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.

West Nile virus (WNV) is a recently re-emerged health problem in Europe. In Italy, an increasing number of outbreaks of West Nile disease, with occurrences of human cases, have been reported since 2008. This is particularly true in northern Italy, where entomological surveillance systems have been implemented at a regional level. The aim of this study was to use, for the first time, all the entomological data collected in the five regions undergoing surveillance for WNV in northern Italy to characterize the viral circulation (at a spatial and temporal scale), identify potential mosquito vectors, and specify relationships between virus circulation and meteorological conditions. In 2013, 286 sites covering the entire Pianura Padana area were monitored. A total of 757,461 mosquitoes were sampled. Of these, 562,079 were tested by real-time PCR in 9,268 pools, of which 180 (1.9%) were positive for WNV. The largest part of the detected WNV sequences belonged to lineage II, demonstrating that, unlike those in the past, the 2013 outbreak was mainly sustained by this WNV lineage. This surveillance also detected the Usutu virus, a WNV-related flavivirus, in 241 (2.6%) pools. The WNV surveillance systems precisely identified the area affected by the virus and detected the viral circulation approximately two weeks before the occurrence of onset of human cases. Ninety percent of the sampled mosquitoes were Culex pipiens, and 178/180 WNV-positive pools were composed of only this species, suggesting this mosquito is the main WNV vector in northern Italy. A significantly higher abundance of the vector was recorded in the WNV circulation area, which was characterized by warmer and less rainy conditions and greater evapotranspiration compared to the rest of the Pianura Padana, suggesting that areas exposed to these conditions are more suitable for WNV circulation. This observation highlights warmer and less rainy conditions as factors able to enhance WNV circulation and cause virus spillover outside the sylvatic cycle.

Background. A new strain of WNV lineage 1 (WNV − 1) emerged in the Veneto Region, northern Italy, in 2021, eight years after the last outbreak of WNV − 1 in Italy. The virus, which co-circulates with WNV-2, has become endemic in the Region, where, in 2022, most human cases of neuroinvasive disease (WNND) reported in Europe have occurred. Methods. Comparative analysis of the epidemiology and clinical presentation of WNV-1 and WNV-2 infection in humans, as well as the temporal and geographic distribution of WNV-1 and WNV-2 among wild birds and Culex pipiens mosquitoes in Veneto, from May 16^th to August 21^st, 2022, to determine if the high number of WNND cases was associated with WNV-1. Results. As of August 21^st, 2022, 222 human cases of WNV infection were confirmed by molecular testing, including 103 with fever (WNF) and 119 with WNND. WNV lineage was determined in 201 (90.5%) cases, comprising 138 WNV-1 and 63 WNV-2 infections. During the same period, 35 blood donors tested positive, including 30 in whom WNV lineage was determined (13 WNV-1 and 17 WNV-2). Comparative analysis of the distribution of WNV-1 and WNV-2 infections among WNND cases, WNF cases and WNV-positive blood donors showed that patients with WNND were more likely to have WNV-1 infection than blood donors (odds ratio 3.44; 95% CI 95% 1.54 to 8.24; p = 0.0043). As observed in humans, in wild birds WNV-1 had higher infectious rate (IR) and showed a more rapid expansion than WNV-2. At variance, the distribution of the two lineages was more even in mosquitoes, but with a trend of rapid increase of WNV-1 IR over WNV-2. Conclusions. Comparative analysis of WNV-1 vs WNV-2 infection in humans, wild birds, and mosquitos showed a rapid expansion of WNV-1 and suggested that WNV-1 infected patients might have an increased risk to develop severe disease.

Background: The recent spread of West Nile Virus in temperate countries has raised concern. Predicting the likelihood of transmission is crucial to ascertain the threat to Public and Veterinary Health. However, accurate models of West Nile Virus (WNV) expansion in Europe may be hampered by limited understanding of the population dynamics of their primary mosquito vectors and their response to environmental changes.Methods: We used data collected in north-eastern Italy (2009–2011) to analyze the determinants of the population growth rate of the primary WNV vectorCulex pipiens. A series of alternative growth models were fitted to longitudinal data on mosquito abundance to evaluate the strength of evidence for regulation by intrinsic density-dependent and/or extrinsic environmental factors. Model-averaging algorithms were then used to estimate the relative importance of intrinsic and extrinsic variables in describing the variations of per-capita growth rates.Results: Results indicate a much greater contribution of density-dependence in regulating vector population growth rates than of any environmental factor on its own. Analysis of an average model ofCx. pipiensgrowth revealed that the most significant predictors of their population dynamics was the length of daylight, estimated population size and temperature conditions in the 15 day period prior to sampling. Other extrinsic variables (including measures of precipitation, number of rainy days, and humidity) had only a minor influence onCx. pipiensgrowth rates.Conclusions: These results indicate the need to incorporate density dependence in combination with key environmental factors for robust prediction ofCx. pipienspopulation expansion and WNV transmission risk. We hypothesize that detailed analysis of the determinants of mosquito vector growth rate as conducted here can help identify when and where an increase in vector population size and associated WNV transmission risk should be expected.