Studies on the pathogenicity of enterovirus-like viruses in chickens.

FP3 and 612 viruses are enterovirus-like viruses. Antibody to these viruses is widespread in chicken flocks, but nothing is known about their pathogenicity. Seven experiments were carried out to investigate the tissue tropism and associated pathology of these novel fowl enterovirus-like viruses and to compare these with the effects of the previously studied enterovirus-like viruses, ELV-1 and avian nephritis (ANV). ANV is now classified as an astrovirus. Preliminary experiments were carried out with FP3 virus, 612 virus and ELV-1 to determine the distribution of viral antigen. Each preliminary experiment was followed by a larger experiment that included more birds and in which a greater range of tissues was studied. It was shown that all four viruses studied replicated in the intestine and had differing abilities to spread to other tissues. Histological changes were present in most antigen-positive tissues but they were usually relatively mild. ELV-1 was associated with the most severe intestinal lesions, followed by FP3 virus. FP3 virus produced lesions in the kidney that were marginally more severe than those caused by the G-4260 strain of ANV. FP3 virus also caused pancreatic lesions. The 612 virus was found to be only mildly pathogenic in specific pathogen free chickens.

Chicken anaemia virus inoculated by the oral route causes lymphocyte depletion in the thymus in 3-week-old and 6-week-old chickens.

There have been many reports of the severe clinical disease and pathology seen in young chicks that have been vertically infected with chicken anaemia virus (CAV). The disease is characterized by anaemia, and atrophy of the thymus and bone marrow. However, while it has been suggested that horizontally acquired infections of older birds are common, to date there has been no description in the literature of the pathology of this type of infection. In the present study, 3-week-old and 6-week-old chickens were infected by the oral route, as is likely to occur naturally, and a wide range of tissues were examined immunocytochemically for the presence of CAV antigen. Histological examination was carried out on the thymus, spleen and bone marrow of all birds, and on all other tissue samples in which CAV antigen was found. CAV antigen and associated pathological change were detected in the thymus of both 3-week-old and 6-week-old birds. However, CAV antigen was rarely found in other tissues, which is in contrast to what is found in birds infected when 1-day-old. In particular, very few infected cells were found in the bone marrow. Anaemia and bone marrow atrophy, which are typically found in chicks infected vertically or when 1-day-old, did not develop in the 3-week-old or 6-week-old birds. The findings of this study show that CAV is capable of infecting thymocytes of older birds, in contrast to previous belief, and that it is associated with lymphocyte depletion. There was only limited evidence of viral replication in the other tissues examined.

Investigation of the unstable attenuation exhibited by a chicken anaemia virus isolate.

An attenuated chicken anaemia virus (CAV) isolate, cloned isolate 10, which was molecularly cloned from the Cuxhaven-1 CAV after 173 cell-culture passages, was shown previously to recover pathogenicity following 10 passages in young chicks. The consensus nucleotide sequence of the 'revertant' (Rev) virus, present as a tissue homogenate, differed from cloned isolate 10 at a single nucleotide residue (nucleotide 1739) that changed amino acid 287 of the capsid protein from alanine to aspartic acid. Subjecting Rev virus to 10 cell-culture passages reselected viruses with an alanine at this amino acid position. Experimental infections using a molecularly cloned Rev virus isolate demonstrated that the mutation at nucleotide 1739 was not in itself responsible for the recovery of pathogenicity exhibited by the Rev virus. Additional sequence analyses of cloned amplicons provided evidence that the Rev virus population comprised minor, genetically different subpopulations, and provided an indication of CAV's potential for genetic change.

Characterisation of a chicken anaemia virus variant population that resists neutralisation with a group-specific monoclonal antibody.

A variant population of chicken anaemia virus (CAV), termed P310 2A9-resist, that resists neutralisation by the monoclonal antibody (MAb) 2A9, was selected from Cux-1 virus that had been passaged 310 times (P310) in MDCC-MSB1 cells. Substantially higher concentrations of MAb 2A9 were required to neutralise the selected virus compared to those required to neutralise a low-passage (P13) Cux-1 isolate. Virus neutralisation tests showed that serum from chickens infected with the P310 2A9-resist virus neutralised P13 virus and that serum from chickens infected with P13 virus conversely neutralised the P310 2A9-resist virus. MDCC-MSB1 cells infected with the P310 2A9-resist virus produced no staining with low dilutions (1:100) of Mab 2A9 in an indirect immunofluorescence (IF) test, whereas cells infected with P13 virus reacted positively at high MAb dilutions (1:80,000). Experimental infections of 1-day-old SPF chicks showed that the P310 2A9-resist virus was substantially attenuated. Chimaeric viruses constructed using PCR-amplified regions from the P310 2A9-resist and a pathogenic low-passage cloned Cux-1 isolate showed that the reduced MAb reactivity and attenuation exhibited by the P310 2A9-resist virus were mainly associated with a region encoding the N-terminal half of the 50 kDa capsid protein VP1 and C-terminal regions of VP2 and VP3. The nucleotide sequence of the protein-coding region of the P310 2A9-resist virus is reported and the amino acid sequences of the 3 encoded proteins compared with those of other Cux-1 isolates.

Effect of parainfluenza-3 virus challenge on cell-mediated immune function in parainfluenza-3 vaccinated and non-vaccinated calves.

A group of four conventional, colostrum-fed calves was vaccinated with live parainfluenza type 3 (PI-3) virus vaccine at 1 and 5 weeks of age. A group of four control calves was treated with cell culture medium at the same time. Two weeks after the second vaccination, both groups of calves were challenged with PI-3 virus by a combined respiratory route. Blood and nasal mucus samples were collected at intervals, and alveolar macrophages were recovered before and after challenge by bronchoalveolar lavage. The results demonstrated that clearance of virus, as indicated by presence of virus antigen was more rapid in previously vaccinated calves. Several alveolar macrophage functions were markedly reduced in all calves 5 to 7 days following virus challenge, although microbicidal activity was unaffected, compared to the controls. The production of neutrophil chemotactic factors by alveolar macrophages occurred more rapidly after virus challenge in the previously vaccinated calves and this correlated with a more rapid neutrophil influx into the lungs in these animals.

Antigenicity and pathogenicity characteristics of molecularly cloned chicken anaemia virus isolates obtained after multiple cell culture passages.

The Cux-1 isolate of chicken anaemia virus (CAV), which had received 310 (P310) cell culture passages, was substantially less pathogenic than virus that had been passaged 13 times (P13). Molecularly cloned virus isolates, selected from the P310 and P13 virus populations using recombinant DNA cloning and transfection procedures, reacted differently with 4 CAV-specific monoclonal antibodies (MAbs), which had been raised to low-passage Cux-1 virus. In contrast to the strong immunofluorescence (IF) reactivities exhibited by all P13 cloned isolates tested, 80% and 57% of the P310 cloned isolates reacted weakly with MAbs 2A9 and 4H4, which are directed against conformational epitopes on the capsid protein, VP1. Sequence analysis of the VP1 coding regions possessed by ten P310 and two P13 cloned isolates showed that 6 amino acid changes within VP1 had been selected by multiple-cell culture passage. One of these at position 89 in VP1 appeared to be crucial for determining reactivity with MAb 2A9. Of nine P310 cloned isolates evaluated, 8 were substantially attenuated compared to the low-passage Cux-1 virus pool. It is concluded that the individual virus variants comprising the P310 virus pool differ with regards to their antigenicity and pathogenicity.

Studies on the efficacy of intranasal vaccination for the prevention of experimentally induced parainfluenza type 3 virus pneumonia in calves.

The efficacy of intranasal vaccination in preventing or limiting disease of the lower respiratory tract induced by parainfluenza 3 (PI3) virus was evaluated under experimental conditions, using a commercially available live vaccine containing a temperature-sensitive strain of PI3 virus. In a preliminary study four colostrum-deprived calves were vaccinated intranasally at one week and again at two months of age, and two similar calves were given an intranasal placebo. After the second vaccination serum antibodies to PI3 virus were detected in all four vaccinated calves, but not in the control animals. Seventeen days after the second vaccination all six calves were challenged with virulent PI3 virus, and they were killed six days later. The clinical scores and the extent of pulmonary consolidation were reduced in the vaccinated animals; PI3 virus was detected in the upper and lower respiratory tract of the control calves but in none of the vaccinated calves. In a larger scale study with 14 colostrum-fed calves, seven were vaccinated at one week and again at five weeks of age, and seven were given an intranasal placebo. Two weeks after the second vaccination all 14 calves were challenged with virulent PI3 virus. The clinical scores and lung consolidation were significantly reduced in the vaccinated calves in comparison with the controls. Six days after infection, 10 of the 14 calves were killed; PI3 virus was detectable in the nasal secretions of all seven control calves but in only one of the vaccinated animals, and PI3 viral antigen was detected in the lungs of the control calves but not in those of the vaccinated animals. One of the vaccinated calves had developed a severe clinical response after the challenge, but it had only minor lung consolidation when killed.

Cytotoxic interactions between bovine parainfluenza type 3 virus and bovine alveolar macrophages.

This paper describes an investigation of the cytotoxic activity of bovine alveolar macrophages for parainfluenza type 3 (PI-3) virus-infected target cells, using 51Cr release assays. Alveolar macrophages from uninfected calves were shown to be capable of killing PI-3 virus infected cells without the presence of antibody or complement (antibody-independent cell-mediated cytotoxicity). The level of killing was shown to vary from animal to animal with specific lysis values ranging from <5% to 70%. Presence of PI-3 virus antiserum was shown to inhibit, rather than enhance macrophage cytotoxicity in a dose-dependent manner, suggesting that bovine alveolar macrophages do not always exhibit antibody-dependent lysis in all cases. Following intranasal and intratracheal inoculation of calves with PI-3 virus, the level of cytotoxicity by macrophages lavaged from the lungs of the calves increased substantially, and by Day 5 post inoculation, levels of 95% to 98% specific lysis were recorded. After Day 5, the killing ability decreased rapidly to low levels. Cell-free lavage fluids, collected from PI-3 virus infected and control calves at various times throughout the experiment, were incubated with aliquots of an alveolar macrophage population from an uninfected donor calf, which initially showed a low level of killing, and were subsequently added to PI-3 virus infected target cells. The recorded levels of cytotoxicity, mirrored those which were seen with the initial macrophage effector cells from the infected and control animals, suggesting that macrophage cytotoxicity was largely controlled by extracellular factors.

Effect of multiple cell culture passages on the biological behaviour of chicken anaemia virus.

The Cux-1 isolate of chicken anaemia virus (CAV) was passaged over 320 times in Marek's disease virus transformed chicken lymphoblastoid (MDCC-MSB1) cells. Comparison of the infectivity titres of virus pools derived from viruses that had received 0 (P0), 49 (P49), 170 (P170) and 320 (P320) passages in our laboratory indicated that the yields of infectious virus increased over 100-fold with passage number from P0 to P170. P320 exhibited unusual cell culture growth characteristics in that, unlike its lower passage counterparts, virus-specific immunofluorescence (IF) and cytopathic effect were detected at very low levels at 2 days post infection, with an additional passage of infected cells into fresh medium being required to produce high levels of infectious virus. Experimental infection of 1-day-old SPF chicks showed that P170 and P320 were substantially attenuated compared to P49 and a pathogenic, low-passage isolate used as control. When assessed by the indirect IF test, infection of 1-day-old chicks with the P49 and P170 isolates elicited similar levels of CAV-specific antibody to those elicited by infection with the pathogenic, low-passage virus and higher than those elicited by infection with the P320 isolate. Experimental infection of 5-week-old chicks indicated that the attenuated P170 and P320 isolates invoked similar CAV-specific antibody levels to those invoked by the pathogenic P49 and control isolates.

Investigation of the attenuation exhibited by a molecularly cloned chicken anemia virus isolate by utilizing a chimeric virus approach.

Molecular cloning of the Cux-1 isolate of chicken anemia virus (CAV), which had been passaged 173 times in cell culture, resulted in the isolation of an attenuated strain, designated cloned isolate 10, which reverted to virulence following 10 passages in young chicks (D. Todd, T. J. Connor, V. M. Calvert, J. L. Creelan, B. M. Meehan, and M. S. McNulty, Avian Pathol. 24:171-187, 1995). The attenuated cloned isolate 10 differs from the molecularly cloned pathogenic Cux-1 isolate in that it possesses a 21-nucleotide insertion within the nontranscribed region of the CAV genome and 17 individual nucleotide substitutions dispersed throughout the genome. Comparative analyses with other published CAV sequences indicated that cloned isolate 10 was unique at nine nucleotide positions and at five amino acid positions. The molecular basis of the attenuation exhibited by cloned isolate 10 was investigated by evaluating the pathogenicities of two sets of complementary chimeric viruses. These sets were produced by transfection with chimeric double-stranded replicative-form (RF) DNA equivalents that contained DNA sequences derived from cloned isolate 10 and the pathogenic cloned Cux-1 isolate. The construction of the chimeric RFs exploited the occurrence of unique EcoRI, PstI, and BamHI restriction sites, which allowed their respective circular CAV RFs to be manipulated as three restriction fragments of 0.58, 0.93, and 0.71 kbp. Examination of the levels of anemia and gross pathology in the thymuses and bone marrows of 14 day-old specific-pathogen-free chicks following infection of 1-day-old chicks with the chimeric and cloned parental isolates indicated that nucleotide changes in each of the three genomic regions contributed towards attenuation. The significance of this result to the development and use of live attenuated CAV vaccines is discussed.

Gordon Memorial Lecture. Chicken anaemia virus--a glimpse of the future?

This paper uses background information about chicken anaemia virus as a guide to how the study and control of virus diseases of poultry may develop in the future. It is predicted that "new' viruses will be discovered in poultry, many of which will be difficult to grow in vitro and whose pathogenicity may appear uncertain. When new diseases/syndromes arise in the future, it should be a priority activity to define their pathology. The limitations of currently available virus diagnostic methods are highlighted. The possibility of vaccinating against economically important subclinical disease is discussed, as is the use of recently developed technologies in differentiating virus strains and in developing new vaccines.

Sequence of porcine circovirus DNA: affinities with plant circoviruses.

The complete nucleotide sequence (1759 nt) of the ssDNA genome of porcine circovirus (PCV) was determined from a cloned dsDNA replicative form isolated from PCV-infected cells. Sequence analysis detected no significant nucleic acid or protein similarity with another animal circovirus, chicken anaemia virus (CAV) but, surprisingly, the highest protein similarity was obtained between the product of the largest predicted PCV ORF (ORF1; encoding a potential protein of 35.7 kDa) and a putative protein encoded by the plant circovirus banana bunchy top virus (BBTV). High protein similarity was also detected with the other plant circoviruses subterranean clover stunt virus (SCSV) and coconut foliar decay virus (CFDV). This region of protein identity corresponds with the putative plant circovirus replication-associated protein (Rep). The presence of a nonanucleotide sequence at the apex of a potential-stem loop structure, identical to that found in the plant circoviruses CFDV and SCSV and similar (one mismatch) to that found in the plant circovirus BBTV and in the geminiviruses, suggests that rolling-circle replication may operate during PCV DNA replication. These findings show that PCV is unique in that it bridges the gap between animal and plant circoviruses. The taxonomic relationship of PCV with other members of the Circoviridae is discussed.

Bovine adenovirus type 10: properties of viruses isolated from cases of bovine haemorrhagic enterocolitis.

The isolation, serological classification and growth properties of adenoviruses isolated from fatal cases of haemorrhagic enterocolitis in calves are described. Four viruses, from different submissions, were isolated in cultures of calf testis cells and were identified as adenoviruses by electron microscopy. The four isolates were serologically identical and were classified as bovine adenovirus type 10 in cross-neutralisation tests with other bovine, ovine and porcine adenovirus species. Their growth in the nucleus of infected cells was accompanied by the production of typical adenovirus-associated inclusions. A serological survey to determine the incidence of infection with the virus in cattle in Northern Ireland demonstrated the presence of low levels of neutralising antibodies in 55 per cent of cattle over two years old, although only 8 per cent were positive at a 1/500 dilution of serum.

Investigation of the transfection capability of cloned tandemly-repeated chicken anaemia virus DNA fragments.

Chicken anaemia virus (CAV) is an icosahedral virus, 25 nm in diameter, which, on the basis of its circular single-stranded DNA genome, has recently been classified in the family, Circoviridae. We have investigated whether infectious, monomeric CAV DNA from recombinant plasmids containing tandemly-repeated CAV replicative form (RF) DNAs, following transfection, was generated by homologous recombination or a replicational release mechanism involving rolling circle replication (RCR) of DNA. Experiments designed to locate the virus strand origin of RCR and/or sites of recombination were performed by sequence analyses of hybrid viruses generated after transfection with cloned tandemly-repeated RFs specified by the sequence-distinct Cux-1 and 26P4 isolates. Positive transfection results obtained from 2 recombinant plasmid constructs were shown to have resulted from homologous recombination occurring at different sites within the RF sequence. Three of 5 hybrid viruses analysed were "circularised" within the same 105 bp sequence, that contains four 19bp repeats and with which promoter/enhancer activity has been associated. This region may represent a novel origin or recombination hot-spot within the CAV genome. A distinctive cruciform-loop structure within the non-coding region was shown to contain an S1 nuclease-sensitive site, detected in CAV RF and in recombinant plasmids containing RF inserts.

Effect of porcine circovirus infection on porcine alveolar macrophage function.

The effect of porcine circovirus (PCV) infection of porcine alveolar macrophage cultures on some of the functional properties of these cells are reported. PCV infection of alveolar macrophages did not effect their ability to phagocytose and kill complement-coated yeast cells or the expression of Fc or complement receptors. A transient increase in major histocompatibility complex (MHC) class I expression in PCV-infected cells were observed 4 days after infection and a decrease in the number of cells expressing MHC class II antigens was observed 8 days after infection. Infection of alveolar macrophages with PCV also resulted in a transient decrease in their ability to act as accessory cells in mitogen-induced lymphocyte proliferation of monocyte-depleted porcine peripheral blood mononuclear cells.

Synergism between chicken anemia virus (CAV) and avian reovirus following dual infection of 1-day-old chicks by a natural route.

One-day-old specific-pathogen-free white leghorn chicks were dually infected with the Cux-1 isolate of chicken anemia virus (CAV) and separate avian reovirus strains (S1133 or Uchida) by an oral route. Fourteen days after inoculation, chicks were bled, and packed cell volumes (PCVs) were determined. Chicks were also weighted and examined for macroscopic changes to the bone marrow, thymus, and bursa of Fabricius. The results obtained following dual inoculation of chicks with CAV and reovirus were compared with results obtained from mock-infected chicks or chicks inoculated with CAV or reovirus alone. Chicks dually infected with CAV and the S1133 reovirus strain had significantly (P < 0.05) lower weight gain and more severe tissue damage than chicks inoculated with either virus alone. In addition, a significant (P < 0.05) reduction in the mean PCV was seen in these dually infected chicks when compared with chicks inoculated with CAV alone. No increase in the severity of the disease signs was observed following dual infection with CAV and reovirus strain Uchida.

Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pig foetal material.

The results of virus and antigen distribution following experimental infection of colostrum deprived pigs with pig circovirus (PCV) by oral/nasal and intravenous routes are reported. PCV and antigen were detected using virus isolation and indirect immunofluorescence on cryostat sections respectively. PCV antigen was detected in tissues throughout the body but primarily in spleen thymus, and lung. No PCV antigen or virus was detected in tissue samples from the central nervous system. Examination of pig foetal material from field cases of abortion/stillbirth resulted in 3 PCV isolates from 2 sera and a spleen sample from 2 groups of stillborn piglets from the same farm. No antibody to PCV alone was detected in 160 foetal sera tested. These results suggest that transplacental infection with PCV does occur, possibly prior to foetal immunocompetance. However, it is probably not a significant cause of reproductive disorders in pigs in Northern Ireland.

Molecular cloning of an attenuated chicken anaemia virus isolate following repeated cell culture passage.

The pathogenicity of the Cux-1 isolate of chicken anaemia virus (CAV) was substantially reduced following large numbers (50 to 173) of passages in MDCC-MSBl cells. Restriction endonuclease analysis of polymerase chain reaction (PCR)- amplified DNAs and recombinant plasmids containing DNA inserts specified by CAV that had been passaged 173 times, indicated that the population of high-passage virus was genetically diverse. A 210-base pair (bp) insertion, containing a 19-bp sequence identical to four repeated sequences that are located in the putative non-coding region of the genome was shown to have become established in the virus population by passage number 30. Individual virus isolates that were selected from the high-passage virus population using recombinant DNA cloning and transfection methodologies varied in their pathogenicities. One cloned virus isolate, designated number 10, produced virtually no anaemia and substantially reduced levels of aplasia of the bone marrow and thymus atrophy. The pathogenicity of this isolate was restored following 10 passages in young chicks.