Can genotype mismatch really affect the level of protection conferred by Newcastle disease vaccines against heterologous virulent strains?

Newcastle disease (ND), caused by virulent class II avian paramyxovirus 1 (Newcastle disease virus, NDV), occurs sporadically in poultry despite their having been immunized with commercial vaccines. These vaccines were all derived from NDV strains isolated around 70 years ago. Since then, class II NDV strains have evolved into 18 genotypes. Whether the vaccination failure results from genotype mismatches between the currently used vaccine strains and field-circulating velogenic strains or from an impaired immune response in the vaccination remains unclear. To test the first hypothesis, we performed a heterologous genotype II vaccine/genotype XI challenge in one-day old specific pathogen free (SPF) chicks and reproduced viral shedding. We then produced two attenuated strains of genotype II and XI by reverse genetics and used them to immunize two-week old SPF chickens that were subsequently challenged with velogenic strains of genotypes II, VII and XI. We found that both vaccines could induce antibodies with hemagglutination inhibition titers higher than 6.5 log2. Vaccination also completely prevented disease, viral shedding in swabs, and blocked viral replication in tissues from different genotypes in contrast to unvaccinated chickens that died shortly after challenge. Taken together, our results support the hypothesis that, in immunocompetent poultry, genotype mismatch is not the main reason for vaccination failure.

Comparison of the efficiency of different newcastle disease virus reverse genetics systems.

Rescue of negative-sense single-stranded RNA viruses ((-)ssRNA virus), generally requires the handling of a large number of plasmids to provide the virus genome and essential components for gene expression and genome replication. This constraint probably renders reverse genetics of (-)ssRNA virus more complex and less efficient. Some authors have shown that the fewer the plasmids, the more efficient reverse genetics is for segmented RNA virus. However, it is not clear if the same applies for (-)ssRNA, such as Newcastle disease virus (NDV). To address this issue, six variants of NDV reverse genetic systems were established by cloning combinations of NP, P and L genes, mini-genome or full-genome in 4, 3, 2 and 1 plasmid. In terms of mini-genome and full-genome rescue, we showed that only the 2-plasmid system, assembling three support plasmids together, was able to improve the rescue efficiency over that of the conventional 4-plasmid system. These results may help establish and/or improve reverse genetics for other mononegaviruses.

Two-plasmid system to increase the rescue efficiency of paramyxoviruses by reverse genetics: The example of rescuing Newcastle Disease Virus.

Within paramyxoviruses, conventional reverse genetics require the transfection of a minimum of four plasmids: three to reconstruct the viral polymerase complex that replicates and expresses the virus genome delivered by a fourth plasmid. The successful transfection of four or more plasmids of different sizes into one cell and the subsequent generation of at least one viable and replicable viral particle is a rare event, which explains the low rescue efficiency, especially of low virulent viruses with reduced replication efficiency in cell lines. In this study, we report on an improved reverse genetics system developed for an avian paramyxovirus, Newcastle Disease Virus (NDV), in which the number of plasmids was reduced from four to two. Compared to the conventional method, the 2-plasmid system enables earlier and increased production of rescued viruses and, in addition, makes it possible to rescue viruses that it was not possible to rescue using the 4-plasmid system.

Diverse gammacoronaviruses detected in wild birds from Madagascar.

To date, infectious bronchitis virus (IBV) is potentially found in wild birds of different species. This work reports the survey of coronaviruses in wild birds from Madagascar based on the targeting of a conserved genome sequence among different groups of CoVs. Phylogenetic analyses revealed the presence of gammacoronaviruses in different species of Gruiformes, Passeriformes, Ciconiiformes, Anseriformes, and Charadriiformes. Furthermore, some sequences were related to various IBV strains. Aquatic and migratory birds may play an important role in the maintenance and spread of coronaviruses in nature, highlighting their possible contribution in the emergence of new coronavirus diseases in wild and domestic birds.

New avian paramyxoviruses type I strains identified in Africa provide new outcomes for phylogeny reconstruction and genotype classification.

Newcastle disease (ND) is one of the most lethal diseases of poultry worldwide. It is caused by an avian paramyxovirus 1 that has high genomic diversity. In the framework of an international surveillance program launched in 2007, several thousand samples from domestic and wild birds in Africa were collected and analyzed. ND viruses (NDV) were detected and isolated in apparently healthy fowls and wild birds. However, two thirds of the isolates collected in this study were classified as virulent strains of NDV based on the molecular analysis of the fusion protein and experimental in vivo challenges with two representative isolates. Phylogenetic analysis based on the F and HN genes showed that isolates recovered from poultry in Mali and Ethiopia form new groups, herein proposed as genotypes XIV and sub-genotype VIf with reference to the new nomenclature described by Diel's group. In Madagascar, the circulation of NDV strains of genotype XI, originally reported elsewhere, is also confirmed. Full genome sequencing of five African isolates was generated and an extensive phylogeny reconstruction was carried out based on the nucleotide sequences. The evolutionary distances between groups and the specific amino acid signatures of each cluster allowed us to refine the genotype nomenclature.

Investigating avian influenza infection hotspots in old-world shorebirds.

Heterogeneity in the transmission rates of pathogens across hosts or environments may produce disease hotspots, which are defined as specific sites, times or species associations in which the infection rate is consistently elevated. Hotspots for avian influenza virus (AIV) in wild birds are largely unstudied and poorly understood. A striking feature is the existence of a unique but consistent AIV hotspot in shorebirds (Charadriiformes) associated with a single species at a specific location and time (ruddy turnstone Arenaria interpres at Delaware Bay, USA, in May). This unique case, though a valuable reference, limits our capacity to explore and understand the general properties of AIV hotspots in shorebirds. Unfortunately, relatively few shorebirds have been sampled outside Delaware Bay and they belong to only a few shorebird families; there also has been a lack of consistent oropharyngeal sampling as a complement to cloacal sampling. In this study we looked for AIV hotspots associated with other shorebird species and/or with some of the larger congregation sites of shorebirds in the old world. We assembled and analysed a regionally extensive dataset of AIV prevalence from 69 shorebird species sampled in 25 countries across Africa and Western Eurasia. Despite this diverse and extensive coverage we did not detect any new shorebird AIV hotspots. Neither large shorebird congregation sites nor the ruddy turnstone were consistently associated with AIV hotspots. We did, however, find a low but widespread circulation of AIV in shorebirds that contrast with the absence of AIV previously reported in shorebirds in Europe. A very high AIV antibody prevalence coupled to a low infection rate was found in both first-year and adult birds of two migratory sandpiper species, suggesting the potential existence of an AIV hotspot along their migratory flyway that is yet to be discovered.

Genetic data from avian influenza and avian paramyxoviruses generated by the European network of excellence (EPIZONE) between 2006 and 2011--review and recommendations for surveillance.

Since 2006, the members of the molecular epidemiological working group of the European "EPIZONE" network of excellence have been generating sequence data on avian influenza and avian paramyxoviruses from both European and African sources in an attempt to more fully understand the circulation and impact of these viruses. This review presents a timely update on the epidemiological situation of these viruses based on sequence data generated during the lifetime of this project in addition to data produced by other groups during the same period. Based on this information and putting it all into a European context, recommendations for continued surveillance of these important viruses within Europe are presented.

Genetic variability in the G protein gene of human respiratory syncytial virus isolated from the Campinas metropolitan region, Brazil.

Human respiratory syncytial virus (hRSV) is recognized as the most important viral agent of serious respiratory tract diseases in the pediatric population worldwide. A prospective study for hRSV was conducted in children ageing less than 1 year admitted in two university hospitals in Campinas, São Paulo, Brazil. The aim of the present study was to investigate the genetic variability of both A and B subgroups of hRSV isolated during an epidemic period in the Campinas metropolitan region, Brazil, by sequencing a variable region of the G protein gene. Phylogenetic trees were constructed from alignments of sequences available in the GenBank database and Brazil isolates for hRSV A and B. The data demonstrate that Brazilian isolates clusters together with A and B viruses from Kenya, New Zealand, South Africa, West Virginia, United States (CH, Rochester), and other Brazilian isolates. Phylogenetic analysis of subgroup A isolates showed that the sequences obtained on the present study falls on three clusters, namely GA2, GA5, and SAA1 that co-circulate during the analyzed period. Subgroup B isolates detected belongs to three genotypes, GB3 (SAB3) and BA (BAIII). Different subgroup B genotypes were detected and BA isolates present in our samples showed some degree of genetic variability. This is one of the first reports on the molecular epidemiology of hRSV strains from the Campinas metropolitan region, São Paulo state, Brazil. And is also the first description of the circulation pattern of hRSV genotypes in two university hospitals, revealing interesting differences between the two subgroups of the virus.

Structure and sequence motifs of siRNA linked with in vitro down-regulation of morbillivirus gene expression.

The most challenging task in RNA interference is the design of active small interfering RNA (siRNA) sequences. Numerous strategies have been published to select siRNA. They have proved effective in some applications but have failed in many others. Nonetheless, all existing guidelines have been devised to select effective siRNAs targeting human or murine genes. They may not be appropriate to select functional sequences that target genes from other organisms like viruses. In this study, we have analyzed 62 siRNA duplexes of 19 bases targeting three genes of three morbilliviruses. In those duplexes, we have checked which features are associated with siRNA functionality. Our results suggest that the intramolecular secondary structure of the targeted mRNA contributes to siRNA efficiency. We also confirm that the presence of at least the sequence motifs U13, A or U19, as well as the absence of G13, cooperate to increase siRNA knockdown rates. Additionally, we observe that G11 is linked with siRNA efficacy. We believe that an algorithm based on these findings may help in the selection of functional siRNA sequences directed against viral genes.

Variant isolates of human metapneumovirus subgroup B genotype 1 in Campinas, Brazil.

BACKGROUND: Human metapneumovirus (HMPV) is a paramyxovirus associated with respiratory illness. The genotypes of HMPV isolates in Brazil have not been well characterized. OBJECTIVES: To investigate the presence of HMPV in clinical samples collected from pediatric patients of two university hospitals in the region of Campinas (São Paulo, Brazil) and to genotype them by partial sequencing of the HMPV F gene. STUDY DESIGN: Nasopharyngeal aspirates were collected from children hospitalized between April and September, 2004 because of acute respiratory infections (ARI). RESULTS: We identified HMPV in 8 of 142 (5.6%) clinical samples. We determined through phylogenetic analysis that HMPV isolates in Campinas during the study were clustered within subgroup B genotype 1. Two of the isolates analyzed showed significant differences from previously isolated B1 viruses, when compared to HMPV isolated in South Africa and Canada, and clustered in a separate branch within this genotype. CONCLUSIONS: In 2004 in our geographic region all HMPV isolates from pediatric patients were in the B1 HMPV genetic group, with two variant isolates.

Inhibition of avian metapneumovirus (AMPV) replication by RNA interference targeting nucleoprotein gene (N) in cultured cells.

Avian metapneumovirus (AMPV) is the primary causative agent of severe rhinotracheitis in turkeys. It is associated with swollen head syndrome in chickens and is the source of significant economic losses to animal food production. In this study, we designed specific short interfering RNA (siRNA) targeting the AMPV nucleoprotein (N) and fusion (F) genes. Three days post-virus infection, virus titration, real time RT-PCR, and RT-PCR assays were performed to verify the effect of siRNA in AMPV replication. A marked decrease in virus titers from transfected CER cells treated with siRNA/N was observed. Also, the production of N, F, and G mRNAs in AMPV was decreased. Results indicate that N-specific siRNA can inhibit virus replication. In future studies, a combination of siRNAs targeting the RNA polymerase complex may be used as a tool to study AMPV replication and/or antiviral therapy.

Susceptibility of different cell lines to infection with bovine respiratory syncytial virus.

The growth of bovine respiratory syncytial virus (BRSV) was evaluated in six different cell lines. Chicken embryo related cells (CER), a chicken embryo fibroblast/baby hamster kidney hybrid and bovine CRIB cells (a bovine viral diarrhea virus-resistant clone of MDBK cells) showed to be the most appropriate for virus multiplication. Both cells provided infectious virus titres of up to 10(5.5) 50% tissue culture infective doses per 100 microl (TCID(50)/100 microl). One-step growth curves revealed no significant differences in the growth of BRSV in these two cell lines. Furthermore, they proved to be susceptible to infection with three different BRSV strains. It was concluded that both CER and CRIB cells may be used for laboratory multiplication of BRSV with optimal results.