A small round virus (SRV) was isolated in 1988 from droppings of enteritis-affected turkeys in North Carolina and tentatively identified as an enterovirus on the basis of size (18–24 nm in diameter), intracytoplasmic morphogenesis, and a single-stranded RNA genome of approximately 7.5 kb. Additional characterization studies based on antigenic and genomic analyses were done to determine the relationship of this turkey enterovirus-like virus (TELV) to turkey astrovirus 2 (TAstV2), a recently characterized SRV of turkeys. Cross-immunofluorescence studies with TELV- and TAstV2-specific antisera indicated a close antigenic relationship between these viruses. TELV RNA was amplified by reverse transcriptase–polymerase chain reaction (RT-PCR) procedures with oligonucleotide primers specific for TAstV2 polymerase gene (open reading frame [ORF] 1b) and capsid protein gene (ORF 2). Subsequent sequence analyses of these TELV-derived RT-PCR products indicated a high degree of similarity with polymerase gene (98.8%) and capsid gene (96.9%) of TAstV2. These studies definitively identify TELV (North Carolina, 1988 isolate) as TAstV2.
The term small round virus (SRV) has been used to designate similar small viruses that are detected by electron microscopy but generally cannot be definitively identified on the basis of morphologic criteria (4,18). The SRVs associated as causes of enteric disease in avian and mammalian species include enterovirus, parvovirus, calicivirus, and astrovirus (2). These viruses often are difficult to differentiate not only on the basis of electron microscopic features but other physiochemical features as well. They are similar in size, ranging from approximately 18 to 38 nm in diameter, and they are all nonenveloped viruses (9). Astroviruses and caliciviruses were named for characteristic morphologic features observed by electron microscopy: the five- or six-pointed starlike surface of astroviruses and the cuplike surface depressions of caliciviruses (4). However, these morphologic features may be absent, potentially leading to misidentification of these viruses. Additionally, these SRVs have similar buoyant densities in cesium chloride, and they are heat resistant and stable at low pH (9). Enterovirus, calicivirus, and astrovirus have similar genomes comprised of single-stranded RNA that range in size from 7 to 8 kb (9).
Astroviruses typically are 28–30 nm in diameter (9). They have a buoyant density in cesium chloride of 1.36–1.39 g/ml, and they possess a linear, single-stranded RNA genome of 7.2–7.9 kb (9). A turkey astrovirus (TAstV) was first described in 1980 by McNulty et al. (12) in the United Kingdom; the virus was associated as a cause of diarrhea and increased mortality in young turkeys. Subsequently, TAstV was identified in turkey flocks in the United States (14). In 2000, Schultz-Cherry et al. isolated a TAstV from thymus of turkeys experiencing high mortality and severe growth depression (poult enteritis–mortality syndrome) (1,15). This TAstV was antigenically and genetically distinct from previously described U.S. isolates (TAstV1); thus, two distinct astroviruses, designated TAstV1 and TAstV2, are now recognized in turkeys (11). Koci et al. (11) recently sequenced the entire TAstV2 genome; these studies demonstrated that TAstV2 genome organization was consistent with other astroviruses; the virus possessed a 5′ untranslated region followed by three open reading frames (ORFs), a 3′ untranslated region, and a poly-A tail. Additionally, these studies resulted in development of reverse transcriptase (RT)–polymerase chain reaction (PCR) procedures for specific amplification of portions of polymerase (ORF 1b) and capsid (ORF 2) genes of the virus (10).
In 1988, during investigations of enteric disease in young turkeys, a SRV was isolated from droppings collected from a turkey flock in western North Carolina (5). The virus tentatively was identified as an enterovirus on the basis of size (18–24 nm in diameter), lack of obvious virion surface structure as determined by direct electron microscopy, intracytoplasmic morphogenesis, and a single-stranded RNA genome of approximately 7.5 kb. However, more recent studies based on antigenic analyses questioned the original classification of this turkey enterovirus-like virus (TELV) and instead suggested a close relationship with TAstV2 (9; Tom Hooper, pers. comm.). The present report describes antigenic and genomic studies to determine the relationship between TELV (North Carolina, 1988 isolate) and TAstV2.
MATERIALS AND METHODS
TELV, isolate NC88, was isolated in 1988 at North Carolina State University College of Veterinary Medicine from droppings collected from enteritis-affected turkeys in North Carolina (5). TAstV2 was obtained from Stacey Shultz-Cherry; this virus was isolated in 2000 at Southeast Poultry Research Laboratory, USDA-ARS, Athens, GA, from thymus of enteritis-affected turkeys (15). These viruses were propagated in embryonated turkey eggs as described (5).
Commercial medium white turkeys were obtained at 1 day of age from a primary breeder company (British United Turkeys of America, Lewisburg, WV). These turkeys were derived from a breeder flock that was free of Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis, Mycoplasma iowae, Salmonella pullorum, Salmonella typhimurium, Salmonella enteritidis, Salmonella arizona, avian influenza virus, turkey coronavirus, and reticuloendotheliosis virus; turkeys were not examined for other extraneous infectious agents. Turkeys were housed in wire-floored, electrically heated brooders in an isolation room with controlled access until inoculation. Nonmedicated game bird starter and water were provided ad libitum.
Convalescent antisera specific for TELV (NC88) and TAstV2 were prepared by oral exposure of 7-day-old turkeys with a 20% suspension of turkey embryo intestines containing the respective virus and collection of antisera 21 days postexposure. Antiserum specific for TAstV2 capsid protein was prepared by immunization of rabbits with a peptide sequence within the TAstV2 capsid protein (K676–R691) as described (8).
Monoclonal antibodies (MAbs) specific for TELV (NC88) were prepared by the procedure described by Carter et al. (3). Briefly, mice were immunized with partially purified TELV (NC88), and splenocytes collected from immunized mice were fused with murine myeloma cells. Hybridoma colonies secreting antibodies specific for TELV (NC88) were detected by assay of cell culture supernatant fluids by an indirect immunofluorescent antibody test (IFAT) with cryostat sections of TELV (NC88)-infected turkey embryo intestines as described below. Each positive hybridoma colony was cloned twice by limiting dilution, and ascites fluid was produced by intraperitoneal injection of approximately 107 hybridoma cells into pristane-primed mice.
TELV (NC88)- and TAstV2-infected turkey embryo intestines were harvested from infected turkey embryos; 20-day-old embryonated turkey eggs were inoculated by yolk sac, and intestines were collected at 3 days postexposure. Intestines were placed in a commercially available cryogenic compound (Tissue-Tek O.C.T. Compound; Miles Laboratories, Elkhart, IN) and immediately frozen. Tissues were sectioned with a cryostat, air dried, and fixed for 10 min in cold (−20 C) absolute acetone. The IFAT was performed as previously described (5). Briefly, turkey-origin antisera were diluted 1:40 in phosphate-buffered saline solution (PBSS); MAb and rabbit-origin antisera were diluted 1:100 in PBSS. Diluted antisera were overlayered onto intestinal sections and incubated at 37 C for 30 min. Slides were washed briefly in three changes of PBSS and tissues were overlayered with a 1:40 dilution of the corresponding antispecies fluorescein isothiocyanate–labeled anti-immunoglobulin G (KPL Inc., Gaithersburg, MD). Slides were incubated at 37 C for 30 min, washed briefly in PBSS, and examined by fluorescent microscopy at 400×. Sections of intestines from uninoculated turkey embryos were treated similarly and served as controls. Antibody controls also were included for each section; nonimmune rabbit sera, turkey convalescent antisera specific for turkey coronavirus, and a MAb specific for feline herpesvirus 1 were used in place of virus-specific antisera.
Total RNA was harvested from TELV (NC88)-infected, TAstV2-infected, and uninfected turkey embryo intestines with the Stratagene Micro RNA Isolation Kit (Stratagene, La Jolla, CA) according to the manufacturer's directions. Viral cDNA was prepared with a commercially available RT reaction kit (Promega, Madison, WI). The RT reaction consisted of RT buffer (100 mM Tris-HCl, pH 8.8, 500 mM KCl, 1% Triton® X-100), 5 mM MgCl2, 1 mM each deoxynucleotide triphosphate, 20 units RNAsin, 12.5 units avian myeloblastosis virus RT, 0.5 μg random primers, 1 μg template RNA, and nuclease-free water to 20 μl. The reaction was completed in a programmable thermal cycler at 42 C for 30 min, 99 C for 5 min, and 4 C for 5 min per the directions in the kit. PCR was conducted in the same tube on the resultant cDNA in a final volume of 100 μl. The PCR reaction consisted of 2.5 units Taq DNA polymerase (Promega), PCR buffer (100 mM Tris-HCl, pH 8.85, 250 mM KCl, 50 mM [NH4]SO4), 2.5 mM MgSO4, 100 ng of each oligonucleotide primer, and nuclease-free water to volume. Oligonucleotide primers were synthesized (Invitrogen Corp., San Diego, CA) on the basis of previously described primer sequences for PCR amplification of TAstV2 RNA: MKCap8 and MKCap19 amplify an 849-bp segment within the TAstV2 capsid gene (ORF 2), and MKPo110 and MKPol11 amplify a 802-bp segment within the viral polymerase gene (ORF 1b) (10). Samples were placed in a hot (94 C) thermal cycler for 1 min, followed by 35 cycles of 94 C for 1 min, 56 C for 30 sec, and 72 C for 2 min, and finished with a final extension at 72 C for 2.5 min. PCR products were analyzed on a 1% Ultra Pure Agarose (Invitrogen Corp.) gel with 0.1 μg/ml ethidium bromide (Sigma Chemical Co., St. Louis, MO).
RT-PCR products were cloned with the use of TOPO TA cloning system (Invitrogen Corp.) according to the manufacturer's directions (15).
DNA was sequenced at the University of North Carolina (Chapel Hill) Automated DNA Sequencing Facility on a Model 373A DNA Sequencer (Applied Biosystems, Foster City, CA) with the Taq DyeDeoxy™ Terminator Cycle Sequencing Kit (Applied Biosystems). All sequences were confirmed by sequencing both strands. Comparative analyses of protein sequences were performed with the MegAlign application of the Lasergene software package (DNASTAR, Madison, WI).
MAb production and characterization
A single hybridoma cell line was identified that secreted antibodies (MAb 91) specific for TELV (NC88). This cell line was selected on the basis of strong reaction of antibody to TELV antigens as determined by IFAT and absence of specific reaction when IFAT was performed with uninfected turkey embryo intestines.
Cross-immunofluorescence studies were done to assess antigenic relationships between TELV (NC88) and TAstV2. Antigenic relationships were examined with homologous and heterologous antibodies. TELV (NC88) and TAstV2 were indistinguishable from each other on the basis of these studies. Strong fluorescence was observed in TELV (NC88)-infected and TAstV2-infected turkey embryo intestines with turkey convalescent antisera prepared against TELV (NC88) and TAstV2 (data not shown). Similarly, strong fluorescence was observed in TELV (NC88)-infected and TAstV2-infected turkey embryo intestines with both TELV (NC88)-specific MAb 91 and rabbit antibodies specific for TAstV2 capsid protein. No fluorescence was observed in uninfected turkey embryo intestines stained with convalescent turkey antisera, MAb 91, or TAstV2 capsid protein-specific antibodies.
RT-PCR and sequencing
RNA obtained from TELV (NC88)-infected turkey embryo intestines was amplified in a RT-PCR with oligonucleotide primers that were based on polymerase gene (ORF 1b) and capsid gene (ORF 2) sequences of TAstV2. The PCR products were approximately 800 bp in size with the TAstV2 polymerase gene (ORF 1b) primers and 850 bp in size with the TAstV2 capsid gene (ORF 2) primers; these products were identical in size with the products obtained with TAstV2 RNA as template (data not shown). No PCR product was observed when RNA was harvested from uninfected turkey embryo intestines, and amplified by RT-PCR, or when RT-PCR was run without RT (data not shown).
The PCR product (approximately 800 bp) obtained from TELV (NC88) RNA with primers specific for TAstV2 polymerase gene (ORF 1b) was cloned and sequenced. The deduced amino acid sequence (240 amino acids) was determined to have 98.8% identity with TAstV2 polymerase protein and 57.1% identity with TAstV1 polymerase protein (Fig. 1).
The PCR product (approximately 850 bp) obtained from TELV (NC88) RNA with primers specific for TAstV2 capsid gene (ORF 2) was cloned and sequenced. The deduced amino acid sequence (261 amino acids) was determined to have 96.9% identity with TAstV2 capsid protein and 19.9% identity with TAstV1 capsid protein (Fig. 2).
Several SRVs, including TELV (NC88), have been detected in avian species and identified as enterovirus-like viruses (7). The term enterovirus-like has been applied to these viruses because they have not been fully characterized. In general, these SRVs are identified as enterovirus-like if they are detected in feces or intestinal contents and have physicochemical properties consistent with members of the family Picornaviridae (13). However, definitive classification of these viruses requires additional antigenic, physicochemical, or molecular characterization studies. In a previous study, a SRV associated with enteric disease in young turkeys (TELV [NC88]) was propagated in embryonated turkey eggs and tentatively identified as an enterovirus (5). In the present study, this enterovirus-like virus was further characterized on the basis of antigenic (cross-immunofluorescence) and genomic (gene sequencing) studies and definitively identified as TAstV2.
On the basis of cross-immunofluorescence procedures with polyclonal and monoclonal antibodies, TELV (NC88) could not be differentiated from TAstV2. These studies indicated a close antigenic relationship between these viruses. Additionally, oligonucleotide primers that previously were described for RT-PCR amplification of TAstV2 RNA within the polymerase (ORF 1b) and capsid (ORF 2) genes were shown in this study to amplify TELV (NC88) RNA. The RT-PCR products with TELV (NC88) RNA and these oligonucleotide primers were very similar in size to the products obtained with TAstV2 RNA. Subsequent nucleotide sequence analyses of the resultant DNA products and comparison of deduced amino acid sequences demonstrated a high degree of identity (>96%) between polymerase and capsid protein sequences of TELV (NC88) and those of TAstV2. In contrast, a much lower degree of identity was observed between polymerase protein of TELV (NC88) and TAstV1 (57.1%), and capsid protein of TELV (NC88) and TAstV1 (19.9%).
The present study demonstrated the difficulties associated with classification of SRVs based only on physicochemical analyses. The initial identification of TELV (NC88) was based on virion size (18–24 nm in diameter), morphology (lack of obvious virion surface structure), intracytoplasmic morphogenesis, buoyant density of 1.33 g/ml in CsCl, and a single-stranded RNA genome of approximately 7.5 kb (5). These characteristics suggested an identification as an enterovirus; however, these same characteristics, with the exception of virion size and buoyant density, also were consistent with astroviruses. Reported size determinations for astroviruses (28–30 nm) and buoyant density (1.36–1.39 g/ml in CsCl) were distinctly different from the values determined for TELV (NC88) during the initial characterization studies (5). The reasons for these discrepancies were not examined in the present study and remain unexplained; however, measurement of virion size and buoyant density are inherently prone to error, and likely this accounts for the observed discrepancies.
TAstV2 was isolated in 2000 from enteritis-affected turkeys and subsequently characterized (9,10,11,15). Molecular characterization of TAstV2 included nucleotide sequence analyses of the entire viral genome and development of RT-PCR procedures for virus detection (10,11). Additionally, virus pathogenicity studies resulted in production of antisera specific for TAstV2 capsid protein (8). These TAstV2 characterization studies provided the reagents and sequence information that were utilized in the present study for definitive identification of TELV (NC88) and reclassification of this virus as TAstV2.
Other poultry viruses that are now classified as picornaviruses (enteroviruses), picornavirus-like viruses, and enterovirus-like viruses likely will be reclassified as astroviruses. Avian nephritis virus (ANV), like TELV (NC88), initially was identified as a picornavirus on the basis of virion size and morphology (17). However, subsequent molecular characterization studies led to reclassification of ANV as an astrovirus (6).