Infectivity of Wild-Bird Origin Influenza A Viruses in Minnesota Wetlands across Seasons.

The environmental tenacity of influenza A viruses (IAVs) in the environment likely plays a role in their transmission; IAVs are able to remain infectious in aquatic habitats and may have the capacity to seed outbreaks when susceptible wild bird hosts utilize these same environments months or even seasons later. Here, we aimed to assess the persistence of low-pathogenicity IAVs from naturally infected ducks in Northwestern Minnesota through a field experiment. Viral infectivity was measured using replicate samples maintained in distilled water in a laboratory setting as well as in filtered water from four natural water bodies maintained in steel perforated drums (hereafter, mesocosms) within the field from autumn 2020 to spring 2021. There was limited evidence for the extended persistence of IAVs held in mesocosms; from 65 initial IAV-positive samples, only six IAVs persisted to at least 202 days in the mesocosms compared to 17 viruses persisting at least this long when held under temperature-controlled laboratory settings in distilled water. When accounting for the initial titer of samples, viruses detected at a higher concentration at the initiation of the experiment persisted longer than those with a lower starting titer. A parallel experimental laboratory model was used to further explore the effects of water type on viral persistence, and the results supported the finding of reduced tenacity of IAVs held in mesocosms compared to distilled water. The results of this investigation provide evidence that many factors, including temperature and physicochemical properties, impact the duration of viral infectivity in natural settings, further extending our understanding of the potential and limitations of environmental-based methodologies to recover infectious IAVs.

North American wintering mallards infected with highly pathogenic avian influenza show few signs of altered local or migratory movements.

Avian influenza viruses pose a threat to wildlife and livestock health. The emergence of highly pathogenic avian influenza (HPAI) in wild birds and poultry in North America in late 2021 was the first such outbreak since 2015 and the largest outbreak in North America to date. Despite its prominence and economic impacts, we know relatively little about how HPAI spreads in wild bird populations. In January 2022, we captured 43 mallards (Anas platyrhynchos) in Tennessee, USA, 11 of which were actively infected with HPAI. These were the first confirmed detections of HPAI H5N1 clade 2.3.4.4b in the Mississippi Flyway. We compared movement patterns of infected and uninfected birds and found no clear differences; infected birds moved just as much during winter, migrated slightly earlier, and migrated similar distances as uninfected birds. Infected mallards also contacted and shared space with uninfected birds while on their wintering grounds, suggesting ongoing transmission of the virus. We found no differences in body condition or survival rates between infected and uninfected birds. Together, these results show that HPAI H5N1 clade 2.3.4.4b infection was unrelated to body condition or movement behavior in mallards infected at this location during winter; if these results are confirmed in other seasons and as HPAI H5N1 continues to evolve, they suggest that these birds could contribute to the maintenance and dispersal of HPAI in North America. Further research on more species across larger geographic areas and multiple seasons would help clarify potential impacts of HPAI on waterfowl and how this emerging disease spreads at continental scales, across species, and potentially between wildlife and domestic animals.

Naturally Acquired Antibodies to Influenza A Virus in Fall-Migrating North American Mallards.

Although waterfowl are the primary reservoir for multiple subtypes of influenza A virus (IAV), our understanding of population immunity in naturally infected waterfowl is poorly understood. Population immunity may be an important driver of seasonal subtype predominance in waterfowl populations and may affect the potential for establishment of introduced IAV such as the Eurasian-like A/Goose/Guangdong/1/1996 lineage in these populations. Here, we examine the prevalence of naturally acquired antibodies to nucleoprotein (NP), hemagglutinin (H3, H4, H5), and neuraminidase (N1, N2, N6, N8) in early migrating mallards (Anas platyrhynchos) sampled in Northwest Minnesota during staging and early fall migration in September 2014, 2015, 2017, and 2018. Serologic results were compared to historic and contemporary virus isolation results from these same study sites. The prevalence of antibodies to NP ranged from 60.8−76.1% in hatch-year (HY) birds and from 86.0−92.7% in after-hatch-year (AHY, >1-year-old) mallards indicating a high level of previous infection with IAV early in the fall migration season. Neutralizing antibodies were detected against H3, H4, and H5 in all years as were antibodies to N1, N2, N6, and N8. A high proportion of NP seropositive ducks tested positive for antibodies to multiple HA and NA subtypes, and this was more common in the AHY age class. Antibody prevalence to the HA and NA subtypes included in this study were consistent with the predominance of H4N6 in these populations during all years and reflected a broadening of the antibody response with age. Additional work is needed to document the longevity of these immune responses, if and how they correlate with protection against IAV transmission, infection, and disease, and if, as detected in this study, they adequately describe the true extent of exposure to IAV or specific HA or NA subtypes.

Experimental Infection of Domestic Piglets (Sus scrofa) with Rift Valley Fever Virus.

Rift Valley fever phlebovirus (RVFV) is a mosquito-transmitted phlebovirus (Family: Phenuiviridae, Order: Bunyavirales) causing severe neonatal mortality and abortion primarily in domestic ruminants. The susceptibility of young domestic swine to RVFV and this species' role in geographic expansion and establishment of viral endemicity is unclear. Six commercially bred Landrace-cross piglets were inoculated subcutaneously with 105 plaque-forming units of RVFV ZH501 strain and two piglets received a sham inoculum. All animals were monitored for clinical signs, viremia, viral shedding, and antibody response for 14 days. Piglets did not develop evidence of clinical disease, become febrile, or experience decreased weight gain during the study period. A brief lymphopenia followed by progressive lymphocytosis was observed following inoculation in all piglets. Four piglets developed a brief viremia for 2 days post-inoculation and three of these had detectable virus in oronasal secretions three days post-inoculation. Primary inoculated piglets seroconverted and those that developed detectable viremias had the highest titers assessed by serum neutralization (1:64-1:256). Two viremic piglets had a lymphoplasmacytic encephalitis with glial nodules; RVFV was not detected by immunohistochemistry in these sections. While young piglets do not appear to readily develop clinical disease following RVFV infection, results suggest swine could be subclinically infected with RVFV.

Influenza A Viruses in Whistling Ducks (Subfamily Dendrocygninae).

As compared to other Anseriformes, data related to influenza A virus (IAV) detection and isolation, and IAV antibody detection in whistling ducks (Dendrocygna spp. and Thalassornis leuconotus; subfamily Dendrocygninae) are limited. To better evaluate the potential role of whistling ducks in the epidemiology of IAV, we (1) conducted surveillance for IAV from black-bellied whistling ducks (BBWD, Dendrocygnaautumnalis) sampled in coastal Louisiana, USA, during February 2018 and 2019, and (2) reviewed the published literature and Influenza Resource Database (IRD) that reported results of IAV surveillance of whistling ducks. In the prospective study, from 166 BBWD sampled, one H10N7 IAV was isolated (0.6% prevalence), and overall blocking enzyme-linked immunosorbent assay (bELISA) antibody seroprevalence was 10%. The literature review included publications and data in the IRD from 1984 to 2020 that reported results from nearly 5000 collected samples. For any given collection, the IAV isolation rate never exceeded 5.5%, and seroprevalence estimates ranged from 0 to 42%. Results from our prospective study in Louisiana are consistent with this historic literature; however, although all data consistently demonstrated a low prevalence of infection, the potential role of this species in the epidemiology of IAV should not be totally discounted. In sum, whistling ducks can be infected with IAV, they represent important species on many areas where waterfowl winter, and their distribution across the globe appears to be changing.

Genome Sequence of a Novel H14N7 Subtype Influenza A Virus Isolated from a Blue-Winged Teal (Anas discors) Harvested in Texas, USA.

We report here the complete genome sequence of a novel H14N7 subtype influenza A virus (IAV) isolated from a blue-winged teal (Anas discors) harvested in Texas, USA. The genomic characteristics of this IAV strain with a previously undetected subtype combination suggest recent viral evolution within the New World wild-bird IAV reservoir.

Effect of Temperature on Replication of Epizootic Hemorrhagic Disease Viruses in Culicoides sonorensis (Diptera: Ceratopogonidae).

Replication of arboviruses, including orbiviruses, within the vector has been shown to be temperature dependent. Cooler ambient temperatures slow virus replication in arthropod vectors, whereas viruses replicate faster and to higher titers at warmer ambient temperatures. Previous research with epizootic hemorrhagic disease virus (EHDV) serotype 1 demonstrated that higher temperatures were associated with shorter extrinsic incubation periods in Culicoides sonorensis Wirth & Jones, a confirmed vector of EHDV in North America. To further our understanding of the effect of temperature on replication of EHDV within the vector, C. sonorensis were experimentally infected with one of three EHDV strains representing three serotypes (1, 2, and 7). Midges were fed defibrinated white-tailed deer (Odocoileus virginianus) blood spiked with EHDV (≥10(6.5) TCID(50)/ml) through a parafilm membrane using an artificial feeding device and were then held at 20, 25, or 30°C. In addition to this in vitro method, a white-tailed deer experimentally infected with EHDV-7 was used to provide an infectious bloodmeal to determine if the results were comparable with those from the in vitro feeding method. Whole midges were processed for virus isolation and titration at regular intervals following feeding; midges with ≥10(2.7) TCID(50) were considered potentially competent to transmit virus. The virus recovery rates were high throughout the study and all three viruses replicated within C. sonorensis to high titer (≥ 10(2.7) TCID(50)/midge). Across all virus strains, the time to detection of potentially competent midges decreased with increasing temperature: 12-16 d postfeeding (dpf) at 20°C, 4-6 dpf at 25°C, and 2-4 dpf at 30°C. Significant differences in replication of the three viruses in C. sonorensis were observed, with EHDV-2 replicating to a high titer in a smaller proportion of midges and with lower peak titers. The findings are consistent with previous studies of related orbiviruses, showing that increasing temperature can shorten the apparent extrinsic incubation period for multiple EHDV strains (endemic and exotic) in C. sonorensis.

Experimental infection of Holstein cows and calves with EHDV­7 and preliminary evaluation of different inoculation methods.

Infection of cattle with epizootic haemorrhagic disease (EHD) virus (EHDV) is frequently subclinical, yet reports of disease have increased in recent years. In 2006, a widespread EHDV­7 epidemic caused disease and economic loss in the Israeli dairy industry. In this study, the main objective was to infect cattle with EHDV­7 and replicate disease observed in Israel during 2006. Two infection studies were performed. Experiment 1, 4 cows inoculated with intradermal (ID) and subcutaneous (SC) injections with an EHDV­7 blood inoculum. Experiment 2, 6 calves inoculated using 1 of the following 3 methods (2 calves/method): (1) mammalian cell culture supernatant by ID and SC injection; (2) culture supernatant by ID, SC, and intravenous injection; and (3) bite transmission from Culicoides sonorensis. Further, during experiment 2, C. sonorensis were fed on 4 infected calves (18 days post-inoculation) and processed for virus isolation 10 days later in order to evaluate infectivity of low­titer viraemia. Three cows had detectable viraemia and all 4 seroconverted. No clinical signs were observed. All 6 calves developed viraemia, peaking 7­10 dpi and all calves seroconverted. No differences in virus kinetics were observed between the inoculation groups. Calves in group 2 had transiently elevated rectal temperatures but no other clinical abnormalities were observed. The 124 midge pools processed after feeding on calves with low­titer viraemia were virus isolation negative. Detectable viraemia was more consistent in calves than adult cows. This study demonstrates US­origin cattle are susceptible to EHDV­7 infection by multiple inoculation methods; however, as reported in other studies, the disease was not replicated experimentally.

Evidence for seasonal patterns in the relative abundance of avian influenza virus subtypes in blue-winged teal (Anas discors).

Seasonal dynamics of influenza A viruses (IAVs) are driven by host density and population immunity. Through an analysis of subtypic data for IAVs isolated from Blue-winged Teal (Anas discors), we present evidence for seasonal patterns in the relative abundance of viral subtypes in spring and summer/autumn.

Infectivity of avian influenza virus-positive field samples for mallards: what do our diagnostic results mean?

Most surveillance programs for avian influenza (AI) virus in wild birds utilize molecular tests such as real-time reverse transcription-PCR (RRT-PCR) or virus isolation (VI) in embryonating chicken eggs. To provide insight into the relationship between positive diagnostic test results and infectivity for an avian host, we challenged Mallards (Anas platyrhynchos) with Mallard-derived cloacal swab field samples found positive by VI or RRT-PCR. Six of 11 samples that were both RRT-PCR positive and VI positive infected Mallards. Sample infectivity for Mallards appeared to be dependent on concentration of infectious virus in the sample; five of the six samples that replicated in Mallards had a measurable virus titer, whereas four of the five samples that did not infect Mallards had titers below the limit of detection (10(0.9) median embryo infectious dose/0.2 mL). None of seven samples that were RRT-PCR positive and VI negative infected Mallards. These results indicate that embryonating chicken eggs are a sensitive diagnostic tool for detecting Mallards excreting infectious AI virus at a high enough concentration to infect another Mallard; however, not all cloacal swab field samples that are positive by VI or RRT-PCR are infective to another Mallard. Additionally, our results indicate that Mallards are susceptible to Mallard-origin AI viruses that have not been propagated in embryonating chicken eggs and that some of these virus strains can infect birds at titers that are lower than those typically used in experimental challenge studies. These data highlight a need to examine the effects of using egg-propagated AI viruses in experimental trials.

Intestinal excretion of a wild bird-origin H3N8 low pathogenic avian influenza virus in mallards (Anas Platyrhynchos).

Mallards (Anas platyrhynchos) and other dabbling ducks in the genus Anas are an important component of the wild bird reservoir for avian influenza (AI) virus; these viruses are maintained in migratory duck populations through a fecal-oral transmission route. We provide a detailed characterization of intestinal viral shedding in Mallards infected with a wild bird-origin low pathogenic (LP) AI virus. Five of eight, 1-mo-old Mallards inoculated with a high dose of an H3N8 LP AI virus became infected as determined by reisolation and seroconversion. Infected birds excreted high concentrations of virus for up to 14 days postinoculation (DPI) without exhibiting overt clinical signs of disease. The pattern of viral shedding was relatively consistent between individual birds, with peak shedding on 2-3 DPI and a progressive decline over the remainder of infection. Detection of viral shedding varied depending on sample type (excrement sample or cloacal swab) and diagnostic test (virus isolation or real-time quantitative reverse transcription polymerase chain reaction). Our data provide detailed insights into the intestinal excretion of an H3N8 LP AI virus in Mallards and the performance of diagnostic assays commonly used in wild bird surveillance. Such information is valuable for estimating potential risks for spillover of LP AI viruses from Mallards to domestic animals, developing accurate transmission models for Mallard populations and facilitating the interpretation and comparison of surveillance results from different studies.

Vector competence of Culicoides sonorensis (Diptera: Ceratopogonidae) to epizootic hemorrhagic disease virus serotype 7.

BACKGROUND: Culicoides sonorensis (Diptera: Ceratopogonidae) is a vector of epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2 in North America, where these viruses are well-known pathogens of white-tailed deer (WTD) and other wild ruminants. Although historically rare, reports of clinical EHDV infection in cattle have increased in some parts of the world over the past decade. In 2006, an EHDV-7 epizootic in cattle resulted in economic loss for the Israeli dairy industry. White-tailed deer are susceptible to EHDV-7 infection and disease; however, this serotype is exotic to the US and the susceptibility of C. sonorensis to this cattle-virulent EHDV is not known. The objective of the study was to determine if C. sonorensis is susceptible to EHDV-7 infection and is a competent vector. METHODS: To evaluate the susceptibility of C. sonorensis, midges were fed on EHDV-7 infected WTD, held at 22 ± 1°C, and processed individually for virus isolation and titration on 4-16 days post feeding (dpf). Midges with a virus titer of ≥ 10(2.7) median tissue culture infective doses (TCID(50))/midge were considered potentially competent. To determine if infected C. sonorensis were capable of transmitting EHDV-7 to a host, a susceptible WTD was then fed on by a group of 14-16 dpf midges. RESULTS: From 4-16 dpf, 45% (156/350) of midges that fed on WTD with high titer viremia (>10(7) TCID(50)/ml) were virus isolation-positive, and starting from 10-16 dpf, 32% (35/109) of these virus isolation-positive midges were potentially competent (≥ 10(2.7) TCID(50)/midge). Midges that fed on infected deer transmitted the virus to a susceptible WTD at 14-16 dpf. The WTD developed viremia and severe clinical disease. CONCLUSION: This study demonstrates that C. sonorensis is susceptible to EHDV-7 infection and can transmit the virus to susceptible WTD, thus, C. sonorensis should be considered a potential vector of EHDV-7. Together with previous work, this study demonstrates that North America has a susceptible ruminant and vector host for this exotic, cattle-virulent strain of EHDV-7.

Susceptibility of white-tailed deer (Odocoileus virginianus) to experimental infection with epizootic hemorrhagic disease virus serotype 7.

During the fall of 2006, in Israel, epizootic hemorrhagic disease virus (EHDV) serotype 7 caused an intense and widespread epizootic in domestic cattle that resulted in significant economic losses for the dairy industry. The susceptibility of potential North American vector and ruminant hosts to infection with EHDV-7 is not known but is essential to understanding the potential for establishment of this exotic orbivirus in North America if it were introduced. Our primary objective was to determine whether white-tailed deer (WTD; Odocoileus virginianus) are susceptible to infection with EHDV-7. Six, 8-mo-old WTD were experimentally infected with EHDV-7, and all became infected and exhibited varying degrees of clinical disease. Clinical signs, clinicopathologic abnormalities, and postmortem findings were consistent with previous reports of orbiviral hemorrhagic disease (HD) in this species. Four of six animals died or were euthanized because of the severity of disease, one on postinoculation day (PID) 5 and the remaining WTD on PID 7. All deer had detectable viremia on PID 3, which peaked on PID 5 or 6 and persisted for as long as PID 46 in one animal. Deer surviving the acute phase of the disease seroconverted by PID 10. Based on the 67% mortality rate we observed, this strain of EHDV-7 is virulent in WTD, reaffirming their role as a sentinel species for the detection of endemic and nonendemic EHDV. Further, the observed disease was indistinguishable from previous reports of disease caused by North American EHDV and bluetongue virus serotypes, highlighting the importance of serotype-specific diagnostics during suspected HD outbreaks.