Spatiotemporal changes in influenza A virus prevalence among wild waterfowl inhabiting the continental United States throughout the annual cycle.

Avian influenza viruses can pose serious risks to agricultural production, human health, and wildlife. An understanding of viruses in wild reservoir species across time and space is important to informing surveillance programs, risk models, and potential population impacts for vulnerable species. Although it is recognized that influenza A virus prevalence peaks in reservoir waterfowl in late summer through autumn, temporal and spatial variation across species has not been fully characterized. We combined two large influenza databases for North America and applied spatiotemporal models to explore patterns in prevalence throughout the annual cycle and across the continental United States for 30 waterfowl species. Peaks in prevalence in late summer through autumn were pronounced for dabbling ducks in the genera Anas and Spatula, but not Mareca. Spatially, areas of high prevalence appeared to be related to regional duck density, with highest predicted prevalence found across the upper Midwest during early fall, though further study is needed. We documented elevated prevalence in late winter and early spring, particularly in the Mississippi Alluvial Valley. Our results suggest that spatiotemporal variation in prevalence outside autumn staging areas may also represent a dynamic parameter to be considered in IAV ecology and associated risks.

Nonstructural Protein NSs Activates Inflammasome and Pyroptosis through Interaction with NLRP3 in Human Microglial Cells Infected with Severe Fever with Thrombocytopenia Syndrome Bandavirus.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne febrile disease caused by SFTS virus (SFTSV), or Dabie bandavirus, in the Phenuiviridae family. Clinically neurological disorders in SFTS have been commonly reported, but their neuropathogenesis has rarely been studied. Microglia are a type of neuroglia accounting for 10 to 12% of all cells in the brain. As resident immune cells, microglial cells are the first line of immune defense present in the central nervous system (CNS). Here, we report that SFTSV was able to infect microglial cells and stimulate interleukin 1ß (IL-1ß) secretion in the brains of infected neonatal BALB/c mice. We characterized the cell death induced in infected human microglial HMC3 cells, also susceptible to SFTSV, and found that the NOD-like receptor protein 3 (NLRP3) inflammasome was activated, leading to secretion of IL-1ß and pyroptosis. Knockdown of NLRP3 or inhibition of the NLRP3 inflammasome activation suppressed the viral replication, suggesting that the activation of the NLRP3 inflammasome may support SFTSV replication in microglial cells. Viral nonstructural protein NSs, a known modulator of immune responses, interacted and colocalized with NLRP3 for the inflammasome activation. It appeared that the N-terminal fragment, amino acids 1 to 66, of NSs was critical to promote the assembly of the inflammasome complex by interacting with NLRP3 for its activation in microglial cells. Our findings provide evidence that SFTSV may cause neurological disorders through infecting microglia and activating the inflammasome through its nonstructural protein NSs for neural cell death and inflammation. This study may have revealed a novel mechanism of SFTSV NSs in dysregulating host response. IMPORTANCE Encephalitis or encephalopathy during severe fever with thrombocytopenia syndrome (SFTS) is considered a critical risk factor leading to high mortality, but there have been no studies to date on the pathogenesis of encephalitis or encephalopathy caused by SFTS virus. Here, we report that SFTSV infection can active the NLRP3 inflammasome and induce IL-1ß secretion in the brains of infected newborn mice. In infected human HMC3 microglia, SFTSV activated the NLRP3 inflammasome via the viral nonstructural protein NSs through interaction with its N-terminal fragment. Notably, our findings suggest that the activation of the NLRP3 inflammasome may promote SFTSV replication in infected microglial cells. This study may reveal a novel mechanism by SFTSV to dysregulate host responses through its nonstructural protein, which could help us understand viral neuropathogenesis in SFTS patients.

Intrinsic features of Zika Virus non-structural proteins NS2A and NS4A in the regulation of viral replication.

Zika virus (ZIKV) is a mosquito-borne flavivirus and can cause neurodevelopmental disorders in fetus. As a neurotropic virus, ZIKV persistently infects neural tissues during pregnancy but the viral pathogenesis remains largely unknown. ZIKV has a positive-sense and single-stranded RNA genome, which encodes 7 non-structural (NS) proteins, participating in viral replication and dysregulation of host immunity. Like those in many other viruses, NS proteins are considered to be products evolutionarily beneficiary to viruses and some are virulence factors. However, we found that some NS proteins encoded by ZIKV genome appeared to function against the viral replication. In this report we showed that exogenously expressed ZIKV NS2A and NS4A inhibited ZIKV infection by inhibiting viral RNA replication in microglial cells and astrocytes. To understand how viral NS proteins suppressed viral replication, we analyzed the transcriptome of the microglial cells and astrocytes and found that expression of NS4A induced the upregulation of ISGs, including MX1/2, OAS1/2/3, IFITM1, IFIT1, IFI6, IFI27, ISG15 or BST2 through activating the ISGF3 signaling pathway. Upregulation of these ISGs seemed to be related to the inhibition of ZIKV replication, since the anti-ZIKV function of NS4A was partially attenuated when the cells were treated with Abrocitinib, an inhibitor of the ISGF3 signaling pathway, or were knocked down with STAT2. Aborting the protein expression of NS4A, but not its nucleic acid, eliminated the antiviral activity of NS4A effectively. Dynamic expression of viral NS proteins was examined in ZIKV-infected microglial cells and astrocytes, which showed comparatively NS4A occurred later than other NS proteins during the infection. We hypothesize that NS4A may possess intrinsic features to serve as a unique type of pathogen associated molecular pattern (PAMP), detectable by the cells to induce an innate immune response, or function with other mechanisms, to restrict the viral replication to a certain level as a negative feedback, which may help ZIKV maintain its persistent infection in fetal neural tissues.

Concomitant pyroptotic and apoptotic cell death triggered in macrophages infected by Zika virus.

Zika virus (ZIKV) is a positive-sense RNA flavivirus and can cause serious neurological disorders including microcephaly in infected fetuses. As a mosquito-borne arbovirus, it enters the bloodstream and replicates in various organs. During pregnancy, it can be transmitted from the blood of the viremic mother to the fetus by crossing the placental barrier. Monocytes and macrophages are considered the earliest blood cell types to be infected by ZIKV. As a first line defense, these cells are crucial components in innate immunity and host responses and may impact viral pathogenesis in humans. Previous studies have shown that ZIKV infection can activate inflammasomes and induce proinflammatory cytokines in monocytes. In this report, we showed that ZIKV could infect and induce cell death in human and murine macrophages. In addition to the presence of cleaved caspase-3, indicating that apoptosis was involved, we identified the cleaved caspase-1 and gasdermin D (GSDMD) as well as increased secretion of IL-1ß and IL-18. This suggests that the inflammasome was activated and that may lead to pyroptosis in infected macrophages. The pyroptosis was NLRP3-dependent and could be suppressed in the macrophages treated with shRNA to target and knockdown caspase-1. It was also be inhibited by an inhibitor for caspase-1, indicating that the pyroptosis was triggered via a canonical approach. Our findings in this study demonstrate a concomitant occurrence of apoptosis and pyroptosis in ZIKV-infected macrophages, with two mechanisms involved in the cell death, which may have potentially significant impacts on viral pathogenesis in humans.

Predicting the time to detect moderately virulent African swine fever virus in finisher swine herds using a stochastic disease transmission model.

BACKGROUND: African swine fever (ASF) is a highly contagious and devastating pig disease that has caused extensive global economic losses. Understanding ASF virus (ASFV) transmission dynamics within a herd is necessary in order to prepare for and respond to an outbreak in the United States. Although the transmission parameters for the highly virulent ASF strains have been estimated in several articles, there are relatively few studies focused on moderately virulent strains. Using an approximate Bayesian computation algorithm in conjunction with Monte Carlo simulation, we have estimated the adequate contact rate for moderately virulent ASFV strains and determined the statistical distributions for the durations of mild and severe clinical signs using individual, pig-level data. A discrete individual based disease transmission model was then used to estimate the time to detect ASF infection based on increased mild clinical signs, severe clinical signs, or daily mortality. RESULTS: Our results indicate that it may take two weeks or longer to detect ASF in a finisher swine herd via mild clinical signs or increased mortality beyond levels expected in routine production. A key factor contributing to the extended time to detect ASF in a herd is the fairly long latently infected period for an individual pig (mean 4.5, 95% P.I., 2.4 - 7.2 days). CONCLUSION: These transmission model parameter estimates and estimated time to detection via clinical signs provide valuable information that can be used not only to support emergency preparedness but also to inform other simulation models of evaluating regional disease spread.

Activation of the NLRP3 inflammasome and elevation of interleukin-1ß secretion in infection by sever fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging phlebovirus that causes a hemorrhagic fever known as the severe fever with thrombocytopenia syndrome (SFTS). Inflammasomes are a molecular platform that are assembled to process pro-caspase 1 and subsequently promote secretion of interleukin (IL)-1ß/IL-18 for proinflammatory responses induced upon infection. We hypothesize that inflammasome activation and pyroptosis induced in SFTS results in elevated levels of IL-1ß/IL-18 responsible for high fever and hemorrhage in the host, characteristic of SFTS. Here we report that IL-1ß secretion was elevated in SFTS patients and infected mice and IL-1ß levels appeared to be reversibly associated to disease severity and viral load in patients' blood. Increased caspase-1 activation, IL-1ß/IL-18 secretion, cell death, and processing of gasdermin D were detected, indicating that pyroptosis was induced in SFTSV-infected human peripheral blood monocytes (PBMCs). To characterize the mechanism of pyroptosis induction, we knocked down several NOD-like receptors (NLRs) with respective shRNAs in PBMCs and showed that the NLR family pyrin domain containing 3 (NLRP3) inflammasome was critical for processing pro-caspase-1 and pro-IL-1ß. Our data with specific inhibitors for NLRP3 and caspase-1 further showed that activation of the NLRP3 inflammasome was key to caspase-1 activation and IL-1ß secretion which may be inhibitory to viral replication in PBMCs infected with SFTSV. The findings in this study suggest that the activation of the NLPR3 inflammasome and pyroptosis, leading to IL-1ß/IL-18 secretion during the SFTSV infection, could play important roles in viral pathogenesis and host protection. Pyroptosis as part of innate immunity might be essential in proinflammatory responses and pathogenicty in humans infected with this novel phlebovirus.

African swine fever detection and transmission estimates using homogeneous versus heterogeneous model formulation in stochastic simulations within pig premises.

Background: African swine fever (ASF) is one of the most important foreign animal diseases to the U.S. swine industry. Stakeholders in the swine production sector are on high alert as they witness the devastation of ongoing outbreaks in some of its most important trade partner countries. Efforts to improve preparedness for ASF outbreak management are proceeding in earnest and mathematical modeling is an integral part of these efforts. Aim: This study aimed to assess the impact on within-herd transmission dynamics of ASF when the models used to simulate transmission assume there is homogeneous mixing of animals within a barn. Methods: Barn-level heterogeneity was explicitly captured using a stochastic, individual pig-based, heterogeneous transmission model that considers three types of infection transmission, (1) within-pen via nose-to-nose contact; (2) between-pen via nose-to-nose contact with pigs in adjacent pens; and (3) both between- and within-pen via distance-independent mechanisms (e.g., via fomites). Predictions were compared between the heterogeneous and the homogeneous Gillespie models. Results: Results showed that the predicted mean number of infectious pigs at specific time points differed greatly between the homogeneous and heterogeneous models for scenarios with low levels of between-pen contacts via distance-independent pathways and the differences between the two model predictions were more pronounced for the slow contact rate scenario. The heterogeneous transmission model results also showed that it may take significantly longer to detect ASF, particularly in large barns when transmission predominantly occurs via nose-to-nose contact between pigs in adjacent pens. Conclusion: The findings emphasize the need for completing preliminary explorations when working with homogeneous mixing models to ascertain their suitability to predict disease outcomes.

Simulated Flock-Level Shedding Characteristics of Turkeys in Ten Thousand Bird Houses Infected with H7 Low Pathogenicity Avian Influenza Virus Strains.

Understanding the amount of virus shed at the flock level by birds infected with low pathogenicity avian influenza virus (LPAIV) over time can help inform the type and timing of activities performed in response to a confirmed LPAIV-positive premises. To this end, we developed a mathematical model which allows us to estimate viral shedding by 10,000 turkey toms raised in commercial turkey production in the United States, and infected by H7 LPAIV strains. We simulated the amount of virus shed orally and from the cloaca over time, as well as the amount of virus in manure. In addition, we simulated the threshold cycle value (Ct) of pooled oropharyngeal swabs from birds in the infected flock tested by real-time reverse transcription polymerase chain reaction. The simulation model predicted that little to no shedding would occur once the highest threshold of seroconversion was reached. Substantial amounts of virus in manure (median 1.5×108 and 5.8×109; 50% egg infectious dose) were predicted at the peak. Lastly, the model results suggested that higher Ct values, indicating less viral shedding, are more likely to be observed later in the infection process as the flock approaches recovery.

A Retrospective Study of Early vs. Late Virus Detection and Depopulation on Egg Laying Chicken Farms Infected with Highly Pathogenic Avian Influenza Virus During the 2015 H5N2 Outbreak in the United States.

The 2015 highly pathogenic avian influenza (HPAI) H5N2 outbreak affected more than 200 Midwestern U.S. poultry premises. Although each affected poultry operation incurred substantial losses, some operations of the same production type and of similar scale had differences between one another in their ability to recognize evidence of the disease before formal diagnoses and in their ability to make proactive, farm-level disease containment decisions. In this case comparison study, we examine the effect of HPAI infection on two large egg production facilities and the epidemiologic and financial implications resulting from differences in detection and decision-making processes. Each egg laying facility had more than 1 million caged birds distributed among 18 barns on one premises (Farm A) and 17 barns on the other premises (Farm B). We examine how farm workers' awareness of disease signs, as well as how management's immediate or delayed decisions to engage in depopulation procedures, affected flock mortality, levels of environmental contamination, time intervals for re population, and farm profits on each farm. By predictive mathematical modeling, we estimated the time of virus introduction to examine how quickly infection was identified on the farms and then estimated associated contact rates within barns. We found that the farm that implemented depopulation immediately after detection of abnormal mortality (Farm A) was able to begin repopulation of barns 37 days sooner than the farm that began depopulation well after the detection of abnormally elevated mortality (Farm B). From average industry economic data, we determined that the loss associated with delayed detection using lost profit per day in relation to down time was an additional $3.3 million for Farm B when compared with Farm A.

Estudio retrospectivo de detección viral temprana y tardía y despoblación en granjas de gallinas de postura infectadas con el virus de la influenza aviar altamente patógeno durante el brote de H5N2 del año 2015 en los Estados Unidos. El brote de influenza aviar altamente patógena (HPAI) H5N2 del año 2015 afectó a más de 200 granjas avícolas del medio oeste de los Estados Unidos. Aunque cada operación avícola afectada incurrió en pérdidas sustanciales, algunas operaciones del mismo tipo de producción y de escala similar tuvieron diferencias entre sí en su capacidad para reconocer evidencias de la enfermedad antes de los diagnósticos formales y en su capacidad para realizar decisiones proactivas para la contención de la enfermedad a nivel de granja. En este estudio de caso, se examinó el efecto de la infección por influenza aviar altamente patógena en dos instalaciones grandes de producción de huevo y las implicaciones epidemiológicas y financieras que fueron resultado de los diferentes procesos de detección y toma de decisiones. Cada instalación de postura de huevo tenía más de un millón de aves enjauladas distribuidas en 18 casetas en una granja (Granja A) y 17 casetas en las otras instalaciones (Granja B). Se examinó cómo el conocimiento de los trabajadores agrícolas sobre los signos de la enfermedad, así como cómo las decisiones de manejo inmediatas o tardías para establecer procedimientos de despoblación, afectaron la mortalidad de las parvadas, los niveles de contaminación ambiental, los intervalos de tiempo para la repoblación y las ganancias en cada granja. Mediante un modelo matemático predictivo, se estimó el tiempo de introducción del virus para examinar la rapidez con la que se identificó la infección en las granjas y luego se estimaron las tasas de contacto asociadas dentro de las casetas. Se encontró que la granja que implementó la despoblación inmediatamente después de la detección de mortalidad anormal (Granja A) pudo comenzar la repoblación de las casetas 37 días antes que la granja que comenzó la despoblación mucho después de la detección de mortalidad anormalmente elevada (Granja B). A partir de los datos económicos promedio de la industria, se determinó que la pérdida asociada con la detección tardía utilizando las pérdidas de ganancias por día en relación con el tiempo de inactividad fue de $3.3 millones adicionales para la Granja B en comparación con la Granja A.

RIPK3-Dependent Necroptosis Is Induced and Restricts Viral Replication in Human Astrocytes Infected With Zika Virus.

Apoptosis, pyroptosis and necroptosis are regulated processes of cell death which can be crucial for viral disease outcomes in hosts because of their effects on viral pathogenicity and host resistance. Zika virus (ZIKV) is a mosquito-borne flavivirus, which infects humans and can cause neurological disorders. Neural developmental disorders and microcephaly could occur in infected fetuses. Several types of nervous cells have been reported to be susceptible to ZIKV infection. Human astrocytes play important roles in the nutritional support and defense of neurons. In this study, we show that human astrocytes are susceptible to ZIKV infection and undergo progressive cell death after infection. In infected astrocytes we detected no cleavage or activation of pro-caspase-3 and pro-caspase-1. Apoptotic substrates and increased secretion of interleukin (IL)-1ß or IL-18 were not detected, either. These ruled out the occurrence of apoptosis or pyroptosis in ZIKV-infected astrocytes. We detected, however, an increase of phosphorylated receptor-interacting serine/threonine-protein kinase (RIPK)1, RIPK3, and mixed lineage kinase domain-like (MLKL) protein, indicating that programmed necrosis, or necroptosis, was induced in infected astrocytes. The phosphorylation and cell death were inhibited in cells pre-treated with GSK'872, an inhibitor of RIPK3, while inhibition of RIPK1 with an inhibitor, Necrostatin-1, had no effect, suggesting that ZIKV-induced necroptosis was RIPK1-independent in astrocytes. Consistent with this finding, the inhibition of RIPK1 had no effect on the phosphorylation of MLKL. We showed evidence that MLKL phosphorylation was RIPK3-dependent and ZBP-1, which could stimulate RIPK3, was upregulated in ZIKV-infected astrocytes. Finally, we demonstrated that in GSK'872-pre-treated astrocytes, viral replication increased significantly, which indicates that necroptosis may be protective against viral replication in astrocytes. Our finding that astrocytes uniquely underwent necroptosis in response to ZIKV infection provides insight and helps us better understand the viral pathogenesis in the ZIKV-infected central nervous system.

Intrinsic apoptosis and cytokine induction regulated in human tonsillar epithelial cells infected with enterovirus A71.

Enterovirus A71 (EV-A71) has emerged as a clinically important neurotropic virus following poliovirus eradication. Recent studies have shown that human tonsillar epithelial cell lines (UT-SCC-60A and UT-SCC-60B) were susceptible to EV-A71, suggesting that human tonsillar crypt epithelium could be important in EV-A71 pathogenesis. However, the mechanism about how EV-A71 infects the upper oro-digestive tract remains largely unclear. In this study, we demonstrated that the human tonsillar epithelial cells infected with EV-A71 underwent apoptotic, in which cytochrome c was released from the mitochondria to the cytosol and caspase-9 was activated, while caspase-2 and -8 were not cleaved or activated during the infection. A selective inhibitor of caspase-9, Z-LEHD-FMK, inhibited the cleavage of the executioner caspase-3 and -7, indicating that only mitochondria-mediated intrinsic apoptotic pathway was activated in EV-A71-infected tonsillar epithelial cells. No evidence of pyroptosis or necroptosis was involved in the cell death. EV-A71 infection induced interferon, pro-inflammatory cytokines and chemokines, including IFN-ß, IL-6, CCL5, and TNF-α in tonsillar epithelial cells, which may play a critical role in EV-A71-caused herpangina. Our data indicated that the induction of the cytokines was partially regulated by the mitogen-activated protein kinases (MAPKs) signaling pathway. The findings unveiled the host response to EV-A71 and its regulation mechanism, and will further our understanding the significance about the tonsillar crypt epithelium as the initial and primary portal in viral pathogenesis for EV-A71 infection.

Estimating epidemiological parameters using diagnostic testing data from low pathogenicity avian influenza infected turkey houses.

Limiting spread of low pathogenicity avian influenza (LPAI) during an outbreak is critical to reduce the negative impact on poultry producers and local economies. Mathematical models of disease transmission can support outbreak control efforts by estimating relevant epidemiological parameters. In this article, diagnostic testing data from each house on a premises infected during a LPAI H5N2 outbreak in the state of Minnesota in the United States in 2018 was used to estimate the time of virus introduction and adequate contact rate, which determines the rate of disease spread. A well-defined most likely time of virus introduction, and upper and lower 95% credibility intervals were estimated for each house. The length of the 95% credibility intervals ranged from 11 to 22 with a mean of 17 days. In some houses the contact rate estimates were also well-defined; however, the estimated upper 95% credibility interval bound for the contact rate was occasionally dependent on the upper bound of the prior distribution. The estimated modes ranged from 0.5 to 6.0 with a mean of 2.8 contacts per day. These estimates can be improved with early detection, increased testing of monitored premises, and combining the results of multiple barns that possess similar production systems.

Enterovirus A71 2B Inhibits Interferon-Activated JAK/STAT Signaling by Inducing Caspase-3-Dependent Karyopherin-α1 Degradation.

Enterovirus A71 (EV-A71) is a major pathogen that causes the hand, foot, and mouth disease, which could be fatal with neurological complications in children. The underlying mechanism for the severe pathogenicity remains obscure, but impaired or aberrant innate immunity is considered to play a key role in viral pathogenesis. We reported previously that EV-A71 suppressed type I interferon (IFN) responses by inducing degradation of karyopherin-α1 (KPNA1), a component of the p-STAT1/2 complex. In this report, we showed that 2B, a non-structural protein of EV-A71, was critical to the suppression of the IFN-α-induced type I response in infected cells. Among viral proteins, 2B was the only one that was involved in the degradation of KPNA1, which impeded the formation of the p-STAT1/2/KPNA1 complex and blocked the translocation of p-STAT1/2 into the nucleus upon IFN-α stimulation. Degradation of KPNA1 induced by 2B can be inhibited in the cells pre-treated with Z-DEVD-FMK, a caspase-3 inhibitor, or siRNA targeting caspase-3, indicating that 2B-induced degradation of KPNA1 was caspase-3 dependent. The mechanism by which 2B functioned in the dysregulation of the IFN signaling was analyzed and a putative hydrophilic domain (H1) in the N-terminus of 2B was characterized to be critical for the release of cytochrome c into the cytosol for the activation of pro-caspase-3. We generated an EV-A71 infectious clone (rD1), which was deficient of the H1 domain. In rD1-infected cells, degradation of KPNA1 was relieved and the infected cells were more sensitive to IFN-α, leading to decreased viral replication, in comparison to the cells infected with the virus carrying a full length 2B. Our findings demonstrate that EV-A71 2B protein plays an important role in dysregulating JAK-STAT signaling through its involvement in promoting caspase-3 dependent degradation of KPNA1, which represents a novel strategy employed by EV-A71 to evade host antiviral innate immunity.

Conducting Influenza Virus Pathogenesis Studies in Avian Species.

Avian infection studies with influenza A are an important means of assessing host susceptibility, viral pathogenesis, host responses to infection, mechanisms of transmission, viral pathotype, and viral evolution. Complex systems and natural settings may also be explored with carefully designed infection studies. In this chapter, we explore the elements of infection studies, general guidelines for choosing a virus to use, host selection, and many aspects of study design.

Reassortment and adaptive mutations of an emerging avian influenza virus H7N4 subtype in China.

Human infections with avian influenza viruses including H5, H7 and H9 hemagglutinin subtypes occur at a low rate. Among human infections with H7 viruses, regional outbreaks with H7N2, H7N3, H7N7 and H7N9 have been documented. Early in 2018, a human infection with a novel H7N4 avian influenza virus was reported in Jiangsu, China. This study is aimed at understanding the probable origin and molecular features of this emerging H7N4 virus. Genomic segments encoding hemagglutinin (HA) and neuraminidase (NA) of H7Nx and HxN4 viruses were compared with this H7N4 strain by alignment and phylogenetic tree analysis. Phylogenetic analysis indicated that the human H7N4 virus probably originated from multiple reassortments of avian H7N7 and H8N4 viruses for its HA and NA, respectively, and likely a regional uncharacterized virus for its internal segments. Our data excluded that circulating avian H9N2 viruses were the origin of the H7N4 internal segments, unlike the human H5N1 and H7N9 viruses that both had H9N2 backbones. This index case provided a unique opportunity to examine viral mutations by directly comparing the human isolate with its closest viral relatives isolated from avian species from the patient's farm, which may suggest critical mutations required for viral adaptation in humans. Whole-genome scanning was performed and the sequences of the human and related avian H7N4 isolates were compared. Mutations in PB2 (E627K), PB2 (K683T), PB1-F2 (N47S), HA (N283D), HA(K321E), NA(A137V), NA(K296R) and M2 (C19Y) were identified in the human isolate while no mutations were found in PB1, NP, NS1, and NS2 of the human H7N4 compared to the avian H7N4 viruses. Our data in this report provide further evidence for the genesis of this novel H7N4 virus with a multi-reassortment model and show molecular changes that might be responsible for the transmission of this virus from chickens or ducks to and subsequent replication in humans.

Isolation and Characterization of Newcastle Disease Virus from Live Bird Markets in Tanzania.

Chickens in live bird markets (LBMs) from six different regions of Tanzania were surveyed for Newcastle disease (ND) virus (NDV) and avian influenza virus in 2012. ELISA-based serology, virus isolation, and characterization, including pathotyping was conducted. Virulent NDV was isolated from almost 10% of the tested samples, with two distinct genotypes being detected. One genotype was similar to recent viruses circulating in Kenya and Uganda, which share a northern border with Tanzania. Several viruses of this genotype were also isolated from Tanzania in 1995, the last time surveillance for NDV was conducted in the country. The second genotype of virus from Tanzania was closely related to viruses from Mozambique, a southern neighbor, and more distantly to viruses from South Africa, Botswana, and several European countries. Partial fusion gene sequence from the isolated viruses showed identical fusion cleavage sites that were compatible with virulent viruses. Selected viruses were tested by the intracerebral pathogenicity index, and all viruses tested had scores of >1.78, indicating highly virulent viruses. Serology showed only a third of the chickens had detectable antibody to NDV, suggesting that vaccination is not being commonly used in the country, despite the availability of vaccines in agricultural-related markets. All samples were taken from clinically healthy birds, and it is believed that the birds were sold or slaughtered before showing ND clinical signs. LBMs remain a biosecurity risk for farmers through the return of live infected birds to the farm or village or the movement of virus on fomites, such as uncleaned wooden cages.

Aislamiento y caracterización de virus de la enfermedad de Newcastle de mercados de aves vivas en Tanzania. Se llevó a cabo un muestreo de pollos en mercados de aves vivas (LBM) de seis regiones diferentes de Tanzania para detectar al virus de la enfermedad de Newcastle (NDV) y el virus de la influenza aviar en el año 2012. Se llevaron a cabo la serología basada en la prueba de ELISA, el aislamiento viral y la caracterización, incluyendo la determinación del patotipo. Formas virulentas del virus de Newcastle se aislaron de casi el 10% de las muestras analizadas y se detectaron dos genotipos distintos. Un genotipo era similar a los virus recientes que circulan en Kenia y Uganda, países que comparten una frontera al norte de Tanzania. Varios virus de este genotipo también se aislaron de Tanzania en el año 1995, la última vez que se realizó la vigilancia del virus de Newcastle en el país. El segundo genotipo de virus de Tanzania estaba estrechamente relacionado con virus de Mozambique, país vecino al sur, y más distantemente con virus de Sudáfrica, Botswana y de varios países europeos. Una secuencia parcial del gene de fusión de los virus aislados mostró sitios de disociación en la proteína de fusión idénticos que eran compatibles con los virus virulentos. Los virus seleccionados fueron analizados mediante el índice de patogenicidad intracerebral y todos los virus analizados tuvieron puntajes mayores de 1.78, lo que indica que son virus altamente virulentos. La serología mostró que solo un tercio de los pollos tenían anticuerpos detectables contra el virus de Newcastle, lo que sugiere que la vacunación no se usa comúnmente en el país, a pesar de la disponibilidad de vacunas en los mercados agrícolas. Todas las muestras fueron recolectadas de aves clínicamente sanas y se cree que las aves fueron vendidas o sacrificadas antes de mostrar signos clínicos de la enfermedad de Newcastle. Los mercados de aves vivas siguen siendo un riesgo de bioseguridad para los agricultores mediante el regreso de aves vivas infectadas a la granja o a los pueblos o por el movimiento de virus en fómites, como las jaulas de madera sin limpiar.

Susceptibility of spotted doves (Streptopelia chinensis) to experimental infection with the severe fever with thrombocytopenia syndrome phlebovirus.

BACKGROUND: Severe fever with thrombocytopenia syndrome virus (SFTSV), an emerging human pathogen naturally transmitted by ticks, has spread widely since it was first detected in 2010. Although SFTSV-specific antibodies have been detected in wild birds, these natural reservoir and amplifying hosts for the virus have not been well studied. METHODOLOGY/PRINCIPLE FINDINGS: Here we report an experimental infection of spotted doves (Streptopelia chinensis) with two strains of SFTSV, JS2010-14, a Chinese lineage strain, and JS2014-16, a Japanese lineage strain, which represent the main viral genotypes currently circulating in East Asia. In these studies, we have determined that spotted doves are susceptible to SFTSV and the severity of the viremia is dose-dependent. When challenged with 107 and 105 PFU, all doves developed viremia which peaked 3-5 days post infection (dpi). Only a subset (25-62.5%) of the birds developed viremia when challenged at 103 PFU. Virulence of SFTSV in spotted doves was strain dependent. Infection with 107 PFU of strain JS2014-16 resulted in 12.5% mortality over 6.8 days and mean peak viremia titers of 106.9 PFU/mL in experimentally inoculated birds. All doves inoculated with 107 PFU of the JS2010-14 strain survived infection with relatively lower mean viremia titers (105.6 PFU/mL at peak) over 6.1 days. CONCLUSIONS/SIGNIFICANCE: Our results suggest that spotted doves, one of the most abundant bird species in China, could be a competent amplifying host for SFTSV and play an important role in its ecology. Between the two SFTSV strains, the strain of the Japanese lineage caused mortality, higher viremia titers in infected birds over a longer time period than did the Chinese strain. Our observations shed light on the ecology of SFTSV, which could benefit the implementation of surveillance and control programs.

Analysis of geographic location and pathways for influenza A virus infection of commercial upland game bird and conventional poultry farms in the United States of America.

BACKGROUND: Avian influenza (AI) is an infectious viral disease that affects several species and has zoonotic potential. Due to its associated health and economic repercussions, minimizing AI outbreaks is important. However, most control measures are generic and mostly target pathways important for the conventional poultry farms producing chickens, turkeys, and eggs and may not target other pathways that may be specific to the upland game bird sector. The goal of this study is to provide evidence to support the development of novel strategies for sector-specific AI control by comparing and contrasting practices and potential pathways for spread in upland game bird farms with those for conventional poultry farms in the United States. Farm practices and processes, seasonality of activities, geographic location and inter-farm distance were analyzed across the sectors. All the identified differences were framed and discussed in the context of their associated pathways for virus introduction into the farm and subsequent between-farm spread. RESULTS: Differences stemming from production systems and seasonality, inter-farm distance and farm densities were evident and these could influence both fomite-mediated and local-area spread risks. Upland game bird farms operate under a single, independent owner rather than being contracted with or owned by a company with other farms as is the case with conventional poultry. The seasonal marketing of upland game birds, largely driven by hunting seasons, implies that movements are seasonal and customer-vendor dynamics vary between industry groups. Farm location analysis revealed that, on average, an upland game bird premises was 15.42 km away from the nearest neighboring premises with birds compared to 3.74 km for turkey premises. Compared to turkey premises, the average poultry farm density in a radius of 10 km of an upland game bird premises was less than a half, and turkey premises were 3.8 times (43.5% compared with 11.5%) more likely to fall within a control area during the 2015 Minnesota outbreak. CONCLUSIONS: We conclude that the existing differences in the seasonality of production, isolated geographic location and epidemiological seclusion of farms influence AI spread dynamics and therefore disease control measures should be informed by these and other factors to achieve success.

Mortality-Based Triggers and Premovement Testing Protocols for Detection of Highly Pathogenic Avian Influenza Virus Infection in Commercial Upland Game Birds.

Outbreaks involving avian influenza viruses are often devastating to the poultry industry economically and otherwise. Disease surveillance is critically important because it facilitates timely detection and generates confidence that infected birds are not moved during business continuity intended to mitigate associated economic losses. The possibility of using an abnormal increase in daily mortality to levels that exceed predetermined thresholds as a trigger to initiate further diagnostic investigations for highly pathogenic avian influenza (HPAI) virus infection in the flock is explored. The range of optimal mortality thresholds varies by bird species, trigger type, and mortality thresholds, and these should be considered when assessing sector-specific triggers. The study uses purposefully collected data and data from the literature to determine optimal mortality triggers for HPAI detection in commercial upland game bird flocks. Three trigger types were assessed for the ability to detect rapidly both HPAI (on the basis of disease-induced and normal mortality data) and false alarm rate (on the basis of normal mortality data); namely, 1) exceeding a set absolute threshold on one day, 2) exceeding a set absolute threshold on two consecutive days, or 3) exceeding a multiple of a seven-day moving average. The likelihood of disease detection using some of these triggers together with premovement real-time reverse transcription PCR (rRT-PCR) testing was examined. Results indicate that the performance of the two consecutive days trigger had the best metrics (i.e., rapid detection with few false alarms) in the trade-off analysis. The collected normal mortality data was zero on 66% of all days recorded, with an overall mean of 0.6 dead birds per day. In the surveillance scenario analyses, combining the default protocol that relied only on active surveillance (i.e., premovement testing of oropharyngeal swab samples from dead birds by rRT-PCR) together with either of the mortality-based triggers improved detection rates on all days postexposure before scheduled movement. For exposures occurring within 8 days of movement, the protocol that combined the default with single-day triggers had slightly more detections than that with two consecutive days triggers. However, all assessed protocol combinations were able to detect all infections that occurred more than 10 days before scheduled movement. These findings can inform risk-based decisions pertaining to continuity of business in the commercial upland game bird industry.

Activadores basados en la mortalidad y protocolos de pruebas de premovimiento para la detección de la infección del virus de influenza aviar altamente patógena en aves de caza de tierras altas comerciales Los brotes que involucran virus de influenza aviar a menudo son económicamente devastadores para la industria avícola. La vigilancia de enfermedades es de importancia crítica porque facilita la detección oportuna y genera confianza en que las aves infectadas no serán movilizadas para continuar con la operación de las industrias avícolas para mitigar las pérdidas económicas asociadas. Se explora la posibilidad de utilizar un aumento anormal en la mortalidad diaria a niveles que excedan umbrales predeterminados como un desencadenante para iniciar investigaciones de diagnóstico para la infección del virus de la influenza aviar altamente patógena en la parvada. El rango de umbrales de mortalidad óptimos varían según la especie de ave, el tipo de activador y los umbrales de mortalidad y estos deben considerarse al evaluar los activadores específicos del sector. El estudio utiliza datos recopilados de manera planeada y datos de la literatura para determinar los desencadenantes de mortalidad óptimos para la detección de la influenza aviar altamente patógena en las parvadas comerciales de aves de caza de tierras altas. Se evaluaron tres activadores de acuerdo a su capacidad de detectar rápidamente influenza aviar altamente patógena (en función de los datos de mortalidad normal e inducida por la enfermedad) y la tasa de falsa alarma (en función de los datos de mortalidad normal); como son, 1) que se exceda un umbral absoluto establecido en un día, 2) que se exceda un umbral absoluto establecido en dos días consecutivos, o 3) que excede un múltiplo de un promedio móvil de siete días. Se examinó la probabilidad de detección de la enfermedad utilizando algunos de estos desencadenantes junto con la prueba de transcripción reversa y PCR en tiempo real (rRT-PCR). Los resultados indicaron que el rendimiento del disparador de dos días consecutivos tuvo los mejores resultados (es decir, detección rápida con pocas falsas alarmas) en el análisis costo-beneficio. Los datos de mortalidad normal recopilados fueron cero en el 66% de todos los días registrados, con una media general de 0.6 aves muertas por día. En los análisis de escenarios de vigilancia, la combinación del protocolo predeterminado que se basó únicamente en la vigilancia activa (por ejemplo pruebas antes de movilizaciones con muestras de hisopos orofaríngeos por rRT-PCR de aves muertas) a la par con cualquiera de los desencadenantes basados en la mortalidad mejoraron las tasas de detección en todos los días posteriores a la exposición antes del movimiento programado. Para las exposiciones que ocurrieron dentro de los ocho días de movimiento, el protocolo que combinó el valor predeterminado con los activadores de un solo día tuvo un poco más de detecciones que el de los activadores de dos días consecutivos. Sin embargo, todas las combinaciones de protocolos evaluadas pudieron detectar todas las infecciones que ocurrieron por más de 10 días antes del movimiento programado. Estos hallazgos pueden proveer información para la toma de decisiones basadas en el riesgo relacionadas con la continuidad de operaciones en la industria comercial de aves de caza de tierras altas.

Evaluating the Effect of the Within-Flock Disease Transmission Rate on Premovement Active Surveillance in Low Pathogenicity Avian Influenza-Infected Flocks.

Premovement active surveillance for low pathogenicity avian influenza (LPAI) may be a useful risk management tool for producers during high-risk periods, such as during an LPAI outbreak, or in areas where there is a recognized high risk for LPAI spread. The effectiveness of three active-surveillance protocols in mitigating LPAI spread risk related to the movement of spent broiler breeders to processing was evaluated in this study. Each protocol differed in the amount of real-time reverse transcription polymerase chain reaction (RRT-PCR) and serology testing conducted. The protocols were evaluated with the use of disease transmission and active surveillance simulation models parametrized specifically for broiler breeders to estimate the probability of detecting a current or past infection and the mean proportion of infectious birds at the time of sampling in houses where the infection remains undetected at the time of movement after exposure to the virus. The two values were estimated considering flock infection for 1-28 days prior to the day of scheduled movement. A distribution for the adequate contact rate, a parameter that controls the rate of within-house spread in the disease transmission model, was estimated for this study by a novel forward simulation approach with the use of serology data from three LPAI-infected broiler breeder flocks in the United States. The estimated distribution suggests that the lower contact-rate estimates from previously published studies were not a good fit for the serology results observed in these U.S. flocks, though considerable uncertainty remains in the parameter estimate. The results for the probability of detection and mean proportion of infectious, undetected birds suggest that RRT-PCR testing is most beneficial during the early stages of infection postexposure, and serology testing is most beneficial during the later stages of infection, results that are expected to hold for flocks outside the United States as well. Thus, protocols that combine RRT-PCR and serology testing can offer a more balanced approach with good performance over the disease course in a flock.

Evaluación del efecto de la tasa de transmisión dentro de la parvada en la vigilancia activa previa al movimiento de parvadas infectadas por influenza aviar de baja patogenicidad. La vigilancia activa para la influenza aviar de baja patogenicidad (LPAI) previa al movimiento puede ser una herramienta útil en el manejo de riesgos para los productores durante períodos de alto riesgo, como durante un brote de influenza aviar de baja patogenicidad o en áreas donde se reconoce que existe un alto riesgo de propagación de esta enfermedad. En este estudio, se evaluó la efectividad de tres protocolos de vigilancia activa para mitigar el riesgo de propagación de la influenza aviar de baja patogenicidad relacionado con el movimiento de los reproductores pesados de desecho a la planta de procesamiento. Los protocolos diferían en la cantidad de muestras procesadas por la transcriptasa reversa y reacción en cadena de la polimerasa en tiempo real (rRT-PCR) y por las pruebas serológicas realizadas. Los protocolos se evaluaron utilizando modelos de simulación de vigilancia activa y transmisión de la enfermedad con parámetros específicamente para reproductores pesados, para estimar la probabilidad de detectar una infección actual o pasada y la proporción media de aves con infección activa al momento del muestreo en casetas donde la infección permanecía sin detectar al momento del movimiento después de la exposición al virus. Los dos valores se estimaron considerando la infección de la parvada de uno a 28 días antes de la fecha programada para el movimiento. Una distribución para la tasa de contacto adecuada, un parámetro que controla la tasa de propagación dentro de la caseta en el modelo de transmisión de la enfermedad, se estimó para este estudio mediante un novedoso enfoque de simulación directa utilizando datos serológicos de tres parvadas reproductores pesados infectados con influenza aviar de baja patogenicidad en los Estados Unidos. La distribución estimada sugiere que las estimaciones de la tasa de contacto más baja obtenida de los estudios publicados previamente no fueron una buena opción para los resultados serológicos observados en estas parvadas en los Estados Unidos, aunque sigue existiendo una gran incertidumbre en la estimación del parámetro. Los resultados de la probabilidad de detección y la proporción media de aves con infección no detectadas sugieren que la prueba rRT-PCR es más beneficiosa durante las primeras etapas de la infección después de la exposición, mientras que la serología es más beneficiosa durante las últimas etapas de la infección, resultados que se espera apliquen también para parvadas fuera de los Estados Unidos. Por lo tanto, los protocolos que combinan rRT-PCR y las pruebas de serología pueden ofrecer un enfoque más equilibrado con un buen rendimiento durante el curso de la enfermedad en una parvada.