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.

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.

Suppression of the IFN-α and -ß Induction through Sequestering IRF7 into Viral Inclusion Bodies by Nonstructural Protein NSs in Severe Fever with Thrombocytopenia Syndrome Bunyavirus Infection.

Induction of type I IFNs during viral infection is crucial for host defense. IRF 3 and IRF7 play a critical role as key transcription factors in the activation of the IFN induction. Viruses have evolved a variety of strategies to evade innate immunity. Our previous studies have shown that the nonstructural protein (NSs) of the severe fever with thrombocytopenia syndrome virus (SFTSV) can suppress the IFN-ß induction through its interaction with tank-binding kinase-1 and sequestering the inhibitor of nuclear factor kappa B kinase(IKK) complex into the inclusion bodies formed by NSs. In this study, we characterized the unique function of IRF7 in innate immunity and its role in inducing IFN-α in particular, regulated by NSs during the SFTSV infection in several cell types of human origin. Whereas IRF3 is constitutively expressed, IRF7 was significantly induced differentially in various cell types in response to SFTSV infection, promoted the induction of IFN-α2 and -α4, and further induced IFN-ß, thus contributing to suppressing the viral replication. Our data indicate that NSs directly interacted with and sequestered IRF7 into the inclusion bodies, which is different from IRF3 indirectly interacting with NSs. Although interaction of NSs with IRF7 did not inhibit IRF7 phosphorylation, p-IRF7 was trapped in the inclusion bodies, resulting in a significant reduction of the IFN-α2 and -α4 induction and therefore enhanced viral replication. Interaction of the viral NSs with both IRF7 and IRF3 and subsequent sequestration of these transcription factors into viral inclusion bodies, a unique strategy used by this phlebovirus, may ensure effective evasion and suppression of host innate immunity.

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.

Enterovirus 71 suppresses interferon responses by blocking Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling through inducing karyopherin-α1 degradation.

Enterovirus 71 (EV71) has emerged as one of the most important enteroviruses since the eradication of poliovirus, and it causes severe neurological symptoms for which no effective antiviral drugs are available. Type I interferons (IFN) α/ß have been used clinically as antiviral therapy as the first line of defense against virus infections successfully for decades. However, treatment with type I interferons has not been effective in patients with EV71 infection. In this study, we found that in cells pretreated with IFN-ß, EV71 infection could still lead to a cytopathic effect, and the viral replication was not affected. The mechanism by which EV71 antagonizes interferon signaling, however, has been controversial. Our study indicated that EV71 infection did not inhibit phosphorylation of STAT1/2 induced by IFN-ß stimulation, but p-STAT1/2 transport into the nucleus was significantly blocked. We showed that EV71 infection reduced the formation of STAT/karyopherin-α1 (KPNA1) complex upon interferon stimulation and that the virus down-regulated the expression of KPNA1, a nuclear localization signal receptor for p-STAT1. Using specific caspase inhibitors and siRNA for caspase-3, we demonstrated that EV71 infection induced degradation of cellular KPNA1 in a caspase-3-dependent manner, which led to decreased induction of interferon-inducible genes and IFN response. Viral 2A and 3C proteases did not degrade KPNA1, inhibit the activity of ISRE or suppress the transcription of interferon-inducible genes induced by IFN-ß. Our study demonstrates a novel mechanism by which antiviral signaling is suppressed through degradation of KPNA1 by activated caspase-3 induced in an enteroviral infection.

Quantifying the effect of swab pool size on the detection of influenza A viruses in broiler chickens and its implications for surveillance.

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.

Critical Role of HAX-1 in Promoting Avian Influenza Virus Replication in Lung Epithelial Cells.

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.

Synaptogyrin-2 Promotes Replication of a Novel Tick-borne Bunyavirus through Interacting with Viral Nonstructural Protein NSs.

Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little is known about the function of synaptogyrin-2. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with high mortality, caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that the viral nonstructural protein NSs forms inclusion bodies (IBs) that are involved in viral immune evasion, as well as viral RNA replication. In this study, we sought to elucidate the mechanism by which NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and adipose differentiation-related protein (ADRP). Through a high throughput screening, we identified synaptogyrin-2 to be highly up-regulated in response to SFTS bunyavirus (SFTSV) infection and to be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased, and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which correlated with the reduced number of the large IBs restructured from regular lipid droplets. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs to become virus factories for viral RNA replication through its interaction with NSs. These findings unveil the function of synaptogyrin-2 as an enhancer in viral infection.

Intrinsic apoptosis and proinflammatory cytokines regulated in human astrocytes infected with enterovirus 71.

Enterovirus 71 (EV71) has emerged as a clinically important neurotropic virus following poliovirus eradication. However, the mechanism of EV71-induced neurological manifestation remains largely unclear. In this study, we showed that human astrocytes were susceptible to EV71 and viral RNA was first detected at 12 h post-infection (p.i.), whilst viral proteins were detected at 36 h p.i. EV71-infected astrocytes underwent apoptosis, in which cytochrome c was released from mitochondria to the cytosol and caspase-9 was activated. Interestingly, caspase-2 and -8 were not cleaved or activated during the infection, whilst a selective inhibitor of caspase-9, Z-LEHD-FMK, blocked the cleavage of caspase-3 and -7, indicating that only the mitochondria-mediated intrinsic apoptotic pathway was activated in EV71-infected astrocytes. EV71 infection also induced proinflammatory cytokines, including IL-6, IL-8, CCL5 and IFN-γ-inducible protein (IP)-10 in astrocytes, which may play a critical role in EV71-induced neuroinflammation and neurological complications. By using inhibitors of mitogen-activated protein kinases (MAPKs), we demonstrated that the induction of the cytokines was mainly regulated by the MAPK p38 signalling pathway as a significant reduction of the cytokines was observed when treated with p38 inhibitors. This study demonstrated that human astrocytes were susceptible to EV71, and the infection led to intrinsic apoptosis and induction of p38-regulated proinflammatory cytokines. These findings further our understanding of the neuropathogenesis in severe cases of EV71 infection.

H7N9 influenza A virus in turkeys in Minnesota.

Introductions of H7 influenza A virus (IAV) from wild birds into poultry have been documented worldwide, resulting in varying degrees of morbidity and mortality. H7 IAV infection in domestic poultry has served as a source of human infection and disease. We report the detection of H7N9 subtype IAVs in Minnesota (MN) turkey farms during 2009 and 2011. The full genome was sequenced from eight isolates as well as the haemagglutinin (HA) and neuraminidase (NA) gene segments of H7 and N9 virus subtypes for 108 isolates from North American wild birds between 1986 and 2012. Through maximum-likelihood and coalescent phylogenetic analyses, we identified the recent H7 and N9 IAV ancestors of the turkey-origin H7N9 IAVs, estimated the time and geographical origin of the ancestral viruses, and determined the relatedness between the 2009 and 2011 turkey-origin H7N9 IAVs. Analyses supported that the 2009 and 2011 viruses were distantly related genetically, suggesting that the two outbreaks arose from independent introduction events from wild birds. Our findings further supported that the 2011 MN turkey-origin H7N9 virus was closely related to H7N9 IAVs isolated in poultry in Nebraska during the same year. Although the precise origin of the wild-bird donor of the turkey-origin H7N9 IAVs could not be determined, our findings suggested that, for both the NA and HA gene segments, the MN turkey-origin H7N9 viruses were related to viruses circulating in wild birds between 2006 and 2011 in the Mississippi Flyway.

Evasion of antiviral immunity through sequestering of TBK1/IKKε/IRF3 into viral inclusion bodies.

Cells are equipped with pattern recognition receptors (PRRs) such as the Toll-like and RIG-I-like receptors that mount innate defenses against viruses. However, viruses have evolved multiple strategies to evade or thwart host antiviral responses. Viral inclusion bodies (IBs), which are accumulated aggregates of viral proteins, are commonly formed during the replication of some viruses in infected cells, but their role in viral immune evasion has rarely been explored. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging febrile illness caused by a novel phlebovirus in the Bunyaviridae. The SFTS viral nonstructural protein NSs can suppress host beta interferon (IFN-ß) responses. NSs can form IBs in infected and transfected cells. Through interaction with tank-binding kinase 1 (TBK1), viral NSs was able to sequester the IKK complex, including IKKε and IRF3, into IBs, although NSs did not interact with IKKε or IRF3 directly. When cells were infected with influenza A virus, IRF3 was phosphorylated and active phosphorylated IRF3 (p-IRF3) was translocated into the nucleus. In the presence of NSs, IRF3 could still be phosphorylated, but p-IRF3 was trapped in cytoplasmic IBs, resulting in reduced IFN-ß induction and enhanced viral replication. Sequestration of the IKK complex and active IRF3 into viral IBs through the interaction of NSs and TBK1 is a novel mechanism for viral evasion of innate immunity.

Roles of viroplasm-like structures formed by nonstructural protein NSs in infection with severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) virus is an emerging bunyavirus that causes a hemorrhagic fever with a high mortality rate. The virus is likely tick-borne and replicates primarily in hemopoietic cells, which may lead to disregulation of proinflammatory cytokine induction and loss of leukocytes and platelets. The viral genome contains L, M, and S segments encoding a viral RNA polymerase, glycoproteins G(n) and G(c), nucleoprotein (NP), and a nonstructural S segment (NSs) protein. NSs protein is involved in the regulation of host innate immune responses and suppression of IFNß-promoter activities. In this article, we demonstrate that NSs protein can form viroplasm-like structures (VLSs) in infected and transfected cells. NSs protein molecules interact with one another, interact with NP, and were associated with viral RNA in infected cells, suggesting that NSs protein may be involved in viral replication. Furthermore, we observed that NSs-formed VLS colocalized with lipid droplets and that inhibitors of fatty acid biosynthesis decreased VLS formation or viral replication in transfected and infected cells. Finally, we have demonstrated that viral dsRNAs were also localized in VLS in infected cells, suggesting that NSs-formed VLS may be implicated in the replication of SFTS bunyavirus. These findings identify a novel function of nonstructural NSs in SFTSV-infected cells where it is a scaffolding component in a VLS functioning as a virus replication factory. This function is in addition to the role of NSs protein in modulating host responses that will broaden our understanding of viral pathogenesis of phleboviruses.

Historical and Recent Cases of H3 Influenza A Virus in Turkeys in Minnesota.

Subtype H3 influenza A viruses (IAVs) are abundant in wild waterfowl and also infect humans, pigs, horses, dogs, and seals. In Minnesota, turkeys are important and frequent hosts of IAV from wild waterfowl and from pigs. Over 48 yr of surveillance history, 11 hemagglutinin (HA) subtypes of IAV from waterfowl, as well as two HA subtypes from swine, H1 and H3, have infected turkeys in Minnesota. However, there have only been two cases of avian-origin H3 IAV infections in turkeys during this 48-yr period. The first avian-origin IAV infection was detected in seven breeder and commercial flocks in 1982 and was caused by a mixed H3H4/N2 infection. In 2013, an avian-origin H3H9/N2 outbreak occurred in five flocks of turkeys between 15 and 56 wk of age. Phylogenetic analysis of the HA gene segment from the 2013 isolate indicated that the virus was related to a wild bird lineage H3 IAV. A meta-analysis of historical H3 infections in domesticated poultry demonstrated that avian-origin H3 infections have occurred in chickens and ducks but were rare in turkeys. H9N2 virus was subsequently selected during the egg cultivation of the 2013 H3H9/N2 mixed virus. A growth curve analysis suggested that passage 3 of A/Turkey/Minnesota/13-20710-2/2013(mixed) had a slightly lower replication rate than a similar avian-origin H3N2. The challenge studies indicated that the infectious dose of avian-origin H3N2 for turkey poults was greater than 10(6) 50% egg infective dose. Considered together, these data suggest that avian-origin H3 introductions to turkeys are rare events.

Cellular scent of influenza virus infection.

Volatile organic compounds (VOCs) emanating from humans have the potential to revolutionize non-invasive diagnostics. Yet, little is known about how these compounds are generated by complex biological systems, and even less is known about how these compounds are reflective of a particular physiological state. In this proof-of-concept study, we examined VOCs produced directly at the cellular level from B lymphoblastoid cells upon infection with three live influenza virus subtypes: H9N2 (avian), H6N2 (avian), and H1N1 (human). Using a single cell line helped to alleviate some of the complexity and variability when studying VOC production by an entire organism, and it allowed us to discern marked differences in VOC production upon infection of the cells. The patterns of VOCs produced in response to infection were unique for each virus subtype, while several other non-specific VOCs were produced after infections with all three strains. Also, there was a specific time course of VOC release post infection. Among emitted VOCs, production of esters and other oxygenated compounds was particularly notable, and these may be attributed to increased oxidative stress resulting from infection. Elucidating VOC signatures that result from the host cells response to infection may yield an avenue for non-invasive diagnostics and therapy of influenza and other viral infections.

Novel bunyavirus in domestic and captive farmed animals, Minnesota, USA.

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.

Suppression of the interferon and NF-κB responses by severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, multiorgan dysfunction, and a high fatality rate between 12 and 30%. It is caused by SFTS virus (SFTSV), a novel Phlebovirus in family Bunyaviridae. Although the viral pathogenesis remains largely unknown, hemopoietic cells appear to be targeted by the virus. In this study we report that human monocytes were susceptible to SFTSV, which replicated efficiently, as shown by an immunofluorescence assay and real-time reverse transcription-PCR. We examined host responses in the infected cells and found that antiviral interferon (IFN) and IFN-inducible proteins were induced upon infection. However, our data also indicated that downregulation of key molecules such as mitochondrial antiviral signaling protein (MAVS) or weakened activation of interferon regulatory factor (IRF) and NF-κB responses may contribute to a restricted innate immunity against the infection. NSs, the nonstructural protein encoded by the S segment, suppressed the beta interferon (IFN-ß) and NF-κB promoter activities, although NF-κB activation appears to facilitate SFTSV replication in human monocytes. NSs was found to be associated with TBK1 and may inhibit the activation of downstream IRF and NF-κB signaling through this interaction. Interestingly, we demonstrated that the nucleoprotein (N), also encoded by the S segment, exhibited a suppressive effect on the activation of IFN-ß and NF-κB signaling as well. Infected monocytes, mainly intact and free of apoptosis, may likely be implicated in persistent viral infection, spreading the virus to the circulation and causing primary viremia. Our findings provide the first evidence in dissecting the host responses in monocytes and understanding viral pathogenesis in humans infected with a novel deadly Bunyavirus.

Antigenic and genetic characterization of a European avian-like H1N1 swine influenza virus from a boy in China in 2011.

Cross-species transmission of influenza A viruses from swine to human occurs occasionally. In 2011, an influenza A H1N1 virus, A/Jiangsu/ALS1/2011 (JS/ALS1/2011), was isolated from a boy who suffered from severe pneumonia in China. The virus is closely related antigenically and genetically to avian-like swine H1N1 viruses that have recently been circulating in pigs in China and that were initially detected in European pig populations in 1979. The isolation of JS/ALS1/2011 provides additional evidence that swine influenza viruses can occasionally infect humans and emphasizes the importance of reinforcing influenza virus surveillance in both pigs and humans.

Development and application of quantitative real-time PCR for the rapid detection of hemorrhagic enteritis virus in tissue samples.

Hemorrhagic enteritis virus (HEV) is a type II avian adenovirus that causes intestinal hemorrhages accompanied with immunosuppression in 4-to-12-wk-old turkeys. In the present study, a hexon gene-based, quantitative real-time PCR with TaqMan probe was developed and applied to tissue samples from poultry farms to detect and quantify HEV genome copy numbers. The method was confirmed to be rapid, specific, and sensitive for the detection of HEV. This method is an excellent research and diagnostic tool that can be used to study pathogenesis and to gain insights into different phases of infection on poultry farms and for high-throughput epidemiologic investigations.

Isolation of influenza A viruses from wild ducks and feathers in Minnesota (2010-2011).

We investigated the feasibility of testing feathers as a complementary approach to detect low pathogenic influenza A viruses (IAVs) in wild duck populations. Feathers on the ground were collected at four duck capture sites during 2010 and 2011, in Minnesota, U. S. A. IAVs were isolated from both feathers and cloacal swabs sampled from ducks at the time of capture. Although virus isolation rates from feather and cloacal swabs were inconsistent between collections, the overall rate of isolation was greatest from the feather samples. Viruses isolated from feathers also reflected the subtype diversity observed in cloacal swab isolates but resulted in many more isolates that contained more than one virus. Our study suggests that testing feathers may represent an alternative noninvasive approach to recover viruses and estimate subtype abundance and diversity.

Migration strategy affects avian influenza dynamics in mallards (Anas platyrhynchos).

Studies of pathogen transmission typically overlook that wildlife hosts can include both migrant and resident populations when attempting to model circulation. Through the application of stable isotopes in flight feathers, we estimated the migration strategy of mallards (Anas platyrhynchos) occurring on California wintering grounds. Our study demonstrates that mallards- a principal host of avian influenza virus (AIV) in nature, contribute differently to virus gene flow depending on migration strategy. No difference in AIV prevalence was detected between resident (9.6%), intermediate-distance (9.6%) and long-distance migrants (7.4%). Viral diversity among the three groups was also comparable, possibly owing to viral pool mixing when birds converge at wetlands during winter. However, migrants and residents contributed differently to the virus gene pool at wintering wetlands. Migrants introduced virus from northern breeding grounds (Alaska and the NW Pacific Rim) into the wintering population, facilitating gene flow at continental scales, but circulation of imported virus appeared to be limited. In contrast, resident mallards acted as AIV reservoirs facilitating year-round circulation of limited subtypes (i.e. H5N2) at lower latitudes. This study supports a model of virus exchange in temperate regions driven by the convergence of wild birds with separate geographic origins and exposure histories.