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Novel Picornavirus in Lambs with Severe Encephalomyelitis.

In 2016 in Scotland, and in 2017 in Wales, progressive neurologic signs were observed in young lambs. These cases were associated with nonsuppurative encephalomyelitis predominantly involving gray matter, including neuronal necrosis/neuronophagia and ganglionitis consistent with lesions caused by a neuronotropic viral infection. The target sites included cerebellar roof nuclei in the Purkinje molecular layer of the cerebellum, caudal brainstem nuclei (red nuclei and vestibular complex), and all levels of spinal cord examined (cervical, thoracic, and lumbar segments) located mainly in the ventral horns and dorsal root ganglia (Figure 1, panels A, B). Severe lesions were found consistently in the spinal cord. Louping ill virus, a common etiologic agent of nonsuppurative encephalitis in ruminants in Great Britain, was ruled out on the basis of the clinicopathological presentations and neuropathology, as well as serology. The observed lesion distribution and age of affected sheep were inconsistent with other possible neurotropic viral infections, such as Borna disease virus (1).

The Study

In 2016, we subjected a sample from an affected lamb's cerebrum to metagenomic analysis (Appendix, http://wwwnc. cdc.gov/EID/article/25/5/18-1573-App1.pdf) with read classification using RIEMS (2). From the resulting dataset (2 million reads), 99.97% were classified, but only 1 read was reliably classified as a viral sequence. This 326-bp read showed the highest sequence identity with the 5' untranslated region (UTR) of the genome of human rhinovirus (81.3%), belonging to the family Picornaviiidae, genus Enterovirus. For further validation and sample screening, this viral read was used to design a quantitative reverse transcription PCR (qRTPCR) assay (Appendix). Using this qRT-PCR, we analyzed lambs that, in 2017, received diagnoses of nonsuppurative encephalomyelitis and ganglionitis typical of neuronotropic viral infection; all 3 animals tested positive (Table 1). We detected the highest viral loads in the cerebellum and spinal cord (PCR quantification cycle [[C.sub.q]] 18-21), where the most severe lesions were also observed. We detected lower loads in the cerebrum ([C.sub.q] 30), ileum ([C.sub.q] 29-32), tonsil ([C.sub.q] 32-36), and mesenteric lymph node ([C.sub.q] 34-36). Lung and spleen samples were negative, as were CNS samples from 2 additional lambs from the Scotland flock with compressive spinal cord lesions and no evidence of nonsuppurative encephalitis (Table 1).

We subjected spinal cord samples from selected lambs affected in the 2017 lambing season to metagenomic analysis (Appendix), which yielded datasets with 2.4 million reads each. RIEMS analysis classified 99.9% of the reads, with 900 and 406 reads as sequences related to Picornaviridae (genera Enterovirus and SapeloviruS), and several unclassified species with relatively low sequence identities (64.8%-96.8%). Several reads were classified only after translation into amino acid sequences and comparison to the protein database. We found no evidence of other pathogens that could have resulted in the neuropathological manifestations.

De novo assembly of reads related to Picornaviridae generated a complete genome of a novel virus, tentatively named ovine picornavirus (OvPV). The obtained 7.5 kb OvPV genome is only very distantly related to known picornaviruses, with nucleotide sequence identities of 59% with a bovine picornavirus (International Nucleotide Sequence Data Collaboration [INSDC] accession no. LC006971) (3) and 55% with a canine picornavirus (accession no. KU871312) (4). The OvPV genome comprises a 5' UTR, a predicted 6,885-nt open reading frame encoding the polyprotein with 2,294 aa, a 3'UTR, and a poly(A)-tail. The putative structure of the polyprotein is similar to sapeloviruses with the characteristic order 5'-L-1ABCD-2ABC-3ABCD-3'; 3 consecutive methionins in frame could act as start codons of the polyprotein. The translation initiation site is proposed to be the third, because it is contained in the best Kozak context (5). Pairwise amino acid identities with P1, P2, and P3 of related picornaviruses are <58% (Appendix Table). Within the family Picornaviridae, together with other unclassified viruses (4,6), OvPV forms a putative new genus that is closest related to the genera Sapelovirus, Rabovirus, and Enterovirus (Figure 2; Appendix Figure). All whole-genome sequences we identified are available from the INSDC databases (accession no. PRJEB28719).

We attempted virus propagation from selected samples of affected animals on various mammalian cell lines (Appendix); however, virus could not be isolated. This result is consistent with other described unsuccessful attempts of viral propagation of related picornaviruses in cell culture (4,7).

To investigate whether the newly detected virus emerged in 2016 or was present previously, we examined archived cases from sheep in England with nonsuppurative encephalomyelitis oriented on gray matter that had tested negative for Louping ill virus by immunohistochemistry. Although the impaired RNA extracted from formalin-fixed paraffin-embedded (FFPE) material proved to be challenging for sequencing and amplification, we identified several additional cases of OvPV infection using reverse transcription PCR, and we generated whole-genome sequences from selected cases (Table 2). We detected OvPV in lambs of different breeds [less than or equal to] 3-4 weeks of age with neurologic signs; the earliest case identified dated back to 1998 (Table 2). Compared with the 2017 cases in Wales, the full OvPV genomes of the archive cases had [approximately equal to]90%-96% sequence identity at the nucleotide level but up to 99% sequence identity at the amino acid level.

We performed in situ hybridization on OvPV PCR-positive FFPE tissues, with a subset of neurons within the brain and spinal cord as hybridization targets (Figure 1). In the spinal cord, OvPV in situ hybridization-positive neuronal soma and their projections were dense in the ventral horn in regions of neuronal degeneration and gliosis (Figure 1, panel C). Where dorsal root ganglia were in section, probe hybridization was detected in individual neurons (Figure 1, panels D, G). No hybridization was detected in sequential sections of OvPV-positive tissues under probing with an unrelated probe (Figure 1, panel E), and matched sections of uninfected animals were consistently negative (not shown).

Complete clinical data were not available for all of the archived cases; however, information was available for 5 flocks. In 4 of these flocks, the affected lambs were being fed artificially, usually because of maternal death or insufficient colostrum. Therefore, insufficient colostral antibody intake may predispose to development of neurologic disease similar to that proposed for porcine teschovirus, but this remains unproven. On the basis of the finding that OvPV has already been present in Wales, Scotland, and England for [greater than or equal to] 20 years, we suspect that OvPV could be present in the intestinal tract commonly, similar to porcine teschovirus and porcine sapelovirus (8,9), but only occasionally causing disease in neonatal and early juvenile lambs.

Conclusions

In this study, we report identification of a novel neuroinvasive picornavirus associated with severe nonsuppurative encephalomyelitis and sensory ganglionitis in sheep. The virus has been associated with encephalomyelitis for [greater than or equal to] 20 years, affecting sheep in Scotland, Wales, and England in a range of sheep breeds and management systems, but solely young lambs to date. These findings could suggest that OvPV infection is usually subclinical and self-limiting, particularly in older animals. For some cases, particularly those involving multiple affected lambs in a single flock over 1 lambing season, the clinical histories of snatching at birth or artificial feeding of orphan or pet lambs indicated likely failure of transfer of colostral antibodies. The resulting colostrum deficiency may have acted as a predisposing factor. Further research is needed to determine geographic distribution, disease burden, transmission route, and other factors; a serologic assay is also needed to further analyze the distribution in the field. However, OvPV should be considered in the differential diagnosis of ovine nonsuppurative encephalomyelitis oriented on gray matter, particularly in young lambs with sensory ganglionitis.

Acknowledgments

We gratefully acknowledge Patrick Zitzow, Jenny Lorke, Bianka Hillmann, Joyce Wood. Maria Walker, and Beth Armstrong for their excellent technical assistance.

This work was supported by the German Federal Ministiy of Education and Research within the project DetektiVir (grant no. 13N13783). SRUC Veterinary Services is funded in part by the Scottish Government's Veterinary Services Programme. The Animal and Plant Health Agency (APHA) Livestock Disease Surveillance System is funded by the UK Department for Environment, Food, and Rural Affairs.

Ms. Forth is a biochemist and PhD student at the FriedrichLoeffler-Institut, Greifswald-Insel Riems, Germany. Her research interests include novel and emerging viruses, metagenomics, and high-throughput sequencing.

References

(1.) Watson PJ, Scholes SF. Polioencephalomyelitis of unknown aetiology in a heifer. Vet Rec. 2004;154:766-7.

(2.) Scheuch M, Hoper D, Beer M. RIEMS: a software pipeline for sensitive and comprehensive taxonomic classification of reads from metagenomics datasets. BMC Bioinformatics. 2015;16:69. http://dx.doi.org/10.1186/s12859-015-0503-6

(3.) Nagai M, Omatsu T, Aoki H, Kaku Y, Belsham GJ, Haga K, et al. Identification and complete genome analysis of a novel bovine picornavirus in Japan. Virus Res. 2015;210:205-12. http://dx.doi.org/10.1016/j.virusres.2015.08.001

(4.) Woo PCY, Lau SKP, Choi GKY, Huang Y, Sivakumar S, Tsoi HW, et al. Molecular epidemiology of canine picornavirus in Hong Kong and Dubai and proposal of a novel genus in Picornaviridae. Infect Genet Evol. 2016;41:191-200. http://dx.doi.org/10.1016/ j.meegid.2016.03.033

(5.) Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987;15:8125-48. http://dx.doi.org/10.1093/nar/15.20.8125

(6.) Pankovics P, Boros A, Reuter G. Novel picornavirus in domesticated common quail (Coturnix coturnix) in Hungary. Arch Virol. 2012;157:525-30. http://dx.doi.org/10.1007/s00705-011-1192-8

(7.) Lau SK, Woo PC, Yip CC, Choi GK, Wu Y, Bai R, et al. Identification of a novel feline picornavirus from the domestic cat. J Virol. 2012;86:395-405. http://dx.doi.org/10.1128/JVI.06253-11

(8.) Chiu SC, Hu SC, Chang CC, Chang CY, Huang CC, Pang VF, et al. The role of porcine teschovirus in causing diseases in endemically infected pigs. Vet Microbiol. 2012;161:88-95. http://dx.doi.org/10.1016/j.vetmic.2012.07.031

(9.) Schock A, Gurrala R, Fuller H, Foyle L, Dauber M, Martelli F, et al. Investigation into an outbreak of encephalomyelitis caused by a neuroinvasive porcine sapelovirus in the United Kingdom. Vet Microbiol. 2014;172:381-9. http://dxdoi.org/10.1016/ j.vetmic.2014.06.001

Address for correspondence: Martin Beer, Friedrich-Loeffler-Institut, Institute of Diagnostic Virology, Sudufer 10, 17493 Greifswald-Insel Riems, Germany; email: martin.beer@fli.de

Author affiliations: Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany (L.F. Forth, K. Schlottau, K. Wernike, A. Pohlmann, D. Hoper, M. Beer); SRUC Veterinary Services, Edinburgh, Scotland, UK (S.F.E. Scholes, F. Howie); University of California-Davis, Davis, California, USA (P.A. Pesavento, K. Jackson); APHA Veterinary Investigation Centre, Carmarthen, Wales, UK (A. Mackintosh); APHA Veterinary Investigation Centre, Penrith, UK (A. Carson)

DOI: https://doi.org/10.3201/eid2505.181573

Caption: Figure 1. Histologic and in situ hybridization findings in cervical segment 7 of the spinal cord of 3-week-old lamb in Scotland in study of picornavirus in lambs I with severe encephalomyelitis. A) Transverse segmental view with hematoxylin and eosin stain under low power. B) Hematoxylin and eosin stain under high power. Nonsuppurative myelitis is oriented on the ventral horn involving neuronal degeneration with satellitosis (black arrow), neuronophagia (arrowhead), and glial nodule formation (blue arrow), accompanied by perivascular mononuclear cell I accumulation and gliosis. Scale bar indicates 60 pm. C) By in situ hybridization, ovine picornavirus (OvPV) probe hybridization (red) predominates in the region of ventral horn poliomyelitis. D) Viral probe hybridization is dense in neuronal cytoplasm (black arrows) and within presumed neuronal extensions within the neuropil. Scale bar indicates 60 pm. E) Using an unrelated probe, no hybridization is detectable. F, G) Probe hybridization within scattered individual neurons within a spinal ganglion (F, hematoxylin and eosin stain; G, OvPV probe in situ hybridization). Original magnification x400.

Caption: Figure 2. Phylogenetic relation of ovine picornavirus to other picornaviruses of the genera Sapelovirus, Rabovirus, and Enterovirus, as well as unclassified picornaviruses. The maximum-likelihood phylogenetic tree is based on complete coding sequences and calculated by IQ-TREE version 1.6.5 (http://www.iqtree.org) with the best-fit model general time reversible plus empirical base frequencies plus free rate model 5. Teschovirus was included as an outgroup. Statistical supports of 100,000 ultrafast bootstraps are indicated at the nodes. Bold indicates sequences generated in this study; INSDC or GenBank accession numbers are provided. Scale bar indicates nucleotide substitutions per site.
Table 1. Investigations of neurologic signs in lambs Scotland and
Wales, 2016 and 2017 *

Year     Flock    Case information                            Case no.

2016     I        Progressive neurologic signs including     S012722-1
                  tetraparesis, recumbency, ataxia, and
                  death were observed in several lambs       S012722-2
                  beginning at 2-3 weeks of age in a flock
                  of 200 ewes that was accredited free of
                  maedi-visna virus. The lamb submitted
                  for investigation (S012722-2) was being
                  artificially reared. Additional lambs
                  showed similar clinical signs, but they
                  recovered apart from residual slight
                  paresis in one limb.

2017     I        Neurologic signs resulting from spinal      S014148
                  cord compression by a vertebral abscess.

2017     II       Approximately one third of a group of 60    S014175
                  young pet lambs (mainly orphan lambs or
                  triplet lambs because of insufficient
                  milk to rear 3 lambs) being artificially    S014176
                  reared were affected in a flock of 650
                  ewes. Clinical signs reported were stiff
                  back legs, tremors that became              S014177
                  exaggerated on handling, progressing to
                  lateral recumbency, terminal seizures,
                  and death or euthanasia. One lamb with
                  mild clinical signs recovered.

Year     EM     Tested tissue    [C.sub.q]        OvPV reads,
                                               no. (%) ([dagger])

2016      -       Cerebrum           NA                0

          +       Cerebrum          29.2          1 (0.00005)

2017      -       Cerebrum         NA NA             NP NP
                 Spinal cord

2017      +       Cerebrum       28.1 18.2      NP 1,383 (0.06)
                 Spinal cord

          +       Cerebrum       26.1 27.6           NP NP
                 Spinal cord

          +       Cerebrum       27.9 19.7       NP 590 (0.02)
                 Spinal cord

* Testing by quantitative reverse transcription PCR for ovine
picornavirus and by metagenomics. [C.sub.q], PCR cycle
quantification; EM, encephalomyelitis; NA, not applicable (Cq value
in RT-qPCR >45); NP, not performed; OvPV, ovine picornavirus; +,
confirmed nonsuppurative encephalomyelitis and ganglionitis; -, no
evidence of nonsuppurative encephalomyelitis and ganglionitis;
instead, compressive lesions in the spinal cord.

([dagger]) Number of OvPV reads in the metagenomics dataset, total
(proportion).

Table 2. Clinical history of lambs in England with nonsuppurative
encephalomyelitis and results of testing of archived samples for
presence of OvPV in spinal cord and brain *

Year    Signalment        Case information
        (case
        reference)

1998    3/week/old mule   Clinical signs of head tilt, rolling
        lamb (1454/98)    eyes and star gazing, and rapid onset of
                          ataxia, recumbency, and paddling; no
                          response to antimicrobial drugs,
                          multivitamins including B1. Negative for
                          Louping ill virus antibodies in
                          cerebrospinal fluid and serum.

2000    2/4/week/old      One of [approximately equal to] 12 lambs
        milking breed     fed artificial colostrum (snatched at
        lamb (960/00)     birth to prevent transmission of maedi-
                          visna virus) that developed neurologic
                          signs at [approximately equal to] 2
                          weeks of age, first weak in forelimbs,
                          then hindlimbs, and by the second day,
                          tremor and incoordination.

2004    Two 7/8/day/      Triplets fed proprietary powdered
        old triplet       colostrum because the ewe had
        lambs (E1028/     insufficient milk. Signs of fine tremor
        04 and E1029/     progressing to recumbency; the third
        04)               triplet developed similar neurologic
                          signs at 3 weeks of age (not submitted
                          for investigation).

2008    2-week-old        Neurologic signs not otherwise
        Texel X           specified.
        Swaledale
        (S313-04-08-1)

2010    1-week-old mule   Approximately 25 lambs have been
        X female lamb     similarly affected. Portions of fixed
        (S78-04-10-1)     juvenile ovine brain were submitted from
                          field postmortem examination with
                          history of suspected neurologic disease
                          before euthanasia.

2014    Breed and age     Preweaned lamb with neurologic signs.
        not known         Field postmortem samples submitted with
        (S247-04-14)      clinical history of "suspect swayback,"
                          thus likely clinical signs of ataxia and
                          paresis.

Year       RT-PCR      OvPV reads, no. (%)     Reference
         ([dagger])     ([double dagger])      coverage
                                              ([section])

1998      Positive         570 (0.003)            92%

2000       Cq 35           1061 (0.01)            98%

2004      Cq 32;         747,777 (3.02);       Full; full
          positive        238,627 (1.11)

2008       Cq 36           242 (0.0006)           89%

2010       Cq 24          18,572 (0.07)           Full

2014       Cq 34                NP                 NP

Year    Nucl. seq.
          identity
        ([section])

1998      90%-96%

2000      95%-96%

2004      95%; 95%

2008      93%-95%

2010        95%

2014         NP

* Cq, PCR quantification cycle; NP, not performed; nucl. seq.,
nucleotide sequence; OvPV, ovine picornavirus; RT-PCR, reverse
transcription PCR; qRT-PCR, quantitative RT-PCR.

([dagger]) RT-PCR was performed as prescreening for sample
selection for high-throughput sequencing. Where feasible, Cq values
of the qRT-PCR are indicated; positive means positive in
semi-nested PCR.

([double dagger]) Number (proportion) of OvPV reads in the complete
high-throughput sequencing dataset.

([section]) Genome coverage and nucleotide sequence identity of
the full genome or several contigs in relation to the OvPV
reference genome strain OvPV/Wales/2017/S014175 (International
Nucleotide Sequence Data Collaboration accession no. LR216008
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Title Annotation:DISPATCHES
Author:Forth, Leonie F.; Scholes, Sandra F.E.; Pesavento, Patricia A.; Jackson, Kenneth; Mackintosh, Adrien
Publication:Emerging Infectious Diseases
Article Type:Report
Date:May 1, 2019
Words:2782
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