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Despite the efforts to achieve a consistent classification scheme based on the complete S1 gene, the genetic characterization of infectious bronchitis virus (IBV) is often performed on partial S1 regions due to economic and time constraints in the diagnostic routine. Sanger sequencing remains the most common and cost-effective option even if the analysis of samples where multiple field and vaccine strain populations coexist can lead to partial or misleading results. The present study aimed to evaluate the different diagnostic outcomes of three commonly used RT-PCR methods targeting two regions of the S1 gene. A possible bias in IBV detection and characterization was investigated in relation to the adopted method, the strain concentration as well as their ratio in mixed samples. Thirty samples were prepared by artificially mixing two vaccine strains, combined at different ratios and selected among four different IBV lineages, i.e. GI-1 (Mass), GI-13 (793/B), GI-19 (QX), GI-23 (Israeli Variant 2). Sequence analysis was conducted both manually and with bioinformatic methods. The result agreement among methods, replicates and analysis approaches was statistically evaluated. Consistent results emerged among the three assays, with a few discrepancies likely caused by primer affinity and target amount. This study confirms the complexity of IBV strain identification and highlights the importance of evaluating and updating the available diagnostic assays for a reliable detection of all circulating IBV strains.

Phylogenetic analysis of influenza viruses collected during December 2009-February 2010 from chickens in live poultry retail shops in Lahore, Pakistan, showed influenza A(H9N2) lineage polymerase and nonstructural genes generate through inter- and intrasubtypic reassortments. Many amino acid signatures observed were characteristic of human isolates; hence, their circulation could enhance inter- or intrasubtypic reassortment.

Avian influenza (AI) viruses of the H7 subtype have the potential to evolve into highly pathogenic (HP) viruses that represent a major economic problem for the poultry industry and a threat to global health. However, the emergence of HPAI viruses from low-pathogenic (LPAI) progenitor viruses currently is poorly understood. To investigate the origin and evolution of one of the most important avian influenza epidemics described in Europe, we investigated the evolutionary and spatial dynamics of the entire genome of 109 H7N1 (46 LPAI and 63 HPAI) viruses collected during Italian H7N1 outbreaks between March 1999 and February 2001. Phylogenetic analysis revealed that the LPAI and HPAI epidemics shared a single ancestor, that the HPAI strains evolved from the LPAI viruses in the absence of reassortment, and that there was a parallel emergence of mutations among HPAI and later LPAI lineages. Notably, an ultradeep-sequencing analysis demonstrated that some of the amino acid changes characterizing the HPAI virus cluster were already present with low frequency within several individual viral populations from the beginning of the LPAI H7N1 epidemic. A Bayesian phylogeographic analysis revealed stronger spatial structure during the LPAI outbreak, reflecting the more rapid spread of the virus following the emergence of HPAI. The data generated in this study provide the most complete evolutionary and phylogeographic analysis of epidemiologically intertwined high- and low-pathogenicity viruses undertaken to date and highlight the importance of implementing prompt eradication measures against LPAI to prevent the appearance of viruses with fitness advantages and unpredictable pathogenic properties. IMPORTANCE The Italian H7 AI epidemic of 1999 to 2001 was one of the most important AI outbreaks described in Europe. H7 viruses have the ability to evolve into HP forms from LP precursors, although the mechanisms underlying this evolutionary transition are only poorly understood. We combined epidemiological information, whole-genome sequence data, and ultradeep sequencing approaches to provide the most complete characterization of the evolution of HPAI from LPAI viruses undertaken to date. Our analysis revealed that the LPAI viruses were the direct ancestors of the HPAI strains and identified low-frequency minority variants with HPAI mutations that were present in the LPAI samples. Spatial analysis provided key information for the design of effective control strategies for AI at both local and global scales. Overall, this work highlights the importance of implementing rapid eradication measures to prevent the emergence of novel influenza viruses with severe pathogenic properties.

We describe the identification and characterization of the H9N2 influenza subtype reported in Egyptian broiler and broiler breeder farms for the first time. Circulation of this subtype in a highly pathogenic H5N1 influenza virus endemic population provides an opportunity for genetic reassortment and emergence of novel viruses.

International audience; Canine distemper virus (CDV) infection represents an important conservation threat to many carnivore species and has contributed to the population decline of several wild terrestrial and aquatic mammalian species. Since 2006, the Alpine region of North-Eastern (NE) Italy has been experiencing a severe and widespread outbreak of CDV affecting the wild carnivore population. In this study we performed an extensive phylogenetic and molecular evolutionary analysis of CDV identified during the recent wildlife epidemic in the Alpine region. Our analysis yielded data on the evolutionary dynamics of the Alpine wildlife CDV epidemic and revealed the emergence and spread of a single genetic cluster of CDV. The wide distribution of the novel cluster combined with the identification of a specific amino acid mutation, which is believed to increase the ability of the virus to replicate in a wider host range, raises concerns over the possible implications of the spread of this virus on the conservation of endangered wildlife species.

Avian influenza viruses of the H9N2 subtype have seriously affected the poultry industry of the Far and Middle East since the mid-1990s and are considered one of the most likely candidates to cause a new influenza pandemic in humans. To understand the genesis and epidemiology of these viruses, we investigated the spatial and evolutionary dynamics of complete genome sequences of H9N2 viruses circulating in nine Middle Eastern and Central Asian countries from 1998 to 2010. We identified four distinct and cocirculating groups (A, B, C, and D), each of which has undergone widespread inter- and intrasubtype reassortments, leading to the generation of viruses with unknown biological properties. Our analysis also suggested that eastern Asia served as the major source for H9N2 gene segments in the Middle East and Central Asia and that in this geographic region within-country evolution played a more important role in shaping viral genetic diversity than migration between countries. The genetic variability identified among the H9N2 viruses was associated with specific amino acid substitutions that are believed to result in increased transmissibility in mammals, as well as resistance to antiviral drugs. Our study highlights the need to constantly monitor the evolution of H9N2 viruses in poultry to better understand the potential risk to human health posed by these viruses.