Population genomic transformations induced by isolation of wild bird avian influenza viruses (Orthomyxoviridae) in embryonated chicken eggs.

Isolation and cultivation of wild-type viruses in model organism cells or tissues is standard practice in virology. Oftentimes, the virus host species is distantly related to the species from which the culture system was developed. Thus, virus culture in these tissues and cells basically constitutes a host jump, which can lead to genomic changes through genetic drift and/or adaptation to the culture system. We directly sequenced 70 avian influenza virus (Orthomyxoviridae) genomes from oropharyngeal/cloacal swabs collected from wild bird species and paired virus isolates propagated from the same samples following isolation in specific-pathogen-free embryonated chicken eggs. The data were analyzed using population genetic approaches including evaluation of single nucleotide polymorphism (SNP) frequencies and divergence with pooled-sequencing analyses, consensus sequence placement in neighbor-joining trees, and haplotype reconstruction and networks. We found that propagation of virus in eggs leads to skewed SNP mutation spectra with some SNPs going to fixation. Both synonymous and nonsynonmous SNP frequencies shifted. We found multiple consensus sequences that differed between the swabs and the isolates, with some sequences from the same sample falling into divergent genetic clusters. Twenty of 23 coinfections detected had different dominant subtypes following virus isolation, thus sequences from both the swab and isolate were needed to obtain full subtype data. Haplotype networks revealed haplotype frequency shifts and the appearance or loss of low-frequency haplotypes following isolation. The results from this study revealed that isolation of wild bird avian influenza viruses in chicken eggs leads to skewed populations that are different than the input populations. Consensus sequence changes from virus isolation can lead to flawed phylogenetic inferences, and subtype detection is biased. These results suggest that for genomic studies of wild bird influenza viruses the biological field should move away from chicken egg isolation towards directly sequencing the virus from host samples.

Predicting whole genome sequencing success for archived avian influenza virus (Orthomyxoviridae) samples using real-time and droplet PCRs.

Long-term viral archives are valuable sources of research data. Each archive can store hundreds of thousands of diverse sample types. In the current era of whole genome sequencing, archived samples become a rich source of evolutionary and epidemiological data that can span years, and even decades. However, the ability to obtain high quality viral whole genome sequences from samples of various types, age, and quality is inconsistent. A minimum quality threshold that helps predict the best success of obtaining high quality genomic sequences for both recent and archived samples is highly valuable. Real-time reverse transcription PCR (rrt-PCR) and droplet digital PCR (ddPCR) are useful tools to evaluate nucleic acid integrity. We hypothesized that diagnostic rrt-PCR and ddPCR data for avian influenza virus (AIV) can predict viral whole genome sequencing success. To test this hypothesis we used RNA extracted from cloacal and oropharyngeal swabs stored in the USDA-APHIS National Wildlife Disease Program Wildlife Tissue Archive. We determined that a specific rrt-PCR Cq value or ddPCR copies/µL resulted in recovery of complete sequences of all eight AIV gene segments. We used logistic regression to estimate probabilities of whole genome recovery at 0.95 (Cq = 15, copies/µL = 49,350), 0.75 (Cq = 24, copies/µL = 16,800), 0.50 (Cq = 29, copies/µL = <1), and 0.25 (Cq = 235, copies/µL = <1). We also identified values at which we predictably recovered HA and NA segments for diagnosing subtypes (Cq = 27.29; copies/µL = 757.50). This approach will allow researchers to assess the potential success of AIV whole genome recovery from diagnostic samples collected in routine AIV surveillance.

Blood Lactate Concentration Is Not Related to the Increase in Cardiorespiratory Fitness Induced by High Intensity Interval Training.

BACKGROUND: There is individual responsiveness to exercise training as not all individuals experience increases in maximal oxygen uptake (VO2max), which does not benefit health status considering the association between VO2max and mortality. Approximately 50% of the training response is genetic, with the other 50% accounted for by variations in dietary intake, sleep, recovery, and the metabolic stress of training. This study examined if the blood lactate (BLa) response to high intensity interval training (HIIT) as well as habitual dietary intake and sleep duration are associated with the resultant change in VO2max (ΔVO2max). METHODS: Fourteen individuals (age and VO2max = 27 ± 8 years and 38 ± 4 mL/kg/min, respectively) performed nine sessions of HIIT at 130% ventilatory threshold. BLa was measured during the first and last session of training. In addition, sleep duration and energy intake were assessed. RESULTS: Data showed that VO2max increased with HIIT (p = 0.007). No associations occurred between ΔVO2max and BLa (r = 0.44, p = 0.10), energy intake (r = 0.38, p = 0.18), or sleep duration (r = 0.14, p = 0.62). However, there was a significant association between training heart rate (HR) and ΔVO2max (r = 0.62, p = 0.02). CONCLUSIONS: When HIIT is prescribed according to a metabolic threshold, energy intake, sleep status, and BLa do not predict ΔVO2max, yet the HR response to training is associated with the ΔVO2max.

Change in VO2max and time trial performance in response to high-intensity interval training prescribed using ventilatory threshold.

Completion of high-intensity interval training (HIIT) leads to significant increases in maximal oxygen uptake (VO2max) and oxidative capacity. However, individual responses to HIIT have been identified as approximately 20-40% of individuals show no change in VO2max, which may be due to the relatively homogeneous approach to implementing HIIT. PURPOSE: This study tested the effects of HIIT prescribed using ventilatory threshold (VT) on changes in VO2max and cycling performance. METHODS: Fourteen active men and women (age and VO2max = 27 ± 8 year and 38 ± 4 mL/kg/min) underwent nine sessions of HIIT, and 14 additional men and women (age and VO2max = 22 ± 3 year and 40 ± 5 mL/kg/min) served as controls. Training was performed on a cycle ergometer at a work rate equal to 130%VT and consisted of eight to ten 1 min bouts interspersed with 75 s of recovery. At baseline and post-testing, they completed progressive cycling to exhaustion to determine VO2max, and on a separate day, a 5 mile cycling time trial. RESULTS: Compared to the control group, HIIT led to significant increases in VO2max (6%, p = 0.007), cycling performance (2.5%, p = 0.003), and absolute VT (9 W, p = 0.005). However, only 57% of participants revealed meaningful increases in VO2max and cycling performance in response to training, and two showed no change in either outcome. CONCLUSIONS: A greater volume of HIIT may be needed to maximize the training response for all individuals.

LEPTOSPIRA ANTIBODIES DETECTED IN WILDLIFE IN THE USA AND THE US VIRGIN ISLANDS.

From 2011 to 2017, 4,534 serum samples from 13 wildlife species collected across the US and in one territory (US Virgin Islands) were tested for exposure to Leptospira serovars Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, and Pomona. Of 1,759 canids, 1,043 cervids, 23 small Indian mongooses ( Herpestes auropunctatus), 1,704 raccoons ( Procyon lotor), and five striped skunks ( Mephitis mephitis), 27.0, 44.4, 30.4, 40.8, and 60%, respectively, were antibody positive for any of the six serovars. The most commonly detected serovars across all species were Bratislava and Grippotyphosa. Our results indicate that Leptospira titers are very common in a wide variety of wildlife species. These species may act as important reservoirs in the epidemiological cycle of the pathogen. Additional studies to determine the relationship between serologic evidence and shedding of the pathogen by wildlife are necessary to better understand the risk.

LIMITED ANTIBODY EVIDENCE OF EXPOSURE TO MYCOBACTERIUM BOVIS IN FERAL SWINE (SUS SCROFA) IN THE USA.

Bovine tuberculosis is a chronic disease of cattle ( Bos taurus ) caused by the bacterium Mycobacterium bovis . Efforts have been made in the US to eradicate the disease in cattle, but spillover into wildlife and subsequent spillback have impeded progress in some states. In particular, infection in white-tailed deer ( Odocoileus virginianus ) has been followed by infection in cattle in some Midwestern states. Infection has also been documented in feral swine ( Sus scrofa ) on the Hawaiian island of Molokai and in various European countries, but no large-scale survey of antibody exposure to the bacteria has been conducted in feral swine in the US. We tested 488 sera from feral swine collected near previously documented outbreaks of bovine tuberculosis in cattle and captive cervids, in addition to 2,237 feral swine sera collected across the US from 1 October 2013 to 30 September 2014. While all but one of the samples were antibody negative, the results are important for establishing baseline negative data since feral swine are capable reservoirs and could be implicated in future outbreaks of the disease.

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.

Molecular surveillance of low pathogenic avian influenza viruses in wild birds across the United States: inferences from the hemagglutinin gene.

A United States interagency avian influenza surveillance plan was initiated in 2006 for early detection of highly pathogenic avian influenza viruses (HPAIV) in wild birds. The plan included a variety of wild bird sampling strategies including the testing of fecal samples from aquatic areas throughout the United States from April 2006 through December 2007. Although HPAIV was not detected through this surveillance effort we were able to obtain 759 fecal samples that were positive for low pathogenic avian influenza virus (LPAIV). We used 136 DNA sequences obtained from these samples along with samples from a public influenza sequence database for a phylogenetic assessment of hemagglutinin (HA) diversity in the United States. We analyzed sequences from all HA subtypes except H5, H7, H14 and H15 to examine genetic variation, exchange between Eurasia and North America, and geographic distribution of LPAIV in wild birds in the United States. This study confirms intercontinental exchange of some HA subtypes (including a newly documented H9 exchange event), as well as identifies subtypes that do not regularly experience intercontinental gene flow but have been circulating and evolving in North America for at least the past 20 years. These HA subtypes have high levels of genetic diversity with many lineages co-circulating within the wild birds of North America. The surveillance effort that provided these samples demonstrates that such efforts, albeit labor-intensive, provide important information about the ecology of LPAIV circulating in North America.

Shedding and serologic responses following primary and secondary inoculation of house sparrows (Passer domesticus) and European starlings (Sturnus vulgaris) with low-pathogenicity avian influenza virus.

Waterfowl and shorebirds are well-recognized natural reservoirs of low-pathogenicity avian influenza viruses (LPAIV); however, little is known about the role of passerines in avian influenza virus ecology. Passerines are abundant, widespread, and commonly come into contact with free-ranging birds as well as captive game birds and poultry. We inoculated and subsequently challenged house sparrows (Passer domesticus) and European starlings (Sturnus vulgaris) with wild-bird origin LPAIV H3N8 to evaluate their potential role in transmission. Oropharyngeal shedding was short lived, and was detected in more starlings (97.2%) than sparrows (47.2%; n=36 of each). Cloacal shedding was rare in both species (8.3%; n=36 of each) and no cage-mate transmission occurred. Infectious LPAIV was cultured from oropharyngeal and cloacal swabs and gastrointestinal and respiratory tissues from both species. Seroconversion was detected as early as 3 days post inoculation (d.p.i.) (16.7% of sparrows and 0% of starlings; n=6 each); 50% of these individuals seroconverted by 5 d.p.i., and nearly all birds (97%; n=35) seroconverted by 28 d.p.i. In general, pre-existing homologous immunity led to reduced shedding and increased antibody levels within 7 days of challenge. Limited shedding and lack of cage-mate transmission suggest that passerines are not significant reservoirs of LPAIV, although species differences apparently exist. Passerines readily and consistently seroconverted to LPAIV, and therefore inclusion of passerines in epidemiological studies of influenza outbreaks in wildlife and domestic animals may provide further insight into the potential involvement of passerines in avian influenza virus transmission ecology.

Base composition profiling of human mitochondrial DNA using polymerase chain reaction and direct automated electrospray ionization mass spectrometry.

We describe an automated system for high-resolution profiling of human mitochondrial DNA (mtDNA) based upon multiplexed polymerase chain reaction (PCR) followed by desolvation and direct analysis using electrospray ionization mass spectrometry (PCR/ESI-MS). The assay utilizes 24 primer pairs that amplify targets in the mtDNA control region, including the hypervariable regions typically sequenced in a forensic analysis. Profiles consisting of product base compositions can be stored in a database, compared to each other, and compared to sequencing results. Approximately 94% of discriminating information obtained by sequencing is retained with this technique. The assay is more discriminating than sequencing minimum HV1 and HV2 regions because it interrogates more of the mitochondrial genome. A profile compared to a population database can be subjected to the same statistics used for assessing the significance of concordant mtDNA sequences. The assay is not hindered by length heteroplasmy, can directly analyze template mixtures, and has a sensitivity of <25 pg of total DNA per reaction. Analysis of 3331 independent trials of the same sample over 28 months produced an average mass measurement uncertainty of 10.1 +/- 8.0 ppm, with >99% of trials producing a full profile with automated analysis. The technique has direct application to analysis of forensic biological evidence.

Increased detection of influenza A H16 in the United States.

As a result of an US interagency avian influenza surveillance effort in wild birds, four isolates of influenza A viruses were initially identified as H7 by hemagglutination inhibition (HI) but subsequently identified as H16 through genetic sequence analysis. We report the development of internal primers for amplification and cycle-sequencing of the full-length H16 gene, increased detection of H16 within the US, and possible steric inhibition or cross-reaction between H7 and H16 antigens during the conventional HI assay. The latter could have critical implications for poultry operations if H16 viruses are detected and mistakenly reported as H7 viruses.