Highly pathogenic avian influenza A(H5N1) virus infections on fur farms connected to mass mortalities of black-headed gulls, Finland, July to October 2023.

Highly pathogenic avian influenza (HPAI) has caused widespread mortality in both wild and domestic birds in Europe 2020-2023. In July 2023, HPAI A(H5N1) was detected on 27 fur farms in Finland. In total, infections in silver and blue foxes, American minks and raccoon dogs were confirmed by RT-PCR. The pathological findings in the animals include widespread inflammatory lesions in the lungs, brain and liver, indicating efficient systemic dissemination of the virus. Phylogenetic analysis of Finnish A(H5N1) strains from fur animals and wild birds has identified three clusters (Finland I-III), and molecular analyses revealed emergence of mutations known to facilitate viral adaptation to mammals in the PB2 and NA proteins. Findings of avian influenza in fur animals were spatially and temporally connected with mass mortalities in wild birds. The mechanisms of virus transmission within and between farms have not been conclusively identified, but several different routes relating to limited biosecurity on the farms are implicated. The outbreak was managed in close collaboration between animal and human health authorities to mitigate and monitor the impact for both animal and human health.

One health surveillance strategy for coronaviruses in Italian wildlife.

The recent reinforcement of CoV surveillance in animals fuelled by the COVID-19 pandemic provided increasing evidence that mammals other than bats might hide further diversity and play critical roles in human infectious diseases. This work describes the results of a two-year survey carried out in Italy with the double objective of uncovering CoV diversity associated with wildlife and of excluding the establishment of a reservoir for SARS-CoV-2 in particularly susceptible or exposed species. The survey targeted hosts from five different orders and was harmonised across the country in terms of sample size, target tissues, and molecular test. Results showed the circulation of 8 CoV species in 13 hosts out of the 42 screened. Coronaviruses were either typical of the host species/genus or normally associated with their domestic counterpart. Two novel viruses likely belonging to a novel CoV genus were found in mustelids. All samples were negative for SARS-CoV-2, with minimum detectable prevalence ranging between 0.49% and 4.78% in the 13 species reaching our threshold sample size of 59 individuals. Considering that within-species transmission in white-tailed deer resulted in raising the prevalence from 5% to 81% within a few months, this result would exclude a sustained cycle after spillback in the tested species.

Highly pathogenic avian influenza A(H5N1) virus infection in farmed minks, Spain, October 2022.

In October 2022, an outbreak in Europe of highly pathogenic avian influenza (HPAI) A(H5N1) in intensively farmed minks occurred in northwest Spain. A single mink farm hosting more than 50,000 minks was involved. The identified viruses belong to clade 2.3.4.4b, which is responsible of the ongoing epizootic in Europe. An uncommon mutation (T271A) in the PB2 gene with potential public health implications was found. Our investigations indicate onward mink transmission of the virus may have occurred in the affected farm.

Asymptomatic infection with clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) in carnivore pets, Italy, April 2023.

In April 2023, an outbreak of clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses carrying the T271A mammalian adaptive mutation in the PB2 protein was detected in a backyard poultry farm in Italy. Five domestic dogs and one cat living on the premises had seroconverted in the absence of clinical signs. Virological and serological monitoring of individuals exposed to the virus proved the absence of human transmission, however, asymptomatic influenza A(H5N1) infections in mammalian pets may have important public health implications.

Outbreak of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus in cats, Poland, June to July 2023.

BackgroundOver a 3-week period in late June/early July 2023, Poland experienced an outbreak caused by highly pathogenic avian influenza (HPAI) A(H5N1) virus in cats.AimThis study aimed to characterise the identified virus and investigate possible sources of infection.MethodsWe performed next generation sequencing and phylogenetic analysis of detected viruses in cats.ResultsWe sampled 46 cats, and 25 tested positive for avian influenza virus. The identified viruses belong to clade 2.3.4.4b, genotype CH (H5N1 A/Eurasian wigeon/Netherlands/3/2022-like). In Poland, this genotype was responsible for several poultry outbreaks between December 2022 and January 2023 and has been identified only sporadically since February 2023. Viruses from cats were very similar to each other, indicating one common source of infection. In addition, the most closely related virus was detected in a dead white stork in early June. Influenza A(H5N1) viruses from cats possessed two amino acid substitutions in the PB2 protein (526R and 627K) which are two molecular markers of virus adaptation in mammals. The virus detected in the white stork presented one of those mutations (627K), which suggests that the virus that had spilled over to cats was already partially adapted to mammalian species.ConclusionThe scale of HPAI H5N1 virus infection in cats in Poland is worrying. One of the possible sources seems to be poultry meat, but to date no such meat has been identified with certainty. Surveillance should be stepped up on poultry, but also on certain species of farmed mammals kept close to infected poultry farms.

Intercontinental Spread of Eurasian Highly Pathogenic Avian Influenza A(H5N1) to Senegal.

In January 2021, Senegal reported the emergence of highly pathogenic avian influenza virus A(H5N1), which was detected on a poultry farm in Thies, Senegal, and in great white pelicans in the Djoudj National Bird Sanctuary. We report evidence of new transcontinental spread of H5N1 from Europe toward Africa.

First detection of avian metapneumovirus subtype C Eurasian lineage in a Eurasian wigeon (Mareca penelope) wintering in Northeastern Italy: an additional hint on the role of migrating birds in the viral epidemiology.

Avian metapneumovirus (aMPV) economically affects the global poultry industry causing respiratory and reproductive disorders. Considering the paucity of data on aMPV occurrence in European free-ranging avifauna, a molecular survey was conducted on wild birds of 23 species belonging to the orders Anseriformes, Charadriiformes or Passeriformes, captured alive and sampled in Northeast Italy as part of the national avian influenza virus (AIV) surveillance activities. A total of 492 oropharyngeal swabs, collected from 2007-2010, all AIV-negative, were screened from aMPV by subtype-specific qRT-PCR. An aMPV-C strain, named aMPV/C/IT/Wigeon/758/07, was found in a wintering young Eurasian wigeon (Mareca penelope) sampled in November 2007. The matrix, fusion, and attachment glycoprotein genes of the detected strain were subsequently amplified by specific independent RT-PCRs, then sequenced, and compared in a phylogenetic framework with known aMPV homologous sequences retrieved from GenBank. Close genetic relationships were found between the aMPV/C/IT/Wigeon/758/07 strain and subtype C Eurasian lineage strains isolated in the late 1990s in French domestic ducks, suggesting epidemiological links. Eurasian wigeons are medium/long-range migrant dabbling ducks that move along the Black Sea/Mediterranean flyway; our finding might, therefore, be related to migratory bridges between countries. To our knowledge, this is the first molecular evidence of the occurrence of aMPV subtype C in Italy and backdates the aMPV-C circulation to 2007. Moreover, the results suggest the susceptibility of Eurasian wigeons to aMPV. Broader investigations are needed to assess the role of wild ducks and the significance of the wildfowl/poultry interface in aMPV-C epidemiology.RESEARCH HIGHLIGHTSWild birds live-captured in Italy were tested for aMPV detection and characterization.aMPV-C Eurasian lineage was found for the first time in a wintering Eurasian wigeon.Migratory birds could be involved in the aMPV epidemiology.

The first familial cluster of the B.1.1.7 variant of SARS-CoV-2 in the northeast of Italy.

PURPOSE: We report on the first identified cluster of the B.1.1.7 variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in the northeast of Italy. METHODS: The cluster was recognized in January 2021 with an epidemiological started from the hospitalization of a 68-year-old man suffering from coronavirus disease 2019 (COVID-19) related pneumonia and we surprisingly found three families involved in the same cluster. RESULTS: We retrospectively rebuilt the pathway of infection and performed a virological analysis. CONCLUSION: This allow us to make clear the very high attack rate and the great infective capacity of this B.1.1.7 variant of SARS-CoV-2.

Effect of assay choice, viral concentration and operator interpretation on infectious bronchitis virus detection and characterization.

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.

Prevalence of SARS-CoV-2 RNA on inanimate surfaces: a systematic review and meta-analysis.

Coronavirus disease (COVID-19) is a respiratory disease affecting many people and able to be transmitted through direct and perhaps indirect contact. Direct contact transmission, mediated by aerosols or droplets, is widely demonstrated, whereas indirect transmission is only supported by collateral evidence such as virus persistence on inanimate surfaces and data from other similar viruses. The present systematic review aims to estimate SARS-CoV-2 prevalence on inanimate surfaces, identifying risk levels according to surface characteristics. Data were obtained from studies in published papers collected from two databases (PubMed and Embase) with the last search on 1 September 2020. Included studies had to be papers in English, had to deal with coronavirus and had to consider inanimate surfaces in real settings. Studies were coded according to our assessment of the risk that the investigated surfaces could be contaminated by SARS-CoV-2. A meta-analysis and a metaregression were carried out to quantify virus RNA prevalence and to identify important factors driving differences among studies. Thirty-nine out of forty retrieved paper reported studies carried out in healthcare settings on the prevalence of virus RNA, five studies carry out also analyses through cell culture and six tested the viability of isolated viruses. Overall prevalences of SARS-CoV-2 RNA on high-, medium- and low-risk surfaces were 0.22 (CI95 [0.152-0.296]), 0.04 (CI95 [0.007-0.090]), and 0.00 (CI95 [0.00-0.019]), respectively. The duration surfaces were exposed to virus sources (patients) was the main factor explaining differences in prevalence.

Sub-Saharan Africa and Eurasia Ancestry of Reassortant Highly Pathogenic Avian Influenza A(H5N8) Virus, Europe, December 2019.

We report detection of a highly pathogenic avian influenza A(H5N8) clade 2.3.4.4b virus in Europe. This virus was generated by reassortment between H5N8 subtype virus from sub-Saharan Africa and low pathogenicity avian influenza viruses from Eurasia.

A G1-lineage H9N2 virus with oviduct tropism causes chronic pathological changes in the infundibulum and a long-lasting drop in egg production.

Since 1997, G1-lineage H9N2 avian influenza viruses have been circulating in Asia and later on in the Middle East, and they have been associated to mild respiratory disease, drops in egg production and moderate mortality in chickens, in particular in the presence of concurrent infections. In this study, we investigated the importance of the G1-lineage H9N2 A/chicken/Israel/1163/2011 virus as a primary pathogen in layers, analyzing its tropism and binding affinity for the oviduct tissues, and investigating the long-term impact on egg production. Besides causing a mild respiratory infection, the virus replicated in the oviduct of 60% of the hens causing different degrees of salpingitis throughout the organ, in particular at the level of the infundibulum, where the detection of the virus was associated with severe heterophilic infiltrate, and necrosis of the epithelium. Binding affinity assays confirmed that the infundibulum was the most receptive region of the oviduct. The drop in egg production was at its peek at 2 weeks post-infection (pi) (60% decrease) and continued up to 80 days pi (35% decrease). On day 80 pi, non-laying birds showed egg yolk peritonitis, and histopathological analyses described profound alteration of the infundibulum architecture, duct ectasia and thinning of the epithelium, while the rest of the oviduct and ovary appeared normal. Our results show that this H9N2 virus is a primary pathogen in layer hens, and that its replication in the infundibulum is responsible for acute and chronic lesions that limits the effective functionality of the oviduct, compromising the commercial life of birds.

Genetic Diversity of Highly Pathogenic Avian Influenza A(H5N8/H5N5) Viruses in Italy, 2016-17.

In winter 2016-17, highly pathogenic avian influenza A(H5N8) and A(H5N5) viruses of clade 2.3.4.4 were identified in wild and domestic birds in Italy. We report the occurrence of multiple introductions and describe the identification in Europe of 2 novel genotypes, generated through multiple reassortment events.

Synergy or interference of a H9N2 avian influenza virus with a velogenic Newcastle disease virus in chickens is dose dependent.

Field observations indicate that the impact of velogenic Newcastle disease virus (vNDV) is more severe in countries with concomitant circulation of low pathogenicity avian influenza virus, as is the case in the Middle East, in particular in Israel, where H9N2 and NDV are endemic. In our study, we evaluated how the exposure of chickens to an H9N2 challenge either favours or interferes with a subsequent vNDV infection and its transmission to sentinels. For this purpose, single vNDV and sequential H9/NDV challenges were performed with increasing doses of vNDV (101-106 EID50). The H9N2 challenge made birds more susceptible to the vNDV, lowering the minimum dose required to cause an infection, exacerbating the clinical outcome, while delaying the onset of the disease and time of death. Interestingly, the presence and degree of these seemingly contrasting effects were dose-dependent and not mutually exclusive.

A genetically engineered H5 protein expressed in insect cells confers protection against different clades of H5N1 highly pathogenic avian influenza viruses in chickens.

The evolution of highly pathogenic H5N1 avian influenza viruses (HPAI-H5N1) has resulted in the appearance of a number of diverse groups of HPAI-H5N1 based on the presence of genetically similar clusters of their haemagglutinin sequences (clades). An H5 antigen encoded by a recombinant baculovirus and expressed in insect cells was used for oil-emulsion-based vaccine prototypes. In several experiments, vaccination was performed at 10 days of age, followed by challenge infection on day 21 post vaccination (PV) with HPAI-H5N1 clades 2.2, 2.2.1, and 2.3.2. A further challenge infection with HPAI-H5N1 clade 2.2.1 was performed at day 42 PV. High haemagglutination inhibition titres were observed for the recH5 vaccine antigen, and lower haemagglutination inhibition titres for the challenge virus antigens. Nevertheless, the rate of protection from mortality and clinical signs was 100% when challenged at 21 days PV and 42 days PV, indicating protection over the entire broiler chicken rearing period without a second vaccination. The unvaccinated control chickens mostly died between two and five days after challenge infection. A low level of viral RNA was detected by reverse transcription followed by a quantitative polymerase chain reaction in a limited number of birds for a short period after challenge infection, indicating a limited spread of HPAI-H5N1 at flock level. Furthermore, it was observed that the vaccine can be used in a differentiation infected from vaccinated animals (DIVA) approach, based on the detection of nucleoprotein antibodies in vaccinated/challenged chickens. The vaccine fulfilled all expectations of an inactivated vaccine after one vaccination against challenge with different clades of H5N1-HPAI and is suitable for a DIVA approach.

Phylogenetically distinct equine influenza viruses show different tropism for the swine respiratory tract.

Influenza A viruses circulate in a wide range of animals. H3N8 equine influenza virus (EIV) is an avian-origin virus that has established in dogs as canine influenza virus (CIV) and has also been isolated from camels and pigs. Previous work suggests that mutations acquired during EIV evolution might have played a role in CIV emergence. Given the potential role of pigs as a source of human infections, we determined the ability of H3N8 EIVs to replicate in pig cell lines and in respiratory explants. We show that phylogenetically distinct EIVs display different infection phenotypes along the pig respiratory tract, but not in cell lines. Our results suggest that EIV displays a dynamic host range along its evolutionary history, supporting the view that evolutionary processes play important roles in host range and tropism and also underscoring the utility of using explant cultures to study influenza pathogenesis.

Histopathological and immunohistochemical study of exocrine and endocrine pancreatic lesions in avian influenza A experimentally infected turkeys showing evidence of pancreatic regeneration.

In order to investigate the pancreatic lesions caused by the infection with either H7N1 or H7N3 low-pathogenicity avian influenza viruses, 28 experimentally infected turkeys were submitted for histopathology, immunohistochemistry, haematobiochemistry and real-time reverse transcriptase polymerase chain reaction after different days post-infection (DPI). The localization of viral antigen and the measurement of insulin and glucagon expression in the pancreas were assessed to verify the progression from pancreatitis to metabolic disorders, such as diabetes. At the early infection phase (4-7 DPI), a severe acute necrotizing pancreatitis was recognized. During the intermediate phase (8-17 DPI), a mixed acute/chronic change associated with regenerative ductular proliferation was observed. A loss of pancreatic islets was detected in most severe cases and viral antigen was found in the pancreas of 11/28 turkeys (4-10 DPI) with the most severe histological damage. In turkeys euthanized at 39 DPI (late phase), a chronic fibrosing pancreatitis was observed with the reestablishment of both the exocrine and the endocrine pancreas. Insulin and glucagon expression manifested a progressive decrease with subsequent ductular positivity. Haematobiochemistry revealed increased lipasemia in the first week post-infection and hyperglycaemia in the second, with a progressive normalization within 21 DPI. This study allowed the identification of progressive virus-associated exocrine and endocrine pancreatic damage, suggesting that influenza virus might be responsible for metabolic derangements. Moreover, it highlighted a remarkable post-damage hyperplastic and reparative process from a presumptive common exocrine/endocrine precursor. This potential regeneration deserves further investigation for its relevance in a therapeutic perspective to replace lost and non-functional cells in diabetes mellitus.

Influenza A viruses grow in human pancreatic cells and cause pancreatitis and diabetes in an animal model.

Influenza A viruses commonly cause pancreatitis in naturally and experimentally infected animals. In this study, we report the results of in vivo investigations carried out to establish whether influenza virus infection could cause metabolic disorders linked to pancreatic infection. In addition, in vitro tests in human pancreatic islets and in human pancreatic cell lines were performed to evaluate viral growth and cell damage. Infection of an avian model with two low-pathogenicity avian influenza isolates caused pancreatic damage resulting in hyperlipasemia in over 50% of subjects, which evolved into hyperglycemia and subsequently diabetes. Histopathology of the pancreas showed signs of an acute infection resulting in severe fibrosis and disruption of the structure of the organ. Influenza virus nucleoprotein was detected by immunohistochemistry (IHC) in the acinar tissue. Human seasonal H1N1 and H3N2 viruses and avian H7N1 and H7N3 influenza virus isolates were able to infect a selection of human pancreatic cell lines. Human viruses were also shown to be able to infect human pancreatic islets. In situ hybridization assays indicated that viral nucleoprotein could be detected in beta cells. The cytokine activation profile indicated a significant increase of MIG/CXCL9, IP-10/CXCL10, RANTES/CCL5, MIP1b/CCL4, Groa/CXCL1, interleukin 8 (IL-8)/CXCL8, tumor necrosis factor alpha (TNF-α), and IL-6. Our findings indicate that influenza virus infection may play a role as a causative agent of pancreatitis and diabetes in humans and other mammals.