Deborah L. Carter

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.

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

Abstract 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.

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

BackgroundCulicoides 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.MethodsTo 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 ≥102.7 median tissue culture infective doses (TCID50)/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.ResultsFrom 4–16 dpf, 45% (156/350) of midges that fed on WTD with high titer viremia (>107 TCID50/ml) were virus isolation-positive, and starting from 10–16 dpf, 32% (35/109) of these virus isolation-positive midges were potentially competent (≥102.7 TCID50/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.ConclusionThis 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 Avian Species to North American H13 Low Pathogenic Avian Influenza Viruses

SUMMARY. Gulls are widely recognized reservoirs for low pathogenic avian influenza (LPAI) viruses; however, the subtypes maintained in these populations and/or the transmission mechanisms involved are poorly understood. Although, a wide diversity of influenza viruses have been isolated from gulls, two hemagglutinin subtypes (H13 and H16) are rarely detected in other avian groups, and existing surveillance data suggests they are maintained almost exclusively within gull populations. In order to evaluate the host range of these gull-adapted influenza subtypes and to characterize viral infection in the gull host, we conducted a series of challenge experiments, with multiple North American strains of H13 LPAI virus in ring-billed gulls (Larus delawarensis), mallards (Anas platyrhynchos), chickens (Gallus domesticus), and turkeys (Meleagris gallopavo). The susceptibility to H13 LPAI viruses varied between species and viral strain. Gulls were highly susceptible to H13 LPAI virus infection and excreted virus via the oropharynx and cloaca for several days. The quantity and duration of shedding was similar between the two routes. Turkeys and ducks were resistant to infection with most strains of H13 LPAI virus, but low numbers of inoculated birds were infected after challenge with specific viral strains. Chickens were refractory to infection with all strains of H13 LPAI virus they were challenged with. The experimental results presented herein are consistent with existing surveillance data on H13 LPAI viruses in birds, and indicate that influenza viruses of the H13 subtype are strongly host-adapted to gulls, but rare spill-over into aberrant hosts (i.e., turkeys and ducks) can occur.

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.

Co-infection of mallards with low-virulence Newcastle disease virus and low-pathogenic avian influenza virus

Waterfowl are considered the natural reservoir of low-virulence Newcastle disease viruses (loNDVs) and low-pathogenic avian influenza viruses (LPAIVs). The objective of this study was to investigate the effect of co-infections with loNDV and LPAIV on the infectivity and excretion of these viruses in mallards. One-month-old mallards were inoculated intranasally with 106 median embryo infectious doses of a wild-bird-origin loNDV and A/Mallard/MN/199106/99 (H3N8) LPAIV on the same day or received the LPAIV 2 or 5 days after loNDV inoculation. All mallards became infected with both viruses based on detection of seroconversion and viral shedding. Co-infection resulted in a higher number of cloacal swabs detected positive for LPAIV and a lower number of cloacal swabs detected positive for loNDV in some groups, although differences between groups were not statistically significant. Co-infection did not affect replication of LPAIV in epithelial cells of the lower intestine and bursa of Fabricius. In summary, the results of this study indicate that co-infection with LPAIV and loNDV does not affect the ability of mallards to be infected with either virus although it may have minimal effects on patterns (source and timing) of viral shedding.

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 (100.9 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.

Effect of Different Routes of Inoculation on Infectivity and Viral Shedding of LPAI Viruses in Mallards

SUMMARY. We studied the effect of different routes of inoculation on the infectivity and duration of viral shedding in mallards (Anas platyrhynchos) infected with wild bird-origin low pathogenic avian influenza viruses (LPAIVs). One-month-old mallards were inoculated with 106 median embryo infectious doses of either A/mallard/MN/199106/99 (H3N8) or A/mallard/MN/355779/00 (H5N2) via 1 of 5 different routes: intranasal (IN), intratracheal (IT), intraocular (IO), intracloacal (IC), or intra-ingluvial (II). Birds in all routes of inoculation groups became infected with LPAIV as detected by virus isolation, real time reverse transcription polymerase chain reaction, and serology. Mallards in different route of inoculation groups had similar viral shedding through oropharynx and cloaca from 1 day postinoculation (dpi). The peak of oropharyngeal (OP) viral shedding was reached between 2 and 3 dpi in all routes of inoculation groups infected with either virus. The peak of cloacal (CL) viral excretion was reached between 2 and 3 dpi in all routes of inoculation groups infected with H3N8 LPAIV and in the IO-, IC-, and II-inoculated groups infected with H5N2 LPAIV, with a delayed and shorter peak for the IN- and IT-inoculated groups. The birds inoculated via the II route had more productive OP and CL viral shedding after infection with either LPAIV, as evidenced by higher number of swabs testing positive over the study period. In conclusion, mallards can be infected with LPAIV by various routes of inoculation, and this corroborates their high susceptibility to infection by these viruses.

Mechanical Transmission of Vesicular Stomatitis New Jersey Virus by Simulium vittatum (Diptera: Simuliidae) to Domestic Swine (Sus scrofa)

ABSTRACT Biting flies have been suggested as mechanical vectors of vesicular stomatitis New Jersey Virus (family Rhabdoviridae, genus Vesiculovirus, VSNJV) in livestock populations during epidemic outbreaks in the western United States. We conducted a proof-of-concept study to determine whether biting flies could mechanically transmit VSNJV to livestock by using a black fly, Simulium vittatum Zetterstedt (Diptera: Simuliidae), domestic swine, Sus scrofa L., model. Black flies mechanically transmitted VSNJV to a naïve host after interrupted feeding on a vesicular lesion on a previously infected host. Transmission resulted in clinical disease in the naïve host. This is the first demonstration of mechanical transmission of VSNJV to livestock by insects.

Competition between influenza A virus subtypes through heterosubtypic immunity modulates re-infection and antibody dynamics in the mallard duck

Our overall hypothesis is that host population immunity directed at multiple antigens will influence the prevalence, diversity and evolution of influenza A virus (IAV) in avian populations where the vast subtype diversity is maintained. To investigate how initial infection influences the outcome of later infections with homologous or heterologous IAV subtypes and how viruses interact through host immune responses, we carried out experimental infections in mallard ducks (Anas platyrhynchos). Mallards were pre-challenged with an H3N8 low-pathogenic IAV and were divided into six groups. At five weeks post H3N8 inoculation, each group was challenged with a different IAV subtype (H4N5, H10N7, H6N2, H12N5) or the same H3N8. Two additional pre-challenged groups were inoculated with the homologous H3N8 virus at weeks 11 and 15 after pre-challenge to evaluate the duration of protection. The results showed that mallards were still resistant to re-infection after 15 weeks. There was a significant reduction in shedding for all pre-challenged groups compared to controls and the outcome of the heterologous challenges varied according to hemagglutinin (HA) phylogenetic relatedness between the viruses used. There was a boost in the H3 antibody titer after re-infection with H4N5, which is consistent with original antigenic sin or antigenic seniority and suggest a putative strategy of virus evasion. These results imply competition between related subtypes that could regulate IAV subtype population dynamics in nature. Collectively, we provide new insights into within-host IAV complex interactions as drivers of IAV antigenic diversity that could allow the circulation of multiple subtypes in wild ducks.