# Patent

## Title
Avian reoviridae and vaccines thereof

## Abstract

This invention relates to novel unique strains avian reoviridae that were isolated from severe cases of Runting Stunting Syndrome. The present invention also relates to the isolation and uses of novel unique avian reoviridae, diagnostic assays using nucleotide or amino acid specific components of such viruses, such as the sequence encoding Sigma C capsid protein and to vaccines that protect birds from diseases caused by such viruses.

## Background
This application is the National Stage of Int'l App'l No. PCT/US2009/048860, filed Jun. 26, 2009, which claims priority of U.S. Ser. No. 61/076,102, filed Jun. 26, 2008.
Throughout this application, various references or publications are cited. Disclosures of these references or publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.


FIELD OF THE INVENTION
This invention relates to novel unique strains of avian reoviridae that were isolated from severe cases of Runting Stunting Syndrome. The present invention also relates to the isolation and uses of novel unique avian reoviridae, diagnostic assays using nucleotide or amino acid specific components of such viruses, such as the sequence encoding Sigma C capsid protein and to vaccines that protect birds from diseases caused by such viruses.
BACKGROUND OF THE INVENTION
Reoviridae such as inter alia reovirus, which replicate in the cytoplasm, are nonenveloped with an icosahedral symmetry and a double-shelled capsid. The viral genome consists of 10 double-stranded RNA (dsRNA) segments, which can be separated into three size classes: L (large), M (medium), and S (small). Proteins encoded by the genome are also separated into three sizes. The specific genome segments responsible for protein coding have been identified for the S1133 strain of avian reovirus.
Avian reoviruses are members of the orthoreovirus genus in the Reoviridae family. Ubiquitous in commercial poultry, they can be differentiated by antigenic configuration, pathotype, relative pathogenicity, growth in cell culture, embryonated chicken eggs sensitivity to trypsin, and host and tissue specificity.
Classic reoviruses have been isolated from a variety of tissues in chickens affected by assorted disease conditions, including viral arthritis/tenosynovitis, stunting syndrome, respiratory disease, enteric disease, immunosuppression, and malabsorption syndrome. They have frequently been found in chickens that were clinically normal. The nature of the disease that occurs following reovirus infection is very much dependent upon host age, immune status, virus pathotype, and route of exposure. Reoviruses have also been isolated from turkeys, ducks, geese, pigeons, pheasants, parrots, and other exotic avian species.
In young meat-type chickens, economic losses related to reovirus infections are frequently associated with increased mortality, viral arthritis/tenosynovitis, and a general lack of performance including diminished weight gains, poor feed conversions, uneven growth rates, and reduced marketability of affected birds.
Avian Malabsorption Syndrome
Avian Malabsorption Syndrome (MAS) also known as Runting Stunting Syndrome (RSS) is a disease of growing poultry, especially chickens or turkeys, with meat-type or broilers being affected most commonly. This disease has been reported in the Netherlands as Runting and Stunting Syndrome (RSS) but also under different names for example infectious stunting syndrome, enteritis, pale bird syndrome, helicopter disease, infectious proventriculitis, brittle bone disease and femoral head necrosis.
Kouwenhoven et al. (Avian Pathology 17:879-892, (1988)) further defined MAS by five criteria: 1) growth impairment up to 3 weeks after infection of one-day old chicks; 2) excretion of yellow orange mucoid to wet droppings; 3) increased plasma alkaline phosphatase (ALP) activity; 4) decreased plasma carotenoid concentration (PCC); and 5) macroscopically widened epiphyseal growth plates of the proximal tibia. The condition has been further characterized by stunted growth, poor feathering, maldigested feed, lack of skin pigmentation, enteritis, pancreatic atrophy, proventriculitis, thymic and bursal atrophy and bone changes.
The transmission of the disease is effected by oral inoculation of intestinal homogenates from affected chicks into one-day-old broilers. In that experiment, it was demonstrated that low plasma carotenoid levels and elevated ALP activities are suitable tools for the diagnosis of MAS or RSS. In further experiments, MAS was transmitted by oral inoculation of liver homogenates from affected chicks into one-day-old broilers. Despite years of research, the etiology of MAS has not yet been fully established, and the condition is still a major economic problem for the poultry industry. It is believed that viruses are responsible, but bacteria or other microorganisms have not been excluded as causal agents.
Viruses that have been found in birds with outbreaks of MAS or RSS possibly include reoviruses, rotaviruses, parvoviruses, entero-like viruses and a toga-like virus (McNulty and McFerran, 1993, and Pantin-Jackwood MJ- and et al., AVIAN DISEASES 52:235-244, 2008). McNulty (World Poultry 14:57-58 (1998); and Pantin-Jackwood MJ (2008)), summarized the state of art on MAS, and have postulated that identification of the viral causative agent is unknown and recommends control by careful management of production sites.
SUMMARY OF THE INVENTION
The present invention provides newly isolated unique avian reoviridae serotypes, designated AVS-A, AVS-B, and AVS-A/B, which have been deposited with the American Type Culture Collection (“ATCC” 10801 University Boulevard, Manassas, Va. 20110-2209 USA) under Budapest Treaty on Jun. 26, 2008 and have accession numbers PTA-9301, PTA-9302, and PTA-9297 respectively. Also, nucleotide sequences encoding antigenic epitopes of these novel serotypes, corresponding to the inner core proteins, e.g., σA proteins of the novel avian reoviridae serotypes have been characterized. AVS-A and AVS-B reoviridae serotypes comprise an ORF sequence encoding an inner core protein (σA proteins) as set forth in one or more of the following nucleotide sequences: SEQ ID NO.1 and 2, respectively, or a sequence having at least 85%, 90%, 93%, 95%, or 97% homology with a nucleotide sequence as set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2. Preferably, novel isolated avian reoviridae serotypes of the invention comprise a nucleotide sequence capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
The present invention also relates to newly characterized nucleotide sequences of reoviridae genome, comprising more precisely the sequence of the genomic L1 segment (3958 base pairs) with a predicted open reading frame between nucleotides 21-3882 encoding the λA protein (SEQ ID NO: 3), the sequence of the genomic L2 segment (3829 base pairs) with a predicted open reading frame between nucleotides 14-3793 encoding the λB protein (SEQ ID NO: 4), the sequence of the genomic L3 segment (3907 base pairs) with a predicted open reading frame between nucleotides 13 to 3870 encoding the λC protein (SEQ ID NO: 5), the sequence of the genomic M1 segment (2283 base pairs) with a predicted open reading frame between nucleotides 13 to 2211 encoding the μA protein (SEQ ID NO: 6), the sequence of the genomic M2 segment (2158 base pairs) with a predicted open reading frame between nucleotides 30 to 2060 encoding the μB protein (SEQ ID NO: 7), the sequence of the genomic M3 segment (1996 base pairs) with a predicted open reading frame between nucleotides 25 to 1932 encoding the μNS protein (SEQ ID NO: 8), the sequence of the genomic S1 segment (1645 base pairs) with a predicted open reading frame between nucleotides 632 to 1612 encoding the σC protein (SEQ ID NO: 9), the sequence of the genomic S2 segment (1324 base pairs) with a predicted open reading frame between nucleotides 16 to 1266 encoding the GA protein (SEQ ID NO: 10), the sequence of the genomic S3 segment (1202 base pairs) with a predicted open reading frame between nucleotides 31 to 1134 encoding the GB protein (SEQ ID NO: 11), and the sequence of the genomic S4 segment (1192 base pairs) with a predicted open reading frame between nucleotides 24 to 1127 encoding the σNS protein (SEQ ID NO: 12). Accordingly, the present invention also provides nucleotide sequences from novel unique reoviridae as set forth in SEQ ID NOs: 3-12, as well as portions thereof encoding for the above listed reoviral proteins, sequences having at least 85%, 90%, 93%, 95%, or 97% homology with nucleotide sequences as set forth in SEQ ID NOs: 3-12, and/or sequences capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 3-12.
The present invention further relates to a composition comprising novel isolated unique avian reoviridae serotypes comprising nucleotide sequences, portions thereof as set forth in SEQ ID NOs: 3-12, or a sequence having at least 85%, 90%, 93%, 95%, or 97% homology with nucleotide sequences as set forth in SEQ ID NOs: 3-12, or a sequence capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 3-12.
In another embodiment, a vaccine is provided for use in the protection of poultry against disease conditions resulting from avian reovirus infections, comprising the reoviridae isolates or a part of the nucleotide sequence of reoviridae isolates disclosed herein, and a pharmaceutically acceptable carrier.
The invention also relates to a vaccine comprising antigenic materials derived from the novel isolated unique avian reoviridae serotypes wherein said materials comprise amino acid sequences encoded by nucleotide sequences as set forth in SEQ ID NOs: 1-12, or by a sequence having at least 85%, 90%, 93%, 95%, or 97% homology with nucleotide sequences as set forth in SEQ ID NOs: 1-12, or by a sequence capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-12.
The present invention also provides antiserum that is induced in an animal by the unique avian reoviridae serotypes disclosed herein. In another embodiment, there is also provided antibodies that bind to the unique avian reoviridae isolates disclosed herein.
The present invention further provides a method of eliciting an immune response in poultry comprising administering a composition comprising the novel unique avian reoviridae serotypes disclosed herein or amino acid sequence thereof encoded by nucleotide sequences as set forth in SEQ ID NOs: 1-12, or by a sequence having at least 85%, 90%, 93%, 95%, or 97% homology with nucleotide sequences as set forth in SEQ ID NOs: 1-12, or by a sequence capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-12.
The present invention also provides a method of protecting poultry against disease conditions resulting from an avian reovirus infection, comprising the step of administering the vaccine disclosed herein to the poultry.
The present invention also provides a method of detecting the presence of novel avian reoviridae isolates in poultry, comprising the steps of: obtaining a sample from the poultry; contacting the sample with antibodies or probes that bind to the avian reoviridae isolates disclosed herein; and detecting binding of the antibodies or probes to the sample, wherein detected binding would indicate the presence of reoviridae isolates of the present invention in the poultry.
The present invention also relates to a method of quantifying novel avian reoviridae isolates in poultry comprising the steps of contacting a sample from poultry with an antibody which binds to the novel reoviridae isolates or the amino acid sequence encoded by nucleotide sequences as set forth in SEQ ID NOs: 1-12, or by a sequence having at least 85%, 90%, 93%, 95%, or 97% homology with nucleotide sequences as set forth in SEQ ID NOs: 1-12, or by a sequence capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-12, and measuring the amount of binding of the antibody to a component of the sample.
The present invention also provides a container comprising at least one dose of the vaccine composition as disclosed herein. The present invention further provides a kit comprising the container and an instruction manual, including information for the administration of at least one dose of the vaccine to poultry for lessening the severity of the MAS or RSS and sequelae including immune suppression.
The present invention further provides diagnostic kits comprising probes that are capable of hybridizing under stringent conditions with nucleotide sequences as disclosed, herein or comprising antibodies that are capable of binding with reoviridae serotypes of the invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the nucleotide sequence encoding σA protein (inner core protein) of reoviridae serotype AVS-A of the present invention.
FIG. 2 shows the nucleotide sequence encoding σA protein (inner core protein) of reoviridae serotype AVS-B of the present invention.
FIG. 3 shows the genome segment designation (L1-L3, M1-M3, S1-S4; on the left), lengths of each genome segment (expressed in base pair; numbers on the right), the location and size of the predicted open reading frames and the protein names are indicated (shaded boxes).
FIG. 4 shows the sequence of the genomic L1 segment (3958 base pairs) with a predicted open reading frame between nucleotides 21 to 3882 encoding the λA protein (SEQ ID NO: 3).
FIG. 5 shows the sequence of the genomic L2 segment (3829 base pairs) with a predicted open reading frame between nucleotides 14 to 3793 encoding the λB protein (SEQ ID NO: 4).
FIG. 6 shows the sequence of the genomic L3 segment (3907 base pairs) with a predicted open reading frame between nucleotides 13 to 3870 encoding the λC protein (SEQ ID NO: 5).
FIG. 7 shows the sequence of the genomic M1 segment (2283 base pairs) with a predicted open reading frame between nucleotides 13 to 2211 encoding the μA protein (SEQ ID NO: 6).
FIG. 8 shows the sequence of the genomic M2 segment (2158 base pairs) with a predicted open reading frame between nucleotides 30 to 2060 encoding the μB protein (SEQ ID NO: 7).
FIG. 9 shows the sequence of the genomic M3 segment (1996 base pairs) with a predicted open reading frame between nucleotides 25 to 1932 encoding the μNS protein (SEQ ID NO: 8).
FIG. 10 shows the sequence of the genomic S1 segment (1645 base pairs) with a predicted open reading frame between nucleotides 632 to 1612 encoding the σC protein (SEQ ID NO: 9).
FIG. 11 shows the sequence of the genomic S2 segment (1324 base pairs) with a predicted open reading frame between nucleotides 16 to 1266 encoding the σA protein (SEQ ID NO: 10).
FIG. 12 shows the sequence of the genomic S3 segment (1202 base pairs) with a predicted open reading frame between nucleotides 31 to 1134 encoding the σB protein (SEQ ID NO: 11).
FIG. 13 shows the sequence of the genomic S4 segment (1192 base pairs) with a predicted open reading frame between nucleotides 24 to 1127 encoding the σNS protein (SEQ ID NO: 12).
FIG. 14 shows the phylogenic relationships among avian reovirus. The novel serotypes isolated by the inventors from clinical cases of Runting and Stunting Syndrome are underlined.



DETAILED DESCRIPTION OF THE INVENTION
Avian reoviridae display displays considerable antigenic heterogeneity and the emergence of new antigenic and pathogenic classes of avian reoviridae may have important implications for the use of reoviridae vaccines in poultry. The present specification now identifies a novel antigenic class of avian reoviridae. Furthermore, it is demonstrated that avian reoviridae isolates belonging to this novel antigenic class are able to induce pronounced disease conditions associated with Avian Malabsorption Syndrome (MAS) also known as Runting and Stunting Syndrome (RSS).
In one embodiment, the present invention provides a new antigenic class of avian reoviridae as a causative agent of a variety of disease conditions in affected poultry, i.e., RSS disease, including enteritis. In another embodiment, the present invention provides a vaccine which effectively affords protection in poultry against disease caused by avian reoviridae of the new antigenic class. In yet another embodiment, the present invention provides a vaccine which effectively affords protection in poultry against variety of disease conditions in affected poultry, i.e., RSS disease, including enteritis.
These newly isolated unique avian reoviridae serotypes, designated AVS-A, AVS-B, and AVS-A/B, have been deposited with the American Type Culture Collection (“ATCC” 10801 University Boulevard, Manassas, Va. 20110-2209 USA) under Budapest Treaty on Jun. 26, 2008 and have accession numbers PTA-9301, PTA-9302, and PTA-9297 respectively. The reoviridae serotype AVS-A/B presents antigenic epitopes common to serotypes AVS-A and AVS-B, and thus corresponds to a natural reassortment thereof.
The present invention thus relates to compositions comprising the newly isolated unique avian reoviridae serotypes as described above. Compositions of the invention may further comprise a pharmaceutically acceptable carrier, adjuvant, or diluents. The composition may comprise an isolated reovirus, a live reovirus that is attenuated, or an isolated reovirus that is inactivated. Compositions may comprise at least 102 titration units of the reoviridae isolates.
The invention also relates to nucleotide sequences encoding antigenic epitopes of the novel serotypes, corresponding to the inner core proteins of the novel reoviridae serotypes have been characterized. AVS-A and AVS-B reoviridae serotypes comprise an ORF sequence encoding an inner core protein as set forth in one or more of the following nucleotide sequences: SEQ ID NOs: 1 and 2, or a sequence wising having at least 85%, 90%, 93%, 95%, or 97% homology with a nucleotide sequence as set forth in SEQ ID NO: 1 and/or SEQ ID NO: 2. Preferably, novel isolated avian reoviridae serotypes of the invention comprise a nucleotide sequence capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.
The present invention also relates to novel isolated nucleotide sequences of reoviridae genome, comprising more precisely the sequence of the genomic L1 segment (3958 base pairs) with a predicted open reading frame between nucleotides 21 to 3882 encoding the inner core λA protein (SEQ ID NO: 3), the sequence of the genomic L2 segment (3829 base pairs) with a predicted open reading frame between nucleotides 14 to 3793 encoding the inner core λB protein (SEQ ID NO: 4), the sequence of the genomic L3 segment (3907 base pairs) with a predicted open reading frame between nucleotides 13 to 3870 encoding the λC protein (SEQ ID NO: 5), the sequence of the genomic M1 segment (2283 base pairs) with a predicted open reading frame between nucleotides 13 to 2211 encoding the inner core μA protein (SEQ ID NO: 6), the sequence of the genomic M2 segment (2158 base pairs) with a predicted open reading frame between nucleotides 30 to 2060 encoding the outer capsid protein, μB protein (SEQ ID NO: 7), the sequence of the genomic M3 segment (1996 base pairs) with a predicted open reading frame between nucleotides 25 to 1932 encoding the non structural protein, μNS protein (SEQ ID NO: 8), the sequence of the genomic S1 segment (1645 base pairs) with a predicted open reading frame between nucleotides 632 to 1612 encoding the outer capsid sigma C protein, σC protein (SEQ ID NO: 9), the sequence of the genomic S2 segment (1324 base pairs) with a predicted open reading frame between nucleotides 16 to 1266 encoding the inner core protein, σA protein (SEQ ID NO: 10), the sequence of the genomic S3 segment (1202 base pairs) with a predicted open reading frame between nucleotides 31 to 1134 encoding the outer capsid protein or σB protein (SEQ ID NO: 11), and the sequence of the genomic S4 segment (1192 base pairs) with a predicted open reading frame between nucleotides 24 to 1127 encoding the σNS protein (SEQ ID NO: 12). Accordingly, the present invention also provides nucleotide sequences from novel unique reoviridae as set forth in SEQ ID NOs: 3-12, as well as portions thereof encoding reoviridae peptides or portions thereof, sequences having at least 85%, 90%, 93%, 95%, or 97% homology with nucleotide sequences as set forth in SEQ ID NOs: 3-12, and/or sequences capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 3-12.
The present invention also relates to nucleotide sequences or portions thereof encoding antigenic epitopes of the avian reoviridae serotypes as set forth in SEQ ID NOs: 3-12, or a sequence having at least 85%, 90%, 93%, 95%, or 97% homology with nucleotide sequences as set forth in SEQ ID NOs: 3-12, or a sequence capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 3-12.
A most preferred nucleotide sequence according to the present invention comprises a nucleotide sequence as set forth in SEQ ID NO: 9 encoding the outer capsid protein, σC protein of reoviridae, or nucleotide sequence as set forth in SEQ ID NO: 11 encoding the outer capsid σB protein, and/or the nucleotide sequence as set forth in SEQ ID NO: 7 encoding the outer capsid μB protein. According to a preferred embodiment, antigenic epitopes are encoded by nucleotide sequences chosen among SEQ ID NOs: 7, 9, or 11, or a sequence having at least 85%, 90%, 93%, 95%, or 97% homology with nucleotide sequences as set forth in SEQ ID NOs: 7, 9, or 11, and/or a sequence capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 7, 9, or 11. According to a most preferred embodiment, antigenic epitopes are encoded by a nucleotide sequence as set forth in SEQ ID NO: 9, or a sequence having at least 85%, 90%, 93%, 95%, or 97% homology with a nucleotide sequence as set forth in SEQ ID NO: 9, and/or a sequence capable of hybridizing under stringent conditions with SEQ ID NO: 9.
The present invention further relates to a composition comprising novel isolated unique avian reoviridae serotypes comprising antigenic amino acid sequences encoded by nucleotide sequences, or portions thereof as set forth in SEQ ID NOs: 1-12, or a sequence having at least 85%, 90%, 93%, 95%, or 97% homology with nucleotide sequences as set forth in SEQ ID NOs: 1-12, or a sequence capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-12.
In general, appropriate antiserum raised against the live reoviridae isolates disclosed herein can be prepared by inoculating 3 to 4 weeks old chickens subcutaneously, intramuscularly or intravenously with a live virus strain having an infectious titre between 102-109 TC ID50/animal; more preferably between 103-106 TC D50/animal. Blood can be collected 3 to 4 weeks after infection, preferably 4 weeks after infection. Chickens may also be reinfected with the same live virus strain 3 to 4 weeks after the first infection with approximately the same dose as used in the first infection. Blood is collected between 2 and 4 weeks after the second infection.
Appropriate antiserum can also be raised against inactivated avian reoviridae strains disclosed herein by inoculating 3 to 4 weeks old chickens subcutaneously or intramuscularly with the inactivated virus preparation. The infectious titre of the preparation before inactivation may be between 107-1011 TC ID50/animal; more preferably between 108-1010 TC ID50/animal. Blood can be collected 4 to 6 weeks after inoculation, preferably 5 weeks after inoculation. Chickens may also be re-inoculated with the same inactivated virus preparation 3 to 6 weeks after the first inoculation. Blood is collected between 3 and 5 weeks after the second inoculation.
The present invention also provides a vaccine for use in the protection of poultry against disease conditions resulting from an avian reoviridae infection, such as RSS disease including enteric disease conditions, comprising an avian reoviridae according to the invention and a pharmaceutically acceptable carrier or diluent.
The reoviridae isolates or a part of its genome according to the present invention can be incorporated into the vaccine as a live attenuated or inactivated virus or as subunit vaccines or as part of a recombinant vaccine. The property of the avian reoviridae isolates to induce RSS-associated disease conditions as described herein are significantly reduced or completely absent if the avian reoviridae isolate is in a live attenuated or inactivated form or as a subunit vaccine or as part of a recombinant vaccine.
The vaccine may comprise antigenic materials derived from the novel isolated unique avian reoviridae serotypes wherein said materials comprise amino acid sequences which are encoded by nucleotide sequences as set forth in SEQ ID NOs: 1-12, portions thereof, or by a sequence having at least 85%, 90%, 93%, 95%, or 97% homology with nucleotide sequences as set forth in SEQ ID NOs: 1-12, or by a sequence capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-12.
Attenuation of an avian reoviridae isolate according to the invention can be achieved by methods well known in the art for this purpose, such as disclosed in Gouvea et al. (Virology 126:240-247, (1983)). Briefly, after the isolation of the virus from a target animal, a virus suspension is inoculated onto primary (avian) cell cultures, such as chicken embryo liver cells (CEL), Chicken kidney cells (CK), or mammalian cell lines such as the VERO cell line, BGM-70 cell line or avian cell line such as QT-35, QM-7, LMH, or other susceptible cell culture. If the isolate is not able to produce cytopathic effect (CPE), then the virus is passaged repeatedly (e.g. 3-10 times) until CPE is observed. As soon as CPE is visible, cells and cell culture fluids are collected, frozen and thawed, clarified by centrifugation and the supernatant containing the avian reoviridae isolate is aliquoted and stored at −20° C. This process may be repeated (e.g. 10-100 times) to further attenuate the virus.
In one embodiment, a vaccine according to the present invention can be prepared by conventional methods such as, for example, commonly used for the commercially available live- and inactivated reoviridae vaccines. For example, the preparation of veterinary vaccine compositions is described in “Vaccines for Veterinary Applications” (ed.: Peters, A. R. et al., Butterworth-Heinemann Ltd, 1993).
In one embodiment, the vaccine according to the present invention containing live attenuated virus can be prepared and marketed in the form of a (frozen) suspension or in a lyophilised form. The vaccine additionally may contain a pharmaceutically acceptable carrier or diluent customarily used for such compositions. Carriers include stabilisers, preservatives and buffers. Suitable stabilisers are, for example, SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried milk serum, albumin or casein) or degradation products thereof. Suitable buffers are for example alkali metal phosphates. Suitable preservatives are thimerosal, merthiolate and gentamicin. Diluents include water, aqueous buffer (such as buffered saline), alcohols and polyols (such as glycerol).
In another embodiment, the live vaccines according to the present invention may contain an adjuvant. Examples of suitable compounds and compositions with adjuvant activity are generally known in the art for the preparation of vaccines.
Although administration by injection, e.g., intramuscular, subcutaneous of the live vaccine according to the present invention is possible. The live vaccine is preferably administered by the inexpensive mass application techniques commonly used for avian reovirus vaccination. These techniques include drinking water and spray vaccination. Alternative methods for administration of the live vaccine include in ovo, eye drop and beak dipping administration.
In another embodiment, the present invention provides a vaccine against enteric disease conditions, such as those observed with RSS, comprising the reoviridae isolates disclosed herein in an inactivated form. The major advantage of an inactivated vaccine is the elevated levels of protective antibodies of long duration that can be obtained. This property makes such an inactivated vaccine in particular suited for vaccination of chicken and turkey breeder stocks.
The aim of inactivation of the viruses harvested after the propagation step is to eliminate reproduction and infectiousness of the viruses. In general, this can be achieved by chemical or physical means. Chemical inactivation can be effected by treating the viruses with, for example, enzymes, formaldehyde, beta-propiolactone, ethylene-imine or a derivative thereof. If necessary, the inactivating compound is neutralized afterwards. Material inactivated with formaldehyde can, for example, be neutralized with thiosulphate. Physical inactivation can preferably be carried out by subjecting the viruses to energy-rich radiation, such as UV light or γ-rays. If desired, after treatment the pH can be adjusted to a value of about 7.
A vaccine containing the inactivated avian reoviridae isolates disclosed herein can, for example, comprise one or more of the above-mentioned pharmaceutically acceptable carriers or diluents suited for this purpose. Preferably, an inactivated vaccine according to the present invention comprises one or more compounds with adjuvant activity. Suitable compounds or compositions for this purpose include aluminium hydroxide, -phosphate or -oxide, oil-in-water or water-in-oil emulsion based on, for example, a mineral oil, such as Bayol F® or Marcol 52® or a vegetable oil such as vitamin E acetate, and saponins. In general, inactivated vaccines are usually administered parenterally, e.g., intramuscularly or subcutaneously.
The vaccine according to the present invention comprises an effective dosage of the reoviridae isolates disclosed herein as the active component, i.e., an amount of immunizing avian reoviridae material that will induce immunity in the vaccinated birds or their progeny against challenge by a virulent virus. Immunity is defined herein as the induction of a significantly higher level of protection in a population of birds after vaccination compared to an unvaccinated group. Typical doses and schedule for vaccination are generally known in the art.
The reoviridae vaccines according to the present invention may be used effectively in poultry, i.e., chickens as well as other poultry such as turkeys, guinea fowl and quail. Chickens include broilers, reproduction stock and laying stock. Turkeys include meat-type birds and breeder stock.
The present invention also provides combination vaccines comprising, in addition to the avian reoviridae of the present invention, one or more vaccine components of other pathogens infectious to poultry. In general, such other pathogens infectious to poultry are antigenically distinct from the unique avian reoviridae of the present invention. In one embodiment, the vaccine components in the combination vaccine are live attenuated or inactivated forms of the pathogens infectious to poultry. In another embodiment, the present invention provides a combination vaccine wherein all of the vaccine components are in an inactivated form.
In another embodiment, a vaccine is provided for use in the protection of poultry against disease conditions resulting from an avian reoviridae infection, comprising the reoviridae isolates disclosed herein, and a pharmaceutically acceptable carrier. In general, the vaccine may comprise the avian reoviridae in a live attenuated or inactivated form. The vaccine may further comprise an adjuvant.
In another embodiment, the vaccine may be a combination vaccine and comprises one or more vaccine components against a second pathogen infectious to poultry. The combination vaccine comprises, in addition to the novel reoviridae serotypes, one or more of an astrovirus, other classical avian reovirus, an infectious bronchitis virus (IBV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), fowl adenovirus (FAV), EDS virus (type III adenovirus) and turkey rhinotracheitis virus (TRTV). Preferably, the combination vaccine comprises astrovirus as a second pathogen.
The invention thus also provides a vaccine for use in the protection of poultry against disease conditions and particularly syndromes of MAS or nephritis resulting from an avian reoviridae and astrovirus co-infection, comprising the reoviridae disclosed herein, an astrovirus and a pharmaceutical acceptable carrier.
In general, the vaccine may comprise the reoviridae isolates and astrovirus and in live attenuated or inactivated forms. The vaccine may further comprise an adjuvant.
The present invention also provides subunit vaccine comprising the reoviridae isolates of the present invention. Genes from the reoviridae isolates disclosed herein producing protective proteins against the disease could be cloned into expression vectors or carriers generally known in the art. Expression of these protective proteins could then be produced as subunit vaccines according to standard procedures well-known in the art.
The present invention also provides recombinant vaccines comprising the reoviridae isolates of the present invention. Genes from the reoviridae isolates disclosed herein producing protective proteins against disease could be cloned into live or inactivated vectors or other carriers generally known in the art for expression of these proteins as vaccines. In addition, these viruses themselves could be used as live attenuated vaccines (vectors) to carry genes producing protective proteins from other agents allowing for protection against the vector as well as the inserted gene. More precisely, the invention relates to nucleotide sequences: SEQ ID NOs: 1-12, or a sequence comprising having at least 85%, 90%, 93%, 95%, or 97% homology with a nucleotide sequence as set forth in SEQ ID NOs: 1-12. Preferably, novel isolated avian reoviridae serotypes of the invention comprise a nucleotide sequence capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-12.
The present invention also provides antiserum that is induced in an animal by the reoviridae isolates and an astrovirus disclosed herein. The present invention also provides antibodies that bind to the reoviridae isolates and astrovirus disclosed herein. In one embodiment, the antibodies are monoclonal antibodies, polyclonal antibodies, chimeric antibodies, or single chain antibodies, bispecific antibodies, synthetic antibodies, antibodies fragment such as Fab, F(ab)2, Fv, or scFv fragments, or chemically modified derivatives thereof.
Monoclonal antibodies can be prepared, for example, by the techniques which are well known to a skilled person in the art. Such techniques involve the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. Furthermore, antibodies, or fragments thereof, can be obtained by using methods which are described, for example, in Harlow and Lane, Antibodies, A Laboratory Manual, CSH Press, Cold Spring Harbor, 1988. Useful antibodies, or their corresponding immunoglobulin chain(s), can be further modified using conventional techniques known in the art, for example by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art (see, e.g., Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.).
The present invention also provides a method of protecting poultry against disease conditions, particularly severe RSS and sequelae resulting from an avian reoviridae and astrovirus co-infection, comprising the step of administering the vaccine disclosed herein to the poultry. Examples of poultry include, but are not limited to, chickens, turkeys, guinea fowl, and quail.
The present invention also provides a method of detecting the presence of unique avian reoviridae in poultry, comprising the steps of: obtaining a sample from the poultry; contacting the sample with antibodies or probes that bind to the reoviridae disclosed herein; and detecting binding of the antibodies or probes to the sample, wherein detected binding would indicate the presence of the novel avian unique reoviridae of the present invention in the poultry. In general, the sample is prepared from intestine, liver, or feces of the poultry. Examples of poultry include, but are not limited to, chickens, turkeys, guinea fowl, and quail.
The method of detecting and/or method of quantifying novel avian reoviridae isolates in poultry may comprise the steps of contacting a sample from poultry with an antibody which binds to the novel reoviridae isolates or the amino acid sequence encoded by nucleotide sequences as set forth in SEQ ID NOs: 1-12, or by a sequence having at least 85%, 90%, 93%, 95%, or 97% homology with nucleotide sequences as set forth in SEQ ID NOs: 1-12, or by a sequence capable of hybridizing under stringent conditions with one or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-12, and measuring the amount of binding of the antibody to a component of the sample.
By way of example, the amount of novel reoviridae isolates in poultry may be measured using the ELISA assays. In practice, standards and samples to be tested are incubated in a in microtiter wells pre-coated with antibody that captures the reovirus. The standards and samples are diluted in a buffer containing detergent for 1 hour at room temperature prior to loading the microtiter wells. The captured reoviridae particles are then detected, for example, with biotinylated second antibody that binds to the reoviridae which is then detected, for example, by using a streptavidin-peroxidase conjugate and colored substrate. The concentration of the reoviridae particles is then determined using a standard curve prepared from known amounts of the reoviridae particles. ELISA techniques are well known to those of ordinary skill in the art.
The present invention further provides a diagnostic kit comprising antibodies or probes that bind to the reoviridae disclosed herein, means for detecting antibodies or probes, thereby allowing following the presence of avian reoviridae and the level of infection of poultry.
The present invention also relates to a novel propagation cell line which is derived from original LMH cell lines (ATCC accession number CRL 2117), which has been adapted as non-collagen dependent cell line and to grow on plastic plates. The novel propagation cell line LMH-KJ has been deposited with the American Type Culture Collection (“ATCC” 10801 University Boulevard, Manassas, Va. 20110-2209 USA) under Budapest Treaty on Jun. 26, 2008, and has accession number PTA-9299.
The present invention further relates to a method of propagating novel isolated reoviridae serotypes consisting in infecting the non-collagen dependent cell lines LMH-KJ, culturing the cells under same growing conditions as that of cells LMH (ATCC accession number CRL 2117) with gentamicin, and harvesting reoviridae when CPE is visible.
The age of the animals receiving a live or inactivated vaccine according to the invention is the same as that of the animals receiving the presently commercially available live- or inactivated avian reoviridae vaccines. For example, broilers may be vaccinated directly from one-day-old onwards with the live attenuated vaccine according to the invention. Vaccination of parent stock, such as broiler breeders, can be done with a live attenuated or inactivated vaccine according to the invention or combinations of both. The advantages of this type of immunization program include the immediate protection of one-day-old progeny provided by maternally derived antibodies vertically transmitted to the young birds. A typical breeder vaccination program includes the vaccination of the breeders at 6-weeks of age with a live attenuated vaccine, followed by a vaccination between 14-18 weeks of age with an inactivated vaccine. Alternatively, the live vaccination may be followed by two vaccinations with inactivated vaccines on 10-12 weeks and 16-18 weeks of age.
The present invention finally provides a container comprising at least one dose of the vaccine as described herein, and a kit comprising such container with an instruction manual, including information for the administration of at least one dose of the vaccine to poultry for lessening the severity of the RSS and sequalae.
The invention being generally described, will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.
EXAMPLES
Example 1
Isolation of Reoviridae
The reoviridae isolates disclosed herein were isolated from several diseased chickens, from Delmarva USA. The reoviridae isolates were cultivated in an adapted hepatocellular carcinoma cell line (CH-SAH, which is alternatively called the LMH cell line). The original LMH cell line was obtained from the American Type Culture Collection (ATCC), accession number ATCC CRL 2117.
Example 2
PCR and Sequencing
Double stranded RNA was isolated from reoviridae isolates serotypes AVS-A, AVS-B and AVS-A/B using the Quiagen RNA purification kit. Multiple RT-PCRs were conducted according to published reovirus procedures. Using the procedures of Xie et al. (AVIAN DISEASES 41:654-660 (1997). Amplification of the unique avian reoviridae RNA using the Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) to amplify the sigma C protein gene, an outer capsid protein gene on segment S1, no PCR products were detected for either serotypes AVS-A, AVS-B, or AVS-A/B, while known avian reovirus strains S1133, 2408 and SS412 amplified the expected 523 bp product.
RT-PCR was conducted following the procedures of Zhang et al. (Arch Virol (2006) 151:1525-1538). Detection and identification of avian, duck, and goose reoviruses by RT-PCR: goose and duck reoviruses are part of the same genogroup in the genus Orthoreovirus) to amplify the inner core protein gene, sigma A gene on the S2 segment. PCR products of approximately 600 bp were amplified for serotypes A and B, as well as S1133, 2408, and SS412.
These RT-PCR primers for the reoviridae sigma A, inner core protein gene and the reoviridae sigma C outer capsid protein gene were used to identify the causative viral agents as the RSSV. The reoviridae outer capsid primers do not amplify genes from these reoviridae isolates while the reoviridae inner core primers do. In addition, the rotavirus outer capsid primers did amplify the avian rotavirus but not the RSSV (Table 1).







TABLE 1







Differential identification of viruses


involved in RSS using RT-PCR













Reovirus Inner
Reovirus Outer
Rotavirus


Avian
Strain
Core RT-PCR
Capsid RT-PCR
Outer Capsid


Virus
Name
for σA
for σC
RT-PCR





Reovirus
S1133
Positive
Positive
Negative


Reovirus
2408
Positive
Positive
Negative


Reovirus
SS412
Positive
Positive
Negative


Rotavirus
Ch2/5
Negative
Negative
Positive


Reoviridae
AVS-A
Positive
Negative
Negative


Reoviridae
AVS-B
Positive
Negative
Negative


Reoviridae
AVS-A/B
Positive
Negative
Negative


Astrovirus
AVS-1
Negative
Negative
Negative


IBDV
Standard
Negative
Negative
Negative


IBDV
Variant E
Negative
Negative
Negative









Example 3
RT-PCR and Sequencing
Using the procedures of Zhang et al ((Arch Virol (2006) 151:1525-1538) to amplify the inner core protein gene, sigma A gene on the S2 segment, RT-PCR products of approximately 600 bp were amplified for AVS-A, AVS-B, AVS-A/B, S1133, 2408, and SS412. These RT-PCR products were purified and sequenced using these primers. The sequences of the RT-PCR products for AVS-A and AVS-B were 100% similar to one another. When compared to strains S1133 and SS412, the sequences were highly conserved. When the sequences were blasted in PudMed, the reoviridae were 90% similar to avian reovirus accessions for ARV17 and 99G. Avian reovirus strain S1133 was 100% similar to 99G and SS412 was 93.7% similar to OS161.
Example 4
Efficacy of inactivated RSSV Vaccine
The efficacy of an inactivated RSSV vaccine was evaluated in progeny chicks of vaccinated breeder hens. The RSSV vaccine consists of reoviruses (designated AVS-A, AVS-B) and an astrovirus (designated AVS-1) that were inactivated using 0.2% formalin (final concentration) and is adjuvanted with an oil based adjuvant. Groups 1 and 2 comprised of specific pathogen free chickens were vaccinated at 32 weeks of age with 0.5 ml of the vaccine subcutaneously in the back of the neck. Group 2 was revaccinated with 1.0 ml of the same vaccine at 4 weeks post 1st vaccination. The other group of the chickens (Group 1) was not revaccinated. The hens were bled right before the 1st vaccination and at 4 and 8 weeks post the 1st vaccination. Starting from four weeks after the 2nd vaccination, eggs were collected from the vaccinated hens and set in an incubator for hatching. After hatching, progeny chicks at day of age were challenged with 0.5 ml of AVS-A. Day of age specific pathogen free chicks were used as challenge controls (Group 3). The chicks that were challenged were necropsied for gross lesions associated with reovirus such as extended ceca with gaseous/yellow contents, pale intestines and distended, watery, gas-filled intestines.
After challenge with AVS-A, 80% ( 16/20) of the non-vaccinated, challenge controls developed gross lesions associated with reovirus (Table 2). For the progeny chicks from the hens vaccinated once (Group 1), only 21% ( 3/14) of chicks were protected against AVS-A. For the progeny chicks from the hens vaccinated twice (Group 2), 56% ( 10/18) of chicks were protected against AVS-A. This result suggests that vaccinating hens twice with the inactivated RSSV vaccine gives adequate protection in progeny chicks against reovirus.
In summary, the RSSV vaccine provided adequate efficacy against AVS-A in the progeny chicks from the hens vaccinated with the vaccine twice.







TABLE 2







Treatment groups and protection against challenge with AVS-A















1st
2nd






vaccination
vaccination
Protection of progeny


Group
Treatment
# of
(0.5 ml, 32
(1.0 ml, 36
chicks, #protected/


#
Group
hens
weeks of age)
weeks of age)
#total (% protection)





1
RSSV vaccine
15
Yes
No
3/14 (21%)



(once)


2
RSSV vaccine
15
Yes
Yes
10/18 (56%) 



(twice)


3
Non-vaccinated,
15
N/A1
N/A
4/20 (20%)



challenge



controls





N/A1= not applicable






Example 5
The Genomic Constellation of the Avian Orthoreovirus Strain AVS-B
The genomic RNA was extracted by the TRIzol method. Preliminary sequence information was obtained by gene specific RT-PCR amplification of fragments of each genome segment. A short oligonucleotide was ligated to the 3′ ends of the genomic RNA, and then its complementary primers were used in combination with gene specific primers to determine the 5′ and 3′ ends of the genomic segments. Nucleotide sequences were determined by the dideoxy chain termination method.
The full-length genome (23,494 bp) of strain AVS-B was determined by the primer walking sequencing strategy. The length of untranslated regions ranged 12 nt to 30 nt at the 5′ end, and 30 nt to 98 nt at the 3′ end. The most 5′ and 3′ end sequences were conserved′ in all genome segments (5′ end, GCUUUU(U). 3′ end, UCAUC). The L1 genome segment of AVS-B strain was most closely related to strain 918 (91% identity), while the L3 genome segment shared the highest nucleotide identity with strains 1017-1 and R2 (89%). Only two L2 genome segments of chicken avian orthoreovirus have been published so far (strains 138 and 176); AVS-B strain was more closely related to strain 138 (91% nt identity). Comparison of the M1, M2, and M3 genome segments revealed moderate to high sequence identities to other avian orthoreovirus strains, the most closely related isolates being 138 (M1, 90% identity), 601G (M2, 85% identity) and OS161 (M3, 88% identity). The S1 genome segment was found to be tricistronic with partially overlapping open reading frames (ORFs); the position of the 3 ORFs (p10, p17 and σC) was similar to that seen in other chicken avian orthoreovirus strains. The σC coding region shared low nucleotide identity to the majority of strains (30%-70%), except for GA 40973/2005, (93%) isolated from a similar case of RSS in the USA. With regard to the S2, S3 and S4 genome segments, AVS-B strain shared the highest nucleotide identity to 138 (S2, 93%; S3, 92%), and to recent US chicken avian orthoreovirus strains (up to 93%).
While the availability sequence information on avian orthoreovirus allowed gaining insight into the overall structure of avian orthoreovirus strains, this is the first to report on a full-length avian orthoreovirus genome sequence. The length of the AVS-B strain genome fell in the size range predicted based on full-length cognate genome segment sequences available for a handful of reference avian orthoreovirus strains. The majority of genes of AVS-B strain shared significant degrees of sequence conservation with related genes of prototype avian orthoreovirus strains (>85%). However, the S1 segment of the genome represented an exception. This genome segment shared only <67% nucleotide identity with corresponding genome segment of other avian orthoreovirus strains. On the other hand, the σC protein encoding region of the S1 genome segment was closely related to another US avian orthoreovirus strain isolated also from RSS. The low number of avian orthoreovirus gene sequences and the lack of absolute sequence identities with related strains did not permit to identify the possible parental strains of this novel strain. Nonetheless, there seems to be convincing evidence that accumulation of point mutations and reassortment of cognate genome segments played key role in the origin and evolution of the AVS-B strain.

## Claims

1. A vaccine comprising a peptide encoded by SEQ ID NO: 1 or 2 and an adjuvant selected from aluminum hydroxide, aluminum phosphate, aluminum oxide, an oil-in-water or a water-in-oil emulsion.

2. The vaccine according to claim 1, wherein said oil is a mineral oil, a vegetable oil, vitamin E acetate or a saponin.

3. An inactivated avian reoviridae produced by contacting an avian reoviridae deposited at the ATCC under accession number PTA-9301, PTA-9302 or PTA-9297 with formaldehyde, beta-propriolactone or ethylene-imine and inactivating said avian reoviridae.

4. A vaccine comprising the inactivated reoviridae of claim 3 and a pharmaceutically acceptable carrier or diluent.

5. The vaccine of claim 4 further comprising an adjuvant.

6. The vaccine according to claim 5, wherein said adjuvant is aluminum hydroxide, aluminum phosphate, aluminum oxide, an oil-in-water or water-in-oil emulsion.

7. The vaccine according to claim 6, wherein said oil is a mineral oil, a vegetable oil, vitamin E acetate or a saponin.

8. The vaccine of claim 4, wherein the vaccine further comprises one or more vaccine components against a second pathogen infectious to poultry.

9. A kit for poultry vaccination comprising a container comprising a vaccine according to claim 4 and an instruction manual, the instruction manual including information for the administration of at least one dose of said vaccine to poultry for lessening the severity of MAS, RSS or sequelae.

10. An attenuated avian reoviridae produced by attenuation of an avian reoviridae deposited at the ATCC under accession number PTA-9301, PTA-9302 or PTA-9297, said attenuation comprising inoculating a primary cell culture with an avian reoviridae deposited at the ATCC under accession number PTA-9301, PTA-9302 or PTA-9297, or passaging said avian reoviridae on said cell culture until a cytopathic effect is obtained, and subsequent freezing, thawing and isolation of said attenuated reoviridae.

11. A vaccine comprising the attenuated reoviridae of claim 10 and a pharmaceutically acceptable carrier or diluent.

12. The vaccine according to claim 11 further comprising an adjuvant.

13. The vaccine according to claim 12, wherein said adjuvant is aluminum hydroxide, aluminum phosphate, aluminum oxide, an oil-in-water or water-in-oil emulsion.

14. The vaccine according to claim 13, wherein said oil is a mineral oil, a vegetable oil, vitamin E acetate or a saponin.

15. The vaccine according to claim 14, wherein said oil is a mineral oil, a vegetable oil, vitamin E acetate or a saponin.

16. The vaccine according to claim 14, wherein said oil is a mineral oil, a vegetable oil, vitamin E acetate or a saponin.

17. The vaccine according to claim 11, wherein the vaccine further comprises one or more vaccine components against a second pathogen infectious to poultry.

18. A kit for poultry vaccination comprising a container comprising a vaccine according to claim 11 and an instruction manual, the instruction manual including information for the administration of at least one dose of said vaccine to poultry for lessening the severity of MAS, RSS or sequelae.