Distinct mechanisms of non-autonomous UPRER mediated by GABAergic, glutamatergic, and octopaminergic neurons.

The capacity to deal with stress declines during the aging process, and preservation of cellular stress responses is critical to healthy aging. The unfolded protein response of the endoplasmic reticulum (UPRER) is one such conserved mechanism, which is critical for the maintenance of several major functions of the ER during stress, including protein folding and lipid metabolism. Hyperactivation of the UPRER by overexpression of the major transcription factor, xbp-1s, solely in neurons drives lifespan extension as neurons send a neurotransmitter-based signal to other tissue to activate UPRER in a non-autonomous fashion. Previous work identified serotonergic and dopaminergic neurons in this signaling paradigm. To further expand our understanding of the neural circuitry that underlies the non-autonomous signaling of ER stress, we activated UPRER solely in glutamatergic, octopaminergic, and GABAergic neurons in C. elegans and paired whole-body transcriptomic analysis with functional assays. We found that UPRER-induced signals from glutamatergic neurons increased expression of canonical protein homeostasis pathways and octopaminergic neurons promoted pathogen response pathways, while minor, but statistically significant changes were observed in lipid metabolism-related genes with GABAergic UPRER activation. These findings provide further evidence for the distinct role neuronal subtypes play in driving the diverse response to ER stress.

Mechanical stress through growth on stiffer substrates impacts animal health and longevity in C. elegans.

Mechanical stress is a measure of internal resistance exhibited by a body or material when external forces, such as compression, tension, bending, etc. are applied. The study of mechanical stress on health and aging is a continuously growing field, as major changes to the extracellular matrix and cell-to-cell adhesions can result in dramatic changes to tissue stiffness during aging and diseased conditions. For example, during normal aging, many tissues including the ovaries, skin, blood vessels, and heart exhibit increased stiffness, which can result in a significant reduction in function of that organ. As such, numerous model systems have recently emerged to study the impact of mechanical and physical stress on cell and tissue health, including cell-culture conditions with matrigels and other surfaces that alter substrate stiffness and ex vivo tissue models that can apply stress directly to organs like muscle or tendons. Here, we sought to develop a novel method in an in vivo, model organism setting to study the impact of mechanical stress on aging, by increasing substrate stiffness in solid agar medium of C. elegans. To our surprise, we found shockingly limited impact of growth of C. elegans on stiffer substrates, including limited effects on cellular health, gene expression, organismal health, stress resilience, and longevity. Overall, our studies reveal that altering substrate stiffness of growth medium for C. elegans have only mild impact on animal health and longevity; however, these impacts were not nominal and open up important considerations for C. elegans biologists in standardizing agar medium choice for experimental assays.

Fatal West Nile Virus Infection in Horse Returning to United Kingdom from Spain, 2022.

We report fatal West Nile virus (WNV) infection in a 7-year-old mare returning to the United Kingdom from Spain. Case timeline and clustering of virus sequence with recent WNV isolates suggest that transmission occurred in Andalusía, Spain. Our findings highlight the importance of vaccination for horses traveling to WNV-endemic regions.

Open science, the replication crisis, and environmental public health.

Concerns about a crisis of mass irreplicability across scientific fields ("the replication crisis") have stimulated a movement for open science, encouraging or even requiring researchers to publish their raw data and analysis code. Recently, a rule at the US Environmental Protection Agency (US EPA) would have imposed a strong open data requirement. The rule prompted significant public discussion about whether open science practices are appropriate for fields of environmental public health. The aims of this paper are to assess (1) whether the replication crisis extends to fields of environmental public health; and (2) in general whether open science requirements can address the replication crisis. There is little empirical evidence for or against mass irreplicability in environmental public health specifically. Without such evidence, strong claims about whether the replication crisis extends to environmental public health - or not - seem premature. By distinguishing three concepts - reproducibility, replicability, and robustness - it is clear that open data initiatives can promote reproducibility and robustness but do little to promote replicability. I conclude by reviewing some of the other benefits of open science, and offer some suggestions for funding streams to mitigate the costs of adoption of open science practices in environmental public health.

Comparative pathogenesis of two genotype VI.2 avian paramyxovirus type-1 viruses (APMV-1) in pheasants, partridges and chickens.

Newcastle disease (ND) is caused by virulent forms of avian paramyxovirus-1 (APMV-1) and is an economically important disease of poultry world-wide. Pigeon paramyxovirus 1 (PPMV-1), a sub-group of APMV-1 is endemic in Columbiformes and can cause infections of poultry. An outbreak of ND in partridges in Scotland, UK, in 2006 (APMV-1/partridge/UK(Scotland)/7575/06) was identified as a class II, genotype VI.2.1.1.2.1, more commonly associated with PPMV-1. It has been hypothesized that game birds may be a route of transmission into commercial poultry settings due to the semi-feral rearing system, which potentially brings them into contact with both wild-birds and poultry species. Therefore, the pathogenesis and transmission of APMV-1/partridge/UK(Scotland)/7575/06 in game birds and chickens was investigated, and compared to a contemporary PPMV-1 isolate, PPMV-1/pigeon/UK/015874/15. Viral shedding and seroconversion profiles demonstrated that pheasants were susceptible to infection with APMV-1/partridge/UK(Scotland)/7575/06 with limited clinical signs observed although they were able to excrete and transmit virus. In contrast, partridges and pheasants showed limited infection with PPMV-1/pigeon/UK/015874/15, causing mild clinical disease. Chickens, however, were productively infected and were able to transmit virus in the absence of clinical signs. From the data, it can be deduced that whilst game birds may play a role in the transmission and epidemiology of genotype VI.2 APMV-1 viruses, the asymptomatic nature of circulation within these species precludes evaluation of natural infection by clinical surveillance. It therefore remains a possibility that genotype VI.2 APMV-1 infection in game birds has the potential for asymptomatic circulation and remains a potential threat to avian production systems.RESEARCH HIGHLIGHTS Demonstration of infection of game birds with Pigeon paramyxovirus-1 (PPMV-1).There are differing dynamics of infection between different game bird species.Differing dynamics of infection between different PPMV-1 isolates and genotypes in game birds and chickens.

Syndromic Surveillance 2.0: Emerging Global Surveillance Strategies for Infectious Disease Epidemics.

The U.S. Army has a long history of preventing, detecting, and treating infectious diseases. Like other organizations and agencies involved in public health, the Army is increasingly interested in syndromic surveillance strategies-those designed to identify outbreaks before clinical data are available. Researchers use various methods to identify surveillance strategies across the globe, investigate these strategies' benefits and limitations, and recommend actions to aid the Army in their efforts to detect emerging epidemics and pandemics.

The P value plot does not provide evidence against air pollution hazards.

A number of papers by Young and collaborators have criticized epidemiological studies and meta-analyses of air pollution hazards using a graphical method that the authors call a P value plot, claiming to find zero effects, heterogeneity, and P hacking. However, the P value plot method has not been validated in a peer-reviewed publication. The aim of this study was to investigate the statistical and evidentiary properties of this method. Methods: A simulation was developed to create studies and meta-analyses with known real effects δ , integrating two quantifiable conceptions of evidence from the philosophy of science literature. The simulation and analysis is publicly available and automatically reproduced. Results: In this simulation, the plot did not provide evidence for heterogeneity or P hacking with respect to any condition. Under the right conditions, the plot can provide evidence of zero effects; but these conditions are not satisfied in any actual use by Young and collaborators. Conclusion: The P value plot does not provide evidence to support the skeptical claims about air pollution hazards made by Young and collaborators.

Evaluation of Lesions and Viral Antigen Distribution in Domestic Pigs Inoculated Intranasally with African Swine Fever Virus Ken05/Tk1 (Genotype X).

The understanding of the pathogenic mechanisms and the clinicopathological forms caused by currently circulating African swine fever virus (ASFV) isolates is incomplete. So far, most of the studies have been focused on isolates classified within genotypes I and II, the only genotypes that have circulated outside of Africa. However, less is known about the clinical presentations and lesions induced by isolates belonging to the other twenty-two genotypes. Therefore, the early clinicopathological identification of disease outbreaks caused by isolates belonging to, as yet, not well-characterised ASFV genotypes may be compromised, which might cause a delay in the implementation of control measures to halt the virus spread. To improve the pathological characterisation of disease caused by diverse isolates, we have refined the macroscopic and histopathological evaluation protocols to standardise the scoring of lesions. Domestic pigs were inoculated intranasally with different doses (high, medium and low) of ASFV isolate Ken05/Tk1 (genotype X). To complement previous studies, the distribution and severity of macroscopic and histopathological lesions, along with the amount and distribution of viral antigen in tissues, were characterised by applying the new scoring protocols. The intranasal inoculation of domestic pigs with high doses of the Ken05/Tk1 isolate induced acute forms of ASF in most of the animals. Inoculation with medium doses mainly induced acute forms of disease. A less severe but longer clinical course, typical of subacute forms, characterised by the presence of more widespread and severe haemorrhages and oedema, was observed in one pig inoculated with the medium dose. The severity of vascular lesions (haemorrhages and oedema) induced by high and medium doses was not associated with the amount of virus antigen detected in tissues, therefore these might be attributed to indirect mechanisms not evaluated in the present study. The absence of clinical signs, lesions and detectable levels of virus genome or antigen in blood from the animals inoculated with the lowest dose ruled out the existence of possible asymptomatic carriers or persistently infected pigs, at least for the 21 days period of the study. The results corroborate the moderate virulence of the Ken05/Tk1 isolate, as well as its capacity to induce both the acute and, occasionally, subacute forms of ASF when high and medium doses were administered intranasally.

Science and Technology Solutions for Scalable SARS-CoV-2 Testing to Inform Return to Full Capacity Strategy in United States Air Force Workforce Personnel.

SARS-CoV-2 has highlighted the requirement for a drastic change in pandemic response. While cases continue to rise, there is an urgent need to deploy sensitive and rapid testing in order to identify potential outbreaks before there is an opportunity for further community spread. Currently, reverse transcription quantitative polymerase chain reaction (RT-qPCR) is considered the gold standard for diagnosing an active infection, using a nasopharyngeal swab; however, it can take days after symptoms develop to properly identify and trace the infection. While many civilian jobs can be performed remotely, the Department of Defense (DOD) is by nature a very fluid organization which requires in-person interaction and a physical presence to maintain effectiveness. In this commentary, we examine several current and emergent technologies and their ability to identify both active and previous SARS-CoV-2 infection, possibly in those without symptoms. Further, we will explore an ongoing study at the Air Force Research Laboratory, utilizing Reverse Transcription Loop-mediated isothermal amplification (RT-LAMP), next-generation sequencing, and the presence of SARS-CoV-2 antibodies through Lateral Flow Immunoassays. The ability to identify SARS-CoV-2 through volatile organic compound biomarker identification will also be explored. By exploring and validating multiple testing strategies, and contributing to Operation Warp Speed, the DOD is postured to respond to SARS-CoV-2, and future pandemics.

Temperate conditions restrict Japanese encephalitis virus infection to the mid-gut and prevents systemic dissemination in Culex pipiens mosquitoes.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the main cause of viral encephalitis in Asia. However, with changing climate JEV has the potential to emerge in novel temperate regions. Here, we have assessed the vector competence of the temperate mosquito Culex pipiens f. pipiens to vector JEV genotype III at temperatures representative of those experienced, or predicted in the future during the summer months, in the United Kingdom. Our results show that Cx. pipiens is susceptible to JEV infection at both temperatures. In addition, at 25 °C, JEV disseminated from the midgut and was recovered in saliva samples, indicating the potential for transmission. At a lower temperature, 20 °C, following an incubation period of fourteen days, there were reduced levels of JEV dissemination and virus was not detected in saliva samples. The virus present in the bodies of these mosquitoes was restricted to the posterior midgut as determined by microscopy and viable virus was successfully recovered. Apart from the influence on virus dissemination, mosquito mortality was significantly increased at the higher temperature. Overall, our results suggest that temperature is a critical factor for JEV vector competence and infected-mosquito survival. This may in turn influence the vectorial capacity of Cx. pipiens to vector JEV genotype III in temperate areas.

The Emergence of H7N7 Highly Pathogenic Avian Influenza Virus from Low Pathogenicity Avian Influenza Virus Using an in ovo Embryo Culture Model.

Outbreaks of highly pathogenic avian influenza virus (HPAIV) often result in the infection of millions of poultry, causing up to 100% mortality. HPAIV has been shown to emerge from low pathogenicity avian influenza virus (LPAIV) in field outbreaks. Direct evidence for the emergence of H7N7 HPAIV from a LPAIV precursor with a rare di-basic cleavage site (DBCS) was identified in the UK in 2008. The DBCS contained an additional basic amino acid compared to commonly circulating LPAIVs that harbor a single-basic amino acid at the cleavage site (SBCS). Using reverse genetics, outbreak HPAIVs were rescued with a DBCS (H7N7DB), as seen in the LPAIV precursor or an SBCS representative of common H7 LPAIVs (H7N7SB). Passage of H7N7DB in chicken embryo tissues showed spontaneous evolution to a HPAIV. In contrast, deep sequencing of extracts from embryo tissues in which H7N7SB was serially passaged showed retention of the LPAIV genotype. Thus, in chicken embryos, an H7N7 virus containing a DBCS appears naturally unstable, enabling rapid evolution to HPAIV. Evaluation in embryo tissue presents a useful approach to study AIV evolution and allows a laboratory-based dissection of molecular mechanisms behind the emergence of HPAIV.

The effect of BCG vaccination on macrophage phenotype in a mouse model of intranasal Mycobacterium bovis challenge.

In order to develop improved vaccinations against tuberculosis, it is essential to understand the effect of vaccination on the immune response, and to overcome the mechanisms by which mycobacteria regulate this immune response. In this study, we examine the effect of intradermal vaccination with Mycobacterium bovis bacille Calmette-Guèrin on macrophage phenotype following intranasal challenge with virulent Mycobacterium bovis. Preserved lung tissues used in the present study were obtained from a previous vaccination trial in BALB/c mice. Vaccinated mice showed less extensive pulmonary lesions along with a significant decrease in bacterial lung burden when compared to control mice. Immunohistochemical markers of classically activated macrophages (iNOS) and alternatively activated macrophages (Arg1, FIZZ1) were applied to lung sections. Vaccination led to a statistically significant decrease in the number of Arg1+ macrophages. The presence of macrophages that expressed Arginase 1 in pulmonary lesions was much smaller than the presence of macrophages expressing iNOS. The low presence of Arg1+ macrophages induced by vaccination may be caused by Th1 polarization and may reduce alternative activation of macrophages, with an overall more effective intracellular killing of bacteria.

Census Demographics and Chlorpyrifos Use in California's Central Valley, 2011-15: A Distributional Environmental Justice Analysis.

Chlorpyrifos, an acetylcholinesterase inhibitor (ACI), is one of the most widely used insecticides in the world, and is generally recognized to be a moderate human neurotoxin. This paper reports a distributional environmental justice (dEJ) analysis of chlorpyrifos use in California's Central Valley, examining the way distributions of environmental risks are associated with race, ethnicity, class, gender, and other systems of structural oppression. Spatial data on chlorpyrifos use were retrieved from California's Department of Pesticide Registration public pesticide use records for 2011-2015. These data were combined with demographic data for the Central Valley from the American Community Survey (ACS). Spatial regression models were used to estimate effects of demographic covariates on local chlorpyrifos use. A novel bootstrap method was used to account for measurement error in the ACS estimates. This study finds consistent evidence that Hispanic population proportion is associated with increased local chlorpyrifos use. A 10-point increase in Hispanic proportion is associated with an estimated 1.05-1.4-fold increase in local chlorpyrifos use across Census tract models. By contrast, effects of agricultural employment and poverty on local chlorpyrifos use are ambiguous and inconsistent between Census tracts and Census-designated places.

Inductive Risk, Science, and Values: A Reply to MacGillivray.

The argument from inductive risk (AIR) is perhaps the most common argument against the value-free ideal of science. Brian MacGillivray rejects the AIR (at least as it would apply to risk assessment) and embraces the value-free ideal. We clarify the issues at stake and argue that MacGillivray's criticisms, although effective against some formulations of the AIR, fail to overcome the essential concerns that motivate the AIR. There are inevitable trade-offs in scientific enquiry that cannot be resolved with any formal methods or general rules. Choices must be made, and values will be involved. It is best to recognize this explicitly. Even so, there is more work to be done developing methods and institutional support for these choices.

West Nile Virus spread and differential chemokine response in the central nervous system of mice: Role in pathogenic mechanisms of encephalitis.

West Nile virus (WNV) is a zoonotic mosquito-borne flavivirus able to cause severe neurological disease in humans, horses and various avian species. The more severe pathological changes of neurotropic WNV infection are caused by virus neuroinvasion and/or the immunological response in the central nervous system (CNS). The extent in which inflammatory cell trafficking orchestrated by chemokines is involved in the pathogenesis of CNS lesions has not been entirely elucidated. To understand the sequence of pro-inflammatory chemokine induction during WNV encephalitis, a murine intranasal inoculation model was used. The relationship between lesional patterns in the mice CNS, the viral antigen distribution and the expression of pro-inflammatory chemokine (CCL2, CCL5 and CXCL10) were evaluated. Viral antigen was first observed in olfactory tract and pyriform cortex neurons, suggesting a retrograde neuronal infection from the olfactory nerve. A spatio-temporal association between WNV antigen and perivascular cuffs development was observed. Chemokine immunostaining was widely distributed in the brain from early stages. CCL2 immunolabelling was localised in neurons, astrocytes, microglia and endothelial cells as well as mononuclear leucocytes within perivascular cuffs. In contrast, CCL5 and CXCL10 immunostaining were mainly observed in astroglia and neurons, respectively. A strong correlation was demonstrated between the presence of perivascular cuffs and CCL2 and CCL5 expression in most brain areas, while CXCL10 was only associated with inflammatory lesions in few specific regions. Importantly, a strong correlation between WNV and CCL5 distribution was observed. However, no correlation was observed between CXCL10 and viral antigen. Neurons were confirmed as the main target cells of WNV, as well as one of the sources of CCL2, CCL5 and CXCL10. This study shows the sequence and comparative distribution pattern between histological lesions, WNV antigen and chemokine expression over the infection process. Furthermore, it identifies potential targets for immune intervention to suppress damaging chemokine responses.

Multi-Institutional Validation of Deep Learning for Pretreatment Identification of Extranodal Extension in Head and Neck Squamous Cell Carcinoma.

PURPOSE: Extranodal extension (ENE) is a well-established poor prognosticator and an indication for adjuvant treatment escalation in patients with head and neck squamous cell carcinoma (HNSCC). Identification of ENE on pretreatment imaging represents a diagnostic challenge that limits its clinical utility. We previously developed a deep learning algorithm that identifies ENE on pretreatment computed tomography (CT) imaging in patients with HNSCC. We sought to validate our algorithm performance for patients from a diverse set of institutions and compare its diagnostic ability to that of expert diagnosticians. METHODS: We obtained preoperative, contrast-enhanced CT scans and corresponding pathology results from two external data sets of patients with HNSCC: an external institution and The Cancer Genome Atlas (TCGA) HNSCC imaging data. Lymph nodes were segmented and annotated as ENE-positive or ENE-negative on the basis of pathologic confirmation. Deep learning algorithm performance was evaluated and compared directly to two board-certified neuroradiologists. RESULTS: A total of 200 lymph nodes were examined in the external validation data sets. For lymph nodes from the external institution, the algorithm achieved an area under the receiver operating characteristic curve (AUC) of 0.84 (83.1% accuracy), outperforming radiologists' AUCs of 0.70 and 0.71 (P = .02 and P = .01). Similarly, for lymph nodes from the TCGA, the algorithm achieved an AUC of 0.90 (88.6% accuracy), outperforming radiologist AUCs of 0.60 and 0.82 (P < .0001 and P = .16). Radiologist diagnostic accuracy improved when receiving deep learning assistance. CONCLUSION: Deep learning successfully identified ENE on pretreatment imaging across multiple institutions, exceeding the diagnostic ability of radiologists with specialized head and neck experience. Our findings suggest that deep learning has utility in the identification of ENE in patients with HNSCC and has the potential to be integrated into clinical decision making.

Network analysis to evaluate the impact of research funding on research community consolidation.

In 2004, the Alfred P. Sloan Foundation launched a new program focused on incubating a new field, "Microbiology of the Built Environment" (MoBE). By the end of 2017, the program had supported the publication of hundreds of scholarly works, but it was unclear to what extent it had stimulated the development of a new research community. We identified 307 works funded by the MoBE program, as well as a comparison set of 698 authors who published in the same journals during the same period of time but were not part of the Sloan Foundation-funded collaboration. Our analysis of collaboration networks for both groups of authors suggests that the Sloan Foundation's program resulted in a more consolidated community of researchers, specifically in terms of number of components, diameter, density, and transitivity of the coauthor networks. In addition to highlighting the success of this particular program, our method could be applied to other fields to examine the impact of funding programs and other large-scale initiatives on the formation of research communities.

Legitimizing Values in Regulatory Science.

BACKGROUND: Over the last several decades, scientists and social groups have frequently raised concerns about politicization or political interference in regulatory science. Public actors (environmentalists and industry advocates, politically aligned public figures, scientists and political commentators, in the United States as well as in other countries) across major political-regulatory controversies have expressed concerns about the inappropriate politicization of science. Although we share concerns about the politicization of science, they are frequently framed in terms of an ideal of value-free science, according to which political and economic values have no legitimate role to play in science. For several decades, work in philosophy of science has identified serious conceptual and practical problems with the value-free ideal. OBJECTIVES: Our objectives are to discuss the literature regarding the conceptual and practical problems with the value-free ideal and offer a constructive alternative to the value-free ideal. DISCUSSION: We first discuss the prevalence of the value-free ideal in regulatory science, then argue that this ideal is self-undermining and has been exploited to delay protective regulation. To offer a constructive alternative, we analyze the relationship between the goals of regulatory science and the standards of good scientific activity. This analysis raises questions about the relationship between methodological and practical standards for good science, tensions among various important social goods, and tensions among various social interests. We argue that the aims of regulatory science help to legitimize value-laden choices regarding research methods and study designs. Finally, we discuss how public deliberation, adaptive management, and community-based participatory research can be used to improve the legitimacy of scientists as representatives of the general public on issues of environmental knowledge. CONCLUSIONS: Reflecting on the aims of regulatory science-such as protecting human health and the environment, informing democratic deliberation, and promoting the capacities of environmental justice and Indigenous communities-can clarify when values have legitimate roles in regulatory science. https://doi.org/10.1289/EHP3317.

Impact of obesity on outcomes for patients with head and neck cancer.

OBJECTIVES: The prognostic role of obesity in head and neck squamous cell carcinoma (HNSCC) is not well defined. This study aims to determine its effect on disease-specific outcomes such as recurrence-free survival (RFS), locoregional recurrence-free survival (LRRFS), and distant metastasis-free survival (DMFS) in addition to overall survival (OS). METHODS: For patients with newly diagnosed HNSCC undergoing radiation therapy (RT) at a single institution, body mass index (BMI) at diagnosis was categorized as normal (18.5 to 24.9 kg/m2), overweight (25 to 29.9 kg/m2) and obese (≥30 kg/m2). Outcomes were compared by BMI group using Cox regression. RESULTS: 341 patients of median age 59 (range, 20-93) who underwent curative RT from 2010 to 2017 were included. 58% had oropharynx cancer, 17% larynx and 15% oral cavity. 72% had stage IVA/B disease and 28% stage I-III. At diagnosis, 33% had normal BMI, 40% overweight, and 28% obese. 59% had definitive RT and 41% had postoperative RT. Alcoholic/smoking status, advanced tumor stage, hypopharynx/larynx tumors, and feeding tube placement were more common in patients with lower BMI (P < .05 for each). Median follow-up was 30 months (range, 3-91). Higher BMI was associated with improved OS (P < .05) and obesity was associated with longer RFS (P < .05) and DMFS (P < .05), but not LRRFS (P = .07) after adjusting for confounding variables. CONCLUSION: Being overweight/obese at the time of HNSCC diagnosis is an independent prognostic factor conferring better survival, while obesity is independently associated with longer time to recurrence, primarily by improving distant control.

Unexpected infection outcomes of China-origin H7N9 low pathogenicity avian influenza virus in turkeys.

The China-origin H7N9 low pathogenicity avian influenza virus (LPAIV) emerged as a zoonotic threat in 2013 where it continues to circulate in live poultry markets. Absence of overt clinical signs in poultry is a typical LPAIV infection outcome, and has contributed to its insidious maintenance in China. This study is the first description of H7N9 LPAIV (A/Anhui/1/13) infection in turkeys, with efficient transmission to two additional rounds of introduced contact turkeys which all became infected during cohousing. Surprisingly, mortality was observed in six of eight (75%) second-round contact turkeys which is unusual for LPAIV infection, with unexpected systemic dissemination to many organs beyond the respiratory and enteric tracts, but interestingly no accompanying mutation to highly pathogenic AIV. The intravenous pathogenicity index score for a turkey-derived isolate (0.39) affirmed the LPAIV phenotype. However, the amino acid change L235Q in the haemagglutinin gene occurred in directly-infected turkeys and transmitted to the contacts, including those that died and the two which resolved infection to survive to the end of the study. This polymorphism was indicative of a reversion from mammalian to avian adaptation for the H7N9 virus. This study underlined a new risk to poultry in the event of H7N9 spread beyond China.