Genetic variation within a species of parasitic nematode, Skrjabingylus chitwoodorum, in skunks.

Carnivores in the families Mustelidae and Mephitidae are essential hosts for the cranial roundworm genus Skrjabingylus. A high prevalence of Skrjabingylus chitwoodorum has been observed in the striped skunk, Mephitis mephitis. Genetic barcoding studies of other nematodes have successfully used the cytochrome oxidase I (COI) mitochondrial gene to analyze genetic variation and divergence. We tested the hypothesis that low population structuring occurs within S. chitwoodorum because M. mephitis is widespread across much of North America and has high levels of gene flow. We extracted DNA from 38 samples of Skrjabingylus removed from the sinuses of M. mephitis and one from the plains spotted skunk, Spilogale putorius interrupta, for amplification and sequencing of COI. Analysis of 492 base pairs confirmed all samples were S. chitwoodorum and showed low genetic divergence (1.0%) within Texas, but high haplotype diversity. Supporting our hypothesis, no obvious divergent lineages based on geographic location were recovered within the samples based on Maximum Likelihood analysis and median joining haplotype network analysis. In fact, samples of Skrjabingylus from New York and South Dakota showed little difference compared with samples from Texas.

PREVALENCE AND INTENSITY OF THE SINUS ROUNDWORM ( SKRJABINGYLUS CHITWOODORUM) IN RABIES-NEGATIVE SKUNKS OF TEXAS, USA.

: Estimates of the distribution and prevalence of the sinus roundworm ( Skrjabingylus chitwoodorum) have been based largely on the inspection of skunk (Mephitidae) skulls showing damage from infections. We examined 595 striped skunks ( Mephitis mephitis) and nine hog-nosed skunks ( Conepatus leuconotus) that had tested negative for rabies by the Texas Department of State Health Services (US) between November 2010 and April 2015 to determine species of Skrjabingylus, prevalence and intensity of infection, and distribution of infection in Texas by county. We expected ecoregions with more precipitation to have higher rates of infection than more-arid ecoregions. Prevalence of S. chitwoodorum in striped skunks was 48.7%, with a mean intensity of 19.4 (SD=24.44, range=1-181) nematodes. There was a bias for the left sinus. The prevalence of infection varied among ecoregions of Texas, but it was not correlated with precipitation. Infection intensity did not vary among ecoregions. The prevalence of sinus roundworms in rabies-negative skunks suggested that behavioral changes because of skrjabingylosis might have been responsible for the submission by the public of some skunks for rabies testing.

Susceptibility of avian influenza viruses of the N6 subtype to the neuraminidase inhibitor oseltamivir.

Avian influenza viruses are a source of genetic material that can be transmitted to humans through direct introduction or reassortment. Although there is a wealth of information concerning global monitoring for antiviral resistance among human viruses of the N1 and N2 neuraminidase (NA) subtypes, information concerning avian viruses of these and other NA subtypes is limited. We undertook a surveillance study to investigate the antiviral susceptibility of avian influenza N6 NA viruses, the predominant subtype among wild waterfowl. We evaluated 73 viruses from North American ducks and shorebirds for susceptibility to the NA inhibitor oseltamivir in a fluorescence-based NA enzyme inhibition assay. Most (90%) had mean IC(50) values ranging from <0.01 to 5.0nM; 10% were from 5.1 to 50.0nM; and none were >50.0nM. Susceptibility to oseltamivir remained stable among all isolates collected over approximately three decades (P⩽0.74). Two isolates with I222V NA substitution had moderately reduced susceptibility to oseltamivir in vitro (IC(50), 30.0 and 40.0nM). One field sample was a mixed population containing an avian paramyxovirus (APMV) and H4N6 influenza virus, as revealed by electron microscopy and hemagglutination inhibition assays with a panel of anti-APMV antisera. This highlights the importance of awareness and careful examination of non-influenza pathogens in field samples from avian sources. This study showed that oseltamivir-resistant N6 NA avian influenza viruses are rare, and must be tested both phenotypically and genotypically to confirm resistance.

Live bird markets of Bangladesh: H9N2 viruses and the near absence of highly pathogenic H5N1 influenza.

Avian influenza surveillance in Bangladesh has been passive, relying on poultry farmers to report suspected outbreaks of highly pathogenic H5N1 influenza. Here, the results of an active surveillance effort focusing on the live-bird markets are presented. Prevalence of influenza infection in the birds of the live bird markets is 23.0%, which is similar to that in poultry markets in other countries. Nearly all of the isolates (94%) were of the non-pathogenic H9N2 subtype, but viruses of the H1N2, H1N3, H3N6, H4N2, H5N1, and H10N7 subtypes were also observed. The highly pathogenic H5N1-subtype virus was observed at extremely low prevalence in the surveillance samples (0.08%), and we suggest that the current risk of infection for humans in the retail poultry markets in Bangladesh is negligible. However, the high prevalence of the H9 subtype and its potential for interaction with the highly pathogenic H5N1-subtype, i.e., reassortment and attenuation of host morbidity, highlight the importance of active surveillance of the poultry markets.

Inactivated seasonal influenza vaccines increase serum antibodies to the neuraminidase of pandemic influenza A(H1N1) 2009 virus in an age-dependent manner.

Levels of preexisting antibodies to the hemagglutinin of pandemic influenza A(H1N1) 2009 (hereafter pandemic H1N1) virus positively correlate with age. The impact of contemporary seasonal influenza vaccines on establishing immunity to other pandemic H1N1 proteins is unknown. We measured serum antibodies to the neuraminidase (NA) of pandemic H1N1 in adults prior to and after vaccination with seasonal trivalent inactivated influenza vaccines. Serum antibodies to pandemic H1N1 NA were observed in all age groups; however, vaccination elevated levels of pandemic H1N1 NA antibodies predominately in elderly individuals (age, ⩾60 years). Therefore, contemporary seasonal vaccines likely contribute to reduction of pandemic H1N1-associated disease in older individuals.

Coincident ruddy turnstone migration and horseshoe crab spawning creates an ecological 'hot spot' for influenza viruses.

Since 1985, avian influenza virus surveillance has been conducted annually from mid-May to early June in charadriiform species from the families Scolopacidae and Laridae (shorebirds and gulls) at Delaware Bay in the northeast United States. The mass migrations of shorebirds, gulls and horseshoe crabs (Limulus polyphemus) coincide at that time, and large numbers of migrating birds pause at Delaware Bay to feed on horseshoe crab eggs deposited at the high-tide line. Influenza viruses are consistently isolated from charadriiform birds at Delaware Bay, at an overall rate approximately 17 times the combined rate of isolation at all other surveillance sites worldwide (490 isolates/9474 samples, 5.2% versus 49 isolates per 15,848 samples, 0.3%, respectively; Proportion test, p < 0.0001). The likelihood of isolating influenza viruses at Delaware Bay is dependent on the presence of ruddy turnstone (Arenaria interpres) at the sampling site (G-test of independence, p < 0.001). The convergence of host factors and environmental factors results in a unique ecological 'hot spot' for influenza viruses in Charadriiformes.

Thermostability of subpopulations of H2N3 influenza virus isolates from mallard ducks.

Maintenance of avian influenza virus in waterfowl populations requires that virions remain infectious while in the environment. Temperature has been shown to negatively correlate with persistence time, which is the duration for which virions are infectious. However, thermostability can vary between isolates regardless of subtype, and it is not known whether this variation occurs when host and geographic location of isolation are controlled. In this study, we analyzed the thermostabilities of 7 H2N3 viruses isolated from mallard ducks in Alberta, Canada. Virus samples were incubated at 37 degrees C and 55 degrees C, and infectivity titers were calculated at different time points. Based on the rate of infectivity inactivation at 37 degrees C, isolates could be grouped into either a thermosensitive or thermostable fraction for both egg- and MDCK-grown virus populations. Titers decreased more rapidly for isolates incubated at 55 degrees C, and this loss of infectivity occurred in a nonlinear, 2-step process, which is in contrast with the consensus on thermostability. This suggests that stock samples contain a mixture of subpopulations with different thermostabilities. The rate of decrease for the sensitive fraction was approximately 14 times higher than that for the stable fraction. The presence of subpopulations is further supported by selection experiments and plaque purification, both of which result in homogenous populations that exhibit linear decreases of infectivity titer. Therefore, variation of thermostability of influenza virus isolates begins at the level of the population. The presence of subpopulations with high thermostability suggests that avian viruses can persist in water longer than previously estimated, thus increasing the probability of transmission to susceptible hosts.

Antiviral susceptibility of avian and swine influenza virus of the N1 neuraminidase subtype.

Influenza viruses of the N1 neuraminidase (NA) subtype affecting both animals and humans caused the 2009 pandemic. Anti-influenza virus NA inhibitors are crucial early in a pandemic, when specific influenza vaccines are unavailable. Thus, it is urgent to confirm the antiviral susceptibility of the avian viruses, a potential source of a pandemic virus. We evaluated the NA inhibitor susceptibilities of viruses of the N1 subtype isolated from wild waterbirds, swine, and humans. Most avian viruses were highly or moderately susceptible to oseltamivir (50% inhibitory concentration [IC(50)], <5.1 to 50 nM). Of 91 avian isolates, 7 (7.7%) had reduced susceptibility (IC(50), >50 nM) but were sensitive to the NA inhibitors zanamivir and peramivir. Oseltamivir susceptibility ranged more widely among the waterbird viruses (IC(50), 0.5 to 154.43 nM) than among swine and human viruses (IC(50), 0.33 to 2.56 nM). Swine viruses were sensitive to oseltamivir, compared to human seasonal H1N1 isolated before 2007 (mean IC(50), 1.4 nM). Avian viruses from 2007 to 2008 were sensitive to oseltamivir, in contrast to the emergence of resistant H1N1 in humans. Susceptibility remained high to moderate over time among influenza viruses. Sequence analysis of the outliers did not detect molecular markers of drug-resistance (e.g., H275Y NA mutation [N1 numbering]) but revealed mutations outside the NA active site. In particular, V267I, N307D, and V321I residue changes were found, and structural analyses suggest that these mutations distort hydrophobic pockets and affect residues in the NA active site. We determined that natural oseltamivir resistance among swine and wild waterbirds is rare. Minor naturally occurring variants in NA can affect antiviral susceptibility.

Puzzling inefficiency of H5N1 influenza vaccines in Egyptian poultry.

In Egypt, efforts to control highly pathogenic H5N1 avian influenza virus in poultry and in humans have failed despite increased biosecurity, quarantine, and vaccination at poultry farms. The ongoing circulation of HP H5N1 avian influenza in Egypt has caused >100 human infections and remains an unresolved threat to veterinary and public health. Here, we describe that the failure of commercially available H5 poultry vaccines in Egypt may be caused in part by the passive transfer of maternal H5N1 antibodies to chicks, inhibiting their immune response to vaccination. We propose that the induction of a protective immune response to H5N1 is suppressed for an extended period in young chickens. This issue, among others, must be resolved and additional steps must be taken before the outbreaks in Egypt can be controlled.

Three amino acid changes in PB1-F2 of highly pathogenic H5N1 avian influenza virus affect pathogenicity in mallard ducks.

Despite reports that the PB1-F2 protein contributes to influenza virus pathogenicity in the mouse model, little is known about its significance in avian hosts. In our previous study, the A/Vietnam/1203/04 (H5N1) wild-type virus (wtVN1203) was more lethal to mallard ducks than a reverse genetics (rg)-derived VN1203. In search of potential viral factors responsible for this discrepancy, we found that synonymous mutations (SMs) had been inadvertently introduced into three genes of the rgVN1203 (rgVN1203/SM-3). Of 11 SMs in the PB1 gene, three resided in the PB1-F2 open reading frame, caused amino acid (aa) substitutions in the PB1-F2 protein, and reduced virus lethality in mallard ducks. The wtVN1203 and recombinant viruses with repairs to these three aa's (rgVN1203/R-PB1-F2) or with repairs to all 11 SMs (rgVN1203/R-PB1) were significantly more pathogenic than rgVN1203/SM-3. In cultured cells, repairing three mutations in PB1-F2 increased viral polymerase activity and expression levels of viral RNA.

Extensive mammalian ancestry of pandemic (H1N1) 2009 virus.

We demonstrate that the novel pandemic influenza (H1N1) viruses have human virus-like receptor specificity and can no longer replicate in aquatic waterfowl, their historic natural reservoir. The biological properties of these viruses are consistent with those of their phylogenetic progenitors, indicating longstanding adaptation to mammals.

Ducks: the "Trojan horses" of H5N1 influenza.

Wild ducks are the main reservoir of influenza A viruses that can be transmitted to domestic poultry and mammals, including humans. Of the 16 hemagglutinin (HA) subtypes of influenza A viruses, only the H5 and H7 subtypes cause highly pathogenic (HP) influenza in the natural hosts. Several duck species are naturally resistant to HP Asian H5N1 influenza viruses. These duck species can shed and spread virus from both the respiratory and intestinal tracts while showing few or no disease signs. While the HP Asian H5N1 viruses are 100% lethal for chickens and other gallinaceous poultry, the absence of disease signs in some duck species has led to the concept that ducks are the "Trojan horses" of H5N1 in their surreptitious spread of virus. An important unresolved issue is whether the HP H5N1 viruses are maintained in the wild duck population of the world. Here, we review the ecology and pathobiology of ducks infected with influenza A viruses and ducks' role in the maintenance and spread of HP H5N1 viruses. We also identify the key questions about the role of ducks that must be resolved in order to understand the emergence and control of pandemic influenza. It is generally accepted that wild duck species can spread HP H5N1 viruses, but there is insufficient evidence to show that ducks maintain these viruses and transfer them from one generation to the next.

TNF/iNOS-producing dendritic cells are the necessary evil of lethal influenza virus infection.

Respiratory infection with highly pathogenic influenza A viruses is characterized by the exuberant production of cytokines and chemokines and the enhanced recruitment of innate inflammatory cells. Here, we show that challenging mice with virulent influenza A viruses, including currently circulating H5N1 strains, causes the increased selective accumulation of a particular dendritic cell subset, the tipDCs, in the pneumonic airways. These tipDCs are required for the further proliferation of influenza-specific CD8(+) T cells in the infected lung, because blocking their recruitment in CCR2(-/-) mice decreases the numbers of CD8(+) effectors and ultimately compromises virus clearance. However, diminution rather than total elimination of tipDC trafficking by treatment with the peroxisome proliferator-activated receptor-gamma agonist pioglitazone moderates the potentially lethal consequences of excessive tipDC recruitment without abrogating CD8(+) T cell expansion or compromising virus control. Targeting the tipDCs in this way thus offers possibilities for therapeutic intervention in the face of a catastrophic pandemic.