Experimental infection of domestic turkeys with lymphoproliferative disease virus of North American origin.

Lymphoproliferative disease virus (LPDV) was first documented in wild turkeys in North America in 2009. LPDV infection is often subclinical but can manifest as lymphoid proliferation or round cell neoplasia. Despite high prevalence across many sampled areas corresponding to declining populations of wild turkeys, knowledge regarding LPDV pathogenesis, risk factors for disease development, and associated impacts on population dynamics are unknown. To understand transmission, viral shedding, and tissue tropism, we inoculated 21 domestic turkeys via the oral cavity, crop, nasal cavity, subcutis, or coelomic cavity. For 12 weeks, oropharyngeal swabs, cloacal swabs, and whole blood were collected weekly. At 1 week postinoculation, 3 turkeys (3/21; 14%) had detectable LPDV proviral DNA in blood by polymerase chain reaction, and 10 developed DNAemia (50%; 10/20) by 12 weeks. LPDV proviral DNA was intermittently detected in oropharyngeal and cloacal swabs. Splenomegaly was the most consistent gross finding in DNAemic birds (8/11; 73%). Lymphoid hyperplasia in the spleen was the most significant microscopic finding (9/11; 82%). Three turkeys (3/11; 27%) developed round cell neoplasia characterized by sheets of pleomorphic, round to polygonal cells in the adrenal gland, bone marrow, skin, small intestine, and/or spleen. LPDV was detected in the spleen and bone marrow from all turkeys with DNAemia and all neoplasms. Our study establishes that infection and disease with North American LPDV from wild turkeys can be experimentally reproduced in domestic turkeys, laying the groundwork for future investigations into LPDV pathogenesis, development of diagnostic techniques, and understanding the impacts of LPDV on wild turkey populations.

Limited Intrahost Diversity and Background Evolution Accompany 40 Years of Canine Parvovirus Host Adaptation and Spread.

Canine parvovirus (CPV) is a highly successful pathogen that has sustained pandemic circulation in dogs for more than 40 years. Here, integrating full-genome and deep-sequencing analyses, structural information, and in vitro experimentation, we describe the macro- and microscale features that accompany CPV's evolutionary success. Despite 40 years of viral evolution, all CPV variants are more than ∼99% identical in nucleotide sequence, with only a limited number (<40) of substitutions becoming fixed or widespread during this time. Notably, most substitutions in the major capsid protein (VP2) gene are nonsynonymous, altering amino acid residues that fall within, or adjacent to, the overlapping receptor footprint or antigenic regions, suggesting that natural selection has channeled much of CPV evolution. Among the limited number of variable sites, CPV genomes exhibit complex patterns of variation that include parallel evolution, reversion, and recombination, compromising phylogenetic inference. At the intrahost level, deep sequencing of viral DNA in original clinical samples from dogs and other host species sampled between 1978 and 2018 revealed few subconsensus single nucleotide variants (SNVs) above ∼0.5%, and experimental passages demonstrate that substantial preexisting genetic variation is not necessarily required for rapid host receptor-driven adaptation. Together, these findings suggest that although CPV is capable of rapid host adaptation, a relatively low mutation rate, pleiotropy, and/or a lack of selective challenges since its initial emergence have inhibited the long-term accumulation of genetic diversity. Hence, continuously high levels of inter- and intrahost diversity are not necessarily required for virus host adaptation.IMPORTANCE Rapid mutation rates and correspondingly high levels of intra- and interhost diversity are often cited as key features of viruses with the capacity for emergence and sustained transmission in a new host species. However, most of this information comes from studies of RNA viruses, with relatively little known about evolutionary processes in viruses with single-stranded DNA (ssDNA) genomes. Here, we provide a unique model of virus evolution, integrating both long-term global-scale and short-term intrahost evolutionary processes of an ssDNA virus that emerged to cause a pandemic in a new host animal. Our analysis reveals that successful host jumping and sustained transmission does not necessarily depend on a high level of intrahost diversity nor result in the continued accumulation of high levels of long-term evolution change. These findings indicate that all aspects of the biology and ecology of a virus are relevant when considering their adaptability.

Impact of anaemia at discharge following colorectal cancer surgery.

OBJECTIVES: Preoperative anaemia is common in patients with colorectal cancer and increasingly optimised prior to surgery. Comparably little attention is given to the prevalence and consequences of postoperative anaemia. We aimed to investigate the frequency and short- or long-term impact of anaemia at discharge following colorectal cancer resection. METHODS: A dedicated, prospectively populated database of elective laparoscopic colorectal cancer procedures undertaken with curative intent within a fully implemented ERAS protocol was utilised. The primary endpoint was anaemia at time of discharge (haemoglobin (Hb) < 120 g/L for women and < 135 g/L for men). Patient demographics, tumour characteristics, operative details and postoperative outcomes were captured. Median follow-up was 61 months with overall survival calculated with the Kaplan-Meier log rank method and Cox proportional hazard regression based on anaemia at time of hospital discharge. RESULTS: A total of 532 patients with median 61-month follow-up were included. 46.4% were anaemic preoperatively (cohort mean Hb 129.4 g/L ± 18.7). Median surgical blood loss was 100 mL (IQR 0-200 mL). Upon discharge, most patients were anaemic (76.6%, Hb 116.3 g/L ± 14, mean 19 g/L ± 11 below lower limit of normal, p < 0.001). 16.7% experienced postoperative complications which were associated with lower discharge Hb (112 g/L ± 12 vs. 117 g/L ± 14, p = 0.001). Patients discharged anaemic had longer hospital stays (7 [5-11] vs. 6 [5-8], p = 0.037). Anaemia at discharge was independently associated with reduced overall survival (82% vs. 70%, p = 0.018; HR 1.6 (95% CI 1.04-2.5), p = 0.034). CONCLUSION: Anaemia at time of discharge following elective laparoscopic colorectal cancer surgery and ERAS care is common with associated negative impacts upon short-term clinical outcomes and long-term overall survival.

Complex and Dynamic Interactions between Parvovirus Capsids, Transferrin Receptors, and Antibodies Control Cell Infection and Host Range.

Antibody and receptor binding are key virus-host interactions that control host range and determine the success of infection. Canine and feline parvovirus capsids bind the transferrin receptor type 1 (TfR) to enter host cells, and specific structural interactions appear necessary to prepare the stable capsids for infection. Here, we define the details of binding, competition, and occupancy of wild-type and mutant parvovirus capsids with purified receptors and antibodies. TfR-capsid binding interactions depended on the TfR species and varied widely, with no direct relationship between binding affinity and infection. Capsids bound feline, raccoon, and black-backed jackal TfRs at high affinity but barely bound canine TfRs, which mediated infection efficiently. TfRs from different species also occupied capsids to different levels, with an estimated 1 to 2 feline TfRs but 12 black-backed jackal TfRs binding each capsid. Multiple alanine substitutions within loop 1 on the capsid surface reduced TfR binding but substitutions within loop 3 did not, suggesting that loop 1 directly engaged the TfR and loop 3 sterically affected that interaction. Binding and competition between different TfRs and/or antibodies showed complex relationships. Both antibodies 14 and E competed capsids off TfRs, but antibody E could also compete capsids off itself and antibody 14, likely by inducing capsid structural changes. In some cases, the initial TfR or antibody binding event affected subsequent TfR binding, suggesting that capsid structure changes occur after TfR or antibody binding and may impact infection. This shows that precise, host-specific TfR-capsid interactions, beyond simple attachment, are important for successful infection.IMPORTANCE Host receptor binding is a key step during viral infection and may control both infection and host range. In addition to binding, some viruses require specific interactions with host receptors in order to infect, and anti-capsid antibodies can potentially disrupt these interactions, leading to neutralization. Here, we examine the interactions between parvovirus capsids, the receptors from different hosts, and anti-capsid antibodies. We show that interactions between parvovirus capsids and host-specific TfRs vary in both affinity and in the numbers of receptors bound, with complex effects on infection. In addition, antibodies binding to two sites on the capsids had different effects on TfR-capsid binding. These experiments confirm that receptor and antibody binding to parvovirus capsids are complex processes, and the infection outcome is not determined simply by the affinity of attachment.

Factors Predicting Operative Difficulty of Laparoscopic Total Mesorectal Excision.

BACKGROUND: Laparoscopic total mesorectal excision is a challenging procedure requiring high-quality surgery for optimal outcomes. Patient, tumor, and pelvic factors are believed to determine difficulty, but previous studies were limited to postoperative data. OBJECTIVE: This study aimed to report factors predicting laparoscopic total mesorectal excision performance by using objective intraoperative assessment. DESIGN: Data from a multicenter laparoscopic total mesorectal excision randomized trial (ISRCTN59485808) were reviewed. SETTING: This study was conducted at 4 centers in the United Kingdom. PATIENTS AND INTERVENTION: Seventy-one patients underwent elective laparoscopic total mesorectal excision for rectal adenocarcinoma with curative intent: 53% were men, mean age was 69 years, body mass index was 27.7, tumor height was 8.5 cm, 24% underwent neoadjuvant therapy, and 25% had previous surgery. MAIN OUTCOME MEASURES: Surgical performance was assessed through the identification of intraoperative adverse events by using observational clinical human reliability analysis. Univariate analysis and multivariate binomial regression were performed to establish factors predicting the number of intraoperative errors, surgeon-reported case difficulty, and short-term clinical and histopathological outcomes. RESULTS: A total of 1331 intraoperative errors were identified from 365 hours of surgery (median, 18 per case; interquartile range, 16-22; and range, 9-49). No patient, tumor, or bony pelvimetry measurement correlated with total or pelvic error count, surgeon-reported case difficulty, cognitive load, operative data, specimen quality, number or severity of 30-day morbidity events and length of stay (all r not exceeding ±0.26, p > 0.05). Mesorectal area was associated with major intraoperative adverse events (OR, 1.09; 95%CI, 1.01-1.16; p = 0.015) and postoperative morbidity (OR, 1.1; 95% CI, 1.01-1.2; p = 0.033). Obese men were subjectively reported as harder cases (24 vs 36 mm, p = 0.042), but no detrimental effects on performance or outcomes were seen. LIMITATIONS: Our sample size is modest, risking type II errors and overfitting of the statistical models. CONCLUSION: Patient, tumor, and bony pelvic anatomical characteristics are not seen to influence laparoscopic total mesorectal excision operative difficulty. Mesorectal area is identified as a risk factor for intraoperative and postoperative morbidity. See Video Abstract at http://links.lww.com/DCR/B35. FACTORES QUE PREDICEN LA DIFICULTAD OPERATIVA DE LA ESCISIÓN MESORRECTAL TOTAL LAPAROSCÓPICA: La escisión mesorrectal total laparoscópica es un procedimiento desafiante. Para obtener resultados óptimos, se requiere cirugía de alta calidad. Se cree que, factores como el paciente, el tumor y la pelvis, determinan la dificultad, pero estudios previos solamente se han limitado a datos postoperatorios.Informar de los factores que predicen el resultado de la escisión mesorrectal total laparoscópica, mediante una evaluación intraoperatoria objetiva.Datos de un ensayo multicéntrico y randomizado de escisión mesorrectal total laparoscópica (ISRCTN59485808).Cuatro centros del Reino Unido.Un total de 71 pacientes fueron sometidos a escisión mesorrectal total laparoscópica electiva, para adenocarcinoma rectal con intención curativa. 53% hombres, edad media, índice de masa corporal y altura del tumor 69, 27.7 y 8.5 cm respectivamente, 24% terapia neoadyuvante y 25% cirugía previa.Rendimiento quirúrgico evaluado mediante la identificación de eventos intraoperatorios adversos, mediante el análisis clínico observacional de confiabilidad humana. Se realizaron análisis univariado y la regresión binomial multivariada para establecer factores que predicen el número de errores intraoperatorios, reportes del cirujano sobre la dificultad del caso y los resultados clínicos e histopatológicos a corto plazo.Se identificaron un total de 1,331 errores intraoperatorios en 365 horas de cirugía (media de 18 por caso, IQR 16-22, rango 9-49). Ningún paciente, tumor o medición de pelvimetría pélvica, se correlacionó con la cuenta de errores pélvicos o totales, reporte del cirujano sobre dificultad del caso, carga cognitiva, datos operativos, calidad de la muestra, número o gravedad de eventos de morbilidad de 30 días y duración de la estadía (todos r <± 0.26, p > 0.05). El área mesorrectal se asoció con eventos adversos intraoperatorios importantes (OR, 1.09; IC 95%, 1.01-1.16; p = 0.015) y morbilidad postoperatoria (OR, 1.1; IC 95%, 1.01-1.2; p = 0.033). Como información subjetiva, hombres obesos fueron casos más difíciles (24 mm frente a 36 mm, p = 0.042) pero no se observaron efectos perjudiciales sobre el rendimiento o los resultados.Nuestro tamaño de muestra es un modesto riesgo de errores de tipo II y el sobreajuste de los modelos estadísticos.No se observa que las características anatómicas del paciente, tumor y pelvis ósea influyan en la dificultad operatoria de la escisión mesorrectal laparoscópica total. El área mesorrectal se identifica como un factor de riesgo para la morbilidad intraoperatoria y postoperatoria. Vea el resumen del video en http://links.lww.com/DCR/B35.

Evolution and Cryo-electron Microscopy Capsid Structure of a North American Bat Adenovirus and Its Relationship to Other Mastadenoviruses.

Since the first description of adenoviruses in bats in 2006, a number of micro- and megabat species in Europe, Africa, and Asia have been shown to carry a wide diversity of adenoviruses. Here, we report on the evolutionary, biological, and structural characterization of a novel bat adenovirus (BtAdV) recovered from a Rafinesque's big-eared bat (Corynorhinus rafinesquii) in Kentucky, USA, which is the first adenovirus isolated from North American bats. This virus (BtAdV 250-A) exhibits a close phylogenetic relationship with Canine mastadenovirus A (CAdV A), as previously observed with other BtAdVs. To further investigate the relationships between BtAdVs and CAdVs, we conducted mass spectrometric analysis and single-particle cryo-electron microscopy reconstructions of the BtAdV 250-A capsid and also analyzed the in vitro host ranges of both viruses. Our results demonstrate that BtAdV 250-A represents a new mastadenovirus species that, in contrast to CAdV, has a unique capsid morphology that contains more prominent extensions of protein IX and can replicate efficiently in a phylogenetically diverse range of species. These findings, in addition to the recognition that both the genetic diversity of BtAdVs and the number of different bat species from disparate geographic regions infected with BtAdVs appears to be extensive, tentatively suggest that bats may have served as a potential reservoir for the cross-species transfer of adenoviruses to other hosts, as theorized for CAdV. IMPORTANCE: Although many adenoviruses are host specific and likely codiverged with their hosts over millions of years, other adenoviruses appear to have emerged through successful cross-species transmission events on more recent time scales. The wide geographic distribution and genetic diversity of adenoviruses in bats and their close phylogenetic relationship to Canine mastadenovirus A (CAdV A) has raised important questions about how CAdV A, and possibly other mammalian adenoviruses, may have emerged. Although most adenoviruses tend to cause limited disease in their natural hosts, CAdV A is unusual in that it may cause high morbidity and sometimes fatal infections in immunocompetent hosts and is thus an important pathogen of carnivores. Here, we performed a comparative evolutionary and structural study of representative bat and canine adenoviruses to better understand the relationship between these two viral groups.

Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages.

Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by >100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ∼10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids. IMPORTANCE: Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction are mediated by the viral capsid, but the structure-function correlates of the capsids and their constituent proteins are still incompletely understood, especially in relation to identifying capsid processes responsible for infection and release from the cell. Here, we characterize the functional effects of capsid protein mutations that result in the loss of virus infectivity, giving a better understanding of the portions of the capsid that mediate essential steps in successful infection pathways and how they contribute to viral infectivity.

Does the number of operating specialists influence the conversion rate and outcomes after laparoscopic colorectal cancer surgery?

BACKGROUND: Laparoscopic techniques in colorectal surgery have been widely utilised due to short-term patient benefits but conversion to open surgery is associated with adverse short- and long-term patient outcomes. The aim of this study was to investigate the influence of dual specialist operating on the conversion rate and patient outcomes following laparoscopic colorectal surgery. METHODS: A prospectively populated colorectal cancer surgery database was reviewed. Cases were grouped into single or dual consultant procedures. Cluster analysis and odds ratio (OR) were used to identify risk factors for conversion. Primary outcome measures were conversion to open and five year overall survival (OS) calculated using the Kaplan-Meier log-rank method. RESULTS: 750 patients underwent laparoscopic colorectal cancer resection between 2002 and 2015 (median age 73, 319 (42.5%) female, 282 (37.6%) rectal malignancies, 135 patients (18%) had two consultants). The single surgeon conversion rate was 20.4% compared to 5.5% for dual operating (OR 4.4, 95% CI 1.87-10.2, p < 0.001). There were no demographic or tumour differences between the laparoscopic/converted and number of surgeon groups. Two-step cluster analysis identified cluster I (lower risk) 406 patients, 8% converted and cluster II (higher risk) 261 patients, conversion rate 30%. Median follow-up was 48 months (range 0-168). Five-year OS was significantly inferior for both converted and single surgeon cases (63% vs. 77%, p < 0.001 and 61% vs. 70%, p = 0.033, respectively). CONCLUSION: In selected colorectal cancer patients operated by fully trained laparoscopic surgeons, we observed a reduction in conversion with associated long-term survival benefit from dual operating specialists.

Experimental Infection of Common Eider Ducklings with Wellfleet Bay Virus, a Newly Characterized Orthomyxovirus.

Wellfleet Bay virus (WFBV), a novel orthomyxovirus in the genus Quaranjavirus, was first isolated in 2006 from carcasses of common eider (Somateria mollissima) during a mortality event in Wellfleet Bay (Barnstable County, Massachusetts, USA) and has since been repeatedly isolated during recurrent mortality events in this location. Hepatic, pancreatic, splenic, and intestinal necrosis was observed in dead eiders. We inoculated 6-week-old common eider ducklings with WFBV in an attempt to recreate the naturally occurring disease. Approximately 25% of inoculated eiders had onset of clinical disease and required euthanasia; an additional 18.75% were adversely affected based on net weight loss during the trial. Control ducklings did not become infected and did not have clinical disease. Infected ducklings with clinical disease had pathologic lesions consistent with those observed during natural mortality events. WFBV was reisolated from 37.5% of the inoculated ducklings. Ducklings surviving to 5 days postinoculation developed serum antibody titers to WFBV.

Single-Particle Tracking Shows that a Point Mutation in the Carnivore Parvovirus Capsid Switches Binding between Host-Specific Transferrin Receptors.

Determining how viruses infect new hosts via receptor-binding mechanisms is important for understanding virus emergence. We studied the binding kinetics of canine parvovirus (CPV) variants isolated from raccoons-a newly recognized CPV host-to different carnivore transferrin receptors (TfRs) using single-particle tracking. Our data suggest that CPV may utilize adhesion-strengthening mechanisms during TfR binding and that a single mutation in the viral capsid at VP2 position 300 can profoundly alter receptor binding and infectivity.

Single Mutations in the VP2 300 Loop Region of the Three-Fold Spike of the Carnivore Parvovirus Capsid Can Determine Host Range.

UNLABELLED: Sylvatic carnivores, such as raccoons, have recently been recognized as important hosts in the evolution of canine parvovirus (CPV), a pandemic pathogen of domestic dogs. Although viruses from raccoons do not efficiently bind the dog transferrin receptor (TfR) or infect dog cells, a single mutation changing an aspartic acid to a glycine at capsid (VP2) position 300 in the prototype raccoon CPV allows dog cell infection. Because VP2 position 300 exhibits extensive amino acid variation among the carnivore parvoviruses, we further investigated its role in determining host range by analyzing its diversity and evolution in nature and by creating a comprehensive set of VP2 position 300 mutants in infectious clones. Notably, some position 300 residues rendered CPV noninfectious for dog, but not cat or fox, cells. Changes of adjacent residues (residues 299 and 301) were also observed often after cell culture passage in different hosts, and some of the mutations mimicked changes seen in viruses recovered from natural infections of alternative hosts, suggesting that compensatory mutations were selected to accommodate the new residue at position 300. Analysis of the TfRs of carnivore hosts used in the experimental evolution studies demonstrated that their glycosylation patterns varied, including a glycan present only on the domestic dog TfR that dictates susceptibility to parvoviruses. Overall, there were significant differences in the abilities of viruses with alternative position 300 residues to bind TfRs and infect different carnivore hosts, demonstrating that the process of infection is highly host dependent and that VP2 position 300 is a key determinant of host range. IMPORTANCE: Although the emergence and pandemic spread of canine parvovirus (CPV) are well documented, the carnivore hosts and evolutionary pathways involved in its emergence remain enigmatic. We recently demonstrated that a region in the capsid structure of CPV, centered around VP2 position 300, varies after transfer to alternative carnivore hosts and may allow infection of previously nonsusceptible hosts in vitro. Here we show that VP2 position 300 is the most variable residue in the parvovirus capsid in nature, suggesting that it is a critical determinant in the cross-species transfer of viruses between different carnivores due to its interactions with the transferrin receptor to mediate infection. To this end, we demonstrated that there are substantial differences in receptor binding and infectivity of various VP2 position 300 mutants for different carnivore species and that single mutations in this region can influence whether a host is susceptible or refractory to virus infection.

Global displacement of canine parvovirus by a host-adapted variant: structural comparison between pandemic viruses with distinct host ranges.

Canine parvovirus type 2 (CPV-2) emerged in 1978 and spread worldwide within 2 years. Subsequently, CPV-2 was completely replaced by the variant CPV-2a, which is characterized by four specific capsid (VP2) mutations. The X-ray crystal structure of the CPV-2a capsid shows that each mutation confers small local changes. The loss of a hydrogen bond and introduction of a glycine residue likely introduce flexibility to sites that control interactions with the host receptor, antibodies, and sialic acids.

Cyclic avian mass mortality in the northeastern United States is associated with a novel orthomyxovirus.

UNLABELLED: Since 1998, cyclic mortality events in common eiders (Somateria mollissima), numbering in the hundreds to thousands of dead birds, have been documented along the coast of Cape Cod, MA, USA. Although longitudinal disease investigations have uncovered potential contributing factors responsible for these outbreaks, detecting a primary etiological agent has proven enigmatic. Here, we identify a novel orthomyxovirus, tentatively named Wellfleet Bay virus (WFBV), as a potential causative agent of these outbreaks. Genomic analysis of WFBV revealed that it is most closely related to members of the Quaranjavirus genus within the family Orthomyxoviridae. Similar to other members of the genus, WFBV contains an alphabaculovirus gp64-like glycoprotein that was demonstrated to have fusion activity; this also tentatively suggests that ticks (and/or insects) may vector the virus in nature. However, in addition to the six RNA segments encoding the prototypical structural proteins identified in other quaranjaviruses, a previously unknown RNA segment (segment 7) encoding a novel protein designated VP7 was discovered in WFBV. Although WFBV shows low to moderate levels of sequence similarity to Quaranfil virus and Johnston Atoll virus, the original members of the Quaranjavirus genus, additional antigenic and genetic analyses demonstrated that it is closely related to the recently identified Cygnet River virus (CyRV) from South Australia, suggesting that WFBV and CyRV may be geographic variants of the same virus. Although the identification of WFBV in part may resolve the enigma of these mass mortality events, the details of the ecology and epidemiology of the virus remain to be determined. IMPORTANCE: The emergence or reemergence of viral pathogens resulting in large-scale outbreaks of disease in humans and/or animals is one of the most important challenges facing biomedicine. For example, understanding how orthomyxoviruses such as novel influenza A virus reassortants and/or mutants emerge to cause epidemic or pandemic disease is at the forefront of current global health concerns. Here, we describe the emergence of a novel orthomyxovirus, Wellfleet Bay virus (WFBV), which has been associated with cyclic large-scale bird die-offs in the northeastern United States. This initial characterization study provides a foundation for further research into the evolution, epidemiology, and ecology of newly emerging orthomyxoviruses, such as WFBV, and their potential impacts on animal and/or human health.

Host-specific parvovirus evolution in nature is recapitulated by in vitro adaptation to different carnivore species.

Canine parvovirus (CPV) emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV), a parvovirus of cats and related carnivores. Although both viruses have wide host ranges, analysis of viral sequences recovered from different wild carnivore species, as shown here, demonstrated that>95% were derived from CPV-like viruses, suggesting that CPV is dominant in sylvatic cycles. Many viral sequences showed host-specific mutations in their capsid proteins, which were often close to sites known to control binding to the transferrin receptor (TfR), the host receptor for these carnivore parvoviruses, and which exhibited frequent parallel evolution. To further examine the process of host adaptation, we passaged parvoviruses with alternative backgrounds in cells from different carnivore hosts. Specific mutations were selected in several viruses and these differed depending on both the background of the virus and the host cells in which they were passaged. Strikingly, these in vitro mutations recapitulated many specific changes seen in viruses from natural populations, strongly suggesting they are host adaptive, and which were shown to result in fitness advantages over their parental virus. Comparison of the sequences of the transferrin receptors of the different carnivore species demonstrated that many mutations occurred in and around the apical domain where the virus binds, indicating that viral variants were likely selected through their fit to receptor structures. Some of the viruses accumulated high levels of variation upon passage in alternative hosts, while others could infect multiple different hosts with no or only a few additional mutations. Overall, these studies demonstrate that the evolutionary history of a virus, including how long it has been circulating and in which hosts, as well as its phylogenetic background, has a profound effect on determining viral host range.

Effect of Temperature on Replication of Epizootic Hemorrhagic Disease Viruses in Culicoides sonorensis (Diptera: Ceratopogonidae).

Replication of arboviruses, including orbiviruses, within the vector has been shown to be temperature dependent. Cooler ambient temperatures slow virus replication in arthropod vectors, whereas viruses replicate faster and to higher titers at warmer ambient temperatures. Previous research with epizootic hemorrhagic disease virus (EHDV) serotype 1 demonstrated that higher temperatures were associated with shorter extrinsic incubation periods in Culicoides sonorensis Wirth & Jones, a confirmed vector of EHDV in North America. To further our understanding of the effect of temperature on replication of EHDV within the vector, C. sonorensis were experimentally infected with one of three EHDV strains representing three serotypes (1, 2, and 7). Midges were fed defibrinated white-tailed deer (Odocoileus virginianus) blood spiked with EHDV (≥10(6.5) TCID(50)/ml) through a parafilm membrane using an artificial feeding device and were then held at 20, 25, or 30°C. In addition to this in vitro method, a white-tailed deer experimentally infected with EHDV-7 was used to provide an infectious bloodmeal to determine if the results were comparable with those from the in vitro feeding method. Whole midges were processed for virus isolation and titration at regular intervals following feeding; midges with ≥10(2.7) TCID(50) were considered potentially competent to transmit virus. The virus recovery rates were high throughout the study and all three viruses replicated within C. sonorensis to high titer (≥ 10(2.7) TCID(50)/midge). Across all virus strains, the time to detection of potentially competent midges decreased with increasing temperature: 12-16 d postfeeding (dpf) at 20°C, 4-6 dpf at 25°C, and 2-4 dpf at 30°C. Significant differences in replication of the three viruses in C. sonorensis were observed, with EHDV-2 replicating to a high titer in a smaller proportion of midges and with lower peak titers. The findings are consistent with previous studies of related orbiviruses, showing that increasing temperature can shorten the apparent extrinsic incubation period for multiple EHDV strains (endemic and exotic) in C. sonorensis.

Screening wild and semi-free ranging great apes for putative sexually transmitted diseases: Evidence of Trichomonadidae infections.

Sexually transmitted diseases (STDs) can persist endemically, are known to cause sterility and infant mortality in humans, and could have similar impacts in wildlife populations. African apes (i.e., chimpanzees, bonobos, and to a lesser extent gorillas) show multi-male mating behavior that could offer opportunities for STD transmission, yet little is known about the prevalence and impact of STDs in this endangered primate group. We used serology and PCR-based detection methods to screen biological samples from wild and orphaned eastern chimpanzees and gorillas (N = 172 individuals, including adults, and juveniles) for four classes of pathogens that either commonly cause human STDs or were previously detected in captive apes: trichomonads, Chlamydia spp., Treponema pallidum (syphilis and yaws), and papillomaviruses. Based on results from prior modeling and comparative research, we expected STD prevalence to be highest in females versus males and in sexually mature versus immature individuals. All samples were negative for Chlamydia, Treponema pallidum, and papillomaviruses; however, a high percentage of wild chimpanzee urine and fecal samples showed evidence of trichomonads (protozoa). Analysis revealed that females were more likely than males to have positive urine-but not fecal-samples; however, there was no evidence of age (sexual maturity) differences in infection status. Sequence analysis of chimpanzee trichomonad samples revealed a close relationship to previously described trichomonads within the genus Tetratrichomonas. Phylogenetic comparisons to archived sequences from multiple vertebrate hosts suggests that many of the chimpanzee parasites from our study are likely transmitted via fecal-oral contact, but the transmission of some Tetratrichomonas sequence-types remains unknown and could include sexual contact. Our work emphasizes that only a fraction of infectious agents affecting wild apes are presently known to science, and that further work on great ape STDs could offer insights for the management of endangered great apes and for understanding human STD origins.

Frequent cross-species transmission of parvoviruses among diverse carnivore hosts.

Although parvoviruses are commonly described in domestic carnivores, little is known about their biodiversity in nondomestic species. A phylogenetic analysis of VP2 gene sequences from puma, coyote, gray wolf, bobcat, raccoon, and striped skunk revealed two major groups related to either feline panleukopenia virus ("FPV-like") or canine parvovirus ("CPV-like"). Cross-species transmission was commonplace, with multiple introductions into each host species but, with the exception of raccoons, relatively little evidence for onward transmission in nondomestic species.

Evolutionary reconstructions of the transferrin receptor of Caniforms supports canine parvovirus being a re-emerged and not a novel pathogen in dogs.

Parvoviruses exploit transferrin receptor type-1 (TfR) for cellular entry in carnivores, and specific interactions are key to control of host range. We show that several key mutations acquired by TfR during the evolution of Caniforms (dogs and related species) modified the interactions with parvovirus capsids by reducing the level of binding. These data, along with signatures of positive selection in the TFRC gene, are consistent with an evolutionary arms race between the TfR of the Caniform clade and parvoviruses. As well as the modifications of amino acid sequence which modify binding, we found that a glycosylation site mutation in the TfR of dogs which provided resistance to the carnivore parvoviruses which were in circulation prior to about 1975 predates the speciation of coyotes and dogs. Because the closely-related black-backed jackal has a TfR similar to their common ancestor and lacks the glycosylation site, reconstructing this mutation into the jackal TfR shows the potency of that site in blocking binding and infection and explains the resistance of dogs until recent times. This alters our understanding of this well-known example of viral emergence by indicating that canine parvovirus emergence likely resulted from the re-adaptation of a parvovirus to the resistant receptor of a former host.

Arkansas's alternative to Medicaid expansion raises important questions about how HHS will implement new ACA waiver authority in 2017.

This essay presents Arkansas's alternative to Medicaid expansion as a case study motivating John McDonough's assessment of the recommendations states may want to make to the Department of Health and Human Services regarding the implementation of statewide Patient Protection and Affordable Care Act-alternative waivers scheduled to begin in 2017. Arkansas's private option uses federal funds to purchase marketplace silver-level qualified health plans for low-income, low-risk participants, while "medically frail" adults are covered through Medicaid. By improving the size and risk profile of Arkansas's health insurance marketplace, the private option will also encourage entry of and competition among private carriers. If it succeeds in keeping insurance premiums below the level they would otherwise be in the marketplace, Arkansas's private option could reduce subsidy costs for the federal government. Under the broadened scope of section 1332 waivers, states will be able to capture such savings and use them to support innovation across both Medicaid-funded and Treasury-subsidized programs and populations.

Arkansas's novel approach to expanding health care coverage.

The state of Arkansas is implementing a novel approach to expanding health care coverage for individuals newly eligible for Medicaid under the Patient Protection and Affordable Care Act (ACA). Through a section 1115 demonstration waiver, the state will use federal funding via a premium assistance model to secure private health insurance offered through the newly formed health insurance marketplace to those individuals aged nineteen to sixty-four who have incomes at or below 138 percent of the federal poverty level. As of April 2014, the Health Care Independence Program (HCIP), as it is formally known, had over 155,000 individuals who had been determined eligible. The HCIP premium assistance approach is commonly referred to as the "private option" and was designed to achieve comparable access, network availability, quality of care, and opportunities for improved outcomes for HCIP enrollees (i.e., those who would be eligible for traditional, fee-for-service Medicaid through ACA expansion) when compared with their privately insured counterparts. This article provides the background, political discourse, policy development, evaluation strategy, and progress report for this innovative new program.