bluuebunny/crossref_metadata_2025 huggingface dataset:
Using Stem Char to Predict Mortality and Insect Infestation of Fire-Damaged Slash Pines
Authors: Dixon Wayne N. , Corneil Jeffrey A. , Wilkinson Robert C. , Foltz John L.
Published: August 2019

Monthly observations of planted slash pines (Pinus elliottii Engelm.) damaged by a March wildfire showed that the magnitude and rapidity of tree mortality were directly related to severity of stem char. Cumulative tree mortalities by November were 5, 25, and 79 percent for 7- to 8-year-old trees in plots with light, medium, and severe stem char classifications (average stem char <61 percent, 61 to 80 percent, and >80 percent of total tree height, respectively). In 15-year-old plantations the mortality rates were 46, 95, and 100 percent for the three damage classes. Most tree mortality occurred rapidly (before mid-May) in the younger plantations and in the severely damaged portions of the older plantations; mortality rates were slower and continued until August in lightly and moderately damaged areas of the older plantations. Ips engraver beetles and the southern pine sawyer were the principal insects infesting trees in May. Reproduction weevil broods were common in tree roots during November. Implications for pest management are discussed.
Seed Source Testing of Alabama Loblolly Pine:
Implications for Seed Movements and Tree Improvement Programs
Authors: Duba Stuart E. , Goggans James F. , Patterson Richard M.
Published: August 2019

Eight Alabama loblolly pine (Pinus taeda L.) seed sources were compared by establishing one plantation near each source of seed. On the basis of growth after 14 years, the eight sources segregated into three groups that corresponded with southern, central, and northern geographic areas. Southern sources generally had the largest heights and volumes, while central sources were intermediate and northern sources smallest. Although some source-by-location interactions did occur, southern sources could be utilized in the central portion of the state and central sources could be utilized in northern areas to provide above-average growth. Implications for tree improvement programs are made.
Hardwood Eradication Improves Productivity of Thinned Loblolly Pine Stands
Authors: Clason T. R.
Published: August 2019

A single hardwood eradication treatment in a 7-year-old loblolly pine (Pinus taeda L.) stand along with pine stocking control increased growth and yield over a 10-year period. Herbaceous vegetation control had no detectable effect at ages 12 and 17. Treated plots had smaller stems, which could be controlled more readily by fire, although the number of understory stems was similar for all treatments. Mean annual radial and merchantable volume growth on treated plots surpassed controls by 30%. Future stand values were enhanced by removing hardwood competition. Residual stand sawtimber volumes on the treated plots were twice that of the control.
Five-Year Performance of Selected Woody Species on an Upper Coastal Plain Spoil Bank
Authors: McMinn James W. , Crane Walter H.
Published: August 2019

Fifth-year survival and height are compared for autumn-olive (Elaeagnus umbellata Thunb.), European alder (Alnus glutinosa (L.) Gaertn.), Japanese flowering crab apple (Malus floribunda Sieb.), sawtooth oak (Quercus acutissima Carruthers), and Virginia pine (Pinus virginiana Mill.) on a sandy clay loam spoil bank in South Carolina. When both survival and height growth are considered, autumn-olive performed best and was a good fruit producer for wildlife. Virginia pine ranked second and may have potential as a fiber producer on disturbed Coastal Plain sites.
Growth Through 13 Years of Two Pruned Cottonwood Plantations Spaced for Sawtimber Production
Authors: Krinard R. M. , Burkhardt E. C.
Published: August 2019

Cottonwood trees (Populus deltoides Bart R. ex Marsh.) pruned in either spring or summer in the third or fourth year to a 1-log height did not differ in growth from unpruned trees during the fifth through thirteenth year interval. At age 13, 84% of the pruned trees had no limbs to a 17-ft. height compared to only 29% of the unpruned trees. Largest trees, pruned and unpruned, averaged 103 ft. tall with a 54% live crown ratio.
Decay Following Thinning of Sweetgum Sprout Clumps: 26-Year Results
Authors: McCracken F. I.
Published: August 2019

Butt rot in living sweetgum (Liquidambar styraciflua) stems was observed 15 and 26 years after the companion living stems were cut in a 55-year-old stand of sprout origin near Tallulah, Louisiana. Living stems had insignificant or no decay 15 years after thinning. Twenty-six years after thinning, an extensive amount of decay had developed in all living stems originating from cut stubs that had split longitudinally down the side during thinning. However, the amount of decay was insignificant or absent when clean cuts had been made which resulted in grown-over stubs that contained water, decay, and anaerobic bacteria. Split stubs did not retain water, and decay had spread into the living stems. Decay risk associated with thinning sprout clumps can apparently be eliminated by undercutting and taking care to prevent splitting during felling.
Schnur's Site-Index Curves Formulated for Computer Applications
Authors: Farrar Robert M.
Published: August 2019

Two systems of interpolation equations are offered for the graphic site-index curves of Schnur for even-aged upland oak stands. One is Wiant's equation system, adjusted to pass through site index at index age. The second is similar but some-what more precise. For both, the average absolute difference between equation system and table values is about 0.73 foot and the maximum absolute difference is about 1.9 feet.
Response of Slash Pine Progenies to Fertilization
Authors: Rockwood D. L. , Windsor C. L. , Hodges J. F.
Published: August 2019

P, NP, and NPK fertilizers were generally effective in increasing growth of some 168 slash pine (Pinus elliottii var. elliottii Engelm.) progenies established in four fertilizer tests in northeast Florida and southeast Georgia. Rust incidence response to fertilization was variable. P and NP additions resulted in higher wood densities. Progeny-fertilizer interaction was observed for only one trait in one test. Variation among progenies was observed for all traits. Fast-growing progenies can be expected to respond well to fertilization.
Honduras Pine Spacing Trial Results in Puerto Rico
Authors: Liegel L. H. , Balmer W. E. , Ryan G. W.
Published: August 2019

Puerto Rico has one of the oldest, continuously measured spacing trials of Honduras pine (Pinus caribaea mor. hondurensis) in existence. At 18 to 20 years, mean survival for all the trial sites except one that suffered hurricane damage in 1979 was 82%. Survival was not related to the triangular spacing treatments of 5, 7, 10, and 14 feet. But spacing did influence diameter growth at all four sites and affected height growth at all but one site Each site had trees ≥100 feet tall at 18 to 20 years; on the two best sites, individual trees reached 90 feet by 12 years. At close spacings, mean annual height increments peaked at 4.0 to 4.5 years on two sites and at 7.2 years on the other two sites. Mean annual diameter increments peaked even earlier, at 3.0 to 3.5 years on the two good sites and at 6.0 years on the two poorer sites. Early effects of competition in reducing both mean annual height and diameter growth, particularly at close spacings, have several managerial implications. The most striking are: (1) underestimating site quality or site index if growth assessments are made only in dense stands, and (2) significant loss of wood volume over long rotations when managers fail to time thinnings before mean annual height and diameter increments decrease.
Beaver Damage to Nonimpounded Timber in Mississippi
Authors: Bullock James F. , Arner Dale H.
Published: August 2019

Beaver (Castor canadensis) damage to nonimpounded timber was investigated in selected stands of merchantable timber located in six major physiographic regions of Mississippi. Sweetgum (Liquidambar styraciflua) was the woody species most used by beaver throughout Mississippi. Values of beaver-damaged merchantable timber ranged from $63.43 to $291.50 per acre; median loss was $164.71 per acre. Of all stems damaged, 72% were girdled, while 28% were felled. Nearly 79% of all stems felled were less than 3 inches dbh and 95% of all stems felled were less than 10 inches dbh.
A Guide to Using Snares for Beaver Capture
Authors: Weaver Keith M. , Arner Dale H. , Mason Carl , Hartley Jeanne J.
Published: August 2019

Extensive field testing indicated that beaver could be captured as effectively with snares as with Conibear or leg-hold traps. Snares were less expensive and did less physical damage to beaver than Conibear or leg-hold traps. A trapping procedure is outlined to help inexperienced trappers capture beaver consistently with snares.
A Simulation-Based Approach to Evaluating Commercial Thinning Decisions in Loblolly Pine Plantations
Authors: Reisinger Thomas W.
Published: August 2019

Computer simulation techniques are described which evaluate the impact of various stand conditions, management regimes, harvesting systems, and future market scenarios on current thinning decisions in pine plantations. Four simulation programs (PTAEDAR, PTAEDA, GENMAC, and HSS) were linked to predict tree growth/stand development and to model mechanized thinnings and final harvests. Simulation results were then combined with an investment analysis program to calculate the net present value of each strategy. An example analysis for a "typical" loblolly pine (Pinus taeda) plantation is presented. As described here, simulation is a practical method of evaluating the profitability of a wide range of alternatives, including planting spacings, thinning regimes, and rotation ages for managing future plantations of loblolly pine.
Planted Longleaf Pine Seedlings Respond to Herbaceous Weed Control Using Herbicides
Authors: Nelson Larry R. , Zutter Bruce R. , Gjerstad Dean H.
Published: August 2019

Height initiation, and height and diameter growth of longleaf pine (Pinus palustris) seedlings were compared on plots receiving herbaceous weed control treatments and on unweeded check plots during the first 4 years after planting. Treatments initiated during the spring following planting included: broadcast weed control for 1 and 2 years, banded weed control in 5-foot bands for 1 and 2 years, and no weed control (check). Weed control had a positive effect on fourth-year height, groundline diameter, and the percentage of seedlings out of the grass stage, while survival was unaffected. The duration of weed control (2 years vs 1 year) had a similar effect on the same response variables, while the method of weed control (broadcast vs. band) had no effect. Trees on plots receiving 2 years of weed control were approximately 3 feet taller and 0.5 inch greater in groundline diameter than trees receiving no weed control. One year of weed control resulted in trees approximately 2 feet taller and 0.3 inch greater in groundline diameter than with no weed control. Weed control treatments shortened the time seedlings were in the grass stage by approximately 1 year, decreasing the time period during which a serious brown-spot needle blight infection could develop.¹
Determining the Initial Annual Payment for a Long-Term Timberland Lease
Authors: Shaffer Robert M. , Klemperer W. David , Meyer Richard D.
Published: August 2019

A method to determine the initial lease payment for a long-term timberland lease is presented from the viewpoint of both the landowner and the firm. The firm's analysis determines the maximum annual lease payment with an after-tax present value equal to the after-tax present value of the anticipated harvest income less all contract costs. The landowner's analysis equates the present value of a series of (less risky) indexed lease payments to the present value of a (more risky) timber production investment alternative.
Pensacola Bahiagrass Can Be Used to Improve the Forage Resource when Regenerating Southern Pines
Authors: Lewis Clifford E. , Manson Warren G. , Bonyata Richard J.
Published: August 2019

Many native forage plants in the South are low quality, poor producers, and unpalatable to cattle. Replacement of these plants with more desirable species would improve the forage resource. One approach is to seed grasses during site preparation when regenerating southern pines. Following site preparation by shearing and strip-disking, Pensacola bahiagrass (Paspalum notatum) was seeded at 15 pounds per acre in the spring and longleaf pine (Pinus palustris) was row-seeded on 12-foot centers in December of the next year. The bahiagrass became established among the residual native plants and was heavily utilized by cattle grazing yearlong. A light application of fertilizer after 3 years tripled bahiagrass yields the first year and doubled it the next year compared to unfertilized plots. Fertilizer improved some nutritional qualities of bahiagrass but digestibility was lowered. Longleaf pine seedlings came out of the grass stage more rapidly and were 50% taller at age 9 with grazing than without it; and in spite of heavier mortality with grazing (36% vs. 21%), stocking was 967 trees per acre at age 11.
Evaluation of the Relative Performance of Pancreas-Specific MicroRNAs in Rat Plasma as Biomarkers of Pancreas Injury
Authors: Erdos Zoltan , Barnum John E , Wang Erjia , DeMaula Christopher , Dey Paritosh Markus , Forest Thomas , Bailey Wendy J , Glaab Warren E
Published: August 2019

Drug-induced pancreatic injury (DIPI) has become linked in recent years to many commonly prescribed medications from several pharmacological classes. Diagnosis is currently most often focused on identification of acute pancreatitis and generally based on subjective clinical assessment and serum amylase and lipase enzymatic activity, which have been criticized as being insufficiently sensitive and specific. The lack of novel noninvasive biomarkers of DIPI can impede the advancement of drug candidates through nonclinical development and translation into clinical settings. Pancreas-specific microRNAs (miRNAs) are currently being evaluated as biomarkers of DIPI that may outperform and/or add value to the interpretation of amylase and lipase. To assess the relative performance of these novel miRNAs, a comprehensive evaluation was conducted to determine the sensitivity and specificity of detecting DIPI in rats. Four miRNAs were evaluated (miR-216a-5p, miR-216b-5p, miR-217-5p, and miR-375-3p) in plasma from 10 studies in which rats were treated with known pancreatic toxicants to assess sensitivity, and from 10 different studies in which toxicity was evident in tissues other than pancreas to assess specificity. The candidate miRNA biomarker performance was compared with amylase and lipase, and receiver operator characteristics (ROC) were determined. Analysis of ROCs demonstrated that all four miRNAs outperformed amylase and lipase in monitoring acute pancreatic injury defined as acinar cell degeneration/necrosis. Specifically, miR-217-5p had the highest performance among all biomarkers assessed. The increased sensitivity and specificity of these miRNAs support their use as biomarkers of DIPI, thereby adding value to the interpretation of amylase and lipase measurements in nonclinical studies. The potential for miRNAs to serve as translational biomarkers in the clinic for the monitoring of DIPI is also supported by this investigation.
A1 The role of virus-antibody co-evolution in the development of a V3-glycan-directed HIV-1 broadly neutralizing antibody lineage
Authors: Kitchin D , Bhiman J , Mvududu D , Oosthuysen B , Lambson B , Madzorera S , Anthony C , Abdool Karim S S , Garrett N J , Doria-Rose N A , Mascola J R , Morris L , Moore P L
Published: August 2019

Broadly neutralizing antibodies (bNAbs) are essential for a preventative HIV-1 vaccine but have not been elicited through vaccination. bNAbs develop in 20–30 per cent of HIV-1 infections and often target the V3-glycan epitope of the HIV envelope protein (Env). In these individuals, virus-antibody co-evolution is thought to drive the maturation of antibody lineages to neutralization breadth. We used deep sequencing of env genes and antibody transcripts from fourteen time points spanning the first 3 years of infection to characterize the virus-antibody co-evolution in donor CAP255 who developed V3-glycan-specific bNAbs. Sequencing and cloning of env genes, followed by neutralization assays, were used to identify Env mutations associated with neutralization escape from two bNAbs (CAP255.G3 and CAP255.C5) isolated at 149 weeks post-infection (wpi). Sequencing data indicated that CAP255 was co-infected by three related viral variants, all of which had an intact N332 glycan, which persisted in > 90 per cent of later viruses. A recombinant V3-region became fixed from 8 wpi, conferring slight neutralization resistance to CAP255.G3/C5 and other V3-glycan bNAbs. Later, T415R/K substitutions in V4 emerged by 51 wpi and were associated with complete viral escape from CAP255.G3/C5, though not from the polyclonal plasma response. All 93-week and later Envs were resistant to CAP255.G3/C5 and V3-glycan bNAb PGT135. Viral escape by 51 wpi suggested that the CAP255 bNAbs arose earlier, driving escape, but persisted to 149 weeks. This was supported by preliminary deep sequencing of the antibody repertoire that indicated bNAb lineage members were already present in the plasma at 39 wpi. Escape from V3-glycan bNAbs via T415R/K mutations have not previously been shown, suggesting a novel mode of recognition within the V3-glycan supersite. Further work will focus on identifying the bNAb-initiating Env and intermediate bNAb lineage members that were capable of engaging contemporaneous Env neutralization escape mutants. Characterization of Envs that engaged bNAb precursors, as well as those that selected for broader members of the bNAb lineage, will inform the design of immunogens capable of eliciting V3-glycan bNAbs in a novel HIV-1 vaccine regimen.
A5 Near full-length HIV-1 genome sequencing in newly diagnosed individuals in Sweden
Authors: Andersson E , Aralaguppe S , Thebo L , Sönnerborg A , Neogi U , Albert J
Published: August 2019

The Swedish HIV-1 epidemic is characterized by a high diversity in HIV subtypes and recombinants as a result of migration. To study the time from infection through viral diversification, transmission patterns, and drug resistance in minor quasispecies, a robust protocol for pan-genotypic near full-length HIV-1 genome (HIV-NFLG) next-generation sequencing (NGS) is key. Our group has established two protocols for HIV-NFLG on the Illumina platform that we aim to compare and, if necessary, modify to find a method with optimized coverage, depth, and subtype inclusivity. Zanini et al. (https://doi.org/10.7554/eLife.11282.001) have developed a method with one-step RT-PCR with six overlapping primer sets, followed by NGS and quality filtering and assembly with in-house methods. Aralaguppe et al. (https://doi.org/10.1016/j.jviromet.2016.07.010) have designed amplification in two fragments, followed by multiplexed NGS and quality control and assembly with Iterative Virus Assembler and VICUNA. Both methods have high coverage per nucleotide and low error rates in amplification and sequencing and can reliably identify SNPs at 1 per cent of the viral population with linkage within the quasispecies. Subtype inclusivity remains a challenge even though both methods show success in amplifying and sequencing subtypes B, C, and the common recombinants 01_AE and 02_AG. Therefore, we aim to evaluate and optimize our NFLG NGS methods on a panel of patient samples that more completely reflects HIV-1 diversity in Sweden. Patient samples from fifty treatment-naïve viremic individuals representing the genotypic HIV-1 panorama in Sweden, including CRFs, are being amplified and sequenced by both protocols. Coverage of the genome, error rate, and possible depth of quasispecies analysis is being evaluated. We will compare number of reads, coverage across the HIV genome, and representation of minor single nucleotide variants as well as subtype inclusivity and impact of plasma RNA levels. To do this we will use an in-house bioinformatic pipeline. The NFLG sequences will also be analyzed with phylogenetic tools for determination of subtypes including CRFs and URFs.
A9 A method to obtain full-length HIV proviral sequences and their sites of integration
Authors: Patro Sean C , Wu Xiaolin , Guo Shuang , Bale Michael J , Wiegand Ann , Spindler Jonathan , Fennessey Christine M , Halvas Elias K , Keele Brandon F , Hughes Stephen H , Mellors John W , Coffin John M , Rausch Jason W , Kearney Mary F
Published: August 2019

Accurate definition of the HIV-1 reservoir on antiretroviral therapy (ART) is of paramount importance to the development of curative strategies. Much of this reservoir is derived from clonal expansion of latently infected CD4+ T cells. Methods used to characterize the reservoir include near full-length single-genome sequencing (NFL-SGS) and integration site analysis (ISA). However, current technologies do not link the intact proviruses detected by NFL-SGS to their sites of integration. Therefore, we developed a method to obtain both near full-length single-proviral sequences and their sites of integration. We call our method full-length integrated proviral single-genome sequencing (FLIP-SGS). Genomic DNA from ACH2 and CEM cells mixed at 1:1,000, or patient samples were diluted to a single proviral endpoint. An in-house, optimized whole genome amplification (WGA) method was performed on wells at the endpoint, generating multiple copies of all DNA molecules within each well. The number of proviral copies after WGA was determined by droplet digital PCR targeting the long terminal region (LTR). Forty per cent of each WGA reaction was used to obtain the provirus–host integration sites with ISA (linker ligation, nested PCR, and Illumina sequencing). The remaining fraction was used to amplify the full-length proviruses in four overlapping fragments (LTR-pol, gag-int, int-env, and env-LTR) for Sanger sequencing. WGA performed on the endpoint-diluted ACH2:CEM DNA amplified single-copy HIV-1 proviral templates greater than 500-fold, making it possible to obtain unique integration sites from single proviruses in ACH2 cells, including one that was previously reported (in the NT5C3A gene on chromosome 7) and two that were not previously reported (in the EIF4ENIF1 gene of chromosome 22 and an unknown region of chromosome 6). Near full-length PCR amplification and Sanger sequencing was performed on proviruses integrated in the NT5C3A gene. FLIP-SGS was applied to peripheral blood mononuclear cells from one HIV-1 infected donor with viremia suppressed on ART and yielded integration sites of four genomes that appear to contain large internal deletions. We report a method for near full-length HIV-1 single-genome sequencing combined with host integration site detection that we call FLIP-SGS. This assay will further define clonal expansion of infected CD4+ T cells as a mechanism that maintains the HIV-1 reservoir and as the source of identical sequences observed during therapy and rebound, rather than from ongoing replication.
A16 Next-generation sequencing to detect transmitted drug resistance mutations in Romanian people who inject drugs
Authors: Surleac Marius , Paraschiv Simona , Nicolae Ionelia , Banica Leontina , Vlaicu Ovidiu , Jipa Raluca , Abagiu Adrian , Otelea Dan
Published: August 2019

Romania has faced an HIV outbreak among people who inject drugs (PWID) since 2011. The introduction of so-called ‘legal highs’ (amphetamine-type stimulants) on the drug market a few years prior contributed substantially to this outbreak. Next-generation sequencing (NGS) provides the possibility to detect drug resistance mutations with higher sensitivity than Sanger sequencing. The aim of this study was to search for transmitted drug resistance (TDR) mutations in strains from PWID recently diagnosed with HIV infection by parallel use of Sanger sequencing and NGS. The study was conducted on strains from 34 PWID diagnosed with HIV infection between 2016 and 2017. Sequencing was performed for the pol (PR, RT, and INT) and env (V2-V3 loop) regions. Sanger sequencing was performed with the commercial ViroseqTMHIV-1 Genotyping system (Abbott Laboratories) and with an in-house protocol for the env gene. NGS was performed in the same genomic regions using Nextera DNA Library Preparation Kit (Illumina) and the Miseq instrument (Illumina). NGS data were processed for error correction, read mapping, and detection of drug resistance mutations with HIV-1 Deepchek analysis software. Geno2pheno algorithm was used for viral tropism prediction and the WHO 2009 list for TDRM analysis. By using NGS, we detected seven cases (20.6%) of TDR in PWID and only two cases (5.8%) with standard sequencing. The TDR mutations detected by NGS were K103N, K101EN, Y181C, T215S in RT gene, I54V and M46L in PR, and none in INT. Two NNRTI mutations (K103N and K101EN) were detected in the same sample. Most of the TDR identified were present in the minority population (between 1% and 2% of the total reads) explaining the higher sensitivity of NGS method compared with standard sequencing. No significant differences were observed between these two methods when tropism prediction was analyzed. The majority of the viruses circulating in this group were R5-tropic. All strains showed more resistance mutations when analyzed by deep sequencing than by Sanger sequencing and more than previously observed in other risk groups. NGS proved to be a sensitive tool to detect TDR in newly infected PWID.
A28 Spatial spread of highly pathogenic avian influenza A (H5N8) virus in Italy, 2017–8
Authors: Zecchin B , Fusaro A , Zamperin G , Milani A , Schivo A , Salomoni A , Salviato A , Ormelli S , Marciano S , Terregino C , Monne I
Published: August 2019

In winter 2016–7 the highly pathogenic avian influenza (HPAI) virus, H5N8 subtype, clade 2.3.4.4 group B, circulated extensively both in wild and domestic birds in Europe. Northern Italy was hit by three epidemic waves: the first in January–May 2017, the second in July–December 2017, and the latest in March 2018. To genetically characterize the viruses circulating in Italy we used the Illumina MiSeq platform to sequence the complete genome of representative viruses from each infected farm, for a total of 86 cases in poultry and 17 in wild birds. Maximum likelihood phylogenetic analyses performed using PhyML version 3.1 identified multiple viral introductions of distinct genotypes of HPAI H5N8 viruses in Italy at the beginning of the epidemic (January–February 2017). During the second epidemic wave a single genetic group originating from the virus A/wild duck/Poland/82A/2016 seemed to have been selected, further evolving into two different clusters, namely Italy-A and Italy-B. We identified four clusters of secondary outbreaks, the largest being the epidemic in the province of Brescia between October 2017 and March 2018, which had affected 26 farms. Evolutionary and phylogeographic analyses performed with the BEAST v1.8.4 package (applying a Bayesian Markov chain Monte Carlo approach, using a constant size coalescent tree prior and a SRD06 model of nucleotide substitution) indicated that different viral introductions had probably occurred through migratory birds from West Russia, Siberia, Central and East Europe. The discrete and continuous phylogeographic analyses showed that group Italy-A had probably emerged between February and April 2017 in the province of Mantua and had then spread eastwards, circulating in the Veneto region and eastern Lombardy; on the contrary, Italy-B had arisen between March and July 2017 in the central part of Lombardy and had spread westwards, circulating in the western part of Lombardy, Emilia Romagna, and Piedmont regions. This study was instrumental to reconstruct the virus dissemination routes and indicated that wild and domestic birds from Lombardy most likely represented the key source for the re-emergence and spread of the HPAI virus during the second and the third epidemic waves. This key spatial information will help to define appropriate disease control strategies.
A31 Diversity change of influenza A (H3N2) strains circulating in Brazil during 2017–8: What to expect in the coming winter?
Authors: Resende P C , Caetano B , Matos A , Lopes J , Garcia C , Miranda M , Born P , Motta F C , Brown D , Siqueira M
Published: August 2019

The H3N2 subtype of influenza A (H3N2) was the predominant strain during the early months of the 2017 influenza epidemic in Brazil. In Australia, it was responsible for a strong and prolonged 2017 season and reached the Northern hemisphere causing an intense 2017/8 influenza season. Several genetic and antigenic A(H3N2) variants were circulating, which made the decision about which strain to incorporate into the influenza vaccine challenging. For 2018, the WHO selected a new H3N2 strain, A/Singapore/INFIMH-16-0019/2016-like, to replace the strain A/HongKong/4801/2014-like in the Southern Hemisphere trivalent vaccine. The aim of this study was to describe the genetic diversity of influenza A (H3N2) viruses circulating in Brazil between January 2017 and January 2018, checking the match between the vaccines and worldwide circulating strains with the Brazilian influenza strains. Hemagglutinin gene sequencing of the influenza A (H3N2) was performed, followed by a phylogenetic reconstruction using additional database sequences to define genetic groups and compare with other worldwide circulating strains. We observed a large diversity of H3N2 genetic clusters, including 3C.2a, 3C.2a1, 3C.3a, and their subgroups. During the 2016–7, inter-epidemic and 2017 epidemic period the cluster most frequently detected belonged to clade 3C.2a1 (148/185; 80.0%), a distinct group related to the 2017 vaccine strain A/HongKong/4801/2014-like (3C.2a). However, the genetic profile changed during the study period and in the inter-epidemic season 2017–8 the most commonly detected genetic group was the 3C.2a cluster (43/58; 74.1%). Inside this cluster, the majority (34/43; 79.1%) of strains belonged to a single genetic 3C.2a subgroup 2 (3C.2a2), bearing antigenic substitutions T131K and R142K (site A) and R261Q (site E). The dominance of this 3C.2a2 in the 2017–8 inter-epidemic period in Brazil was similar to the 2017–8 season in Europe and Canada according their surveillance data. The new vaccine strain has five to six antigenic changes in comparison to the predominant 3C.2a2 circulating in South America since September 2017 until now. It is possible that the vaccine mismatch will not protect the population against a majority of circulating strains. Surveillance of the vaccine effectiveness supported by antigenic and serological analysis are necessary to prove this hypothesis. However, this highlights the difficulty of vaccine strain selection and highlights the need for of a universal influenza vaccine.
A32 Genomic surveillance of Zika virus transmission in the Amazonas State, Brazil
Authors: Giovanetti Marta , de Jesus Jaqueline Goes , Joshua Quick , Claro Ingra Morales , Junior Joilson Xavier , Hill Sarah C , Renato Souza , Paz Paola , Lourenco Jose , Dellicour Simon , Sabino Esther , Loman Nicholas J , Naveca Felipe , Pybus Oliver G , Faria Nuno Rodrigues , Carlos Junior Alcantara Luiz
Published: August 2019

Zika virus (ZIKV) has caused an unprecedented epidemic linked to severe congenital syndromes. Transmission of ZIKV in the Americas was first confirmed in May 2015 in northeast Brazil, though the virus was likely introduced 1–2 years prior to its detection. Manaus, the capital city of the Amazonas State, the largest territory of any state in Brazil and the main economic center in the northern region, reported between 2016 and 2017 more than 2,327 suspected cases of ZIKV infection. To gain insights into the timing, source, and likely route(s) of ZIKV introduction in the Amazonas State, we tracked the virus by sequencing ZIKV genomes from infected patients. Using nanopore sequencing technology, we generated 56 Brazilian ZIKV genomes from Manaus city in the Amazonas state, sampled from human cases. On the basis of available sequences of isolates from the Americas, the Manaus sequences, we analyzed fell within a single strongly supported monophyletic clade (bootstrap support = 99%, posterior support = 1.00) that belongs to the Asian genotype. Genetic analysis suggests the outbreak most likely originated from transmission cycles not previously identified in North Brazil and not from a separate introduction into the Americas. Molecular dating analysis indicates that the outbreak was caused by a single founder strain that is estimated to have arrived in Manaus around February 2015. By analyzing surveillance and genetic data, we discovered that ZIKV moved among transmission zones in Manaus. Geographical analysis further indicates that the Northern part of the Manaus regions has a high transmission potential for ZIKV. Our work illustrates that near-real time genomics in the field can augment traditional approaches to infectious disease surveillance and control. Estimated dates for the international spread of ZIKV from the north region indicate the persistence of the virus transmission in recipient regions. Our study provides an understanding of how ZIKV initiates transmission in new regions.
A37 Transmission success of dengue virus type 1 lineages in a dynamic virus population: An evolutionary view
Authors: Koo Carmen , Xu Helen , Rajarethinam Jayanthi , Lai Yee Ling , Ng Lee-Ching , Hapuarachchi Hapuarachchige Chanditha
Published: August 2019

Arbovirus transmission involves an interplay between host, virus, and environmental factors. Because of the complexity of interactions, the transmission success of arboviruses could either be a function of viral fitness or be stochastic. In the present study, using 1,963 envelope (E) gene sequences and 239 whole genomes, we conducted a large-scale molecular epidemiological analysis of a dengue virus type 1 (DENV-1) population to understand the transmission success, evolution, and dispersal patterns of different lineages of DENV-1 circulating in Singapore from 2011 to 2016. The study population was highly dynamic and heterogeneous. However, only a handful of genetically distinct strains (n = 6) established sustained transmission, but at variable levels of dominance. Phylogeographic analysis revealed a weak spatial clustering and 35 well-supported diffusion pathways, implying widespread and complex dispersal of these strains in local settings. Yet, the dominant strains were neither evolving faster than less dominant ones nor under positive selection. These observations suggested that lineage dominance was likely to be stochastic and opportunistically driven by non-viral factors such as host immune pressure and vector abundance. Our findings, therefore, emphasize the implications of understanding the vector and human factors in parallel to virus dynamics on continuing efforts to control the arbovirus disease transmission in endemic regions.
A40 Estimation of Lassa virus emergence in Upper Guinea through a time-calibrated phylogeny
Authors: Fichet-Calvet Elisabeth , Magassouba N’Faly , Günther Stephan
Published: August 2019

Lassa fever is a hemorrhagic fever caused by an arenavirus, the Lassa virus (LASV), and can affect 150–200,000 persons per year in West Africa. The virus is hosted by several rodents, Mastomys natalensis and M. erythroleucus, Hylomyscus pamfi, and Mus baoulei. People can be contaminated at home or in the farms, by touching contaminated surfaces, eating contaminated food, or breathing aerosolized viral particles. Human-to-human transmission is occurring as well through infected bodily fluids. In Upper Guinea in particular, M. natalensis is the main host, with LASV prevalence of 14 per cent and IgG prevalence of 27 per cent. In humans, IgG prevalence is 40 per cent. This is, therefore, a hot spot for LASV transmission. In a previous phylogenetic study including 132 partial nucleoprotein (NP) sequences isolated from rodents, we showed that LASV could have emerged 90 years ago in the area. Here, we aim to revise the time of emergence upon analyzing the complete NP and polymerase genes of two strains coming from Upper Guinea: ‘Bantou 366’, a strain isolated from M. natalensis in 2003, and ‘Faranah’, a strain isolated from a human in 1996. They were aligned with 22 other LASV sequences belonging to all lineages and dated by their day of collection. In BEAST (v1.10) tree reconstruction, the following settings were used: GTR+gamma distributed rate variation (four discrete categories) across each codon position and constant population size demographic model. Four clock models were tested: strict, uncorrelated relaxed, random local, and fixed local. The best model was determined by comparing the resulting likelihoods using AICM model testing. Markov chain Monte Carlo (MCMC) sampling was performed for a total of 20 million states (sampling every 10,000 states) to obtain an effective sample size above 200 for all parameters. Results of MCMC sampling were examined in Tracer 1.6. The results showed that the Upper Guinea clade emerged 153 years ago when the phylogeny was reconstructed for partial NP (nt = 754, better model fit with strict clock), 208 years ago with complete NP (nt = 1,707, better model fit with random local clock), and 350 years ago with complete polymerase (nt = 6,681, better model fit with strict clock). The difference of emergence 1, 2, or 3 centuries ago, can be explained by the inclusion of some parts of the genome evolving slower than the partial NP. Therefore, the longer the sequence, the greater the divergence time. In order to have an accurate time of divergence, we suggest to use complete genes to perform a time-calibrated phylogeny.
A44 Genome analysis of bovine enterovirus variants isolated from cattle in Thailand
Authors: Kosoltanapiwat N , Income N , Cadar D , Schmidt-Chanasit J , Tongshoob J
Published: August 2019

Bovine enteroviruses (BEV) are non-enveloped RNA viruses of the genus Enterovirus, family Picornaviridae, which are commonly found in cattle. They have been classified into two species, enterovirus E (EV-E) and enterovirus F (EV-F). The viruses were previously considered non-pathogenic, but recent evidences suggest their association with pathogenesis in cattle. BEV-like enteroviruses have also been increasingly isolated from a wide range of animals, such as sheep, goats, horses, geese, possum, and deer, from many countries. The isolation and characterization of novel enteroviruses expands the range of the genus. Our data show that both EV-E and EV-F are circulating in cattle in Thailand. The viruses have been detected in 35–67 per cent of dairy and meat cattle feces in Kanchanaburi Province. Recently, we retrieved EV-E isolates from cattle feces by virus isolation in Madin-Darby Bovine Kidney cells. Four virus isolates were subjected to whole-genome sequencing using Illumina next-generation sequencing. A phylogenetic analysis of VP1 capsid protein, which is used for virus genotyping, suggested that there are at least two EV-E genotypes circulating in cattle in the area of study. Two virus strains, closely related to EV-E1 with amino acid sequence identities >88 per cent were identified as EV-E1. The other two strains, closely related to EV-E2 with amino acid sequence identities < 85 per cent, were likely to constitute a new EV-E genotype separate from the existing EV-E2.
A45 Genetic diversity of anelloviruses in the blood virome
Authors: Thijssen Marijn , Beller Leen , Yinda Kwe Claude , Deboutte Ward , Maes Piet , Matthijnssen Jelle , Van Ranst Marc , Pourkarim Mahmoudreza
Published: August 2019

The microbiome has an important impact on human health. The microbiome is a complex ecosystem that contains of a wide variety of microorganisms shaped by the immune system, host genetic factors, and the environment. Studies of the human virome have identified a diverse group of viruses in different compartments of the body, including viruses of the Anelloviridae family. These viruses are widespread among the general population. In various clinical conditions an association has been found between the Anelloviridae abundance and the patient’s immune status. However, no pathological consequences have been identified for this viral family. In this study, we analyzed the Anelloviridae diversity in plasma samples of liver transplant recipients. The virome contents of plasma samples from liver transplant recipients were sequenced by next-generation sequencing techniques on an Illumina platform (NextSeq). Complete Anelloviridae ORF1 contigs were extracted from metagenomic data and aligned with 66 RefSeq anellovirus sequences for phylogenetic analysis. The study included 144 plasma samples of 24 liver transplant recipients who had been infected by the hepatitis B virus and developed end-stage liver disease. The identified Anelloviridae viruses belong to the Alphatorquevirus, Betatorquevirus, and Gammatorquevirus genera. In total, we were able to retrieve 142 unique anellovirus contigs that were less than 95 per cent identical on the nucleotide level. A phylogenetic tree was constructed from these contigs with 65 RefSeq sequences retrieved from GenBank. The majority of the identified Anelloviridae sequences were assigned to the Alphatorquevirus genus, which represents the largest group of anelloviruses. We were able to identify a high diversity of Anelloviridae viruses in serum samples of liver transplant recipients. Phylogenetic analysis showed that the majority of anelloviruses belonged to the Alphatorque genus. Future research should focus at elucidating the role of these commensal viruses in both immunocompromised and healthy individuals.
A46 Hand, foot, and mouth disease in Vietnam
Authors: Nhu Le Nguyen Truc , Van Hoang Minh Tu , Nhan Le Nguyen Thanh , Anh Nguyen To , Thanh Tran Tan , Hang Vu Thi Ty , Ny Nguyen Thi Han , Hong Nguyen Thi Thu , Hung Nguyen Thanh , Khanh Truong Huu , Quy Du Tuan , Tuan Ha Manh , Viet Ho Lu , Viet Do Chau , Nam Nguyen Tran , Thanh Nguyen Thi My , Sabanathan Saraswathy , Qui Phan Tu , Chau Nguyen Van Vinh , Thwaites Guy E , Wills Bridget , Thwaites C Louise , van Doorn H Rogier , Van Tan Le
Published: August 2019

Hand, Foot, and Mouth Disease (HFMD) is a major public health issue in the Asia-Pacific region. Our research program aims to address unanswered questions about clinical, epidemiology, pathogen evolution, cost of illness, and host-genetic makers associated with severe HFMD in Vietnam. A multi-hospital-based observational study has been conducted at three referral hospitals in Ho Chi Minh City, Vietnam since 2013. Demographic, clinical data, and cost of illness were collected alongside clinical specimens. Multiplex PCR and next-generation sequencing were employed to identify enterovirus serotypes and to study pathogen evolution, respectively. A genome-wide association-based approach was used to explore genetic markers of disease severity. From 2013 to 2017, 2,191 HFMD patients were enrolled. More than twenty enterovirus serotypes were detected in 84.3 per cent of patients. EV-A71 was the major cause, accounting for 22 per cent of total number of cases, followed by CV-A6 (21%), CV-A16 (13%), and CV-A10 (8%). Interestingly, these four common enteroviruses replaced each other during the study period. EV-A71 and CV-A6 were the two most predominant viruses detected in 2013 and 2014. However, CV-A6 was replaced by CV-A16 and CV-A10 in 2015 and 2016, respectively. A total of 396 whole-genome sequences (EV-A71 (n = 200), CV-A6 (n = 98), CV-A10 (n = 66), and CV-A16 (n = 32)) were obtained. Phylogenetic analysis showed that EV-A71 subgenogroup B5 has replaced C4 in 2012, and, since then, B5 has continued to circulate predominantly, while C4 has been sporadically detected. All Vietnamese CV-A6 isolates belonged to genogroup A, which has caused large outbreaks of HFMD worldwide. Costs of illness varied between disease severities, ranging from $USD 244 [95% confidence interval (95% CI): 230–258] per patient for grade 2A (mild) to $USD 1984 (95% CI: 1,752–2,227) for grade 3 (severe). The genome-wide association study identified two genetic markers potentially associated with severe HFMD. The results highlight that active surveillance and understanding pathogen evolution are essential to inform public health in prioritizing the development of intervention strategies. Efforts to unravel the evolutionary process of Vietnamese CV-A10 and CV-A16 in relation to global strains are ongoing. An independent cohort is needed to replicate the preliminary findings of the genome-wide association study.
A52 MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity
Authors: Chu D K W , Hui K P Y , Perera R A P M , Miguel E , Oladipo J O , Traore A , Fassi-Fihri O , Chan M C W , Zhou Z , So R T Y , Chevalier V , Peiris J S M
Published: August 2019

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of this zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa and the Arabian Peninsula, the continuous appearance of zoonotic MERS cases in humans is confined to the Arabian Peninsula. MERS-CoV from Africa has hitherto been poorly studied. Here, we report the genetic and phenotypic characterization of MERS-CoV from dromedaries in African countries. Phylogenetically, viruses from dromedaries in Africa formed a monophyletic clade, which we have provisionally designated as virus clade C. Molecular dating analyses of MERS-CoV, including clade C viruses, suggests that the ancestral MERS-CoV in dromedaries could have spread to the two continents within a short timeframe. Camel MERS-CoVs from west and north African countries form a subclade (C1) that shares genetic signatures of a major deletion in the accessory gene ORF4b. Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung, and BF785 replicated to lower titer in lungs of human DPP4-transduced mice. However, it is still inconclusive whether ORF4b deletions may lead to the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to the zoonotic potential of MERS-CoV.
A55 Molecular systematics of sturgeon nucleocytoplasmic large DNA viruses
Authors: Clouthier S , Anderson E , Kurath G , Breyta R
Published: August 2019

Namao virus (NV) is a sturgeon nucleocytoplasmic large DNA virus (sNCLDV) that can cause a lethal disease of the integumentary system in lake sturgeon Acipenser fulvescens. As a group, the sNCLDV have not been assigned to any currently recognized taxonomic family of viruses. In this study, a dataset of NV DNA sequences was generated and assembled as two non-overlapping contigs of 306 and 448 base pairs (bp) and then used to conduct a comprehensive systematics analysis using Bayesian phylogenetic inference for NV, other sNCLDV, and representative members of six families of the NCLDV superfamily. The phylogeny of NV was reconstructed using protein homologues encoded by nine nucleocytoplasmic virus orthologous genes (NCVOGs): NCVOG0022—mcp, NCVOG0038—DNA polymerase B elongation subunit, NCVOG0076—VV A18-type helicase, NCVOG0249—VV A32-type ATPase, NCVOG0262—AL2 VLTF3-like transcription factor, NCVOG0271—RNA polymerase II subunit II, NCVOG0274—RNA polymerase II subunit I, NCVOG0276—ribonucleotide reductase small subunit, and NCVOG1117—mRNA capping enzyme. The accuracy of our phylogenetic method was evaluated using a combination of Bayesian statistical analysis and congruence analysis. Stable tree topologies were obtained with datasets differing in target molecule(s), sequence length, and taxa. Congruent topologies were obtained in phylogenies constructed using individual protein datasets and when four proteins were used in a concatenated approach. The major capsid protein phylogeny indicated that ten representative sNCLDV form a monophyletic group comprised of four lineages within a polyphyletic Mimi-Phycodnaviridae group of taxa. Overall, the analyses revealed that Namao virus is a member of the Mimiviridae family with strong and consistent support for a clade containing NV and CroV as sister taxa.