Genome-scale phylogeny of the alphavirus genus suggests a marine origin.

The genus Alphavirus comprises a diverse group of viruses, including some that cause severe disease. Using full-length sequences of all known alphaviruses, we produced a robust and comprehensive phylogeny of the Alphavirus genus, presenting a more complete evolutionary history of these viruses compared to previous studies based on partial sequences. Our phylogeny suggests the origin of the alphaviruses occurred in the southern oceans and spread equally through the Old and New World. Since lice appear to be involved in aquatic alphavirus transmission, it is possible that we are missing a louse-borne branch of the alphaviruses. Complete genome sequencing of all members of the genus also revealed conserved residues forming the structural basis of the E1 and E2 protein dimers.

Silent circulation of arboviruses in Cameroon.

OBJECTIVES: To investigate the silent circulation and transmission of arthropod-borne viruses (arboviruses) in the Fako Division of Cameroon. DESIGN: This survey was conducted based on clinical observations and laboratory diagnosis; field collections of mosquitoes. SETTING: This study was conducted in the Fako Division of South West Cameroon. SUBJECTS: One hundred and two sera were obtained from febrile patients (with negative laboratory findings for malaria and typhoid fever) at clinics in the Fako Division, and diurnal anthropophilic mosquitoes (4,764) collected. INTERVENTIONS: Virus isolation was attempted from these, and sera were screened for antibodies against 18 African arboviruses by haemagglutination inhibition (HI) and complement fixation (CF) tests. RESULTS: No virus was isolated. Fifty three of 79 (67.1%) sera reacted with one or more viral antigens. Twenty nine sera (36.7%) reacted with members of the genus Alphavirus, with Chikungunya (CHIKV) and O'nyong-nyong (ONNV) viruses as the most frequent (34.2%). Forty six sera (58.2%) reacted with members of the genus Flavivirus: 24 (30.4%) were cross-reactive, but 11.4% reacted monotypically with Zika, 5.1% with yellow fever virus (YFV), 5.1% with dengue virus-2 (DENV-2), 2.5% with DENV-1 and 1.3% with Wesselsbron virus, respectively. The plaque reduction neutralisation test used to specify the agent that elicited the response could not resolve 33.3% of the cross reactions between CHIKV and ONNV. Neutralising antibody titres against ONNV and CHIKV were very high indicating probable re-infection. CONCLUSION: Our results indicate previously undetected circulation of arboviruses in Cameroon, and suggest that they are important, overlooked public health problems.

Sibling Species in Montastraea annularis, Coral Bleaching, and the Coral Climate Record.

Measures of growth and skeletal isotopic ratios in the Caribbean coral Montastraea annularis are fundamental to many studies of paleoceanography, environmental degradation, and global climate change. This taxon is shown to consist of at least three sibling species in shallow waters. The two most commonly studied of these show highly significant differences in growth rate and oxygen isotopic ratios, parameters routinely used to estimate past climatic conditions; unusual coloration in the third may have confused research on coral bleaching. Interpretation or comparison of past and current studies can be jeopardized by ignoring these species boundaries.

Ecological effects of a major oil spill on panamanian coastal marine communities.

In 1986 more than 8 million liters of crude oil spilled into a complex region of mangroves, seagrasses, and coral reefs just east of the Caribbean entrance to the Panama Canal. This was the largest recorded spill into coastal habitats in the tropical Americas. Many population of plants and animals in both oiled and unoiled sites had been studied previously, thereby providing an unprecedented measure of ecological variation before the spill. Documenation of the spread of oil and its biological begun immediately. Intertidal mangroves, algae, and associated invertebrates were covered by oil and died soon after. More surprisingly, there was also extensive mortality of shallow subtidal reef corals and infauna of seagrass beds. After 1.5 years only some organisms in areas exposed to the open sea have recovered.

Evolution of Socioeconomic Conditions and Its Relation to Spatial-Temporal Changes of Giardiasis and Helminthiasis in Amazonian Children.

This study analyzed the evolution of socioeconomic, sanitary, and personal factors as well as spatiotemporal changes in the prevalence of helminthiasis and giardiasis in urban Amazonian children between 2003 and 2011. Child age, lack of sanitation, and lack of access to bottled water were identified as significant associated factors for helminthiasis and giardiasis. There was an overall improvement in socioeconomic and sanitary conditions in the city resulting in decreased helminth prevalences from 12.42 to 9.63% between 2003 and 2010, but the prevalence increased to 15.03% in 2011 due to migratory movement and unstable sanitary conditions. As for Giardiasis, socioeconomic and environmental changes were not enough to reduce prevalence (16% in 2003 and 23% in 2011). Spatial analysis identified a significant cluster for helminthiasis in an area of poor housing conditions. Control programs in the Amazon need to target high-risk areas focusing changes in sanitation, water usage, and health education.

Isolation of viruses from mosquitoes (Diptera: Culicidae) collected in the Amazon Basin region of Peru.

As part of a comprehensive study on the ecology of arthropod-borne viruses in the Amazon Basin region of Peru, we assayed 539,694 mosquitoes captured in Loreto Department, Peru, for arboviruses. Mosquitoes were captured either by dry ice-baited miniature light traps or with aspirators while mosquitoes were landing on human collectors, identified to species, and later tested on Vero cells for virus. In total, 164 virus isolations were made and included members of the Alphavirus (eastern equine encephalomyelitis, Trocara, Una, Venezuelan equine encephalomyelitis, and western equine encephalomyelitis viruses), Flavivirus (Ilheus and St. Louis encephalitis), and Orthobunyavirus (Caraparu, Itaqui, Mirim, Murutucu, and Wyeomyia viruses) genera. In addition, several viruses distinct from the above-mentioned genera were identified to the serogroup level. Eastern equine encephalomyelitis virus was associated primarily with Culex pedroi Sirivanakarn & Belkin, whereas Venezuelan equine encephalomyelitis virus was associated primarily with Culex gnomatos Sallum, Huchings & Ferreira. Most isolations of Ilheus virus were made from Psorophora ferox (Von Humboldt). Although species of the Culex subgenus Melanoconion accounted for only 45% of the mosquitoes collected, 85% of the virus isolations were made from this subgenus. Knowledge of the viruses that are being transmitted in the Amazon Basin region of Peru will enable the development of more effective diagnostic assays, more efficient and rapid diagnoses of clinical illnesses caused by these pathogens, risk analysis for military/civilian operations, and development of potential disease control measures.

Mortality and infertility in adult mosquitoes after the ingestion of blood containing ivermectin.

Mosquitoes of 3 species (Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus) were fed on human blood containing various concentrations of ivermectin. Three effects (death, decreased egg production, and reduced egg hatching) were observed in the insects, depending upon the concentration of ivermectin ingested. The LD50 of ivermectin in human blood for the 3 mosquito species was estimated to be 126, 208, and 698 ng/ml, respectively. Mosquitoes dying after ingestion of ivermectin developed signs of acute toxicity including paralysis, lethargy, incoordination, and difficulty in movement. Death usually occurred within 48-72 hr. With sublethal blood concentrations of the chemical, mosquitoes survived, but there was a marked reduction in both the number and viability of their eggs. This infertility was only temporary, however, as subsequent refeeding of the insects on uncontaminated blood resulted in the production of normal numbers of fertile eggs. Blood levels of ivermectin which made 50% of the eggs infertile in Ae. aegypti and Ae. albopictus were calculated 3.4 and 4.3 ng/ml, respectively. These latter concentrations of the chemical are within the range found in blood of humans and domestic animals receiving ivermectin for treatment of parasitic infections. This finding suggests that the widespread use of ivermectin in veterinary and human medicine may have an unrecognized effect on mosquito populations.

Simulation of arbovirus overwintering: survival of Toscana virus (Bunyaviridae:Phlebovirus) in its natural sand fly vector Phlebotomus perniciosus.

A series of experiments were done to study the effect of simulated summer and winter temperatures on the development of Phlebotomus perniciosus (Diptera:Psychodidae) and on the survival of Toscana virus in transovarially infected insects. Sand flies maintained at 28 degrees C developed relatively fast, with adults emerging from 40 to 55 days after initial oviposition. Similar results were obtained with insects reared at 25 degrees C. In contrast, sand flies maintained at 15 degrees C developed slowly up to the fourth larval instar; at that point, further development ceased and the insects entered diapause. Diapause could be terminated by increasing the ambient temperature to 25 degrees C. The ambient temperatures at which the immature forms were reared (15 degrees C, 25 degrees C, and 28 degrees C) had no effect on the subsequent F1 adult filial infection rates with Toscana virus (49.1%, 47.5%, and 46.5%, respectively). The results of these experiments provide a model of how Toscana virus survives the winter in endemic areas by maintenance in diapausing P. perniciosus larvae. In another experiment, venereal transmission of Toscana virus was shown from transovarially infected males to non-infected virgin females. This is the first demonstration of sexual transmission of a phlebovirus by sand flies. If venereal transmission occurs in nature, it would provide an alternative method of virus amplification in the vector population, in the absence of viremic vertebrates.

Ultrastructural aspects of replication of the New Jersey serotype of vesicular stomatitis virus in a suspected sand fly vector, Lutzomyia shannoni (Diptera: Psychodidae).

Transmission electron microscopy was used to examine replication of the New Jersey serotype of vesicular stomatitis virus (VSNJ) (Rhabdoviridae: Vesiculovirus) in Lutzomyia shannoni (Diptera: Psychodidae), a recently implicated sand fly vector. Following ingestion of an infectious blood meal, female sand flies were fixed and examined at approximately 12-hr intervals for six days. The New Jersey serotype of vesicular stomatitis virus was first detected in the abdominal midgut after 34 hr of incubation. Virus next appeared in fat body and the thoracic midgut at 48 hr, while salivary glands first contained visible virus in apical cavities 5-6 days after infection. Flight muscles and nervous tissue occasionally contained small numbers of VSNJ virions, while virus was never detected in the ovaries or malphigian tubules. The midgut and fat body appeared to be major sites of VSNJ virus replication. In all tissues examined, virus matured primarily by budding from the plasma membrane. Virions were occasionally observed within vacuoles, along with nucleocapsids. In the midgut, budding occurred exclusively from the basolateral plasma membrane, while maturation in salivary gland cells involved apical budding. Accumulation of virions adjacent to basal laminae surrounding several tissues suggested that this structure physically impedes virus dissemination within the sandfly. The paucity of virus budding 120-144 hr after infection suggested that the VSNJ virus infection was modulated in Lu. shannoni.

Characterization of Leishmania colombiensis sp. n (Kinetoplastida: Trypanosomatidae), a new parasite infecting humans, animals, and phlebotomine sand flies in Colombia and Panama.

Characterization of Leishmania colombiensis sp.n. is presented, which on the basis of biological and molecular criteria, appears to be a new member of the L. braziliensis complex. A total of nine isolates of the new parasite were made in Colombia and Panama between 1980 and 1986: two from human cases of cutaneous leishmaniasis, six from phlebotomine sand flies, and one from a sloth. Although most closely related to L. lainsoni, L. colombiensis sp.n. is clearly distinguishable from other members of the genus by its reactivity with monoclonal antibodies, isoenzyme electrophoresis, and restriction endonuclease fragment patterns of kinetoplast DNA (k-DNA).

Isolation and genetic characterization of a hantavirus (Bunyaviridae: Hantavirus) from a rodent, Oligoryzomys microtis (Muridae), collected in northeastern Peru.

This paper describes the isolation and partial genetic characterization of a hantavirus from a pygmy rice rat, Oligoryzomys microtis, collected within the urban area of Iquitos, Loreto Department, Peru. The virus, designated HTN-007, exhibited the highest degree of genetic similarity to Rio Mamore virus, which was originally described from the same rodent species in eastern Bolivia. Comparison of small and medium segment nucleotide sequence data from HTN-007 and Rio Mamore virus revealed 87% and 85% sequence identity, respectively. Based on these analyses, HTN-007 appears to be a variant of Rio Mamore virus. As such, it represents the first successful isolation of Rio Mamore virus and the first evidence for the existence of a hantavirus in Peru. Serologic studies done by immunofluorescence on blood samples of 56 O. microtis trapped at the collection site indicated that 21.4% had antibodies to hantavirus. In view of the proximity of this rodent species to humans and the close phylogenetic relationship of Rio Mamore virus to hantaviruses that have been associated with human disease, Rio Mamore virus may be a hantavirus of some public health importance in tropical South America.

Trocara virus: a newly recognized Alphavirus (Togaviridae) isolated from mosquitoes in the Amazon Basin.

This report describes Trocara virus, a newly recognized member of the genus Alphavirus, that has been isolated from Aedes serratus mosquitoes collected at two widely separated sites in the Amazon Basin. Biological, antigenic and genetic characteristics of the new virus are given. Results of these studies indicate that Trocara virus is the first member of a newly discovered antigenic complex within the family Togaviridae genus Alphavirus. The public health and veterinary importance of Trocara virus is still unknown.

Trans-beta-farnesene as a feeding stimulant for the sand fly Lutzomyia longipalpis (Diptera: Psychodidae).

The aphid alarm pheromone, trans-beta-farnesene (TBF), was found to stimulate feeding in both male and female Lutzomyia longipalpis Lutz & Neiva. Four other structurally related compounds (farnesol; 808 farnesene; trans, trans-farnesyl acetate; farnesyl methyl ether) were slightly less stimulating to these insects. The effect of TBF varied with sand fly age and the concentration of the chemical used. In contrast, TBF did not stimulate feeding in either sex of four other sand fly species (L. shannoni Dyar, Phlebotomus papatasi (Scopoli), P. argentipes Annandale & Brunetti, P. perniciosus Newstead). TBF might be useful in enhancing L. longipalpis field collections or in developing a poison bait for the control of this species.

Histologic detection of multiple blood meals in Phlebotomus duboscqi (Diptera: Psychodidae).

A histologic technique was used to detect multiple hamster blood meals taken by Phlebotomus duboscqi Neveu-Lemaire during a 5-d period. Forty-eight flies were fed two or three blood meals separated by 48, 72, or 120 h and sampled immediately; multiple meals were detected in 27 flies (56%). Double meals separated by 72 h within a single gonotrophic cycle were documented in 11/19 (58%) flies; double meals separated by 120 h were detected in only 4/17 (24%) flies. Triple blood meals taken at 0, 72, and 120 h were detected in 5/12 (42%) flies; all of these flies contained the second and third meals. Early blood meals were detected clearly within later blood meals as a delimited body of dark digested blood, heme (sometimes also with pink undigested blood), the presence of an associated pale pink-staining peritrophic plug, the presence and appearance of the peritrophic membrane surrounding the meals, and a physical space between meals; the first two characteristics were the most important. Development of the ovarian follicles including apparent dilatations was also observable using this histologic technique. The results of this study indicate that the rate of multiple feeding can be determined using histology. The technique would be useful in evaluating the blood feeding frequency of field-caught sand flies in endemic areas of leishmaniasis, bartonellosis, and phleboviruses.

Peritrophic envelopes of Lutzomyia spinicrassa (Diptera: Psychodidae).

The secretion, morphology, and chemical composition of the peritrophic envelope were studied in the phlebotomine sand fly, Lutzomyia spinicrassa Morales, OsornoMesa, Osorno & Hoyos, a suspected vector of Leishmania braziliensis in Colombia and Venezuela. Viewed under light microscopy, the envelope matured rapidly and could be dissected from the blood bolus as early as 12 h and until 36 h after feeding; subsequently it began to degrade. The envelope was initially a closed sac around the blood meal, but opened posteriorly in most flies by 6 h. The posterior opening may facilitate the migration and establishment of Le. braziliensis in the hindgut. Secretion of envelope precursors was from the entire midgut epithelium. Electron microscopy revealed that electron-dense precursor material (possibly chitin) was present, bathing the microvilli during the first 12 h after blood feeding. This secretion appeared to originate from the bases of the microvilli. From 1 to 36 h, an electron-lucid precursor material (possibly protein) was secreted from the entire length of microvilli and from their bases. Both precursors appeared to be formed at the epithelial surface, not associated with secretory vesicles. The envelope developed rapidly from precursor material, and by 6 h a defined electron-lucid structure was present above the microvilli. Most mature envelopes (12-36 h) were 0.5-2.1 microns thick, multilayered, wholly electron-lucid, and composed of microfibrils and granules. Electron-dense components were seen in some envelopes at 24-36 h. An anterior hyaline plug was present from 12 to 36 h. Envelopes were composed of chitin, protein, and glycoprotein, based on chemical and histochemical tests. The likely presence of several amino acids (lysine, aspartic acid, and glutamic acid) that may cross-link chitin and protein was demonstrated by a positive ninhydrin-Schiff test. This study constitutes the first ultrastructural investigation of peritrophic envelope development by a New World sand fly.

Effect of ivermectin on the ovarian development of Aedes aegypti (Diptera: Culicidae).

The effect of ivermectin (0.1 microgram/ml) on blood digestion, ovarian development, and ovipositional attributes of Aedes aegypti was studied using standard morphological and histological techniques. Uncoordinated movements and paralysis were observed in most ivermectin-treated females within 1 h after ingestion of blood containing the chemical. Eight days after the blood meal, 23.5% of the treated females had died, whereas no mortality occurred in controls. Formation of the peritrophic membrane and digestion of the blood meal were delayed in the surviving treated mosquitoes. The most striking effect of ivermectin on Ae. aegypti at this dosage was on ovarian development. Changes observed among ivermectin-treated mosquitoes included: blood digestion without development of ovarian follicles; degeneration of primary follicles and formation of ovarian dilatations within 24 h after ingestion of the chemical; significant reduction in the rate of vitellogenesis and follicle development; decreased egg production; reduced egg hatching; abnormal egg size and shape; and increased percentages of unhatched embryonated and sterile eggs. Although the precise action of ivermectin on Ae. aegypti is unknown, our studies indicate that the chemical directly or indirectly affects at least three major organ systems (nervous, digestive, and reproductive).

Formation and composition of the peritrophic membrane in the sand fly, Phlebotomus perniciosus (Diptera: Psychodidae).

The secretion, morphology, and chemical composition of the peritrophic membrane was studied in the sand fly, Phlebotomus perniciosus Newstead. The membrane was secreted from the entire midgut epithelium. An electron-dense fine granular secretion, possibly chitin, was present along the length of the microvilli immediately until 24 h after feeding. From 12-48 h, an electron-lucid coarse granular component, possibly protein, was also secreted from the microvillar surface. By light microscopy, the mature 36-h membrane characteristically consisted of a dark anterior cap and posterior open ring, with a transparent intervening membrane and anterior plug. Ultrastructure of the fully formed membrane at 24-48 h was highly variable. Undifferentiated membranes appeared as a single electron-lucid layer; differentiated membranes were more complex, sometimes two-layered, containing electron-lucid and -dense fibers and granules. Results of binding to succinylated wheat germ agglutinin, histochemistry, and amino acid analysis indicated that the membrane was composed of chitin, glycoprotein, and protein. Eighteen amino acids were identified in membrane proteins; aspartic-glutamic acids (and amides), serine, glycine, and lysine (45% by weight) may be important in cross-linking membrane components.

Development of Endotrypanum (Kinetoplastida:Trypanosomatidae) in experimentally infected phlebotomine sand flies (Diptera:Psychodidae).

The developmental biology (parasite establishment, migration, and differentiation) of Brazilian strains of Endotrypanum are reported for 3 sand fly species: Lutzomyia longipalpis Lutz & Neiva, L. shannoni Dyar, and Phlebotomus papatasi Scopoli. Laboratory-reared sand flies were infected by feeding on a promastigote suspension through a chick-skin membrane. Infections within the insect gut were examined at various times after feeding by staining fresh and fixed specimens. Development of Endotrypanum varied for each parasite-host species association. After feeding on culture forms of E. schaudinni Mesnil & Brimont (strain ISHA/BR/80/IM1111), significantly more L. shannoni (100%, 9/9) became infected than did L. longipalpis (62.3%, 33/53) or P. papatasi (27.3%, 15/55). The greatest number of infections were in the midgut and hindgut from 6 to 16 d after feeding, but flagellates also were present in the Malpighian tubules. Moreover, distinct development patterns in the sand fly gut were obtained when the Callejon L. longipalpis colony was fed on cultures of other Endotrypanum strains. Significantly fewer sand flies became infected with strain MCHO/BR/85/IM2259 (18.2%, 4/22) than with strain ISHA/BR/80/IM1111 (55.6%, 20/36). There were also individual variation in the distribution and survival of parasites within the guts of flies in each group. These data indicate that there is variation in the susceptibility to infection with Endotrypanum among and within sand fly species.

Head, neck, and mandible dynamics generated by 'whiplash'.

That injuries to the temporomandibular joint (TMJ) can be imposed by short-acting forces generated during rear-end collisions of motor vehicles was first proposed more than 50 years ago. Since that time, numerous anecdotal and clinical reports relating the onset of TMJ symptoms to low-velocity rear-end collisions have appeared in the literature. Various mechanisms of injury to the TMJ occurring during extension and flexion phases of 'whiplash' have been proposed. However, transient forces developed at the TMJ in impact velocity changes on the order of 8.0 kilometers per hour (km/h) have been shown to be well within typical physiologic ranges. This study applies current head/neck extension-flexion dynamic data to develop linear and angular force-time histories experienced at the TMJ. Fourteen test collisions of motor vehicles utilizing seven live test subjects were conducted in July 1993. Linear and angular accelerometers and high-speed photographic cameras recorded the vehicle and human-subject responses. Head accelerations and forces generated at the TMJ bore a generally linear relationship to the impact velocity changes in the range tested (3.9-10.9 km/h). Mandibular opening responses were measured on three test subjects. Neither neck hyperflexion nor hyperextension occurred for any subject on any trial. At some point in the series, all test subjects experienced neck muscle strain symptoms lasting 1-3 days. No TMJ symptoms were experienced. The head, neck, and mandible motions occurring in the 'whiplash' maneuver are more complex than previously described. The cervical muscle injury threshold appears to be reached in the 8.0 km/h range. Linear and rotational forces generated at the TMJ in rear-end impacts below the 11.0 km/h velocity-change level do not appear to be injurious.

Bioindication potential of carbonic anhydrase activity in anemones and corals.

Activity levels of carbonic anhydrase (CA) were assessed in anemones Condylactis gigantea and Stichodactyla helianthus with laboratory exposures to copper, nickel, lead, and vanadium, and also in animals collected from polluted vs pristine field sites. CA activity was found to be decreased with increase in metal concentration and also in animals collected from the polluted field site. Preliminary assessments to adapt the CA assay for use in the widespread coral Montastraea cavernosa show decreased CA activity in specimens from the polluted field site and provide an avenue for future research aimed at more thoroughly describing coral CA activity for potential application in bioindication.