Survey of Rickettsia spp. in ticks (Acari: Ixodidae) infesting opossums (Didelphis albiventris) and capybaras (Hydrochoerus hydrochaeris) from an urban park in southeastern Brazil.

Ticks are hematophagous arthropods and, during feeding, may transmit pathogens to vertebrate hosts, including humans. This study aimed to investigate the presence of Rickettsia spp. in ticks collected between 2010 and 2013 from free-ranging capybaras (Hydrochoerus hydrochaeris) and opossums (Didelphis albiventris) that inhabit Sabiá Park in Uberlândia, Brazil. Overall, 1,860 ticks were collected: 1,272 (68.4%) from capybaras (487 of the species Amblyomma sculptum, 475 adults and 12 nymphs; 778 Amblyomma dubitatum, 727 adults and 51 nymphs; and seven larva clusters of the genus Amblyomma); and 588 (31.6%) from opossums (21 A. sculptum, one adult and 20 nymphs; 79 A. dubitatum, all nymphs; 15 Ixodes loricatus, 12 adults and three nymphs; 457 Amblyomma sp. larva clusters; 15 Ixodes sp. larva clusters; and one Argasidae larva cluster). Out of 201 DNA samples tested for the presence of Rickettsia spp. DNA using polymerase chain reaction (PCR) 12 showed amplification of a gtlA gene segment that was specific to Rickettsia bellii, a bacterium non-pathogenic to humans. As there has been a report showing serological evidence of infections caused by Rickettsia species of the spotted fever group (SFG) in capybaras and opossums in the park, including Rickettsia rickettsii, the etiological agent of Brazilian spotted fever, and considering the presence of A. sculptum ticks, which are aggressive to humans, as well as these vertebrate hosts, which are amplifiers of R. rickettsii, it is important to monitor the presence of SFG rickettsiae in the Sabiá Park, which is visited daily by thousands of people.

Molecular detection of Borrelia sp. in Ornithodoros cavernicolous (Acari: Argasidae) in midwestern Brazil.

Ticks are obligate hematophagous parasites that can transmit to vertebrate hosts several pathogens, including viruses, bacteria, protozoa and helminths. Among these agents, some Borrelia species some Borrelia species cause disease in humans and other vertebrate hosts; therefore, they have medical and veterinary health importance. To gather additional information on Borrelia species in Brazil, the current study aimed to detect the presence of these species in Ornithodoros cavernicolous ticks collected in September 2019 from cement pipes that are used by bats as shelter in a farm located in the midwestern region of Brazil. DNA samples obtained from 18 specimens of O. cavernicolous were subjected of two polymerase chain reactions, targeting a segment of the Borrelia fla B gene. Of the samples tested, only one (6 %, 1/18) showed amplification. The nucleotide sequence of the amplified DNA showed more than 97 % (293/300) identity with a sequence of a Borrelia sp. detected in blood collected from a bat from Macaregua Cave, Colombia, and more than 97 % (292/300) detected in lungs from vampire bats from northeastern Brazil. The deduced amino acid sequences were identical to each other. Phylogenetic analysis indicated that these sequences formed a group of Borrelia species (putatively associated with bats) that is closely related to sequences of Borrelia species of the Lyme borreliosis group. Further investigations should be carried out in order to determine whether the sequence of the Borrelia sp. we found belongs to a new taxon. It will also be of great importance to determine which vertebrate hosts, besides bats, O. cavernicolous ticks can parasitize in order to investigate whether the Borrelia sp. we found may be transmitted and cause disease to the other vertebrate hosts.

Rickettsia communities and their relationship with tick species within and around the national park of Iguaçu, Brazil.

We report Rickettsia species from 2,334 ticks collected from environment (1,939 ticks) and animals (395 ticks) in the largest inland fragment of the Atlantic rainforest of southern Brazil and its fragments. Additionally, the DNA infection rates of Amblyomma ovale tick populations in the Neotropics with Rickettsia bellii and Rickettsia parkeri strain Atlantic rainforest were calculated using data from scientific publications, and their correlation was evaluated. From 11 tick species Rickettsia DNA was detected in seven (Amblyomma brasiliense, Amblyomma coelebs, Amblyomma incisum, Amblyomma longirostre, A. ovale, Haemaphysalis juxtakochi, Ixodes fuscipes) and was not detected in four species (Amblyomma dubitatum, Ixodes loricatus, Rhipicephalus microplus and Rhipicephalus sanguineus sensu lato). DNA of five Rickettsia species was detected (R. bellii, Rickettsia amblyommatis, Rickettsia rhipicephali, Rickettsia felis and Rickettsia sp. Aragaoi). To determine the prevalence of Rickettsia DNA positivity according to vector species, ticks were processed individually or in pools of 2-10 individuals (samples). The most prevalent Rickettsia species was R. bellii, found in 112 samples, followed by R. amblyommatis, R. rhipicephali, R. felis and Rickettsia sp. Aragaoi, found in 16, five, two and one sample, respectively. Rickettsia bellii DNA was found in five tick species with the highest infection rate in A. ovale and A. brasiliense. Absence of R. parkeri strain Atlantic rainforest in A. ovale ticks was an unexpected result. Furthermore, a negative correlation was identified between the infection rates (DNA) of R. bellii and/or R. parkeri strain Atlantic rainforest within A. ovale tick populations in the Neotropics. Putting together current knowledge, it can be proposed that, within natural settings, the diversity of rickettsiae and ticks creates a buffering effect on the overgrowth of rickettsiae and episodes of bacteremia in the hosts.

Evaluation of a mimotope of the Rickettsia outer membrane protein A (OmpA) as an antigen in enzyme-linked immunosorbent assay to detect rickettsiosis in capybaras (Hydrochoerus hydrochaeris), horses (Equus caballus), and opossums (Didelphis sp.).

Rickettsia rickettsii is the etiological agent of Rocky Mountain spotted fever, which is an important tick-borne zoonosis and, in Brazil, it causes Brazilian spotted fever, which has high lethality rate. This study aimed to evaluate a synthetic peptide corresponding to a segment of the outer membrane protein A (OmpA) as an antigen in a serological test for the diagnosis of rickettsial infections. The amino acid sequence of the peptide was selected by predicting B cell epitopes using B Cell Epitope Prediction (Immune Epitope Database and Analysis Resource) and Epitopia and OmpA sequences of Rickettsia rickettsii strain 'Brazil' and Rickettsia parkeri strains 'Maculatum 20' and 'Portsmouth'. A peptide with amino acid sequence common to both Rickettsia species was synthesized and arbitrarily named OmpA-pLMC. To evaluate this peptide in enzyme-linked immunosorbent assay (ELISA), serum samples of capybara (Hydrochoerus hydrochaeris), horse (Equus caballus), and opossum (Didelphis albiventris) that had been previously tested by indirect immunofluorescence assay (IFA) for rickettsial infection were separated into IFA-positive and IFA-negative groups and used in the assay. There were no significant differences in ELISA optical density (OD) values between IFA-positive and IFA-negative groups with horse samples. The mean OD values were significantly higher in the IFA-positive capybara serum samples (IFA-pos vs. IFA-neg = 2.389 ± 0.761 vs. 1.760 ± 0.840). However, receiver operating characteristic (ROC) curve analysis did not show significant diagnostic parameters. On the other hand, 12 out of 14 (85.7%) opossum samples of the IFA-positive group showed reactivity in ELISA, and this was significantly higher than of the IFA-negative group (0.7196 ± 0.440 vs. 0.2318 ± 0.098, respectively; 85.7% sensitivity, 100% specificity). Therefore, our results show that OmpA-pLMC has a potential to be used in immunodiagnostic assays to detect spotted fever group rickettsial infections.

Effect of BaltPLA2, a phospholipase A2 from Bothrops alternatus snake venom, on the viability of cells infected with dengue virus.

Dengue fever is considered a major public health problem in tropical and subtropical regions. Our study analyzed the effect of BaltPLA2, a phospholipase A2 from Bothrops alternatus snake venom, on the viability of cells infected with Dengue virus. In presence of BaltPLA2, the viability of infected cells increased significantly in virucidal, post-treatment, and adsorption assays. Although preliminary these results reveal the need for further studies to investigated whether BaltPLA2 has antiviral activity against Dengue virus.

Respiratory virus infections in hospitalized and non-hospitalized children: determinants of severe course of the disease.

INTRODUCTION: Viral respiratory disease constitutes a great burden worldwide mainly among children. OBJECTIVE: One pursued to compare disease characteristics of children who required hospitalization from those who did not require hospitalization due to a viral respiratory disease. METHODOLOGY: Medical and demographic data were collected through questionnaires and nasopharyngeal aspirates were tested for detection of respiratory disease viruses of in and outpatients up to five years old, presenting acute respiratory infection. RESULTS: Respiratory syncytial virus predominated among hospitalized children while other viruses (Human rhinovirus, Influenza virus, Parainfluenza virus, Adenovirus, and Human metapneumovirus) together predominated among non-hospitalized patients. Although children with underlying risk condition required longer hospitalization, previously healthy children presented severe disease and required hospitalization as well. Also, clinical characteristics were not found that may distinguish RSV infected children who had comorbidities from those previously healthy. CONCLUSIONS: Children who were hospitalized due to respiratory distress had well defined characteristics: early age, respiratory syncytial virus infection, bronchiolitis and presence of comorbidity. Nevertheless, rapid respiratory syncytial virus identification among early age children may be of great value in order to avoid medical misconduct, such as unnecessary antibiotic prescription and preventive health care before an eventual clinical worsening encompassing previous health status.

Molecular detection of a Borrelia sp. in nymphs of Amblyomma brasiliense ticks (Acari: Ixodidae) from Iguaçu National Park, Brazil, genetically related to Borrelia from Ethiopia and Côte d'Ivoire.

Gram-negative spirochetes of the genus Borrelia are transmitted to vertebrate hosts through the tick bite during blood intake. Pathogenic Borrelia species may cause relapsing fever or Lyme borreliosis in humans. Our study aimed to molecularly detect and characterize bacteria of this genus in ticks collected in the Iguaçu National Park, located in southern Brazil. Ticks were collected from the environment (free living) and from hosts from May 2015 to July 2017. In total, 829 ticks were tested, being 741 from the environment and 88 from hosts; 128 ticks were larvae, 523 were nymphs, and 178 were adults (80 males and 98 females). The species identified were: Amblyomma brasiliense (42.9 %), Amblyomma coelebs (16.8 %), Amblyomma sp. (15.0 %), Amblyomma incisum (10.3 %), Amblyomma ovale (8.7 %), Haemaphysalis juxtakochi (5.5 %), Rhipicephalus microplus (0.5 %), Amblyomma longirostre (0.2 %), and Rhipicephalus sanguineus sensu lato (0.1 %). DNA extraction was performed with pools of larvae or nymphs, or individually with adults, for a total of 394 samples. The PCR technique used to detect Borrelia DNA was performed with two rounds of amplification reactions targeting a segment of the flagellin B gene (flaB). Amplification occurred in only one DNA sample, which was obtained from nymphs of A. brasiliense collected from the vegetation of a trail. The nucleotide sequence analysis revealed 90.8 % identity to a sequence of Borrelia sp. from Côte d'Ivoire, and 89.1 % identity to a sequence from Ethiopia. Phylogenetic analysis revealed that the Borrelia sequence from A. brasiliense was distinct from the Borrelia species of the groups belonging to Lyme borreliosis, relapsing fever and the one associated with reptile and echidna as hosts. The sequence is likely from a putative new species of Borrelia and was detected for the first time in A. brasiliense ticks.

Ticks and Rickettsia on anteaters from Southeast and Central-West Brazil.

The giant anteater (Myrmecophaga tridactyla) and the collared anteater (Tamandua tetradactyla) are widespread in Brazil and found in all Brazilian biomes. These hosts frequently use domestic animal environments such as pastures, where tick and related microorganism interchange may occur between hosts. Reports of tick infestations of these animals are scattered and refer to small samples and/or are geographically restricted. We herein present data on a wide geographic distribution of ticks and their Rickettsia collected from 72 giant and 30 collared anteaters, mostly road killed, over a period of 18 years, from Southeast and Central-West Brazil encompassing four States and 46 Municipalities. Overall nine tick species (Amblyomma auricularium, A. calcaratum, A. nodosum, A. ovale, A. parvum, A. sculptum, A. triste, Rhipicephalus microplus and R. sanguineus sensu lato) were collected from anteaters. Amblyomma sculptum, A. nodosum, and A. calcaratum were the most prevalent corresponding to, respectively, 48.8%, 39.3% and 2.7% of all ticks (n = 1775). However, A. nodosum tick numbers on collared anteaters were significantly higher (P < 0.001) than those on giant anteaters. At the same time, an abundance of A. sculptum adults on giant anteaters was significantly higher (Z = 2.875; P = 0.004) than that of A. nodosum and only eight A. sculptum nymphs were found on collared anteaters. DNA samples from 20 ticks from nine different animals yielded a visible amplicon in PCR targeting gltA. The PCR products targeting spotted-fever Rickettsia gene (ompA) from five adults of A. nodosum were sequenced and were shown to be 100% identical to Rickettsia parkeri strain NOD (MF737635.1). The product of one nymph and one adult of A. sculptum yielded a sequence 99% identical to R. parkeri strain NOD. Further, Rickettsia bellii genes were found in three A. nodosum adults. Ecological, behavioral and anatomical traits of anteaters are discussed to explain reported tick infestations and Rickettsia DNA found.

Detection and molecular characterization of Mogiana tick virus (MGTV) in Rhipicephalus microplus collected from cattle in a savannah area, Uberlândia, Brazil.

In Brazil, recent studies have reported viruses detected in ticks with pathogenic potential in vertebrate hosts. Ticks of the species Rhipicephalus microplus collected from bovines in a savannah area were tested by RT-PCR for the presence of RNA targetting a segment of NS3-like protein gene of Mogiana tick virus, a member of the recently-described Jingmenvirus group. Amplification with size similar to the expected was observed with 25% (7/28) RNA samples of ticks that were collected from 39% (7/18) of the bovines. Nucleotide sequence analysis of three PCR products revealed divergence that varied from 3.3 to 5.0% in a single farm. Although Jingmen tick virus, another member belonging to the Jingmenvirus group, has been detected in human patients with Crimean-Congo hemorrhagic fever in Kosovo, whether or not MGTV causes disease in cattle and other animals remains to be investigated.

Ticks (Acari: Ixodidae) in the Serra da Canastra National Park in Minas Gerais, Brazil: species, abundance, ecological and seasonal aspects with notes on rickettsial infection.

The Cerrado Biome is the second largest in Brazil covering roughly 2 million km2, with varying features throughout its area. The Biome is endangered but it is also source of animal species for rural, green urban and degraded rainforest areas. Ticks are among Cerrado species that establish at anthropogenic sites and although information about them is steadily increasing, several features are unknown. We herein report tick species, abundance and some ecological relationships within natural areas of the Cerrado at higher altitudes (800-1500 m) within and around Serra da Canastra National Park, in Minas Gerais State Brazil. In total of 1196 ticks were collected in the environment along 10 campaigns held in 3 years (2007-2009). Amblyomma sculptum was the most numerous species followed by Amblyomma dubitatum and Amblyomma brasiliense. Distribution of these species was very uneven and an established population of A. brasiliense in the Cerrado is reported for the first time. Other tick species (Amblyomma ovale, Amblyomma nodosum, Amblyomma parvum, Ixodes schulzei and Haemaphysalis leporispalustris) were found in lesser numbers. Domestic animals displayed tick infestations of both rural and urban origin as well as from natural areas (Rhipicephalus sanguineus sensu lato, Rhipicephalus microplus, Dermacentor nitens, A. sculptum, A. ovale, Amblyomma tigrinum, Argas miniatus). Amblyomma sculptum had the widest domestic host spectrum among all tick species. DNA of only one Rickettsia species, R. bellii, was found in an A. dubitatum tick. Several biological and ecological features of ticks of the studied areas are discussed.

Influence of microhabitat use and behavior of Amblyomma sculptum and Amblyomma dubitatum nymphs (Acari: Ixodidae) on human risk for tick exposure, with notes on Rickettsia infection.

Brazilian spotted fever (BSF) is a potentially lethal human disease caused by Rickettsia rickettsii transmitted by ticks, including Amblyomma sculptum. However, in Southeast Brazil, where most BSF cases occur, capybaras are key hosts for both A. sculptum and Amblyomma dubitatum. We therefore compared the risk of human exposure to these ticks at a non-endemic anthropogenic site in Southeast Brazil where both tick species are maintained by capybaras and occur at high abundance. Cloth dragging, human baits and CO2 traps were used to assess tick abundance and risk for human exposure. The two tick species displayed profound differences in behavior and microhabitat use. Notably, A. sculptum but not A. dubitatum quested for hosts openly from vegetation (ambush behavior) and infested human baits. Furthermore, A. dubitatum was more aggregated at a specific site whereas A. sculptum was more widespread along differing and drier microhabitats. Adults and nymphs of both species were infected with Rickettsia bellii. Overall, the results indicate that even though both species co-existed in the same area, A. sculptum posed a greater risk for biting humans and thus also for transmitting tick-borne pathogens.

Humoral Immune Response in Mice Elicited by Combined Yeast-Derived Hepatitis B Virus Core, Surface, and Mosaic Surface Antigens.

BACKGROUND: Although successful, the second-generation hepatitis B vaccine programs around the world have a small group of immunized individuals that does not respond efficiently to the vaccination. Other issues of these vaccines are individuals that are low or nonresponders and/or have incomplete protection against heterologous hepatitis B virus (HBV) genotypes/subtypes and against HBV escape mutants. In addition, there are approximately 240 million people chronically infected with HBV worldwide and 620,000 deaths per year caused by the infection. METHODS: In this study we developed three Hansenula polymorpha plasmids containing the following sequences: (a) HBsAg subtype ayw, (b) HBcAg sequence subtype adw2, and (c) chimeric HBsAg (adw4/ ayw) - preS1 (adw2) - 3 repetitions of preS2 (genotypes A, B, and C). The sequences were successfully expressed and the antigens purified. Using Balb/c mice the antigens were tested in different dosage combinations. RESULTS: Three antigens were obtained at a high purity level and with high reproducibility. We also assessed their immunogenic properties, showing that the antigens, individually or in combination, generated anti-HBs, anti-preS1, anti-preS2, and anti-HBc antibodies efficiently in mice. CONCLUSIONS: The formulation tests showed that a combination of 0.02 µg of HBs, 0.2 µg of preS1-preS2-HBs, and 0.02 µg of HBc was effective in eliciting specific antibodies in mice.

Characterization of oligonucleotide aptamers targeting the 5'-UTR from dengue virus.

AIM: The dengue virus is responsible for a high worldwide incidence of infections, aggravated by late diagnosis, and often confused with other tropical diseases. Results/methodology: Oligonucleotide aptamers binding to the 5'-UTR from dengue virus selected after eight rounds by systematic evolution of ligands by exponential enrichment technology were analyzed by dot-blot assay and in silico prediction of secondary structures, demonstrating the presence of stem-loops that may have the potential for interaction with the viral genome, which can lead to loss of their original conformation. CONCLUSION: This is the first description of RNA aptamers against functional RNA elements of the dengue virus genome with implications for disease control, which may have potential as tools in the future of antiviral therapies and for diagnostics.

Clinical and Molecular Epidemiology of Multidrug-Resistant P. aeruginosa Carrying aac(6')-Ib-cr, qnrS1 and blaSPM Genes in Brazil.

We described a comprehensive analysis of the molecular epidemiology of multidrug-resistant (MDR) P. aeruginosa. Molecular analysis included typing by Pulsed Field Gel Electrophoresis, identification of genes of interest through PCR-based assays and sequencing of target genes. Case-control study was conducted to better understand the prognostic of patients and the impact of inappropriate therapy in patients with bacteremia, as well as the risk factors of MDR infections. We observed a high rate of MDR isolates (40.7%), and 51.0% of them was independently associated with inappropriate antibiotic therapy. Bacteremia was detected in 66.9% of patients, and prolonged hospital stay was expressive in those resistant to fluoroquinolone. Plasmid-mediated quinolone resistance genes (PMQR), qnrS1 and aac(6')Ib-cr, were detected in two different nosocomial isolates (5.3%), and the aac(6')-Ib7 variant was detected at a high frequency (87.5%) in those negative to PMQR. The presence of mutations in gyrA and parC genes was observed in 100% and 85% of selected isolates, respectively. Isolates harboring PMQR genes or mutations in gyrA and parC were not closely related, except in those containing SPM (São Paulo metallo-ß-lactamase) clone. In addition, there is no study published in Brazil to date reporting the presence of Pseudomonas aeruginosa isolates harboring both qnrS1 and aac(6')Ib-cr genes, with alarming frequency of patients with inappropriate therapy.

Amblyomma sculptum (Amblyomma cajennense complex) tick population maintained solely by domestic pigs.

In Brazil Amblyomma sculptum from Amblyomma cajennense complex is a tick species with a rather broad distribution encompassing the Cerrado Biome and devastated areas of the Atlantic rainforest. This species is also the major vector to humans of Rickettsia rickettsii, agent of the Brazilian spotted fever. Main primary hosts for the adult stage and frequently associated to high environmental infestation levels are the native capybaras and tapirs and a domestic host, the horse. Recent, however, solely circumstantial evidence, has shown that pigs (Sus scrofa) domestic, feral or exotic (wild boars) as well as wild suidae may maintain A. sculptum populations as well. We herein describe the maintenance of an A. sculptum tick population by domestic pigs for three years when raised in paddocks. Observations herein described occurred in the facilities of an intensive outdoor pig production system within a farm in Uberaba municipality southeast Brazil. Initial evaluation was done upon request due to human tick-bite complains in June 2013. Later tick infestations were evaluated at each of eight consecutive seasons from August 2014 to May 2016. In the first evaluation, 104 nymphs and one female A. sculptum were collected from four pigs and 269 nymphs of A. sculptum and 12 Amblyomma spp larva clusters from the paddocks. Only one A. sculptum nymph was found in each of the surrounding bovine pasture and a nearby riparian forest. Seven Rhipicephalus spp and three Amblyomma spp larva were found in the former as well. Infestations were greatly reduced with elimination of the two most infested paddocks and mowing of vegetation. Still along the inspection of eight consecutive seasons A sculptum ticks were collected overwhelmingly adults from pigs. Thus, observations herein described show that outdoor pig raising may provide both a permissive environment and adequate host allowing for a minimal threshold for the maintenance of A. sculptum populations. Since raising domestic pigs in paddocks to increase animal welfare is an increasing practice, it may indicate a new trend in this parasitism, with yet an unknown effect upon animal and human health.

Molecular characterization of influenza viruses collected from young children in Uberlandia, Brazil - from 2001 to 2010.

BACKGROUND: Influenza remains a major health problem due to the seasonal epidemics that occur every year caused by the emergence of new influenza virus strains. Hemagglutinin (HA) and neuraminidase (NA) glycoproteins are under selective pressure and subjected to frequent changes by antigenic drift. Therefore, our main objective was to investigate the influenza cases in Uberlândia city, Midwestern Brazil, in order to monitor the appearance of new viral strains, despite the availability of a prophylactic vaccine. METHODS: Nasopharyngeal samples were collected from 605 children less than five years of age presenting with acute respiratory disease and tested by immunofluorescence assay (IFA) for detection of adenovirus, respiratory syncytial virus, parainfluenza virus types 1, 2, and 3 and influenza virus types A and B. A reverse transcription-PCR (RT-PCR) for influenza viruses A and B was carried out to amplify partial segments of the HA and NA genes. The nucleotide sequences were analyzed and compared with sequences of the virus strains of the vaccine available in the same year of sample collection. RESULTS: Forty samples (6.6%) were tested positive for influenza virus by IFA and RT-PCR, with 39 samples containing virus of type A and one of type B. By RT-PCR, the type A viruses were further characterized in subtypes H3N2, H1N2 and H1N1 (41.0%, 17.9%, and 2.6%, respectively). Deduced amino acid sequence analysis of the partial hemagglutinin sequence compared to sequences from vaccine strains, revealed that all strains found in Uberlândia had variations in the antigenic sites. The sequences of the receptor binding sites were preserved, although substitutions with similar amino acids were observed in few cases. The neuraminidase sequences did not show significant changes. All the H3 isolates detected in the 2001-2003 period had drifted from vaccine strain, unlike the isolates of the 2004-2007 period. CONCLUSIONS: These results suggest that the seasonal influenza vaccine effectiveness could be reduced because of A H3N2 variants that circulated in 2001-2003 years. Thus, an early monitoring of variants circulating in the country or in a region may provide important information about the probable efficacy of the vaccine that will be administered in an influenza season.

Human rhinovirus and disease severity in children.

OBJECTIVE: To evaluate retrospectively human rhinovirus (HRV) infections in children up to 5 years old and factors involved in disease severity. METHODS: Nasopharyngeal aspirates from 434 children presenting a broad range of respiratory infection symptoms and severity degrees were tested for presence of HRV and 8 other respiratory viruses. Presence of host risk factors was also assessed. RESULTS: HRV was detected in 181 (41.7%) samples, in 107 of them as the only agent and in 74 as coinfections, mostly with respiratory syncytial virus (RSV; 43.2%). Moderate to severe symptoms were observed in 28.9% (31/107) single infections and in 51.3% (38/74) coinfections (P = .004). Multivariate analyses showed association of coinfections with lower respiratory tract symptoms and some parameters of disease severity, such as hospitalization. In coinfections, RSV was the most important virus associated with severe disease. Prematurity, cardiomyopathies, and noninfectious respiratory diseases were comorbidities that also were associated with disease severity (P = .007). CONCLUSIONS: Our study showed that HRV was a common pathogen of respiratory disease in children and was also involved in severe cases, causing symptoms of the lower respiratory tract. Severe disease in HRV infections were caused mainly by presence of RSV in coinfections, prematurity, congenital heart disease, and noninfectious respiratory disease.

Influence of the CCR-5/MIP-1 α axis in the pathogenesis of Rocio virus encephalitis in a mouse model.

Rocio virus (ROCV) caused an outbreak of human encephalitis during the 1970s in Brazil and its immunopathogenesis remains poorly understood. CC-chemokine receptor 5 (CCR5) is a chemokine receptor that binds to macrophage inflammatory protein (MIP-1 α). Both molecules are associated with inflammatory cells migration during infections. In this study, we demonstrated the importance of the CCR5 and MIP-1 α, in the outcome of viral encephalitis of ROCV-infected mice. CCR5 and MIP-1 α knockout mice survived longer than wild-type (WT) ROCV-infected animals. In addition, knockout mice had reduced inflammation in the brain. Assessment of brain viral load showed mice virus detection five days post-infection in wild-type and CCR5-/- mice, while MIP-1 α-/- mice had lower viral loads seven days post-infection. Knockout mice required a higher lethal dose than wild-type mice as well. The CCR5/MIP-1 α axis may contribute to migration of infected cells to the brain and consequently affect the pathogenesis during ROCV infection.

Efficient error correction for next-generation sequencing of viral amplicons.

BACKGROUND: Next-generation sequencing allows the analysis of an unprecedented number of viral sequence variants from infected patients, presenting a novel opportunity for understanding virus evolution, drug resistance and immune escape. However, sequencing in bulk is error prone. Thus, the generated data require error identification and correction. Most error-correction methods to date are not optimized for amplicon analysis and assume that the error rate is randomly distributed. Recent quality assessment of amplicon sequences obtained using 454-sequencing showed that the error rate is strongly linked to the presence and size of homopolymers, position in the sequence and length of the amplicon. All these parameters are strongly sequence specific and should be incorporated into the calibration of error-correction algorithms designed for amplicon sequencing. RESULTS: In this paper, we present two new efficient error correction algorithms optimized for viral amplicons: (i) k-mer-based error correction (KEC) and (ii) empirical frequency threshold (ET). Both were compared to a previously published clustering algorithm (SHORAH), in order to evaluate their relative performance on 24 experimental datasets obtained by 454-sequencing of amplicons with known sequences. All three algorithms show similar accuracy in finding true haplotypes. However, KEC and ET were significantly more efficient than SHORAH in removing false haplotypes and estimating the frequency of true ones. CONCLUSIONS: Both algorithms, KEC and ET, are highly suitable for rapid recovery of error-free haplotypes obtained by 454-sequencing of amplicons from heterogeneous viruses.The implementations of the algorithms and data sets used for their testing are available at: http://alan.cs.gsu.edu/NGS/?q=content/pyrosequencing-error-correction-algorithm.

Hepatitis C virus antigenic convergence.

Vaccine development against hepatitis C virus (HCV) is hindered by poor understanding of factors defining cross-immunoreactivity among heterogeneous epitopes. Using synthetic peptides and mouse immunization as a model, we conducted a quantitative analysis of cross-immunoreactivity among variants of the HCV hypervariable region 1 (HVR1). Analysis of 26,883 immunological reactions among pairs of peptides showed that the distribution of cross-immunoreactivity among HVR1 variants was skewed, with antibodies against a few variants reacting with all tested peptides. The HVR1 cross-immunoreactivity was accurately modeled based on amino acid sequence alone. The tested peptides were mapped in the HVR1 sequence space, which was visualized as a network of 11,319 sequences. The HVR1 variants with a greater network centrality showed a broader cross-immunoreactivity. The entire sequence space is explored by each HCV genotype and subtype. These findings indicate that HVR1 antigenic diversity is extensively convergent and effectively limited, suggesting significant implications for vaccine development.