goats and pigs with HoBi-like viruses by direct inoculation or exposure to persistently infected calves. Vet. Microbiol., 181(3–4): 289–293.
Bauermann FV, Flores EF, Falkenberg SM, Weiblen R, Ridpath JF (2014). Lack of evidence for the presence of emerging HoBi-like viruses in North American fetal bovine serum lots. J. Vet. Diagn. Invest., 26(1): 10–17.
Cortez A, Heinemann MB, de Castro AMMG, Soares RM, Pinto AMV, Alfieri AA, Flores EF, Leite RC, and Richtzenhain LJ (2006). Genetic characterization of Brazilian bovine viral diarrhea virus isolates by partial nucleotide sequencing of the 5’-UTR region. Pesqui. Vet. Bras., 26(4): 211–216.
Decaro N, Lucente MS, Losurdo M, Larocca V, Elia G, Occhiogrosso L, Marino PA, Cirone F, Buonavoglia C (2016). HoBi-like pestivirus and its impact on cattle productivity. Transb. Emerg. Dis., 63(5): 469–473.
Decaro N, Sciarretta R, Lucente MS, Mari V, Amorisco F, Colaianni ML, Cordioli P, Parisi A, Lelli R, Buonavoglia C (2012). A nested PCR approach for unambiguous typing of pestiviruses infecting cattle. Mol. Cell. Probes, 26(1): 42–46.
Fulton RW, Ridpath JF, Confer AW, Saliki JT, Burge LJ, Payton ME (2003). Bovine viral diarrhoea virus antigenic diversity: Impact on disease and vaccination programmes. Biol. J. Int. Assoc. Biol. Stand., 31(2): 89–95.
Giammarioli M, Ridpath JF, Rossi E, Bazzucchi M, Casciari C, De Mia GM (2015). Genetic detection and characterization of emerging HoBi-like viruses in archival foetal bovine serum batches. Biologicals, 43(4): 220–224.
Haider N, Rahman MS, Khan SU, Mikolon A, Gurley ES, Osmani MG, Shanta IS, Paul SK, Macfarlane-Berry L, Islam A (2014). Identification and epidemiology of a rare HoBi-like pestivirus strain in Bangladesh. Transbound. Emerg. Dis., 61(3): 193–198.
Lindenbach BD, Prágai BM, Montserret R, Beran RKF, Pyle AM, Penin F, Rice CM (2007). The C terminus of hepatitis C virus NS4A encodes an electrostatic switch that regulates NS5A hyperphosphorylation and viral replication. J. Virol., 81(17): 8905–8918.
Magar R, Minocha HC, Montpetit C, Carman PS, Lecomte J (1988). Typing of cytopathic and noncytopathic bovine viral diarrhea virus reference and Canadian field strains using a neutralizing monoclonal antibody. Can. J. Vet. Res., 52(1): 42.
Ridpath JF, Bendfeldt S, Neill JD, Liebler-Tenorio E (2006). Lymphocytopathogenic activity in vitro correlates with high virulence in vivo for BVDV type 2 strains: Criteria for a third biotype of BVDV. Virus Res., 118(1): 62–69.
Schirrmeier H, Strebelow G, Depner K, Hoffmann B, Beer M (2004). Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species. J. Gen. Virol., 85(12): 3647–3652.
Smith DB, Meyers G, Bukh J, Gould EA, Monath T, Scott Muerhoff A, Pletnev A, Rico-Hesse R, Stapleton JT, Simmonds P, Becher P (2017). Proposed revision to the taxonomy of the genus Pestivirus, family Flaviviridae. J. Gen. Virol., 98(8): 2106–2112.
Stalder HP, Meier PH, Pfaffen G, Wageck-Canal C, Rüfenacht J, Schaller P, Bachofen C, Marti S, Vogt HR, Peterhans E (2005). Genetic heterogeneity of pestiviruses of ruminants in Switzerland. Prev. Vet. Med., 72(1–2): 37–41.
Vilček Š, Herring AJ, Herring JA, Nettleton PF, Lowings JP, Paton DJ (1994). Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch. Virol., 136(3): 309–323.
Weber MN, Mósena ACS, Simões SVD, Almeida LL, Pessoa CRM, Budaszewski RF, Silva TR, Ridpath JF, Riet-Correa F, Driemeier D (2016). Clinical presentation resembling mucosal disease associated with ‘HoBi’-like pestivirus in a field outbreak. Transbound. Emerg. Dis., 63(1): 92–100.
To share on other social networks, click on any share button. What are these?