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We report an unknown taxon of bot fly (Diptera: Oestridae: Oestrinae) in red river hogs (Potamochoerus porcus Linnaeus, 1758) in Cross River State, Nigeria. From direct observation and interviews with local hunters, we document that, remarkably, the parasite typically occurs within the intracranial supra-meningeal space – i.e., between the inner wall of the skull and the brain – but without causing visible inflammation or clinical signs. The parasite is most similar (up to 87.9%) to Rhinoestrus usbekistanicus based on cytochrome oxidase subunit 1 DNA sequencing but is sufficiently divergent phylogenetically to represent a new or previously un-sequenced taxon. Morphologically, the parasite shares some, but not all, features with R. nivarleti. Local cultural belief systems attribute aspects of red river hog behavior (e.g. intelligence, elusiveness) to the parasite, suggesting a prolonged presence in the red river hog population. The parasite's unusual anatomic location may be aberrant, or it may be a protective adaptation to life in red river hogs, which forage vigorously with their snouts.

Since 1814, when rubella was first described, the origins of the disease and its causative agent, rubella virus (Matonaviridae: Rubivirus), have remained unclear1. Here we describe ruhugu virus and rustrela virus in Africa and Europe, respectively, which are, to our knowledge, the first known relatives of rubella virus. Ruhugu virus, which is the closest relative of rubella virus, was found in apparently healthy cyclops leaf-nosed bats (Hipposideros cyclops) in Uganda. Rustrela virus, which is an outgroup to the clade that comprises rubella and ruhugu viruses, was found in acutely encephalitic placental and marsupial animals at a zoo in Germany and in wild yellow-necked field mice (Apodemus flavicollis) at and near the zoo. Ruhugu and rustrela viruses share an identical genomic architecture with rubella virus2,3. The amino acid sequences of four putative B cell epitopes in the fusion (E1) protein of the rubella, ruhugu and rustrela viruses and two putative T cell epitopes in the capsid protein of the rubella and ruhugu viruses are moderately to highly conserved4–6. Modelling of E1 homotrimers in the post-fusion state predicts that ruhugu and rubella viruses have a similar capacity for fusion with the host-cell membrane5. Together, these findings show that some members of the family Matonaviridae can cross substantial barriers between host species and that rubella virus probably has a zoonotic origin. Our findings raise concerns about future zoonotic transmission of rubella-like viruses, but will facilitate comparative studies and animal models of rubella and congenital rubella syndrome. Ruhugu virus and rustrela virus are the first close relatives of rubella virus, providing insights into the zoonotic origin of rubella virus and the epidemiology and evolution of all three viruses.

Understanding the emergence of novel viruses requires an accurate and comprehensive annotation of their genomes. Overlapping genes (OLGs) are common in viruses and have been associated with pandemics but are still widely overlooked. We identify and characterize ORF3d, a novel OLG in SARS-CoV-2 that is also present in Guangxi pangolin-CoVs but not other closely related pangolin-CoVs or bat-CoVs. We then document evidence of ORF3d translation, characterize its protein sequence, and conduct an evolutionary analysis at three levels: between taxa (21 members of Severe acute respiratory syndrome-related coronavirus), between human hosts (3978 SARS-CoV-2 consensus sequences), and within human hosts (401 deeply sequenced SARS-CoV-2 samples). ORF3d has been independently identified and shown to elicit a strong antibody response in COVID-19 patients. However, it has been misclassified as the unrelated gene ORF3b, leading to confusion. Our results liken ORF3d to other accessory genes in emerging viruses and highlight the importance of OLGs.

In March 2017, a wild-caught female common mudpuppy Necturus maculosus from Iowa, USA, with an enlarged posterior abdomen was submitted for diagnostic assessment. The cause of the abdominal distension was a large fluid-filled abdominal mass, diagnosed as a nephroblastoma. Parasites and numerous bacteria were isolated and identified from the mudpuppy but were determined to be incidental. Samples of the neoplasm inoculated onto an American toad Anaxyrus americanus cell line (BufoTad) yielded cytopathic effect during several passages. However, standard molecular testing of the cell culture supernatant failed to identify any viruses. Next-generation sequencing identified the replicating agent as a bacterium of the genus Acholeplasma. Immunohistochemistry confirmed the presence of Acholeplasma within the nephroblastoma, including within tumor cells. This is the first report of nephroblastoma and the second report of neoplasia in this species. The results also suggest that certain bacteria of the genus Acholeplasma might be oncogenic.

Hepatocystis is a genus of single-celled parasites infecting, amongst other hosts, monkeys, bats and squirrels. Although thought to have descended from malaria parasites (Plasmodium spp.), Hepatocystis spp. are thought not to undergo replication in the blood–the part of the Plasmodium life cycle which causes the symptoms of malaria. Furthermore, Hepatocystis is transmitted by biting midges, not mosquitoes. Comparative genomics of Hepatocystis and Plasmodium species therefore presents an opportunity to better understand some of the most important aspects of malaria parasite biology. We were able to generate a draft genome for Hepatocystis sp. using DNA sequencing reads from the blood of a naturally infected red colobus monkey. We provide robust phylogenetic support for Hepatocystis sp. as a sister group to Plasmodium parasites infecting rodents. We show transcriptomic support for a lack of replication in the blood and genomic support for a complete loss of a family of genes involved in red blood cell invasion. Our analyses highlight the rapid evolution of genes involved in parasite vector stages, revealing genes that may be critical for interactions between malaria parasites and mosquitoes., Author summary Hepatocystis parasites are single-celled organisms, closely related to the Plasmodium species which cause malaria. But Hepatocystis are distinct–unlike Plasmodium they are thought not to replicate in the blood and cause little or no disease in their mammalian hosts. They are transmitted from one host to the next, not by mosquitoes, but by biting midges. In this study we generated a genome sequence for Hepatocystis–the first time this data has ever been produced and analysed for this species. We compared genome sequences of Hepatocystis and Plasmodium, confirming that Hepatocystis is descended from Plasmodium. We strengthened support for the absence of replication in the blood and, in line with this finding, discovered that genes involved in interaction with red blood cells have been lost in Hepatocystis. Our analyses revealed rapid evolution of genes which are active when the parasite is in the insect vector, highlighting those which might be important for understanding interaction between malaria parasites and mosquitoes. Hepatocystis has a fascinating evolutionary story and is a powerful comparator for understanding malaria parasite biology.

Use of antimicrobials in animal agriculture is under increasing scrutiny, but the quantity of antimicrobials used on large US dairy farms has not been evaluated using data from large farms and different metrics. This study investigated total antimicrobial usage (AMU) in adult dairy cows and preweaned calves (PWC) and contrasted 2 metrics used for measurement of AMU. Wisconsin dairy farms were eligible if they had >250 lactating cows, maintained computerized animal health records, and were willing to allow researchers access to treatment records. Animal health data for a 1-yr period was retrospectively collected from computerized records, and a farm visit was performed to verify case definitions and recording accuracy. Both dose-based (animal daily doses; ADD) and mass-based (total mg of antimicrobials per kg of body weight; BW) metrics were calculated at the herd, cow, and PWC levels. Descriptive statistics for AMU were examined for both age groups. Mean AMU was compared among active ingredients and route of usage using ANOVA models that included farm as a random variable. At enrollment, farms (n = 40) contained approximately 52,639 cows (mean: 1,316 ± 169; 95% CI: 975, 1657) and 6,281 PWC (mean: 180 ± 33; 95% CI: 112, 247). When estimated using ADD, total herd AMU was 17.2 ADD per 1,000 animal-days (95% CI: 14.9, 19.5), with 83% of total herd-level AMU in adult cows. When estimated using the mass-based metric, total herd AMU was 13.6 mg of antimicrobial per kilogram of animal BW (95% CI: 10.3, 17.0), with 86% of total AMU used in adult cows. For cows, 78% of total ADD (15.8 ADD per 1,000 cow-d) was administered as intramammary (IMM) preparations. In contrast, when AMU was estimated using a mass-based metric, IMM preparations represented only 24% of total AMU (12.1 mg of antimicrobial/kg of cow BW). For cows, ceftiofur was the primary antimicrobial used and accounted for 53% of total ADD, with 80% attributed to IMM and 20% attributed to injectable treatments. When estimated using a mass-based metric, ampicillin was the predominant antimicrobial used in cows and accounted for 33% of total antimicrobial mass per kilogram of BW. When AMU was estimated for PWC using ADD, injectable antimicrobials represented 79% of total usage (28.3 ADD per 1,000 PWC-d). In contrast, when AMU was estimated for PWC using a mass-based metric, injectable products represented 42% of total AMU, even though more farms administered antimicrobials using this route. When AMU in PWC was summarized using ADD, penicillin represented 32% of AMU, and there were no significant differences in ADD among ampicillin, oxytetracycline or enrofloxacin. When a mass-based metric was used to estimate AMU in PWC, oral products (sulfadimethoxine and trimethoprim-sulfa) represented more than half of the total AMU given to this group. Overall, these results showed that choice of metric and inclusion of different age groups can substantially influence interpretation of AMU on dairy farms.

Male lower urinary tract symptoms (LUTS) comprise a common syndrome of aging that negatively impacts quality of life. The etiology of LUTS is multifactorial, involving benign prostatic hyperplasia, smooth muscle and neurologic dysfunction, inflammation, sexually transmitted infections, fibrosis, and potentially dysbiosis, but this aspect remains poorly explored. We investigated whether the presence of infectious agents in urine might be associated with LUTS by combining next-generation DNA sequencing for virus discovery, microbiome analysis for characterization of bacterial communities, and mass spectrometry-based metabolomics. In urine from 29 LUTS cases and 9 controls from Wisconsin, we found a statistically significant association between a diagnosis of LUTS and the presence of JC virus (JCV), a common neurotropic human polyomavirus (Polyomaviridae,Betapolyomavirus) linked to severe neurologic disease in rare cases. This association (based on metagenomics) was not borne out when specific polymerase chain reaction (PCR) testing was applied to this set of samples, likely due to the greater sensitivity of PCR. Interestingly, urine metabolomics analysis identified dysregulation of metabolites associated with key LUTS processes. Microbiome analysis found no evidence of microbial community dysbiosis in LUTS cases, but JCV-positive samples contained moreAnaerococcusspecies, which are involved in polymicrobial infections of the urinary tract. Neither age nor body mass index were significantly associated with the presence of urinary JCV—in the initial group or in an additional, regionally distinct group. These data provide preliminary support the hypothesis that viruses such as JCV may play a role in the development or progression of LUTS, together with other infectious agents and host metabolic responses.

Gyroviruses are small, single stranded DNA viruses in the family Anelloviridae. In chickens, the type virus (chicken anemia virus; CAV) causes epidemic disease in poultry flocks worldwide. In 2007 and 2008, young crested screamers (Chauna torquata) at a zoo in Wisconsin, USA, died of neurologic disease with clinical and pathological features resembling CAV infection. Conventional diagnostics were negative, but molecular analyses revealed coinfection of an affected bird with three variants of a novel Gyrovirus lineage, GyV10. Analysis of ten additional screamers from this and another zoo revealed infection in all but one bird, with co-infections and persistent infections common. The association between GyV10 ("screamer anemia virus," provisionally) and the disease remains unproven, but certain immunological and neurologic features of the syndrome would expand the known pathologic consequences of Gyrovirus infection. To control the virus, autogenous vaccines, environmental decontamination, and management strategies to limit vertical and horizontal transmission might prove effective.

West Nile virus (WNV) and Flanders virus (FLAV) co-occur in regions of North America. Because both viruses are maintained in a transmission cycle involving Culex mosquitoes and birds, screening mosquitoes for FLAV has been suggested as an enhancement to WNV surveillance and epidemic prediction. Using samples collected in 2010 and 2012 in Chicago, IL, USA, we demonstrate the presence of FLAV in four out of 287 (1.4%) Culex pools. We estimated minimum infection rates for WNV and FLAV to be 5.66 and 1.22 in 2010 and 8.74 and 0.61 in 2012, respectively. FLAV occurred 1 and 3 wk prior to the peak of WNV transmission in 2010 and 2012, respectively. FLAV sequences from Chicago were genetically diverse and phylogenetically representative of lineage A viruses from across the United States.

We describe a lethal respiratory outbreak among wild chimpanzees in Uganda in 2013 for which molecular and epidemiologic analyses implicate human rhinovirus C as the cause. Postmortem samples from an infant chimpanzee yielded near-complete genome sequences throughout the respiratory tract; other pathogens were absent. Epidemiologic modeling estimated the basic reproductive number (R0) for the epidemic as 1.83, consistent with the common cold in humans. Genotyping of 41 chimpanzees and examination of 24 published chimpanzee genomes from subspecies across Africa showed universal homozygosity for the cadherin-related family member 3 CDHR3-Y529 allele, which increases risk for rhinovirus C infection and asthma in human children. These results indicate that chimpanzees exhibit a species-wide genetic susceptibility to rhinovirus C and that this virus, heretofore considered a uniquely human pathogen, can cross primate species barriers and threatens wild apes. We advocate engineering interventions and prevention strategies for rhinovirus infections for both humans and wild apes.

Globally, habitat degradation is accelerating, especially in the tropics. Changes to interface habitats can increase environmental overlap among nonhuman primates, people, and domestic animals and change stress levels in wildlife, leading to changes in their risk of parasite infections. However, the direction and consequences of these changes are unclear, since animals may benefit by exploiting human resources (e.g., improving nutritional health by eating nutritious crops) and decreasing susceptibility to infection, or interactions with humans may lead to chronic stress and increased susceptibility to infection. Vervet monkeys are an excellent model to understand parasitic disease transmission because of their tolerance to anthropogenic disturbance. Here we quantify the gastrointestinal parasites of a group of vervet monkeys (Chlorocebus aethiops) near Lake Nabugabo, Uganda, that frequently overlaps with people in their use of a highly modified environment. We compare the parasites found in this population to seven other sites where vervet monkey gastrointestinal parasites have been identified. The vervets of Lake Nabugabo have the greatest richness of parasites documented to date. We discuss how this may reflect differences in sampling intensity or differences in the types of habitat where vervet parasites have been sampled.

Culex flavivirus (CxFV) is an insect-specific flavivirus infecting Culex mosquitoes, which are important vectors of West Nile virus (WNV). CxFV and WNV cocirculate in nature and coinfect Culex mosquitoes, including in a WNV “hotspot” in suburban Chicago. We previously identified a positive association between CxFV and WNV in mosquito pools collected from suburban Chicago in 2006. To further investigate this phenomenon, we compared the spatial and temporal distribution of CxFV during an interepidemic year (2011) and an epidemic year (2012) for WNV. Both viruses were more prevalent in mosquito pools in 2012 compared to 2011. During both years, the CxFV infection status of mosquito pools was associated with environmental factors such as habitat type and precipitation frequency rather than coinfection with WNV. These results support the idea that WNV and CxFV are ecologically associated, perhaps because both viruses respond to similar environmental drivers of mosquito populations.

An 11-d-old Holstein bull calf was presented to the Veterinary Medical Teaching Hospital at the University of Wisconsin–Madison because of a 4-d history of diarrhea and persistent low-grade fever. Initial diagnosis was enteritis caused by Cryptosporidium and rotavirus. During hospitalization, the calf became stuporous and was only responsive to noxious stimuli, with hypotonia of all 4 limbs, tail, head, and neck. A cerebrospinal fluid analysis revealed xanthochromia, with marked lymphocytic pleocytosis, which was suggestive of viral meningitis and/or encephalitis. Aichivirus B, which belongs to the Kobuvirus genus, was tentatively identified in spinal fluid by next-generation DNA sequencing. This virus can affect a multitude of species, including humans and cattle, and has been isolated from both healthy and diarrheic individuals. However, to date, a possible connection with neurologic disease has not been described, to our knowledge.

“Village dogs” in developing economies are assumed to be heavily burdened by infectious disease. We followed a cohort of 61 village dogs in rural western Uganda prospectively for fifteen months to measure changes in health and demographic outcomes, and to examine risk factors for morbidity and mortality. The mean (± standard deviation) number of dogs per household was 2.4 (± 2.0), of which 56.0% were male and 44.0% female. For females, average age at first estrus was 1.7 (± 0.6) years with a mean litter size of 3.8 (± 1.5). In the first, second and third parities, average puppy mortality per litter was 3.2 (± 2.5), 2.4 (± 2.1) and 3.4 (± 2.9), respectively. The main causes of morbidity and mortality were infectious disease (46.1%), culling (euthanasia) by owners (30.8%), and attacks by baboons, Papio anubis (23.1%). Cox proportional hazard regression showed that a clinical diagnosis of anemia significantly predicted morbidity (HR = 4.3 (95% CI: 1.1–17.8); p < 0.05), and younger age significantly predicted mortality (HR = 3.6 (95% CI: 1.2–10.6); p < 0.05). Our results indicate that infectious disease is indeed important to the health and survival in village dogs in this setting, but that cultural practices related to ownership and interactions with wildlife also contribute substantially to morbidity and mortality.

Hunting and consumption of wild animals, colloquially known as “bushmeat,” is associated with health trade-offs. Contact with wildlife increases exposure to wildlife-origin zoonotic diseases yet bushmeat is an important nutritional resource in many rural communities. In this study, we test the hypothesis that bushmeat improves food security in communities that hunt and trade bushmeat regularly. We conducted 478 interviews with men and women in six communities near Cross River National Park in Nigeria. We used interview responses to relate prevalence and diversity of bushmeat consumption to household food security status. Animal-based foods were the most commonly obtained items from the forest, and 48 types of wild vertebrate animals were consumed within the past 30 days. Seventy-five percent of households experienced some degree of food insecurity related to food access. Bushmeat consumption was significantly associated with relatively higher household food security status. Rodents were more important predictors of food security than other animal taxa. Despite increased bushmeat consumption in food-secure households, food-insecure households consumed a higher diversity of bushmeat species. Results show that consumption of bushmeat, especially rodents, is uniquely related to improved food security. Reliance on a wider diversity of species in food-insecure households may in turn affect their nutrition, exposures to reservoirs of zoonotic infections, and impact on wildlife conservation. Our results indicate that food security should be addressed in conservation and public health strategies aimed at reducing human–wildlife contact, and that improved wildlife protection, when combined with alternative animal-based foods, would positively affect food security in the long term.

Viral hemorrhagic septicemia virus (VHSV) is an ongoing cause of disease and mortality in freshwater fishes across the Great Lakes region of the Midwestern United States. Antibody detection assays such as enzyme-linked immunosorbent assay (ELISA) are nonlethal serological methods that can have significantly shorter turnaround times than the current validated viral detection diagnostic methodology for VHSV: cell culture with confirmation by polymerase chain reaction (PCR). This study evaluated an ELISA that detects nonneutralizing antinucleocapsid antibodies to VHSV in Northern Pike Esox lucius. Juvenile Northern Pike were experimentally infected with VHSV by intraperitoneal injection. The infected fish were monitored for 12 weeks for signs of disease, and weekly serum samples were obtained. An analysis of the survival data showed that mortality occurred significantly more quickly in inoculated fish than in control fish. Fish that were infected by injection showed a significant increase in antibody response by 2 weeks postinfection. However, variation in the rate and pattern of antibody response among the infected fish was high at any given point. The optimum window for detecting antibodies in Northern Pike is 2-12 weeks postinfection, which generally follows the median time to appearance of clinical signs (21 d postinfection). The receiver-operating characteristic curve analysis showed the ELISA to have a sensitivity of 80.5% and a specificity of 63.2% in Northern Pike, but these values can be adjusted by choosing different percent inhibition cutoffs, which may facilitate the use of the test for specific management goals. The results of this study offer insights into the disease progression and immune kinetics of VHSV, including interindividual variation, which will aid in the management of this economically important virus.

Multiple vector-borne pathogens often circulate in the same vector and host communities, and seasonal infection dynamics influence the potential for pathogen interactions. Here, we explore the seasonal infection patterns of avian malaria (Haemosporida) parasites (Plasmodium and Haemoproteus) and West Nile virus (WNV) in birds and mosquitoes in suburban Chicago. We show that both pathogens vary seasonally in Culex mosquitoes and avian hosts, but that patterns of covariation are complex. Different putative Plasmodium species varied asynchronously across the season in mosquitoes and birds, suggesting that different forces may govern their transmission. Infections of Culex mosquitoes with Plasmodium parasites were positively associated with WNV infections in pools of individuals aggregated from the same time and site, suggesting that these pathogens respond to common environmental drivers and co-circulate among the same host and vector populations. Future research should focus on these common drivers, and whether these pathogens interact in vectors and hosts.

The local abundance of Culex mosquitoes is a central factor adding to the risk of West Nile virus transmission, and vector abundance data influence public health decisions. This study evaluated differences in abundance estimates from mosquitoes trapped using two common methods: CO2- baited CDC light traps and infusion-baited gravid traps in suburban, Chicago, Illinois. On a weekly basis, the two methods were modestly correlated ( r = 0.219) across 71 weeks over 4 years. Lagged weather conditions of up to four weeks were associated with the number of mosquitoes collected in light and gravid traps. Collections in light traps were higher with higher temperature in the same week, higher precipitation one, two, and four weeks before the week of trapping, and lower maximum average wind speed. Collections in gravid traps were higher with higher temperature in the same week and one week earlier, lower temperature four weeks earlier, and with higher precipitation two and four weeks earlier. Culex abundance estimates from light traps were significantly higher in semi-natural areas compared to residential areas, but abundance estimates from gravid traps did not vary by the landscape type. These results highlight the importance of the surveillance methods used in the assessment of local Culex abundance estimates. Measures of risk of exposure to West Nile virus should assess carefully how mosquito abundance has been estimated and integrated into assessments of transmission risk.

Mosquito-based surveillance is a practical way to estimate the risk of transmission of West Nile virus (WNV) to people. Variations in temperature and precipitation play a role in driving mosquito infection rates and transmission of WNV, motivating efforts to predict infection rates based on prior weather conditions. Weather conditions and sequential patterns of meteorological events can have particularly important, but regionally distinctive, consequences for WNV transmission, with high temperatures and low precipitation often increasing WNV mosquito infection. Predictive models that incorporate weather can thus be used to provide early indications of the risk of WNV infection. The purpose of this study was first, to assess the ability of a previously published model of WNV mosquito infection to predict infection for an area within the region for which it was developed, and second, to improve the predictive ability of this model by incorporating new weather factors that may affect mosquito development. The legacy model captured the primary trends in mosquito infection, but it was improved considerably when calibrated with local mosquito infection rates. The use of interaction terms between precipitation and temperature improved model performance. Specifically, temperature had a stronger influence than rainfall, so that lower than average temperature greatly reduced the effect of low rainfall on increased infection rates. When rainfall was lower, high temperature had an even stronger positive impact on infection rates. The final model is practical, stable, and operationally valid for predicting West Nile virus infection rates in future weeks when calibrated with local data., Journal of Medical Entomology, 53(4), pp.935-944; 2016

Foot-and-mouth disease (FMD) is a highly contagious viral disease that severely impacts global food security and is one of the greatest constraints on international trade of animal products. Extensive viral population diversity and rapid, continuous mutation of circulating FMD viruses (FMDVs) pose significant obstacles to the control and ultimate eradication of this important transboundary pathogen. The current study investigated mechanisms contributing to within-host evolution of FMDV in a natural host species (cattle). Specifically, vaccinated and non-vaccinated cattle were infected with FMDV under controlled, experimental conditions and subsequently sampled for up to 35 days to monitor viral genomic changes as related to phases of disease and experimental cohorts. Consensus-level genomic changes across the entire FMDV coding region were characterized through three previously defined stages of infection: early, transitional, and persistent. The overall conclusion was that viral evolution occurred via a combination of two mechanisms: emergence of full-genomic minority haplotypes from within the inoculum super-swarm, and concurrent continuous point mutations. Phylogenetic analysis indicated that individuals were infected with multiple distinct haplogroups that were pre-existent within the ancestral inoculum used to infect all animals. Multiple shifts of dominant viral haplotype took place during the early and transitional phases of infection, whereas few shifts occurred during persistent infection. Overall, this work suggests that the establishment of the carrier state is not associated with specific viral genomic characteristics. These insights into FMDV population dynamics have important implications for virus sampling methodology and molecular epidemiology.

Antibiotic resistance is a global concern, although it has been studied most extensively in developed countries. We studied Escherichia coli and class 1 integrons in western Uganda by analyzing 1,685 isolates from people, domestic animals, and wild nonhuman primates near two national parks. Overall, 499 isolates (29.6%) were resistant to at least one of 11 antibiotics tested. The frequency of resistance reached 20.3% of isolates for trimethoprim-sulfamethoxazole but was nearly zero for the less commonly available antibiotics ciprofloxacin (0.4%), gentamicin (0.2%), and ceftiofur (0.1%). The frequency of resistance was 57.4% in isolates from people, 19.5% in isolates from domestic animals, and 16.3% in isolates from wild nonhuman primates. Isolates of livestock and primate origin displayed multidrug resistance patterns identical to those of human-origin isolates. The percentage of resistant isolates in people was higher near Kibale National Park (64.3%) than near Bwindi Impenetrable National Park (34.6%), perhaps reflecting local socioeconomic or ecological conditions. Across antibiotics, resistance correlated negatively with the local price of the antibiotic, with the most expensive antibiotics (nalidixic acid and ciprofloxacin) showing near-zero resistance. Among phenotypically resistant isolates, 33.2% harbored class 1 integrons containing 11 common resistance genes arranged into nine distinct gene cassettes, five of which were present in isolates from multiple host species. Overall, these results show that phenotypic resistance and class 1 integrons are distributed broadly among E. coli isolates from different host species in this region, where local socioeconomic and ecological conditions may facilitate widespread diffusion of bacteria or resistance-conferring genetic elements. IMPORTANCE Antibiotic resistance is a global problem. This study, conducted in rural western Uganda, describes antibiotic resistance patterns in Escherichia coli bacteria near two forested national parks. Resistance was present not only in people, but also in their livestock and in nearby wild nonhuman primates. Multidrug resistance and class 1 integrons containing genes that confer resistance were common and were similar in people and animals. The percentage of resistant isolates decreased with increasing local price of the antibiotic. Antibiotic resistance in this setting likely reflects environmental diffusion of bacteria or their genes, perhaps facilitated by local ecological and socioeconomic conditions.

Over 40 species of nonhuman primates host simian immunodeficiency viruses (SIVs). In natural hosts, infection is generally assumed to be nonpathogenic due to a long coevolutionary history between host and virus, although pathogenicity is difficult to study in wild nonhuman primates. We used whole-blood RNA-seq and SIV prevalence from 29 wild Ugandan red colobus (Piliocolobus tephrosceles) to assess the effects of SIV infection on host gene expression in wild, naturally SIV-infected primates. We found no evidence for chronic immune activation in infected individuals, suggesting that SIV is not immunocompromising in this species, in contrast to human immunodeficiency virus in humans. Notably, an immunosuppressive gene, CD101, was upregulated in infected individuals. This gene has not been previously described in the context of nonpathogenic SIV infection. This expands the known variation associated with SIV infection in natural hosts and may suggest a novel mechanism for tolerance of SIV infection in the Ugandan red colobus.

Respiratory illnesses have caused significant mortality in African great ape populations. While much effort has been given to identifying the responsible pathogens, little is known about the factors that influence disease transmission or individual susceptibility. In the Kanyawara community of wild chimpanzees, respiratory illness has been the leading cause of mortality over 31 years, contributing to 27% of deaths. Deaths were common in all age groups except juveniles. Over 22 years of health observations, respiratory signs were rare among infants and most common among older adults of both sexes. Respiratory signs were also common among males during the transition to adulthood (ages 10–20 years), particularly among those of low rank. Respiratory signs peaked conspicuously in March, a pattern that we could not explain after modelling climatic factors, group sizes, diet or exposure to humans. Furthermore, rates of respiratory illness in the chimpanzees did not track seasonal rates of illness in the nearby village. Our data indicate that the epidemiology of chimpanzee respiratory illness warrants more investigation but clearly differs in important ways from humans. Findings on individual susceptibility patterns suggest that respiratory signs are a robust indicator for investigating immunocompetence in wild chimpanzees.

Wild nonhuman primates are immediate sources and long-term reservoirs of human pathogens. However, ethical and technical challenges have hampered the identification of novel blood-borne pathogens in these animals. We recently examined RNA viruses in plasma from wild African monkeys and discovered several novel, highly divergent viruses belonging to the family Arteriviridae . Close relatives of these viruses, including simian hemorrhagic fever virus, have caused sporadic outbreaks of viral hemorrhagic fever in captive macaque monkeys since the 1960s. However, arterivirus infection in wild nonhuman primates had not been described prior to 2011. The arteriviruses recently identified in wild monkeys have high sequence and host species diversity, maintain high viremia, and are prevalent in affected populations. Taken together, these features suggest that the simian arteriviruses may be “preemergent” zoonotic pathogens. If not, this would imply that biological characteristics of RNA viruses thought to facilitate zoonotic transmission may not, by themselves, be sufficient for such transmission to occur.

In 49 patients with known Ebola virus disease outcomes during the ongoing outbreak in Sierra Leone, 13 were coinfected with the immunomodulatory pegivirus GB virus C (GBV-C). Fifty-three percent of these GBV-C + patients survived; in contrast, only 22% of GBV-C − patients survived. Both survival and GBV-C status were associated with age, with older patients having lower survival rates and intermediate-age patients (21 to 45 years) having the highest rate of GBV-C infection. Understanding the separate and combined effects of GBV-C and age on Ebola virus survival may lead to new treatment and prevention strategies, perhaps through age-related pathways of immune activation.

To the Editor: Hepatitis A (HAV; family Picornaviridae; genus Hepatovirus) is an ≈7.5-kb single-stranded positive-sense RNA virus that causes acute inflammation of the liver in humans and nonhuman primates. Although HAV is most commonly transmitted by food and water contaminated with feces, humans have acquired HAV from handling infected nonhuman primates in captivity (1). HAV has been detected in recently imported captive primates after spontaneous outbreaks of acute hepatitis in animal facilities, but the definitive hosts of this virus have remained obscure (2,3). We identified by next-generation sequencing HAV in the blood of a free-living olive baboon (Papio anubis) from Kibale National Park, Uganda, sampled in September 2010. Subsequent testing of a separate Kibale olive baboon troop in 2014 indicated the virus was prevalent and shed in feces. As part of a long-term study of nonhuman primate health and ecology, 23 animals were immobilized and sampled in 2010 as previously described (4). All animal protocols received prior approval from the Uganda National Council for Science and Technology (Kampala, Uganda), the Uganda Wildlife Authority (Kampala, Uganda), and the University of Wisconsin–Madison Animal Care and Use Committee (Madison, WI, USA). All samples were shipped in accordance with international laws under Convention on International Trade in Endangered Species of Wild Fauna and Flora Ugandan permit no. 002290. During May 2012, we subjected total RNA from 1 mL of blood plasma of each animal to next-generation sequencing as previously described (4); results showed HAV-like sequences in 1 of 23 baboons. De novo assembly of these reads yielded a nearly complete HAV genome, which we term KibOB-1. KibOB-1 is most similar (94.2% nt identity; Figure) to AGM-27, an HAV originally detected in an African green monkey (Chlorocebus aethiops) imported to a Russian primate facility from Kenya (3). Figure Whole-genome phylogenetic reconstruction of representative HAVs. HAVs are grouped into 6 genotypes based on 168 bp of the C-terminal extension of the viral protein 1 gene. Baboon HAV detected in Kibale National Park, Uganda, in 2010 and 2014 (GenBank ... For 11 baboons, we also collected a paired fecal sample, which we analyzed for evidence of viral shedding. Samples were preserved in RNAlater (Ambion Inc., Austin, TX, USA) at −20°C, and viral RNA was isolated by using the ZR Soil/Fecal RNA Microprep kit (Zymo Research, Irvine, CA, USA) following manufacturer’s protocols. Reverse transcription PCR (RT-PCR) of RNA was primed with random hexamers by using the RNA to cDNA Ecodry Premix (Random Hexamers) (Clontech Laboratories, Inc., Mountain View, CA, USA), and diagnostic PCR was conducted with primers flanking the C-terminal extension of the HAV viral protein (VP) 1 gene (pX) by using the High Fidelity PCR Master Mix-Ecodry Premix (Clontech Laboratories, Inc.). Five of 11 paired fecal samples tested positive for HAV by RT-PCR, indicating a higher prevalence of the virus in feces than in blood. We then surveyed a second troop of habituated olive baboons at the same field site during February–April 2014 (5). From these baboons, 7 of 19 fecal samples tested positive by RT-PCR. Confirmatory Sanger sequencing of RT-PCR amplicons was successful for 3 of these 7 animals (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"KT819576-KT819578","start_term":"KT819576","end_term":"KT819578","start_term_id":"1036222291","end_term_id":"1036222295"}}KT819576-KT819578). Phylogenetic analyses of these sequences demonstrate monophyly and a low degree of interhost variability (>94% nt identity). The risk to humans posed by KibOb-1 remains unknown. Although human infection with HAV genotype V has not been reported, evidence suggests that HAV variants might be capable of infecting a diversity of primate hosts (6). Although it is not known whether the closely related AGM-27 strain was discovered infecting its natural host, the similarity of KibOB-1 and AGM-27 raises the possibility of a recent host transfer. Major host shifts characterize the evolutionary histories of recently discovered bat and rodent hepatoviruses (7). Host fidelity of KibOB-1 is similarly unknown, but experimental infection of several nonhuman primate species with the similar AGM-27 virus found varying pathogenicity in different species (6). In particular, the AGM-27 caused productive infection in chimpanzees, with stimulation of a broadly reactive HAV immunoglobulin response (6). Human and simian HAVs are considered a single serotype (6); thus, serosurveillance for HAV in humans might be unable to distinguish between human and zoonotic simian HAV infection, enabling the possibility of cryptic zoonotic transmission. Similarly, detection of HAV antibodies in wild primates, such as in a recent study of baboons in South Africa living in close proximity to humans (8), might not indicate anthroponotic transmission of human viruses but rather infection with an endemic HAV. Prior studies have documented cross-species transmission between the primates of Kibale National Park and neighboring human populations, especially of gastrointestinal pathogens (9). A study tracking food-crop–raiding events on 97 farms within 0.5 km of Kibale’s forest edge found that 72% of households faced baboon raids over a 23-month period, including 228 discrete baboon raids (10). This finding suggests that a major portion of the local community remains at risk for exposure to potentially infectious baboon excreta. Such exposure, in addition to the evidence presented here that HAV is prevalent in wild baboons of Uganda and is shed into the environment, merits increased attention to the zoonotic risk for simian hepatoviruses.

Arthropod-borne viruses are among the most genetically constrained RNA viruses, yet they have a remarkable propensity to adapt and emerge. We studied wild birds and mosquitoes naturally infected with West Nile virus (WNV) in a ‘hot spot’ of virus transmission in Chicago, IL, USA. We generated full coding WNV genome sequences from spatiotemporally matched bird and mosquito samples using high-throughput sequencing, allowing a molecular evolutionary assessment with deep coverage. Mean FST among samples was 0.66 (±0.02 SE) and was bimodal, with mean nucleotide diversity being higher between samples (interhost πN = 0.001; πS = 0.024) than within them (intrahost πN 1.01 (in the PrM, NS2a, NS3, NS4b, and 5'-noncoding genomic regions) showed bird versus mosquito variant frequency differences of >30 per cent and/or polymorphisms fixed in ≥5 host or vector individuals, suggesting host tropism for these variants. However, phylogenetic analyses demonstrated a lack of grouping by bird or mosquito, most inter-sample differences were synonymous (mean interhost πN/πS = 0.04), and there was no significant difference between hosts and vectors in either their nucleotide diversities or levels of purifying selection (mean intrahost πN/πS = 0.28 in birds and πN/πS = 0.21 in mosquitoes). This finding contrasts with the ‘trade-off’ and ‘selective sieve’ hypotheses that have been proposed and tested in the laboratory, which predict strong host versus vector effects on WNV genetic variation, with heightened selective constraint in birds alternating with heightened viral diversity in mosquitoes. Overall, our data show WNV to be highly selectively constrained within and between both hosts and vectors but still able to vary at a limited number of sites across the genome. Such site-specific plasticity in the face of overall selective constraint may offer a mechanism whereby highly constrained viruses such as WNV and its relatives can still adapt and emerge.

Simian hemorrhagic fever virus (SHFV) causes a severe and almost uniformly fatal viral hemorrhagic fever in Asian macaques but is thought to be nonpathogenic for humans. To date, the SHFV life cycle is almost completely uncharacterized on the molecular level. Here, we describe the first steps of the SHFV life cycle. Our experiments indicate that SHFV enters target cells by low-pH-dependent endocytosis. Dynamin inhibitors, chlorpromazine, methyl-β-cyclodextrin, chloroquine, and concanamycin A dramatically reduced SHFV entry efficiency, whereas the macropinocytosis inhibitors EIPA, blebbistatin, and wortmannin and the caveolin-mediated endocytosis inhibitors nystatin and filipin III had no effect. Furthermore, overexpression and knockout study and electron microscopy results indicate that SHFV entry occurs by a dynamin-dependent clathrin-mediated endocytosis-like pathway. Experiments utilizing latrunculin B, cytochalasin B, and cytochalasin D indicate that SHFV does not hijack the actin polymerization pathway. Treatment of target cells with proteases (proteinase K, papain, α-chymotrypsin, and trypsin) abrogated entry, indicating that the SHFV cell surface receptor is a protein. Phospholipases A2 and D had no effect on SHFV entry. Finally, treatment of cells with antibodies targeting CD163, a cell surface molecule identified as an entry factor for the SHFV-related porcine reproductive and respiratory syndrome virus, diminished SHFV replication, identifying CD163 as an important SHFV entry component. IMPORTANCE Simian hemorrhagic fever virus (SHFV) causes highly lethal disease in Asian macaques resembling human illness caused by Ebola or Lassa virus. However, little is known about SHFV's ecology and molecular biology and the mechanism by which it causes disease. The results of this study shed light on how SHFV enters its target cells. Using electron microscopy and inhibitors for various cellular pathways, we demonstrate that SHFV invades cells by low-pH-dependent, actin-independent endocytosis, likely with the help of a cellular surface protein.

Bats are natural reservoir hosts of highly virulent pathogens such as Marburg virus, Nipah virus, and SARS coronavirus. However, little is known about the role of bat ectoparasites in transmitting and maintaining such viruses. The intricate relationship between bats and their ectoparasites suggests that ectoparasites might serve as viral vectors, but evidence to date is scant. Bat flies, in particular, are highly specialized obligate hematophagous ectoparasites that incidentally bite humans. Using next-generation sequencing, we discovered a novel ledantevirus (mononegaviral family Rhabdoviridae, genus Ledantevirus) in nycteribiid bat flies infesting pteropodid bats in western Uganda. Mitochondrial DNA analyses revealed that both the bat flies and their bat hosts belong to putative new species. The coding-complete genome of the new virus, named Kanyawara virus (KYAV), is only distantly related to that of its closest known relative, Mount Elgon bat virus, and was found at high titers in bat flies but not in blood or on mucosal surfaces of host bats. Viral genome analysis indicates unusually low CpG dinucleotide depletion in KYAV compared to other ledanteviruses and rhabdovirus groups, with KYAV displaying values similar to rhabdoviruses of arthropods. Our findings highlight the possibility of a yet-to-be-discovered diversity of potentially pathogenic viruses in bat ectoparasites.

Simian arteriviruses are a diverse clade of viruses infecting captive and wild nonhuman primates. We recently reported that Kibale red colobus virus 1 (KRCV-1) causes a mild and self-limiting disease in experimentally infected crab-eating macaques, while simian hemorrhagic fever virus (SHFV) causes lethal viral hemorrhagic fever. Here we characterize how these viruses evolved during replication in cell culture and in experimentally infected macaques. During passage in cell culture, 68 substitutions that were localized in open reading frames (ORFs) likely associated with host cell entry and exit became fixed in the KRCV-1 genome. However, we did not detect any strong signatures of selection during replication in macaques. We uncovered patterns of evolution that were distinct from those observed in surveys of wild red colobus monkeys, suggesting that these species may exert different adaptive challenges for KRCV-1. During SHFV infection, we detected signatures of selection on ORF 5a and on a small subset of sites in the genome. Overall, our data suggest that patterns of evolution differ markedly among simian arteriviruses and among host species. IMPORTANCE Certain RNA viruses can cross species barriers and cause disease in new hosts. Simian arteriviruses are a diverse group of related viruses that infect captive and wild nonhuman primates, with associated disease severity ranging from apparently asymptomatic infections to severe, viral hemorrhagic fevers. We infected nonhuman primate cell cultures and then crab-eating macaques with either simian hemorrhagic fever virus (SHFV) or Kibale red colobus virus 1 (KRCV-1) and assessed within-host viral evolution. We found that KRCV-1 quickly acquired a large number of substitutions in its genome during replication in cell culture but that evolution in macaques was limited. In contrast, we detected selection focused on SHFV ORFs 5a and 5, which encode putative membrane proteins. These patterns suggest that in addition to diverse pathogenic phenotypes, these viruses may also exhibit distinct patterns of within-host evolution both in vitro and in vivo .

Infectious disease transmission is a cost of sociality in humans and other animals. Nevertheless, the mechanisms linking social behaviour to infection risk are poorly known. We conducted a field experiment to examine how host intrinsic traits, behaviour and physiology affect infection of nonhuman primates with gastrointestinal parasites. We measured rate to reinfection in a social group of red-capped mangabeys, Cercocebus torquatus, following chemotherapeutic treatment for parasite infections. By measuring behaviour, infection and glucocorticoid levels, we compared the relative effects of space sharing, directional contact and physiological stress on risk of acquiring new infections. We found that, within proximity networks, individuals that were central and well connected and that had a tendency to switch groups were at increased risk of infection with helminths. Protozoan infections, however, were acquired more uniformly across the population. In general, position in the social network and, in particular, space sharing appears to be more important than the immunosuppressive effects of physiological stress or host traits in determining risk of infection. Our results suggest that future studies of disease ecology within wildlife populations should focus on measures of network association in addition to individual host traits.

A critical failure in our preparedness for an influenza pandemic is the lack of a universal vaccine. Influenza virus strains diverge by 1 to 2% per year, and commercially available vaccines often do not elicit protection from one year to the next, necessitating frequent formulation changes. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. We have constructed a recombinant modified vaccinia virus Ankara (MVA) that expresses an H5N1 mosaic hemagglutinin (H5M) (MVA-H5M). This mosaic was generated in silico using 2,145 field-sourced H5N1 isolates. A single dose of MVA-H5M provided 100% protection in mice against clade 0, 1, and 2 avian influenza viruses and also protected against seasonal H1N1 virus (A/Puerto Rico/8/34). It also provided short-term (10 days) and long-term (6 months) protection postvaccination. Both neutralizing antibodies and antigen-specific CD4 + and CD8 + T cells were still detected at 5 months postvaccination, suggesting that MVA-H5M provides long-lasting immunity. IMPORTANCE Influenza viruses infect a billion people and cause up to 500,000 deaths every year. A major problem in combating influenza is the lack of broadly effective vaccines. One solution from the field of human immunodeficiency virus vaccinology involves a novel in silico mosaic approach that has been shown to provide broad and robust protection against highly variable viruses. Unlike a consensus algorithm which picks the most frequent residue at each position, the mosaic method chooses the most frequent T-cell epitopes and combines them to form a synthetic antigen. These studies demonstrated that a mosaic influenza virus H5 hemagglutinin expressed by a viral vector can elicit full protection against diverse H5N1 challenges as well as induce broader immunity than a wild-type hemagglutinin.

Ticks in the nostrils of humans visiting equatorial African forests have been reported sporadically for decades, but their taxonomy and natural history have remained obscure. We report human infestation with a nostril tick in Kibale National Park, Uganda, coincident with infestation of chimpanzees in the same location with nostril ticks, as shown by high-resolution digital photography. The human-derived nostril tick was identified morphologically and genetically as a nymph of the genus Amblyomma, but the mitochondrial 12S ribosomal RNA or the nuclear intergenic transcribed spacer 2 DNA sequences of the specimen were not represented in GenBank. These ticks may represent a previously uncharacterized species that is adapted to infesting chimpanzee nostrils as a defense against grooming. Ticks that feed upon apes and humans may facilitate cross-species transmission of pathogens, and the risk of exposure is likely elevated for persons who frequent ape habitats.

Nonhuman primates (NHPs) are a historically important source of zoonotic viruses and are a gold-standard model for research on many human pathogens. However, with the exception of simian immunodeficiency virus (SIV) (family Retroviridae ), the blood-borne viruses harbored by these animals in the wild remain incompletely characterized. Here, we report the discovery and characterization of two novel simian pegiviruses (family Flaviviridae ) and two novel simian arteriviruses (family Arteriviridae ) in wild African green monkeys from Zambia (malbroucks [ Chlorocebus cynosuros ]) and South Africa (vervet monkeys [ Chlorocebus pygerythrus ]). We examine several aspects of infection, including viral load, genetic diversity, evolution, and geographic distribution, as well as host factors such as age, sex, and plasma cytokines. In combination with previous efforts to characterize blood-borne RNA viruses in wild primates across sub-Saharan Africa, these discoveries demonstrate that in addition to SIV, simian pegiviruses and simian arteriviruses are widespread and prevalent among many African cercopithecoid (i.e., Old World) monkeys. IMPORTANCE Primates are an important source of viruses that infect humans and serve as an important laboratory model of human virus infection. Here, we discover two new viruses in African green monkeys from Zambia and South Africa. In combination with previous virus discovery efforts, this finding suggests that these virus types are widespread among African monkeys. Our analysis suggests that one of these virus types, the simian arteriviruses, may have the potential to jump between different primate species and cause disease. In contrast, the other virus type, the pegiviruses, are thought to reduce the disease caused by human immunodeficiency virus (HIV) in humans. However, we did not observe a similar protective effect in SIV-infected African monkeys coinfected with pegiviruses, possibly because SIV causes little to no disease in these hosts.

Parasites are ubiquitous in wildlife populations, but physiological and behavioural responses of hosts to infection are difficult to measure. We experimentally treated semi-free-ranging red-capped mangabeys ( Cercocebus torquatus ) in Nigeria with antiparasitic drugs and examined subsequent changes in glucocorticoid production and individual behaviour. Because both parasites and stress impact energy balance and health, we measured (i) behavioural time re-allocation via activity budgets, (ii) social relationships (e.g. social connectivity and dominance hierarchy stability) and (iii) body condition. We collected triplicate faecal samples ( n = 441) from 49 individuals prior to and following treatment. Cortisol levels fluctuated in parallel with parasite abundance. Elevations in cortisol, but not parasitism, were related to reduced body condition. Behaviour also shifted according to infection status, with uninfected individuals spending more time foraging and less time resting and vigilant compared with when they were infected. Time spent feeding, travelling or socializing did not differ between pre- and post-treatment time periods. Group cohesion, but not dominance stability, changed following treatment, suggesting parasite-induced social avoidance. Together, these findings show a coordinated response to infection that promotes host tolerance through stress and energy conservation, reduces transmission risk and increases protection when infected hosts are vulnerable.

No longer do you have to visit rural areas to find ticks; birds are flying them directly to you. When researchers sampled several thousand birds in Chicago, they found that some carried ticks and that some of these ticks carried the organism that spreads Lyme disease. Although the number of infected ticks on these birds was low, risk for their invading an area and spreading infection to humans cannot be ignored. If conditions are favorable, a few infected ticks can quickly multiply. Migratory birds also carried tick species only known to be established in Central and South America. Limited introduction and successful establishment of ticks and disease-carrying organisms pose a major health risk for humans, wildlife, and domestic animals in urban environments worldwide., Bird-facilitated introduction of ticks and associated pathogens is postulated to promote invasion of tick-borne zoonotic diseases into urban areas. Results of a longitudinal study conducted in suburban Chicago, Illinois, USA, during 2005–2010 show that 1.6% of 6,180 wild birds captured in mist nets harbored ticks. Tick species in order of abundance were Haemaphysalis leporispalustris, Ixodes dentatus, and I. scapularis, but 2 neotropical tick species of the genus Amblyomma were sampled during the spring migration. I. scapularis ticks were absent at the beginning of the study but constituted the majority of ticks by study end and were found predominantly on birds captured in areas designated as urban green spaces. Of 120 ticks, 5 were infected with Borrelia burgdorferi, spanning 3 ribotypes, but none were infected with Anaplasma phagocytophilum. Results allow inferences about propagule pressure for introduction of tick-borne diseases and emphasize the large sample sizes required to estimate this pressure.