Metagenomic studies characterize both the composition and diversity of uncultured viral and microbial communities. BLAST-based comparisons have typically been used for such analyses; however, sampling biases, high percentages of unknown sequences, and the use of arbitrary thresholds to find significant similarities can decrease the accuracy and validity of estimates. Here, we present Genome relative Abundance and Average Size (GAAS), a complete software package that provides improved estimates of community composition and average genome length for metagenomes in both textual and graphical formats. GAAS implements a novel methodology to control for sampling bias via length normalization, to adjust for multiple BLAST similarities by similarity weighting, and to select significant similarities using relative alignment lengths. In benchmark tests, the GAAS method was robust to both high percentages of unknown sequences and to variations in metagenomic sequence read lengths. Re-analysis of the Sargasso Sea virome using GAAS indicated that standard methodologies for metagenomic analysis may dramatically underestimate the abundance and importance of organisms with small genomes in environmental systems. Using GAAS, we conducted a meta-analysis of microbial and viral average genome lengths in over 150 metagenomes from four biomes to determine whether genome lengths vary consistently between and within biomes, and between microbial and viral communities from the same environment. Significant differences between biomes and within aquatic sub-biomes (oceans, hypersaline systems, freshwater, and microbialites) suggested that average genome length is a fundamental property of environments driven by factors at the sub-biome level. The behavior of paired viral and microbial metagenomes from the same environment indicated that microbial and viral average genome sizes are independent of each other, but indicative of community responses to stressors and environmental conditions.

A new viral sequence likely belonging to a virus of the family Astroviridae was determined using the gut content of chickens affected with the runting-stunting syndrome (RSS) in chickens. Since the appropriate virus could not be isolated in cell culture the open reading frame of the viral capsid protein was cloned to generate a recombinant baculovirus. The protein was purified and used as an experimental vaccine in broiler breeders to provide maternal derived antibodies for the protection of the offspring. The presence of specific antibodies was monitored by an ELISA. The offspring of vaccinated breeder hens were partially protected in a RSS challenge model.

A comparative study examining replication and disease pathogenesis associated with low-pathogenic H5N1, H5N2, or H5N3 avian influenza virus (AIV) infection of chickens and ducks was performed. The replication and pathogenesis of highly pathogenic AIV (HPAIV) has received substantial attention; however, the behavior of low-pathogenic AIVs, which serve as precursors to HPAIVs, has received less attention. Thus, chickens or ducks were inoculated with an isolate from a wild bird [A/Mute Swan/MI/451072/06 (H5N1)] or isolates from chickens [A/Ck/PA/13609/93 (H5N2), A/Ck/TX/167280-4/02 (H5N3)], and virus replication, induction of a serological response, and disease pathogenesis were investigated, and the hemagglutinin and neuraminidase (NA) gene sequences of the isolates were determined. Virus isolated from tracheal and cloacal swabs showed that H5N1 replicated better in ducks, whereas H5N2 and H5N3 replicated better in chickens. Comparison of the NA gene sequences showed that chicken-adapted H5N2 and H5N3 isolates both have a deletion of 20 amino acids in the NA stalk region, which was absent in the H5N1 isolate. Histopathological examination of numerous organs showed that H5N2 and H5N3 isolates caused lesions in chickens in a variety of organs, but to a greater extent in the respiratory and intestinal tracts, whereas H5N1 lesions in ducks were observed mainly in the respiratory tract. This study suggests that the H5N1, H5N2, and H5N3 infections occurred at distinct sites in chicken and ducks, and that comparative studies in different model species are needed to better understand the factors influencing the evolution of these viruses.

Avian paramyxoviruses (APMVs) other than Newcastle disease virus have been isolated from feral avian species and have the potential to cause clinical disease. The objective of this study was to investigate the APMV antibody prevalence in commercial poultry settings. Sera from 100 commercial, layer-type and broiler-type (broiler-breeder and broiler) chicken flocks were analyzed on a random basis. Pooled serum samples from each flock were tested for the presence of antibodies to APMV-1,-2,-3,-4,-6,-7,-8, and -9 by hemagluttination inhibition (HI) test. Reactions with HI titers > or = 1:64 were shown by APMV-1 (71%), APMV-2 (15%), APMV-3 (35%), APMV-4 (26%), APMV-6 (45%), APMV-7 (27%), APMV-8 (31%), and APMV-9 (48%). In the presence of a high HI titer for APMV-1 (1024), we obtained positive HI titers for most of the other APMV subtypes, thus implicating that sensitive techniques need to be developed to detect the prevalence of these subtypes in commercial poultry.

Infectious bursal disease virus (IBDV) serotype 1 is the causative agent of a highly contagious immunosuppressive disease of young chickens. In the past, a number of antigenic, as well as pathogenic, subtypes have been described. The determination of the antigenic makeup of circulating strains is of vital interest to the poultry industry because changes in the antigenicity of circulating field strains have an impact on the use of vaccines. To obtain a more comprehensive overview of the relationship between the nucleotide and amino acid sequence and the antigenic makeup of field isolates, a system based on reverse genetics of IBDV was established. Using this approach, a database for field isolates from three different states in the United States (Georgia, Alabama, and Louisiana), consisting of nucleotide sequence, amino acid sequence, and a reaction pattern based on a panel of monoclonal antibodies, was established. The obtained results showed that phylogenic analysis, which is based on the similarity of sequences, would lead to false conclusions regarding a possible antigenic makeup of the particular isolate. Sequences of field samples were divided into three groups: 1) those that grouped with variant strain E/Del sequences but were antigenically different, 2) those that did not group with sequences of E/Del but were similar in their antigenic makeup, and 3) those that did not group with E/Del sequences and were antigenically different. In addition, using the reverse-genetics approach, a number of field isolates showed no reactivity with any of the used monoclonal antibodies, indicating that an unknown, antigenic subtype of IBDV serotype 1 is circulating in the field.

Based on the haemagglutination inhibition assay, nine antigenically distinct serotypes of avian paramyxoviruses (APMV) are described. Isolates from APMV 2, 3, 6 and 7 can cause respiratory symptoms and/or problems of the reproductive tract that may produce complications if secondary infections occur, while isolates from APMV 4, 5, 8 and 9 rarely produce clinical signs in species from which they are isolated. Isolates belonging to the APMV 1 subtype induce a wide range of disease symptoms varying from mild symptoms to a disease with devastating consequences as caused by velogenic Newcastle disease virus. In this report, one isolate each of APMV 2, 4, and 6 were isolated from wild birds and subsequently characterized in specific pathogen free chickens. All three isolates caused no clinical symptoms but showed microscopic lesions in the trachea, lungs, gut, and pancreas characteristic for a viral infection. Interestingly, only APMV 2 induced haemagglutination inhibition antibodies, while haemagglutination inhibition antibodies of chickens infected with APMV 4 and 6 were not detected. The replication of the virus in the birds was confirmed by isolation of the virus in embryonated eggs.

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is responsible for a highly contagious and economically important disease causing immunosuppression in chickens. IBDV variants isolated in the USA exhibit an antigenic drift affecting neutralizing epitopes in the capsid protein VP2. To understand antigenic determinants of the virus, we have used a reverse genetics approach to introduce selected amino acid changes - individually or in combination - into the VP2 gene of the classical IBDV strain D78. We thus generated a total of 42 mutants with changes in 8 aa selected by sequence comparison and their location on loops PBC and PHI at the tip of the VP2 spikes, as shown by the crystal structure of the virion. The antibody reactivity of the generated mutants was assessed using a panel of five monoclonal antibodies (mAbs). Our results show that a few amino acids of the projecting domain of VP2 control the reactivity pattern. Indeed, the binding of four out of the five mAbs analysed here is affected by mutations in these loops. Furthermore, their importance is highlighted by the fact that some of the engineered mutants display an identical reactivity pattern but have different growth phenotypes. Finally, this analysis shows that a new field strain isolated from a chicken flock in Belgium (Bel-IBDV) represents an IBDV variant with a hitherto unobserved antigenic profile, involving one change (P222S) in the PBC loop. Overall, our data provide important new insights for devising efficient vaccines that protect against circulating IBDV strains.

Segment B of bisegmented infectious bursal disease virus (IBDV) encodes virus protein 1 (VP1), possessing RNA-dependent RNA polymerase (RdRp) activity. This multidomain protein includes an RdRp domain with a non-canonical order of three sequence motifs forming the active site: C-A-B. The A-B-C order of the motifs, as found in RdRps of the majority of viruses, was converted by relocation (permutation) of motif C to a C-A-B order. Due to the unusual location and unproven significance, the motif was named 'C?'. This motif includes an Ala-Asp-Asn tripeptide that replaces the C motif Gly-Asp-Asp sequence, widely considered a hallmark of RdRps. In this study, functional significance of the C? motif was investigated by using purified His-tagged VP1 mutants with either a double replacement (ADN to GDD) or two single-site mutants (ADD or GDN). All mutants showed a significant reduction of RdRp activity in vitro, in comparison to that of VP1. Only the least-affected GDN mutant gave rise to viable, albeit partially impaired, progeny using a reverse-genetics system. Experiments performed to investigate whether the C motif was implicated in the control of metal dependence revealed that, compared with Mn(2+) and Mg(2+), Co(2+) stimulated RdRp unconventionally. No activity was observed in the presence of several divalent cations. Of two Co(2+) salts with Cl(-) and anions, the former was a stronger stimulant for RdRp. When cell-culture medium was supplemented with 50 muM Co(2+), an increase in IBDV progeny yield was observed. The obtained results provide evidence that the unusual Co(2+) dependence of the IBDV RdRp might be linked to the permuted organization of the motif.

The interferon-induced human MxA protein belongs to the dynamin superfamily of large GTPases and accumulates in the cytoplasm. MxA is a key component of the innate antiviral response and has previously been shown to inhibit several viruses with single-stranded RNA genomes of both polarities and a DNA virus. In addition, MxA also targets two double-stranded RNA viruses, Infectious bursal disease virus and a mammalian reovirus as shown in this study. Thus, the antiviral spectrum of human MxA is broader than hitherto suspected. Interestingly, virus growth was not affected in cells expressing MxA(E645R), a mutant form of MxA that showed antiviral activity against orthomyxoviruses.