Mucosal Disease-Like Syndrome in a Calf Persistently Infected byHobi-Like Pestivirus

Nicola Decaro,a Gianvito Lanave,a Maria Stella Lucente,a Viviana Mari,a Katia Varello,b Michele Losurdo,a Vittorio Larocca,a

Elena Bozzetta,b Nicola Cavaliere,c Vito Martella,a Canio Buonavogliaa

Department of Veterinary Medicine, University of Bari Aldo Moro, Bari, Italya; Istituto Zooprofilattico Sperimentale di Piemonte, Liguria e Valle d’Aosta, Turin, Italyb; IstitutoZooprofilattico Sperimentale di Puglia e Basilicata, Foggia, Italyc

A calf persistently infected with Hobi-like pestivirus displayed severe clinical signs and subsequently died. Gross lesions and his-topathological changes were suggestive of hemorrhagic and necrotic inflammation involving several tissues. A Hobi-like pestivi-rus pair was isolated from the dead calf, i.e., cytopathogenic (CP) and noncytopathogenic (NCP) strains strictly related to eachother and to Italian prototype isolates at the genetic level. Two biotype-specific real-time reverse transcription-PCR assays deter-mined the time of the emergence of the CP virus as 1 month before the calf’s death. This highest RNA titers were reached in lym-phoid and nervous system tissues, whereas only traces of CP viral RNA were found in blood. In contrast, great NCP virus loadswere present in all tissues and biological fluids. The present report provides new insights into the pathogenesis and molecularmechanisms of this emerging group of pestiviruses.

Hobi-like pestivirus is a new tentative species of the genus Pes-tivirus, whose prototype, strain D32/00_Hobi, was detected

in a batch of commercial fetal bovine serum of Brazilian origin (1).Hobi-like viruses were subsequently recovered from other com-mercial fetal calf serum batches worldwide but also from serumsamples from naturally infected cattle (2–7). To date, natural in-fections caused by this new bovine pestivirus have been reportedonly sporadically (2, 8), but in most cases they were associatedwith overt disease consisting of respiratory distress (9, 10) or re-productive failure (11, 12). Hobi-like pestiviruses were able toinduce respiratory disease even under experimental conditions(13, 14). Experimental infection of cattle, sheep, and swineshowed that the Hobi-like pestivirus is able to infect all of thosespecies, although only ruminants displayed clinical signs and virusshedding (13). Through immunization studies with ruminants,poor cross-neutralization between extant pestiviruses and Hobi-like isolates was evident, which raised some concerns about theability of currently available vaccines to protect against the newpestivirus species (14–16).

Mucosal disease (MD) is a lethal disease of cattle that is causedby the emergence of a cytopathogenic (CP) pestivirus in an animalpersistently infected (PI) with a noncytopathogenic (NCP) strain.This disease is usually characterized by hemorrhagic, necrotic, andulcerative lesions (17, 18).

Although a Hobi virus CP-NCP pair was isolated from a heiferthat died of respiratory distress (10) and Hobi virus persistentinfection was recently reported (19), no clear evidence of MD hasbeen obtained so far. In this note, we report the clinical, immuno-logical, postmortem, and histopathological findings of MD-likesyndrome naturally occurring in a Hobi-like virus PI calf.

MATERIALS AND METHODSClinical case. The disease involved a calf PI with Hobi-like pestivirus thathad been monitored at the Infectious Diseases Unit of the Animal Hospi-tal of the University of Bari (Italy) since March 2012 (19). The PI calfdisplayed recurrent clinical signs of respiratory disease, enteritis, and der-matomycosis due to concurrent pathogens likely as a consequence of pes-tivirus-induced immunosuppression. In late March 2013 at the age of 17months, the PI calf underwent exacerbation of clinical signs, showing

hemorrhages in the nasal cavities, conjunctivitis, and an ocular discharge,followed by watery diarrhea that rapidly led to death in a week despiteintensive treatment with fluids and antibiotics. Rectal temperatures werehigh (up to 41°C) between 8 and 26 March 2013, decreasing below base-line values (38 to 39.3°C) a few days before death (Fig. 1). While the totalnumber of leukocytes was generally within the normal range (5.0 � 109 to12.0 � 109 cells liter�1), with a single exception on March 30 (13.8 � 109

leukocytes liter�1), differential counts underwent remarkable changes(Fig. 1). Lymphopenia reached minimal values on 8 March, with 1.7 � 109

lymphocytes liter�1 (lower reference limit, 3.0 � 109 lymphocytes li-ter�1), and neutrophilia peaked on 26 March at 10.4 � 109 cells liter�1

(upper reference limit, 5.0 � 109 neutrophils liter�1) (Fig. 1).Histopathology. At postmortem examination, the major organs were

sampled, fixed in 10% neutral buffered formalin, and routinely processedfor histopathological examination. Formalin-embedded samples were cutinto 3-�m sections. Sections for microscopic examination were deparaf-finized, rehydrated, and stained with hematoxylin and eosin (HE) forevaluation of pathological changes.

Pestivirus detection, characterization, isolation, and purification.Detection of pestiviruses in biological fluids and tissue samples from thedead calf was carried out with a real-time reverse transcription (RT)-PCRassay specific for Hobi-like pestivirus (20) and a nested PCR protocol forsimultaneous detection and characterization of all of the pestiviruses thatinfect cattle (21).

Virus was isolated from the brain of the dead calf. Tissue samples werehomogenized in Dulbecco’s minimal essential medium containing anti-biotics (penicillin, 5,000 IU/ml; streptomycin, 2,500 �g/ml; amphotericinB, 10 �g/ml). After centrifugation at 3,000 � g for 15 min, the supernatantwas used to inoculate confluent monolayers of Madin-Darby bovine kid-ney cells. Viral growth was monitored by an immunofluorescence assaywith an anti-NS3 monoclonal antibody pool (3A3, 3H4, IF2) (9) and goat

Received 4 April 2014 Returned for modification 27 April 2014Accepted 28 May 2014

Published ahead of print 4 June 2014

Editor: E. Munson

Address correspondence to Nicola Decaro, nicola.decaro@uniba.it.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.00986-14

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anti-mouse IgG conjugated with fluorescein isothiocyanate (Sigma-Al-drich Srl, Milan, Italy). CP and NCP Hobi-like viruses were separatedthrough successive plaque and endpoint dilution assays, respectively, aspreviously described (10, 22).

Genomic characterization of the Hobi-like pestivirus pair. Thenearly full-length genomes of the CP and NCP Hobi-like strains (Italy-68/13cp and Italy-68/13ncp) isolated from the dead calf were determinedfrom the purified viral isolates by means of RT-PCR amplification withprimers described by Liu et al. (3) under PCR conditions reported byDecaro et al. (10). The PCR products generated from the atypical pes-tivirus were directly sequenced by BaseClear BV (Leiden, The Nether-lands), and a consensus sequence was obtained for each virus withGeneious version 6.1.5 (created by Biomatters). Additional RT-PCRassays with strain-specific primers and sequencing attempts were per-formed to close gaps between the assembled contigs. The consensusnucleotide sequences were manually refined with GeneDoc (http://www.nrbsc.org/gfx/genedoc/) and aligned with the ClustalW tool ofthe European Molecular Biology Laboratory (http://www.ebi.ac.uk).The nearly full-length sequences of the CP and NCP viruses were com-pared to the analogous sequences of pestivirus CP and NCP referencestrains, including the Italian Hobi-like strains (9, 10, 23). The samesequences were subjected to phylogenetic analysis by the parsimonyand neighbor-joining methods of the MEGA 4.1 software (24), sup-plying statistical support with bootstrapping of �1,000 replicates.

Real-time RT-PCR monitoring of CP and NCP viruses. Two real-time RT-PCR assays were developed in order to discriminate and quantifythe CP and NCP Hobi-like strains detected in the dead calf separately.Oligonucleotides were designed by taking into account the Jiv (bovine J-do-main protein interacting with viral protein) insertion detected in the NS2-3gene of the CP strain in order to detect either the CP or the NCP virus specif-ically. The oligonucleotide used for the CP and NCP assays were primersCP-for (5=-TCATAAGAGAGGGAGAATTGGACG-3=) and CP-rev (5=-GTCCATTTCAAACCTCCTATGCTT-3=), primers NCP-For (5=-AGAATTGGACGGGCCTTTTAGGCA-3=) and NCP-Rev (5=-CTTTAGTTGCCAGTATTGGTAAATTCC-3=), and probes CP-Pb (6-carboxyfluorescein–CCTGGC

ATCGGCTGCACATCATCG-Black Hole Quencher 1) and NCP-Pb (6-carboxyfluorescein–CTGTCCTCTTGCTATGTACTGTAAATACCCTCTGG-Black Hole Quencher 1), respectively. These assays were used to calcu-late the CP and NCP viral loads in tissues collected postmortem. Samplescollected in vivo during the monitoring period (19) were also processed inorder to determine the date of the emergence of the CP strain and assess itsshedding and viremia. The absolute number of RNA copies of each virus wascalculated by means of standard curves obtained from 10-fold dilutions ofRNA transcripts from plasmid containing the target region of the relatedbiotype. The same dilutions of the standard RNAs were also used to assess thetest sensitivities.

Detection of other pathogens. Tissue samples from the dead calf weresubjected to molecular assays for detection of the main viruses of cattle aspreviously described (10). RT-PCR and PCR assays were performed withSuperScript One-Step RT-PCR for Long Templates (Life Technologies)and LA PCR kit Ver. 2.1 (TaKaRa Bio Inc., Shiga, Japan), respectively.

Standard procedures for the in vitro isolation of common bacteriawere used. Samples were plated on 5% sheep blood agar and culturedaerobically at 37°C for 24 h for detection of aerobic pathogens. Bacteriawere identified by standard biochemical procedures and analytical profileindex (bioMérieux Italia S.p.A., Rome, Italy). For isolation of mycoplas-mas, samples were inoculated into modified Hayflick broth at 37°C aspreviously described (25). Detection of parasites was achieved by fecalflotation and histopathologic examination.

Nucleotide sequence accession numbers. The full-length genomes ofthe virus pair were determined and deposited in the GenBank databaseunder accession numbers KJ627179 (Italy-68/13ncp) and KJ627180(Italy-68/13cp).

RESULTSPostmortem findings. At postmortem examination, the calf dis-played focal areas of necrosis in the oral cavity with hemorrhageson the gum lines (Fig. 2A), hemorrhagic tracheitis (Fig. 2B), pneu-monia with interstitial emphysema, thinning of the spleen

FIG 1 Monitoring of the white blood cell (WBC) count (A) and rectal temperature (B) of the calf with MD-like syndrome. Total and differential WBC countsand temperatures are reported as cell numbers per liter of blood and degrees Celsius, respectively.

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(Fig. 2C), severe enteritis (Fig. 2D), and enlargement of the mes-enteric lymph nodes. However, typical findings of MD, includingerosive lesions and ulcerations in the mucosa of the gastrointesti-nal tract and hemorrhage and necrosis of the mucosa over thePeyer’s patches, were not observed.

At microscopic examination, the calf displayed alterations ofthe major organs involving mainly the lymphoid tissues, suchas necrosis of the red pulp in the spleen (Fig. 2E) and lymphoiddepletion and hemorrhagic foci in the tonsils, prescapular (Fig.2F) and mesenteric lymph nodes, and thymus. Subpleural areasof inflammation, interstitial edema, and hyperemia affectedthe lungs, whereas focal hemorrhages and leukocyte infiltra-tion were the main histopathologic findings in the myocar-dium. Lymphoplasmocellular enteritis with fusion of the intes-tinal villi and moderate multifocal nephritis were alsoobserved. Neuropathologic lesions were evident in the brain

that included neuronophagy and spongiosis of the gray cortex.Sarcocystis species cysts were detected in the muscular fibers ofthe esophagus and myocardium.

Detection of Hobi-like CP and NCP viruses. The dead calftested positive for Hobi-like pestivirus by both real-time RT-PCR(20) and nested PCR (21) assays. No additional common patho-gens of cattle were detected by traditional or molecular assays,with the exception of the bovine sarcosporidiosis diagnosedthrough histopathology and Mycoplasma bovirhinis already de-tected in the respiratory tract during the monitoring period.

Two distinct bands were obtained from the major organs bymeans of an RT-PCR assay amplifying the full-length NS2-3region, thus accounting for the presence of a Hobi-like pesti-virus pair (10). The two viruses, Italy-68/13cp and Italy-68/13ncp, were isolated and separated in cell cultures as previouslydescribed (10).

FIG 2 Gross lesions (A to D) and histopathological findings (E and F) in the calf with MD-like syndrome. Panels: A, focal necrosis in the oral cavity andhemorrhages on the gum lines; B, hemorrhagic tracheitis; C, enteritis and enlargement of the mesenteric lymph nodes; D, thinning of the spleen; E, photomi-crograph of a spleen section showing necrosis of the red pulp (HE staining; magnification, �10); F, photomicrograph of a prescapular lymph node sectionshowing lymphoid depletion and hemorrhagic areas (HE staining; magnification, �10).

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Full-genomic characterization of the Hobi-like virus pair.The CP and NCP Hobi-like isolates were found to be strictly re-lated to each other, displaying 97.17% nucleotide sequence iden-tity in their full-length sequences. Also evident was a high genetic

relatedness, ranging from 95.21 to 98.50% nucleotide sequenceidentity, to Hobi-like strains previously detected in the same cattleherd (9, 10), as well as to Brazilian prototype strain D32/00_HoBi(93.92 to 96.16% nucleotide sequence identity), whose full-length

FIG 3 Neighbor-joining tree based on the full-length genomes of members of the genus Pestivirus. Statistical support was provided by bootstrapping of �1,000replicates. The scale bar represents the estimated number of nucleotide substitutions per 1,000 bases. The following pestivirus strains were aligned (GenBankaccession numbers are in parentheses): Hobi-like pestivirus Italy-1/10-1 (HQ231763), Italy-83/10ncp (JQ612704), Italy-83/10cp (JQ612705), Italy-129/07(KC788748), D32/00_HoBi (AB871953), JS12/01 (JX469119), Th/04_KhonKaen (FJ040215), BVD H2121 (GU270877), Gifhorn (GQ902940), X818(NC_003679), Reindeer (AF144618), CSFV Brescia X (AY578687), HCLV (AF531433), Brescia (AF091661), Alfort-A19 (U90951), Shimen/HVRI (AY775178),Riems (AY259122), Pestivirus of giraffe H138 (AF144617), BVDV-1 ILLNC (U86600), ZM-95 (AF526381), Oregon-C24V (AF091605), CP7-5A (AF220247),SD1 (M96751), Singer_Arg (DQ088995), KE9 (EF101530), NADL (M31182), VEDEVAC (AJ585412), BVDV-2 JZ05-1 (GQ888686), New York’93 (AF502399),XJ-04 (FJ527854), C413 (NC_002032), and Hokudai Lab/09 (AB567658).

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genome was recently determined. In contrast, Thai Hobi-like iso-late Th/04_KhonKaen was more distantly related, displaying only87.54 to 89.65% nucleotide sequence identity.

The close relatedness to other Italian isolates, as well as to Bra-zilian strains, was also evident in phylogenetic trees constructedon the basis of the full-length genome and/or informative regions(Fig. 3).

The CP and NCP viruses differed by the presence in the ge-nome of the former strain of an insertion of cellular origin corre-sponding to Jiv (26) (Fig. 4). Retrospective analysis of previouslycollected samples showed that the Jiv sequence incorporated intothe CP viral genome did not vary during the period between itsfirst detection and the death of the animal.

Surprisingly, the same insertion had been found in the Hobi-like pestivirus CP strain previously detected in a heifer from thesame herd that died of respiratory disease (10). The nucleotidesequence identity between the two CP strains was 99.88% whenconsidering the full-length genomes, whereas artificial removal ofthe Jiv insertion resulted in a nucleotide sequence identity of99.51% between the CP and NCP Italy-68/13 virus strains. Whenanalysis was restricted to the polyprotein-encoding sequence,strain Italy-68/13-cp displayed 97.09 and 99.90% nucleotide se-quence identities to isolates Italy-68/13-ncp and Italy-83/10cp,respectively. In this region, the nucleotide sequence identity be-

tween the two biotypes of Italy-68/13 virus pairs increased to99.63% after removal of the Jiv insertion. Analysis of the genomicregions upstream and downstream of the Jiv insertion confirmedthe higher genetic relatedness of strain Italy-68/13cp to the CPvirus isolated a few years before (99.85 and 99.88% nucleotidesequence identities) than to the NCP virus infecting the same an-imal (99.25 and 99.82% nucleotide sequence identities).

Apart from the Jiv sequence, there were 21 amino acid muta-tions in the polyprotein sequences of the NCP and CP strains ofthe Italy-68/13 virus pair, whereas only 11 differences betweenthe two CP viruses isolated from the same herd were detected(Table 1).

Real-time RT-PCR monitoring of the Hobi-like virus pair.Two real-time RT-PCR assays were designed to discriminate be-tween the CP and NCP viruses in order to assess the distribution ofthe two viruses in tissues collected postmortem and to determinethe date of the emergence of the CP virus in samples collected invivo during the monitoring period (19). The TaqMan assays werefound to be biotype specific, showing a detection limit of 10 stan-dard RNA copies for both biotypes. The standard curves gener-ated covered a linear range of 9 orders of magnitude (from 101 to109 copies of standard DNA) and showed linearity over the entirequantitation range (slopes of �3.459 and �3.511 for the CP and

FIG 4 Comparison of Jiv inserts from 16 pestiviruses. The amino acid sequence of the portion of the Jiv insert found within the NS2-3 coding region of 16pestiviruses was compared to the cellular Jiv sequence (top sequence, accession number AY027882). Dots indicate conserved residues with respect to the cellularJiv sequence. The following pestivirus strains were aligned (GenBank accession numbers are in parentheses): BDV strains Cumnock (U43603) and Moredun(U43602); BVDV-1 strains NADL (AJ133738), MD1 (Z54332), and Indiana (Z54331); BVDV-2 strains 125c (U25053), 296c (AF268172), 5912c (AF268179),6082c (AF268180), Galena (AF268176), ND8799c (AF268175), 297c (AF268177), and SD11630c (AF268178); Pestivirus of giraffe 1-H138 (AF268178); andHobi-like pestivirus strains Italy-83/10cp (JQ612705) and Italy-68/13cp (KJ627180).

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NCP strains, respectively), providing an accurate measurementover a very large variety of starting target amounts.

The two biotypes were widely distributed among the internalorgans of the dead calf, displaying greater loads of RNA for theNCP Hobi-like pestivirus than for the CP strain, with the excep-tion of the intestine, where the viruses reached similar titers (Table2). Other tissues harboring great CP viral loads were the nervoussystem and lymphoid tissues, where this biotype exceeded a titer of105 RNA copies �l�1 of template.

Interestingly, retrospective analysis of specimens collected invivo during the monitoring period determined that the CP strainemerged about 1 month before the death of the animal and 3weeks before the onset of severe clinical signs. However, fever andlymphopenia (Fig. 1) occurred as early as the emergence of the CPstrain. As depicted in Fig. 5, shedding and viremia of the NCPstrain were constant and occurred at high titers, reaching maximalvalues in urine, as previously reported (19). In contrast, the shed-ding of the CP virus increased rapidly after strain emergence andreached titers comparable to those detected for the NCP strainonly in feces, whereas the viral loads observed in nasal swabs and

TABLE 1 Substitutions in the polyprotein sequences of Italian Hobi-likevirus pairs

Coding region andcodon

Italy-68/13ncp

Italy-68/13cpa

Italy-83/10ncp

Italy-83/10cpa

Npro

39 I I I T46 R K K K

C203 R R K R

Erns

305 K E K E361 W R R R442 C G G G476 Q R Q R479 V I I I495 Y H H H

E1502 N N N D528 M V V V586 G E E E628 Q E E E654 N S N S690 Q Q Q H

E2722 Y Y F Y729 Y H H H760 V I V I847 A A V A864 V V E V870 E K K K941 E E K E946 T R R R

NS2/31178 V I V I1208 C S S S1239 Q H Q H1266 Y Y H Y1292 L L I L1378 T T I T1423 K K E E1428 P P L L1962 M M M K2041 I I I V2178 T T A A

NS4A2295 V I I I

NS4B2355 E E G E2485 Y Y H Y2537 R R R K2576 A A V A

NS5A2753 E E E G2837 R R G R2923 E E E G

TABLE 1 (Continued)

Coding region andcodon

Italy-68/13ncp

Italy-68/13cpa

Italy-83/10ncp

Italy-83/10cpa

3054 N N D N3123 W W C W3135 Q Q H Q

NS5B3608 T T A T3611 S P P P3738 K R R R3864 I I V I

a The Jiv insert of CP strains is not included (see Fig. 3 for details).

TABLE 2 NCP and CP Hobi-like pestivirus loads in tissues of the calfwith MD-like syndromea

Tissue

Hobi-like pestivirus load

NCP CP

Brain 3.85 � 107 1.52 � 105

Cerebellum 3.53 � 107 2.64 � 105

Brain stem 6.96 � 106 3.06 � 105

Trigeminal ganglion 2.05 � 107 1.09 � 105

Tonsils 9.64 � 106 8.86 � 105

Prescapular lymph node 9.44 � 107 1.63� 107

Tongue 1.28 � 107 7.44 � 103

Parotid gland 1.26 � 108 2.84 � 104

Thymus 5.50 � 107 3.32 � 105

Trachea 2.43 � 107 6.44 � 104

Lungs 1.64 � 107 8.99 � 105

Myocardium 3.03 � 105 1.06 � 101

Liver 4.07 � 105 3.35 � 102

Spleen 2.70 � 107 1.66 � 105

Small intestine 1.70 � 106 3.69 � 106

Large intestine 5.87 � 105 1.03 � 105

Mesenteric lymph node 7.24 � 104 5.62 � 102

Kidney 2.79 � 106 4.86 � 101

Urinary bladder 3.89 � 107 1.02 � 102

a Viral loads are expressed as RNA copy numbers per �l of template.

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FIG 5 In vivo detection and quantification of CP and NCP Hobi-like pestiviruses. Viral RNA titers in blood, urine (A), feces, and nasal secretions (B) weredetermined by biotype-specific real-time RT-PCR assays and are expressed as copy numbers per �l of template.

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urine were much lower. A striking finding for the CP strain was thedetection of very low titers in the animal’s blood, even in the lastdays of its life (Fig. 5B).

DISCUSSION

Two biotypes of pestiviruses, NCP and CP, are known, which aredistinguished by the ability to cause a cytopathic effect in a tissueculture. In cattle, both biotypes are involved in the pathogenesis ofMD, a severe clinical form caused by the emergence of a bovineviral diarrhea virus (BVDV) CP strain in calves PI with an NCPvirus (17, 18). The CP and NCP viruses isolated from the sameanimal are called a virus pair. Virus pairs of BVDV-1, BVDV-2,border disease virus (27), and (more recently) Hobi-like pestivi-rus (10) have been isolated. However, it was not known whetherthe animal harboring the Hobi-like virus pair was PI with the NCPvirus before being superinfected with the CP strain.

The present paper reports a case of MD-like syndrome in a calfthat harbored the Hobi-like virus pair and had been monitored forabout 1 year after the assessment of its PI status (19). This moni-toring allowed us to determine the date of the emergence of the CPstrain in the NCP PI animal. The CP virus was detected in all of thespecimens collected about 3 weeks before the onset of severe clin-ical signs, reaching the highest titers in urine and feces. In contrast,only traces of CP virus RNA were detected in blood, suggestingthat this biotype displays an affinity for the gastroenteric tract.Accordingly, the greatest CP loads in the dead animal were foundin the intestine, followed by nervous system and lymphoid tissues.The real-time PCR assays developed to trace the NCP and CPviruses were highly specific since oligonucleotides were designedin order to rule out cross amplification. In fact, CP oligonucleo-tides amplified a genomic fragment spanning the region from justupstream of the Jiv sequence to the very 5= end of the cellularinsertion, whereas the NCP oligonucleotides targeted the regionsjust upstream and downstream of this sequence, thus preventingthe amplification of a very long product or at least probe hydro-lysis in the case of the CP strain. However, it has been shown thata significant amount of different recombinants can be detected inPI animals long before the onset of MD, many of which containthe CP-specific marker (28, 29). Since many of these recombi-nants are intermediate products of the recombination process,thus not representing genomes of fully viable viruses, the realloads of the CP viruses calculated in the present study could havebeen overestimated.

The isolated CP strain was strictly related to and, interestingly,presented the same insertion as strain Italy-83/10cp, which hadbeen isolated from the same herd about 3 years before (10). Alongwith other insertions of bovine origin, Jiv has been identified as acausative marker of pestivirus cytopathogenicity (30). The pres-ence of the same Jiv sequence in CP Hobi-like pestiviruses, whichare so distant at the temporal and spatial levels, is a striking findingwith no obvious explanation. It could be speculated that the in-sertion of Jiv sequences into the Hobi-like pestivirus genome has apreferential pattern in terms of position and sequence length.However, this finding, along with the higher genetic relatedness ofthe isolated CP virus to isolate 83/10cp than to the NCP strainisolated from the same animal, seems to account for its derivationfrom the former virus. Therefore, superinfection of the PI animalwith strain Italy-83/10cp rather than de novo generation of the CPvirus from the NCP strain found in the same calf should be con-sidered as the cause of the MD-like syndrome. As a possible expla-

nation, contamination of the animal with isolate Italy-83/10cp bythe same personnel having contact with the calf and handling thevirus in the lab could be considered. However, the number ofdifferences among the sequences of the three viruses analyzed isvery low, and therefore it might be that the observed higher ho-mology between 68/13cp and 83/10cp results from convergentevolution, i.e., from introduction of adaptive mutations impor-tant for the CP biotype. Anyway, regardless of the mode of infec-tion (natural or incidental), the development of fatal disease as aconsequence of superinfection with a CP Hobi-like virus of a PIcalf provides new insights into the pathogenesis and molecularmechanisms of this emerging group of pestiviruses.

The clinical signs and postmortem findings in this case wereslightly different from those commonly observed in calves withMD caused by extant BVDV-1 or BVDV-2, i.e., hemorrhagic, ne-crotic, and ulcerative lesions involving all mucosal tissue (17, 18).In the case presented here, hemorrhages and necrosis were re-stricted to the oral cavity and upper respiratory tract and even thehistopathologic findings were less severe than those described forclassical MD. There was a weak relationship between the viralloads in different organs and the clinical presentation, and moreremarkable gross and microscopic changes would have been ex-pected on the basis of the detected CP viral RNA titers. A certaincorrelation between viral loads and severity of histopathologicalchanges was evident only for lymphoid tissues, where the extent ofnecrosis and hemorrhage seemed to parallel the amounts of CPviral RNA. In contrast, this association was not so clear for othertissues displaying great virus loads, such as the gastroenteric tract,where only moderate alterations were found upon histopatho-logic examination.

It is noteworthy that the first CP-NCP Hobi-like pestivirus pairhad been isolated from a heifer that died of respiratory disease(10). Thus, even considering the fact that MD can occur withdifferent clinical pictures, the first reported detection of a Hobi-like pestivirus pair in cattle seems to account for an atypical MDthat is characterized mainly by respiratory distress. Only furtherfield study may provide additional evidence for this MD-like syn-drome induced by Hobi-like pestivirus.

ACKNOWLEDGMENTS

This work was supported by grants from the Italian Ministry of Health toN.D. and N.C. (Ricerca corrente 2011, project Epidemiologia del virusdella diarrea virale bovina tipo 3 [BVDV-3] nel Sud Italia) and from theItalian Ministry of Education, University, and Research to C.B. (PRIN2010-2011, project Pestivirus dei ruminanti: virus emergenti, aspetti di-agnostici e profilattici).

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Mucosal Disease Associated with Hobi-Like Pestivirus

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