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Outbreaks of Neuroinvasive Astrovirus Associated with Encephalomyelitis, Weakness, and Paralysis among Weaned Pigs, Hungary.

Astroviruses are small, nonenveloped viruses with single-stranded 6.2-7.8 kb RNA genome of positive polarity (1,2). The family Astroviridae is currently divided into 2 genera: the genus Mamastrovirus of mammal-infecting viruses and the genus Avastrovirus of avian viruses (3,4). The genetically heterogenic astroviruses that are widespread among mammals and birds are generally associated with gastroenteritis, less commonly with respiratory disease, and rarely encephalitis or disseminated infections (2,5-19). Astrovirus infections with central nervous system (CNS) involvement were reported recently in mink, human, bovine, ovine, and swine hosts (the latter in certain cases of AII type congenital tremors) (5,6,12-14). Most neuroinvasive astroviruses belong to the Virginia/Human-Mink-Ovine (VA/HMO) phylogenetic clade and cluster with enteric astroviruses identified from asymptomatic or diarrheic humans and animals (15,16). Recent research shows that pigs harbor one of the highest astrovirus diversities among mammals examined (3,15,20). Porcine astroviruses (PoAstVs) were identified mainly from diarrheic fecal specimens, less commonly from respiratory specimens, although the etiologic role of astrovirus infection in gastroenteritis or in other diseases among swine is not settled (3,9,20-23). We report the detection of neuroinvasive porcine astrovirus type 3 (Ni-PoAstV-3) by reverse transcription PCR (RT-PCR) and in situ hybridization (ISH) in recent and archived CNS samples of newly weaned paraplegic pigs from 3 highly prolific swine farms in Hungary.

Materials and Methods

Sample Collection and Handling

During November 2015-July 2017, we collected multiple tissue samples from 5 paraplegic and 5 asymptomatic pigs at the index farm located in Hungary (GD; specific location redacted) (Table 1). We also tested nasal and anal swab pairs collected by using polyester-tipped swabs from another 5 paraplegic and 13 healthy animals. We washed tissue samples twice in 10 mmol/L phosphate buffered saline (PBS) to remove excess blood and held them at -80[degrees]C until total RNA extraction. For formalin-fixed, paraffin-embedded (FFPE) blocks, we fixed the dissected samples (Table 2) with buffered 8% formaldehyde, dehydrated and embedded into paraffin.

We also analyzed archived FFPE specimens from paraplegic pigs from earlier outbreaks of posterior paraplegia in Tazlar in 2011 and in Balmazujvaros in 2014 (Table 2). The 3 swine farms are located in the central and eastern parts of Hungary, [much greater than]100 km from each other, without known connection.

Previous Laboratory Diagnostics

CNS homogenates from the index farm tested negative by PCR for the following pathogens (families in parentheses): porcine reproductive and respiratory syndrome virus (Arteriviridae); porcine circovirus 2 (Circoviridae); hemagglutinating encephalitis virus (Coronaviridae); and porcine parvovirus 1, 2, 4, and porcine bocavirus (Parvoviridae). Immunohistochemical detection of Toxoplasma gondii and West Nile virus and bacterial cultivation attempts from the CNS samples were also negative. Virus isolation attempts using brain homogenates of affected animals in swine kidney (PK-15) and Caucasian colon adenocarcinoma (Caco-2) cell lines were not successful (no cytopathic effects were visible). We detected no PoAsV type 3 (PoAstV-3) in the cell culture supernatants by nested RT-PCR with RNAdependent RNA polymerase (RdRp) primer pairs.

Total RNA Extraction and RT-PCR Screening

Treatment of FFPE samples included the deparaffination and rehydration steps, proteinase K digestion, and total RNA extraction. We used the same treatment protocols and the same reaction conditions and reagents used in the RT-PCR and nested RT-PCR reactions as are described previously, with minor modifications (24-26) (online Technical Appendix, https://wwwnc.cdc.gov/EID/article/23/13/170804-Techapp1.pdf). For the RT-PCR screening of CNS samples for the presence of pestiviruses (Flaviviridae) and swine picornaviruses (Picornaviridae), including teschovirus, enterovirus, sapelovirus, Seneca Valley virus, pasivirus, kobuvirus and encephalomyocarditis, we used virus-specific primer pairs as well as the outer and inner primer pairs targeting the RdRp or the capsid regions of PoAstV-3 (Figure 1; online Technical Appendix Tables 1, 2).

Absolute Quantification Using Quantitative RT-PCR

For the absolute quantification of viral RNA present in different tissue, urine, and fecal samples, we used the SYBR Green-based quantitative RT-PCR (RT-qPCR) method (Maxima SYBR Green qPCR Master Mix; Thermo Scientific, Waltham, MA, USA). For the generation of standard curve, we used 10-fold dilution series of purified and spectrophotometrically quantified RNA transcripts in the reactions. The RT-qPCR assays contained 3 technical repeats of all samples and standards. The slope of the standard curve was -3.4228 and the calculated PCR efficiency was 99.96%. The detailed protocol is provided in the online Technical Appendix.

Long-range Amplification, 5'/3' RACE-PCR, and Sanger Sequencing

For the complete genome (or complete 3' open reading frame [ORF] 1b-ORF2-3' untranslated region [UTR]) acquisitions of the PoAstVs, we used different long-range and 5'/3' rapid amplification of cDNA ends RT-PCRs according to previously described protocols (26,27). We designed the sequence-specific primers used for the amplification of overlapping genome fragments based on the genome of PoAstV-3 strain US-MO123 (GenBank accession no. JX556691) and closely related sequences downloaded from the GenBank database (online Technical Appendix Table 3). We sequenced PCR products directly with the BigDye Terminator v1.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Stafford, TX, USA) using the primer-walking method with an automated sequencer (ABI Prism 310 Genetic Analyzer; Applied Biosystems). We have submitted the nucleotide sequences of study astrovirus strains to GenBank under accession nos. KY073229-32.

Sequence and Phylogenetic Analyses

We aligned astrovirus sequences by using the MUSCLE web tool of EMBL-EBI (28) and performed pairwise nucleotide and amino acid identity calculations of the aligned sequences with GeneDoc version 2.7 (http://iubio.bio.indiana.edu/soft/ molbio/ibmpc/genedoc-readme.html). We constructed phylogenetic trees of deduced amino acid sequence alignments by using MEGA version 6.06 software (29) and the neighbor-joining method with the Jones-Taylor-Thornton matrixbased model. Bootstrap values were set to 1,000 replicates, and only likelihood percentages of [greater than or equal to]50% were indicated.

Histology and In Situ Hybridization

We performed chromogenic (with 3,3'-diaminobenzidine/ DAB) in situ hybridization in FFPE slides (RNAscope 2.0, Brown Kit; Advanced Cell Diagnostics, Newark, CA, USA) according to the manufacturer's instructions for viral RNA detection of Ni-PoAstV-3. We used 30 probe pairs generated at Advanced Cell Diagnostics designed to hybridize native viral Ni-PoAstV-3 RNA. Negative controls included Dap-B (dihydrodipicolinate reductase gene from Escherichia coli probe); an unrelated viral probe; and normal porcine brain region-matched sections.

Results

Clinical Observations

There are [approximately equal to]2,000 sows and their offspring in the investigated highly prolific index farm (GD). Episodes of neurologic disease of unknown etiology have persisted in the past 2 years. The syndrome affects an average of 30-40 weaned pigs monthly (1.5%-2% of total), although the number of monthly cases infrequently rose to [approximately equal to]80 pigs (4%) in the autumn-winter seasons. The clinical signs of posterior leg weakness or paraplegia and pitching (stage 1); later paralysis of both legs and skin pain (stage 2); or loss of consciousness, paresis, and serious flaccid paralysis of muscles (stage 3) typically appear among weaned pigs 25-35 days old, 1 week after the weaning procedure (Video). We did not observe gastroenteric symptoms. All of the affected pigs in stage 3 of the disease were unable to eat or drink; they died due to exsiccosis (dehydration) or were euthanized. Signs persisted typically for 1 week before death or euthanasia. Postmortem examination results showed no signs of mechanical damage (fractures, abscesses, or hemivertebrae). Pigs are vaccinated against porcine circovirus 2, Mycoplasma hyopneumoniae, and Actinobacillus pleuropneumoniae. Preventive amoxicillin treatment of the piglets was done routinely at weaning. Due to the preventive measures in effect as of spring 2017, which included extensive decontamination of the piggeries and the physical separation of the newly weaned pigs from different litters, the number of encephalomyelitis cases among weaned pigs decreased with only 1-2 cases/month observed on the index farm.

The 2 additionally examined swine farms located in Tazlar and Balmazujvaros each held approximately 500 sows and their offspring. Similar symptoms of staggering and paralysis appeared among pigs 3-5 weeks old in outbreaks in 2011 (Tazlar) and 2014 (Balmazujvaros).

Detection and Analysis of Astroviruses from CNS Samples of Affected Animals

In March 2016, we collected brain stem, spinal cord, nasal swab, and fecal samples from a newly weaned pig from index farm GD (GD-1, index animal) that showed signs of encephalomyelitis and posterior paraplegia (stage 1). The brain stem and spinal cord samples tested negative by RT-PCR for pestivirus (family Flaviviridae) and several swine-infecting picornaviruses (family Picornaviridae) (online Technical Appendix Table 1). On the basis of the increasing evidence of the pathogenic role of neurotropic astroviruses among humans and farm animals (5,6,14,17,30) we investigated the presence of astrovirus using panastrovirus PCR primers (online Technical Appendix Table 1) (31). The brain stem and spinal cord samples showed strong RT-PCR positivity. The panastrovirus PCR products were sequenced using panastrovirus PCR primers (online Technical Appendix Table 1) and compared to each other and to the available astroviruses using blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The 397-nt sequences of brain stem and spinal cord were identical and showed 89% nt identity to PoAstV-3 isolate US-MO123 (GenBank accession no. JX556691) as the closest match (32).

We sequenced 2 samples from the index animal: the full-length genome of the neuroinvasive astrovirus strain NI-Brain/9-2016a/HUN (GenBank accession no. KY073229) from the brain stem sample and the complete capsid-encoding ORF2 from the spinal cord sample NI-SC/9-2016a/HUN (GenBank accession no. KY073230). The 6393-nt (without the poly[A] tail) complete genome showed the typical astrovirus genome organization with 3 putative ORFs, 2529 nt (ORF1a), 1527 nt (ORF1b), and 2265 nt (ORF2), flanked by short 5' and 3' UTRs (Figure 1). We identified the conserved proteolytic cleavage site ([V.sub.561]H[Q.sup.-]TNT) of serine protease (ORF1a) and the conserved [Y.sub.358]GDD motif of the RdRp (ORF 1b) (33). The nonstructural proteins of ORF 1a (842 aa) and ORF 1b (508 aa) and the capsid protein of ORF2 (754 aa) showed 93%, 95%, and 93% aa identity, respectively, to the corresponding genome parts of the closest known relative PoAstV-3 strain, US-MO123. All of the conserved genomic features of mamastroviruses were present in strain NI-Brain/9-2016a/HUN: the conserved [C.sub.1]CAAA pentamer at the 5' end of the genome; the frame-shift heptamer motif ([A.sub.2511]AAAAAC) followed by a stem-loop structure at the 3' end of ORFla; the conserved sgRNA promoter sequence motif of [U.sub.4048]UUGGAGgGGaGGACCaAA[N.sub.g][AUG.bar]gC (variable nts are in lowercase, start codon of ORF2 is underlined) at the junction of ORF1b/ORF2; and the stem loop II--like motif (s2m) in the 3' end of the genome between nt position=s 6322 and 6353. The 3' UTR of NI-Brain/9-2016a/HUN is 27 nt shorter and did not contain the short sequence repeat found at the 3' end of strain US-MO123 ([G.sub.6381/6392]AUUUCUUUNA). Based on the high sequence identity and the similar genomic features, the NI-Brain/9-2016a/HUN strain most likely belongs to the PoAstV-3 genotype. The ORF2 of NI-Brain/9-2016a/HUN shares 99% nt/aa identity with the corresponding capsid gene of NI-SC/9-2016a/HUN from the spinal cord of the same animal, suggesting that the same virus was present in both regions of the CNS.

We detected Ni-PoAstV-3 using RT-PCR in all CNS samples collected from another 4 affected newly weaned pigs held in the index farm (Table 1). All of the samples from the asymptomatic control animals were Ni-PoAstV-3 negative.

We determined the complete genomes of 2 Ni-PoAstV-3 strains (NI-Brain/173-2016a/HUN, GenBank accession no. KY073231; and NI-Brain/386-2015/HUN, accession no. KY073232) that originated from 2 affected animals (GD-3 and GD-5) in stage 3 of the disease, chosen at different times (July 2016 and November 2015) of the outbreak (Table 1). These isolates showed 99.5%, 100%, and 98.7%-99.2% aa identities, respectively, to NI-Brain/9-2016a/HUN in the ORF1a, ORF1b, and ORF2 (capsid) regions.

Most of the aa differences between the Ni-PoAstV-3 study strains and the other enteric PoAstV-3 strains are located in the N-terminal part of ORF1a and in the C-terminal part of ORF2 (Table 3). Phylogenetic analysis showed a close relationship between the identified Ni-PoAstV-3 sequences and the known PoAstV-3 strains located within the same larger clade containing most other mamastroviruses with known neurotropic potential (Figure 2).

Detection of Ni-PoAstV-3 in Non-CNS Samples

We detected Ni-PoAstV-3 in multiple non-CNS samples from the respiratory system, lymphoid system, circulatory system, and salivary glands of affected animals (Table 1). We detected virus only in the second PCR round in 1 ileum sample and in 2 of the 3 analyzed fecal samples using nested RT-PCR (Table 1). Samples from internal organs (spleen and kidney) and urine samples tested negative by nested RT-PCR (Table 1).

We determined the copy number of Ni-PoAstV-3 using SYBR Green-based -qPCR. All of the samples that showed nested RT-PCR positivity only in the second (nested) PCR round had negative test results by RT-qPCR, indicating low copy number (<100 copies/[micro]g total RNA) of the virus in that tissue sample. The highest copy number was detected in the brain stem, followed by the spinal cord (Figure 3). Of note, we detected relatively high copy numbers in the tonsil and nasal mucosa samples (Figure 3). The serum of animal GD-3 contained 2.07 x [10.sup.6] virus copies/mL and of animal GD-4 1.64 x [10.sup.3] virus copies/mL.

To validate the general presence of Ni-PoAstV-3 in the respiratory system and the absence of the virus in the feces during the acute phase of the illness, we collected additional nasal and anal swab pairs from 5 affected pigs and 13 clinically healthy pigs of the same age ([approximately equal to]25-35 days) from the index farm. Four (80%) of the 5 nasal swab samples from affected animals tested positive but all of the anal swab samples tested negative using nested RT-PCR with primers targeting the RdRp region of Ni-PoAstV-3. The nasal and anal swab samples of the asymptomatic animals were all negative by nested RT-PCR. Because we collected varying amounts of samples by polyester-tipped swabs, we did not perform absolute quantification of Ni-PoAstV-3 by RT-qPCR.

Detection of Ni-PoAstV-3 in Archived FFPE Samples

All but 1 archived FFPE samples from Tazlar and Balmazujvaros were positive by nested RT-PCR for Ni-PoAstV-3 using 2 sets of primer pairs targeting the RdRp and capsid genes of Ni-PoAstV-3 (Table 2; Figure 1; online Technical Appendix Table 2). The spinal cord FFPE sample from Balmazujvaros had a negative result using both nested RT-PCR primer sets. The nested RT-PCR positive samples had positive results by ISH (data not shown).

Histology and ISH

Histologically, shared CNS lesions among the animals examined were moderate to marked lymphohistiocytic cell perivascular cuffing with marked vasculitis and neuronal degeneration, necrosis, and neurophagia with multifocal microgliosis and satellitosis (Figure 4). The neuronal necrosis was especially evident in the dorsal and ventral horns of the cervical spinal cord gray matter, although it was also detected in neurons of the Purkinje layer (cerebellum), the medulla oblongata, cerebellar peduncles, and midbrain (Figure 5). Necrotic neurons were variously swollen and hypereosinophilic or shrunken with tinctorial changes including faded, amphophilic, or eosinophilic cytoplasm (Figure 5). Nuclei of affected neurons are pyknotic, karyorrhectic, or losing border definition within the cytoplasm. We performed ISH on 5 affected animals (Table 2). Ni-PoAstV-3 hybridization was predominantly restricted to neurons, including those with visible necrosis and, in the cerebellum in particular, some that were histologically unaffected, although some regions of gliosis (presumed inflammation after neuronal necrosis) also contained viral RNA (Figure 5, panel M). Hybridization was distinct, with punctate to diffuse cytoplasmic staining throughout the cytoplasm. The unique microarchitecture of the Purkinje layer of the cerebellum offered the clear demonstration that viral nucleic acid was present within dendritic processes coursing through the molecular layer (Figure 5, panels G, J). We found no pathologic lesions in other samples from kidneys, liver, gastrointestinal tract, or immune system (data not shown). The samples from the immune system were also negative by Ni-PoAstV-3 ISH (Table 2).

Discussion

We detected astrovirus RNA in multiple tissues collected during 2015-2017 from newly weaned pigs with encephalomyelitis and posterior paraplegia of unknown origin, with the highest viral load detected in brain stem and spinal cord samples. We detected the same virus in archived brain and spinal cord FFPE samples from similarly affected animals from 2 additional swine herds collected in 2011 and 2014. These data indicate that a genetically similar, neurovirulent astrovirus is circulating in multiple swine farms since 2011 or earlier in Hungary.

According to the refined classification for the assessment of causation (34), the Ni-PoAstV-3 and the observed encephalitis and paraplegia are in a probable causal relationship (Level 2). Paraplegia associated with astrovirus neuroinfection is not unprecedented; minks had astrovirus-induced "shaking mink syndrome" and were reported paraplegic at the final stage of the disease (12,35).

Neurologic signs were observable mainly among newly weaned pigs (Video, https://wwwnc.cdc.gov/EID/ article/23/12/17-0804-V1.htm). The time of weaning, which involves nutritional (from milk to solid feed), social (mixing with different litters without the sow), and environmental (moving to a new pen) changes, is known to be the most stressful period in a pig's lifetime and is associated with dysfunction of the immune system (36). Furthermore, the inadequate quantity and quality of colostrum intake of sucking piglets, and therefore the presumably low level of specific maternal antibodies due to highly prolific sows with large litters in the index farm, might also contribute to the emergence of the clinical disease. Decreased immune status was frequently present with extraintestinal dissemination of astroviruses in humans and in mice (5,37-41).

Our sequence analyses indicate that the identified astrovirus strains belong to the PoAstV-3 genotype, which clusters within the VA/HMO phylogenetic clade (Figure 2), as do most mammalian strains with known neurotropic potential (6,14,19). However, other canonical human astroviruses outside of the VA/HMO clade could also be associated with CNS disease (41). At the molecular level, the most conspicuous difference between the genomes of neuroinvasive virus and the enteric PoAstV-3 strain U.S.-MO123 is the 27 nt deletions found in the 3' UTR of the CNS-associated astroviruses. The possible impact of this 3' UTR deletion on viral tropism is unknown, although neuroinvasive bovine astroviruses also possess 3' UTR architecture that differs from the diarrhea-associated astroviruses (42).

At the amino acid level, one of the most divergent regions between the neuroinvasive and other PoAstV-3 strains was found at the receptor-interaction domain of ORF2 (Table 3), which contains potential receptor binding sites (43,44). This finding could indicate an altered receptor spectrum and therefore altered tissue tropism of neuroinvasive and enteric PoAstV-3 strains.

PoAstV-3 strains were previously detected only from fecal samples of healthy or diarrheic piglets worldwide (20,22,45). We found that Ni-PoAstV-3 was either undetectable or detected only at low viral loads in the analyzed fecal samples, whereas the virus was generally detectable in the respiratory system of paraplegic pigs. This finding may indicate that CNS infection and replication occur later than enteric replication or that initial replication occurs extraintestinally (e.g., in the respiratory tract). Multiple types of astroviruses were recently identified from nasopharyngeal swabs or lung tissue samples from swine, bovines, and humans with respiratory symptoms including the neurotropic human VA1 strain from a patient with febrile acute respiratory disease (9-11,23), although neither the respiratory tropism nor the airborne transmission of astroviruses has been experimentally confirmed. Therefore, testing of only fecal samples from sick animals may result in underestimation of the incidence of astrovirus in pigs.

We measured the highest viral loads of Ni-PoAstV-3 in brain and spinal cord samples, similar to those found in diseased ovine and human patients with astrovirus-associated encephalitis (5,14). Ni-PoAstV-3 was also detectable in serum specimens and multiple organs of the respiratory, lymphoid, and cardiovascular systems of diseased swine. These results indicate that Ni-PoAstV-3 can result in viremia and disseminated infection involving the brain, spinal cord, and multiple organs during the acute phase of encephalomyelitis and posterior paraplegia. Astroviruses seem to play a role in a common and severe disease (encephalomyelitis and paralysis) in pigs.

The observable histopathologic changes, as well as the neuronal localizations of Ni-PoAstV-3 RNA in CNS samples of paraplegic pigs, are comparable to astrovirus-associated encephalitic cases of minks, humans, and cattle. Similar neuronal degeneration or necrosis with microgliosis in the brain or cerebellum, as well as inflammation of gray matter of the spinal cord, were previously described in cattle with astrovirus-associated nonsuppurative encephalitis (6,35,46,47), which suggests the general course of an astrovirus neuroinfection.

While some astroviruses are known to cause outbreaks of gastroenteritis, astrovirus-associated encephalitis cases have been reported only sporadically among humans, cattle, and sheep (6,14-16,47). The constant presence with recurrent increases of neurologic disease cases in swine farms indicates that natural neuroinvasive astrovirus infections may cause common, severe, persistent epidemics among domestic pigs and constitute an economically important agent threatening livestock and even humans, considering the possible zoonotic and recombinant potential of astroviruses (48).

Our results must be interpreted in the light of some potential limitations, which are currently true for other astrovirus-associated encephalitis studies: the absence of experimental evidence such as in vivo inoculation experiments, which could clarify the true causality between the astrovirus neuroinfection and the manifested CNS symptoms; and the roles of presumed respiratory replication and decreased immune state. Therefore, despite a growing body of scientific data regarding the presence of astroviruses in CNS in different animals, the direct association of astrovirus neuroinfection and encephalomyelitis should be treated with caution. Newly weaned pigs could potentially provide an in vivo animal model to study and clarify this association.

Acknowledgments

We thank Peter Engelmann for help in the cloning experiments.

This work was supported by grants from the Hungarian Scientific Research Fund (OTKA/NKFIH K111615) and Blood System Research Institute. A.B. and P.P. were supported by the Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences.

Dr. Boros is a molecular virologist at the Regional Laboratory of Virology, ANTSZ Regional Institute of State Public Health Service, Pecs, Hungary. His research interests include virus discovery and viral infectious diseases.

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Address for correspondence: Gabor Reuter, Department of Medical Microbiology and Immunology, University of Pecs, Szigeti ut 12, H-7624 Pecs, Hungary; email: reuter.gabor@gmail.com

Akos Boros, Mihaly Albert, Peter Pankovics, Hunor Biro, Patricia A. Pesavento, Tung Gia Phan, Eric Delwart, Gabor Reuter

Author affiliations: ANTSZ Regional Institute of State Public Health Service, Pecs, Hungary (A. Boros, P Pankovics, G. Reuter); University of Pecs, Pecs (A. Boros, P. Pankovics, G. Reuter); Ceva Phylaxia Ltd., Budapest, Hungary (M. Albert); SHP Ltd., Kaposvar, Hungary (H. Biro); University of California, Davis, California, USA (P.A. Pesavento); Blood Systems Research Institute, San Francisco, California, USA (T.G. Phan, E. Delwart); University of California, San Francisco (E. Delwart)

DOI: https://doi.org/10.3201/eid2312.170804

Caption: Figure 1. Genome map of the neuroinvasive PoAstV-3 strain NI-Brain/9-2016a/HUN (GenBank accession no. KY073229) from a symptomatic newly weaned pig from a farm in Hungary together with the location of RT-PCR products used for different astrovirus screening reactions and quantitative RT-PCR analyses. The black arrow indicates the possible localization of a ribosomal frame-shift during the synthesis of ORF1ab peptide. The first and last nucleotide positions of the ORFs are marked with numbers at the top and bottom of each box. ORF, open reading frame; PanAstV, panastrovirus; PoAstV-3, porcine astrovirus type 3; RT-PCR, reverse transcription PCR; UTR, untranslated region.

Caption: Figure 2. Phylogenetic analyses of the amino acid sequences of PoAstV-3 isolates (bold) from 3 symptomatic newly weaned pigs (GD-1, GD-3, and GD-5; see Table 1) from a farm in Hungary compared with reference isolates. A) ORF1a; B) ORF1b; C) ORF2. We included in the analysis available strains of the closest relatives (identified by blastx search [https://blast.ncbi.nlm.nih.gov/ Blast.cgi]) of neuroinvasive PoAstV-3, all of the known porcine astroviruses with available complete coding sequences, all of the representative astrovirus strains with neurotrophic potential (white arrows), and some representative astrovirus sequences; GenBank accession numbers are in brackets. Gray boxes indicate the Virginia/Human-Mink-Ovine clade, which contains most of the neurotrophic astroviruses. Scale bars indicate amino acid substitutions per site. AAstV, avastrovirus; AstV, astrovirus; GG, genogroup; MAstV: mamastrovirus; ORF, open reading frame; PoAstV-3, porcine astrovirus type 3.

Caption: Figure 3. Logarithmic graph of the viral copy numbers of porcine astrovirus type 3 (PoAstV-3) in different organs determined by SYBR Green-based quantitative reverse transcription PCR (RT-qPCR) of samples from 5 symptomatic newly weaned pigs (GD-1-5; see Table 1) from a farm in Hungary. All the samples, which were positive for PoAstV-3 only by nested RT-PCR, were found negative by quantitative RT-PCR. BS, brain stem; CNS, central nervous system; NM, nasal mucosa; SC-C/T/L, cervical, thoracic, or lumbar spinal cord; SG, salivary gland.

Caption: Figure 4. Tissue sections of cervical spinal cord (A), brain stem (B, C) and cerebellum (D) stained with hematoxylin and eosin from a symptomatic newly weaned pig from a farm in Hungary show the signs of stage 3 encephalomyelitis. Mononuclear perivascular cuffs with vasculitis (black arrowheads), neuronal necrosis (white arrowheads), neurophagia (white double arrowheads), multifocal microgliosis, and signs of meningitis (black arrows) are shown. Asterisk (*) indicates blood vessel. Scale bars indicate 50 [micro]m (panels A, D) or 20 [micro]m (panel A inset; panels B, C).

Caption: Figure 5. Results of histopathologic testing of central nervous system tissues from 2 symptomatic newly weaned pigs from a farm in Hungary. Sections of the cervical spinal cord (A-E), cerebellum (F-J), and cortex (L, M) from the index animal (GD-1) and the brain stem (K) from an additional affected stage 1 animal (GD-11). A, D, F, I, L) Hematoxylin and eosin stain. Gliosis (black arrows) is multifocal within the gray matter (panels A, D) and in the molecular layers (panels F, I, L, and M). Neuronal degeneration and necrosis are evident by hypereosinophilia, angular degeneration, loss of neuronal detail, and vacuolation (double arrows in panels A, D). Some Purkinje neurons are slightly angular with mild vacuolation (double arrowheads in panel I). B, E, G, J, K, M) In situ hybridization of neuroinvasive porcine astrovirus. Hybridization of the neuroinvasive porcine astrovirus probe is restricted to neurons (white arrowheads in panels B, E, K) or limited to Purkinje neurons (double black arrowheads in panels G, J) with extension into dendritic processes that course through the molecular layer (black arrowheads in panels G, J). Hybridization of the neuroinvasive porcine astrovirus type 3 probe (black arrowhead in panel M) is present in the gliosis (black arrows in panels L, M). C, H) Using a control probe on a serial section, no hybridization is detectable. In situ hybridization. Scale bars indicate 500 [micro]m (panels A-C, F-H) or 50 [micro]m (panels D, E, I-M).
Table 1. Data on 5 symptomatic and 5 control newly weaned pigs
from a farm in Hungary and results of PoAstV-3 screening by
nested RT-PCR of samples collected during 2015-2017 *

Data                 Symptomatic animals ([dagger])

                                GD-1

Collection month                2016
                                 Mar
Age, d                           25
Clinical signs                   PP
(disease stage)                  (1)
Brain stem                        +
Spinal cord
  Cervical                       NA
  Thoracic                       NA
  Lumbar                          +
Nasal mucosa          - (+) ([double dagger])
Lung                             NA
Tonsils                          NA
Salivary glands                  NA
Myocardium                       NA
Feces                           - (-)
Ileum                            NA
Lymph nodes
  Mesenterial                    NA
  Submandibular                  NA
Urine                            NA
Kidney                           NA
Liver                            NA
Spleen                           NA
Serum                            NA

Data                           Symptomatic animals ([dagger])

                              GD-2            GD-3        GD-4

Collection month              2016            2016        2016
                              Mar              Jul         Jul
Age, d                         25              25          25
Clinical signs                 PP              PP          PP
(disease stage)               (1)              (3)         (3)
Brain stem                     +                +           +
Spinal cord
  Cervical                     NA               +           +
  Thoracic                     NA               +           +
  Lumbar                       NA               +           +
Nasal mucosa          + ([double dagger])       +           +
Lung                           NA               +           +
Tonsils                      - (-)              +           +
Salivary glands                NA             - (+)         +
Myocardium                     +               NA          NA
Feces                          NA             - (+)       - (+)
Ileum                          NA             - (-)       - (+)
Lymph nodes
  Mesenterial                - (-)            - (-)       - (-)
  Submandibular                NA             - (+)         +
Urine                          NA             - (-)       - (-)
Kidney                         NA             - (-)       - (-)
Liver                          NA             - (+)       - (+)
Spleen                         NA             - (-)       - (-)
Serum                          NA               +           +

Data                  Symptomatic         Asymptomatic control
                    animals ([dagger])     animals ([dagger])

                          GD-5                    GD-6

Collection month          2015                    2016
                          Nov                     Jul
Age, d                     35                      35
Clinical signs           PP (3)                   None
(disease stage)
Brain stem                 +                     - (-)
Spinal cord
  Cervical                 NA                      NA
  Thoracic                 NA                      NA
  Lumbar                   +                     - (-)
Nasal mucosa               NA                    - (-)
Lung                       NA                      NA
Tonsils                    +                     - (-)
Salivary glands            NA                      NA
Myocardium                 +                       NA
Feces                      NA                      NA
Ileum                    - (-)                   - (-)
Lymph nodes
  Mesenterial              NA                    - (-)
  Submandibular            NA                      NA
Urine                      NA                      NA
Kidney                     NA                      NA
Liver                      NA                      NA
Spleen                     NA                      NA
Serum                      NA                      NA

Data                   Asymptomatic control
                        animals ([dagger])

                                GD-7

Collection month                2017
                                 Jun
Age, d                           25
Clinical signs                  None
(disease stage)
Brain stem                      - (-)
Spinal cord
  Cervical                      - (-)
  Thoracic                      - (-)
  Lumbar                        - (-)
Nasal mucosa          - (-) ([double dagger])
Lung                             NA
Tonsils                          NA
Salivary glands                  NA
Myocardium                       NA
Feces                           - (-)
Ileum                            NA
Lymph nodes
  Mesenterial                    NA
  Submandibular                  NA
Urine                            NA
Kidney                           NA
Liver                            NA
Spleen                           NA
Serum                            NA

Data                     Asymptomatic control
                          animals ([dagger])

                                GD-8

Collection month                2017
                                 Jun
Age, d                           25
Clinical signs                  None
(disease stage)
Brain stem                      - (-)
Spinal cord
  Cervical                      - (-)
  Thoracic                      - (-)
  Lumbar                        - (-)
Nasal mucosa          - (-) ([double dagger])
Lung                             NA
Tonsils                          NA
Salivary glands                  NA
Myocardium                       NA
Feces                           - (-)
Ileum                            NA
Lymph nodes
  Mesenterial                    NA
  Submandibular                  NA
Urine                            NA
Kidney                           NA
Liver                            NA
Spleen                           NA
Serum                            NA

Data                    Asymptomatic control
                         animals ([dagger])

                                GD-9

Collection month                2017
                                 Jun
Age, d                           25
Clinical signs                  None
(disease stage)
Brain stem                      - (-)
Spinal cord
  Cervical                       NA
  Thoracic                       NA
  Lumbar                         NA
Nasal mucosa          - (-) ([double dagger])
Lung                             NA
Tonsils                          NA
Salivary glands                  NA
Myocardium                       NA
Feces                           - (-)
Ileum                            NA
Lymph nodes
  Mesenterial                    NA
  Submandibular                  NA
Urine                            NA
Kidney                           NA
Liver                            NA
Spleen                           NA
Serum                            NA

Data                    Asymptomatic control
                         animals ([dagger])

                                GD-10

Collection month                2017
                                 Jun
Age, d                           35
Clinical signs                  None
(disease stage)
Brain stem                      - (-)
Spinal cord
  Cervical                       NA
  Thoracic                       NA
  Lumbar                         NA
Nasal mucosa          - (-) ([double dagger])
Lung                             NA
Tonsils                          NA
Salivary glands                  NA
Myocardium                       NA
Feces                           - (-)
Ileum                            NA
Lymph nodes
  Mesenterial                    NA
  Submandibular                  NA
Urine                            NA
Kidney                           NA
Liver                            NA
Spleen                           NA
Serum                            NA

* We collected tissues from 5 affected pigs with encephalomyelitis
and PP and 5 asymptomatic control animals from the index farm.
The screening nested RT-PCR primers are designed to the
RNA-dependent RNA polymerase region of PoAstV-3. NA, no available
sample; PoAstV-3, porcine astrovirus type 3; PP, posterior
paraplegia; RT-PCR, reverse transcription PCR; +, positive;
-, negative.

([dagger]) Symbols indicate results from first PCR reactions;
symbols in parentheses indicate results from second (nested)
RT-PCR reactions.

([double dagger]) Nasal swab sample.

Table 2. Results of PoAstV-3 detection, histology, and ISH analyses
using formalin-fixed, paraffin-embedded blocks of samples from 3
symptomatic newly weaned pigs from a farm in Hungary and samples
from 2 other farms with symptomatic pigs *

                  Collection
Farm ID               year            Animal ID

GD                    2016              GD-1
                                        GD-2
                      2015        GD-11 ([section])

Tazlar                2011              TAZ-1

Balmazujvaros         2014              BAM-1

                                        Nested RT-PCR ([dagger])
                  FFPE block
Farm ID                ID               RdRp              Capsid

GD                   GD-1A              - (+)             - (+)
                     GD-2A              - (-)             - (-)
                     GD-11A             - (+)             - (+)

Tazlar               TAZ-1A             - (+)             - (+)
                     TAZ-1B             - (+)             - (+)

Balmazujvaros        BAM-1A             - (-)             - (-)
                     BAM-1B             - (+)             - (+)

                                        ISH
                                      ([double
Farm ID           Tissue samples      dagger])

GD                Spinal cord             +
                   Brainstem              +
                   Cerebellum             +
                  Medulla oblonga         -
                   Lymph node             -
                     Tonsil               -
                   Myocardium             -
                     Spleen               -
                     Thymus               -
                   Brainstem              +
                   Cerebellum             +

Tazlar            Hippocampus             -
                   Brainstem              +
                  Spinal cord             +

Balmazujvaros     Spinal cord             -
                   Brainstem              +
                   Cerebellum             +

* The screening RT-PCR primers are designed to either the
RdRp or the capsid region of PoAstV-3. GD, index farm;
ID, identification; ISH, in situ hybridization; PoAstV-3,
porcine astrovirus type 3; RdRp: RNA-dependent RNA polymerase;
RT-PCR, reverse transcription PCR; +, positive; -, negative.

([dagger]) Symbols indicate the results of the first screening
PCRs; symbols in parentheses indicate the results of the
second (nested) RT-PCR. The results of nested RT-PCR refer
to a mixture of tissues embedded into the total of 7 paraffin
blocks.

([double dagger]) Indicates results for neuroinvasive PoAstV-3.

([section]) FFPE samples were the only specimens taken from
this animal.

Table 3. Amino acid differences between neuroinvasive PoAstV-3
strains from 3 symptomatic newly weaned pigs from a farm in
Hungary and reference enteric PoAstV-3 strains detected from
fecal samples *

                                        Genomic region

Category                    ORF1a       ORF1a    ORF1a      ORF1a

Amino acid position         1-400       1-400   401-844    401-844

PoAstV-3 type                 Ni         Ent       Ni        Ent
Amino acid changes           M25         S/L      F408        L
                             Y41          F       I434        V
                             R117         K       S481        P
                             T120        S/L      S576       T/V
                             T122        S/L      G608        N
                            K151G        RC       N646        H
                             L170         M       E679        D
                             L179         M
                             M185         L
                             D208        E/N
                         D202S[P/Q]-    NPTDG
                            P217A        TT
                          T220[V/A]      IS
                             P224        H/R
                             I299         V
                             E332         D
                             V338        L/I
                             L346         F
                             I369         V

                                       Genomic region

Category                    ORF1a       ORF1b   ORF1b    ORF2

Amino acid position         1-400       1-508   1-508    1-415
                                                         (AD)
PoAstV-3 type                 Ni         Ni      Ent      Ni
Amino acid changes           M25         N54      D      R29N
                             Y41        D106G   [A/E]D    S34
                             R117       A181      S       R38
                             T120       I206      V       V55
                             T122       R213      K      T57R
                            K151G       Y293     H/N      T61
                             L170       E343      D
                             L179       K375      R
                             M185       I378      T
                             D208       N382      D
                         D202S[P/Q]-    I415     A/T
                            P217A
                          T220[V/A]
                             P224
                             I299
                             E332
                             V338
                             L346
                             I369

                                      Genomic region

Category                    ORF1a          ORF2        ORF2

Amino acid position         1-400         1-415      416-754
                                           (AD)       (RID)
PoAstV-3 type                 Ni           Ent          Ni
Amino acid changes           M25        KT[I/A/V]      L439
                             Y41            R          S453
                             R117           Y          F457
                             T120           T          Y559
                             T122           SK       A570[P]
                            K151G           A        N572[Y]
                             L170                      D581
                             L179                      I601
                             M185                      S617
                             D208                      T628
                         D202S[P/Q]-                   S678
                            P217A                      I696
                          T220[V/A]
                             P224
                             I299
                             E332
                             V338
                             L346
                             I369

                               Genomic region

Category                    ORF1a         ORF2

Amino acid position         1-400       416-754
                                         (RID)
PoAstV-3 type                 Ni          Ent
Amino acid changes           M25         H[P/V]
                             Y41           D
                             R117          Y
                             T120          F
                             T122          N
                            K151G          D
                             L170          N
                             L179          V
                             M185          N
                             D208          S
                         D202S[P/Q]-       T
                            P217A          V
                          T220[V/A]
                             P224
                             I299
                             E332
                             V338
                             L346
                             I369

* We identified 3 PoAstV-3 isolates: NI-Brain/9-2016a/HUN (GenBank
accession no. KY073230); NI-Brain/173-2016a/HUN (accession no.
KY073231); and NI-Brain/386-2015/HUN (accession no. KY073232).
We compared these with enteric strains from GenBank (accession
nos. JX556691, LC201595-7, and LC201599). AD, presumed particle
assembly domain; Ent, enteric; Ni, neuroinvasive; ORF, open reading
frame; PoAstV-3, porcine astrovirus type 3; RID, presumed
receptor-interaction domain.
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Title Annotation:RESEARCH
Author:Boros, Akos; Albert, Mihaly; Pankovics, Peter; Biro, Hunor; Pesavento, Patricia A.; Phan, Tung Gia;
Publication:Emerging Infectious Diseases
Geographic Code:4EXHU
Date:Dec 1, 2017
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