IMMUNOGENIC COMPOSITIONS FOR NOVEL REASSORTANT MAMMALIAN ORTHEOVIRUS FROM PIGS
20220193220 · 2022-06-23
Inventors
Cpc classification
C12N2720/12032
CHEMISTRY; METALLURGY
C12N7/00
CHEMISTRY; METALLURGY
A61K2039/555
HUMAN NECESSITIES
C12N2720/12211
CHEMISTRY; METALLURGY
International classification
Abstract
An immunogenic composition for reducing the incidence or severity of subclinical and clinical signs of orthoreovirus infection is provided. The composition(s) includes at least one segment or portion thereof of orthoreovirus that is derived from a different serotype, host, or strain than at least one other segment or portion thereof. The present disclosure also provides methods for treating, preventing, and reducing the subclinical and clinical signs of orthoreovirus infection in a subject or group of subjects.
Claims
1. A composition comprising: at least two segments or portions thereof of orothoreovirus, wherein said segment is selected from the group consisting of L1, L2, L3, M1, M2, M3, S1, S2, S3, S4, of orthoreovirus and any combination thereof and wherein said portion thereof is selected from the group consisting of λ1, λ2, λ3, μ1, μ2, σ1, σ2, σ3, μNS, μNSC, σNS, σ1s of orthoreovirus; and an additional component selected from the group consisting of a stabilizer, an adjuvant, an antimicrobial, an antifungal, a preservative, and any combination thereof; wherein at least one segment or portion thereof is from a different serotype, strain, or host than another segment or portion thereof.
2. The composition of claim 1, wherein the composition comprises 10 segments.
3. The composition of claim 2, wherein the composition comprises 3 large segments, 3 medium segments, and 4 small segments.
4. The composition of claim 3, wherein said large segments comprise the L1, L2, and L3 segments, or wherein said medium segments comprise the M1, M2, and M3 segments, or wherein said small segments comprise the S1, S2, S3, and S4 segments.
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7. The composition of claim 1, wherein said portion thereof includes regions encoding for 8 structural proteins.
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9. The composition of claim 1, wherein said portion thereof includes regions encoding for 4 nonstructural proteins.
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11. The composition of claim 1, wherein said segment has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 4-13 or includes a portion thereof that encodes an amino acid sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 14-25.
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15. The composition of claim 1, wherein said composition comprises the protein encoded by the segment(s) or portion(s) thereof.
16. The composition of claim 1, wherein said segments or portions of thereof of orothoreovirus are in a killed or inactivated orthoreovirus; and wherein at least one segment or portion thereof is from a different serotype, strain, or host than another segment or portion thereof.
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31. A method of reducing the incidence and/or severity of at least one clinical or subclinical sign of infection with orthoreovirus comprising the step of administering the composition of claim 1 to a subject or group of subjects in need thereof.
32. The method of claim 31, wherein the orthoreovirus comprises 10 segments.
33. The method of claim 31, wherein the orthoreovirus comprises 3 large segments, 3 medium segments, and 4 small segments.
34. The method of claim 33, wherein said large segments comprise the L1, L2, and L3 segments, or wherein said medium segments comprise the M1, M2, and M3 segments, or wherein said small segments comprise the S1, S2, S3, and S4 segments.
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37. The method of claim 31, wherein said portion thereof includes regions encoding for 8 structural proteins.
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39. The method of claim 31, wherein said portion thereof includes regions encoding for 4 nonstructural proteins.
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41. The method of claim 31, wherein said segment has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 4-13 or includes a portion thereof that encodes an amino acid sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 14-25.
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45. The method of claim 31, wherein said composition comprises the protein encoded by the segment(s) or portion(s) thereof.
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61. A method of treating or preventing orthoreovirus comprising the step of administering the composition of claim 1 to a subject or group of subjects in need thereof.
62. The method of claim 61, wherein the orthoreovirus comprises 10 segments.
63. The method of claim 61, wherein the orthoreovirus comprises 3 large segments, 3 medium segments, and 4 small segments.
64. The method of claim 63, wherein said large segments comprise the L1, L2, and L3 segments, or wherein said medium segments comprise the M1, M2, and M3 segments, or wherein said small segments comprise the S1, S2, S3, and S4 segments.
65. (canceled)
66. (canceled)
67. The method of claim 61, wherein said portion thereof includes regions encoding for 8 structural proteins.
68. (canceled)
69. The method of claim 61, wherein said portion thereof includes regions encoding for 4 nonstructural proteins.
70. (canceled)
71. The method of claim 61, wherein said segment has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 4-13 or includes a portion thereof that encodes an amino acid sequence having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 14-25.
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75. The method of claim 61, wherein said composition comprises the protein encoded by the segment(s) or portion(s) thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0060] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.
[0061] Materials and Methods
[0062] Ethics Statement
[0063] The animal experiment to investigate pathogenicity and transmissibility of the MRV isolate in pigs was reviewed and approved by the Institutional Animal Care and Use Committee at Kansas State University and was performed in Biosafety Level 2+ animal facilities under guidance from the Comparative Medicine Group at Kansas State University.
[0064] Pigs
[0065] Twenty-one 5-week-old pigs, which were seronegative to swine influenza virus and porcine respiratory and reproductive syndrome virus, were used in this study. We also confirmed that these pigs were seronegative to MRV by testing blood samples using hemagglutinin inhibition assay, and negative to porcine epidemic diarrhea virus, transmissible gastroenteritis Virus and porcine group A rotavirus by testing rectal swab samples collected from each pig using the specific real-time qPCR assays.
[0066] Clinical Case
[0067] In March 2018, a U.S. Midwest swine farm with approximately one 1,000 3-month-old pigs experienced a severe disease event, in which more than 300 pigs showed neurological signs “down and peddling” without diarrhea and 120 pigs died. The resident veterinarian euthanized and necropsied two diseased pigs, and the tissue samples including brain, kidney, spleen, lung, liver, heart, intestine and stomach fundus from each pig were collected and submitted to Kansas State Veterinary Diagnostic Laboratory for diagnosis.
[0068] Virus Isolation and Preparation
[0069] A homogenous mixture of brain, kidney, spleen, lung, liver, heart, intestine, and stomach fundus sample from each pig was made for routine diagnostics and virus isolation. Homogenized tissues were filtered and inoculated onto a monolayer of MDCK cells, which were grown in Minimal Essential Medium (MEM), supplemented with 3% bovine serum albumin (BSA) (Sigma-Aldrich), 1 μg/ml N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich) and 1% antibiotic-antimycotic (Gibco) at 37° C. in the atmosphere with 5% CO.sub.2. The inoculated cells were monitored for cytopathic effects (CPE) every 12 hours and were blind-passaged for three passages.
[0070] Cell culture supernatant was collected and centrifuged at 5,000 rpm for 10 minutes at 4° C. to remove cellular debris after clear CPE were observed. The centrifuged sample was then processed for ultra-centrifugation with 40% sucrose at 25,000 rpm at 4° C. for 2 hours. Both RNA and DNA were extracted from the ultra-centrifuged samples (QIAamp cador Pathogen Mini Kit, QIAGEN, USA) for target-specific PCR assays and next generation sequencing.
[0071] Pan-Viral Group PCR
[0072] Pan-viral family/genus PCRs and sequencing were performed for the following viral families/genera: Coronaviridae, Herpesviridae, Orthomyxoviridae (Influenza viruses A, B and C), and Reoviridae (Aquareovirus and Orthoreovirus) (Phaneuf et al., 2013; Tong et al., 2009). First round reverse transcription PCR for RNA viruses was performed with Superscript III/Platinum Taq One Step kits (Invitrogen) and Titanium Taq (Clontech) kits for the second round PCR. First and second round PCR for DNA viruses was performed with Hot Start Ex Taq kits (Takara). A positive PCR control containing mutation-engineered synthetic RNA transcript or DNA amplicon and a negative control using nuclease-free water were included in each run. PCR products were visualized on 2% agarose gels. Positive bands of the expected size that had strong signal and without additional bands were purified using Exonuclease I (New England Biolabs) and Shrimp Alkaline Phosphatase (Roche). Samples were incubated at 37° C. for 15 minutes followed by 80° C. for 15 minutes to inactivate the Exonuclease and Shrimp Alkaline Phosphatase. Purified PCR amplicons were sequenced with the PCR primers in both directions on an ABI Prism 3130 Automated Capillary Sequencer (Applied Biosystems) using Big Dye 3.1 cycle sequencing kits (Life Technologies).
[0073] Next Generation Sequencing (NGS) and Analysis
[0074] Extracted nucleic acids (NA) were pre-amplified using a modified random amplification protocol as described previously (Tong et al., 2012). Briefly, NA samples are reverse-transcribed using a primer containing both a known sequence and a random nanomer, followed by a second primer extension reaction using the same primer. These extension fragments are then amplified by PCR using the known sequence of the extension primer. PCR amplicons obtained from the pre-amplification were purified, fragmented and used to construct libraries with dual index barcoding for Illumina sequencing on a MiSeq instrument as previously described (Li et al., 2017).
[0075] Initially, reads were assembled and classified using SURPI (Naccache et al., 2014). Subsequently, reovirus reads identified by SURPI were extracted and further analyzed by de novo assembly as well reference-based assembly with Geneious v11.1.4. Consensus sequences of all genomic segments were generated by Geneious and used for phylogenetic analysis.
[0076] Sequence and Phylogenetic Analysis
[0077] Each segment sequence of the isolate was blasted and compared with available sequences in GenBank, and the hit with the best identity for each gene was recorded. Each segment sequences of the closely related and other typical reovirus strains were downloaded for further phylogenetic analysis. Maximum likelihood phylogenetic tree of each segment based on the open reading frame was built with MEGA 5.0 using the Jukes-Cantor model with a bootstrap value of 1,000. GenBank accession numbers for each segment of this isolate are pending.
[0078] Immunofluorescence (IFA) Assay
[0079] IFA assay was employed to confirm the MRV isolate using a mouse monoclonal antibody against reovirus capsid protein μ1C (10F6; DSHB, USA). A monolayer of MDCK cells was infected with the isolated MRV for 48 hours, and then fixed with 10% methanol for 10 minutes following 3 washes using phosphate buffered saline with tween 20 (PBST). After blocking with 5% fetal bovine serum (FBS) for 1 hour at room temperature and washing cells with PBST for 3 times, the cell monolayer was incubated with anti-reovirus capsid protein μ1C monoclonal antibody with 1:50 dilution at room temperature for 2 hours. The cell monolayer was then washed for 3 times and incubated with the second antibody FITC-labeled goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, USA) with 1:200 dilution for 2 hours. After washing the cell monolayer for 3 times with PBST, fluorescence signals was observed under the microscope. Uninfected cells were processed in parallel and used as negative controls.
[0080] MRV Real-Time RT-PCR Assay
[0081] In order to detect all serotypes of MRVs, a quantitative real-time PCR (RT-qPCR_L1) was developed by targeting the conserved region of MRV L1 genes based on sequence information available in GenBank. The probe was labeled with 6-carboxyfluorescein and with 6-carboxytetramethylrhodamine at the 5′ and 3′ ends, respectively; and nucleotide information of primers and probe is summarized in Table 1. The RT-qPCR_L1 assay was performed by using qScript XLT 1-Step RT-qPCR ToughMix (Quantabio, Beverly, Mass., USA) according to the manufacturer's instructions with a total reaction system of 10 μl, which contained 5 μl of ToughMix, 0.48 μl of L1-F (12 μM), 0.48 μl of L1-R (12 μM), 0.16 μl of L1-Probe (4 μM), 1.38 μl of H.sub.2O, and 2.5 μl of RNA template. The thermos-cycling conditions were set as follows: 50° C. for 10 min, 95° C. for 1 min, then 45 cycles of at 95° C. for 10 seconds and 60° C. for 45 seconds. The analytic sensitivity of the RT-qPCR_L1 assay was assessed using 10-fold serially diluted in vitro-transcribed RNA of the MRV isolate L1, gene ranging from 1×10.sup.−2 to 1×10.sup.9 RNA molecules by two independent experiments according to above conditions.
TABLE-US-00001 TABLE 1 Primers and probe used in real-time RT-PCR assay Location of Amplicon Primers/probe sequence primers (bp) L1- 5′-TGGCAGCGDTGGATACGTTATTC-3′ 2521-2543 137 bp probe (SEQ ID NO. 1) L1-F 5′-GCGAAYTCTTCAGCRGAGGAGC-3′ 2434-2455 (SEQ ID NO. 2) L1-R 5′-CGTGARAAAGCACAGCATARAGCC-3′ 2547-2570 (SEQ ID NO. 3)
[0082] Pig Study Design
[0083] Twenty-one 5-week-old pigs were randomly divided into 2 groups including one infected and one control group. Nine pigs were inoculated with the MRV isolate (1.0×10.sup.7 TCID.sub.50/pig in 3 ml) intranasally (1.5 ml through the intranasal route) and orally (1.5 ml through the oral route), and three naïve pigs were commingled with infected pigs at 2 days post infection (dpi) to investigate virus transmission. Another nine pigs were mock-inoculated controls. Clinical signs and body temperature were monitored daily. Rectal swab sample was collected from each pig daily and nasal swabs were collected from each pig every two days. Blood samples were collected from each infected pigs at 0, 3, 5, 7, 9 and 14 dpi and from contact pigs at 0, 3, 5 and 7 days post contact (dpc). Three infected and control pigs were necropsied at 4 and 7 dpi, three contact pigs were necropsied at 7 dpc. During necropsy, tissue lesions was evaluated by an experienced pathologist; the tissues including duodenum, jejunum, ileum, colon, brain stem, lung, kidney, heart, liver and spleen were collected for further virological and histopathological analysis. The remaining three infected and control pigs were kept for 14 days to determine seroconversion.
[0084] Histopathology and Immunohistochemistry (IHC) Analysis
[0085] Tissues from brain, lung, liver, kidney, spleen, pancreas, stomach duodenum, jejunum, ileum ileocolic junction, spiral colon and descending colon collected from each pig were fixed in 10% neutral buffered formalin for further histopathological analysis. Based on initial PCR screening and clinical signs, a subset of positive tissues from infected pigs (n=3) along with matching tissues from the control animal at 4 dpi were routinely processed, and stained with hematoxylin and eosin and IHC. A board-certified veterinary pathologist evaluated histopathological lesions of each slide of stained tissues in a blinded fashion. IHC was conducted to detect MRV antigens by using the anti-reovirus capsid protein μ1C monoclonal antibody (1:80 diluted) and the second antibody Power-Vision Poly-AP anti-mouse IgG.
[0086] Hemagglutination Inhibition (HI) Assay
[0087] A total of 207 swine serum samples were collected from three states including Kansas, Texas and Minnesota. Serum sample collected from gnotobiotic, caesarian derived colostrum deprived piglets, which are MRV negative, was used for the negative control. All serum samples were treated by receptor destroying enzyme (RDE II) (DENKA SEIKEN, Japan) and adsorbed by using swine red blood cells (RBCs) prior to the HI assay. Briefly, serum samples were added into RDE II solution in the ratio of 1:3, and mixed thoroughly and incubated for 18 to 20 hours. After incubation, six volumes of 0.85% saline were added into the mixture and heated at 56° C. for 30 to 60 minutes to deactivate the RDE II. For each serum sample, 200 μl RDE II-treated serum was transferred to 96-well microtiter plates and 10 μl of 25% (v/v) swine RBCs was added into each well. After a gentle vortex, the plate was put at 4° C. for 1 hour and then centrifuged at 400×g for 10 min. The final 1:10 diluted serum samples were transferred to new 96 well plates. Four HA units of MRVs were used in the HI assay and added into serially diluted serum samples. The plates were gently mixed and incubated at room temperature for 1 hour, then 50 μl of 1% swine RBCs was added to each well and incubated at room temperature for another 1 hour. The HI titer for each sample was then determined.
[0088] Results
[0089] Initial Screening of Clinical Samples from Pigs with Neurological Signs
[0090] After receiving the tissue samples from two diseased pigs with neurological signs, the Kansas State Veterinary Diagnostic Laboratory performed a panel of routine molecular diagnosis, aerobic culture and histopathological analysis. Molecular diagnosis of tissue homogenate mixtures using RT-PCR or RT-qPCR assays showed that both porcine reproductive and respiratory syndrome virus and atypical porcine pestivirus were negative. Results of aerobic culture showed that Bordetella bronchiseptica was detected in lungs of one pig and Staphylococcus aureus was found in the brain of one pig after culturing for five days. Histopathological analysis revealed both pigs had moderate lymphohistiocytic, interstitial pneumonia in the lungs, moderate multifocal atrocytic hypertrophy and swelling with minimal gliosis in the brain and mild, lymphocytic, plasmacytic and eosinophilic enterocolitis in small intestine.
[0091] We used the tissue homogenates to test influenza A, B, C and D viruses, porcine teschovirus, sapelovirus and encephalomyocarditis virus by RT-PCR or real-time PCR assays. Results of these assays were all negative to tested pathogens. Virus isolation was performed on MDCK cells, and obvious CPE were observed at 48 hours post-infection at the third passages of the tissue homogenates from both pigs. The supernatant collected from infected cells showing CPE was tested above pathogens again, and results were negative to all these pathogens tested.
[0092] Isolation and Characterization of a Novel Reassortant MRV
[0093] Both RNA and DNA were extracted from the ultra-centrifuged supernatants collected from infected MDCK cells displaying CPE for further testing. Pan-viral group PCR for Reoviridae was positive to mammalian orthoreovirus and negative to other viral families tested. Results of NGS revealed a novel MRV strain present in the sample, and full genome sequences of all 10 segments were obtained. Sequence analysis showed that the 51 segment displays 92% homology with a bovine-derived MRV1 (C/bovine/Indiana/MRV00304/2014) detected in bovine calves in the U.S.A. in 2014 (Anbalagan, Spaans, and Hause, 2014), the M2 segment is closely related to the human D5/Jones MRV2 strain (94% homology), while the remaining eight segments are highly homologous to the swine-origin MRV3 (T3/Swine/FS03/USA/2015 and T3/Swine/BM100/USA/2015) detected in pigs in the U.S.A. (Thimmasandra Narayanappa et al., 2015). The highest nucleotide homology for each segment of the isolate compared to available sequences of MRV strains in GenBank were depicted in Table 2. Phylogenetic tree of each segment of the virus was generated and shown in
TABLE-US-00002 TABLE 2 Highest nucleotide identities of MRV strains with each gene segment of the novel reassortant MRV/Porcine/USA/2018. MRV/porcine/ ldentity MRV GenBank USA/2018 % strain Serotype Host No. L1 94 FS-03/Porcine/USA/2014 3 Pig KM820754.1 94 BM-100/Porcine/USA/2014 3 Pig KM820744.1 L2 94 T3/Bovine/Maryland/1961 3 Bovine AF378008.1 91 FS-03/Porcine/USA/2014 3 Pig KM820755.1 L3 95 FS-03/Porcine/USA/2014 3 Pig KM820756.1 95 BM-100/Porcine/USA/2014 3 Pig KM820746.1 M1 95 FS-03/Porcine/USA/2014 3 Pig KM820757.1 95 BM-100/Porcine/USA/2014 3 Pig KM820747.1 M2 94 MRV2 D5/Jones 2 Human M19355.1 91 MRV2 sR1590 2 Pig LC482242.1 M3 94 FS-03/Porcine/USA/2014 3 Pig KM820759.1 94 BM-100/Porcine/USA/2014 3 Pig KM820749.1 S1 92 C/bovine/Indiana/MRV00304/2014 1 Bovine KJ676385.1 92 T1/bovine/Maryland/Clone23/59 1 Bovine AY862134.1 S2 95 BM-100/Porcine/USA/2014 3 Pig KM820751.1 94 FS-03/Porcine/USA/2014 3 Pig KM820761.1 S3 94 FS-03/Porcine/USA/2014 3 Pig KM820762.1 93 BM-100/Porcine/USA/2014 3 Pig KM820752.1 S4 93 FS-03/Porcine/USA/2014 3 Pig KM820763.1 93 BM-100/Porcine/USA/2014 3 Pig KM820753.1
[0094] The MRV isolate was able to replicate efficiently and its titer could reach was 8 log.sub.10 TCID.sub.50 per mL in MDCK cells supplementing with TPCK-trypsin (1 μg/mL). The MRV antigen was mainly detected in the cytoplasm of infected MDCK cells by using an anti-reovirus μ1C protein monoclonal antibody 10F6 (
[0095] Sensitivity of Developed MRV Real-Time PCR
[0096] A real-time PCR (RT-qPCR_L1) was developed by targeting the conserved region of MRV L1 gene in order to quantify virus loads in samples collected from the pig study. To determine the sensitivity of the developed RT-qPCR_L1 assay, the L1 gene of the MRV isolate was cloned into a T7 promoter vector and in vitro transcribed to produce viral RNA. The analytic sensitivity of RT-qPCR_L1 assay showed a detection limit of 10 RNA copies and the cutoff of threshold cycle (CT) of this assay was 40. Further analysis showed a linear correlation of a series of RNA dilutions with R.sup.2 higher than 0.999, indicating that this assay is reproducible and quantitative. We also detected RNA samples extracted from serially diluted MRV isolate with a known titer, and results showed that the assay was able to detect 100 TCID.sub.50 per mL of the MRV virus. These results indicate that the developed RT-qPCR_L1 can be used to determine virus titer of samples collected in following pig studies.
[0097] Pathogenicity and Transmissibility of MRV in Pigs
[0098] Pigs in control group didn't show clinical signs through the length of the study. Clinical signs including fever, diarrhea and nasal discharge were observed in both infected and contact pigs. Six out of 9 infected pigs displayed fever (over 104° F.) starting at 2, 3 or 4 dpi, lasting for 2 to 3 days, and 7 out of 9 infected pigs showed diarrhea starting at 1 or 4 dpi, lasting for 1 to 4 days. Interestingly, 1 out of 9 infected pigs showed “walking discordant” (a neurological sign), starting at 2 dpi and lasting for 3 days, and 2 out of 9 infected pigs showed nasal discharge, starting at 6 dpi and lasting for 2 days. Noticeably, 2 out of 3 contact pigs developed fever, starting at 1 or 5 dpc and lasting for 1 day, and 2 out of 3 contact pigs had diarrhea, starting at 3 or 6 dpi and lasting for 2 to 3 days; 1 out of 3 showed nasal discharge, starting at 4 dpc and lasting for 2 days.
[0099] We employed the develop RT-qPCR_L1 to determine virus loads in collected samples during the pig study. No MRV viral RNA was detected in both nasal and rectal swab samples collected from control or contact pigs. MRV viral RNA was detected in rectal swab samples collected from one infected pig at 2, 4 and 5 dpi, and from one infected pig at 3 and 4 dpi and from another 2 infected pigs at 4 dpi (
[0100] MRV Viral RNA was only detected in the intestine, not in other tissues collected from both infected and contact pigs. Two infected pigs necropsied at 4 dpi and 2 contact pigs necropsied at 7 dpc showed MRV viral RNA positive in their intestine tissues. Viral RNA of 10.sup.7.69 molecules (per gram) was detected in the duodenum sample from one infected pig whose nasal swabs were positive to MRV at 4 dpi, while the intestine samples including duodenum, colon and ileum collected from another infected pigs were positive to MRV with a titer of 10.sup.5.20. 10.sup.6.80 and 10.sup.7.93 RNA molecules per gram. In contrast, viral RNA of 10.sup.5.32 molecules (per gram) was detected in the colon sample of one contact pig, while the duodenum sample from another contact pig were positive to MRV with a titer of a titer of 10.sup.7.82 RNA molecules per gram. All infected pigs seroconverted at 7 dpi with HI titer ranging from 40 to 320 (the geometric mean was 71), and the HI titer increased at 9 and 14 dpi (the geometric mean was 101 and 202 at 9 and 14 dpi, respectively). However, no HI titer was detected in contact and control pigs.
[0101] We found that 2 of 3 infected pigs which were necropsied at 4 and 7 respectively, had large and swollen mesenteric lymph nodes in contrast to control pigs. Other organs had no obvious changes in infected pigs compared to those of control pigs. Based on the positive results of initial PCR screening and clinical signs, the intestine sections from duodenum, mid jejunum and ileum and ileocecocolic junction, and bilateral sections of rostral cerebrum, thalamus, hippocampus, midbrain (colliculi), brainstem (obex, cerebral peduncles) and cerebellum from 3 infected pigs necropsied at 4 dpi were further examined. All intestine sections of 3 infected pigs had missing or exfoliating villous tip epithelium, and mild to moderate populations of lymphocytes and plasma cells and multifocally neutrophils in lamina propria (
[0102] In sections of brain, one inoculated animal (pig #20) with clinical neurological sign described as “walking discordant”, had mild to moderate, multifocal, perivascular cuffs of lymphocytes, rare plasma cells, and macrophages along with scattered minimal to moderate collections of irregular glial cells (gliosis) throughout the thalamus, midbrain, and base of cerebellum (
[0103] Sero-Prevalence of MRV in Pigs with Different Ages
[0104] To determine prevalence of MRV in pigs, a total of 228 serum samples were collected from pigs at Kansas, Texas, and Minnesota and tested by the HI assay using the novel isolate MRV/Porcine/USA/2018. These samples included 110 serum samples collected in 2018 from pigs at Minnesota and 70 samples collected in 2014 from pigs at Texas, which were approximately three-week-old; while the 47 samples collected in 2018 from pigs at Kansas which were approximately 3-month-old. The Kansas swine farm had experienced an outbreak of neurological disease while no obvious clinical signs was observed in pigs from both Texas and Minnesota. The 98% of serum samples from Kansas were positive to MRV, while serum samples from Texas and Minnesota displayed 46% and 63% positive to this virus, respectively. This results suggest the MRV is likely widespread in swine herds.
TABLE-US-00003 TABLE 3 MRV sero-prevalence in pigs at different geographical regions in the U.S. No. of No. of MRV- Positive Geometric Collection samples positive Percent HI titer Mean Time Kansas 47 46 98% 10-320 50.1 2018 Minnesota 111 70 63% 10-160 47.8 2018 Texas 70 32 46% 10-80 24.3 2014
[0105] Discussion
[0106] In this study, we isolated a novel MRV/Porcine/USA/2018 from pigs in US Midwest swine farm in which approximately 300 pigs displayed neurological signs with approximately 40% mortality. Sequence and phylogenetic analysis revealed that this isolate was a reassortant strain with the 51 gene from a bovine-derived ressortant MRV1 (Anbalagan, Spaans, and Hause, 2014) and the M2 gene close to the human MRV2 D/Jones and the remaining eight genes from a swine-derived MRV3 that caused diarrhea in piglets in the U.S. in 2015 (Thimmasandra Narayanappa et al., 2015). Like influenza A viruses, the segmented nature of MRVs leads to reassortment among different serotypes or strains to produce novel viral strains. One former study shows that a novel reassortant bat MRV virus has the 51 gene similar to those from the bovine-derived MRV1 viruses and other remaining genes from bat MRV viruses (Lelli et al., 2015) and another study reveals that MRV isolates in different bat species in China are closely related to human, swine and mink orthoreoviruses (Yang et al., 2015). The novel reassortant MRV isolate reported in this study provides further evidence on reassortment among three MRV serotypes. However, how this novel virus was generated and whether an intermediates host was needed remains unknown and needs to be investigated. Importantly, bat-origin orthoreoviruses such as Melaka virus and Kampar virus have been associated with human infections (Chua et al., 2007; Chua et al., 2008; Chua et al., 2011). The MRV3 has been recorded to infect human and multiple animal hosts, and was recently identified in alpine chamois (Besozzi et al., 2019). In addition, a novel orthoreovirus that results in acute gastroenteritis in a hospitalized child has been isolated in Europe and revealed that the virus most likely originates from bats (Steyer et al., 2013). All these results demonstrate interspecies transmission and frequent reassortment events of MRVs.
[0107] Our pig studies indicate that the MRV isolate is pathogenic and transmissible in pigs, evidenced by disease, and virus replication and transmission found in both infected and contact pigs. Noticeably, only one of 9 infected pigs showed a neurological sign “walking discordant”, while most infected and contact pigs had diarrhea. Encephalitis was histologically observed in the brain of this pig although MRV antigens were not detected. In contrast, MRV antigens were detected in the FAE in the Peyers Patch of the ileum of infected pigs with diarrhea, which corresponds to the reported M cell distribution in pigs (Kido et al., 2003) and staining patterns seen in mouse reovirus experiments (Amerongen et al., 1994), suggesting the MRV adheres to the same cells to start replication in different species. The results of the pig study are not consistent with high mortality and neurological disease observed in diseased pigs in the outbreak farm. In addition, viral RNA and antigens were only detected in the intestine rather than brain of infected pigs in our study. This discrepancy suggests that other factors might be needed in order to reproduce the disease, such as unknown pathogens, environmental factor and stress etc. as cofactors. Thus, further studies are needed to understand what kind of co-factors are required to reproduce the neurological disease observed in pigs.
[0108] Encephalitis and meningitis caused by MRV infection has been documented in infected humans (Ouattara et al., 2011; Tyler et al., 2004). The pathway of MRV infection of CNS in newborn mice has been systemically investigated. Serotype 1 reovirus spreads to CNS via the hematogenous route, resulting in self-limiting hydrocephalus, while serotype 3 strain enters CNS by neutral routes and causes lethal encephalitis (Tyler, McPhee, and Fields, 1986; Weiner et al., 1977). Further studies to investigate dissemination pathways and neural tropism using serotype 1 and 3 reassortant clones suggest that reovirus virulence to CNS may be related to specific interactions between hemagglutinin and neuronal surface receptor (Dichter and Weiner, 1984; Tardieu and Weiner, 1982; Weiner, Powers, and Fields, 1980). Based on our knowledge, the novel MRV strain described in this study is the second isolate associated with neurological disease in pigs. The first MRV related to neurological disease is a MRV2 virus which was isolated from a pig with encephalitis in Austria. In contrast to two former swine MRV3 stains identified in the U.S. in 2015 that caused diarrhea in piglets (Thimmasandra Narayanappa et al., 2015), the novel MRV isolate has a different 51 and M2 gene, suggesting that they might be responsible for inducing different phenotypes of disease in pigs. Further studies are needed to investigate their roles in virus pathogenicity and tissue tropism using reverse genetics.
[0109] We have shown a high MRV sero-prevalence in pigs in the USA at different ages, suggesting the MRV could be widespread and an important pathogen for swine industry. In humans, there is a very high MRV sero-prevalence in infants, likely related to maternal antibody because seropositive rates can be up to 75% in 0-3 month old infants and 11% in 3-6 month old babies, while it decreases to 0% in children at 6-12 months of age (Tai et al., 2005). Approximately 50% sero-positivity in post-weaned (3-week-old) piglets at two farms located in different states suggests that the MRV-seropositive could also be associated with maternal antibodies. However, 3 to 6-month-old pigs in the disease outbreak farm were 98% MRV-seropositive, indicating that MRV is capable of causing severe infections in pigs. Additionally, previous studies have revealed that MRV3 σ1-based indirect ELISA assay can also detect MRV serotype 1 strains (Li et al., 2018) and the feline MRV cross-reacts with three MRV serotypes based on the neutralization testing (Csiza, 1974). These facts suggest a potential serological cross-reaction among different serotypes of MRVs. Therefore, whether the high sero-prevalence in pigs we found is MRV1-specific or due to cross-reactivity with other serotypes, or due to maternal antibodies needs to be determined in future studies.
[0110] In conclusion, we isolate and characterize a novel reassortant MRV virus that is pathogenic and transmissible in pigs although we did not reproduce the neurological disease in pigs. Our results combined with previous studies indicate that MRV is an important pathogen for the swine industry (Thimmasandra Narayanappa et al., 2015). Therefore, further surveillance and pathogenicity studies on MRVs in pigs should be performed in order to understand viral pathogenicity and transmissibility as well as reassortment of MRVs as the novel reassortant MRV might emerge to threaten animal and public health.