Domain III peptides from flavivirus envelope protein are useful antigens for serologic diagnosis and targets for immunization

The Flavivirus genus of the Flaviviridae family includes 70 enveloped single-stranded-RNA positive-sense viruses transmitted by arthropods. Among these viruses, there are a relevant number of human pathogens including the mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV), Langat virus (LGTV) and Omsk hemorrhagic fever (OHFV). The flavivirus envelope (E) protein is a dominant antigen inducing immunologic responses in infected hosts and eliciting virus-neutralizing antibodies. The domain III (DIII) of E protein contains a panel of important epitopes that are recognized by virus-neutralizing monoclonal antibodies. Peptides of the DIII have been used with promising results as antigens for flavivirus serologic diagnosis and as targets for immunization against these viruses. We review here some important aspects of the molecular structure of the DIII as well as its use as antigens for serologic diagnosis and immunization in animal models.     

Clinical infection of Cantabrian chamois (Rupicapra pyrenaica parva) by louping ill virus: new concern for mountain ungulate conservation?

Louping ill virus (LIV) was recently involved in an outbreak of encephalitis in domestic goats from Asturias region, northwestern Spain. Since livestock and wildlife in Asturias are frequently in close contact, we designed a retrospective survey for LIV antibody prevalence in wild ungulates by testing sera from 51 red deer (Cervus elaphus), 19 Cantabrian chamois (Rupicapra pyrenaica parva) and 8 roe deer (Capreolus capreolus) by the haemagglutination inhibition (HI) test. Only two Cantabrian chamois out of the 78 tested (2.6?±?3.5 %) gave positive results. Seroprevalence in chamois was 10.5?±?13.8 %. One of these chamois was found dead after falling down a cliff and the other one was found alive but with neurological signs. Histological examination of brain samples revealed that both animals showed severe inflammatory lesions compatible with a viral encephalitis caused by LIV, but LIV antigen was not detectable by specific immunohistochemistry. Real time RT-PCR was performed on formalin-fixed paraffin embedded sections of brain but was unable to confirm the presence of LIV RNA due to poor sample quality. By testing one of two HI positive sera from chamois by virus neutralization test and plaque reduction neutralization test against West Nile virus, Bagaza virus, Usutu virus, LIV and tick-borne encephalitis virus, we confirmed the presence of high antibody titres (1:10240) against LIV in the absence of antibodies to another Flavivirus. This work describes the first association between LIV and clinical encephalitis in chamois, which suggests that special attention should be paid to the impact on chamois conservation and management in Asturias, and perhaps in other European regions.     

Newly recognized mosquito-associated viruses in mainland China, in the last two decades

There are four principal arboviruses in mainland China. Two kinds of them are mosquito-borne viruses, namely Japanese encephalitis virus and dengue virus, which lead to Japanese encephalitis, and dengue fever/dengue hemorrhagic fever respectively; the other two are tick-borne viruses, namely tick-borne encephalitis virus and Crimean-Congo hemorrhagic fever virus (also known as Xinjiang hemorrhagic fever virus), which contribute to tick-borne encephalitis and Xinjiang hemorrhagic fever respectively. With exception of these four main arboviruses, many other mosquito-associated viruses have been isolated and identified in recent years. These newly isolated and identified mosquito-associated viruses are probably responsible for human and animal infections and diseases. The purpose of this review is to describe the newly isolated mosquito-associated viruses in mainland China which belong to five viral families, including their virological properties, phylogenetic relationships, serological evidence, as well as to appeal the public health concentration worldwide.     

A Sero-epidemiological Study of Arboviral Fevers in Djibouti,Horn of Africa

Arboviral infections have repeatedly been reported in the republic of Djibouti, consistent with the fact that essential vectors for arboviral diseases are endemic in the region. However, there is a limited recent information regarding arbovirus circulation, and the associated risk predictors to human exposure are largely unknown. We performed, from November 2010 to February 2011 in the Djibouti city general population, a cross-sectional ELISA and sero-neutralisation-based sero-epidemiological analysis nested in a household cohort, which investigated the arboviral infection prevalence and risk factors, stratified by their vectors of transmission. Antibodies to dengue virus (21.8%) were the most frequent. Determinants of infection identified by multivariate analysis pointed to sociological and environmental exposure to the bite of Aedes mosquitoes. The population was broadly naïve against Chikungunya (2.6%) with risk factors mostly shared with dengue. The detection of limited virus circulation was followed by a significant Chikungunya outbreak a few months after our study. Antibodies to West Nile virus were infrequent (0.6%), but the distribution of cases faithfully followed previous mapping of infected Culex mosquitoes. The seroprevalence of Rift valley fever virus was 2.2%, and non-arboviral transmission was suggested. Finally, the study indicated the circulation of Toscana-related viruses (3.7%), and a limited number of cases suggested infection by tick-borne encephalitis or Alkhumra related viruses, which deserve further investigations to identify the viruses and vectors implicated. Overall, most of the arboviral cases'' predictors were statistically best described by the individuals'' housing space and neighborhood environmental characteristics, which correlated with the ecological actors of their respective transmission vectors'' survival in the local niche. This study has demonstrated autochthonous arboviral circulations in the republic of Djibouti, and provides an epidemiological inventory, with useful findings for risk mapping and future prevention and control programs.     

Multi-Gene Detection and Identification of Mosquito-Borne RNA Viruses Using an Oligonucleotide Microarray

Background

Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae), Alphavirus (Togaviridae), Orthobunyavirus (Bunyaviridae), and Phlebovirus (Bunyaviridae).

Methodology/Principal Findings

The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012.

Conclusions/Significance

We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish public health priorities, detect disease outbreaks, and evaluate control programs.     

Laboratory testing of clinically approved drugs against Balamuthia mandrillaris

Balamuthia mandrillaris is a free-living protist pathogen that can cause life-threatening granulomatous amoebic encephalitis. Given the lack of effective available drugs against B. mandrillaris encephalitis with a mortality rate of more than 90 %, here we screened drugs, targeting vital cellular receptors and biochemical pathways, that are already in approved clinical use for their potential clinical usefulness. Amoebicidal assays were performed by incubating B. mandrillaris with drugs (3 × 10⁵ cells/0.5 mL/well) in phosphate buffered saline for 24 h and viability was determined using Trypan blue exclusion staining. For controls, amoebae were incubated with the solvent alone. To determine whether effects are reversible, B. mandrillaris were pre-exposed to drugs for 24 h, washed twice, and incubated with human brain microvascular endothelial cells, which constitute the blood–brain barrier as food source, for up to 48 h. Of the ten drugs tested, amlodipine, apomorphine, demethoxycurcumin, haloperidol, loperamide, prochlorperazine, procyclidine, and resveratrol showed potent amoebicidal effects, while amiodarone and digoxin exhibited minimal effectiveness. When pre-treated with these drugs, no viable trophozoites re-emerged, suggesting that drugs destroyed parasite irreversibly. Based on the in vitro assay, amlodipine, apomorphine, demethoxycurcumin, haloperidol, loperamide, prochlorperazine, procyclidine, and resveratrol are potential antimicrobials for further testing against B. mandrillaris encephalitis. These findings may provide novel strategies for therapy but further research is needed to determine clinical usefulness of aforementioned drugs against granulomatous amoebic encephalitis caused by B. mandrillaris, and other free-living amoebae, such as Acanthamoeba spp., and Naegleria fowleri.     

Microbiological Identification and Analysis of Swine Lungs Collected from Carcasses in Swine Farms, China

The primary objective of this 3 years study was to determine the prevalence of porcine pathogens of the lungs of swine in swine farms in southern China. A total of 5,420 samples were collected from 200 swine farms. The bacterium that was most commonly isolated was Streptococcus suis, with 10.24 % of the samples being positive, 114 lungs (2.1 %) were positive for pseudorabies virus and 263 (4.85 %) were positive for classical swine fever virus; much lower than positive for PRRSV (15.1 %, p = 0.023) and PCV2 (13.8 %, p = 0.038). lungs that were positive for PRRSV and/or PCV-2 have significantly increased odds of being positive for any of the S. suis (9.79 vs. 0.44 %, p = 0.003).     

Chromosome numbers and karyotype of five species from Seriphidium (Asteraceae)

Chromosome data are important for elucidating intergeneric relationships and delimiting infrafamilial tribes in plants. This paper reports karyological data for 12 Seriphidium (Bess.) Poljak. species from China, of these, five count as new reports and the others have been reported elsewhere. We also report the tetraploid level in S. schrenkianum for the first time. The karyotype formulae and types for the five new reports are: S. schrenkianum (Ledeb.) Poljak. 2n = 4x = 36 = 22m + 12sm + 2st, S. sublessingianum (Kell.) Poljak. 2n = 2x = 18 = 14m + 4sm and S. transiliense (Poljak.) Poljak. 2n = 2x = 18 = 4M + 6m + 2m(SAT) + 4sm + 2sm(SAT), belong to 2A type; S. cinum (Berg. ex Poljak.) Poljak. 2n = 2x = 18 = 8m + 10sm and S. sawanense Y. R. Ling et C. J. Humphries 2n = 2x = 18 = 10m + 2m(SAT) + 6sm, belong to 2B type. Furthermore, we estimated karyotype asymmetry in the 12 species by using an intrachromosomal asymmetry index and an interchromosomal asymmetry index. The data increase information about the karyosystematics of Seriphidium.     

Seroprevalence of Japanese encephalitis virus infection in captive Japanese macaques (Macaca fuscata)

Japanese encephalitis virus (JEV), which is transmitted by mosquitoes, infects many animal species and causes serious acute encephalitis in humans and horses. In this study, a serosurvey of JEV in Japanese macaques (Macaca fuscata) reared in Aichi Prefecture was conducted using purified JEV as an antigen for ELISA. The results revealed that 146 of 332 monkeys (44 %) were seropositive for JEV. In addition, 35 of 131 monkeys (27 %) born in the facility were seropositive, and the annual infection rate in the facility was estimated as 13 %. Our results provide evidence of the frequent exposure of many Japanese macaques to JEV, suggesting that there is a risk of JEV transmission to humans by mosquitoes.     

Development and validation of a 5-day-ahead hay fever forecast for patients with grass-pollen-induced allergic rhinitis

One-third of the Dutch population suffers from allergic rhinitis, including hay fever. In this study, a 5-day-ahead hay fever forecast was developed and validated for grass pollen allergic patients in the Netherlands. Using multiple regression analysis, a two-step pollen and hay fever symptom prediction model was developed using actual and forecasted weather parameters, grass pollen data and patient symptom diaries. Therefore, 80 patients with a grass pollen allergy rated the severity of their hay fever symptoms during the grass pollen season in 2007 and 2008. First, a grass pollen forecast model was developed using the following predictors: (1) daily means of grass pollen counts of the previous 10 years; (2) grass pollen counts of the previous 2-week period of the current year; and (3) maximum, minimum and mean temperature (R ²?=?0.76). The second modeling step concerned the forecasting of hay fever symptom severity and included the following predictors: (1) forecasted grass pollen counts; (2) day number of the year; (3) moving average of the grass pollen counts of the previous 2 week-periods; and (4) maximum and mean temperatures (R ²?=?0.81). Since the daily hay fever forecast is reported in three categories (low-, medium- and high symptom risk), we assessed the agreement between the observed and the 1- to 5-day-ahead predicted risk categories by kappa, which ranged from 65 % to 77 %. These results indicate that a model based on forecasted temperature and grass pollen counts performs well in predicting symptoms of hay fever up to 5 days ahead.     

Flow cytometric and karyological analyses of Calendula species from Iberian Peninsula

Calendula L. (Asteraceae) is a taxonomically and cytologically complex genus due to its high morphological and karyological variation. To gather consistent cytological information aiming to consolidate the existing knowledge, sustain the taxonomic revision of the genus and explore the evolutionary relationships among species, the genome size and chromosome number of the Iberian Peninsula representatives of this genus were assessed. The study included 11 taxa that occur in the Iberian Peninsula, one in Madeira and two from Morocco. Chromosome counts were made using the squash technique in root tips and flower buds, while nuclear DNA contents were assessed using propidium iodide flow cytometry. The following chromosome numbers are reported: 2n = 44 for C. arvensis, 2n = 30 for C. tripterocarpa, and 2n = 32 for the remaining Iberian taxa. The genome size of Calendula species was assessed for the first time and ranged from 1.75 pg/2C in C. maroccana to 5.41 pg/2C in C. arvensis. Within the complex formed by C. incana and C. suffruticosa, a gradient of genome size values was obtained. Intraspecific variation in genome size was detected in some taxa. The obtained genome size values and their variation are discussed in the light of the theories proposed for the speciation of the genus, with events of hybridization, genome duplication and dysploidy being hypothesized to play a major role in the evolution of this genus.     

The epidemiology and disease burden of children hospitalized for viral infections within the family Flaviviridae in China: A national cross-sectional study

BackgroundViruses of the family Flaviviridae, including Japanese encephalitis virus (JEV), dengue virus (DENV), yellow fever virus (YFV) and hepatitis C virus (HCV), are widely distributed worldwide. JEV, DENV and YFV belong to the genus Flavivirus, whereas HCV belongs to the genus Hepacivirus. Children’s symptoms are usually severe. As a result, rates of hospitalization due to infection with these viruses are high. The epidemiology and disease burden of hospitalized children have rarely been described in detail to date. The objective of this study was to report the general epidemiological characteristics, clinical phenotype, length of stay (LOS), burden of disease, and potential risk factors for hospitalized children infected with JEV, DENV, YFV, or HCV in Chinese pediatric hospitals.MethodologyA cross-sectional study of epidemiology and disease burden of children hospitalized for Flaviviridae virus infections between December 2015 and December 2020 in China was performed. Face sheets of discharge medical records (FSMRs) were collected from 27 tertiary children’s hospitals in the Futang Research Center of Pediatric Development and aggregated into FUTang Update medical REcords (FUTURE). Information on sociodemographic variables, clinical phenotype, and LOS as well as economic burden was included in FSMRs and compared using appropriate statistical tests.FindingsThe study described 490 children aged 0–15 years hospitalized for infections with Flaviviridae viruses. Japanese encephalitis (JE) cases are the highest, accounting for 92.65% of the total hospitalization cases caused by Flaviviridae virus infection. The incidence of JE peaked from July to October with a profile of a high proportion of severe cases (68.06%) and low mortality (0.44%). Rural children had a significantly higher incidence than urban children (91.63%). Most hospitalized dengue cases were reported in 2019 when dengue outbreaks occurred in many provinces of China, although only 14 dengue cases were collected during the study period. Yellow fever (YF) is still an imported disease in China. The hospitalizations for children with hepatitis C (HC) were not high, and mild chronic HC was the main clinical phenotype of patients. Among the four viral infections, JE had the highest disease burden (LOS and expenditure) for hospitalized children.ConclusionFirst, the present study reveals that JE remains the most serious disease due to Flaviviridae virus infection and threatens children’s health in China. Many pediatric patients have severe illnesses, but their mortality rate is lower, suggesting that existing treatment is effective. Both JEV vaccination and infection control of rural children should represent a focus of study. Second, although the dual risks of indigenous epidemics and imports of DENV still exist, the prevalence of DENV in children is generally manageable. Third, YFV currently shows no evidence of an epidemic in China. Finally, the proportion of children with chronic hepatitis C (CHC) is relatively large among hospitalized children diagnosed with HCV. Thus, early and effective intervention should be offered to children infected with HCV to ease the burden of CHC on public health.     

Exploring of Primate Models of Tick-Borne Flaviviruses Infection for Evaluation of Vaccines and Drugs Efficacy

Tick-borne encephalitis virus (TBEV) is one of the most prevalent and medically important tick-borne arboviruses in Eurasia. There are overlapping foci of two flaviviruses: TBEV and Omsk hemorrhagic fever virus (OHFV) in Russia. Inactivated vaccines exist only against TBE. There are no antiviral drugs for treatment of both diseases. Optimal animal models are necessary to study efficacy of novel vaccines and treatment preparations against TBE and relative flaviviruses. The models for TBE and OHF using subcutaneous inoculation were tested in Cercopithecus aethiops and Macaca fascicularis monkeys with or without prior immunization with inactivated TBE vaccine. No visible clinical signs or severe pathomorphological lesions were observed in any monkey infected with TBEV or OHFV. C. aethiops challenged with OHFV showed massive hemolytic syndrome and thrombocytopenia. Infectious virus or viral RNA was revealed in visceral organs and CNS of C. aethiops infected with both viruses; however, viremia was low. Inactivated TBE vaccines induced high antibody titers against both viruses and expressed booster after challenge. The protective efficacy against TBE was shown by the absence of virus in spleen, lymph nodes and CNS of immunized animals after challenge. Despite the absence of expressed hemolytic syndrome in immunized C. aethiops TBE vaccine did not prevent the reproduction of OHFV in CNS and visceral organs. Subcutaneous inoculation of M. fascicularis with two TBEV strains led to a febrile disease with well expressed viremia, fever, and virus reproduction in spleen, lymph nodes and CNS. The optimal terms for estimation of the viral titers in CNS were defined as 8–16 days post infection. We characterized two animal models similar to humans in their susceptibility to tick-borne flaviviruses and found the most optimal scheme for evaluation of efficacy of preventive and therapeutic preparations. We also identified M. fascicularis to be more susceptible to TBEV than C. aethiops.     

Tick survey and detection of Crimean-Congo hemorrhagic fever virus in tick species from a non-endemic area, South Marmara region, Turkey

Crimean-Congo hemorrhagic fever (CCHF) is an increasing health concern in Turkey since 2002. There were also some recent human cases from the South Marmara region of Turkey; thus, a tick survey was performed, and possible vector tick species for the CCHF virus were determined in the region. A total of 740 adult ticks were collected from infested livestock from five locations: Çanakkale-Biga, Bursa-Orhaneli, Bursa-Keles, Bal?kesir and Bilecik. Total of 11 tick species from the genera Hyalomma, Rhipicephalus, Dermacentor, Ixodes and Haemaphysalis were identified. Rhipicephalus ticks were dominant in the region; the most frequently observed tick species was R. turanicus, (53.1 %), and only 15.4 % of the identified ticks were H. marginatum. The occurrence of H. rufipes infestation in the region fort he first time. A total of 73 pools of adult ticks were tested with both an antigen-detecting ELISA and RT real-time PCR (RT rt PCR). The presence of the CCHF virus was demonstrated in 9 (12.3 %) of the tested tick pools. Although seven of the tick pools were positive for the CCHF virus with both of the methods, one pool was positive only with RT rt PCR and the other pool was only positive with the ELISA. Positive results were obtained from ticks collected from cattle, sheep and goats from two locations, Bursa-Orhaneli and Bilecik. The CCHF virus was detected in R. turanicus (n = 3), R. bursa (n = 2), H. marginatum (n = 2) and D. marginatus (n = 2) ticks. The results of this study confirm the presence of the CCHF virus and present preliminary data on the vector tick species in the southern Marmara region of Turkey.     

Phylogeny of the Genus Flavivirus

We undertook a comprehensive phylogenetic study to establish the genetic relationship among the viruses of the genus Flavivirus and to compare the classification based on molecular phylogeny with the existing serologic method. By using a combination of quantitative definitions (bootstrap support level and the pairwise nucleotide sequence identity), the viruses could be classified into clusters, clades, and species. Our phylogenetic study revealed for the first time that from the putative ancestor two branches, non-vector and vector-borne virus clusters, evolved and from the latter cluster emerged tick-borne and mosquito-borne virus clusters. Provided that the theory of arthropod association being an acquired trait was correct, pairwise nucleotide sequence identity among these three clusters provided supporting data for a possibility that the non-vector cluster evolved first, followed by the separation of tick-borne and mosquito-borne virus clusters in that order. Clades established in our study correlated significantly with existing antigenic complexes. We also resolved many of the past taxonomic problems by establishing phylogenetic relationships of the antigenically unclassified viruses with the well-established viruses and by identifying synonymous viruses.The genus Flavivirus of the family Flaviviridae comprises over 70 viruses, many of which, such as the dengue (DEN) viruses, Japanese encephalitis (JE) virus, St. Louis encephalitis (SLE) virus, and yellow fever (YF) virus are important human pathogens (22, 31). Dengue and its severe and sometimes fatal forms, dengue hemorrhagic fever and dengue shock syndrome, alone affect nearly 80 million people a year (30). As demonstrated in recent outbreaks of meningitis by West Nile (WN) virus in Algeria and Romania, viruses of this group sometimes cause serious public health concern in unexpected locations (27).Most of these viruses were serologically classified into eight antigenic complexes, but many viruses, including the prototype of this group, YF virus, could not be affiliated with any complexes (6). Furthermore, many new viruses have been documented since the establishment of the serological classification, but their overall relationship with the other viruses has not been determined. The difficulty encountered with flavivirus classification partly derives from the extensive geographic distribution and the diversity of the arthropod vectors or vertebrates hosts associated with biological transmission of these viruses. Also, it derives from a confusion in virus nomenclature. For example, tick-borne encephalitis virus strains isolated primarily in western parts of Eurasia have been called TBE viruses, but, as clearly pointed out by Calisher (5), no such virus as tick-borne encephalitis virus (or TBE) has ever been registered to an international body dedicated to virus taxonomy. To compound the problem further, an increasing number of viruses have been added as new members of so-called TBE complex without a virus definition provided (16, 18, 29, 42, 52). This practice clearly demonstrates a need for establishing objective criteria for a better classification of those viruses.Molecular genetic classification of these viruses has been attempted before. In all previous studies, fewer than one-third of the members, primarily mosquito-borne and tick-borne viruses, were used to create phylogenetic trees, which showed evolution of mosquito-borne and tick-borne viruses from the presumed ancestor (3, 11). Since few sequence data were available from other viruses, in particular the viruses without known vectors (hereafter called the non-vector group), those phylogenetic trees provided only partial information.To establish a comprehensive phylogeny of the genus Flavivirus, we attempted to obtain the genomic sequence of a 1.0-kb segment at the 3′ terminus of the NS5 gene from all viruses whose sequences were not available. We analyzed, together with the other sequence data already published, the genetic relationships among the members of this group. Quantitative criteria based on a combination of the bootstrap support level and the pairwise nucleotide sequence identity were established to define subgeneric taxa. These included cluster, clade, and species. With our new taxonomic definitions, we then compared our genetic classification with the traditional system based on serological data.     

Discovery of novel multi-target indole-based derivatives as potent and selective inhibitors of chikungunya virus replication

We recently identified indole derivatives (IIIe and IIIf) with anti-chikungunya virus (CHIKV) activities at lower micro molar concentrations and a selective index of inhibition higher than the lead compound Arbidol. Here we highlight new structural information for the optimization of the previously identified lead compounds that contain the indole chemical core. Based on the structural data, a series of indole derivatives was synthesized and tested for their antiviral activity against chikungunya virus in Vero cell culture by a CPE reduction assay.Systematic optimization of the lead compounds resulted in tert-butyl-5-hydroxy-1-methyl-2-(2-trifluoromethysulfynyl)methyl)-indole-3-carboxylate derivative IIc with a 10-fold improved anti-CHIKV inhibitory activity (EC₅₀ = 6.5 ± 1 μM) as compared to Arbidol demonstrating a potent, selective and specific inhibition of CHIKV replication with only a moderate cell protective effect against other related alphaviruses. The reported computational insights, together with the accessible synthetic procedure, pave the road towards the design of novel synthetic derivatives with enhanced anti-viral activities.     

Seroprevalence for selected pathogens of zoonotic importance in wild nutria (Myocastor coypus)

Little information is present in the literature on diseases of the nutria (Myocastor coypus) in the wild. Serum samples obtained from 176 trapped animals were tested for antibody reactivity against 11 pathogens of significant zoonoses. Sera were positive against Leptospira (38.0 %, odds relative risk?=?0.03, Taylor series 95 % confidence limits 0.01–0.06), Toxoplasma gondii (27.8 %, odds relative risk?=?2.30, Taylor series 95 % confidence limits 1.23–4.31), Chlamydophila psittaci (21.0 % odds relative risk?=?4.94, Taylor series 95 % confidence limits 2.75–8.89), Streptococcus equi subspecies zooepidemicus (15.9 % odds relative risk?=?1.87, Taylor series 95 % confidence limits 1.04–3.37), and encephalomyocarditis virus (3.4 %, odds relative risk?=?0.05, Taylor series 95 % confidence limits 0.02–0.12). Some of the rodents showed antibodies at high titers, mostly indicating recent exposure. Seroprevalence rates varied among the location and age groups, although not by season or gender. All samples were seronegative for Brucella spp., Francisella tularensis, vesicular stomatitis virus, rabies virus, foot-and-mouth disease virus, and Encephalitozoon cuniculi.     

Analysis of microRNAs induced by Venezuelan equine encephalitis virus infection in mouse brain

MicroRNAs (miRNA) are small RNA (∼22nts) molecules that are expressed endogenously in cells and play an important role in regulating gene expression. Recent studies have shown that cellular miRNA plays a very important role in the pathogenesis of viral infection. Venezuelan equine encephalitis virus (VEEV) is an RNA virus and is a member of the genus Alphavirus in the family Togaviridae. VEEV is infectious in aerosol form and is a potential biothreat agent. In this study, we report for the first time that VEEV infection in mice brain causes modulation of miRNA expression. Pathway analyses showed that majority of these miRNAs are involved in the neuronal development and function. Target gene prediction of the modulated miRNAs correlates with our recently reported mRNA expression in VEEV infected mice brain.     

Relationships between soybean shoot nitrogen components and soybean aphid populations

Defining the relationships between soybean (Glycine max [L.] merr.) shoot nitrogen (N) components and soybean aphid (Aphis glycines Matsumura) populations will increase understanding of the biology of this important insect pest. In this 2-year field study, caged soybean plants were infested with soybean aphids (initial infestation of 0, 10, 50, or 100 aphids plant^?1) at the fifth node developmental stage. Soybean aphid populations, soybean shoot dry weight, and shoot concentrations of nitrate-N, ureide-N, and total N were measured starting at full bloom through full seed soybean development stages. Soybean aphid population as well as shoot concentration of ureide-N increased rapidly starting at full bloom, peaked at beginning seed, and dramatically decreased by full seed soybean reproductive stages. Regression analysis indicated significant relationships (P = 0.01; r = 0.71) between soybean aphid populations and shoot ureide-N concentration. Thus, soybean aphid population levels appear to coincide with shoot ureide-N concentrations in the soybean plant.     

Comparison of Acyclovir and Multistrain <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> in Women with Recurrent Genital Herpes Infections: a Double-Blind,Randomized, Controlled Study

We performed a randomized double-blind controlled trial to compare the efficacy and safety of multistrain probiotic and acyclovir in women patients with recurrent genital herpes simplex virus type 2 (HSV-2) infections. Eighty-one patients enrolled in the study were being treated with multistrain Lactobacillus brevis one vaginal capsule every 12 h and oral acyclovir 400 mg twice daily for 6 months. Of 53 patients who completed both treatment courses, no important differences were identified between acyclovir and probiotic for the primary and secondary efficacy endpoint, resolution of episode (hazard ratio, 0.60; 95% CI, 0.3429 to 1.0663; P = 0.08), lesion healing time (hazard ratio, 0.57; 95% CI, 0.3034 to 1.0717, P = 0.08), viral shedding (hazard ratio, 0.54; 95% CI, 0.3027 to 0.9750, P = 0.04), and percentage of pain (hazard ratio, 0.48; 95% CI, 0.2708 to 0.8545, P = 0.01). The median time to first and second recurrence after treatment were 43 and 121 days in patients receiving acyclovir and 33 and 118 days in patients receiving probiotic (HR 2.61; 95% CI, 1.4427 to 4.7546, P = 0.001, and HR 0.62; 95% CI, 0.3500 to 1.1133, P = 0.1, respectively). No clinically important effects happened during the probiotic treatment but some of adverse events reported in patients taking acyclovir. Easy availability, low cost, and no side effect of L. brevis are valuable properties of probiotic therapy compared with acyclovir. Therefore, we concluded that multistrain L. brevis could play an important role in suppression of recurrent genital herpes simplex virus infection.