Patent Application
20180326034
The Sequence Listing associated with the application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is SequenceListing.txt. The text file is 213 kilobytes, was created on Nov. 16, 2015 and is being submitted electronically via EFS-Web.
This invention relates generally to compositions and methods for diagnosis and prophylactic vaccines for newly emerging mammalian orthoreoviruses that cause considerable mortality and morbidity in swine farms.
Without limiting the scope of the invention, its background is described in connection with recent outbreaks of epidemic diarrhea in swine populations. In May 2013, a devastating outbreak of epidemic diarrhea in young piglets commenced in swine farms of the United States, causing immense economic concerns. The mortality can reach up to 100% in piglets less than 10 days of age, with a recorded loss of at least 8 million neonatal pigs since 2013. Enteric coronaviruses, such as swine enteric coronaviruses (SECoVs), porcine epidemic diarrhea virus (PEDV), and porcine deltacoronavirus (PDCoV), were isolated from these outbreaks and characterized. However, despite intensive biosecurity measures adopted to prevent the spread of SECoV in many farms and the use of two U.S. Department of Agriculture (USDA) conditionally licensed vaccines against PEDV, the outbreaks have continued and have now spread to many other countries, including Mexico, Peru, Dominican Republic, Canada, Columbia, and Ecuador in the Americas and Ukraine. Repeated outbreaks have also been reported on the same farms that were previously infected with PEDV. In June 2014, the USDA issued a federal order to report, monitor, and control swine enteric coronavirus disease (SECD).
Porcine orthoreoviruses are also known to cause diarrhea in swine populations and outbreaks have been reported in China and Korea but not in the United States. The family Reoviridae comprises 15 genera of double-stranded RNA (dsRNA) viruses. Orthoreoviruses are a genus within the Reovirus family in the subfamily Spinareovirinae. There are five species within the Orthoreovirus genus with Mammalian ortheoreovirus (MRV) being the type species. This virus species is characterized by a segmented double stranded RNA genome within a nonenveloped, icosahedral virion with a double capsid structure. The segmented MRV genome has 10 discrete RNA segments that have been isolated from a wide variety of animal species, including bats, civet cats, birds, reptiles, pigs, and humans. Most orthoreoviruses are recognized to cause respiratory infections, gastroenteritis, hepatitis, myocarditis, and central nervous system disease in humans, animals, and birds. Orthoreovirus genomes are prone to genetic reassortment and intragenic rearrangement. The exchange of RNA segments between viruses can lead to molecular diversity and evolution of viruses with increased virulence and host range. MRV serotypes 1 to 3 were associated with enteritis, pneumonia, or encephalitis in swine around the world, including China and South Korea. The zoonotic potential of MRV3 has been reported recently. However, porcine orthoreovirus infection of pigs was unknown previously in the United States.
From the foregoing, it appeared to the present inventors that a new infectious agent might be involved in the outbreaks. Provided herein is the discovery of novel infectious agents causing epidemic diarrhea in swine as well as assays for detection and preventive vaccines.
The present invention is directed to assays for diagnosis and prevention of a novel porcine orthoreovirus type 3 (POV3-VT) that the present inventors determined to be a causative agent in diarrheic piglet outbreaks in three states. The agent was identified in ring-dried swine blood meal from multiple sources. In order to combat this new agent, the present inventors have developed methods for detection of the virus in multiple samples, antibodies to the virus in pig populations and vaccines for prevention of the disease.
In certain embodiments a vaccine that confers immunity to POV3-VT is provided that includes an immunogenic amount of one or more type specific POV3-VT proteins or immunogenic portions thereof. In certain embodiment the type specific POV3-VT proteins are selected from the group consisting of σ1, σ1s, μ1 and μ2 proteins and immunogenic portions thereof. The immunogenic proteins may be presented a a number of different ways including via a live attenuated virus vaccine, a killed virus vaccine and a subunit vaccine. Preferable subunit vaccines are generated by in vitro production of the immunogenic proteins in bacterial or baculovirus cells.
Also provided are vaccines that confers immunity to POV3-VT including an immunogenic MRV3 σ1 protein, or an immunogenic polypeptide portion thereof, wherein the MRV3 σ1 protein has at least 92% identity with amino acid residues 1 to 455 of SEQ. ID. NO: 20.
Attenuated live virus vaccines are provided wherein the vaccine is developed by passage of a POV3-VT virus in a non-porcine host until the passaged virus is capable of conferring immunity when inoculated into pigs but incapable of causing epidemic diarrhea.
In certain embodiments, a method of detecting an infection of an animal by a POV3-VT virus is provided including providing a sample from the animal, and detecting the presence or absence in the sample of an antibody that specifically binds to a polypeptide comprising a POV3-VTt σ1 protein, or an immunogenic polypeptide portion thereof (SEQ ID NO: 20), wherein the detecting of the presence or absence in the sample of an antibody that specifically binds to the polypeptide comprises use of an antibody-based technique capable of detecting the specific binding of an antibody to a protein, and the detecting of the specific binding of an antibody in the sample to the polypeptide detects infection of the animal by the POV3-VT virus. The method may be an immunohistochemistry assay, a radioimmunoassay, an ELISA (enzyme linked immunosorbant assay), a sandwich immunoassay, an immuno-radiometric assay, a gel diffusion precipitation reaction, a immunodiffusion assay, an in situ immunoassay, a Western blot, a precipitation reaction, an agglutination assay, a complement fixation assays, a immunofluorescence assay, a protein A assay, and an immunoelectrophoresis assay.
In other embodiments a process of detecting POV3-VT in a biological sample is provided including producing an amplification product by amplifying a POV3-VT S1 segment nucleotide sequence using forward and reverse primers homologous to regions within the S1 segment of POV3-VT under conditions suitable for a polymerase chain reaction and measuring said amplification product to detect POV3-VT in said biological sample. In other embodiments the method of detection of POV3-VT in a biological sample includes producing an amplification product by amplifying a plurality of targets including a POV3-VT S1 segment and at least one additional POV3-VT segment selected from the group consisting of S2, S3, S4, L1, L2, L3, M1, M2 and M3 segments, each amplification using forward and reverse primers homologous to regions within each respective segment of POV3-VT under conditions suitable for a polymerase chain reaction; and detecting the amplification products to detect POV3-VT in said biological sample.
In certain embodiments, the POV3-VT is detected in feed supplements and by detecting the presence of the virus in feed supplements, contamination with live virus can be avoid either by refusing use of the contaminated supplements or by further testing the supplements to determine whether live virus is present. Combinations of sensitive testing for the presence of viral DNA/RNA coupled with further selective testing for live virus not only allows avoidance of contaminated feed but also allows the development of techniques able to fully inactivate potentially contaminated feed supplements.
Also provided herein is a probe for the detection of a POV3-VT virus nucleic acid that comprises a nucleotide sequence having at least 98% sequence homology with the unique S1 segment (SEQ. ID. NO: 19) of POV3-VT together with a label. The probe may thus be radiolabeled, fluorescently-labeled, biotin-labeled, enzymatically-labeled, or chemically-labeled. The POV3-VT virus nucleic acid may be amplified for detection by polymerase chain reaction (PCR), real-time PCR, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time reverse transcriptase-polymerase chain reaction (rt RT-PCR), ligase chain reaction, or transcription-mediated amplification (TMA).
For a more complete understanding of the present invention, including features and advantages, reference is now made to the detailed description of the invention along with the accompanying figures:
Disclosed herein is a novel porcine orthoreovirus type 3 (POV3) isolated from diarrheic feces of piglets from outbreaks in three states and ring-dried swine blood meal from multiple sources. Genetic and phylogenetic analyses of two POV3 isolates revealed that they are identical but differed significantly from nonpathogenic mammalian orthoreoviruses circulating in the United States. Provided herein are diagnostics and vaccines to identify control and prevent this new infectious agent, including through the detection and inactivation of the virus in porcine blood products.
Despite strict biosecurity and vaccination measures against swine enteric coronavirus, the disease identified by the present inventors has continued to spread to at least 32 states of USA and other countries including Mexico, Peru, Dominican Republic, Canada, Columbia and Ecuador in the Americas and Ukraine with repeated outbreaks. As disclosed herein, the present inventors have demonstrated the association and pathogenicity of porcine Orthoreovirus type 3 (POV3) with these outbreaks in pigs. As used herein the novel virus isolates are also referred to as POV3-VT (Virginia Tech), which includes the isolates FS03 and BM100 as well as POV3 strains having a σ1 capsid protein with 98% sequence homology to the σ1 capsid protein (SEQ. ID. 20) encoded by segment S1 as well as nucleic acids that encode a protein having a 98% sequence homology to the σ1 capsid protein of SEQ. ID. 20.
As disclosed herein, the present inventors have isolated and characterized a novel porcine POV3 from fecal samples in cases of epidemic piglet diarrhea and have shown that the high pathogenicity of these novel POV3 strains in neonatal pigs leads to lethal enteric disease. We have also isolated these novel POV3 strains from swine blood meal, which is a by-product of the slaughtering industry and is used as a protein source in the diets of livestock. A chloroform extract of blood meal and a virus derived from the same sample caused similar disease in experimental pigs, suggesting blood meal as a source of infection. Indeed, more than 80% of ring-dried blood meal feed supplements were found positive for the novel POV3 virus. Importantly, while the World Organization for Animal Health Office International des Epizooties (OIE) ad hoc group on porcine epidemic diarrhea virus (PEDV) recently concluded that contaminated pig blood products, including spray-dried plasma, are not a likely source of infectious PEDV because spray-drying typically inactivates enveloped coronaviruses. In contrast to PEDV, the novel POV3 virus disclosed herein is particularly heat resistant such that, if present in pig blood products, it will not be property inactivated according to standard procedures.
Our results showed the POV3 isolates are thermostable and trypsin resistant, kill developing chicken embryos, and produce syncytium in BHK-21 cells but not in Vero cells. Fusogenic orthoreoviruses, including MRVs, encode a fusion-associated small transmembrane (FAST) protein that is responsible for syncytiogenesis. However, the POV3 strains identified herein lack this protein but nonetheless produce syncytium in infected BHK-21 cells or intestinal epithelium. The virions were double layered with a mean diameter of 82 nm, in concordance with the reported size for MRVs, but are larger than the reported sizes of 70 to 72 nm for bat orthoreoviruses. Size differences in MRV particle forms, such as virions, intermediate subvirion particles (ISVPs), and core particles, have been reported. Viral factories with paracrystalline arrays of virions in infected Vero cells are an important characteristic of these strains, unlike the tubular viral factories seen in T3D type strains. Our results suggest that POV3 may use intestinal microvilli to release complete virions as arrays in addition to cell lysis.
Deep sequencing analysis of the purified cell culture or developing chicken embryo isolates revealed a novel POV3 sequence. The sequencing data from two selected porcine POV3 isolates (one each from feces and blood meal) revealed a high sequence homology, thus strongly suggesting that blood meal could be a possible mode of transmission along with other undetermined modes. The thermostability of these POV3 strains at 56, 80, and 90° C. for 1 hour lends further credence to this notion. Ring drying of blood meal entails coagulation of blood by heating to 90° C., which may not be sufficient to inactivate these heat-resistant POV3 strains. The current European Union regulation for pig blood products for use in pig feeds (EU 483/2014) requiring treatment at 80° C. and storage for 2 weeks at room temperature to inactivate PEDV appears to be insufficient to inactivate the novel POV3 disclosed herein.
The genome sequences of the 10 segments of the strains disclosed herein, revealed interesting features in a unique and novel combination. For example, they carry specific mutations in σ1 protein that would impart trypsin resistance and neurotropism, in μ2 protein for interferon antagonism, and possessed multiple basic residues in the σ1s protein for hematogenous dissemination. The observed nine unique amino acid substitutions on the μ1 protein may have a role in conferring thermostability to these strains as has been found in associated with thermostability in T3-type strains.
Even though MRVs are not generally common in causing severe disease outbreaks in livestock, several strains of porcine MRVs have been isolated from diarrheic pigs in China and Korea. Similarly, certain MRV3 strains have been reported from bats in Europe suffering from clinical disease and in children with bat origin nonfusogenic MRV3 in Europe. All of these studies and our results confirm that the novel POV3 strains reported here are pathogenic. At necropsy, all infected piglets had accumulation of fluid in the intestine. The reproducibility of severe diarrhea and clinical disease with mortality in experimentally infected piglets with isolated POV3 confirms the pathogenic nature of these strains. Villous blunting is a consistent feature of piglets affected by neonatal diarrhea syndrome. The observed protein casts in the renal tubules and mild hepatic lipidosis could be attributed to the metabolic disorder. The presence of isoleucine at position 249 probably prevented the cleavage of σ1 protein by intestinal luminal proteases, enabling efficient viral growth and migration to other tissues compared to the trypsin-sensitive σ1 protein (threonine at 249) in endemic T3D type strains with attenuated virulence.
Provided herein are diagnostic methods able to detect viral infections and infectious material including animal derived protein supplements. In some embodiments, the proteins expressed by the segments listed in Table 2 are detected. Protein expression can be detected by any suitable method. In some embodiments, proteins are detected by immunohistochemistry. In other embodiments, proteins are detected by their binding to an antibody raised against the protein. Antibody binding is detected by techniques known in the art (e.g., radioimmunoassay, ELISA (enzyme linked immunosorbant assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
In certain embodiments, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
For purposes of ELISA assays for detection of viral antigens, provided herein are useful diagnostic reagents for detecting the POV3 infection using an antibody purified from a natural host such as, for example, by inoculating a pig with the porcine TTV or the immunogenic composition of the invention in an effective immunogenic quantity to produce a viral infection and recovering the antibody from the serum of the infected pig. Alternatively, the antibodies can be raised in experimental animals against the natural or synthetic polypeptides derived or expressed from the amino acid sequences or immunogenic fragments encoded by the nucleotide sequence of the isolated POV3. For example, monoclonal antibodies may be produced according to procedures known in the art that are directed to antigens of the isolated novel POV3.
In other embodiments, POV3 proteins were expressed and used in immunodetection assays to detect the presence of POV3 specific antibodies. In particular, serological testing using POV3-specific hemagglutination-inhibition and ELISA assay provide accurate and simple tools for revealing the association of this novel virus infection with diseases. Assay for detection of antibody to purified or partially purified culture derived vPOV3-VT can also be detected by techniques known in the art (e.g., radioimmunoassay, “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
In other embodiments, molecular assays are employed to detect the presence of minute amounts of the virus in pig populations but also in feed supplements. According to one embodiment of the present invention, real-time PCR using POV3 specific primers is used specifically to detect the presence of U.S. porcine POV3, in feed supplements. In other embodiment, chip based hybridization assays are employed to test multiple lots of feed supplements after PCR application. When detected, the feed supplements can be quarantined and further tested for the presence of live virus. In particular, according to the surprising findings of the present inventors, the POV3 disclosed herein is particularly heat resistant thus allowing live virus to survive heat treatments currently employed to generate ring-dried swine blood meal. Through the diagnostics disclosed herein, methods of treatment of swine blood meal are adapted to provide for complete inactivation of the U.S. porcine MRV3 (“POV3”).
Also provided herein are vaccines for prevention of disease. Such vaccine include killed virus vaccines, live attenuated virus vaccines as well as subunit vaccines. Also included in the scope of the present invention are nucleic acid vaccines. Inoculated pigs are protected from viral infection and associated diseases caused by U.S. porcine POV3 infection. The methods protect pigs in need of protection against viral infection by administering to the pig an immunologically effective amount of a vaccine according to the invention, such as, for example, a vaccine comprising an immunogenic amount of the infectious POV3RNA, a plasmid or viral vector containing an infectious DNA clone of POV3, recombinant POV3 DNA, polypeptide expression products, bacteria-expressed or baculovirus-expressed purified recombinant proteins, etc. Other antigens such as other infectious swine agents and immune stimulants may be given concurrently to the pig to provide a broad spectrum of protection against viral infections.
The vaccines comprise, for example, the infectious viral and molecular nucleic acid clones, cloned POV3 infectious DNA genome segments in suitable plasmids or vectors, avirulent live virus, inactivated virus, expressed recombinant capsid subunit vaccine, etc. in combination with a nontoxic, physiologically acceptable carrier and, optionally, one or more adjuvants. Alternatively, DNA derived from the RNA of segments of the isolated POV3 that encode one or more capsid proteins may be inserted into live vectors, such as a poxvirus or an adenovirus and used as a vaccine.
Adjuvants, which may be administered in conjunction with vaccines of the present invention, are substances that increases the immunological response of the pig to the vaccine. The adjuvant may be administered at the same time and at the same site as the vaccine, or at a different time, for example, as a booster. Adjuvants also may advantageously be administered to the pig in a manner or at a site different from the manner or site in which the vaccine is administered. Suitable adjuvants include, but are not limited to, aluminum hydroxide (alum), immunostimulating complexes (ISCOMS), non-ionic block polymers or copolymers, cytokines, saponins, monophosphoryl lipid A (MLA), muramyl dipeptides (MDP) and the like. Other suitable adjuvants include, for example, aluminum potassium sulfate, heat-labile or heat-stable enterotoxin isolated from Escherichia coli, cholera toxin or the B subunit thereof, diphtheria toxin, tetanus toxin, pertussis toxin, Freund's incomplete or complete adjuvant, etc. Toxin-based adjuvants, such as diphtheria toxin, tetanus toxin and pertussis toxin may be inactivated prior to use, for example, by treatment with formaldehyde.
The new vaccines of this invention are not restricted to any particular type or method of preparation. The cloned viral vaccines include, but are not limited to, infectious DNA vaccines (i.e., using plasmids, vectors or other conventional carriers to directly inject DNA into pigs), live vaccines, modified live vaccines, inactivated vaccines, subunit vaccines, attenuated vaccines, genetically engineered vaccines, etc. These vaccines are prepared by standard methods known in the art.
Additional genetically engineered vaccines, which are desirable in the present invention, are produced by techniques known in the art. Such techniques involve, but are not limited to, further manipulation of recombinant DNA, modification of or substitutions to the amino acid sequences of the recombinant proteins and the like
Genetically engineered vaccines based on recombinant DNA technology are made, for instance, by identifying alternative portions of the viral gene encoding proteins responsible for inducing a stronger immune or protective response in pigs (e.g., proteins derived from unique portions of the novel virus as disclosed herein, etc.). Such identified genes or immuno-dominant fragments can be cloned into standard protein expression vectors, such as the baculovirus vector, and used to infect appropriate host cells (see, for example, O'Reilly et al., “Baculovirus Expression Vectors: A Lab Manual,” Freeman & Co., 1992). The host cells are cultured, thus expressing the desired vaccine proteins, which can be purified to the desired extent and formulated into a suitable vaccine product. In one embodiment, the recombinant subunit vaccines are based on bacteria-expressed or baculovirus-expressed capsid proteins of the novel POV3 strains disclosed herein.
If the clones retain any undesirable natural abilities of causing disease, it is also possible to pinpoint the nucleotide sequences in the viral genome responsible for any residual virulence, and genetically engineer the virus avirulent through, for example, site-directed mutagenesis. Site-directed mutagenesis is able to add, delete or change one or more nucleotides (see, for instance, Zoller et al., DNA 3:479-488, 1984). An oligonucleotide is synthesized containing the desired mutation and annealed to a portion of single stranded viral DNA. The hybrid molecule, which results from that procedure, is employed to transform bacteria. Then double-stranded DNA, which is isolated containing the appropriate mutation, is used to produce full-length DNA by ligation to a restriction fragment of the latter that is subsequently transfected into a suitable cell culture. Ligation of the genome into the suitable vector for transfer may be accomplished through any standard technique known to those of ordinary skill in the art. Transfection of the vector into host cells for the production of viral progeny may be done using any of the conventional methods such as calcium-phosphate or DEAE-dextran mediated transfection, electroporation, protoplast fusion and other well-known techniques (e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 1989). The cloned virus then exhibits the desired mutation.
Immunologically effective amounts of the vaccines of the present invention are administered to pigs in need of protection against viral infection. The immunologically effective amount or the immunogenic amount that inoculates the pig can be easily determined or readily titrated by routine testing. An effective amount is one in which a sufficient immunological response to the vaccine is attained to protect the pig exposed to POV3. Preferably, the pig is protected to an extent in which one to all of the adverse physiological symptoms or effects of the viral disease are significantly reduced, ameliorated or totally prevented.
The vaccine may be administered in a single dose or in repeated doses. Dosages may range, for example, from about 1 microgram to about 1,000 micrograms of the plasmid DNA containing an infectious chimeric DNA genome (dependent upon the concentration of the immuno-active component of the vaccine), but should not contain an amount of virus-based antigen sufficient to result in an adverse reaction or physiological symptoms of viral infection. Methods are known in the art for determining or titrating suitable dosages of active antigenic agent to find minimal effective dosages based on the weight of the pig, concentration of the antigen and other typical factors. In certain embodiments, the infectious viral DNA clone is used as a vaccine, or a live infectious virus can be generated in vitro and then the live virus is used as a vaccine. In that case, from about 50 to about 10,000 of the 50% tissue culture infective dose (TCID50) of live virus, for example, can be given to a pig.
The advantages of live vaccines are that all possible immune responses are activated in the recipient of the vaccine, including systemic, local, humoral and cell-mediated immune responses. The disadvantages of live virus vaccines, which may outweigh the advantages, lie in the potential for contamination with live adventitious viral agents or the risk that the virus may revert to virulence in the field.
To prepare inactivated virus vaccines, for instance, the virus propagation and virus production can occur in cultured porcine cell lines such as, without limitation PK-15 cells as well as BHK-21 cells, Vero cells, etc. Virus inactivation is then optimized by protocols generally known to those of ordinary skill in the art or, preferably, by the methods described herein. Inactivated virus vaccines may be prepared by treating the virus with inactivating agents such as formalin or hydrophobic solvents, acids, etc., by irradiation with ultraviolet light or X-rays, by heating, etc. Inactivation is conducted in manners understood in the art. For example, in chemical inactivation, a suitable virus sample or serum sample containing the virus is treated for a sufficient length of time with a sufficient amount or concentration of inactivating agent at a sufficiently high (or low, depending on the inactivating agent) temperature or pH to inactivate the virus. Inactivation by heating is conducted at a temperature and for a length of time sufficient to inactivate the virus, considering the particular heat stability of the virus as disclosed herein. Inactivation by irradiation is conducted using a wavelength of light or other energy source for a length of time sufficient to inactivate the virus. The virus is considered inactivated if it is unable to infect a cell susceptible to infection.
Attenuated vaccines are prepared by serial passage in a host that affects the virulence of the virus in pigs such that the virus is able to replicate in the pig and generate a full immune response without causing significant morbidity. For instance, attenuated viruses may be prepared by the technique of the present invention which involves the novel serial passage through embryonated chicken eggs.
The preparation of subunit vaccines typically differs from the preparation of a modified live vaccine or an inactivated vaccine. Prior to preparation of a subunit vaccine, the protective or antigenic components of the vaccine must be identified. DNA encoding the antigenic components are cloned and expressed in and purified from bacterial hosts such as E. coli, or other expression systems, such as baculovirus expression systems, for use as subunit recombinant capsid vaccines. Such protective or antigenic components include certain amino acid segments or fragments of the viral capsid proteins which raise a particularly strong protective or immunological response in pigs; single or multiple viral capsid proteins themselves, oligomers thereof, and higher-order associations of the viral capsid proteins which form virus substructures or identifiable parts or units of such substructures; oligoglycosides, glycolipids or glycoproteins present on or near the surface of the virus or in viral substructures such as the lipoproteins or lipid groups associated with the virus, etc. These immunogenic components are readily identified by methods known in the art. Once identified, the protective or antigenic portions of the virus (i.e., the “subunit”) are subsequently purified and/or cloned by procedures known in the art.
If the subunit vaccine is produced through recombinant genetic techniques, expression of the cloned subunit genes, for example, may be expressed by the method provided above, and may also be optimized by methods known to those in the art (see, for example, Maniatis et al., “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory, Cold Spring Harbor, Mass. (1989)).
Genetically engineered vaccines, which are also desirable in the present invention, are produced by techniques known in the art. Such techniques involve, but are not limited to, the use of RNA, recombinant DNA, recombinant proteins, live viruses and the like. Genetically engineered proteins, useful in vaccines, for instance, may be expressed in insect cells, yeast cells or mammalian cells. The genetically engineered proteins, which may be purified or isolated by conventional methods, can be directly inoculated into a porcine or mammalian species to confer protection.
For baculovirus expression, an insect cell line (such as sf9, sf21, or HIGH-FIVE) is transformed with a transfer vector containing genetic material obtained from the virus that encodes one or more of the unique and immuno-dominant proteins of the virus.
The vaccine can be administered in a single dose or in repeated doses. Dosages may contain, for example, from 1 to 1,000 micrograms of virus-based antigen (dependent upon the concentration of the immuno-active component of the vaccine), but should not contain an amount of virus-based antigen sufficient to result in an adverse reaction or physiological symptoms of viral infection. Methods are known in the art for determining or titrating suitable dosages of active antigenic agent based on the weight of the bird or mammal, concentration of the antigen and other typical factors. Desirably, the vaccine is administered directly to a porcine or other mammalian species not yet exposed to the virus. The vaccine can conveniently be administered orally, intrabuccally, intranasally, transdermally, parenterally, etc. The parenteral route of administration includes, but is not limited to, intramuscular, intravenous, intraperitoneal and subcutaneous routes.
When administered as a liquid, the present vaccine may be prepared in the form of an aqueous solution, a syrup, an elixir, a tincture and the like. Such formulations are known in the art and are typically prepared by dissolution of the antigen and other typical additives in the appropriate carrier or solvent systems. Suitable carriers or solvents include, but are not limited to, water, saline, ethanol, ethylene glycol, glycerol, etc. Typical additives are, for example, certified dyes, flavors, sweeteners and antimicrobial preservatives such as thimerosal (sodium ethylmercurithiosalicylate). Such solutions may be stabilized, for example, by addition of partially hydrolyzed gelatin, sorbitol or cell culture medium, and may be buffered by conventional methods using reagents known in the art, such as sodium hydrogen phosphate, sodium dihydrogen phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, a mixture thereof, and the like.
Liquid formulations also may include suspensions and emulsions which contain suspending or emulsifying agents in combination with other standard co-formulants. These types of liquid formulations may be prepared by conventional methods. Suspensions, for example, may be prepared using a colloid mill. Emulsions, for example, may be prepared using a homogenizer.
Parenteral formulations, designed for injection into body fluid systems, require proper isotonicity and pH buffering to the corresponding levels of mammalian body fluids. Isotonicity can be appropriately adjusted with sodium chloride and other salts as needed. Suitable solvents, such as ethanol or propylene glycol, can be used to increase the solubility of the ingredients in the formulation and the stability of the liquid preparation. Further additives which can be employed in the present vaccine include, but are not limited to, dextrose, conventional antioxidants and conventional chelating agents such as ethylenediamine tetraacetic acid (EDTA). Parenteral dosage forms must also be sterilized prior to use.
While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts which can be employed in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
The following examples are include for the sake of completeness of disclosure and to illustrate the methods of making the compositions and composites of the present invention as well as to present certain characteristics of the compositions. In no way are these examples intended to limit the scope or teaching of this disclosure.
Nine out of 11 ring-dried swine blood meal (RDSB) samples from different manufacturing sources (82%) and 18 out of 48 fecal samples (37%) from neonatal pigs from farms with epidemic diarrhea outbreaks in North Carolina, Minnesota, and Iowa amplified a 326-bp S1 fragment with orthoreovirus group-specific primers. Among the 18 orthoreovirus positive fecal samples, 11 samples were further sequence verified using MRV3-S1 gene-specific primers amplifying a 424-bp fragment. CPE including syncytium formation and rounding of individual cells, were evident at 48 h postinfection (hpi) in BHK-21 cells inoculated with chloroform-extracted samples of feces and blood meal (
To determine whether normal, healthy pigs harbor orthoreoviruses, 36 samples of feces and matched samples of plasma from different states (Indiana, Ohio, Iowa, and Illinois) were obtained from farms with or without a PEDV outbreak. Six samples of feces and plasma each were obtained from uninfected farms in Indiana and Ohio, 12 samples of feces and plasma each were obtained from a farm in Illinois collected 6 weeks post-epidemic diarrhea, and 12 samples of feces and plasma each were obtained from a farm in Iowa collected 6-month post-epidemic diarrhea. None of these samples were found to be positive for orthoreovirus by RT-PCR. Furthermore, chloroform extracts of feces from a few randomly selected MRV3-negative samples were blindly passaged twice on BHK-21 cells, and no CPE was observed.
Viral RNA Isolation.
Viral RNA was isolated from fecal and ring dried swine blood meal samples using the QIAmp RNA kit (Qiagen, United States), and reverse transcription-PCR (RT-PCR) was performed using MRV3-S1 gene-specific primers. The following MRV3 S1 segment specific primers were used (D. Lelli et al., Identification of Mammalian orthoreovirus type 3 in Italian bats. Zoonoses and public health 60, 84-92 (2013)):
The amplified PCR products were analyzed by electrophoresis on a 1.5% (wt/vol) agarose gel, and the PCR products were purified and directly sequenced.
Virus Isolation.
Virus isolation was performed on RT-PCR-positive fecal and blood meal samples. Chloroform extracts of a 20% fecal suspension and 10% ring-dried blood meal samples were filtered through 0.2-μm-pore membrane filters (Millipore, United States) and inoculated into 9 to 11 day old, specific-pathogen-free (SPF), developing chicken embryos (via the chorioallantoic membrane [CAM] route) and BHK-21 cells. Embryos and cells were incubated at 37° C. for 5 days and monitored daily for mortality and cytopathic effects (CPE), respectively. At 5 days postinfection (dpi), allantoic fluid and CAM were harvested from eggs, and the cell culture supernatant was collected from BHK-21 cultures, chloroform extracted, and further passaged in SPF chicken embryos or BHK-21 cells, respectively. Viral RNA was detected by RT-PCR using MRV3 S1 segment-specific primers. Amplified MRV3-S1 PCR products were sequenced to confirm the viral genome. The virus isolates obtained from BHK-21 cells were further confirmed using an indirect immunofluorescence assay (IFA), employing a mouse monoclonal antibody directed against type 3 orthoreovirus σ1 protein (clone 2Q2048; Abcam, United States).
Virus Purification.
BHK-21 cell monolayers grown in T-175 flasks were infected with the POV3 isolates at a multiplicity of infection (MOI) of 0.1 in Dulbecco's modified Eagle's medium (DMEM) containing 1% fetal calf serum (FCS). The cells were harvested at 3 dpi and subjected to three freeze-thaw cycles. The cellular debris was clarified by centrifugation at 3,700×g at 4° C. Crude virus was pelleted from the clarified supernatant by ultracentrifugation at 66,000×g for 2 h using an SW-28 rotor (Beckman Coulter, US). The virus pellet was resuspended in 1 ml TN buffer (20 mM Tris, 400 mM NaCl, 0.01% N-lauryl sarcosine [pH 7.4]). The virus suspension was then layered onto a 15 to 45% (wt/vol) discontinuous sucrose gradient and centrifuged at 92,300×g for 2 h at 4° C. using an SW-41 Ti swing-out rotor (Beckman Coulter, US). The virus band at the interface was collected and used for characterization and genomic studies.
The novel porcine orthoreovirus is unique in morphology and biological characteristics. Genomic RNA from sucrose density gradient-purified virions was resistant to S1 nuclease treatment, confirming the double-stranded nature of the viral genome. SDS-PAGE indicated that the viral genome consists of 10 segments (
In infected Vero cells, the presence of paracrystalline arrays of virus particles free of organelles and viral factories in the cytoplasm was evident. The mean diameter of the virus particles was 82 nm (
Virus Characterization.
Hemagglutination (HA) and hemagglutination inhibition (HI) assays were performed. Briefly, the viruses were serially diluted in 50 μl of phosphate-buffered saline (PBS [pH 7.4]) in 96-well V-bottom microtiter plates (Corning-Costar, US) followed by 50 μl of 1% pig erythrocytes (Lampire Biological Laboratories, US). The plates were incubated for 2 h at 37° C. to record the HA titer. The HI assay was performed using mouse monoclonal antibody directed against type 3 orthoreovirus_1 protein (clone 2Q2048; Abcam, US) and 4 HA units of the virus. The HI assay plates were incubated initially at 37° C. for 1 h and then at 4° C. overnight before scoring. For electron microscopy, ultrathin sections of virus-infected BHK-21 cells (3 dpi), intestines of experimentally infected pigs, or purified virions were placed on Formvar-carbon-coated electron microscope grids and negatively stained with 2% (wt/vol) uranyl acetate or 1% sodium phosphotungstic acid for 30 s. The specimens were then examined in a JEOL 1400 transmission electron microscope (JEOL, US) at an accelerating voltage of 80 kVA.
To determine the temperature sensitivity, the virus strains were subjected to five different temperature treatments at 34, 37, 56, 80, and 90° C. for 1 h. Serial dilution of the virus was then made in DMEM, which was then titrated for infectivity in BHK-21 cells. For trypsin sensitivity, virus was incubated with 1 μg/ml tosyl phenylalanyl chloromethyl ketone (TPCK) trypsin in DMEM for 1 h at 37° C. and titrated for infectivity in BHK-21 cells. To demonstrate the double-stranded nature of the viral genome, total RNA extracted from purified virions was subjected to S1 nuclease digestion and 7.5% SDS-PAGE and silver nitrate staining. For protein profiling, the purified virus was denatured in protein sample buffer and analyzed by standard 7.5% SDS-PAGE and Coomassie blue staining.
Deep sequencing (MiSeq) of purified viral RNAs from two selected POV3 isolates (FS03 from a pig fecal sample and BM100 from a porcine blood meal) confirmed their genomic identity with MRV3. No other contaminating viral sequences were detected in the deep sequence data. The high level of sequence identity between FS03 and BM100 sequences validated our immunofluorescence, gel electrophoresis and virus protein profile data. The total length of the porcine orthoreovirus genome is 23,561 nucleotides (nt). The two porcine isolates have consensus genome termini at the 5′ and 3′ ends similar to other MRVs. The 5′ untranslated region (UTR) ranged in length from 12 to 31 nt, and the 3′ UTR ranged in length from 32 to 80 nt, with variations from prototype MRV3 T3D (Table 1). The 5′ UTRs of both POV3 FS03 and BM100 have a 6-nt deletion in L1 and a 1-nt deletion in each of the L2 and S4 segments. In addition, a deletion of 3 nt in the M2 segment open reading frame (ORF) was noticed. The genome of these novel viruses contains reassorted gene segments from other MRVs.
GCUACA
GCUAUU
GCUAAU
GCUAUU
U
GCUAAU
GCUAAA
U
GCUAUU
GCUAUU
GCUAAA
GCUAUU
aThe 5′ and 3′ untranslated regions (UTRs) of U.S. porcine strains FS03 and BM100 show mutations on the M2, S1, and S2 segments. The conserved terminal sequences are shown in boldface, and mutations are italicized.
Predicted functions of different proteins encoded by the 10 segments analogous to known members of the Orthoreovirus genus are shown in Table 2 below:
Orthoreovirus protein functions
The deduced amino acid sequences of POV3 FS03 and BM100 are homologous except for the σ1 protein, with 1 amino acid (aa) change between them. The percentage of homology of each of the different proteins coded by these two viruses with prototype MRV 1-4 is provided in Table 3 below:
On comparison of the deduced amino acids, it appears that with proteins of the L class segment, λ2 protein was homologous to MRV1, while the λ1 and λ3 proteins were highly similar to the MRV 1 and 3 prototypes, T1-Lang (T1L) and T3/Dearing (T3D), respectively. In M class proteins, only μNS was identical to T3D, while μ1 and μ2 were identical to T1L. As shown in
The highest diversity among all proteins was observed for the S1 segment encoded σ1 protein, with closest homology to T3/Bat/Germany virus (91%). Deduced amino acid sequence analysis of σ1 protein revealed that the sialic acid binding domain (NLAIRLP), and protease resistance (2491) and neurotropism (340 D and 419E) residues were conserved in the U.S. porcine orthoreovirus strains. As depicted in
Deep Sequencing.
The double-stranded RNA (dsRNA) isolated from two purified viruses, FS03 isolated from fecal samples and BM100 isolated from swine ring-dried blood meal, were subjected to NextGen genome sequencing. The NEBNext Ultr directional RNA library prep kit for Illumina (catalog no. e74205; NEB) was used to prepare the RNA library with some modifications. Using a standard protocol, 100 ng of viral RNA was fragmented to 250 nucleotides at 94° C. for 10 min. After adapter ligation, 350- to 375-bp libraries (250- to 275-bp insert) were selected using Pippin Prep (Sage Science, United States). The template molecules with the adapters were enriched by 12 cycles of PCR to create the final library. The generated library was validated using the Agilent 2100 bioanalyzer and quantitated using the Quant-iT dsDNA H.S. kit (Invitrogen) and quantitative PCR (qPCR). Two individually barcoded libraries (FS03 virus with A006-GCCAAT, and BM100 virus with A012-CTTGTA) were pooled and sequenced on Illumina MiSeq. Briefly, the individual libraries were pooled in equimolar amounts, denatured, and loaded onto MiSeq. The pooled library was spiked with 5% phiX and sequenced to 2×250 paired-end reads (PE) on the MiSeq using the MiSeq reagent kit V2 at 500 cycles (MS-102-2003) to generate 24 million PE.
Genome Assembly.
Reference-based mapping and de novo assembly methods were applied to the raw data for assembly into viral genomes. Reference-based mapping was performed against the mammalian orthoreovirus genome by using the CLC Genomics Workbench software (version 7.0.4; CLC Bio, Denmark). The de novo assembly was performed with the following overlap settings: mismatch cost of 2, insert cost of 3, minimum contig length of 1,000 bp, a similarity of 0.8, and a trimming quality score of 0.05. This assembly yielded 3,444 contigs that were annotated according to Gene Ontology terms with the Blast2Go program, which was executed as a plugin of CLC by mapping against the UniprotKB/Swiss-Prot database with a cutoff E value of 1e−05. Furthermore, to determine putative gene descriptions, homology searches were carried out through querying the NCBI database using the tBLASTx algorithm. The de novo-assembled sequences were used to confirm the validity of the reference-based sequence assembly. Both de novo assembly and the reference-based mapping produced identical sequences.
Nucleotide Sequence Accession Numbers.
The complete genome sequences of both viruses FS03 and BM100 are provided herein and have been deposited in GenBank under accession no. KM820744 to KM820763 as shown in Table 4 below. In the Table, the proteins for which alignments are provided in
M1
KM820757
KM820747
AF461682
AF124519
HM159616
SEQ. ID. 13, 14
SEQ. ID. 33, 34
SEQ. ID. 49
M2
KM820758
KM820748
AF490617
M19355
HM159617
SEQ. ID. 15, 16
SEQ. ID. 35, 36
SEQ. ID. 48
S1
KM820760
KM820750
M14779
M35964
HM159619
SEQ. ID. 19, 20
SEQ. ID. 39, 40
SEQ. ID. 47
Phylogenetic analysis of the FS03 and BM100 POV3 isolates revealed a strong evolutionary relationship with MRV3 strains. The ORFs of the nucleotide sequences of the L1, S1, S2, S3, and S4 segments were used to construct the phylogenetic trees. Based on S1 phylogeny, both isolates were monophyletic with MRV3 of bat origin (
Phylogenetic Analysis.
The nucleotide and deduced amino acid sequences of L1 and S class segments (S1, S2, S3, and S4) were compared with those of other closely related orthoreoviruses using the BioEdit sequence alignment editor software (version 7.0.0; BioEdit, Ibis Biosciences, Carlsbad, Calif.). The phylogenetic evolutionary histories for the virus strains were inferred using the maximum likelihood method based on either JTT w/freq model for the S2, S3, S4, and L1 segments in Mega 6.06 or the Jukes-Cantor evolution model with “WAG” (i.e., Whelan and Goldman model) protein substitution for S1 segment in CLC workbench 7.0.4 after testing for their appropriateness to be the best fit. The bootstrap consensus tree inferred from 1,000 replicates was taken to represent the evolutionary history of the taxa analyzed.
Experimental neonatal pigs were screened for swine deltacoronavirus, PEDV, Kobuvirus, swine transmissible gastroenteritis virus (TGEV), rotavirus, and orthoreoviruses by RT-PCR and found to be negative, except for three pigs that were positive for Kobuvirus, whose pathogenicity is yet to be established. Neonatal pigs orally inoculated with purified viruses FS03, BM100, T3/Swine/I03/USA/2014 (103), or a chloroform extract of blood meal 100 (CBM100) developed clinical illness in all infected animals (100%), with loss of physical activity, severe diarrhea, and decrease in body weight. Infected animals had significantly high mean clinical scores compared to the mock-infected group (P<0.01). Piglets infected with FS03 and 103 had the highest clinical scores as early as 1 dpi, which peaked at 3 dpi. Three pigs in the mock-infected group had a slow recovery from parenteral anesthetics, with elevated mean clinical scores for the first 2 days but returned to normal later. Gross lesions, such as catarrhal enteritis and intussusception, were observed in all of the infected animals. The cumulative macroscopic lesion scores of FS03 and 103 were higher than those of other groups on day 4 dpi. Compared to mock-infected pigs, the small intestines of the virus-infected pigs showed mild to severe villous blunting and fusion (crypt/villous ratios of 1:1 to 1:4), occasional villous epithelial syncytial cells, swollen epithelial cells with granular cytoplasm and multifocal necrosis of mucosal epithelium, and round to oval vacuoles in the intestinal epithelial cells. In a few pigs, protein casts in renal tubules, minimal to mild hepatic lipidosis and hepatocellular vacuolar changes, and mild to moderate suppurative bronchopneumonia were also seen.
Ultrastructural examination revealed multinucleated cells with apoptotic nuclei, and in some cells, dark granular bodies resembling stress granules were seen. Viral particles were localized in regions of the cytoplasm that lacked typical cytoplasmic organelles. Large numbers of viral particles egressed by cell lysis or as a string of beads through microvilli from infected villous epithelial cells into the lumen of the intestine. Multinucleated cells with virions egressing through microvilli were evident. Virions disrupt microvilli before release and were still surrounded by the cell membrane of microvilli, and after release were devoid of membranes in the lumen of the intestine.
Virus replication in the intestines and fecal virus shedding in infected pigs were also confirmed by virus isolation in cell culture and by S1-segment-specific RT-PCR. The intestinal contents had POV3 virus in 80% of the infected piglets through RT-PCR, suggesting the virus replication in the intestine is consistent with electron microscopic findings of virus replication within the enterocytes.
Pathogenicity Study in Neonatal Pigs.
All animal studies were performed as approved by the Institutional Animal Care and Use Committee of Virginia Tech (IACUC no. 14-105-CVM, 5 Jun. 2014). Thirty-five 2-day-old piglets, purchased from the Virginia Tech Swine Center, were housed as 7 animals/group in HEPA-filtered level 2 biosecurity facility.
Prior to the start of the experiment, pigs were tested for most common enteric RNA viruses, such as rotavirus, PEDV, swine deltacoronavirus, Kobuvirus, and TGEV, by RT-PCR of the fecal samples using specific primers (primer sequences available upon request). The amplified PCR products were analyzed by electrophoresis on 1.5% (wt/vol) agarose gel.
After acclimatizing for a day, the animals were anesthetized, and 2 ml of 5×105 TCID50/ml of each virus strain or chloroform extract of 10% blood meal suspension (2.5 g ring-dried blood meal) was homogenized in 12.5 ml DMEM to get a 20% solution that was extracted with an equal volume of chloroform. The upper aqueous phase obtained was diluted with an equal volume of DMEM to get a final concentration of 10%, and the piglet was orally inoculated using a 5-ml syringe. Mock-infected animals received 2 ml DMEM orally. The animals were monitored two times a day: rectal temperature, body weight, and clinical scores based on physical appearance, activity, respiratory, gastrointestinal, and systemic signs were recorded on a scale of 0 to 3. Fecal swabs were collected daily and suspended in 1 ml of DMEM containing 10× antibiotic solution (Hy-Clone, United States), mixed vigorously, incubated for 1 h, and stored at −80° C. until tested. At 4 dpi, or when they reached the clinical endpoint, all animals were euthanized. Gross and microscopic lesions were scored by a board-certified veterinary pathologist blind to the experimental groups. The S1 gene-specific RT-PCR was performed to confirm the production of orthoreovirus in the intestine using the intestinal contents of the experimentally infected piglets.
Statistical Analysis.
Summary statistics were calculated to assess the overall quality of the data. Analysis of variance (ANOVA) was used for assessment of the mean clinical score and microscopic lesion scores. The significance level was set for a P value of <0.01 and a 95% confidence interval. Statistical analysis was performed using GraphPad Prism software (version 6.0; Graph Pad Software, Inc., San Diego, Calif.).
To identify the prevalence and geographic distribution of this novel orthoreovirus, a retrospective serological surveillance of 1067 sera samples collected from 24 states during 2014-2015 and 28 sera samples from 2008 was performed. Samples were tested by Hemagglutination Inhibition (HI) of pig erythrocytes with plaque purified porcine Orthoreovirus type 3 (POV3) and virus neutralization (VN) test in BHK21 cells. The prevalence of POV3-specific HI antibodies was very high during 2014-2015 but negative for samples from 2008. The HI titers ranged from 2 to 4096 against POV3 with 88.37% of samples above the cut-off titer of 1:8. High HI antibody titers (2048 and above) were recorded only from swine sera samples collected from Iowa, North Carolina Pennsylvania, Texas, South Dakota, Oklahoma, Montana, Michigan, Georgia and Colorado. There were no significant differences in the HI titers with respect to age (1-56 weeks) of pigs. However, serum neutralization assay on 200 randomly selected samples showed low levels of VN antibodies (<1:10). The prevalence of high titer HI antibodies and low level of VN antibodies has warranted the immediate development of vaccines against this pathogenic POV3, as exemplified herein.
One example of a killed virus vaccine was generated by Binary Ethyleneimine (BEI) Inactivation. The virus strain designated POV3-BM100 was originally isolated from swine ring dried blood meal. The virus was initially propagated in BHK-21 culture three times and was plaque purified. Virus plaque no. 2 was further propagated and amplified twice in BHK-21 cells to make a Master Seed virus. The titer of the virus was determined by TCID50 assay. Cell cultures are grown in Dulbecco's modified minimal essential media (Hyclone DMEM/High Glucose Thermo. USA, cat no: SH30243.02) supplemented with 10% FCS and Hyclone 1× penicillin-Streptomycin solution, Thermo, USA cat no V30010) antibiotic and anti-mycotic solution. The serum concentration was reduced to 1% for a maintenance medium and chymotrypsin was added at a concentration of 1 ug/mL to the maintenance medium to promote virus infectivity. BHK-21 cells grown in T-175 flask at 37° C., 5% CO2 with 80-90% confluency are used for virus infection. The growth media is removed. The seed virus is thawed on ice. The cell monolayers are washed thrice in sterile PBS. Sufficient virus is added to achieve a minimum multiplicity of infection (MOI) of 0.01. The fluids are harvested along with the cellular material 72 hours after infection, dispensed and frozen at −80° C. The working seed lot of the virus is sonicated or given 3-4 freeze thaw cycles (at −80° C.) to release the intracellular virions to be used for inactivation. The viral suspension is centrifuges at 3000 rpm for 20 min at 4° C. and the supernatant fluids harvested. The titer of the virus before inactivation is determined using TCID50 method or plaque assay in triplicates. Non-frozen porcine orthoreovirus produced as described above can be further inactivated using binary ethyleneimine (BEI).
BEI Inactivation:
BEI is prepared from 0.1M 2-bromo-ethylamine hydrobromide (2-BEA, Acro Organics, USA, Catalogue no 2576-47-8) in solution of 0.2 N NaOH (Sigma, USA) and the BEA solution is treated in water bath at 37° C. for 1 hour for the cyclization reaction that converts BEA to BEI (0.1M BEI stock solution). A solution of 0.1M BEI is further filter sterilized using 0.22 micron syringe filter and used immediately for Virus inactivation. BEI was used at three different concentrations viz 1 mM, 2.5 mM and 5 mM. Samples are harvested to evaluate the inactivation process. Control samples are also retained for comparison (Mock infected cell culture supernatant). Samples are taken using aseptic technique inside the bio-safety cabinet. At the end of each time point (incubation period) 2% v/v of a sterile 1M sodium thiosulfate solution was added to ensure neutralization of the BEI. The neutralized sample is thoroughly mixed on a vortex mixer and stored at −80° C. until used for testing.
Samples were collected at different time points (0 h, 6 h, 12 h, 24 h, 48 h and 72 h) and neutralized with appropriate volume of 1M sodium thiosulfate and frozen at −80 degree deep freezer. The virus titer in each time point is assayed using TCID50 method at the end of complete inactivation period. The regression curve is plotted to study the inactivation kinetics. From the virus inactivation kinetics study results of which are shown in
Inactivation validation: The samples collected during inactivation, the original virus control (held at −80° C.) and the non-treated virus control held at 37° C. for 48 hours are diluted in appropriate diluent (from neat to 10−8) are titrated in 96 well micro-wells as per standard established technique to determine the TCID50 titers of each samples. Each sample is inoculated in four replicates. The cell cultures are incubated for a prescribed time and titration is read according to CPE or by other established methods such as immunofluorescence or immunoperoxidase staining
In one embodiment a modified live-attenuated virus vaccine is generated from the novel virus isolates. The virus has been propagated in Vero cells and BHK-21 cells as well as chicken embryos and serial passaging is underway to generate a modified live-attenuated vaccine (MLV). By serial passage in non-porcine host cells, the virulence of the virus is gradually affected until the virus losses the ability to cause significant morbidity in adult and juvenile pigs.
The hemagglutination-inhibition (HI) assay is an effective method for assessing immune responses to porcine orthoreovirus hemagglutinin (HA). The HA protein on the surface of swine orthoreovirus/MRV agglutinates erythrocytes. Specific attachment of antibody to the antigenic sites on the HA molecule interferes with the binding between the viral HA and receptors on the erythrocytes. This effect inhibits hemagglutination and is the basis for the HI assay. In general, a standardized quantity of HA antigen (4 HA units) is mixed with serially diluted serum samples and swine red blood cells (sRBCs) are added to detect specific binding of antibody to the HA molecule. The presence of specific anti-HA antibodies will inhibit the agglutination which would otherwise occur between the virus and the RBCs. During adsorption with horse RBCs, non-specific virus inhibitors may be introduced into serum, which will cause a false positive result in HI assay with pig RBC. Such non-specific inhibitors can be eliminated by receptor destroying enzyme (RDE) treatment.
Materials are assembled including: 1) porcine orthoreovirus (POV3)/Mammalian orthoreovirus 3 (MRV3), 2) pig serum samples (serum samples should not be repeatedly freeze-thawed but are ideally aliquoted and stored at −20 to −70° C.), 3) swine RBCs in PBS (Porcine RBCs in Alsever's solution are obtainable from Lampire Biological or equivalent source, and used at a concentration of 1.0% in PBS+0.5% BSA), 4) horse blood cells in Alsever's solution (as fresh as possible), 5) Phosphate buffered saline (PBS) (0.01M PBS, pH 7.2), store at 4° C. and keep on ice during use, 6) Receptor destroying enzyme (RDE), 7) 96-well, V-bottom, polystyrene, microtiter plates (Nunc, cat. #249570).
To determine the HA titer of the test virus, the pig RBC is prepared at 1.0% (v/v). To start preparation of packed RBCs, carefully collect, using a 10 ml pipette, 5-7 ml of pig RBCs from the bottom of the bottle. Remove horse RBCs from the bottom of the container to minimize contamination with cell fragments. Filter through a sterile cotton gauze pad into a 50 ml conical centrifuge tube. Gently fill the conical tube with cold PBS and centrifuge at 800×g for 5 minutes at 4° C. Aspirate the supernatant using a 10 ml pipette, being careful to not disturb the pellet of RBCs. Gently fill the conical tube with cold PBS and mix gently by inversion followed by centrifugation at 800×g for 5 minutes at 4° C. Aspirate the supernatant using a 10 ml pipette, being careful to not disturb the pellet of RBCs. Carefully repeat the cold PBS wash one more time for a total of three PBS washes to prevent hemolysis, always handle the RBCs gently, keep the PBS on ice or at 4° C., and do not wash more than 3 times. Aspirate the remaining supernatant with a P1000 microliter pipette for final packed RBCs and keep the packed RBCs on ice. Prepare a 1.0% v/v suspension of RBCs. For example, add 2.5 ml of the packed, washed to 247.5 ml cold PBS+0.5% BSA in a 500 ml glass bottle (rinse with PBS before use). Mix gently by swirling. For the HA titer determination, mark the V bottom plates with the names of the viruses to be tested. Viruses are tested in duplicate. Add 50 μl of cold PBS to wells 2 through 12 in rows A and B. If more than 1 virus, use the rest of rows as needed. Add 50 μl of cold PBS to the entire H row. This row will serve as the RBC control. Immediately prior to removing virus from vial, gently vortex the vial of virus using three quick pulses. Then add 100 μl of the virus to be tested to wells A1 and B1. Make serial 2-fold dilutions by transferring 50 μl from well 1 successively through well 12. Discard 50 μl from well 12. Add 50 μl of 1.0% pig RBC suspension to all wells in rows A, B (or other rows if more than 1 virus), and H on the plate. Gently tap the plates to mix. Stack plates and cover with an empty plate and incubate at room temperature for 60 minutes. Read the virus HA titers by tilting the plate at a 45 to 60° angle. The settled RBCs in row H should start “running” and form a teardrop-shape due to gravity. Wait until these RBCs finish “running” and then note the RBC buttons in the virus titrations that “run”. These RBCs do not exhibit hemagglutination. The highest dilution of virus that causes complete hemagglutination is considered the HA titration end-point. The HA titer is the reciprocal of the dilution of virus in the last well with complete hemagglutination. Dilute virus in cold PBS to make a working solution containing 8 HAU/50 μl. Verify that the diluted virus contains 8 HAU per 50 μl by performing a second HA test as described above. The titer of the virus should be 8. If not 8, then adjust the virus concentration by adding virus if <8 HAU or cold PBS if >8 HAU. Store the working dilution of virus on ice and use within the same day.
HI Assay with Pig RBCs.
1. Thaw the sera at room temperature and heat inactivate at 56 degree for 30 minutes, then keep on ice during use.
2. Mark the V bottom plates with the plate number and the names of the viruses accordingly based on experiment design.
3. Column 12 of all plates can be reserved for the RBC control. Positive and negative control sera, and back titration can be run in a separate plate or incorporated in available columns of plates.
4. If dilution plates/titer tubes are used, for duplicate test with one virus, make a serial 2-fold dilution of treated sera by adding 110 μl of treated sera (1:10) to titer tubes in rows A, columns 1-11.
5. Add 55 μl of cold PBS to titer tubes in rows B-H, columns 1-11.
6. Transfer 55 μl of sera from row to row (A->B->C . . . H) using a P200 multichannel pipette to make serial 2-fold dilutions.
7. Discard 55 μl from row H after mixing.
8. Positive and negative control with appropriate initial dilution should be serially diluted following the same procedure above.
9. Transfer 25 μl of each diluted serum sample from dilution plate into V-bottom plates starting with row H and going to row A. No need to change tips if transferring from the highest dilution (row H) to the lowest dilution (row A). It is critical that the tips must be changed before beginning to pipet the next set of serum samples.
10. If dilution plate are not available, serial dilution of sera samples can be done directly on plates. For each replicate test with one virus, first, add 25 μl of cold PBS to V-bottom plate in rows B-H, columns 1-11. Second, add 50 μl of heat inactivated sera to row A, columns 1-11. Then, transfer 25 μl RDE-treated sera from row to row (A->B->C . . . H) to make serial 2-fold dilutions. Discard 25 μl from row H after mixing.
11. Add 25 μl of standardized virus containing 4 HAU to wells containing sera. Note this is the same as 50 μl containing 8 HAU.
12. Gently tap the plates to mix. Stack plates and cover with an empty plate.
13. Incubate virus and sera at room temperature (22° to 25° C.) for one hour.
14. Add 50 μl of PBS to column 12. This will serve as the RBC control.
15. Add 50 μl of 1.0% pig RBC suspension to each well.
16. Gently tap the plates to mix. Stack plates and cover with an empty plate.
17. Incubate at room temperature for one hour.
18. Record the HI titers of sera after one hour incubation by tilting the plates at a 45 to 600 angle. The settled RBCs in column 12 should start “pulling” or “running” and form a “teardrop-shape” due to gravity. Wait until these RBC's finish “pulling” and then read the RBC buttons that “run” or “stream” in the same way. A well with complete hemagglutination inhibition will look the same as the RBC controls. The serum HI titer is the reciprocal of the serum dilution in the last well with complete hemagglutination inhibition.
To identify the prevalence and geographic distribution of this novel orthoreovirus, we performed a retrospective serological surveillance of 1067 sera samples collected from 24 states during 2014-2015 and 28 sera samples from 2008 using the above Hemagglutination Inhibition (HI) assay of pig erythrocytes with plaque purified MRV3 as the hemagglutinin. It was determined that the age of the pigs had no significant influence on the HI titers, in animal tested from 1-56 weeks of age. The prevalence of POV3-specific HI antibodies was very high during 2014-2015 but negative for samples from 2008. The HI titers ranged from 2 to 4096 against POV3 with 88.37% of samples above the cut-off titer of 1:8. High HI antibody titers (2048 and above) were recorded only from swine sera samples collected from Iowa, North Carolina Pennsylvania, Texas, South Dakota, Oklahoma, Montana, Michigan, Georgia and Colorado States. The HI titers of 450 samples are plotted in terms of 2 Log scale as depicted on
An indirect ELISA protocol was developed for screening swine or any other species sera samples for the presence or absence of POV3 specific IgG using ultra-purified whole virus or recombinant proteins of the POV3 virus for sero-monitoring of POV3 infection. Generally, dilutions of swine sera are added to purified POV3 coated microtiter plates and antibodies specific for POV3 bind to the microtiter plates. The antibodies bound to the plates are detected using labelled anti-swine IgG such as alkaline phosphatase-labeled antibody followed by a p-nitrophenyl phosphate substrate. The optical density of the colored end product is proportional to the amount of POV3 specific antibody present in the serum.
In one example performed, purified POV3 (1 mg/mL) frozen aliquots stored at −80° C. were thawed at room temperature. The viral antigen was diluted to a predetermined concentration (generally 2.5 μg/ml) with sterile antigen-coating buffer (1×PBS/0.02% NaN3). An aliquot of 100 μl of antigen was pipetted into each well of microtiter plate(s) and covered for incubation at 4° C. overnight. The wells were blocked using 300 uL/well Super Block Blocking buffer in PBS (Thermo Scientific, USA, cat no: 37515) for 1 hour at room temperature and the plates were stored in a humidified chamber kept at 4° C. If sodium azide is used, coated plates may be stored for several months at 4° C., provided that storage conditions are suitable to prevent evaporation and contamination of the Blocking solution. Further reagents prepared included Substrate stop solution: 3M NaOH [1 liter], 2M Sulfuric acid/Stop solution [200 ml], and Coating Buffer 10× (10×PBS/0.2% NaN3 [1L]: NaCl—80 g, KH2PO4—3.14 g, Na2HPO4.7H2P—20.61 g, KCl—1.6 g, NaN3—2 g). When diluted the pH of the 1× coating buffer should be should be 7.2±0.2.
Sera dilution Buffer 10×: 10×PBS/0.2% NaN3/0.5% Tween-20 [1L]: NaCl 80 g, KH2PO4 3.14 g, Na2HPO4.7H2O 20.61 g, KCl 1.60 g, NaN3 2 g is prepared. Add 800 ml of reagent grade water to a 2-liter beaker placed on a magnetic stirrer. Weigh out the dry chemicals listed above and add them to the water. Dissolve the chemicals and bring the volume to 1 L with reagent grade water. Add 5 ml Tween-20. When diluted the pH of the 1× sera dilution buffer should be should be 7.2±0.2. The Wash buffer is 1×PBS/0.05% Tween-20, pH 7.2±0.2.
Procedure for testing swine sera with unknown anti-POV3 antibody concentrations. Retrieve all serum samples, controls and reference sera stored frozen and place them at room temperature to thaw (˜30 minutes). Samples should not be freeze/thawed more than 3 times. Perform serial dilutions (usually 2- or 3-fold) of sera as necessary with dilution buffer and incubate the diluted samples at room temperature for 30 minutes. Wash the antigen-coated microtiter plates 5 times with wash buffer. During the first wash, allow the wash buffer to soak on the plate 30 seconds to 1 minute after filling the wells. Using a multichannel pipettor, transfer 50 μl of each serum dilution from the dilution plates to the washed antigen coated plates. Add only antibody buffer to two wells in each plate to serve as blanks. Cover plates with lids and incubate at room temperature for 2 hours. Prepare the appropriate dilution of anti-swine IgG conjugate in antibody buffer 15 minutes before its use. Wash the plates 5 times with wash buffer. During the first wash, allow the wash buffer to soak on the plate 30 seconds to 1 minute after filling the wells. Add 100 μl of diluted enzyme conjugate to all microtiter plate wells. Cover plates with lids and incubate for 1 hour at room temperature. Prepare a 1 mg/ml solution of p-nitrophenyl phosphate in the diethanolamine substrate buffer 15 minutes before it is required. Mix the substrate solution on the shaker while wrapped in a paper towel to protect it from light. Wash the plates 5 times with wash buffer. During the first wash, allow the wash buffer to soak on the plate 30 seconds to 1 minute after filling the wells. Add 100 μl of substrate solution to all microtiter plate wells. Put lids on plates and incubate for 2 hours at room temperature. Add 50 μl of 3M NaOH to all wells to stop the enzyme reaction. Wait at least 5 minutes, before reading the optical density of the plates on a microtiter plate reader at 450 nm.
To demonstrate that the POV3 purified viral antigen produces comparable results and comparable lower limits of detection using true positive swine serum samples, checkerboard titration was performed with different dilutions of the antigen and antibody. Antigen was adsorbed on to the surface of a microtiter plate in increasing concentrations. Reference serum is added at one dilution across the plate and the ELISA is completed using POV3 specific known antibody. The optimal coating concentration of an antigen lot is determined by inspecting optical density values vs. antigen concentration. Eight different dilution of the known positive sera sample (1:1000 to 1:128000) were tested with three different concentrations of the purified POV3 virus viz 1.25 ug/mL, 2.5 ug/mL and 5 ug/mL as described previously. The results obtained were plotted concentrations of antibody (Y-axis) against the OD values on (X-axis). In one tested preparation, the optimal concentration of purified virus for coating was determined to be 2.5 ug/mL. The sensitivity/lower limits of sera dilution for ELISA may be determined by checkerboard titration of known positive and negative sera samples diluted from 1:100 to 1:51200 with 2.5 ug/mL coated purified POV3 and using an antiMRV S1 monoclonal antibody as a positive control.
To detect POV3 in feces and tissue samples and blood meal samples, a simple RT-PCR was developed targeting the S1 and L1 genes of the pathogenic porcine orthoreovirus. The primers were designed based on the insilico analysis and selection of unique regions that were present on the pathogenic POV3-VT porcine orthoreovirus as characterized by the present inventors. RNA extracted from the specimens was subjected to cDNA synthesis using ABI first strand synthesis kit, employing random primer/reverse primer. RNA was heat denatured at 70° C. for 10 min, snap cooled, mixed with cDNA master mix and incubated at 25° C. for 10 min for binding of primer. RT reaction carried out for 2 hours at 37° C., RT-inactivation at 85° C. for 5 min. cDNA was amplified using PCR using either S1 specific or L1 specific primers as follows:
Initial denaturation at 94° C. for 5 min; 40 cycles consisting of denaturation at 94° C. 30 sec; primer annealing at 58° C. for 30 sec and extension at 72° C. for 30 sec. Final extension at 72° C. for 10 minutes. The amplified length was 424 bp and 537 bp for S1 and L1 gene fragments respectively as seen in
RT-PCR screening of POV3 was conducted in brain and lung tissues of experimentally infected piglets. To detect POV3 in tissue samples, lung and brain samples were selected from experimentally infected piglets. The RNeasy Mini Kit (Qiagen, USA) was used to extract RNA from Fresh, frozen, or RNA later stabilized tissue (up to 30 mg, depending on the tissue type) as per the manufacturer recommendation. RNA was subjected to cDNA synthesis using ABI first strand synthesis kit, employing random primer/reverse primer. RNA heat denatured at 70° C. for 10 min, snap cooled, mixed with cDNA master mix and incubated at 25° C. for 10 min for binding of primer. RT reaction carried out for 2 hours at 37° C., RT-inactivation at 85° C. for 5 min. cDNA was amplified using PCR using S1 specific forward and reverse primers with initial denaturation at 94° C. for 5 min; 40 cycles consisting of denaturation at 94° C. 30 sec; primer annealing at 58° C. for 30 sec and extension at 72° C. for 30 sec. Final extension at 72° C. for 10 minutes. The amplified length was 424 bp. RT-PCR followed here successfully amplified the partial S1 gene fragment of 424 bp in both tissue types as seen in
A further example of a method for detecting the presence or absence of POV3 in a swine biological sample is provided. As POV3 viruses are segmented RNA viruses, the method comprises a reverse transcription step and cDNA amplification cycles using either POV3 S1 or L1 gene specific primers to produce an amplification product if a POV3 nucleic acid molecule is present in the sample. As a result of the methods described herein, the amplification and subsequent detection of the target nucleic acids is possible. A real-time PCR assay was run with the following primer combinations, using POV3 RNA as template. Primer combination S1: POV3_VT_S1 Fwd, SEQ. ID. 3, and POV3_VT_S1 Rev, SEQ. ID. 4. Primer combination L1: POV3 L1 fwd, SEQ. ID. 5 and: POV3 L1 rev, SEQ. ID. 6.
The PCR reaction was set-up according to the parameters below. Two sets of reactions were performed. A Biorad i cycler machine was used to perform the following cycling conditions −55° C. for 5 mins, 60° C. for 5 mins and 65° C. for 5 mins. This is followed by 45 cycles of: 94° C. for 5 s and 60° C. for 40 s. Each reaction was performed in duplicate. The test with POV3 signal will be considered positive if the CT value is below 40.
In a real time PCR assay a positive reaction is detected by accumulation of a fluorescent signal. The CT value (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross a threshold that exceeds background. CT levels are inversely proportional to the amount of target nucleic acid in the sample with the lower the CT level the greater the amount of target nucleic acid in the sample.
The assay is suitable to diagnose both POV3 S1 and L1 segments. As shown in
All publications, patents and patent applications cited herein are hereby incorporated by reference as if set forth in their entirety herein. While this invention has been described with reference to illustrative embodiments, this description is not intended to be construed in a limiting sense. Various modifications and combinations of illustrative embodiments, as well as other embodiments of the invention, will be apparent to persons skilled in the art upon reference to the description. It is therefore intended that the appended claims encompass such modifications and enhancements.
This application claims priority as a 35 U.S.C. § 371 application based on PCT/US2015/061034, filed Nov. 17, 2015, which in turn claims priority based on U.S. Provisional Application Ser. No. 62/080,462 filed Nov. 17, 2014, which is incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2015/061034 | 11/17/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62080462 | Nov 2014 | US |
