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Following confirmation of the first case of the ongoing U.S. HPAI H5N1 epizootic in commercial poultry on February 8, 2022, the virus has continued to devastate the U.S. poultry sector and the pathogen has since managed to cross over to livestock and a few human cases have also been reported. Efficient outbreak management benefits greatly from timely detection and proper identification of the pathways of virus introduction and spread. In this study, we used changes in mortality rates as a proxy for HPAI incidence in a layer, broiler and turkey flock together with diagnostic test results to infer within-flock HPAI transmission dynamics. Mathematical modeling techniques, specifically the Approximate Bayesian Computation algorithm in conjunction with a stochastic within-flock HPAI transmission model were used in the analysis. The time window of HPAI virus introduction into the flock (TOI) and the adequate contact rate (ACR) were estimated. Then, using the estimated TOI together with the day when the first HPAI positive sample was collected from the flock, we calculated the most likely time to first positive sample (MTFPS) which reflects the time to HPAI detection. The estimated joint (i.e., all species combined) median of the MTFPS for different flocks was six days, the joint median most likely ACR was 6.8 newly infected birds per infectious bird per day, the joint median R0 was 13 and the joint median number of test days per flock was two. These results were also grouped by species and by epidemic phase and discussed accordingly. We conclude that this findings from this and other related studies are beneficial for the different stakeholders in outbreak management. We recommend that combining TOI analysis with complementary approaches such as phylogenetic analyses is critically important for improved understanding of disease transmission pathways. The estimated parameters can also be used to parametrize mathematical models that can guide the design of surveillance protocols, risk analyses of HPAI spread, and emergency preparedness for HPAI outbreaks.

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 African swine fever (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. [Open Vet J 2022; 12(6.000): 787-796]

Abstract 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.

Abstract 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.

Abstract 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)-1β/IL-18 for proinflammatory responses induced upon infection. We hypothesize that inflammasome activation and pyroptosis induced in SFTS results in elevated levels of IL-1β/IL-18 responsible for high fever and hemorrhage in the host, characteristic of SFTS. Here we report that IL-1β secretion was elevated in SFTS patients and infected mice and IL-1β levels appeared to be reversibly associated to disease severity and viral load in patients’ blood. Increased caspase-1 activation, IL-1β/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-1β. 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-1β 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-1β/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.

Abstract 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.

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.

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-1β 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.

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.

Abstract 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.

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)-1β 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.

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-β, 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.

Abstract Background Timely diagnosis of influenza A virus infections is critical for outbreak control. Due to their rapidity and other logistical advantages, lateral flow immunoassays can support influenza A virus surveillance programs and here, their field performance was proactively assessed. The performance of real-time polymerase chain reaction and two lateral flow immunoassay kits (FluDETECT and VetScan) in detecting low pathogenicity influenza A virus in oropharyngeal swab samples from experimentally inoculated broiler chickens was evaluated and at a flock-level, different testing scenarios were analyzed. Results For real-time polymerase chain reaction positive individual-swabs, FluDETECT respectively detected 37% and 58% for the H5 and H7 LPAIV compared to 28% and 42% for VetScan. The mean virus titer in H7 samples was higher than for H5 samples. For real-time polymerase chain reaction positive pooled swabs (containing one positive), detections by FluDETECT were significantly higher in the combined 5- and 6-swab samples compared to 11-swab samples. FluDETECT detected 58%, 55.1% and 44.9% for the H7 subtype and 28.3%, 34.0% and 24.6% for the H5 in pools of 5, 6 and 11 respectively. In our testing scenario analysis, at low flock-level LPAIV infection prevalence, testing pools of 11 detected slightly more infections while at higher prevalence, testing pools of 5 or 6 performed better. For highly pathogenic avian influenza virus, testing pools of 11 (versus 5 or 6) detected up to 5% more infections under the assumption of similar sensitivity across pools and detected less by 3% when its sensitivity was assumed to be lower. Conclusions Much as pooling a bigger number of swab samples increases the chances of having a positive swab included in the sample to be tested, this study’s outcomes indicate that this practice may actually reduce the chances of detecting the virus since it may result into lowering the virus titer of the pooled sample. Further analysis on whether having more than one positive swab in a pooled sample would result in increased sensitivity for low pathogenicity avian influenza virus is needed.

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.

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.

BackgroundSevere 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 findingsHere 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/significanceOur 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.

Influenza A virus subtypes in non-human hosts have not been characterized in Kenya. We carried out influenza surveillance in selected domestic animals and compared the virus isolates with isolates obtained in humans during the same period.We collected nasal swabs from pigs, dogs and cats; oropharyngeal and cloacal swabs from poultry; and blood samples from all animals between 2010 and 2012. A standardized questionnaire was administered to farmers and traders. Swabs were tested for influenza A by rtRT-PCR, virus isolation and subtyping was done on all positive swabs. All sera were screened for influenza A antibodies by ELISA, and positives were evaluated by hemagglutination inhibition (HI). Full genome sequencing was done on four selected pig virus isolates.Among 3,798 sera tested by ELISA, influenza A seroprevalence was highest in pigs (15.9%; 172/1084), 1.2% (3/258) in ducks, 1.4% (1/72) in cats 0.6% (3/467) in dogs, 0.1% (2/1894) in chicken and 0% in geese and turkeys. HI testing of ELISA-positive pig sera showed that 71.5% had positive titers to A/California/04/2009(H1N1). Among 6,289 swabs tested by rRT-PCR, influenza A prevalence was highest in ducks [1.2%; 5/423] and 0% in cats and turkeys. Eight virus isolates were obtained from pig nasal swabs collected in 2011 and were determined to be A(H1N1)pdm09 on subtyping. On phylogenetic analysis, four hemagglutinin segments from pig isolates clustered together and were closely associated with human influenza viruses that circulated in Kenya in 2011.Influenza A(H1N1)pdm09 isolated in pigs was genetically similar to contemporary human pandemic influenza virus isolates. This suggest that the virus was likely transmitted from humans to pigs, became established and circulated in Kenyan pig populations during the study period. Minimal influenza A prevalence was observed in the other animals studied.

The spatial spread of highly pathogenic avian influenza (HPAI) H5N2 during the 2015 outbreak in the U.S. state of Minnesota was analyzed through the estimation of a spatial transmission kernel, which quantifies the infection hazard an infectious premises poses to an uninfected premises some given distance away. Parameters were estimated using a maximum likelihood method for the entire outbreak as well as for two phases defined by the daily number of newly detected HPAI-positive premises. The results indicate both a strong dependence of the likelihood of transmission on distance and a significant distance-independent component of outbreak spread for the overall outbreak. The results further suggest that HPAI spread differed during the later phase of the outbreak. The estimated spatial transmission kernel was used to compare the Minnesota outbreak with previous HPAI outbreaks in the Netherlands and Italy to contextualize the Minnesota transmission kernel results and make additional inferences about HPAI transmission during the Minnesota outbreak. Lastly, the spatial transmission kernel was used to identify high risk areas for HPAI spread in Minnesota. Risk maps were also used to evaluate the potential impact of an early marketing strategy implemented by poultry producers in a county in Minnesota during the outbreak, with results providing evidence that the strategy was successful in reducing the potential for HPAI spread.

The PB1-F2 protein of influenza A virus has been considered a virulence factor, but its function in inducing apoptosis may be of disadvantage to viral replication. Host mechanisms to regulate PB1-F2-induced apoptosis remain unknown. We generated a PB1-F2-deficient avian influenza virus (AIV) H9N2 and found that the mutant virus replicated less efficiently in human lung epithelial cells. The PB1-F2-deficient virus produced less apoptotic cells, indicating that PB1-F2 of the H9N2 virus promotes apoptosis, occurring at the early stage of infection, in the lung epithelial cells. To understand how host cells regulate PB1-F2-induced apoptosis, we explored to identify cellular proteins interacting with PB1-F2 and found that HCLS1-associated protein X-1 (HAX-1), located mainly in the mitochondria as an apoptotic inhibitor, interacted with PB1-F2. Increased procaspase-9 activations, induced by PB1-F2, could be suppressed by HAX-1. In HAX-1 knockdown A549 cells, the replication of AIV H9N2 was suppressed in parallel to the activation of caspase-3 activation, which increased at the early stage of infection. We hypothesize that HAX-1 promotes AIV replication by interacting with PB1-F2, resulting in the suppression of apoptosis, prolonged cell survival, and enhancement of viral replication. Our data suggest that HAX-1 may be a promoting factor for AIV H9N2 replication through desensitizing PB1-F2 from its apoptotic induction in human lung epithelial cells.

BACKGROUND:Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a tick-borne phlebovirus in family Bunyaviridae. Studies have found that humans, domestic and wildlife animals can be infected by SFTSV. However, the viral ecology, circulation, and transmission remain largely unknown. METHODOLOGY/PRINCIPAL FINDINGS:Sixty seven human SFTS cases were reported and confirmed by virus isolation or immunofluorescence assay between 2011 and 2014. In 2013-2014 we collected 9,984 ticks from either vegetation or small wild mammals in the endemic area in Jiangsu, China, and detected SFTSV-RNA by real-time RT-PCR in both questing and feeding Haemaphysalis longicornis and H. flava. Viral RNA was identified in larvae of H. longicornis prior to a first blood meal, which has never been confirmed previously in nature. SFTSV-RNA and antibodies were also detected by RT-PCR and ELISA, respectively, in wild mammals including Erinaceus europaeus and Sorex araneus. A live SFTSV was isolated from Erinaceus europaeus captured during the off tick-feeding season and with a high SFTSV antibody titer. Furthermore, SFTSV antibodies were detected in the migratory birds Anser cygnoides and Streptopelia chinensis using ELISA. CONCLUSIONS/SIGNIFICANCE:The detection of SFTSV-RNA in non-engorged larvae indicated that vertical transmission of SFTSV in H. longicornis might occur in nature, which suggests that H. longicornis is a putative reservoir host of SFTSV. Small wild mammals such as Erinaceus europaeus and Sorex araneus could be infected by SFTSV and may serve as natural amplifying hosts. Our data unveiled that wild birds could be infected with SFTSV or carry SFTSV-infected ticks and thus might contribute to the long-distance spread of SFTSV via migratory flyways. These findings provide novel insights for understanding SFTSV ecology, reservoir hosts, and transmission in nature and will help develop new measures in preventing its rapid spread both regionally and globally.

The spread of highly pathogenic avian influenza virus (AIV) (H5N1) underlines the potential for global AIV movement through birds. The phylogenies of AIV genes from avian hosts usually separate into Eurasian and North American clades, reflecting limited bird migration between the hemispheres. However, mounting evidence that some H6 sequences from North America cluster with Eurasian subtype H6 sequences calls the strict hemispheric divide into question. We conducted a comprehensive phylogenetic analysis of the extent and timing of cross-hemisphere movements by the H6 gene. Results suggested that Eurasian H6 subtype has invaded North America several times, with the first invasions occurring 10 years before the first detection of invading isolates. The members of the North American clade decreased from 100% in the 1980s to 20% in the 2000s among H6 isolates from North America. Unraveling the reasons for this large-scale gene movement between hemispheres might identify drivers of global AIV circulation.

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease, which can lead to fatal neurological complications in young children and infants. Few gastrointestinal symptoms are observed clinically, suggesting the presence of a unique immunity to EV71 in the gut. We reported a robust induction of interferons (IFNs) in human intestinal epithelial cells (HT-29), which was suppressed in other types such as RD and HeLa cells. The underlying mechanism for the apparent difference remains obscure. In this study we report that in EV71-infected HT-29 cells, TLR/TRIF signaling was essential to IFN induction; viral replication increased and the induction of IFN-α, -β, -ω, -κ, and -ε decreased markedly in TRIF-silenced HT-29 cells. Importantly, TRIF was degraded by viral 3Cpro in RD cells, but resisted cleavage, and IRF3 was activated and translocated into the nucleus in HT-29 cells. Taken together, our data suggest that IFNs were induced differentially in human HT-29 cells through an intact TLR/TRIF signaling, which differs from other cell types and may be implicated in viral pathogenesis in EV71 infection.

Several RNA viruses such as astrovirus, rotavirus, reovirus and parvovirus have been detected in both healthy and diseased commercial poultry flocks. The aim of this study was to characterize (a) the development of the RNA viral community in the small intestines of healthy broiler chickens from hatch through 6 weeks of age (market age) and (b) the contribution of the breeder source vs. bird age in development of the community structure. Intestinal tissue samples were harvested from breeders and their progeny, processed for viral RNA extraction and sequenced using Illumina Hiseq sequencing technology resulting in 100 bp PE reads. The results from this study indicated that the breeder source influenced the RNA viral community only at hatch but later environment i.e. bird age had the more significant effect. The most abundant RNA viral family detected at 2, 4 and 6 weeks of age was Astroviridae, which decreased in abundance with age while the abundance of Picornaviridae increased with age.

We tested blood samples from domestic and captive farmed animals in Minnesota, USA, to determine exposure to severe fever with thrombocytopenia syndrome virus and Heartland-like virus. We found antibodies against virus nucleoproteins in 10%–18% of samples from cattle, sheep, goats, deer, and elk in 24 Minnesota counties.

Highly pathogenic avian influenza virus (HPAIV) H5N1 has been endemic in Egypt since 2006, and there is increasing concern for its potential to become highly transmissible among humans. Infection by HPAIV H5N1 has been described in experimentally challenged birds. However, the pathogenicity of the H5N1 isolated in Egypt has never been reported in naturally infected chickens and ducks. Here we report a 2013 outbreak of HPAIV H5N1 in commercial poultry farms and backyards in Sharkia Province, Egypt. The main symptoms were ecchymosis on the shanks and feet, cyanosis of the comb and wattles, subcutaneous edema of the head and neck for chickens, and nervous signs (torticollis) for ducks. Within 48-72 hrs of the onset of illness, the average mortality rates were 22.8-30% and 28.5-40% in vaccinated chickens and non-vaccinated ducks, respectively. Tissue samples of chickens and ducks were collected for analyses with cross-section immunohistochemistry and real-time RT-PCR for specific viral RNA transcripts. While viral RNA was detected in nearly all tissues and sera collected, viral nucleoprotein was detected almost ubiquitously in all tissues, including testis. Interestingly, viral antigen was also observed in endothelial cells of most organs in chickens, and clearly detected in the trachea and brain in particular. Viral nucleoprotein was also detected in mononuclear cells of various organs, especially pulmonary tissue. We performed phylogenetic analyses and compared the genomic sequences of the hemagglutinin (HA) and nonstructural proteins (NS) among the isolated viruses, the HPAIV circulated in Egypt in the past and currently, and some available vaccine strains. Further analysis of deduced amino acids of both HA and NS1 revealed that our isolates carried molecular determinants of HPAIV, including the multibasic amino acids (PQGERRRK/KR*GLF) in the cleavage site in HA and glutamate at position 92 (D92E) in NS1. This is the first report of the pathogenicity of the HPAIVH5N1 strain currently circulating in naturally infected poultry in Egypt, which may provide unique insights into the viral pathogenesis in HPAIV-infected chickens and ducks.

Respiratory disease due to influenza virus is common in both human and swine populations around the world with multiple transmission routes capable of transmitting influenza virus, including indirect routes. The objective of this study was to evaluate the role of fomites in influenza A virus (IAV) transmission between pig populations separated by two different biosecurity settings. Thirty-five pigs were divided into four experimental groups: 10 pigs (1 replicate) were assigned to the infected group (I), 10 pigs (2 replicates of 5 pigs) were assigned to the low biosecurity sentinel group (LB), 10 pigs (2 replicates of 5 pigs) were assigned to the medium biosecurity sentinel group (MB), and 5 pigs (1 replicate) were assigned to the negative control group (NC). Eight of 10 pigs in the infected group were inoculated with IAV and 36 hours following inoculation, personnel movement events took place in order to move potentially infectious clothing and personal protective equipment (PPE) to sentinel pig rooms. Following contact with the infected group, personnel moved to the MB group after designated hygiene measures while personnel moved directly to the LB group. Nasal swabs and blood samples were collected from pigs to assess IAV infection status and fomites were sampled and tested via RRT-PCR. All experimentally inoculated pigs were infected with IAV and 11 of the 144 fomite samples collected following contact with infected pigs were low level positive for IAV genome. One replicate of each sentinel groups LB and MB became infected with IAV and all five pigs were infected over time. This study provides evidence that fomites can serve as an IAV transmission route from infected to sentinel pigs and highlights the need to focus on indirect routes as well as direct routes of transmission for IAV.

Swine influenza is an acute respiratory disease in pigs caused by swine influenza virus (SIV). Highly virulent SIV strains cause mortality of up to 10%. Importantly, pigs have long been considered "mixing vessels" that generate novel influenza viruses with pandemic potential, a constant threat to public health. Since its emergence in 2009 and subsequent pandemic spread, the pandemic (H1N1) 2009 (H1N1pdm) has been detected in pig farms, creating the risk of generating new reassortants and their possible infection of humans. Pathogenesis in SIV or H1N1pdm-infected pigs remains poorly characterized. Proinflammatory and antiviral cytokine responses are considered correlated with the intensity of clinical signs, and swine macrophages are found to be indispensible in effective clearance of SIV from pig lungs. In this study, we report a unique pattern of cytokine responses in swine macrophages infected with H1N1pdm. The roles of mitogen-activated protein (MAP) kinases in the regulation of the host responses were examined. We found that proinflammatory cytokines IL-6, IL-8, IL-10, and TNF-α were significantly induced and their induction was ERK1/2-dependent. IFN-β and IFN-inducible antiviral Mx and 2'5'-OAS were sharply induced, but the inductions were effectively abolished when ERK1/2 was inhibited. Induction of CCL5 (RANTES) was completely inhibited by inhibitors of ERK1/2 and JNK1/2, which appeared also to regulate FasL and TNF-α, critical for apoptosis in pig macrophages. We found that NFκB was activated in H1N1pdm-infected cells, but the activation was suppressed when ERK1/2 was inhibited, indicating there is cross-talk between MAP kinase and NFκB responses in pig macrophages. Our data suggest that MAP kinase may activate NFκB through the induction of RIG-1, which leads to the induction of IFN-β in swine macrophages. Understanding host responses and their underlying mechanisms may help identify venues for effective control of SIV and assist in prevention of future influenza pandemics.

Mongolia combines a near absence of domestic poultry, with an abundance of migratory waterbirds, to create an ideal location to study the epidemiology of highly pathogenic avian influenza virus (HPAIV) in a purely wild bird system. Here we present the findings of active and passive surveillance for HPAIV subtype H5N1 in Mongolia from 2005-2011, together with the results of five outbreak investigations. In total eight HPAIV outbreaks were confirmed in Mongolia during this period. Of these, one was detected during active surveillance employed by this project, three by active surveillance performed by Mongolian government agencies, and four through passive surveillance. A further three outbreaks were recorded in the neighbouring Tyva Republic of Russia on a lake that bisects the international border. No HPAIV was isolated (cultured) from 7,855 environmental fecal samples (primarily from ducks), or from 2,765 live, clinically healthy birds captured during active surveillance (primarily shelducks, geese and swans), while four HPAIVs were isolated from 141 clinically ill or dead birds located through active surveillance. Two low pathogenic avian influenza viruses (LPAIV) were cultured from ill or dead birds during active surveillance, while environmental feces and live healthy birds yielded 56 and 1 LPAIV respectively. All Mongolian outbreaks occurred in 2005 and 2006 (clade 2.2), or 2009 and 2010 (clade 2.3.2.1); all years in which spring HPAIV outbreaks were reported in Tibet and/or Qinghai provinces in China. The occurrence of outbreaks in areas deficient in domestic poultry is strong evidence that wild birds can carry HPAIV over at least moderate distances. However, failure to detect further outbreaks of clade 2.2 after June 2006, and clade 2.3.2.1 after June 2010 suggests that wild birds migrating to and from Mongolia may not be competent as indefinite reservoirs of HPAIV, or that HPAIV did not reach susceptible populations during our study.

When avian influenza viruses (AIVs) are transmitted from their reservoir hosts (wild waterfowl and shorebirds) to domestic bird species, they undergo genetic changes that have been linked to higher virulence and broader host range. Common genetic AIV modifications in viral proteins of poultry isolates are deletions in the stalk region of the neuraminidase (NA) and additions of glycosylation sites on the hemagglutinin (HA). Even though these NA deletion mutations occur in several AIV subtypes, they have not been analyzed comprehensively. In this study, 4,920 NA nucleotide sequences, 5,596 HA nucleotide and 4,702 HA amino acid sequences were analyzed to elucidate the widespread emergence of NA stalk deletions in gallinaceous hosts, the genetic polymorphism of the deletion patterns and association between the stalk deletions in NA and amino acid variants in HA. Forty-seven different NA stalk deletion patterns were identified in six NA subtypes, N1-N3 and N5-N7. An analysis that controlled for phylogenetic dependence due to shared ancestry showed that NA stalk deletions are statistically correlated with gallinaceous hosts and certain amino acid features on the HA protein. Those HA features included five glycosylation sites, one insertion and one deletion. The correlations between NA stalk deletions and HA features are HA-NA-subtype-specific. Our results demonstrate that stalk deletions in the NA proteins of AIV are relatively common. Understanding the NA stalk deletion and related HA features may be important for vaccine and drug development and could be useful in establishing effective early detection and warning systems for the poultry industry.

The mycotoxin, aflatoxin B1 (AFB1) is a hepatotoxic, immunotoxic, and mutagenic contaminant of food and animal feeds. In poultry, AFB1 can be maternally transferred to embryonated eggs, affecting development, viability and performance after hatch. Domesticated turkeys (Meleagris gallopavo) are especially sensitive to aflatoxicosis, while Eastern wild turkeys (M. g. silvestris) are likely more resistant. In ovo exposure provided a controlled AFB1 challenge and comparison of domesticated and wild turkeys. Gene expression responses to AFB1 in the embryonic hepatic transcriptome were examined using RNA-sequencing (RNA-seq). Eggs were injected with AFB1 (1 μg) or sham control and dissected for liver tissue after 1 day or 5 days of exposure. Libraries from domesticated turkey (n = 24) and wild turkey (n = 15) produced 89.2 Gb of sequence. Approximately 670 M reads were mapped to a turkey gene set. Differential expression analysis identified 1535 significant genes with |log2 fold change| ≥ 1.0 in at least one pair-wise comparison. AFB1 effects were dependent on exposure time and turkey type, occurred more rapidly in domesticated turkeys, and led to notable up-regulation in cell cycle regulators, NRF2-mediated response genes and coagulation factors. Further investigation of NRF2-response genes may identify targets to improve poultry resistance.