The isolation and characterisation of astroviruses from chickens

Abstract

The isolation, cultivation and characterization of three chicken astroviruses (CAstV) isolates are described. They are antigenically related to each other but unrelated to avian nephritis virus (ANV) and duck hepatitis virus type 2 (DVH2) in neutralization, immunofluorescence and gel diffusion tests. CAstV, ANV and DVH2 all grew well in the LMH cell line, which was used for assay and serological tests. Serological surveys in 1982 and 2001 showed that antibody to CAstV virus was widespread in broiler and broiler breeder flocks and present in some turkey flocks. Infection of 1-day-old specific pathogen free chicks with one isolate in the laboratory resulted in mild diarrhoea and some distention of the small intestine. The virus could be isolated in high titres from all parts of the small intestine but rarely from other organs. Electron microscopic examination of purified particles of this agent revealed the presence of clusters of small round viruses with a diameter ranging from 25 to 30 nm. The amino acid sequence derived from the relatively conserved non-structural polyprotein region of this virus shows 62% identity with the corresponding region of turkey astrovirus 2, 58% identity with turkey astrovirus 1, 55% identity with avian nephritis virus and 33% identity with sheep astroviruses. Taken together, the results indicate that the agent is a new chicken astrovirus belonging to the family Astroviridae.

Introduction

Astroviruses are small round, non-enveloped, viruses with a typical diameter of 28 to 30 nm, possessing a positive-stranded RNA genome. The family Astroviridae is divided into two genera of mammalian astroviruses and avian astroviruses. Astroviruses cause usually a mild gastroenteritis in most species. However, several reports indicate the association of astroviruses with acute disease in the young of multiple species and more severe diseases in poultry have also been described to be caused by astroviruses (Matsui & Greenberg, 2001).

In poultry, astroviruses have been recognized to cause problems in turkeys (McNulty et al., 1980) and have been shown to cause diarrhoea and an increased mortality (Reynolds et al., 1987; Koci et al., 2000; Yu et al., 2000). Two types of turkey astrovirus (TAstV) have been identified, TAstV-1 and TAstV-2, but these turkey astroviruses have been found to be genetically and immunologically distinct (reviewed in Koci & Schultz-Cherry, 2002). Another poultry astrovirus, avian nephritis virus (ANV) isolated from chickens by Yamaguchi et al. (1979), has now been classified as an astrovirus following the complete sequencing of the viral genome (Imada et al., 2000). This virus is associated with mild growth depression and kidney lesions, and some mortality has also been reported. One-day-old chicks are the birds most susceptible to disease caused by ANV. In ducks, in contrast to other avian species, astroviruses have been associated with a fatal hepatitis. The causal agent of duck virus hepatitis type 2 (DVH2) (Asplin, 1965) has a typical astrovirus morphology (Gough et al., 1984).

We report here the isolation and characterization of three isolates of a chicken astrovirus (CAstV) that are antigenically identical and serologically distinct from ANV and DVH2 but show some sequence identity with ANV and TAstV. The viruses can be cultivated in cell culture, and neutralization tests on sera from the field indicate that antibody is widespread in chickens and also antibody is found in some turkey flocks.

Materials and Methods

Cell cultures

Chick embryo liver cell (CEL) cultures were prepared from 14-day-old specific pathogen free (SPF) embryos. The livers were removed and cut into small fragments, washed with phosphate-buffered saline (PBS) and trypsinized in PBS containing 1% trypsin for 3 min at 37°C, being constantly stirred. The supernatant was removed and fresh PBS/trypsin added. This step was repeated five times and then the cells in the supernatant deposited by slow speed centrifugation, and resuspended in growth medium (Eagle's modified essential medium [MEM]) containing 7.5% foetal calf serum (FCS) buffered with bicarbonate. Six-centimetre Falcon petri dishes were seeded with 4×10⁶ cells in 5 ml growth medium, and incubated at 38.5°C in a 5% CO₂ atmosphere.

LMH cells (Kawaguchi et al., 1987), a chicken heptocellular carcinoma cell line, were grown in T25 flasks (Falcon) in 10 ml growth medium, seeded at 10⁶ cells per flask and incubated at 38.5°C. The cells were subcultured after 4 days, when the cells were treated with warm (37°C) 1% trypsin in PBS after the medium had been removed. After 10 min the tryspin solution was poured off and growth medium added. The cells were washed off the surface by pipetting, resuspended in medium and diluted to give the correct seeding rate. The preparation of chick embryo fibroblasts and chick kidney has been described (Baxendale, 1971).

Isolation of chicken astroviruses

Strain VDU/AS1 was isolated in 1981. Tracheal and vent swabs taken from 10 broiler chicks taken at 5 days of age from a flock that appeared to have started to show the early clinical signs of the runting and stunting syndrome (McNulty & McFerran, 1993) were washed with PBS and the total volume (0.2 ml) of fluid from each sample was inoculated on to two CEL cultures on 6 cm petri dishes. After 6 days of incubation in an atmosphere of 5% CO₂ at 38.5°C a cytopathic effect (CPE) was observed in the cultures inoculated with one of the tracheal swab samples. This isolate was passaged twice before being plaque-purified three times, a single plaque being taken from a plate with less than 10 plaques on it. Reovirus and adenovirus was isolated from most of the other samples.

Strain VDU/AS2 was isolated in 1981. Ten 1 ml heparinized blood samples were taken from 1-week-old broiler chicks that showed uneven growth. The white blood cells were separated from the samples after centrifugation (using a bench centrifuge) and approximately 0.1 ml packed white cells were inoculated into the medium of each of two 48 h old CEL cell culture plates. After 24 h incubation in a 5% CO₂ atmosphere at 38.5°C the white cells together with the medium were poured off the cell cultures. The cultures were washed with PBS and the maintenance medium replenished. After a further 6 days of incubation a CPE was observed in two of the cultures inoculated with the white blood cells from one chick, no other virus being isolated.

VDU/AS3 was isolated from a pool of homogenized small intestines with contents (SI) from three 3-week-old broiler chicks exhibiting clinical signs typical of the runting and stunting syndrome together with diarrhoea containing partly digested food. The SI sample was filtered using a coarse filter and diluted 100-fold with PBS containing five times normal cell culture levels of penicillin and streptomycin, centrifuged (bench centrifuge) to remove particulate debris and then the supernatant was diluted in 10-fold steps to 10^–4 and 0.1 ml each dilution inoculated onto four CEL 6 cm petri dishes. After absorption for 40 min two dishes were overlaid with growth medium containing 0.9% agar and two re-fed with normal fluid maintenance medium. Four days later typical reovirus plaques and CPE were observed in dilutions up to 10^–3. Plaques different from reovirus plaques were observed in one dish at the 10^–4 dilution. A single plaque was picked and inoculated onto fresh CEL cultures where a CPE developed by 5 days. The virus was plaque-picked on two further occasions before a pool of virus was laid down.

Other viruses

The ANV strain G4260 was provided kindly by Dr R. Nicholas and the DVH2 (strainM52/A/2) by Ms Barbara Westbury at the Veterinary Laboratories Agency (Addlestone, UK).

Cultivation of viruses

Pools of the CAstVs were prepared by infecting CEL cultures when confluent. When a cytopathic effected was observed, 90% of the culture fluids were removed by pipette and infected cells, and the remaining fluid snap-frozen at −70°C and used as inoculum for further passage.

Both ANV and DVH2 were adapted to LMH cells by passage. LMH cultures were grown in T75 flasks the flasks being seeded with 30×10⁶ cells in 40 ml MEM with 7.5% FCS. When confluent, 0.5 ml viruses was inoculated into the medium and the cultures incubated at 38.5°C and examined for a CPE daily. Both cells and supernatant were snap-frozen when a CPE developed, and were used for further passage.

Tests of CAstV pools for extraneous agents

Hyperimmune antisera to the three CAstV pools were tested for antibodies to Marek's disease virus, reovirus, fowl adenovirus, and fowlpox by the gel precipitation test. The serum neutralization test was used to test for antibodies to infectious laryngotracheitis virus, the avian leucosis viruses, the EDS'76 adenovirus, reticuloendotheliosis virus, infectious bursal disease virus, avian encephalomyelitis virus, turkey rhinotracheitis virus and chick anaemia virus.

Virus assay

The CAstVs were initially titrated on CEL cultures, but LMH cells were later used. ANV and DVH2 were assayed on LMH cells. Each of two confluent pre-drained cell cultures on 6 cm petri dishes were inoculated with 0.1 ml of 10-fold dilutions of virus diluted in MEM. After incubation for 1 h at 38.5°C the plates were re-fed with 6 ml MEM containing 1.0% agar and 7.5% FCS serum. After 2 days of incubation in a 5% CO₂ atmosphere at 38.5°C, the plates received 2 ml MEM with 1.0% agar and 5% FCS. After 2 days further incubation the dishes are stained with 2 ml buffered MEM containing 0.15% neutral red, and the plaques were read 24 h later.

Neutralization tests

The plaque-reduction test was used for CAstV and DVH2 antibody assays. Two-fold dilutions of sera were mixed with an equal volume of virus diluted in MEM to give 200 plaque forming units (PFU) in 0.1 ml. After incubation for 1 h at room temperature the virus/serum mixtures were assayed as already described. A negative serum and virus control was included in every test and the antibody titre was the dilution that neutralized 75 to 80% of plaques.

The microtitre neutralization test was used for ANV antibody assays. Two-fold dilutions of serum were mixed with an equal volume of virus diluted in MEM to give 200 median tissue culture infective doses per 0.1 ml. Following incubation at room temperature for 1 h, 0.1 ml from each dilution was placed into four microtitre wells in a 96-well plate and 0.1 ml growth medium and 3×10⁴ LMH cells added to each well. A virus titration and negative serum was included in each test. The plates were incubated for 5 days at 38.5°C in a CO₂ atmosphere and then read microscopically. The serum titre was the dilution where only 50% of wells showed a CPE.

Immunofluorescence test

LMH cells grown on 6 cm petri dishes were infected with the relevant virus diluted to give approximately 10⁶ PFU/ml, and 0.1 ml was inoculated onto drained plates. After 1 h adsorption the cells were re-fed with 5 ml MEM with 2% calf serum. The cells were then incubated and fixed at 6 h for DVH2 and for 18 h for the other viruses. For fixation 70% cold acetone was used for 20 min after the cultures were washed with PBS. The acetone was removed and the cultures allowed to dry. They were then inoculated with dilutions of specific chicken antisera, 0.4 ml per plate. After 1 h incubation at 37°C they were washed with PBS and 0.4 ml of an appropriate dilution of FITC-conjugated rabbit anti-chicken serum added to each plate. After a further 1 h at 37°C the plates were washed again with PBS and 1 ml of 50% glycerol in PBS added, and they were then examined using a fluorescent microscope.

Gel diffusion test

The method used was as described previously (Baxendale, 1971) except that antigen was prepared from heavily infected either CEL or LMH cultures, the cells and supernatant being mixed with an equal volume of saturated ammonium sulphate and left at 4°C overnight. The precipitate was recovered after centrifugation and sufficient PBS added to just re-dissolve it.

Antiserum preparation

Approximately 10 five-day-old SPF chicks were inoculated orally in the case of ANV and CAstV and were inoculated subcutaneously for DVH2 with 0.1 ml virus with a titre of 10⁵ to 10⁶ PFU/ml. After 3 weeks they received a 0.3 ml subcutaneous inoculation containing high titre purified virus mixed with an equal volume of Freund's incomplete adjuvant. The chicks were bled at 3, 4 and 7 weeks of age.

Tests to establish the immunological relationship of the viruses

Where the CAstVs were compared with each other, antiserum taken 3 weeks post infection was used, as sera taken early in the immune response are likely to be highly specific and differences between the viruses might be detected. In contrast, where CAstV was compared with ANV and DVH2, hyperimmune antiserum, which is often more broadly reactive, was used to detect any possible immunological cross-reaction between viruses.

CAstV concentration and electron microscopy

High titre purified virus was prepared for molecular studies, antibody preparation and electron microscopy. Confluent LMH cultures in T75 flasks were infected with approximately 10⁶ PFU virus after the medium had been removed. The cultures were re-fed with serum-free buffered MEM and incubated at 38.5°C. When 80% of the cells showed a CPE, 90% of the medium was removed and the flask snap-frozen at –70°C. Following thawing, the cells and medium were ultra-sonicated (three bursts of 10 sec) and an equal volume of the fluoro-carbon Arklone added. The samples were shaken vigorously for 2 min, centrifuged at 500g for 5 min on a bench centrifuge. The Arklone layer was removed and the aqueous layer placed on a 60% sucrose cushion. Following centrifugation at 100,000×g for 1 h the supernatant above the cushion interface was discarded and the interface recovered and mixed at a ratio of 1:3 with Tris–NaCl buffer to give a sucrose concentration of 20%. This was layered on to a 20 to 60% sucrose gradient and centrifuged for 2 h at 130,000×g. Ten equal fractions were taken from each tube by pipette and each sample assayed for virus. The highest titre virus was found in the lower half of the gradient, usually fractions 7 and 8. Suspension containing viral particles purified from infected LMH cells was placed on carbon-coated copper grids and subjected to negative staining and electronic microscopic examination.

Reverse transcriptase-polymerase chain reaction and sequence determination

In order to obtain some insight about the nature of the agent, we first examined the concentrated and purified preparation by electron microscopy. This revealed the presence of clusters of small round viruses (SRV). In order to obtain some sequence information from the genome of the agent, the concentrated and purified material described earlier was used for the isolation of total RNA using the Rneasey kit (Qiagen). Due to the morphological resemblance of the agent with SRV, we chose to employ two primers derived from a conserved region situated in ORF1 of small-round-structured virus (SRSV) genomes (Wyn-Jones et al., 2000) instead of priming the polymerase chain reaction (PCR) with random primers. Reverse transcription by avian myeloblastosis virus reverse transcriptase on 1 μg total RNA was primed with a degenerate oligonucleotide (5′-GCRTTGGTGGTGATGACTAT-3′). This primer was also used for DNA amplification together with another degenerate primer (5′-GCCCARGCTGAAATGAC-3′). The PCR product was then analysed on 1% agarose gel and the sequence was determined on both directions using these primers.

Chick experiment 1

The aim of this experiment was to see whether VDU/AS3 was pathogenic for 1-day-old chicks. Twenty 1-day-old SPF White Leghorn chicks were inoculated orally with 0.2 ml containing 10^5.6 PFU of the third CEL cell culture passage of VDU/AS3 after it had been cloned by picking a single plaque three times. They were placed in isolation and examined for clinical signs for 5 days, at which time they were killed and subjected to postmortem examination. A similar group of uninfected chicks were kept in separate isolation.

Chick experiment 2

This experiment was designed to establish in which portion of the bowel VDU/AS1 replicated. Fifteen SPF five-day-old White Leghorn chicks were inoculated orally with 0.3 ml VDU/AS1 with a titre of 10^5.6 PFU/ml. The chicks and a further nine uninfected chicks were housed in separate isolators. On days 2, 4, 6, 8 and 10, two infected and one control were killed and sections of the proventriculus, duodenal loop, jejunum, ileum and caeca were taken and divided into two pieces. One was placed into Bouins fixative for later histological examination and the other was thoroughly washed to remove bowel contents before being snap-frozen at –70°C. These sections were thawed, an equal volume of PBS added and then chopped into small fragments with scissors. They were then sonicated for three bursts of 8 sec, centrifuged for 5 min at 250g on a bench centrifuge, and then the supernatant assayed for virus content on CEL cultures. Samples of the liver, kidney, bursa and thymus were also taken and fixed in Bouins for later examination. The remaining chicks were bled at 3 weeks and the sera tested for CAstV antibodies.

Field surveys for CAstV antibodies

In 1982 sera was taken from broiler flocks in the United Kingdom, Holland, Spain and Australia, and also UK broiler breeder and turkey flocks. Between 10 and 20 sera per flock were tested at 1:32 dilution for CAstV neutralizing antibodies using the VDU/AS1 isolate. Sera taken in 2001 from UK broiler flocks and from broiler breeder flocks were tested at 1:64 for neutralizing antibodies, four to six sera per flock. Six SPF flocks were also tested in 1982 and one in 2002.

Results

Growth in cell culture

The CAstV isolates grew well in CEL cultures, producing a marked CPE after four to five passages. All the viruses grew well in LMH cells producing a marked CPE, with most of the infectious virus being contained within the cells. The plaque morphology of the CAstVs grown under agar was similar with both CEL and LMH cells. ANV, DVH2 and CAstV when assayed in LMH cells, however, produced very different plaques. When viewed with indirect light at 5 days post inoculation, the CAstV ones appeared opaque, the DVH2 ones clear and the ANV ones smaller and less distinct. However, when viewed with direct light, the CAst and DVH2 ones appeared similar (Figure 1). In the immunofluorescence test all the viruses-infected cultures showed cytoplasmic fluorescence when stained with specific antisera (Figure 2). The CAstVs grew only poorly in chick embryo fibroblasts and chick kidney cultures initially, but on passage they grew well in chick kidney and produced a marked CPE.

Figure 1. CAstV (VDU AS1 strain) (1a), DVH2 (1b) and ANV (1c) plaques in LMH cells 5 days post infection, stained with neutral red and viewed with direct light. The CAstV and DVH2 plaques are easily seen whereas the ANV plaques are less distinct.

Figure 2. Immunofluorescence of CAstV strain VDU/AS1 in chick embryo liver cultures showing cytoplasmic staining in the infected liver cells and no fluorescence in fibroblastic cells (arrows). Bar 50 μm.

Tests for extraneous agents

No antibodies to the viruses listed in Materials and Methods were detected in the hyperimmune antisera prepared against the three CAst pools of virus.

Serological relationship

The three CAstV isolates appeared antigenically identical (Table 1), all reacting equally well with antisera prepared against each virus and having r ratios of between 0.7 and 1.0 (Archetti & Horsfall, 1950). There was no cross-reaction between heterologus hyperimmune antisera and the viruses ANV, DVH2 and CAstV in any of the tests (Table 2), with r ratios<0.01 in neutralization tests. Up to three lines of precipitation were observed between the viruses and their homologous antisera in the gel diffusion test but none were shared.

Table 1. Immunological relationship between VDU/AS isolates

		Virus
Antisera	Serological test	VDU/AS1	VDU/AS2	VDU/AS3
VDU/AS1	Neutralisation	256^a	128	256
	IFT	64	64	64
	Gel Diffusion	+^b	+	+
VDU/AS2	Neutralisation	128	128	256
	IFT	32	32	32
	Gel Diffusion	+	+	+
VDU/AS3	Neutralisation	256	512	512
	IFT	128	128	128
	Gel Diffusion	+	+	+
^a serum titre.
^b Positive reaction.

Table 2. Immunological relationship between the astrovirus VDU/AS1, ANV and DVH 2

		Virus
Antisera	Serological Test	VDU/AS1	ANV	DVH 2
VDU/AS1	Neutralisation	1600^a	<10	<10
	IFT	256	<10	<10
	Gel Diffusion	+^b	−^c	−
ANV	Neutralisation	<10	2048	<10
	IFT	<10	64	<10
	Gel Diffusion	−	+	−
DVH 2	Neutralisation	<10	<10	1600
	IFT	<10	<10	128
	Gel Diffusion	−	−	+
^a serum titre.
^b Positive reaction.
^c No reaction.

Morphology of the agent and sequence data

Examination in an electron microscope revealed the presence of clusters of SRVs with a diameter ranging from 25 to 30 nm (Figure 3). Based on this observation, the PCR reaction was primed with two primers derived from a conserved genome region situated in open reading frame 1 of SRSV (Wyn-Jones et al., 2000). The obtained PCR product was approximately 400 base pairs (bp) in size, although the expected product was approximately 350 bp. This suggested that the agent varied considerably from SRSV in the amplified region. The nucleotide sequence determined from both directions (∼320 bp) was then compared with reported sequences in the GenBank database using a basic local alignment search tool (BLAST). Surprisingly, the putative amino acid sequences derived from the PCR product were identified as having high similarities to the non-structural polyproteins of TAstV-2 (62% identities; Figure 4), TAstV-1 (58% identities), ANV (55% identities) and sheep astroviruses (33% identities). Sequence comparison between CAstV and the sheep astrovirus covers positions 642 to 705 of the non-structural protein of sheep astrovirus (GeneBank accession number Y15937) and the first 66 amino acids of CAstV. Due to the very high sequence divergence, the sheep astrovirus sequence was not included in Figure 4. In the region shown in Figure 4, the amino acid identity between the two TAstVs is 58% and both TAstVs share 47% sequence homology with ANV. No significant sequence similarities were found with SRSV. This genetic information indicates that the agent is a chicken astrovirus belonging to the family Astroviridae.

Figure 3. Electron micrograph of purified CAstV particles (diameter 25 to 30 nm).

Figure 4. Alignments of deduced amino acid sequences of CAstV (numbered 1 to 99) with amino acids (one letter code) derived from the polyprotein region of TAstV-1 (GenBank accession number Y15936), TAstV-2 (GenBank accession number AF20663) and ANV (GenBank accession number AB033998). Only amino acid differences are shown, empty space denotes identities and – shows a gap. CAstV showed 62%, 58% and 55% identities to TAstV-2, TAstV-1 and ANV, respectively.

Chick experiment 1

The infected chicks developed mild diarrhoea by day 4 post infection and passed partly digested food in faeces, and when killed 5 days after infection four had some degree of small intestine distension. None of the uninfected chicks showed these signs.

Chick experiment 2

The virus was isolated mainly from the small intestine from day 2 until day 10 (Table 3). Although virus was detected in the caecum and occasionally in the proventriculus, it was of relatively low titre. No virus was isolated from the control chicks.

Table 3. Titres of VDU/AS1 virus in pfu/gm of tissue sample taken from different parts of the intestine between 2 and 10 days post infection of 7-day-old SPF chicks

	Titres on given days post inoculation (log)10
	Day 2		Day 4		Day 6		Day 8		Day 10
Tissue	Bird 1	Bird 2	Bird 3	Bird 4	Bird 5	Bird 6	Bird 7	Bird 8	Bird 9	Bird 10
Proventriculus	<3.30	<3.30	<3.30	4.25	3.60	<3.30	<3.30	4.48	<3.30	<3.30
Duodenal loop	5.08	6.48	6.38	7.20	7.15	7.30	3.78	5.94	<3.30	5.20
Jejunum	4.40	<4.30	7.41	7.60	6.45	7.25	3.30	5.34	<3.30	5.45
Ileum	3.48	4.40	7.08	6.82	6.58	6.20	4.34	4.41	<3.30	4.00
Caeca	–	3.84	5.06	3.78	3.78	4.34	4.30	4.34	<3.30	3.78

On histological examination, the liver, kidneys, spleen and thymus of infected chicks appeared normal. The follicles of a bursa taken 4 days post infection had sparsely populated medullae but it is doubtful that there were lesions of significance as this is seen in uninfected chicks occasionally. All the other bursae appeared normal. Some small areas of limited damage at the base of the villi of the small intestine were seen in some cases with infected chicks. All tissues from the control chicks appeared normal. The five sera taken from the infected group had high titre CAstV antibodies whereas the four sera from the uninfected control chicks were negative.

Field survey for CAstV antibodies.

Antibodies were found to be widespread in most broiler and breeder flocks tested in both 1982 and 2002 (Table 4). There was no correlation between the presence of antibody and uneven growth. Some flocks that displayed the runting and stunting syndrome had no CAstV antibodies. In other flocks where the vast majority of the chicks grew normally all blood samples had CAstV antibodies. Antibody was also found in some of the turkey flocks in 1982, none of which appeared to show clinical signs of the runting and stunting syndrome. No antibody was found in the SPF flocks.

Table 4. Incidence of neutralising antibody to VDU/AS1 virus in broiler flocks bled between 3–6 weeks, broiler breeder flocks bled at various ages and turkey flocks in 1982, and broiler flocks bled at 6 weeks of age and broiler breeder flocks bled between 32 and 57 weeks in 2001

	Incidence of antibody in flock
Negative	Some but not 100% of sera positive	100% positive
10	UK Broiler Flocks 1982, 27	13
1	Dutch Broiler Flocks 1982, 4	11
0	Spanish Broiler Flocks 1982, 3	3
2	USA Broiler Flocks 1982, 12	2
2	Australian Broiler Flocks-1982, 5	0
7	UK Broiler Flocks 2001, 3	10
4	UK Broiler Breeder Flocks 1982 >10 weeks, 2	0
3	UK Broiler Breeder Flocks 1982 <10 weeks, 4	1
2	UK Broiler Breeders 2001 between 32 and 57 weeks, 2	1
1	Turkey Growers 1982, 2	0
1	Turkey Breeders 1982, 1	0

Discussion

The CAstV virus now shown to be an astrovirus is antigenically distinct from ANV and DVH2 although the genomes share some sequence identity with ANV and other astroviruses. Astrovirus-like virions have been seen in the faeces of chickens examined by electron microscopy (McNulty, 2001) and also SRVs have been described (McNulty et al., 1990), but little detailed serology has been reported with the viruses not related to ANV. No sequence data has been published on these viruses. Further work with the TAstVs and their antisera is required to establish whether there is any relationship to the CAstVs. However, in a preliminary study, a turkey antiserum and a monoclonal antibody to TAstV type 2 virus failed to react with the VDU/AS1 virus in both the immunofluorescence and neutralization tests (personal observation).

The isolation of the CAstV is complicated by the presence of reoviruses and adenoviruses in the faeces of young chicks; as these viruses grow well in CEL and LMH cell cultures they often outgrow the more slow growing CAstV. A test such as the reverse transcriptase-PCR would therefore appear to be of value in detecting the astrovirus. In this study, it would have been difficult to reach a definitive diagnosis of the agent using only the electron microscopic examinations; SRV may fall into one of the viral families belonging to Calciviridae, Astroviridae, Picornaviridae, Circoviridae and Parvoviridae. Although morphologies characteristic of each family are usually visible to an experienced electron microscopist, we were unable to detect star-like surface projections characteristic of astroviruses. The pH and isolation protocols have been suggested to affect the ability to visualize the typical star-like structures of astroviruses (reviewed in Koci & Schulz-Cherry, 2002). Since the morphology led us to suspect SRV rather than an astrovirus, the initial primers were designed based on a conserved region of SRSV. However, the amino acid sequence obtained from the PCR product shares more similarity to astroviruses rather than SRSV, indicating that the agent belongs to the family Astroviridae. Sequences covering large parts of the genome of the agent have now been obtained and comparisons of deduced amino acids confirmed the similarity of the agent to avian astroviruses (data not shown).

Detailed comparison of the complete sequence of the CAstV will reveal its evolutionary relationship to the avian astroviruses and astroviruses affecting other species.

These studies demonstrate that CAstV is widespread in the field and was so in 1982. Its possible distribution in turkey flocks needs to be further investigated as only a limited number were tested here and no virus isolation attempted.

A more detailed study is also needed to reveal any association between infection and clinical disease in the field. The CAstVs may vary in virulence, and co-infection with other agents as well as age of infection may affect any clinical manifestation. In the study reported here the virus (VDU/AS3) used to infect 1-day-old chicks had been plaque-picked three times and then passaged in cell culture three times, and following infection only mild signs were seen. It is possible that this passage history affected the virulence of the virus. The CAstV replicated to high titres in the small intestine and VDU/AS1 was isolated up to 10 days post infection, the longest time tested. The titre of virus per gram isolated may have been higher if only the intestinal lining without the muscular layers was used for isolation, as it was only in these lining cells that any changes were observed on histological examination. In an earlier study (Baxendale, personal observation) little or no CAstV was isolated from tissues other than the intestine, some virus occasionally being detected in buffy coats and bursa in laboratory infected chicks.

Recently Koci et al. (2003) demonstrated that TAstV type 2 caused growth depression, a decrease in thymus size and enteric infection of turkeys. Despite severe diarrhoea the intestines of affected turkeys showed only mild histological changes with no inflammation. The virus was recovered from a number of tissues as well as the blood, suggesting a viraemic stage.

In the serological surveys reported here sera were tested at 1:32 or 1:64, these dilutions being selected to avoid any non-specific neutralization. However, now many sera from uninfected SPF chickens have been tested at a 1:16 dilution and no non-specific neutralization observed. As some chicks infected orally take up to 3 weeks to develop antibody titres of 32, a 1:16 dilution would detect early low titre antibodies and could be used in further surveys.

Many of the field sera that tested positive in the serum neutralisation test also had gel precipitating antibodies and hence the gel diffusion test may be of value as an inexpensive, technically simple flock test for these CAstVs.

Acknowledgements

The excellent technical work of Julie Churchill, Nadia Khammash, Tiffany Avory and Leonie de Vaan is gratefully acknowledged. Sera from field flocks were kindly provided by a number of veterinarians and Dr James Guy, North Carolina State University, Raleigh N.C. kindly provided the TAst type 2 virus antisera.