Abstract
A calf persistently infected with Hobi-like pestivirus displayed severe clinical signs and subsequently died. Gross lesions and histopathological changes were suggestive of hemorrhagic and necrotic inflammation involving several tissues. A Hobi-like pestivirus pair was isolated from the dead calf, i.e., cytopathogenic (CP) and noncytopathogenic (NCP) strains strictly related to each other and to Italian prototype isolates at the genetic level. Two biotype-specific real-time reverse transcription-PCR assays determined the time of the emergence of the CP virus as 1 month before the calf's death. This highest RNA titers were reached in lymphoid and nervous system tissues, whereas only traces of CP viral RNA were found in blood. In contrast, great NCP virus loads were present in all tissues and biological fluids. The present report provides new insights into the pathogenesis and molecular mechanisms of this emerging group of pestiviruses.
INTRODUCTION
Hobi-like pestivirus is a new tentative species of the genus Pestivirus, 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 commercial fetal calf serum batches worldwide but also from serum samples from naturally infected cattle (2–7). To date, natural infections caused by this new bovine pestivirus have been reported only sporadically (2, 8), but in most cases they were associated with overt disease consisting of respiratory distress (9, 10) or reproductive failure (11, 12). Hobi-like pestiviruses were able to induce respiratory disease even under experimental conditions (13, 14). Experimental infection of cattle, sheep, and swine showed that the Hobi-like pestivirus is able to infect all of those species, although only ruminants displayed clinical signs and virus shedding (13). Through immunization studies with ruminants, poor cross-neutralization between extant pestiviruses and Hobi-like isolates was evident, which raised some concerns about the ability of currently available vaccines to protect against the new pestivirus species (14–16).
Mucosal disease (MD) is a lethal disease of cattle that is caused by the emergence of a cytopathogenic (CP) pestivirus in an animal persistently infected (PI) with a noncytopathogenic (NCP) strain. This disease is usually characterized by hemorrhagic, necrotic, and ulcerative lesions (17, 18).
Although a Hobi virus CP-NCP pair was isolated from a heifer that died of respiratory distress (10) and Hobi virus persistent infection was recently reported (19), no clear evidence of MD has been obtained so far. In this note, we report the clinical, immunological, postmortem, and histopathological findings of MD-like syndrome naturally occurring in a Hobi-like virus PI calf.
MATERIALS AND METHODS
Clinical case.
The disease involved a calf PI with Hobi-like pestivirus that had been monitored at the Infectious Diseases Unit of the Animal Hospital of the University of Bari (Italy) since March 2012 (19). The PI calf displayed recurrent clinical signs of respiratory disease, enteritis, and dermatomycosis due to concurrent pathogens likely as a consequence of pestivirus-induced immunosuppression. In late March 2013 at the age of 17 months, 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 despite intensive treatment with fluids and antibiotics. Rectal temperatures were high (up to 41°C) between 8 and 26 March 2013, decreasing below baseline values (38 to 39.3°C) a few days before death (Fig. 1). While the total number of leukocytes was generally within the normal range (5.0 × 109 to 12.0 × 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 liter−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).
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 counts and temperatures are reported as cell numbers per liter of blood and degrees Celsius, respectively.
Histopathology.
At postmortem examination, the major organs were sampled, fixed in 10% neutral buffered formalin, and routinely processed for histopathological examination. Formalin-embedded samples were cut into 3-μm sections. Sections for microscopic examination were deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE) for evaluation of pathological changes.
Pestivirus detection, characterization, isolation, and purification.
Detection of pestiviruses in biological fluids and tissue samples from the dead calf was carried out with a real-time reverse transcription (RT)-PCR assay specific for Hobi-like pestivirus (20) and a nested PCR protocol for simultaneous detection and characterization of all of the pestiviruses that infect cattle (21).
Virus was isolated from the brain of the dead calf. Tissue samples were homogenized in Dulbecco's minimal essential medium containing antibiotics (penicillin, 5,000 IU/ml; streptomycin, 2,500 μg/ml; amphotericin B, 10 μg/ml). After centrifugation at 3,000 × g for 15 min, the supernatant was used to inoculate confluent monolayers of Madin-Darby bovine kidney cells. Viral growth was monitored by an immunofluorescence assay with an anti-NS3 monoclonal antibody pool (3A3, 3H4, IF2) (9) and goat anti-mouse IgG conjugated with fluorescein isothiocyanate (Sigma-Aldrich Srl, Milan, Italy). CP and NCP Hobi-like viruses were separated through successive plaque and endpoint dilution assays, respectively, as previously described (10, 22).
Genomic characterization of the Hobi-like pestivirus pair.
The nearly full-length genomes of the CP and NCP Hobi-like strains (Italy-68/13cp and Italy-68/13ncp) isolated from the dead calf were determined from the purified viral isolates by means of RT-PCR amplification with primers described by Liu et al. (3) under PCR conditions reported by Decaro et al. (10). The PCR products generated from the atypical pestivirus were directly sequenced by BaseClear BV (Leiden, The Netherlands), and a consensus sequence was obtained for each virus with Geneious version 6.1.5 (created by Biomatters). Additional RT-PCR assays with strain-specific primers and sequencing attempts were performed to close gaps between the assembled contigs. The consensus nucleotide sequences were manually refined with GeneDoc (http://www.nrbsc.org/gfx/genedoc/) and aligned with the ClustalW tool of the European Molecular Biology Laboratory (http://www.ebi.ac.uk). The nearly full-length sequences of the CP and NCP viruses were compared to the analogous sequences of pestivirus CP and NCP reference strains, including the Italian Hobi-like strains (9, 10, 23). The same sequences were subjected to phylogenetic analysis by the parsimony and neighbor-joining methods of the MEGA 4.1 software (24), supplying 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 quantify the CP and NCP Hobi-like strains detected in the dead calf separately. Oligonucleotides were designed by taking into account the Jiv (bovine J-domain protein interacting with viral protein) insertion detected in the NS2-3 gene of the CP strain in order to detect either the CP or the NCP virus specifically. The oligonucleotide used for the CP and NCP assays were primers CP-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–CCTGGCATCGGCTGCACATCATCG-Black Hole Quencher 1) and NCP-Pb (6-carboxyfluorescein–CTGTCCTCTTGCTATGTACTGTAAATACCCTCTGG-Black Hole Quencher 1), respectively. These assays were used to calculate the CP and NCP viral loads in tissues collected postmortem. Samples collected in vivo during the monitoring period (19) were also processed in order to determine the date of the emergence of the CP strain and assess its shedding and viremia. The absolute number of RNA copies of each virus was calculated by means of standard curves obtained from 10-fold dilutions of RNA transcripts from plasmid containing the target region of the related biotype. The same dilutions of the standard RNAs were also used to assess the test sensitivities.
Detection of other pathogens.
Tissue samples from the dead calf were subjected to molecular assays for detection of the main viruses of cattle as previously described (10). RT-PCR and PCR assays were performed with SuperScript 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 bacteria were used. Samples were plated on 5% sheep blood agar and cultured aerobically at 37°C for 24 h for detection of aerobic pathogens. Bacteria were identified by standard biochemical procedures and analytical profile index (bioMérieux Italia S.p.A., Rome, Italy). For isolation of mycoplasmas, samples were inoculated into modified Hayflick broth at 37°C as previously described (25). Detection of parasites was achieved by fecal flotation and histopathologic examination.
Nucleotide sequence accession numbers.
The full-length genomes of the virus pair were determined and deposited in the GenBank database under accession numbers KJ627179 (Italy-68/13ncp) and KJ627180 (Italy-68/13cp).
RESULTS
Postmortem findings.
At postmortem examination, the calf displayed focal areas of necrosis in the oral cavity with hemorrhages on the gum lines (Fig. 2A), hemorrhagic tracheitis (Fig. 2B), pneumonia with interstitial emphysema, thinning of the spleen (Fig. 2C), severe enteritis (Fig. 2D), and enlargement of the mesenteric lymph nodes. However, typical findings of MD, including erosive lesions and ulcerations in the mucosa of the gastrointestinal tract and hemorrhage and necrosis of the mucosa over the Peyer's patches, were not observed.
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 and hemorrhages on the gum lines; B, hemorrhagic tracheitis; C, enteritis and enlargement of the mesenteric lymph nodes; D, thinning of the spleen; E, photomicrograph of a spleen section showing necrosis of the red pulp (HE staining; magnification, ×10); F, photomicrograph of a prescapular lymph node section showing lymphoid depletion and hemorrhagic areas (HE staining; magnification, ×10).
At microscopic examination, the calf displayed alterations of the major organs involving mainly the lymphoid tissues, such as necrosis of the red pulp in the spleen (Fig. 2E) and lymphoid depletion and hemorrhagic foci in the tonsils, prescapular (Fig. 2F) and mesenteric lymph nodes, and thymus. Subpleural areas of inflammation, interstitial edema, and hyperemia affected the lungs, whereas focal hemorrhages and leukocyte infiltration were the main histopathologic findings in the myocardium. Lymphoplasmocellular enteritis with fusion of the intestinal villi and moderate multifocal nephritis were also observed. 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 of the esophagus and myocardium.
Detection of Hobi-like CP and NCP viruses.
The dead calf tested positive for Hobi-like pestivirus by both real-time RT-PCR (20) and nested PCR (21) assays. No additional common pathogens of cattle were detected by traditional or molecular assays, with the exception of the bovine sarcosporidiosis diagnosed through histopathology and Mycoplasma bovirhinis already detected in the respiratory tract during the monitoring period.
Two distinct bands were obtained from the major organs by means of an RT-PCR assay amplifying the full-length NS2-3 region, thus accounting for the presence of a Hobi-like pestivirus pair (10). The two viruses, Italy-68/13cp and Italy-68/13ncp, were isolated and separated in cell cultures as previously described (10).
Full-genomic characterization of the Hobi-like virus pair.
The CP and NCP Hobi-like isolates were found to be strictly related to each other, displaying 97.17% nucleotide sequence identity in their full-length sequences. Also evident was a high genetic relatedness, ranging from 95.21 to 98.50% nucleotide sequence identity, to Hobi-like strains previously detected in the same cattle herd (9, 10), as well as to Brazilian prototype strain D32/00_HoBi (93.92 to 96.16% nucleotide sequence identity), whose full-length genome was recently determined. In contrast, Thai Hobi-like isolate Th/04_KhonKaen was more distantly related, displaying only 87.54 to 89.65% nucleotide sequence identity.
The close relatedness to other Italian isolates, as well as to Brazilian strains, was also evident in phylogenetic trees constructed on the basis of the full-length genome and/or informative regions (Fig. 3).
FIG 3.
Neighbor-joining tree based on the full-length genomes of members of the genus Pestivirus. Statistical support was provided by bootstrapping of >1,000 replicates. The scale bar represents the estimated number of nucleotide substitutions per 1,000 bases. The following pestivirus strains were aligned (GenBank accession 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).
The CP and NCP viruses differed by the presence in the genome of the former strain of an insertion of cellular origin corresponding to Jiv (26) (Fig. 4). Retrospective analysis of previously collected samples showed that the Jiv sequence incorporated into the CP viral genome did not vary during the period between its first detection and the death of the animal.
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 16 pestiviruses was compared to the cellular Jiv sequence (top sequence, accession number AY027882). Dots indicate conserved residues with respect to the cellular Jiv 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); and Hobi-like pestivirus strains Italy-83/10cp (JQ612705) and Italy-68/13cp (KJ627180).
Surprisingly, the same insertion had been found in the Hobi-like pestivirus CP strain previously detected in a heifer from the same herd that died of respiratory disease (10). The nucleotide sequence identity between the two CP strains was 99.88% when considering the full-length genomes, whereas artificial removal of the Jiv insertion resulted in a nucleotide sequence identity of 99.51% between the CP and NCP Italy-68/13 virus strains. When analysis was restricted to the polyprotein-encoding sequence, strain Italy-68/13-cp displayed 97.09 and 99.90% nucleotide sequence identities to isolates Italy-68/13-ncp and Italy-83/10cp, respectively. In this region, the nucleotide sequence identity between the two biotypes of Italy-68/13 virus pairs increased to 99.63% after removal of the Jiv insertion. Analysis of the genomic regions upstream and downstream of the Jiv insertion confirmed the higher genetic relatedness of strain Italy-68/13cp to the CP virus isolated a few years before (99.85 and 99.88% nucleotide sequence identities) than to the NCP virus infecting the same animal (99.25 and 99.82% nucleotide sequence identities).
Apart from the Jiv sequence, there were 21 amino acid mutations in the polyprotein sequences of the NCP and CP strains of the Italy-68/13 virus pair, whereas only 11 differences between the two CP viruses isolated from the same herd were detected (Table 1).
TABLE 1.
Substitutions in the polyprotein sequences of Italian Hobi-like virus pairs
| Coding region and codon | Italy-68/13ncp | Italy-68/13cpa | Italy-83/10ncp | Italy-83/10cpa |
|---|---|---|---|---|
| Npro | ||||
| 39 | I | I | I | T |
| 46 | R | K | K | K |
| C | ||||
| 203 | R | R | K | R |
| Erns | ||||
| 305 | K | E | K | E |
| 361 | W | R | R | R |
| 442 | C | G | G | G |
| 476 | Q | R | Q | R |
| 479 | V | I | I | I |
| 495 | Y | H | H | H |
| E1 | ||||
| 502 | N | N | N | D |
| 528 | M | V | V | V |
| 586 | G | E | E | E |
| 628 | Q | E | E | E |
| 654 | N | S | N | S |
| 690 | Q | Q | Q | H |
| E2 | ||||
| 722 | Y | Y | F | Y |
| 729 | Y | H | H | H |
| 760 | V | I | V | I |
| 847 | A | A | V | A |
| 864 | V | V | E | V |
| 870 | E | K | K | K |
| 941 | E | E | K | E |
| 946 | T | R | R | R |
| NS2/3 | ||||
| 1178 | V | I | V | I |
| 1208 | C | S | S | S |
| 1239 | Q | H | Q | H |
| 1266 | Y | Y | H | Y |
| 1292 | L | L | I | L |
| 1378 | T | T | I | T |
| 1423 | K | K | E | E |
| 1428 | P | P | L | L |
| 1962 | M | M | M | K |
| 2041 | I | I | I | V |
| 2178 | T | T | A | A |
| NS4A | ||||
| 2295 | V | I | I | I |
| NS4B | ||||
| 2355 | E | E | G | E |
| 2485 | Y | Y | H | Y |
| 2537 | R | R | R | K |
| 2576 | A | A | V | A |
| NS5A | ||||
| 2753 | E | E | E | G |
| 2837 | R | R | G | R |
| 2923 | E | E | E | G |
| 3054 | N | N | D | N |
| 3123 | W | W | C | W |
| 3135 | Q | Q | H | Q |
| NS5B | ||||
| 3608 | T | T | A | T |
| 3611 | S | P | P | P |
| 3738 | K | R | R | R |
| 3864 | I | I | V | I |
The Jiv insert of CP strains is not included (see Fig. 3 for details).
Real-time RT-PCR monitoring of the Hobi-like virus pair.
Two real-time RT-PCR assays were designed to discriminate between the CP and NCP viruses in order to assess the distribution of the two viruses in tissues collected postmortem and to determine the date of the emergence of the CP virus in samples collected in vivo during the monitoring period (19). The TaqMan assays were found to be biotype specific, showing a detection limit of 10 standard RNA copies for both biotypes. The standard curves generated covered a linear range of 9 orders of magnitude (from 101 to 109 copies of standard DNA) and showed linearity over the entire quantitation range (slopes of −3.459 and −3.511 for the CP and NCP strains, respectively), providing an accurate measurement over a very large variety of starting target amounts.
The two biotypes were widely distributed among the internal organs of the dead calf, displaying greater loads of RNA for the NCP Hobi-like pestivirus than for the CP strain, with the exception of the intestine, where the viruses reached similar titers (Table 2). Other tissues harboring great CP viral loads were the nervous system and lymphoid tissues, where this biotype exceeded a titer of 105 RNA copies μl−1 of template.
TABLE 2.
NCP and CP Hobi-like pestivirus loads in tissues of the calf with 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 |
Viral loads are expressed as RNA copy numbers per μl of template.
Interestingly, retrospective analysis of specimens collected in vivo during the monitoring period determined that the CP strain emerged about 1 month before the death of the animal and 3 weeks before the onset of severe clinical signs. However, fever and lymphopenia (Fig. 1) occurred as early as the emergence of the CP strain. As depicted in Fig. 5, shedding and viremia of the NCP strain were constant and occurred at high titers, reaching maximal values in urine, as previously reported (19). In contrast, the shedding of the CP virus increased rapidly after strain emergence and reached titers comparable to those detected for the NCP strain only in feces, whereas the viral loads observed in nasal swabs and urine were much lower. A striking finding for the CP strain was the detection of very low titers in the animal's blood, even in the last days of its life (Fig. 5B).
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) were determined by biotype-specific real-time RT-PCR assays and are expressed as copy numbers per μl of template.
DISCUSSION
Two biotypes of pestiviruses, NCP and CP, are known, which are distinguished by the ability to cause a cytopathic effect in a tissue culture. In cattle, both biotypes are involved in the pathogenesis of MD, a severe clinical form caused by the emergence of a bovine viral diarrhea virus (BVDV) CP strain in calves PI with an NCP virus (17, 18). The CP and NCP viruses isolated from the same animal are called a virus pair. Virus pairs of BVDV-1, BVDV-2, border disease virus (27), and (more recently) Hobi-like pestivirus (10) have been isolated. However, it was not known whether the animal harboring the Hobi-like virus pair was PI with the NCP virus before being superinfected with the CP strain.
The present paper reports a case of MD-like syndrome in a calf that harbored the Hobi-like virus pair and had been monitored for about 1 year after the assessment of its PI status (19). This monitoring allowed us to determine the date of the emergence of the CP strain in the NCP PI animal. The CP virus was detected in all of the specimens collected about 3 weeks before the onset of severe clinical signs, reaching the highest titers in urine and feces. In contrast, only traces of CP virus RNA were detected in blood, suggesting that this biotype displays an affinity for the gastroenteric tract. Accordingly, the greatest CP loads in the dead animal were found in the intestine, followed by nervous system and lymphoid tissues. The real-time PCR assays developed to trace the NCP and CP viruses were highly specific since oligonucleotides were designed in order to rule out cross amplification. In fact, CP oligonucleotides amplified a genomic fragment spanning the region from just upstream of the Jiv sequence to the very 5′ end of the cellular insertion, whereas the NCP oligonucleotides targeted the regions just upstream and downstream of this sequence, thus preventing the amplification of a very long product or at least probe hydrolysis in the case of the CP strain. However, it has been shown that a significant amount of different recombinants can be detected in PI animals long before the onset of MD, many of which contain the CP-specific marker (28, 29). Since many of these recombinants are intermediate products of the recombination process, thus not representing genomes of fully viable viruses, the real loads of the CP viruses calculated in the present study could have been overestimated.
The isolated CP strain was strictly related to and, interestingly, presented the same insertion as strain Italy-83/10cp, which had been isolated from the same herd about 3 years before (10). Along with other insertions of bovine origin, Jiv has been identified as a causative marker of pestivirus cytopathogenicity (30). The presence of the same Jiv sequence in CP Hobi-like pestiviruses, which are so distant at the temporal and spatial levels, is a striking finding with no obvious explanation. It could be speculated that the insertion of Jiv sequences into the Hobi-like pestivirus genome has a preferential pattern in terms of position and sequence length. However, this finding, along with the higher genetic relatedness of the isolated CP virus to isolate 83/10cp than to the NCP strain isolated from the same animal, seems to account for its derivation from the former virus. Therefore, superinfection of the PI animal with strain Italy-83/10cp rather than de novo generation of the CP virus from the NCP strain found in the same calf should be considered as the cause of the MD-like syndrome. As a possible explanation, contamination of the animal with isolate Italy-83/10cp by the same personnel having contact with the calf and handling the virus in the lab could be considered. However, the number of differences among the sequences of the three viruses analyzed is very low, and therefore it might be that the observed higher homology between 68/13cp and 83/10cp results from convergent evolution, i.e., from introduction of adaptive mutations important for the CP biotype. Anyway, regardless of the mode of infection (natural or incidental), the development of fatal disease as a consequence of superinfection with a CP Hobi-like virus of a PI calf provides new insights into the pathogenesis and molecular mechanisms of this emerging group of pestiviruses.
The clinical signs and postmortem findings in this case were slightly different from those commonly observed in calves with MD caused by extant BVDV-1 or BVDV-2, i.e., hemorrhagic, necrotic, and ulcerative lesions involving all mucosal tissue (17, 18). In the case presented here, hemorrhages and necrosis were restricted to the oral cavity and upper respiratory tract and even the histopathologic findings were less severe than those described for classical MD. There was a weak relationship between the viral loads in different organs and the clinical presentation, and more remarkable gross and microscopic changes would have been expected on the basis of the detected CP viral RNA titers. A certain correlation between viral loads and severity of histopathological changes was evident only for lymphoid tissues, where the extent of necrosis and hemorrhage seemed to parallel the amounts of CP viral RNA. In contrast, this association was not so clear for other tissues displaying great virus loads, such as the gastroenteric tract, where only moderate alterations were found upon histopathologic examination.
It is noteworthy that the first CP-NCP Hobi-like pestivirus pair had been isolated from a heifer that died of respiratory disease (10). Thus, even considering the fact that MD can occur with different clinical pictures, the first reported detection of a Hobi-like pestivirus pair in cattle seems to account for an atypical MD that is characterized mainly by respiratory distress. Only further field study may provide additional evidence for this MD-like syndrome induced by Hobi-like pestivirus.
ACKNOWLEDGMENTS
This work was supported by grants from the Italian Ministry of Health to N.D. and N.C. (Ricerca corrente 2011, project Epidemiologia del virus della diarrea virale bovina tipo 3 [BVDV-3] nel Sud Italia) and from the Italian Ministry of Education, University, and Research to C.B. (PRIN 2010-2011, project Pestivirus dei ruminanti: virus emergenti, aspetti diagnostici e profilattici).
Footnotes
Published ahead of print 4 June 2014
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