Detection and characterization of a new astrovirus in chicken and turkeys with enteric and locomotion disorders.

In the present paper, we report the unexpected discovery of a new virus in samples from chicken and turkey flocks with clinical disorders such as tenosynovitis, enteric problems, or runting and/or stunting-like conditions. Since 1987, several virus isolation attempts on samples from these flocks resulted in the same macroscopic characteristic lesions in embryonated specific pathogen free eggs, being mortality with bright-red discolouration of legs and wing-tips, a swollen dark-red liver and oedema. Initial work suggested the presence of an agent with characteristics of a non-enveloped RNA virus. Further work, which is described in this paper, showed that the isolated strains formed a new group of avian nephritis viruses, which is genetically and antigenically distinct from known avian astroviruses. Inoculation of a representative strain (isolate 19) of this new group of avian nephritis viruses, provisionally named avian nephritis virus-3, in specific pathogen free layer chicks resulted in diarrhoea, runting and stunting, and even mortality.

The influence of the inactivating agent on the antigen content of inactivated Newcastle disease vaccines assessed by the in vitro potency test.

An in vitro potency test has recently been included in the European Pharmacopoeia (EP) monograph (01/2007:0870) to assess the potency of inactivated Newcastle disease (ND) vaccines. This enzyme linked immunosorbent assay (ELISA) is an attractive alternative for the existing in vivo potency tests especially with regard to the objective of the European Authorities to Replace, Reduce and Refine the use of laboratory animals for production and quality control of immunobiologicals. In the present study the influence of the inactivant on the antigen content established by ELISA was evaluated. Therefore, oil based vaccines containing similar concentrations of beta-propiolactone (BPL) or formaldehyde inactivated Newcastle disease virus (NDV) were examined by ELISA and in the in vivo potency tests outlined in the EP. The results obtained demonstrate that the use of formaldehyde as inactivant lowered the in vitro potency compared to BPL as inactivant. In contrast, the in vivo potency was not affected. Therefore, the ELISA should not be used to compare the potency of commercial ND vaccines containing formaldehyde inactivated NDV with those containing BPL inactivated NDV. However, the ELISA is considered an attractive alternative for the existing in vivo potency tests since it can be used by vaccine manufacturers for the release of inactivated ND vaccines.

[Avian influenza: eradication from commercial poultry is still not in sight]. / Aviaire influenza: zicht op eradicatie bij commercieel gehouden pluimvee steeds verder weg.

Avian influenza viruses are highly infectious micro-organisms that primarily affect birds. Nevertheless, they have also been isolated from a number of mammals, including humans. Avian influenza virus can cause large economic losses to the poultry industry because of its high mortality. Although there are pathogenic variants with a low virulence and which generally cause only mild, if any, clinical symptoms, the subtypes H5 and H7 can mutate from a low to a highly virulent (pathogenic) virus and should be taken into consideration in eradication strategies. The primary source of infection for commercial poultry is direct and indirect contact with wild birds, with waterfowl forming a natural reservoir of the virus. Live-poultry markets, exotic birds, and ostriches also play a significant role in the epidemiology of avian influenza. The secondary transmission (i.e., between poultry farms) of avian influenza virus is attributed primarily to fomites and people. Airborne transmission is also important, and the virus can be spread by aerosol in humans. Diagnostic tests detect viral proteins and genes. Virus-specific antibodies can be traced by serological tests, with virus isolation and identification being complementary procedures. The number of outbreaks of avian influenza seems to be increasing - over the last 5 years outbreaks have been reported in Italy, Hong Kong, Chile, the Netherlands, South Korea, Vietnam, Japan, Thailand, Cambodia, Indonesia, Laos, China, Pakistan, United States of America, Canada, South Africa, and Malaysia. Moreover, a growing number of human cases of avian influenza, in some cases fatal, have paralleled the outbreaks in commercial poultry. There is great concern about the possibility that a new virus subtype with pandemic potential could emerge from these outbreaks. From the perspective of human health, it is essential to eradicate the virus from poultry; however, the large number of small-holdings with poultry, the lack of control experience and resources, and the international scale of transmission and infection make rapid control and long-term prevention of recurrence extremely difficult. In the Western world, the renewed interest in free-range housing carries a threat for future outbreaks. The growing ethical objections to the largescale culling of birds require a different approach to the eradication of avian influenza.

A recombinant newcastle disease virus with low-level V protein expression is immunogenic and lacks pathogenicity for chicken embryos.

Newcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3'-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5, 000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.

Molecular cloning and sequence analysis of the genome of chicken anaemia agent.

The replicative form (RF) DNA of chicken anaemia agent (CAA) was isolated and cloned into bacterial plasmids. After religation of the cloned CAA DNA and transfection into MDCC-MSB1 cells, the DNA could induce c.p.e. characteristic of that caused by CAA, and an antigen was produced which gave positive immunofluorescence when detected with an anti-CAA serum. Sanger sequencing of the 2298 bp genome revealed several open reading frames (ORFs); the major ORF encoded a polypeptide of 51.8K. In SDS-PAGE of CAA viral particles a 50K protein has been reported as the only detectable viral protein. The genomic region downstream of the major ORF had several predicted GC-rich inverted repeats, a poly(A) signal and four copies of an 18 bp repeat element. Database searches did not reveal any sequence with homology to the viral genomic DNA, nor to the amino acid sequence of any of the ORFs, apart from the N-terminal 40 amino acids of the major ORF which showed a limited similarity to the structure of protamines.