Thomas J. DeLiberto

Highly pathogenic avian influenza viruses and generation of novel reassortants,United States, 2014–2015

Asian highly pathogenic avian influenza A(H5N8) viruses spread into North America in 2014 during autumn bird migration. Complete genome sequencing and phylogenetic analysis of 32 H5 viruses identified novel H5N1, H5N2, and H5N8 viruses that emerged in late 2014 through reassortment with North American low-pathogenicity avian influenza viruses.

Seasonal variation in serum testosterone, testicular volume, and semen characteristics in the coyote (Canis latrans).

The coyote is a seasonally breeding mammal, with most copulations occurring between December and April (depending on location). The objective of this study was to characterize seasonal changes in serum testosterone concentrations, testicular volume, and ejaculate quantity and quality in captive male coyotes. There were seasonal differences in testicular volume, with the greatest volume (20.2+/-5.4cm2), mean+/-S.E.M.) in February, corresponding with peak breeding season. Circulating serum testosterone concentrations peaked (3.31+/-0.9 ng/mL) during January and were positively correlated (P< or =0.001, r=0.413) with testicular volume. Ejaculate volume (1.67+/-0.4 mL) and sperm concentration (549.2 x 10(6)+/-297.7 spermatozoa/mL) both peaked during January and February, consistent with the height of the breeding season. Ejaculate volume and sperm concentrations were positively correlated with testicular size (r=0.679, P< or =0.001 and r=0.499, P< or =0.001, respectively) and with serum testosterone concentrations (r=0.368, P< or =0.01 and r=0.208, P< or =0.05). Progressively motile, viable, and morphologically normal spermatozoa fluctuated seasonally, peaked (90.4+/-4.5, 84.8+/-4.1, and 87.9+/-2.9%) during the breeding season, and then subsequently declined (period of aspermatogenesis). All three of these end points were positively correlated with testicular size (r=0.589, P< or =0.001; r=0.586, P< or =0.001; and r=0.469; P< or =0.001) and serum testosterone (r=0.167, P< or =0.05; r=0.190, P< or =0.05; and r=0.221, P< or =0.01). In conclusion, there were intricate relationships among testosterone concentrations, testicular volume, and the production of both functionally intact and morphologically normal spermatozoa.

Reassortant H9N2 Influenza Viruses Containing H5N1-Like PB1 Genes Isolated from Black-Billed Magpies in Southern China

H9N2 influenza A viruses have become endemic in different types of terrestrial poultry and wild birds in Asia, and are occasionally transmitted to humans and pigs. To evaluate the role of black-billed magpies (Pica pica) in the evolution of influenza A virus, we conducted two epidemic surveys on avian influenza viruses in wild black-billed magpies in Guangxi, China in 2005 and characterized three isolated black-billed magpie H9N2 viruses (BbM viruses). Phylogenetic analysis indicated that three BbM viruses were almost identical with 99.7 to 100% nucleotide homology in their whole genomes, and were reassortants containing BJ94-like (Ck/BJ/1/94) HA, NA, M, and NS genes, SH/F/98-like (Ck/SH/F/98) PB2, PA, and NP genes, and H5N1-like (Ck/YN/1252/03, clade 1) PB1 genes. Genetic analysis showed that BbM viruses were most likely the result of multiple reassortments between co-circulating H9N2-like and H5N1-like viruses, and were genetically different from other H9N2 viruses because of the existence of H5N1-like PB1 genes. Genotypical analysis revealed that BbM viruses evolved from diverse sources and belonged to a novel genotype (B46) discovered in our recent study. Molecular analysis suggested that BbM viruses were likely low pathogenic reassortants. However, results of our pathogenicity study demonstrated that BbM viruses replicated efficiently in chickens and a mammalian mouse model but were not lethal for infected chickens and mice. Antigenic analysis showed that BbM viruses were antigenic heterologous with the H9N2 vaccine strain. Our study is probably the first report to document and characterize H9N2 influenza viruses isolated from black-billed magpies in southern China. Our results suggest that black-billed magpies were susceptible to H9N2 influenza viruses, which raise concerns over possible transmissions of reassortant H9N2 viruses among poultry and wild birds.

Large-Scale Avian Influenza Surveillance in Wild Birds throughout the United States

Avian influenza is a viral disease that primarily infects wild and domestic birds, but it also can be transmitted to a variety of mammals. In 2006, the United States of America Departments of Agriculture and Interior designed a large-scale, interagency surveillance effort that sought to determine if highly pathogenic avian influenza viruses were present in wild bird populations within the United States of America. This program, combined with the Canadian and Mexican surveillance programs, represented the largest, coordinated wildlife disease surveillance program ever implemented. Here we analyze data from 197,885 samples that were collected from over 200 wild bird species. While the initial motivation for surveillance focused on highly pathogenic avian influenza, the scale of the data provided unprecedented information on the ecology of avian influenza viruses in the United States, avian influenza virus host associations, and avian influenza prevalence in wild birds over time. Ultimately, significant advances in our knowledge of avian influenza will depend on both large-scale surveillance efforts and on focused research studies.

NIGHTLY AND SEASONAL MOVEMENTS, SEASONAL HOME RANGE, AND FOCAL LOCATION PHOTO-MONITORING OF URBAN STRIPED SKUNKS (MEPHITIS MEPHITIS): IMPLICATIONS FOR RABIES TRANSMISSION

We followed radio-collared striped skunks (Mephitis mephitis) from January 2004– December 2005 in two urban areas of Flagstaff, Arizona, USA to determine seasonal patterns of movement and home-range size. We also used automated cameras to determine the potential for inter- and intraspecific interaction at skunks diurnal resting sites and nocturnal focal locations. We found no difference between sexes in nightly rates of travel or in size of seasonal home range. Nightly rates of travel were greatest in the postbreeding months (May–July) and smallest from November to February, consistent with larger home ranges (95% kernel estimates) from March– August and smaller home ranges from September–February. Sixty-three percent of monitored males and 38% of monitored females crossed the urban–wildland interface, in at least one direction on at least one night, and some remained outside the urban area for days or weeks, indicating that skunks could act as vectors of disease across the urban–wildland interface. We recorded co-occurrence of skunks with domestic cats (Felis domesticus), raccoons (Procyon lotor), gray foxes (Urocyon cinereoargenteus), and other skunks at focal locations and diurnal retreats used by skunks, suggesting these areas are potential sites for both inter- and intraspecific rabies transmission and could be targeted by wildlife managers during trapping or vaccination programs.

Identification and analysis of the first 2009 pandemic H1N1 influenza virus from U.S. feral swine.

The first case of pandemic H1N1 influenza (pH1N1) virus in feral swine in the United States was identified in Texas through the United States Department of Agriculture (USDA) Wildlife Services’ surveillance program. Two samples were identified as pandemic influenza by reverse transcriptase quantitative PCR (RT‐qPCR). Full‐genome Sanger sequencing of all eight influenza segments was performed. In addition, Illumina deep sequencing of the original diagnostic samples and their respective virus isolation cultures were performed to assess the feasibility of using an unbiased whole‐genome linear target amplification method and multiple sample sequencing in a single Illumina GAIIx lane. Identical sequences were obtained using both techniques. Phylogenetic analysis indicated that all gene segments belonged to the pH1N1 (2009) lineage. In conclusion, we have identified the first pH1N1 isolate in feral swine in the United States and have demonstrated the use of an easy unbiased linear amplification method for deep sequencing of multiple samples.

Surveillance for Avian Influenza Viruses in Wild Birds in Denmark and Greenland, 2007–10

SUMMARY. In Denmark and Greenland, extensive surveillance of avian influenza (AI) viruses in wild bird populations has been conducted from 2007 through 2010. In Denmark, the surveillance consisted of passive surveillance of wild birds found dead or sick across Denmark and active surveillance of apparently healthy live birds in waterfowl reservoirs and along migratory flyways, birds living in proximity to domestic poultry, and hunted game birds. Dead birds were sampled by oropharyngeal swabbing. Healthy live wild birds were captured with nets, traps, or by hand and were sampled by swabbing of the oropharyngeal and cloacal tracts, or swabs were collected from fresh fecal droppings. Hunted game birds were delivered to game-handling establishments, where each bird was sampled by oropharyngeal and cloacal swabbing. During the 2007–10 period, a total of 11,055 wild birds were sampled in Denmark, of which 396 were birds that were found dead. In Greenland, samples were collected mainly from fecal droppings in breeding areas. Samples from 3555 live and apparently healthy wild birds were tested. All swab samples were tested by pan-influenza reverse transcriptase–PCR (RT-PCR), and the positive samples were further tested by H5/H7 specific RT-PCRs. H5/H7-positive samples were subjected to hemagglutination cleavage site sequencing for pathotyping. In addition, all RT-PCR–positive samples were subjected to virus isolation, and the virus isolates were subsequently subtyped. In Denmark, low pathogenic (LP) H5 viruses were detected throughout the period, in addition to a few LPAI H7 and several other subtypes. In Greenland, very few samples were positive for AI. None of them were found to be of the H5 or H7 subtypes by RT-PCR. Isolation of these viruses in eggs was unsuccessful; thus, they were not subtyped further. The findings did, however, demonstrate the presence of LPAI viruses in Greenland. For several water bird species overwintering in North America and northwest Europe, respectively, Greenland constitutes a common breeding area. This raises the possibility that viruses could be transmitted to North America via Greenland and vice versa. In Denmark, the screenings for AI showed LPAI viruses to be naturally occurring in the wild bird population, particularly in waterfowl. The occurrence of AI viruses in the wild bird population may pose a risk for AI infections in Danish poultry.

Diffusion of influenza viruses among migratory birds with a focus on the Southwest United States

The Southwest United States, including Arizona and New Mexico, has a diverse climate and is home to many different avian species. We sequenced the hemagglutinin (HA) gene of twenty influenza specimens for the years 2007-2009. This included four from Arizona, and sixteen from New Mexico. We analyzed the sequences and determined the following HA subtypes: H3, H4, H6, H8, and H11. For each subtype, we combined our virus sequences with those from a public database, and inferred phylogeographic models of influenza diffusion. Statistical phylogeography indicated that overall evolutionary diffusion of avian influenza viruses is geographically structured (p<0.05). In addition, we found that diffusion to the Southwest was often from nearby states including California. For H3, H4 and H6, the intra-flyway gene flow rates were significantly (p<0.001) higher than those of inter-flyway. Such rate difference was also observed in H8 and H11, yet, without statistical significance (p=0.132, p=0.190, respectively). Excluding any one flyway from the calculation generated similar results, suggesting that such barrier effect on gene flow rates is not exclusively produced by any single flyway. We also calculated the Bayes factor test for the significant non-zero rates between states and identified significant routes both within and across flyways. Such inter-flyway spread of influenza was probably the result of birds from four flyways co-mingling on breeding grounds in northern regions or marshaling on staging areas post breeding in Canada or Alaska, before moving south each fall. This study provides an initial analysis of evolutionary diffusion of avian influenza virus to and from the Southwest United States. However, more sequences from this region need to be generated to determine the role of host migration and other factors on influenza diffusion.

Evolutionary Characterization of the Pandemic H1N1/2009 Influenza Virus in Humans Based on Non-Structural Genes

The 2009 influenza pandemic had a tremendous social and economic impact. To study the genetic diversity and evolution of the 2009 H1N1 virus, a mutation network for the non-structural (NS) gene of the virus was constructed. Strains of the 2009 H1N1 pandemic influenza A virus could be divided into two categories based on the V123I mutation in the NS1 gene: G1 (characterized as 123 Val) and G2 (characterized as 123 Ile). Sequence homology analysis indicated that one type of NS sequence, primarily isolated from Mexico, was likely the original type in this pandemic. The two genotypes of the virus presented distinctive clustering features in their geographic distributions. These results provide additional insight into the genetics and evolution of human pandemic influenza H1N1.

Annual Seroprevalence of Yersinia pestis in Coyotes as Predictors of Interannual Variation in Reports of Human Plague Cases in Arizona, United States

Although several health departments collect coyote blood samples for plague surveillance, the association between reported human cases and coyote seroprevalence rates remains anecdotal. Using data from an endemic region of the United States, we sought to quantify this association. From 1974 to 1998, about 2,276 coyote blood samples from four Arizona counties were tested for serological evidence of exposure to Yersinia pestis, the causative agent of plague. Using a titer threshold presumed to be indicative of recent infection (serum titers of ≥1:256), we found a statistically significant relationship between years with >17% sero-positive coyotes and years with two or more human cases reported. Moreover, when the annual coyote seroprevalence rates were dichotomized at 17%, 84% of the years were correctly classified using four biologically relevant meteorological variables in a linear regression. This is the first time a statistically significant temporal association between human plague cases and coyote seroprevalence rates has been shown. However, issues with data resolution and surveillance effort that potentially limit the public health utility of using coyote seroprevalence rates are discussed.