Genetic and Antigenic Characterization of Bungowannah Virus, a Novel Pestivirus

Home , Pestivirus

Veterinary Microbiology 178 (2015) 252–259

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Short communication

Genetic and antigenic characterization of Bungowannah virus, a novel pestivirus

a, a a a a a

P.D. Kirkland *, M.J. Frost , K.R. King , D.S. Finlaison , C.L. Hornitzky , X. Gu ,

b b c b b d

M. Richter , I. Reimann , M. Dauber , H. Schirrmeier , M. Beer , J.F. Ridpath

Virology Laboratory, Elizabeth Macarthur Agriculture Institute, Woodbridge Rd, Menangle, New South Wales 2568, Australia

Institute of Diagnostic Virology, Friedrich-Loefﬂer-Institut, 17493 Greifswald-Insel Riems, Germany

Department of Experimental Animal Facilities and Biorisk Management at the Friedrich-Loefﬂer-Institut, 17493 Greifswald-Insel Riems, Germany

National Animal Disease Centre, 2300 Dayton Avenue, Ames, IA, 50010, United States

A R T I C L E I N F O A B S T R A C T

Article history: Bungowannah virus, a possible new species within the genus Pestivirus, has been associated with a

Received 20 April 2014

disease syndrome in pigs characterized by myocarditis with a high incidence of stillbirths. The current

Received in revised form 8 May 2015

analysis of the whole-genome and antigenic properties of this virus conﬁrms its unique identity, and

Accepted 21 May 2015

further suggests that this virus is both genetically and antigenically remote from previously recognized

pestiviruses. There was no evidence of reactivity with monoclonal antibodies (mAbs) that are generally

Keywords:

considered to be pan-reactive with other viruses in the genus, and there was little cross reactivity with

Pestivirus

polyclonal sera. Subsequently, a set of novel mAbs has been generated which allow detection of

Bungowannah virus

Bungowannah virus. The combined data provide convincing evidence that Bungowannah virus is a

Characterisation

member of the genus Pestivirus and should be ofﬁcially recognized as a novel virus species.

Full-length genome sequence

Monoclonal antibodies

1. Introduction myonecrosis and was named Bungowannah virus (Kirkland

et al., 2007). Subsequent ﬁeld and experimental transmission

The Pestivirus genus of the family Flaviviridae includes four studies conﬁrmed the role of this virus as a potent fetal pathogen

recognized species – bovine viral diarrhea virus type 1 and 2 (Finlaison et al., 2009; Finlaison et al., 2010).

(BVDV-1 and -2), border disease virus (BDV) and classical swine Initial phylogenetic analysis based on comparison of 5 non-

0 pro

fever virus (CSFV; King et al., 2012). Four further putative species translated region (5 NTR), and the N and E2 protein coding

have been proposed: Giraffe virus, Pronghorn virus, a group of regions found it to be only distantly related to the 4 recognized

viruses variously referred to as HoBi-like or BVDV-3, and pestivirus species or any of the other putative pestivirus species

Bungowannah virus, which is the subject of the current study (Kirkland et al., 2007). Selected pestivirus monoclonal antibodies

(Kirkland et al., 2007; Plowright, 1969; Schirrmeier et al., 2004; (mAbs), previously reported to be pan-reactive, did not detect

Vilcek et al., 2005). In June 2003, in New South Wales, Australia, an Bungowannah virus antigens in infected cell cultures.

outbreak of disease was reported, initially presenting as sudden In the current study we have generated and compared the

death in weaner age pigs and later as a marked increase in the birth whole-genome sequence with that of recognized and putative

of stillborn foetuses and elevated preweaning mortalities (McOrist Pestivirus species. In addition, antigenic differences between

et al., 2004). A novel pestivirus was identiﬁed in association with a Bungowannah virus and other pestiviruses have been examined

multifocal non-suppurative myocarditis sometimes with using newly generated mAbs. Collectively these data provide

convincing evidence to consider this virus as a new species within

the genus Pestivirus.

* Corresponding author. Tel.: +61 2 4640 6331; fax: +61 2 4640 6429.

E-mail addresses: [email protected] (P.D. Kirkland), 2. Material and methods

[email protected] (M.J. Frost), [email protected]

(K.R. King), deborah.ﬁ[email protected] (D.S. Finlaison),

2.1. Cell cultures

[email protected] (C.L. Hornitzky), maria.richter@ﬂi.bund.de

(M. Richter), ilona.reimann@ﬂi.bund.de (I. Reimann), malte.dauber@ﬂi.bund.de

For initial studies, Bungowannah virus was grown in BVDV-free

(M. Dauber), horst.schirrmeier@ﬂi.bund.de (H. Schirrmeier),

martin.beer@ﬂi.bund.de (M. Beer), [email protected] (J.F. Ridpath). PK-15 cells (RIE5-1, Collection of Cell Lines in Veterinary Medicine

http://dx.doi.org/10.1016/j.vetmic.2015.05.014

P.D. Kirkland et al. / Veterinary Microbiology 178 (2015) 252–259 253

(CCLV), Friedrich-Loefﬂer-Institut, Insel Riems, Germany). BVDV (MagMAX -96 Viral RNA Isolation Kit AM1836, Ambion, Austin,

strains were grown in secondary bovine testis (BT) cells, while BDV Texas) in accordance with the manufacturer’s instructions. The

was grown in primary lamb testis (LT) cells as described previously magnetic beads were handled, washed and the nucleic acid eluted

(Kirkland et al., 2007). Later, for immunoﬂuorescence (IF) studies using a magnetic particle handling system (KingFisher 96, Thermo

involving chimeric viruses, diploid bovine oesophageal KOP-R cells Fisher Scientiﬁc, Finland). RNA was stored frozen at 80 C until

(RIE244, CCLV) and porcine SK-6 cells (RIE262, CCLV) were grown use.

in Dulbecco’s Modiﬁed Eagle’s Medium (DMEM) with 10% BVDV-

free fetal calf serum (FCS). 2.5. Sequencing of the complete viral genome

2.2. Viruses In order to complete the Bungowannah virus sequence,

rns

fragments containing DNA coding for E to p7, p7 to NS3,

Bungowannah virus grown in PK-15 cells was used at the third NS3 to NS5A and NS5A to NS5B were ampliﬁed as described

passage after isolation from tissues of affected pigs. Viruses previously (Kirkland et al., 2007), using the primers listed in

selected to represent the recognized pestivirus species were: Supplementary Table 1. Sequence data from the 3 NTR was

BVDV-1 (strain NADL or CP7), BVDV-2 (890), BDV (X818), and CSFV generated by adding a poly (A) tail to the viral RNA, using the

(NSW 1966). For testing a panel of Bungowannah virus-speciﬁc A-Plus Poly (A) Polymerase Tailing Kit (EPICENTRE, Madison,

mAbs the primary Bungowannah virus isolate was propagated two Wisconsin, U.S.A.) followed by rapid ampliﬁcation of cDNA ends

more times in SK-6 cells before use. A chimeric virus (vCP7_E1E2- (RACE, BD), as described by BD Biosciences (CLONTECH Laborato-

rns

Bungo) and a bicistronic virus (vCP7_E -Bungo_bi) were also ries, California, U.S.A.) with the following modiﬁcations. Hot start

included to test the protein speciﬁcity of the Bungowannah virus- PCR (Qiagen, Hilden, Germany) was carried out with primers

speciﬁc mAbs (see below and also Richter et al., 2011). ER62F and BD Universal primer A mix with an annealing

temperature of 65 C and an extension time of 2 min. The

2.3. Construction of the chimeric full-length cDNA clone pA/CP7_C- Bungowannah virus-speciﬁc primer NS5BF and BD nested Univer-

Bungo sal Primer A were used for the Hot start nested PCR, with an

annealing temperature of 65 C and an extension time of 2 min. All

The genomic region encoding the Bungowannah virus struc- PCR-products were sequenced as previously described (Kirkland

tural C protein was ampliﬁed from a synthetic open reading frame et al., 2007).

(ORF) Bungo_C-E2mod_pMK-RQ (Geneart AG, Regensburg,

Germany). Subsequently, the puriﬁed amplicon was used as a 2.6. Sequence analysis

megaprimer in a fusion PCR with BVDV CP7 as template (Geiser

et al., 2001; Richter et al., 2011; Stech et al., 2008). Primers and The Bungowannah virus genome sequence was assembled

details for generation of the recombinant construct are available using the Sequencher 4.10.1 software (Gene Codes, Michigan).

upon request. In vitro-transcription of the mutant pA/CP7_E1- Different segments of the genome were compared using sequences

Bungo and RNA transfection by electroporation was performed as obtained from NCBI GenBank.

described previously (Richter et al., 2011). Analyses of these sequences were carried out using MEGA

version 4 (Tamura et al., 2007). Multiple alignments were

2.4. Extraction of viral genome completed using Clustal W. Dendrograms were generated using

the neighbor-joining method. Percentage of nucleotide and amino

RNA was extracted from the supernatant of Bungowannah acid identity between the viral sequences was determined using

virus-infected PK-15 cells by using a magnetic bead based system the DNADist and ProtDist programs, respectively. Statistical

Table 1a

Length of genome, polyprotein, and NTRs of different pestivirus strains.

a a 0 0

Species Strain GenBank accession Genome length Polyprotein (amino 5 NTR 3 NTR References no (nucleotides) acids) (nt) (nt)

BVDV-1 NADL M31182 (12,308) (3898) 385 226 Collett et al., 1988

SD-1 M96751 12,308 3898 385 226 Deng and Brock, 1992

C24V AF091605 12,310 3898 385 228

Osloss M96687 (12,266) (3899) 381 185 De Moerlooze et al., 1993

CP7 AF220247 (12,267) (3898) 382 188 Becher et al., 2000

BVDV-2 890 U18059 (12,285) (3897) 385 206 Ridpath and Bolin, 1995

CSFV Alfort J04358 12,297 3898 372 228 Meyers et al., 1989

Brescia M31768 12,295 3898 372 226 Moormann et al., 1990

C-strain Z46258 12,311 3898 373 241 Moormann et al., 1996

BDV X818 AF037405 12,333 3895 372 273 Becher et al., 1998

Reindeer-1 V60-Krefeld AF144618 12,318 3895 370 260 Avalos-Ramirez et al.,

2001

Giraffe-1 H138 AF144617 (12,308) (3891) 382 250 Avalos-Ramirez et al., 2001

Pronghorn Pronghorn AY781152.3 12,273 3898 369 210 Neill et al., 2014

All Species Summary 12,266-12,333 3891-3898 369-385 185-273

Bungowannah Prototype NC_023176 12,649 3918 391 504 This study

virus B869

Table adapted from Avalos-Ramirez et al. (2001).

Numbers in parentheses indicate the length of the genome or polyprotein without inserted cellular sequences (BVDV-1 NADL, BVDV-1 Osloss, Giraffe-1) or without

inserted viral sequences (BVDV-1CP7, BVDV-2 890).

b 0 0

Including sequence deposited in GenBank and additional nt at the 5 and/or 3 terminus reported by Brock et al. (1992) (BVDV-1 NADL), and Moormann et al. (1996)

(BVDV-1 Osloss, CSFV Brescia).

254 P.D. Kirkland et al. / Veterinary Microbiology 178 (2015) 252–259

Fig. 1. Dendrogram showing the comparison of the genetic diversity of Bungowannah virus with representative pestiviruses: (A) using the deduced amino acid sequence for

the NS3 protein, (B) using the full-length nucleotide sequence.

(HM195188)

Branch lengths are proportional to genetic distances. Numbers indicate the percentage of 1000 bootstrap replicates that support each labelled interior branch.

reliability of the dendrogram and percentage identity tables were BVDV-1: BVDV 22,146, BVDV NADL.

determined using bootstrapping at 1000 times. BVDV-2: Giessen.

BDV: Frijters, Moredun, 137/4, Rudolph, Stolpe, Chemnitz,

2.7. Monoclonal antibodies and polyclonal sera Gifhorn.

CSFV: CSF0650 (#4409), CSF0573, CSF0140 (Diepholz), CSF0634

A broad panel of mAbs and polyclonal antisera were tested for (Vi3837/38), CSF0277 (Paderborn), CSF0902 (Alfort), CSF0573.

their ability to react with Bungowannah virus antigens. The mAbs Putative pestivirus species: Giraffe, HoBi, Pronghorn.

that were used included the broadly reactive antibodies 20.10.6, MAbs detecting Bungowannah virus were generated as

2NB2, P1H11, P3C12, P4A11, P4G11, WB162, WS538; the BVDV- described previously (Dauber, 1999; Jäckel et al., 2013). Numerous

reactive 15c5, 1NB5, N2B12, P1D8, PX 14/2, WS363, WS433; the clones secreting Bungowannah virus-speciﬁc antibodies were

BVDV/BDV-reactive C16/1/2, P3D10, P3F6, WB103, WB105, WB210, obtained and 19 mAbs were selected for further study with wild

rns

WB215; the BVDV/CSFV-reactive CF10 and the CSFV-speciﬁc type Bungowannah virus and the Capsid, E , E1 and

WH211 and WH303. The selected polyclonal sera and the virus E1E2 chimeras. In order to assess their protein speciﬁcity, the

used to immunize the donor animals were: new mAbs generated were subjected to IF analyses using infected

P.D. Kirkland et al. / Veterinary Microbiology 178 (2015) 252–259 255

Table 1b

Comparative sizes of different genomic regions (nucleotides) of representative pestiviruses.

pro rns

Species Strain N C E E1 E2 p7 NS2 NS3 NS4A NS4B NS5A NS5B

(nt) (nt) (nt) (nt) (nt) (nt) (nt) (nt) (nt) (nt) (nt) (nt)

BVDV-1 NADL cp 504 306 681 585 972 210 1662 2049 192 1041 1488 2157

SD-1 504 306 678 585 1122 210 1356 2049 192 1041 1488 2157

C24V 504 306 681 585 1122 210 1359 2049 192 1041 1491 2157

Osloss 504 306 681 585 1125 210 1587 1896 189 1041 1488 2157

CP7 504 306 681 585 1122 210 1386 2049 192 1041 1488 2157

BVDV-2 890 504 303 681 585 1116 210 1587 2049 192 1041 1491 2157

CSFV Alfort 504 294 681 585 1119 210 1371 2049 192 1041 1491 2157

Brescia 504 297 681 585 1119 210 1371 2049 192 1041 1491 2157

C-strain 504 297 681 585 1119 210 1371 2049 192 1041 1491 2157

BDV X818 504 294 681 585 1119 210 1359 2049 192 1041 1491 2157

Reindeer V60 Krefeld 504 294 681 585 1122 210 1641 2049 192 1041 1491 2157

Giraffe H138 504 300 681 585 1119 210 1359 2049 192 1041 1491 2157

Pronghorn Prototype 504 291 681 585 1110 228 1374 2034 189 1044 1500 2154

Bungowannah virus Prototype 531 297 666 588 1131 216 1380 2049 189 1041 1515 2151

Cp cytopathic virus (note that p7/NS2 cleavage site is an estimate based on NADL accession number (NP_776265).

or transfected bovine KOP-R cells. These cell cultures had been further typing. Transfection or infection was detected by IF staining

inoculated with parental BVDV CP7, wild type Bungowannah virus, at three days post-transfection (p.t.) or post-infection (p.i.) using

chimeric viruses or transfected with RNA of the chimeric construct either the Bungowannah virus-speciﬁc mAbs or the pestivirus

pA/CP7_E1-Bungo. To establish which Bungowannah virus struc- NS3-speciﬁc mAb C16 (Institute of Virology, Tierärztliche Hoch-

tural proteins could be detected by the respective mAbs, four schule Hannover, Germany). Transfected cells were ﬁxed as

different chimeras, which either expressed the C protein (vCP7_C- described by Richter et al. (2014a). Infected KOP-R cells were

Bungo), the envelope protein Erns (vCP7_Erns-Bungo_bi) or the heat-ﬁxed for 2 h at 80 C followed by reaction with the antibodies

glycoprotein E1 alone (CP7_E1-Bungo) or E1 and E2 of Bungo- of interest. As secondary antibody a goat anti-mouse mAb

wannah virus in combination (vCP7_E1E2-Bungo), were used. (Invitrogen) was used.

2.8. Peroxidase-linked assay and immunoﬂuorescence analysis

2.9. Peroxidase-linked virus neutralization assay

The reactivity of Bungowannah virus antigens was determined

The polyclonal sera were also tested for their neutralising

using a peroxidase-linked assay or IF staining. The appropriate cell

activity against Bungowannah virus, BVDV-1 (NADL), BVDV-2

cultures in 96-well microplates were inoculated with approxi-

(890) and BDV (X818) in a standard neutralisation test with

mately 100 TCID50 of virus per well at seeding of the cells

100 TCID50 as described previously (Kirkland and Mackintosh,

(Bungowannah virus, BVDV-1 and BVDV-2) or after 24 h (BDV).

2006). For the detection of Bungowannah virus-infected cell

Alternate rows in each microplate remained uninoculated to

cultures, a polyclonal serum from a pig infected with Bungowan-

provide a negative control for each serum. PK-15 cells were ﬁxed in

nah virus was used (1/200 in PBS containing 1% gelatin).

situ as previously described (Kirkland et al., 2007), while bovine

and ovine cells for BVDV and BDV strains, respectively, were ﬁxed

as described by Kirkland and MacKintosh (2006). For comparisons 3. Results and discussion

involving CSFV, infected and ﬁxed PK-15 cells were supplied by the

Australian Animal Health Laboratory (AAHL), Geelong. Immuno- 3.1. Sequence analysis and comparison to other pestivirus genomes

peroxidase staining was completed as described by Kirkland and

MacKintosh (2006). The Bungowannah virus genome is approximately

– ﬁ

For IF analysis KOP-R cells were seeded in 48-well culture plates 300 400 nucleotides longer than has been identi ed for other

and 24 h later cells were inoculated with wild type Bungowannah pestiviruses (Avalos-Ramirez et al., 2001; Becher et al., 1998;

virus at a multiplicity of infection (MOI) of 0.1 to 1.0, or transfected/ Schirrmeier et al., 2004; Vilcek et al., 2005). Despite this, the

rns

infected with the recombinant Capsid, E , E1 or E1E2 chimera for deduced amino acid sequence for the polyprotein indicates that

Fig. 2. Alignment of the highly conserved amino acid region in E2 for selected pestiviruses and Bungowannah virus isolate. The conserved sequence VVRGKFNTTLLNGPAF is

shaded, variations are shown in bold and underlined. Conserved cysteine residues are also shown in bold.

256 P.D. Kirkland et al. / Veterinary Microbiology 178 (2015) 252–259

Fig. 3. 3 NTR alignment of viruses representing the recognized pestivirus species compared to Bungowannah virus. Stop codons are shown in bold, shaded nucleotides

represent the conserved region, the variable region is between the stop codon and constant region, AT-rich regions are shown in italics and the boxed area is the most 3

conserved domain in between stem loop I and II.

the open reading frame is similar in length to other members of the Consistent with other noncytopathic pestiviruses no insertions

genus (Table 1a). were detected in the NS2/3 coding region (Avalos-Ramirez et al.,

The comparison of genomic sequences including 5 NTR and the 2001) or in the capsid coding region (Nagai et al., 2003). The

complete open reading frame of pestiviruses representative of the lengths of protein encoding regions are similar to that of other

BVDV-1, BVDV-2, BDV, CSFV and putative giraffe species with pestiviruses, with the exception of a 27 nucleotide insertion at the

pro

Bungowannah virus showed similarities varying from 30.7 to 33.6% start of the N gene (Table 1b). This insertion has not been

(Fig. 1A and B). In contrast, the similarities among these reported in other pestiviruses (Becher et al., 1999, 2003; Richter

representative pestiviruses ranged from 54.8% to 84.7%. et al., 2014a; Vilcek et al., 2005).

P.D. Kirkland et al. / Veterinary Microbiology 178 (2015) 252–259 257

previous reports (Dekker et al., 1995; Edwards et al., 1991;

Shannon et al., 1991). However, there was no reactivity of any of

these mAbs with Bungowannah virus antigens in the PLA. Of note

was the lack of reactivity with the mAbs (e.g. WB103/105, C16,

2NB2, P1H11, P4G11) that had previously been considered to be

pan-reactive with members of the pestivirus genus (Edwards et al.,

1991; Shannon et al., 1991). This appears to be unique for any of the

pestiviruses identiﬁed to date. Although a virus such as the

pronghorn isolate shows considerable genetic diversity from other

viruses, there was still reactivity with some of the mAbs, for

example, pronghorn with 20.10.6 (Vilcek et al., 2005). Similarly,

other viruses that have also been proposed as new species (e.g.

HoBi and giraffe viruses) have been shown to react with a number

of these mAbs (Schirrmeier et al., 2004).

From the panel of polyclonal antisera examined, strong

reactivity with Bungowannah virus antigens was only observed

Fig. 4. Nucleotide alignment of the conserved sequences at the terminus of the in the PLA using homologous sera from pigs that had been infected

5 NTR for selected pestiviruses and Bungowannah virus isolate. The GTATA element

with Bungowannah virus or the high titred antisera produced

is shaded and the Bungowannah virus insertion is shown in bold.

against the BVDV-2 strain Giessen, the CSFV strains 4409, Diepholz

and Paderborn (kindly supplied by Dr. I. Greiser-Wilke, EU

Examination of the deduced amino acid sequence for the

Reference Laboratory for CSFV, Hannover, Germany), and a

immunodominant E2 envelope glycoprotein revealed the presence

hyperimmune antiserum (R969) that had been raised against

of 18 cysteine residues, similar to other pestiviruses, and four potential

seven NSW pestivirus isolates (Shannon et al., 1991).

glycosylation sites. A region of 18 amino acids in the E2 protein that are

highly conserved among all other pestiviruses (Becher et al., 1999), is

ﬁ

not highly conserved in Bungowannah virus (Fig. 2). 3.3. Evaluation of novel Bungowannah virus-speci c monoclonal

Furthermore, there are several distinctive elements in the antibodies

3 NTR, including an insertion of 234 bases, immediately after the

ﬁrst stop codon of the open reading frame (Fig. 3). A blast search of Nineteen different mAbs having reactivity with Bungowannah

rns

ﬁ ﬁ

GenBank did not reveal a signiﬁcant sequence similarity between virus were identi ed. Eighteen mAbs were speci c for E , and one

this insertion and any GenBank sequences. There are more stop for E2. Fig. 5 depicts examples of the patterns of reactivity. In

codons in the Bungowannah virus 3 NTR, located at nucleotides general, there was reactivity of these mAbs with chimeric

12,246, 12,288, 12,318, 12,387 and 12,628. The fourth stop codon constructs that encoded Bungowannah virus sequences but not

aligns with the 3 NTR stop codon, found in the Moredun strain of those with BVDV. As expected, wild type Bungowannah virus could

rns

BDV. The AT-rich regions, containing ATTTA motifs, which are a be detected by mAbs showing reactivity with either E or E2

consistent feature of the pestivirus 3 NTR, are not present in the (Fig. 5F and L).

Bungowannah virus gene sequence. At 51.5% the proportion of A

and T bases in the 3 NTR is relatively low compared to the 60–74%

3.4. Virus neutralization test

observed for other pestiviruses. The function of the AT-rich regions

and ATTTA motifs are yet not known, but it has been suggested that

The results of the cross neutralization study are summarized in

they have a function in the translational regulation or RNA stability

Table 2. As expected, the highest neutralizing antibody titres

(Vilcek et al., 1999) and that their absence or presence may impact

against Bungowannah virus were observed using sera from pigs

on virus infectivity (Xu et al., 2006). However, similar to other

that had been infected with Bungowannah virus. Although limited

pestiviruses (Deng and Brock, 1992), the conserved domain

0 cross neutralization was observed either of Bungowannah virus or

between stem loop I and II of the 3 NTR is present (Fig. 3).

by Bungowannah virus antibody, the titer was low (32 in all

Bungowannah virus has the GTATA element at the start of the

0 cases). Further, this reactivity was only observed with very high

5 NTR (Fig. 4) that is considered necessary for efﬁcient virus

titered antisera. Less cross-reactivity was observed by virus

replication of pestiviruses (Avalos-Ramirez et al., 2001; Becher

neutralization test compared with the results in the PLA. There

et al., 2000; Frolov et al., 1998).

was no virus neutralization activity against Bungowannah virus

Compared to other pestiviruses, the conservation of amino acid

with antisera from the more divergent pronghorn virus, but this

sequences at the cleavage sites in the Bungowannah virus

serum also showed little or no cross reactivity with recognized

polyprotein varies depending on the site. The trends shown by

pestivirus species such as BVDV. Although not included in the

Bungowannah virus follow those described by Vilcek et al. (2005)

current virus neutralization study, during the original diagnostic

for pronghorn virus. There was complete or nearly complete

pro investigations, sera from sows, naturally infected with Bungo-

conservation of sequences for the cleavage sites for N /C,

rns wannah virus, showed no neutralizing activity against CSFV

NS2/NS3, NS3/NS4A, NS4B/NS5A and NS5A/NS5B. For C/E , E2/

(McOrist et al., 2004).

p7 and NS4A/NS4B proteins the cleavage site was conserved on one

From a diagnostic perspective, failure to detect Bungowannah

side only. The greatest variability among recognized pestiviruses

virus using mAbs and PCR primers previously considered to be

and between Bungowannah and other viruses of the genus was in

rns pan-pestivirus reactive allows this virus to elude established

the sequences surrounding the cleavage sites at E /E1, E1/E2 and

diagnostic assays. However, the newly developed Bungowannah

p7/NS2.

virus-speciﬁc antibodies could be used in the future to avoid this

gap. As some aspects of the clinical presentation of Bungowannah

3.2. Antigenic analyses virus infection are consistent with a low-virulence strain of CSFV

(Finlaison et al., 2010, 2012) if conﬁrmatory diagnostic assays are

Generally, the mAbs included in these studies displayed not used, it is likely that infection of pigs with Bungowannah virus

reactivity with reference viruses that were consistent with would remain undetected.

258 P.D. Kirkland et al. / Veterinary Microbiology 178 (2015) 252–259

Fig. 5. IF analysis of KOP-R cells transfected with in vitro-transcribed RNA of the chimeric construct pA/CP7_E1-Bungo or inoculated with supernatants of parental BVDV-1

rns

CP7, wild type Bungowannah virus (BV), the chimeras vCP7_C-Bungo, vCP7_E -Bungo_bi and vCP7_E1E2-Bungo, respectively. At 72 hp.t. or p.i. expression of the appropriate

rns

proteins was detected by IF staining using either Bungowannah virus-speciﬁc mAbs directed against E (682/43C3) or E2 (682/45F12) or the BVDV NS3-speciﬁc mAb C16.

rns rns

MAb 682/43C3 only reacted with the chimera that expresses Bungowannah virus E and BV (D, F). The expression of Bungowannah virus E and E2 was not detected in the

chimera vCP7_C-Bungo (B, H) but infection of the cultures was conﬁrmed by positive staining with the BVDV-speciﬁc mAb C16 (N). Cells infected with BVDV CP7 only stained

with the BVDV speciﬁc mAb C16 (M), and failed to react with the new Bungowannah virus-speciﬁc mAbs (A, G). IF staining clearly demonstrated that the mAb 682/

45F12 detected Bungowannah virus E2 protein (K), whereas the E1 protein could not be stained (I).

Table 2 recently demonstrated that bat cells are fully susceptible to

ﬁ

Virus neutralization assays with mono-speci c polyclonal antisera. Bungowannah virus and therefore, the hypothesis of an ancestor in

a bat populations and later adaptation to pigs also has to be

Antiserum Viruses

considered (Richter et al., 2014b).

Species Laboratory Bungowannah BVDV- BVDV- BDV code 1 2

4. Conclusions

VNT VNT VNT VNT

Bungowannah R2950 1024 – – –

This study provides important data to support the classiﬁcation

virus R3065A 1024 – – 4

R3065B 1024 16 – 16 of Bungowannah virus as a new pestivirus species. Comparisons

– –

R3065C 512 8 with representative viruses from the recognised pestivirus species,

R3065D 512 – – 16

viruses from proposed new species and also recently recognised

BVDV-1a R3394A – 4096 256 1024

viruses, show that the genome is very different and genetically

BVDV-1b R3394B 8 1024 256 256

remote from any of the other species. While major genetic and

BVDV-1c R3394C – 512 32 64

BVDV-2a R3394D – 32 8192 4 antigenic differences exist, Bungowannah virus contains all of the

BDV R3394E – 64 – 512

genomic and structural elements of a classical pestivirus. The data

Pronghorn R3394F – – – 4

presented by Richter and co-workers (2011, 2014a,b) further

Although not tested here, serum samples from Bungowannah virus-infected support this view. Collectively these data readily meet the criteria

pigs were tested in a CSF VNT during diagnostic investigations with negative results. described for the deﬁnition of a pestivirus species (Becher et al.,

1999). Consequently, Bungowannah virus should be ofﬁcially

One of the most intriguing unanswered questions is the origin recognized as the type virus of a new species of the genus

of Bungowannah virus. The genetic disparity with the other Pestivirus. The newly generated Bungowannah virus-speciﬁc mAbs

pestiviruses known to circulate in Australia (BVDV-1 and BDV) and will support the development of diagnostic assays for the detection

the relatively short history of ruminants and pigs in Australia of both Bungowannah virus antigens and antibodies.

suggest that recent evolution from one of these viruses is unlikely.

The emergence of Bungowannah virus from a wild animal

Conﬂict of interest

population in Australia would also seem unlikely. The fauna of

Australia is unique for its relative scarcity of native placental

The authors conﬁrm that there are no known or apparent

animals, and marsupials occupy many of the ecological niches

conﬂicts of interest related to this research or publication. The

ﬁlled by placental animals elsewhere in the world. In fact, all wild

funding agencies played no role in the decision to publish or the

and domestic mammals from the order Artiodactyla have been

preparation of this publication.

present in Australia for less than 250 years. There are no reports of

pestivirus infections in marsupials and surveys of Tasmanian

wildlife have failed to detect antibodies against pestiviruses such Acknowledgements

as BVDV (Munday, 1972). The possibility of the virus being

introduced via a contaminated vaccine cannot be excluded The authors are indebted to the staff of the Virology Laboratory at

(Finlaison et al., 2010). The more efﬁcient replication of EMAI for their technical assistance throughout the course of these

Bungowannah virus in porcine cells suggests a possible porcine studies. We are indebted to Australian Pork Limited and the NSW

origin or adaptation to the porcine host. Nevertheless, it has been DepartmentofPrimaryIndustriesfortheprovisionoffundingfor this

P.D. Kirkland et al. / Veterinary Microbiology 178 (2015) 252–259 259

project. Parts of the project were supported by the European Union King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J., 2012. Virus Taxonomy. Ninth

Report of the International Committee on Taxonomy of Viruses, Part II.

FP7 project European Management Platform for Emerging and Re-

Amsterdam, Elsevier-Academic Press, pp. 1010–1014.

emerging Infectious Disease Entities (EMPERIE no. 223498).

Kirkland P.D. and MacKintosh S.G., 2006. Ruminant Pestivirus Infections. Australian

and New Zealand Standard Diagnostic Procedures for Animal Diseases. Sub-

Committee on Animal Health Laboratory Standards for Animal Health

Appendix A. Supplementary data

Committee, Australia. www.scahls.org.au.

Kirkland, P.D., Frost, M.J., Finlaison, D.S., King, K.R., Ridpath, J.F., Gu, X., 2007.

Supplementary data associated with this article can be Identiﬁcation of a novel virus in pigs – Bungowannah virus: a possible new

species of pestiviruses. Virus Res. 129, 26–34.

found, in the online version, at http://dx.doi.org/10.1016/j.

vetmic.2015.05.014. McOrist, S., Thornton, E., Peake, A., Walker, R., Robson, S., Finlaison, D., Kirkland, P.D.,

Reece, R., Ross, A., Walker, K., Hyatt, A., Morrissy, C., 2004. An infectious

myocarditis syndrome affecting late-term and neonatal piglets. Aust. Vet. J. 82,

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