In the present study, four suspected virus-infected tomato samples collected in the U.S. and Mexico were used for sRNA library construction and deep sequencing. Each library generated between 5–7 million sRNA reads, of which more than 90% were from the tomato genome. Upon in-silico subtraction of the tomato sRNAs, the remaining highly enriched, virus-like siRNA pools were assembled with or without reference virus or viroid genomes. A complete genome was assembled for Potato spindle tuber viroid (PSTVd) using siRNA alone. In addition, a near complete virus genome (98%) also was assembled for Pepino mosaic virus (PepMV). A common mixed infection of two strains of PepMV (EU and US1), which shared 82% of genome nucleotide sequence identity, also could be differentially assembled into their respective genomes. Using de novo assembly, a novel potyvirus with less than 60% overall genome nucleotide sequence identity to other known viruses was discovered and its full genome sequence obtained. Taken together, these data suggest that the sRNA deep sequencing technology will likely become an efficient and powerful generic tool for virus identification in plants and animals.Small RNAs (sRNA), including microRNAs (miRNA) and small interfering RNAs (siRNA), are produced abundantly in plants and animals and function in regulating gene expression or in defense against virus or viroid infection. Analysis of siRNA profiles upon virus infection in plant may allow for virus identification, strain differentiation, and  Solanum lycopersicum) is the world’s second most economically important vegetable, after potato, with a total world production in 2009 approaching 153 million tons in 173 countries Tomato (Pepino mosaic virus (PepMV), genus Potexvirus and family Alphaflexiviridae, has emerged as one of the most economically important viruses infecting greenhouse tomatoes worldwide In the last decade, a priori knowledge.Timely and accurate detection of phytopathogens is a prerequisite in making effective disease management decisions. Most virus detection methods  depend on prior knowledge of antibody or sequence of the potential virus. Advancement in next generation sequencing (NGS) technologies has provided a powerful alternative for total pathogen characterization without A metagenomic survey of microbes in honey bee colony collapse syndrome demonstrated the successful use of this approach in identifying a virus as the possible causal agent for this disorder de novo assembly of siRNAs to achieve a complete genome for a novel virus or a new genotype where a reference genome sequence is not available or not reliable. To address these challenges, Isakov et al. Human immunodeficiency virus (HIV) and a mycoplasma in human cells. It is believed that such enrichment of pathogen-related sRNAs will enhance the chance to identify these viruses with very low titer.sRNAs are produced abundantly in plants and animals and are involved in regulating gene expression. They also have been shown to function in defense against virus infection in silico subtraction of host plant-related sRNAs, followed by Sanger sequencing of RT-PCR products to allow for an efficient generation and validation of complete virus and viroid genome sequences. We were able to effectively identify both PepMV and Potato spindle tuber viroid (PSTVd) in a mixed infection and to achieve a strain-specific differentiation of PepMV isolates. In addition, by applying this strategy we were able to identify a novel potyvirus from infected tomatoes, obtaining its complete genome sequence without a priori knowledge of its existence.We describe here a general bioinformatics pipeline that incorporates an enrichment procedure through Four virus-infected tomato samples with various disease symptoms, including chlorosis, mosaic stunting, leaf deformation or fruit marbling , were orFour individual sRNA libraries were prepared from the purified sRNAs of these tomato samples using unique barcoded-adapters, which were pooled and sequenced on an Illumina Genome Analyzer IIx. Each library generated between 5–7 million reads , with anTo enrich each pool of siRNAs originating from viruses, we first aligned the sRNA reads to the tomato genome sequence Initial sRNA assembly was conducted against known virus and viroid genome databases based on their alignments to these reference genomes. Both (+) and (−) polarity were considered in siRNA assembly to the reference genomes. A gap was considered when a reference genome region was not covered by either polarity of siRNAs.The siRNAs generated in sample CAHN8 were matched to two PepMV strains  and also to PSTVd . A complValidation of the synthetic virus genome upon assembly of the virus-specific siRNA was performed using RT-PCR with overlapping sequence fragments along the identified virus genome. The 5′ terminal sequences were determined using the Rapid Amplification of cDNA Ends (RACE) system . In this experiment, we confirmed the authenticity of two distinct genotypes of PepMV in each of three U.S. samples , and their complete genome sequences were determined . Furthermore, PSTVd genome sequence in CAHN8 (GenBank Accession No. JQ806338) was also validated through Sanger sequencing to be nearly identical to PSTVd-CA1 (GenBank Accession No. HM753555).To validate a siRNA-assembled virus genome, we designed a step-wise primer walking system  to obtaide novo assembled contigs were used to BLAST against the nt and nr databases.A mixed infection of two genotypes of PepMV was found in all three U.S. greenhouse tomato samples. Upon gap filling and 5′ RACE, six complete PepMV genomes, including both EU and US1, were obtained. These PepMV-EU and PepMV-US1 isolates, originating from the same greenhouse in Arizona (EF09_58 and EF09_60), were nearly identical, sharing 99.6% nucleotide sequence identity. On the other hand, PepMV isolates from the California sample (CAHN8) could be differentiated from the Arizona isolates, with only 98.6% nt identity to their respective genotypes (EU and US1). Phylogenetic relationships of these six new PepMV isolates along with 22 previously published PepMV genome sequences were analyzed, and their genotype assignments were found to be in agreement with convention . In addi−) polarity than those in (+) polarity for PSTVd in tomato sample CAHN8. In fact, nearly two thirds (63.6%) of PSTVd siRNAs were in (−) polarity, with only one third (36.4%) in (+) polarity (−) polarity siRNAs were also prevalent over the (+) polarity (In agreement with a previous observation on siRNA profiles characterized on two pospiviroids in grapevine polarity  A. Interpolarity  A.−) polarities. The third hotspot (HS3) mapped to the Pathogenicity domain of (−) polarity siRNAs  was located in the Central to the Variable domains of a typical pospiviroid. The second hotspot (HS2) covered the Terminal Left region in both (+) and (y siRNAs . The mos−) polarity of the respective genome and were similar in terms of sequence and genome location among various isolates in the same genotype. The most conserved siRNA for the EU genotype (TGACTGTAGAATCAAGATGGT) was located on nt 3990-3970 (CAGGAGATTGTCGACTAGCGGC) was mapped to nt 3473-3452  generated 54 sequence contigs with lengths ranging from 37 to 8,432 nt. A BLASTX search revealed the two longest contigs  had significant alignments (amino acid sequence identity of ∼60%) to polyproteins of several known potyviruses. Closer examination indicated that these two contigs had a 13-nt overlap at ends thus could potentially be joined into one contig (de novo assembled contigs with length of 307 and 79 nt [69 nt after removing the poly(A) tail] can be perfectly aligned to the 3′ end of the ToNSV genome  of the ToNSV genome.Through mechanical inoculation, the Mexican sample (MX9354) could induce plant stunting, necrosis, and leaf and fruit deformation symptoms on the inoculated tomato plants . Howevere contig . To obtae contig . After ce contig . Based oe contig . Phylogee contig . It is wV genome . These tde novo assemblies with and without host sRNA subtraction using datasets from the other three samples provided the similar results (data not shown).It has been suggested that subtraction of host-derived sRNAs can improve the efficiency of virus sequence identification from short sRNA sequences de novo. Complete genomes of six PepMV isolates and a PSTVd isolate were obtained and validated through RT-PCR and Sanger sequencing. Although a similar approach has been used to identify HIV and a mycoplasma in a human cell culture de novo assembly of virus sequences. The strategy of subtraction of host sRNAs for enrichment of virus-derived siRNAs has not been used in previous studies in plant virus identification de novo assembly of virus sequences by subtraction of host-derived sRNAs. In addition, Hagen et al. Here, we describe a bioinformatics pipeline to efficiently identify tomato viruses and viroids in a mixed infection and to achieve a strain-specific differentiation. This bioinformatics pipeline relied on the use of computer-assisted subtraction of host plant-associated sRNAs. In our experiments, nearly 90% of the total sRNAs from the tomato genome were removed. Highly enriched exogenous siRNAs were used for the assembly of the virus genome, either reference-guided or −) polarity for both PSTVd and PepMV in tomato. Such preferential accumulation of the (−) polarity siRNAs for PSTVd is in contrast with two previous studies, using Sanger sequencing of sRNAs in tomato, in which it was reported that (+) polarity siRNAs in PSTVd Analysis of the accumulation of siRNAs has demonstrated that the most prevalent siRNAs were in the (−) polarity siRNAs were also prevalent over that of the (+) polarity. Previous observation in invertebrate viruses showed that both polarities of siRNAs were present in an equal number −) polarity siRNAs, although located in two different genomic regions of the virus in each specific strain, were all targeting the RNA-dependant RNA polymerase gene, suggesting a potential role in host defense against virus replication. Previous study http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). A mechanism for the possible involvement of the hot spot siRNA in RNA silencing for plant recovery is awaiting further investigation. A fundamental understanding of the mechanism of virus-host interactions may lead us to better use the hotspot siRNA information to manage viral disease infection.In the case of PepMV, the , which also allowed us to obtain a complete virus genome for a novel virus without a priori knowledge. We believe that these confirmation procedures are necessary in order to generate a complete and correct genomic sequence when using the current de novo assembly strategy. Even though a synthetic viral genome may serve as a reference sequence for the discovery of a very closely related genotype of a target virus, authenticity of these siRNA generated genomic sequences need to be validated by Sanger sequencing of the RT-PCR products.Gap filling and validation of an assembled virus genome from siRNAs was necessary to obtain complete, authentic virus genome sequences. Although assembly of sRNA sequences has been successfully used by a number of researchers to generate virus genome contigs, a priori knowledge, and associated confirmation of a complete virus genome by Sanger sequencing clearly demonstrates the power and efficiency of our bioinformatics pipeline. ToNSV is causing serious damage in certain Mexican tomato crop production systems. Characterization of ToNSV’s etiology has been hampered by the novelty of this new virus, which has less than 60% nucleotide sequence identity to other known viruses. Availability of the complete viral genome sequence will permit us to further characterize its biological properties, to develop serological and molecular detection technologies, and to better understand the factors influencing disease epidemiology. With this knowledge, we can better devise an effective disease management strategy.Identification of a novel potyvirus (ToNSV) without Even with the rapid advances in sequencing technologies and decrease in cost of sequencing, major challenges still remain in the development of an efficient bioinformatics pipeline for effective virus identification and complete genome assembly and validation. As more plant and animal genome sequences become available, this subtraction and assembly strategy should avail itself to broader applications in timely and thorough detection of existing, emerging, or novel viruses and viroids.Four tomato samples with suspected virus infection were originally collected from California (CAHN8), Arizona (EF09_58 and EF09_60), and Mexico (MX9354) and maintained on tomato (cv. Moneymaker) through mechanical inoculation in a greenhouse. Total plant RNA was purified from 3 g of leaf tissue using the TRIzol method following manufacturer’s instructions . The purified RNA preparations were evaluated on a Bioanalyzer  and shipped in ethanol solution to the University of Illinois Genomic Center for small RNA library construction and sequencing.Small RNA libraries were constructed from 1.0 µg of total RNA using the “Small RNA v1.5 Sample Prep” kit from Illumina . Unique 4 bp barcode sequences were added to the 5′ adaptors so that the libraries could be multiplexed and the reads sorted by samples. The barcoded libraries were pooled in equimolar concentration and sequenced on a single lane on a Genome Analyzer IIx for 41 cycles and the files were processed with Illumina’s CASAVA pipeline (version 1.5).de novo using Velvet de novo assemblies by replacing with the most frequent base covering that specific position. The resulting final contigs were used to query against the GenBank nt and nr databases, respectively, using the BLAST program sRNA sequence processing, assembly, and virus and viroid genome identification were conducted using a custom bioinformatics pipeline. Briefly, raw Illumina sRNA reads were first processed to trim adaptor and barcode sequences, and then assigned to individual samples based on the barcode sequences they contained. Trimmed sRNA sequences shorter than 15 nt were discarded. Subtraction of sRNAs derived from the host genome was performed by aligning sRNAs to the tomato genome sequences using the BWA program In an effort to generate complete virus genome sequences, specific primers were designed to fill the gaps in the assemblies and the 5′ terminal sequence was obtained through rapid amplification of cDNA ends (RACE). The authenticity of a synthetic siRNA assembled virus genome sequences was validated through Sanger sequencing of the RT-PCR products generated with primers derived from the reference genome sequences for PepMV-US1 (Genbank Accession No. DQ000984) or PepMV-EU (GenBank Accession No. AJ438767). RT-PCR products were directly sequenced using the common sequencing adaptors (M13 Forward and Reverse primers) linked to each virus specific primer . In some5′-GTGAAAGTATGCCCCACCAC) and another inner primer  were used for the first and second round of amplification of the virus specific cDNA. PCR products with expected sizes were cloned using TOPO TA cloning kit  and selected clones (4–6 clones) sequenced. Similarly for EU isolates, an outside primer  and an inner primer  were included in the reactions.To determine the 5′terminal sequence for PepMV isolates, the First Choice RLM-RACE kit  was used. For the US1 isolates: an outer primer . To obtain 5′RACE products, two independent experiments were performed in nested PCR using abridged universal amplification primer in combination with two individual virus-specific inner primers .For the novel virus (ToNSV) without a reference sequence, primers were designed based on the two assembled siRNA contigs. RT-PCR was used to validate the assembled contigs with overlapping RT-PCR products . To dete5′-GGTCTCGAGTTTTTTTTTTTTTTT-3′) and their respective virus-specific forward primers .Because both PepMV (a potexvirus) and ToNSV (a potyvirus) were expected to contain poly(A) tails at their 3′ termini, the 3′ terminal sequence was determined by sequencing the cloned RT-PCR products generated using an oligo(dT) primer (DNASTAR (Lasergene 8) was used to assemble and analyze each genomic sequence. A BLAST analysis of the complete genomic sequence and deduced polyprotein sequence was performed to identify closely related viral sequences available in the GenBank. The pairwise sequence comparison (PASC) program Figure S1The four tomato samples exhibiting virus-like symptoms were originally collected from greenhouse grown tomato plants in the U.S. or Mexico. A). CAHN8, with chlorosis and mosaic, in southern California. B). MX 9354, stunting, necrotic spot and leaf and fruit deformation, near Mexico City. C). EF09-58, mosaic and purpling leaf, in Arizona. D). EF09-60, marbling fruit, in Arizona.(TIF)Click here for additional data file.Table S1Oligonucleotide primers used in generating RT-PCR products for Sanger sequencing to obtain full genomic sequences for the two genotypes of Pepino mosaic virus (EU and US1) and for the new potyvirus (Tomato necrotic stunt virus).(DOC)Click here for additional data file.Table S2siRNA hot spot sequences and relative positions on Potato spindle tuber viroid in isolate CAHN8.(DOC)Click here for additional data file.Table S3Conserved siRNAs on the hotspots against Pepino mosaic virus EU strain in the three tomato samples.(DOC)Click here for additional data file.Table S4Conserved siRNA on the hotspots against Pepino mosaic virus US1 strain in the three independent samples.(DOC)Click here for additional data file.Table S5An unusual hot spot with siRNAs of upto 36 nt to PepMV-EU in the isolate EF09-58.(DOC)Click here for additional data file.Table S6Alignments of de novo assembled contigs to the ToNSV genome.(XLSX)Click here for additional data file.


Rift Valley fever virus (RVFV) is a bunyavirus endemic to Africa and the Arabian Peninsula that infects humans and livestock. The virus encodes two glycoproteins, Gn and Gc, which represent the major structural antigens and are responsible for host cell receptor binding and fusion. Both glycoproteins are organized on the virus surface as cylindrical hollow spikes that cluster into distinct capsomers with the overall assembly exhibiting an icosahedral symmetry. Currently, no experimental three-dimensional structure for any entire bunyavirus glycoprotein is available. Using fold recognition, we generated molecular models for both RVFV glycoproteins and found significant structural matches between the RVFV Gn protein and the influenza virus hemagglutinin protein and a separate match between RVFV Gc protein and Sindbis virus envelope protein E1. Using these models, the potential interaction and arrangement of both glycoproteins in the RVFV particle was analyzed, by modeling their placement within the cryo-electron microscopy density map of RVFV. We identified four possible arrangements of the glycoproteins in the virion envelope. Each assembly model proposes that the ectodomain of Gn forms the majority of the protruding capsomer and that Gc is involved in formation of the capsomer base. Furthermore, Gc is suggested to facilitate intercapsomer connections. The proposed arrangement of the two glycoproteins on the RVFV surface is similar to that described for the alphavirus E1-E2 proteins. Our models will provide guidance to better understand the assembly process of phleboviruses and such structural studies can also contribute to the design of targeted antivirals. Bunyaviridae (genus Phlebovirus), transmitted primarily by mosquitoes and is endemic throughout much of Africa and, in recent years in the Arabian Peninsula. The virus causes outbreaks in a wide range of vertebrate hosts, with humans and livestock being the most affected. Infection of livestock can result in economically disastrous abortion storms and high mortality among young animals. In humans, the virus causes a variety of pathologic effects with less than 1% of infections thought to result in fatal hemorrhagic fever or encephalitis  is a member of the family is MMWR, . Howeveris MMWR, . RVFV isRVFV is a typical enveloped bunyavirus and has a tri-segmented, negative-sense RNA genome, and most likely enters the host cells via receptor-mediated endocytosis, which requires an acid-activated membrane fusion step , Sandfly fever virus (Phlebovirus), Andes and Tula viruses (Hantaviruses), and have been used successfully to study the functionality of fusion peptides and the interaction and oligomerization of glycoproteins  was used. The secondary structure of RVFV Gn and Gc, respectively, were examined using Jpred3Initial backbone models were generated using the fold recognition Meta ServerThe 3D models of the RVFV Gn and Gc glycoproteins were fitted into the RVFV vaccine strain MP-12 cryoEM map , glycosylation sites, and consensus secondary structure prediction elements Figure A. Gn is To model the 3D structures of Gn and Gc, and to verify that RVFV Gc adopts a class II fusion protein fold, we initially focused on the near full-length RVFV Gn (530 aa in length) and Gc (507 aa in length) protein sequences Figure A. HoweveThe fold recognition revealed that the best matching profile for RVFV Gn resulted in a hit which had structural similarity to the Influenza 1918 human H1 hemagglutinin, specifically the receptor binding domain HA1 Figure . FurtherThe Gn and Gc model was evaluated in terms of stereochemical and geometric parameters such as bond lengths, bond angles, torsion angles, and packing environment and was found to satisfy all stereochemical criteria . The nomenclature of these three domains has been defined by analogy with the alphavirus E1 protein, domain I , domain II and domain III. Domain II contains two predicted glycosylation sites at positions N794 and N829 and also bears the predicted fusion loop of RVFV Gc that potentially inserts into the target host membrane during the pH-dependent virus fusion step In contrast to many other lipid enveloped RNA viruses, bunyaviruses do not contain a matrix protein that has the function of linking and stabilizing the nucleocapsid and viral envelope proteins. Based on our model, we suggest that a highly organized arrangement of the Gn and Gc glycoprotein ectodomains is responsible for overall virion stability and that the capsomer–capsomer interactions play a central role in defining the icosahedral virion symmetry.Multiple monoclonal antibodies against RVFV Gn and Gc have been described  for the icosahedral shell of RVFV is presented in Figure Bunyaviridae. Such structural studies are hoped also to contribute to the design of effective antivirals.The presented arrangement of Gn and Gc and description of their interactions may play an important role in glycoprotein folding and maturation, capsomer and virus assembly, virus fusion, and neutralization of infection. On-going site-directed mutagenesis experiments using a reverse-genetics system (Ikegami et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Introduction. Pityriasis lichenoides is a rare, acquired spectrum of skin conditions of an unknown etiology. Case Report. A 28-year-old man presented with recurrent outbreaks of herpes simplex virus associated with the onset of red-to-brown maculopapules located predominantly in trunk in each recurrence. Positive serologies to herpes simplex virus type 2 were detected. Histopathological examination of one of the lesions was consistent with a diagnosis of pityriasis lichenoides chronica. Discussion. Pityriasis lichenoides is a rare cutaneous entity of an unknown cause which includes different clinical presentations. A number of infectious agents have been implicated based on the clustering of multiple outbreaks and elevated serum titers to specific pathogens . In our patient, resolution of cutaneous lesions coincided with the administration of antiviral drugs and clinical improvement in each genital herpes recurrence. In conclusion, we report a case in which cutaneous lesions of pityriasis lichenoides chronica and a herpes simplex virus-type 2-mediated disease have evolved concomitantly. Pityriasis lichenoides is a rare, acquired spectrum of skin conditions of an unknown etiology, although infectious agents have been suspected as etiologic factors.A 28-year-old man presented with recurrent outbreaks of herpes simplex virus (HSV) type 2 in the form of ulcers on the glans penis associated with the onset of red-to-brown maculopapules located predominantly in trunk in each recurrence. The patient had four recurrences on a two-year followup .Positive serologies to HSV type 2 were detected in each outbreak (IgM and IgG). He received acyclovir 400 mg five times a day for 5 days, then a prophylactic dose of 400 mg twice a day; however, after discontinuation of antiviral drug, lesions reappeared.Histopathological examination of one of the lesions showed epidermal acanthosis, scanty lymphocytic exocytosis, without spongiosis, or focal necrosis. Perivascular infiltrates of lymphocytes with occasional eosinophils and erythrocytic extravasation were seen in the dermis . HSV DNAPityriasis lichenoides (PL) is a rare cutaneous entity of an unknown cause which includes different clinical presentations. It encompasses a spectrum of clinical presentations ranging from acute papular lesions rapidly evolving to form pseudovesicles and central necrosis (pityriasis lichenoides et varioliformis acuta or PLEVA) to small, scaling, benign-appearing papules (pityriasis lichenoides chronica or PLC). PLEVA and PLC are not distinct diseases but are rather different manifestations of the same process. The pathogenic mechanism behind PL is unclear although infectious or drug-related hypersensitivity reactions versus lymphoproliferative disorders are the mainstay theories. A number of infectious agents have been implicated based on the clustering of multiple outbreaks and elevated serum titers to specific pathogens, such as human immunodeficiency virus, hepatitis C virus, cytomegalovirus, Epstein-Barr virus, Toxoplasma gondii, and HSV –4, but tIn our patient, resolution of cutaneous lesions coincided with the administration of antiviral drugs  and clinical improvement in each genital herpes recurrence. PLC did not become less severe at successive attacks. Whether this association represents HSV reactivation caused by immune dysfunction related to the skin disease or HSV acting as an antigenic trigger is uncertain.In conclusion, we report a case in which cutaneous lesions of pityriasis lichenoides chronica and a herpes simplex virus type 2-mediated disease have evolved concomitantly.


Bovine parainfluenza virus type 3 (BPIV3) is associated with upper respiratory disease in cattle in many countries. Here, we report the complete genome sequences of the BPIV3 BN-1 strain, isolated from cattle in Japan, and the BN-CE vaccine strain, derived from the BN-1 strain by passages in chicken embryo fibroblasts. Respirovirus genus of the Paramyxoviridae family. BPIV3 is one of the most important viruses associated with the bovine respiratory disease complex (BRDC) in cattle. Complete genome sequences of BPIV3 have been determined previously, and isolated BPIV3 viruses were classified as genotypes A, B, and C (Bovine parainfluenza virus type 3 (BPIV3) is an enveloped, single-stranded, negative-sense RNA virus belonging to the B, and C –5. The BB, and C  and atteAB770484 and AB770485, respectively.The genome sequences of the BN-1 and BN-CE strains have been submitted to GenBank under the accession no.


The beads were characterized by size, scanning electron microscopy (SEM), zeta potential, swelling behavior, and degree of erosion. Also, the possible interaction between penicillin and biopolymers was investigated by differential scanning calorimetry (DSC), powder X-ray diffraction (XRD), and Fourier transform infrared (FTIR) analysis. The SEM micrograph results indicated a homogeneous drug distribution in the matrix. Also, based on thermal analyses (TGA/DSC), interactions were detected between microbead components. Although FTIR spectra of penicillin-loaded microbeads did not reveal the formation of new chemical entities, they confirmed the chemical drug stability. XRD patterns showed that the incorporated crystalline structure of penicillin did not significantly alter the primarily amorphous polymeric network. In addition, the results confirmed a prolonged penicillin delivery system profile. These results imply that alginate and OSA starch beads can be used as a suitable controlled-release carrier for penicillin.The aim of this study was to characterize the penicillin-loaded microbeads composed of alginate and octenyl succinic anhydride (OSA) starch prepared by ionotropic pregelation with calcium chloride and to evaluate their  Benzathine penicillin G (PenG) and its semisynthetic derivatives comprise one of the main groups of antimicrobials used in the treatment of major infectious diseases . Since iThe use of natural polymers in the design of drug delivery formulations has received much attention because of their excellent biocompatibility and biodegradability. Among them, alginate and starch are very promising and have been exploited in the pharmaceutical industry for controlled drug delivery. β-D-mannuronic acid (M units) and α-L-guluronic acid (G units) monomers, which vary in amount and sequential distribution along the polymer chain depending on the source of alginate [2+, cooperatively bind between the G-blocks of adjacent alginate chains, producing the so-called “egg-box” structure and creating ionic interchain bridges, which cause gelling of aqueous alginate solutions [Alginates are composed of (1-4)-linked alginate . Divalenolutions . Another potential biopolymer for drug delivery is starch. Starch is regenerated from carbon dioxide and water by photosynthesis in plants. Owing to its complete biodegradability, low cost, and renewability, starch is considered a promising candidate for developing sustainable materials . OctenylMixtures of alginate and OSA starch have been studied as coating materials for ascorbic acid edible film . This apFontes et al.  developeWe have analyzed the thermal and structural properties and drug release kinetics of the microspheres and biopolymers in an attempt to gain a better understanding of the structure-function relationship. The thermal properties were characterized using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The structure of the beads was studied using scanning electron microscopy and X-ray diffraction (XRD). The chemical propriety was analyzed by FTIR studies. The swelling and erosion behaviors were also evaluated because of their effects on the diffusion and release of drugs when microspheres are applied in drug delivery systems. Comprehensive knowledge of the material properties of a product in production and its application conditions is critical for a product success.2·H2O was obtained from Vetec. Penicillin G sodium salt was purchased from Sigma-Aldrich .Sodium alginate was obtained from Keltone LV. The ratio of mannuronic acid to guluronic acid residues (M/G) ranged from 0.4 to 1.9. Octenyl succinic anhydride (OSA) starch was obtained from National Starch, and CaCl2 solution for 10 min. The beads were then removed, washed with Milli-Q water, and dried in the oven at 35°C for 12 h. The beads containing PenG were prepared in triplicate by the extrusion method (dripping method) as described by Fontes et al. . Sodium γ = 220 nm, Waters). The alginate-OSA starch beads loaded with PenG were completely dissolved in phosphate buffer pH 7.4 and measured by HPLC. Preliminary UV studies showed that the presence of dissolved polymers did not interfere with the absorbance of the drug at 220 nm. HPLC analysis of PenG was performed using a Waters 1525 HPLC system with an analytical 4.6 × 150 mm (5 μm) RPC18 YMC-Pack ODS-AQ column, a flow rate of 1 mL/min, and a detector wavelength of 220 nm (Waters 2414). Samples (15 μL) were injected and eluted using A (0.025 M KH2PO4 in water (pH = 3)) and B (acetonitrile) as the mobile phase with an isocratic method (67% of A). PenG showed a retention time of 4.5 ± 0.17 min, and its detection limit was 0.1 μg/mL. The retention percentage (%) of entrapment PenG was calculated using the following equation:The retention percentage of PenG was determined by HPLC analysis  were taken as reference. Each experiment was done in triplicate.The in vitro release data, various kinetic models were used. The zero-order rate equation (Radjusted2) value was used as a criterion to choose the best model to describe drug release:Qt/Q∞ is the amount of drug released in time t, Q0 is the initial amount of the drug, K0 is the zero-order rate constant, K1 is the first order constant, Kh is the rate constant for Higuchi, Kb is the rate constant for Baker-Lonsdale, and Kk is the rate constant for Korsmeyer Peppas.To analyze the equation  describeequation  describeequation . The Bakequation  describeequation . The adjSW) for the beads sample at time t was calculated using  was estimated as follows:Wi is the initial starting dry weight and Wf is the final weight of the same dried and partially eroded sample. All experiments were done in triplicate.The degree of erosion was determined after the immersion of dry beads in 20 mL of Milli-Q water. After a selected time interval, the beads were withdrawn and dried in an oven at 110–120°C for a 24 h time period, allowed to cool in a desiccator, and finally weighed until constant weight was achieved . Three different samples were measured for each time point, and fresh samples were used for each individual time point. The percentage erosion (The microstructures of the beads were studied by scanning electron microscopy (SEM). Randomly selected dry beads were deposited on double-coated carbon conductive tape previously adhered to SEM aluminum stubs. The bead samples were then sputter-coated with a thin gold layer using a coating unit  and analyzed in a JEOL JSM 5310 operated at 15 or 20 kV. For the surface morphology observation, beads with and without PenG addition were utilized.The surface charge of beads was evaluated as a function of pH by zeta potential (Dispersion Technology DT 1200).αCu radiation wavelength of 1.54184 Å [X-diffraction patterns of the individual biomaterials, physical mixtures, and beads were obtained with an X-ray diffractometer  using a K.54184 Å .−1 were recorded using an FTIR spectrometer (Prestige 21-Shimadzu).Individual beads/samples were crushed in a mortar with a pestle. The crushed material was mixed with potassium bromide in a 1 : 100 proportion and dried at 40°C. The mixture was compressed to a 12 mm semitransparent disk by applying a pressure of 10 tons for 2 minutes. The FTIR spectra over the wavelength range from 4100 to 500 cmDifferential Scanning Calorimetry (DSC). The DSC of PenG, sodium alginate, OSA starch, as well as unloaded alginate/OSA starch beads, and PenG/sodium alginate/OSA starch beads were evaluated. The test was carried out by using a thermal analysis system  calibrated with indium as the standard and operated in the temperature range of 30–400°C. The bead sample (5 mg) was heated at a rate of 10°C min−1 in an aluminum pan under a nitrogen atmosphere using an empty pan as the reference. The onsets of melting point and enthalpy of fusion were automatically computed.Thermogravimetric Analysis (TGA). Degradation temperatures were performed in a TGA  using a sample mass of ca. 7 mg and an platinum sample holder at a heating rate of 10°C min−1 under a dynamic nitrogen atmosphere flowing at 20 mL min−1. 2+ is electronically positive, polyelectrolyte complexes can be formed between PenG/alginate and Ca2+ via electrostatic interactions during the microparticle preparation. Furthermore, the interactions between PenG and OSA starch are favorable, as PenG has a hydrophobic portion that can interact with the octenyl-succinate lipophilic component of OSA starch. The dripping technique produced spherical droplets that, after falling into the CaCl2 solution, resulted in spherical thermostable gel particles because of the ionic interactions between guluronate blocks formed from alginate and Ca2+ ions.In this study, microbead drug delivery systems were prepared using alginate and OSA starch as wall materials for PenG microencapsulation. Because both PenG and alginate are electronically negative and CaThe results showed a high retention percentage of PenG (95.4%) when alginate and OSA starch were used as the wall materials .PenG-loaded alginate/OSA starch beads showed a spherical geometry (1 ± 2 mm diameter) and a compact structure as evidenced by the SEM analysis reported in The surface of the beads exhibited a homogeneous microstructure with several wrinkles. The arrangement of the particles formed a surface porosity with pores of a few micrometers in diameter. The surface charge of the beads was evaluated as a function of pH . The resWhen the pH of the medium was increased, the carboxylic acid groups became ionized, resulting in the increase of negative charge. The PenG alginate/OSA starch beads presented higher negative values of zeta potential than the unloaded beads. This observation may be attributed to the negativelycharged PenG on the alginate/OSA starch microbeads, which caused a decrease in zeta potential. This result shows that beads can make ionic interactions with other biomacromolecules that have a positive charge, such as chitosan and polylysine.When hydrophilic polymers come into contact with a liquid hydrate, a gel layer is formed. The formation of the gel layer is essential for sustaining and controlling drug release from polymer solid dosage forms. The thickness of this hydrated layer determines the diffusion of the drug molecules through the polymer mass into the liquid medium, but diffusion is not the only mechanism controlling drug release. The rate and extent of drug release also depend on the swelling and erosion of the hydrated polymer preparation .2+, and OSA starch. When dried, the alginate network shrinks and collapses, and egg-box junctions move close enough to form side-by-side aggregations under the mediation of free calcium ions. This process is equal to the increasing cross-linking density and makes the dried alginate gel structure extremely dense, which is the reason that the alginate gel beads barely swelled in pure water, as shown in According to Davidovich-Pinhas and Bianco-Peled , the degThe beads did not show any visual sign of disintegration in the media over a period of 30 days. Another parameter evaluated was the degree of bead erosion. A decrease in the weight of the beads occurred after 24 hours of water immersion when the maximum degree of hydration was reached. The hydration probably caused the removal and solubilization of the polymer chains with a slight loss in the weight of the polymers. The maximum erosion degree of the beads was 5% over 30 days of experiments.The release behavior of PenG-loaded beads is presented in The kinetics of PenG release in alginate and OSA starch beads was also evaluated in a 0.35 M calcium chloride solution in order to investigate whether increasing ionic strength by the presence of calcium ions would influence the release of PenG. in vitro release data were studied using various kinetic models to predict the drug release kinetic mechanism. R2) values in various kinetic models tested. The release kinetics of PenG was found to be better described by the zero-order equation, which provided good linearity and the best fit line (Radjusted2 = 0.99).In order to understand the kinetics and mechanism of drug release, the According to Kiortsis et al. , for then is the release exponent, indicative of mechanism of drug release. For the case of sphere shape, 0.43 ≤ n corresponds to Fickian diffusion mechanism, 0.43 < n < 0.85 to anomalous (non-Fickian) transport, n = 0.85 to Case II (relaxation) transport, and n > 0.85 to super Case II transport . The value of the release exponent in PenG sustained release obtained was 0.82. This indicates that PenG release from alginate-OSA starch beads followed anomalous transport. In the anomalous processes of drug release, Fickian diffusion through the hydrated layers of the matrix and polymer chain relaxation/erosion are both involved [By incorporating the first 60% of release, data mechanism of release can be indicated according to Korsmeyer, where involved . −1) versus time (h). Linear regression analysis of the data yields the equation of best line as c = 6.45t + 190.67 and R2 = 0.99. For predictive completion of release, the drug release data of PenG obtained from dissolution is plotted as concentration (mg·Lk0 = 6.45 mg·L−1 h−1. The rate of release in terms of amount of PenG dissolved or released per unit time can be obtained as follows:V is volume of dissolution medium (L).According to , the slo−1. Assuming that this rate of release remains constant throughout the release process, the duration of release is calculated as 769.23 h (32 days) using 100 mg. Hence, the formulation allows a gradual release of the antibiotic from the beads. PenG diffuses through an outer gel layer, which erodes and allows the aqueous medium to penetrate further into the core.The rate of release calculated from  is 0.13 θ = 13.7°,  23.0°, and 40°. However, according to Wang et al. [θ = 13.7° and 23.0°. This difference may be because of the amounts of guluronic and manuronic acid present in the different alginate samples. OSA starch also showed three crystalline peaks at 2θ = 16°, 18°, and 22°. It presented a typical semicrystalline structure because of its close molecular packing and regular crystallization. Also, Tukomane et al. [The X-ray diffraction patterns of the individual polymers, physical mixtures of the polymers, PenG alone, and the PenG-loaded beads are displayed in g et al.  and Yangg et al. , the alge et al.  state the et al.  when algθ = 32° and 46°. The regularity of the crystal structure may be because of the egg-box regions along the direction of the alginate chain and its aggregating direction.The X-ray diffraction patterns of the physical mixtures of polymers and beads showed new peaks of crystallinity at 2Thermal analysis is the most common approach to study physicochemical interactions of a two- or more component system .  Table Alginate and OSA starch presented one endothermic peak, which is probably due to the melting point. The exothermic peaks of alginate at 237°C resulted from the decomposition of biopolymer because of depolymerization reactions, which are likely owing to the partial decarboxylation of the protonated carboxylic groups and oxidation reactions of the polyelectrolytes . The PenG thermograms presented two exothermic peaks, which are possibly attributed to oxidation. When alginate was mixed with OSA starch for the production of beads by ionotropic gelation, the thermogram also showed one endothermic peak. The endothermic peak shifted to a higher temperature (196.08°C). This shift corresponded to the interaction of alginate with OSA starch and calcium ions, which showed higher stability of the complexes. The degradation exothermic peak of sodium alginate was absent in the beads; thus, no decomposition event was observed up to 350°C. The thermograms of the PenG-loaded alginate-OSA starch beads did not present any peak during analyses. These results indicate good interaction between both components.Tg) were also determined. This parameter can be used as a measurement for the mobility of the macromolecules and the evaluation of the solid-to-liquid transition [Tg of alginate and OSA starch were 52.10, 67.06°C, respectively , which indicates a predominant energetic interaction between the biopolymers and the antibiotic. This effect causes an increase of the glass transition due to the denser packing in the mixture because of the decreasing mobility and the free volume caused by the local ordering effect of heterocontact formation [The glass transition temperatures . TGA has been proved to be a suitable method to investigate the thermal stability of polymeric systems. The knowledge of the threshold decomposition temperature and mode of decomposition upon heating is recommended to determine the highest processing temperature that can be used . The TGAThe alginate TG curve presents an initial dehydration process followed by decomposition in two overlapping steps under nitrogen, which is in agreement with DSC data. Similar behavior was observed by Soares et al. ; the decThe initial weight loss of OSA starch that began just above room temperature corresponds to water desorption. This process was not observed in the mixtures, an indication that a small quantity of water is adsorbed in their structures. OSA starch shows a single degradation step at 250°C.The unloaded alginate/OSA starch beads showed a two-step degradation inherent to both biopolymers. PenG TGA curves show that weight loss occurred only after 200°C in a single-step degradation.The OSA starch, alginate, and PenG exhibited 84.92, 68.2, and 73.49% of weight loss at 500°C, while the unloaded alginate-OSA starch beads and PenG/alginate/OSA starch beads presented 53.55% of weight loss at 500°C. The lower weight loss of the beads could be the result of strong interactions between the components of the beads. The thermal stability at 100–120°C (the temperature used in the microbead preparation) shows that both samples were stable during the preparation of the beads, indicating the viability of the implant preparation method developed.The possibility of chemical interactions was evaluated by FTIR. − stretching) were present, with a broad asymmetrical band at 1610 cm−1 and a narrower symmetrical band at 1418 cm−1. An even broader absorption was observed near 1030 cm−1, which can be attributed to COH stretching. OSA starch has similar profiles. In the fingerprint region, there are several discernible absorbances at 1155, 1080, 1021, and 930 cm−1, which were attributed to CO bond stretching [−1 is characteristic of the CH stretching vibration. An extremely broad band resulting from vibration of the hydroxyl groups (OH) appeared at 3390 cm−1. These results were confirmed by FTIR spectra obtained from both pure alginate and OSA starch (data not shown) and by the previously reported literature [The FTIR spectra of unloaded alginate/OSA starch beads provide evidence of both alginate and OSA starch structure information. For the alginate, characteristic functional groups (COOretching . The banterature .−1, indicating the presence of carboxyl and carboxylate group stretching. The band at 3350 cm−1 was assigned to the NH stretching. The mode oxazolone showed one characteristic band at 1790 cm−1 for the carbonyl group. The thiazolidine structure in PenG displayed the carbonyl band at 1622 cm−1. The bands at 1502 and 1701 cm−1 are characteristic of primary amide and secondary amide structures, respectively [The FTIR spectra of PenG alone showed major peaks in the wave number ranging from 800 to 1500 cmectively .In FTIR spectra of PenG-loaded alginate/OSA starch beads, no extra bindings or chemical shifts were observed, indicating that there is no strong chemical interaction between polymer and drug into the polymer/drug network. Although DSC data show that there are physical interactions, as mentioned earlier, between the polymer and drug, the FTIR spectra did not reveal the formation of new chemical entities. These results confirm the drug's chemical stability, the permanence of its biological activity, and the possibility of a sustained drug delivery system profile. in vitro release studies proved the capacity of the microbeads to release the drug in a prolonged profile, and the release kinetics of PenG was found to be better described by the zero-order equation. In summary, the delivery system developed and fully characterized in the present work can be applied as a subdermal implant for the treatment of rheumatic fever and other antimicrobial treatments in the future.In the present work, antibiotic-loaded alginate/OSA starch microbeads were successfully characterized using several different techniques. The morphological characterization showed that the homogeneous microbeads had negative surface charge. We have shown, by XRD, PenG distribution onto the polymeric matrix. These findings were confirmed by DSC. Shifts of the endothermic and exothermic peaks observed between individual biopolymers and final microbead carriers were interpreted as interactions with different thermal properties. Also, FTIR analysis showed the absence of detectable chemical interactions between the drug and polymer and the presence of PenG in structural biological activity. The


Leptopilina boulardi, its two hosts Drosophila melanogaster and D. simulans, the wasp virus LbFV, and the endosymbiotic bacterium Wolbachia. The virus is known to manipulate the superparasitism behavior of the parasitoid whereas some Wolbachia strains can reproductively manipulate and/or confer pathogen protection to Drosophila hosts. We used two nuclear backgrounds for both Drosophila species, infected with or cured of their respective Wolbachia strains, and offered them to L. boulardi of one nuclear background, either infected or uninfected by the virus. The main defence mechanism against parasitoids, i.e. encapsulation, and other important traits of the interaction were measured. The results showed that virus-infected parasitoids are less frequently encapsulated than uninfected ones. Further experiments showed that this viral effect involved both a direct protective effect against encapsulation and an indirect effect of superparasitism. Additionally, the Wolbachia strain wAu affected the encapsulation ability of its Drosophila host but the direction of this effect was strongly dependent on the presence/absence of LbFV. Our results confirmed the importance of heritable symbionts in the outcome of antagonistic interactions.Symbionts are widespread and might have a substantial effect on the outcome of interactions between species, such as in host-parasitoid systems. Here, we studied the effects of symbionts on the outcome of host-parasitoid interactions in a four-partner system, consisting of the parasitoid wasp  Drosophila hydeiEndosymbionts are extremely frequent in arthropods, especially in insects. By providing additional heritable genetic material, they may contribute to the adaptation of their insect host Leptopilina boulardi that is able to parasitize both Drosophila melanogaster and D. simulans larvae. Parasitization may lead to three different outcomes: (i) the parasitoid avoids the immune system of the Drosophila larva, reaches the adult stage and ultimately kills the Drosophila; (ii) the Drosophila succeeds in killing the parasitoid by a cascade of immune reactions leading to the encapsulation of the young wasp Whereas most studies have focused on the effect of a single symbiont either in the host or in the parasitoid on the outcome of the host-parasitoid interaction but see , we haveDrosophila species are often infected by the maternally-transmitted bacterium Wolbachia. Different strains have been described, some inducing various reproductive manipulations in their hosts, such as cytoplasmic incompatibility, while others have unknown effect. This raises the question of the mechanism explaining their prevalence in natural populations Wolbachia strains may increase the resistance of their Drosophila host to parasitoid attacks. It has been found that Wolbachia can confer resistance against various parasites such as RNA viruses PlasmodiumWolbachia-infected D. simulans have, for instance, reduced ability to encapsulate parasitoids Wolbachia in the isopod Armidillidium vulgare is able to infect host haemocytes L. boulardi is often infected by a maternally-transmitted DNA virus called LbFV (Leptopilina boulardi Filamentous Virus), whose prevalence may exceed 90% in some locations Drosophila larvae and usually avoid superparasitism, i.e. laying eggs in already parasitized larvae, virus-infected females readily lay eggs in previously parasitized host larvae. Infected offspring are consequently exposed to strong competition, as only one parasitoid is able to fully develop inside a single host larva. Superparasitism is adaptive for the virus as it enables its horizontal transmission among the parasitoid larvae competing within the same fly larva. Theoretical work has shown that the virus is selected for increasing the natural superparasitism tendency of the parasitoid because it allows infection of new parasitoid matrilines Drosophila's immune response.The parasitoid Wolbachia (infecting the Drosophila host) on the outcome of the host-parasitoid interaction using two genetic backgrounds of D. melanogaster and D. simulans and one genetic background of L. boulardi. We measured the successful encapsulation rate by counting adult flies that survived parasitoid attack. In a second experiment, we also controlled for the occurrence and effect of superparasitism by measuring encapsulation in Drosophila larvae. The results showed that symbionts indeed influence the final outcome in this host-parasitoid interaction.In this paper, we tested the combined effect of LbFV (infecting the parasitoid) and of different strains of Drosophila melanogaster (YW-BNE and w1118) and D. simulans (CO and DSR) were used, either infected with or cured of different Wolbachia strains, leading to eight inbred lines as described in Drosophila lines were then reared on antibiotic-free food for several generations before the start of the experiments. Their Wolbachia infection status was checked by PCR detection using the 81F-691R wsp primers specific to WolbachiaTwo nuclear backgrounds of each of L. boulardi, designated NSref and Sref, with the same nuclear genetic background but a different virus-infection status were used (Drosophila larva on average Wolbachia-free D.melanogaster line originating from Lyon (France). Both NSref and Sref have been shown to be Wolbachia-free in a previous study L. boulardi lines was determined by diagnostic PCR using the primers 500-R/102F designed for specific detection of LbFV Drosophila hosts Two reference lines of ere used . NSref iWolbachia on several key traits of the Drosophila-parasitoid interaction. For each line, one hundred eggs were deposited into rearing vials . Twenty-four hours later, a single female parasitoid, either LbFV-infected or not, was introduced into each vial (n = 15 for each parasitoid infection status) and removed 24 hours later. Ten control vials for each Drosophila line (Wolbachia-infected and Wolbachia-free lines) were kept without parasitoid. Experiments were carried out in large incubators at 26°C under 12L∶12D photoperiod and 70% relative humidity.In experiment 1, we addressed the question of the contribution of LbFV and Drosophila flies that were not parasitized, or were parasitized but eliminated the parasitoid, started to emerge and were collected daily and counted at the end of the experiment. In response to parasitism, Drosophila larvae can initiate a protective immune reaction, which can lead to the encapsulation of the parasitoid egg or larva From day 7, Drosophila-parasitoid interaction were measured (iPR), or the proportion of Drosophila larvae parasitized by a single female parasitoid in a given vial i, was estimated by comparing the number of emerged flies in the treatment vial i (iNd) to the mean number of flies in the control vials (cN) of each Drosophila line as follows:iNcap being the number of adult flies containing capsules in vial i.Different key life-history traits influencing the outcome of the measured . The pariSER), defined as the proportion of parasitized Drosophila larvae that survived up to the adult stage, was calculated by dividing the number of flies containing capsules by the estimated number of parasitized larvae:iPS), defined as the proportion of parasitoids that survived up to the adult stage after successfully avoiding encapsulation, was calculated as follows:iNp being the number of adult parasitoid offspring in vial i.From this estimator, the successful encapsulation rate  exposed to parasitoids was thus defined as the fly survival in vial i (iSd) relative to their respective survival in control vials (cSd):We used the number of adult parasitoid offspring Drosophila eggs deposited on day 1 (experiment 2.1), and the other half contained 125 eggs deposited on day 2 (experiment 2.2). We used two Drosophila densities (100 eggs or 125 eggs) in order to vary the host/parasitoid ratio and possibly the frequency of superparasitism. For each larval density, ten additional vials without parasitoid were used as controls. From each treatment vial, ten randomly chosen Drosophila pupae were dissected under a stereomicroscope. We recorded the number of parasitoid eggs, parasitoid larvae and the number of capsules found in each pupa. The larval encapsulation rate (iLER) is the proportion of fly larvae that encapsulated all parasitoids. For this analysis, we only considered fly larvae containing either one or two parasitoids since larvae containing more than two parasitoids were too rare to support strong statistical analyses. The encapsulation rate at adult stage (iSER) was measured as previously described in this paper except that dissected larvae were taken into account by subtracting 10 flies from the mean number of flies in the control vials (Nc).Virus-infected and uninfected parasitoids display contrasting egg-laying strategies (frequent superparasitism for Sref and rare for NSref). The virus' effects on the outcome of host-parasitoid interactions may therefore either result from a direct effect of the virus or from an indirect effect through the occurrence of superparasitism. In order to distinguish between these effects, we performed a second experiment using only the cured DSR line. We chose this particular line for its successful encapsulation rate, clearly dependent on the parasitoid infection status. Forty vials were prepared for both uninfected and virus-infected parasitoid lines. Half of these contained 100 All data sets were analysed with the R software (version 2.11.1) . Except for the larval encapsulation rate, all life-history traits were analysed using linear models after adequate transformation to reach the assumptions of normality and homoscedasticity. Some experimental vials in which parasitoids did not lay any eggs were disregarded from the analyses. Linear models were constructed by putting first the temporal block effect in order to remove the potential stochastic effects before testing the parameters of interest, i.e. the effects of the two symbionts. The larval encapsulation rate was analysed using a generalized linear model with a binomial error structure (logit link function) given the binary nature of the data .Drosophila-parasitoid interaction. Moreover, the Drosophila nuclear background was always significant showing that the outcome of host-parasitoid interaction was highly dependent on the host genotype  and survived to the adult stage . Despite1,52F = 12.98; P<0.0007; Virus-infected parasitoids tended to show a slightly higher parasitism rate but this difference was not significant . The virDrosophila relative survival, consistent with the lower successful encapsulation rate of virus-infected parasitoids , or their ability to successfully encapsulate parasitoids  infection was correlated with a reduction in the successful encapsulation rate of virus-free parasitoids , but with a slight increase in the encapsulation rate of virus-infected parasitoids . Also, within this background, Wolbachia (wAu) infection was correlated with a significant reduction in parasitism rate when Drosophila were attacked by infected parasitoids  but was not correlated with parasitism rate when Drosophila were attacked by uninfected parasitoids . Finally, Wolbachia (wAu) infection had no effect on Drosophila relative survival when CO flies were exposed to virus-free parasitoids  whereas wAu-free flies had a lower survival than wAu-infected flies when exposed to virus-infected parasitoids .Within this background, Wolbachia interactions should be interpreted with care since they were all highly dependent on the temporal block, according to the significant interactions of third order . There was also a high variability between experiments 2.1  and 2.2  with a significantly lower successful encapsulation rate in experiment 2.2 .The most important effect detected in experiment 1 was the decrease in encapsulation rate when parasitoids were infected by LbFV. As the virus modifies the way females distribute their eggs among Drosophila larvae among parasitized ones) were observed with both virus-infected  and uninfected parasitoids .In both experiment 2.1 and 2.2, substantial superparasitism rates . Additionally, superparasitized larvae showed a 11.7% decrease in successful encapsulation events  compared with monoparasitized larvae  and humoral response efficiencies was demonstrated In a second set of experiments using Drosophila immune response is depressed by the presence of the virus, or because infected-parasitoids have an increased virulence ability. The mechanism responsible for this protection, yet unknown, could involve either a virus-driven immune suppression as observed with polydnaviruses In addition to this indirect effect of the virus on encapsulation rate through the induction of superparasitism, we also demonstrated a significant direct effect of the virus. In monoparasitized larvae, the presence of LbFV was associated with a decrease in larval encapsulation rate. This effect may arise either because the Drosophila competition between parasitoid larvae.Whereas the direct protective effect of the virus is clearly advantageous for the parasitoid, the fitness reward from the indirect effect of superparasitism is unclear. In our experiment, one single female was put in each treatment vial, and could therefore directly benefit from self-superparasitism. In nature, however, conspecific-superparasitism is likely to be much more frequent than self-superparasitism. In such conditions, it is unknown if the superparasitizing female would benefit from the protective effect offered by superparasitism since this would depend on the outcome of the within-Besides encapsulation, the virus also negatively affected parasitoid developmental success. This virus effect could be either a direct effect of the physiological cost of infection, or an indirect effect of the increased superparasitism in infected parasitoids, as suggested by a previous study wAu, Wolbachia did not affect any of the tested traits. A positive effect of Wolbachia on the successful encapsulation rate was expected, at least for wMel and wMelPop since these strains have previously been shown to increase hemolymph melanization, a key reaction involved in encapsulation, in both D. melanogaster and D. simulanswMel, wMelPop nor wRi. Fytrou et al. (2006) found that wRi-infected DSR flies were less efficient in encapsulating the parasitoid Leptopilina heterotoma. Their results differ from our findings on the similar DSR Drosophila line, and indicate that the final outcome of host-parasitoid interactions also depends on the parasitoid species.Except for Wolbachia in Drosophila that was only observed with the strain wAu in D. melanogaster. Overall, Wolbachia-free CO flies suffered more from virus-infected parasitoid attacks than wAu-infected flies did. The slight increase in the encapsulation rate of virus-infected parasitoids suggests that a wAu-mediated protection might be activated in presence of LbFV. This is consistent with the strong antiviral protection of wAu in CO flies, allowing resistance against the RNA virus DCV Wolbachia strains tested, although they were also found to have an antiviral activity in previous studies Wolbachia-free than on Wolbachia-infected larvae, whereas virus-free parasitoids displayed a similar parasitism rate whatever the infection status of CO flies. This result suggests that wAu might either influence the ability of infected parasitoids to locate Drosophila larvae, modify their egg-laying preferences or that wAu-infected Drosophila larvae might be better at avoiding parasitoid attacks when the parasitoid is infected by the virus. We must however be cautious as all these interaction effects strongly depended on the temporal block and thus on unknown environmental parameters. Further investigations should be carried out before concluding that wAu can be beneficial to its host, and to determine by which way wAu interacts with LbFV.A surprising result was the complex interaction observed between viral infection in the parasitoid and infection by L. boulardi. A virus-induced increase in L. boulardi's virulence might depict an ongoing evolution towards a mutualistic association between the virus and the parasitoid, similar to what is believed to have occurred between ancestral polydnaviruses and their wasp carriers wAu strain, that Wolbachia might not only be a reproductive parasite in arthropods, but may as well contribute to variation of traits involved in host-parasitoid interactions. Because symbionts benefit from vertical transmission, they produce heritable variation on which natural selection can act and directly contribute to the adaptation of their host. As such, there is a crucial need to view infections by so-called parasites in a broader ecological context by considering several life-history traits of their hosts and their interactions with other species within the community In conclusion, our data confirm that symbionts in hosts and parasitoids contribute to variation in extremely important phenotypes such as resistance and virulence, in addition to classical nuclear factors Table S1Analysis of variance of life-history per Drosophila nuclear background in experiment 1. * Significant effect; Level of significance: α = 0.0125 (Bonferroni correction for multiple comparisons). Successful encapsulation rate: square root-transformed data; Parasitism rate: arcsine square root-transformed data; Parasitoid developmental success: square root-transformed data; Number of parasitoid offspring: square root-transformed data; Drosophila relative survival: log-transformed data.(DOC)Click here for additional data file.


Virus evolution should be monitored because frequent reassortment, which contributes to virus diversity, creates the potential for more severe infections. Several severe respiratory virus infections that have emerged during the past decade originated in animals, including bats. In Indonesia, exposure to bats has been associated with increased risk of acquiring orthoreovirus infection. Although orthoreovirus infections are mild and self-limiting, we explored their potential for evolution into a more virulent form. We used conventional virus culture, electron microscopy, and molecular sequencing to isolate and identify orthoreoviruses from 3 patients in whom respiratory tract infection developed after travel to Indonesia. Virus characterization by plaque-reduction neutralization testing showed antigenic similarity, but sequencing of the small segment genes suggested virus reassortment, which could lead to increased virulence. Bats as a reservoir might contribute to virus evolution and genetic diversity, giving orthoreoviruses the potential to become more virulent. Evolution of this virus should be closely monitored so that prevention and control measures can be taken should it become more virulent. Reoviridae comprises a diverse group of nonenveloped, segmented, double-stranded RNA viruses classified into 10 genera. Members of the genus Orthoreovirus are classified as fusogenic or nonfusogenic, depending on their ability to cause syncytium formation in cell culture. Genetically, orthoreoviruses contain 10 segments: 3 large, 3 medium, and 4 small . The characteristic of the segmented genome facilitates virus reassortment, which contributes to virus diversity. Sequencing studies indicate that reassortment in nature is a major determinant of reovirus evolution , we confirmed infection in 2 of these patients and showed cross-reactivity of antibodies among the 3 virus strains. To track virus evolution and provide evidence of genetic reassortment, we used PCR sequencing and phylogenetic analysis to compare their genetic relatedness.Patient 1 was a 51-year-old male tourist from Bali, Indonesia, who became ill in April 2007. This case has been reported elsewhere (Patient 2 was a 26-year-old woman who sought care at a designated fever clinic during the outbreak of pandemic influenza in July 2009. She had a 2-day history of influenza-like illness (ILI), and oseltamivir was prescribed. Throat and nasal swab specimens were taken 3 days after onset of illness. Serum was collected 59 days after symptom onset. The patient recovered uneventfully. She reported having traveled to Bali, Indonesia, for 5 days and noticing ILI symptoms on the last day of her visit. While in Indonesia, she visited a safari park and entered bat caves on the second day. Her travel companions remained asymptomatic.Patient 3 was a 29-year-old woman who sought care for ILI at the General Outpatient Clinic in Hong Kong in June 2010 after a recent visit to Indonesia. As part of sentinel surveillance for ILI, throat and nasal swab specimens were taken for routine isolation of respiratory viruses  on cell culture, we used immunofluorescence testing with monoclonal antibodies to preliminarily identify viruses , stained with 3% phosphotungstic acid (pH 6.3) (1 min), and inactivated with ultraviolet irradiation before examination at 80 KV with a Philips transmission electron microscope .www.megasoftware.net) with the neighbor-joining method. Sequences were submitted to GenBank and compared with other orthoreovirus sequences for genetic relatedness and evolution . Serum, virus, and cell controls were included in the assay. After 1 h of virus absorption, the inoculum was discarded, plates were washed with 2% MEM, 4 mL of overlay LE agar was added, and the plates were incubated at 37°C in a CO2 incubator for 4 days. The end-point neutralizing antibody titer was determined by serum with the highest dilution that reduced plaques by 70% more than the virus control  and heat inactivation at 56°C for 30 min. We then performed serial double dilutions of serum samples from 1:5 to 1:2,560. Equal volumes of serum and virus mixture were incubated at 37°C for 2 h. Then 200 μL of serum and virus mixture with final dilutions of 1:10 to 1:5,120 was added to each of the 6-well plates containing a monolayer of confluent Vero cells  showed syncytial CPE in culture tubes after 3 to 4 days of incubation in each of the 4 cell lines.  Immunofluorescent testing indicated that the samples were negative for influenza, respiratory syncytial, measles, and mumps viruses. Electron microscopy revealed virus morphologic appearance consistent with that of a reovirus .According to PRNT results, serum of patient 2 had a neutralizing antibody titer of 1,280 against HK46886/09 virus and HK23629/07 virus and a titer of 640 against HK50842/10 virus. Serum of patient 3 had a neutralizing antibody titer of 20 against all 3 viruses. The control serum had a neutralizing antibody titer <10.For the S1 virus cell attachment protein, the HK46886/09 and HK50842/10 viruses were clustered with the Miyazaki virus and related closely to the Kampar virus isolated in Malaysia; the S1 virus cell attachment protein of the HK23629/07 virus has been reported as similar but distinct from Melaka virus . For the,,The isolation and epidemiologic studies of Melaka and Kampar virus in Malaysia provide evidence that a novel group of fusogenic orthoreovirus of bat origin is associated with human respiratory tract infection. The role of bats as reservoirs and sources of potential emerging viral infections has been extensively studied with regard to Nipah, Hendra, lyssavirus, and coronaviruses  virus, severe acute respiratory syndrome coronavirus, Nipah virus, and Hendra virus, have been associated with animal origins. Infections occurred in humans when the pathogens crossed the species barrier to expand their host range. Changes in human activities also bring humans in close contact with animals (Although symptoms of orthoreovirus infection are mild and self-limiting, continued close monitoring of virus evolution and further study are essential. Changes in virulence would signal the need for prevention and control strategies.


Information is scarce about sylvatic rabies virus in Asia and about rabies in palm civets. We report a novel sylvatic rabies virus variant detected in a golden palm civet in Sri Lanka. Evolutionary analysis suggests the virus diverged from canine rabies viruses in Sri Lanka in ≈1933 (range 1886–1963). The ecology of rabies in wildlife populations and natural ecosystems is poorly understood , which lives only in southern India (P. aureus), the golden dry-zone palm civet , and the Sri Lankan brown palm civet (P. montanus)  for testing. We detected rabies virus in the animal’s brain by using the fluorescent antibody test and extracted viral RNA and DNA by using Trizol . The rabies virus from this sample was designated as H-1413-09. The whole genome of the virus was sequenced directly from the sample as described (b (cytb) gene and performed a BLAST search (www.ncbi.nlm.nih.gov/blast/Blast.cgi) for similarity with other sequences. By aligning nucleotide sequences of the cytb gene of mitochondrial DNA of domestic cat, jungle cat, fishing cat, Asiatic golden cat, marbled pole cat, European pole cat, lynx, puma, leopard, African lion, tiger, jaguar, civet, and palm civet with ClustalW2 , we designed primer Felis cytb-F, 5′-ATGACCAACATTCGAAAATCACACC-3′ (nt 1–25), and primer Felis cytb-R, 5′-CAATAATGCCTGAGATGGGTATTAG-3′ . Using these primers, we performed PCR as follows: initial denaturation at 94°C for 2 min, followed by 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min for 35 cycles, followed by a final extension at 72°C for 5 min. PCR generated a 1,117-bp fragment from which a 1,004-nt sequence was determined. Analysis showed that the sequence has 100% identity with the partial (224-nt) sequence of the cytb gene of P. zeylonensis (GenBank accession no. FJ881681); this is the only sequence available for P. zeylonensis. The sequence also has 95% identity with P. jerdoni and 90%–92% identity with P. hermaphroditus.To confirm the species of the rabid animal, we determined the nucleotide sequence of the mitochondrial cytochrome 7 generations and sampled every 3,000 steps. This procedure resulted in an effective sample size of >2,000 for all estimated parameters. The posterior densities were calculated with 10% burn-in and checked for convergence by using Tracer version 1.5 (http://beast.bio.ed.ac.uk/Main_Page). The mean rate of nucleotide substitution estimated for the N gene was 2.2 × 104 substitutions/site/year . This rate is in agreement with previous findings , we determined the genetic distance of the N gene to determine whether strain H-1413-09 is more diverse than other rabies viruses in Sri Lanka . Our study indicates that rabies might be another risk factor for extinction of these animals. Identification of a variant rabies virus in wildlife has serious implications for rabies control in Sri Lanka. Identification of such a virus would help provide epidemiologic data about the spread of rabies and its incursion into new geographic regions and would justify allocation of increased resources to help control rabies (,Palm civets are facing extinction in Sri Lanka because the species is losing its habitat, being hunted for its meat, and dying of parasitic diseases (,–P. zeylonensis is a reservoir of rabies virus or represents spillover from another animal deserves extensive investigation. The detection of rabies in wildlife indicates that much remains to be discovered in the tropical ecosystem of Sri Lanka. The circulation of a sylvatic variant rabies virus may be another hurdle in the rabies-control effort in Sri Lanka.Several rabies virus variants associated with wildlife are known in the Americas and Africa (


Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes. Current methods for detecting real-time alphavirus (Family  Alphaviruses are mosquito-borne pathogens that can cause severe human and veterinary disease, several of which are considered potential biological weapons ,3. AlphaDefining how alphaviruses infect the mosquito vector and transmit to mammalian hosts is an active area of study, but the tools for monitoring alphavirus infection in mosquitoes have largely relied on postmortem analysis or using recombinant viruses engineered to express fluorescent or luminescent proteins from duplicated subgenomic promoters (SGP) . AlthougTogaviridae, genus alphavirus) are positive strand RNA viruses with a genome size of ~12Kb containing a 5’ RNA cap and a 3’ polyadenylated tail ) resulting in pBG471 . Th. ThAe. apromoter  to optimThe reporter RNAs used in this study express fluorescent proteins during infection and allow visual confirmation of Sindbis virus infection. However, a system similar to this could be engineered to express other proteins only during active virus replication. An interesting application for this technology would be to engineer reporter RNAs that encode a cytotoxic or mosquitocidal gene, which would only be expressed concurrent with virus infection. For example, expression of the Saporin ribosomal toxin gene  during iThe subgenomic reporter RNA system we present can detect alphavirus infection in cell culture systems and in transgenic mosquitoes. Once optimized, this novel system will provide a valuable method to visually monitor dissemination of wild-type alphaviruses throughout insect cells and be useful for monitoring the transmission of alphaviruses from mosquitoes to mammalian hosts, increasing our understanding of viral transmission cycle.


We have shown earlier that a single dose of cloned defective interfering (DI) influenza A virus strongly protects mice from disease following a lethal challenge with different subtypes of influenza A virus. These animals suffered no clinical disease but experienced a subclinical infection which rendered them immune to reinfection with the same challenge virus. However, little is known about how DI virus achieves such protection. Here we investigated the role of adaptive immunity in DI virus-mediated protection using severe-combined immunodeficient (SCID) mice, which lack competence in both B- and T-cell compartments but retain NK cell activity. SCID mice which were treated with DI virus and infected with influenza virus initially remained completely well, while infected litter mates that received UV-inactivated DI virus became seriously ill and died. However, after 10 days of good health, the DI virus-protected SCID mice developed a clinical disease that was similar, but not completely identical, to the acute influenza disease. Disease was delayed longer by a higher dose of DI virus. We excluded the possibilities that the DI virus load in the lungs had declined, that the DI RNA sequence had changed so that it no longer interfered with the infectious genome, or that infectious virus had become resistant to the DI virus. These data show that while DI virus provides full protection from the acute disease in the absence of adaptive immunity, that same immunity is essential for clearing the infection. This indicates that the conventional view that DI virus-induced protection is mediated solely by competition for replication with the challenge virus is incorrect for influenza virus. DI virus is thus totally dependent on infectious virus for replication. Interference occurs when the ratio of defective: infectious genomes increases to a level which results in a reduction of the amount of infectious virus produced In the course of replication most viruses make defective-interfering (DI) viruses, which are virus particles composed of a normal set of viral proteins encapsidating a deleted version of the viral genome. Because they lack essential genetic information, DI viruses are replication deficient. Replication of the defective genome is achieved by the presence in the same cell of a genetically compatible infectious genome, usually from the virus that generated the DI genome, and which provides the missing function(s) Influenza A viruses have a genome that comprises 8 separate segments of single stranded, negative sense RNA. DI influenza viruses arise readily and their study has a long history extending back over 60 years Analysis of the mechanism(s) by which DI viruses prevent disease in treated animals is not fully understood. The DI influenza virus particle and its component proteins are indistinguishable from those of infectious virus and, in principle, they may be capable of stimulating a similar adaptive immunity as to that resulting from exposure seen with to a conventional inactivated influenza virus vaccines. The data show that adaptive immunity is not required for DI virus to protect SCID mice from acute influenza. However, in contrast to immune-competent animals, a delayed onset disease occurred about 1 week later, indicating that adaptive immunity is required to act in concert with DI virus to clear the infection.22.12). This is referred to as ‘active DI virus’. The UV inactivation target is viral RNA, and UV has little effect on the DI RNA because of its small target size, 395 nt compared with 13,600 nt for infectious virus. Longer UV irradiation (8 min) inactivated mouse-protecting activity and provided a preparation that controlled for any immune system-stimulating or receptor-blocking effects (‘inactivated DI virus’). However, UV treatment did not completely destroy all DI RNA. UV did not affect haemagglutinin or neuraminidase activities.The 244 DI RNA used here to protect mice was originally generated spontaneously during transfection of 293T cells with plasmids 2.2k) mice (bred in-house), wild type Balb/c (H-2d) mice (Harlan UK Ltd.), and mutant Balb/cJHan™Hsd-Prkdcscid mice (Harlan) with a defect in the Prkdc gene which encodes DNA-PK. This leads to aberrant VDJ recombination and hence deficient B and T cells. SCID mice have a normal complement of NK cells. Wild-type Balb/c mice required 2 × 103 ffu of WSN challenge virus to cause consistent but non-lethal clinical disease; this was twice the dose needed for C3H/He-mg mice scid mice were also infected with 2 × 103 ffu of WSN. Adult mice (4–6 weeks old) were inoculated intranasally under light ether anaesthesia as previously described We used wild type C3H/He-mg (H-22.3−) in infected mouse lung. We used the following primers and probes: segment 1 F (5′ TGCAATGGGACTGAGAATTAGCT 3′), segment 1R (5′ TCCGCTTGTTCTCTTAAATGTGAAT 3′) and probe (5′ VIC-CACCAAAACTGAAGGAT 3′); 244 1F (5′ CATAATCAAGAAGTACACATCAGGAAGAC 3′), 244 1R (5′ CTCTTTGCCCAGAATGAGGAAT 3′) and probe (5′ FAM-CCCTCAGTCTTCTCC 3′); segment 7 1F (5′ CTTCTAACCGAGGTCGAAACGTA 3′), segment 7 1R (5′ GGATTGGTCTTGTCTTTAGCCA 3′) and probe (5′ FAM-CTCGGCTTTGAGGGGGCCTGA 3′) RNA was extracted from the left lungs of mice by grinding with sterile sand and Trizol (Invitrogen). Quantitative real time PCR was performed on an ABI prism 7000 to quantitate virion-sense (RNA2.4The right-hand lung from each infected mouse was homogenised with sand in PBS containing 0.1% (w/v) BSA, and centrifuged to remove debris. A/WSN infectivity was titrated in a focus-forming assay using MDCK cells in 96-well plates in triplicate. Cells were incubated at 33 °C for 18 h, fixed in 4% (v/v) formaldehyde, and blocked with 5% (w/v) milk powder in PBS. Virus-positive cells were detected using a mouse monoclonal antibody specific for the A/WSN haemagglutinin, and a goat anti-mouse IgG–alkaline phosphatase conjugate (Sigma), both in buffered saline containing 0.1% (v/v) Tween, and finally incubated with an alkaline phosphatase substrate . At least 50 stained cells (foci) at an appropriate dilution were counted in each of three wells and averaged to give a titre in focus-forming units (FFU)/lung.33.1Before examining SCID mice we tested the infection parameters of A/WSN in the immune competent Balb/c strain from which they had been derived. Mice inoculated simultaneously with 1.2 μg of active DI virus and infected with A/WSN were either completely protected or suffered only a mild clinical disease of short duration with slight weight loss a and b. SCID mice infected with A/WSN succumbed to a disease similar to that seen in immune-competent Balb/c mice as judged by clinical signs and weight loss from day 3 after infection, progressing to death or to the point at which they had to be euthanized c and d. SCID mice which had been protected from influenza by treatment with 1.2 μg of active DI virus all remained well for 9 days, but on day 10 some started to lose weight and show signs of disease c and d. 3.2To understand how DI virus mediated protection we examined mice for lung consolidation and lung infectivity. Protection conferred by 1.2 μg of active DI virus a and b cOn day 2 post-infection the lung infectivity in SCID mice inoculated with inactivated DI virus + A/WSN was already 10% of the maximum value reached on day 4, while the lung titre in mice receiving active DI virus + A/WSN was 83-fold lower on day 2. Although the infectious load in active DI virus-treated mice increased slowly over the next few days the difference seen with treated with active or inactive DI virus remained at over 10-fold to day 6 post infection. At this time active DI virus-treated, infected mice appeared perfectly normal, while mice that received inactivated DI virus + A/WSN had had lost nearly 20% body mass and were extremely ill. From days 4 to 8 the infectious load in DI treated-mice rose steadily, and at day 8 there was overt lung consolidation c. Consol3.3There are no reports in the literature for the dynamics of influenza full-length or DI RNA synthesis in the mouse lung. To determine if the delayed disease was due to changes in the levels of 244 DI or infectious virus RNA, total RNA was isolated from the lungs of each group of SCID mice described in 8 copies of 244 DI RNA in the lungs of SCID mice inoculated with the active DI virus + A/WSN, and about 100-fold lower in the group that received inactivated DI virus + A/WSN. The latter represents UV-fragmented 244 RNA and residual intact 244 RNA  confirmed that the terminal sequences were also unchanged (data not shown). The same result was obtained in 2 independent experiments, demonstrating that authentic 244 DI was present in substantial amounts in the sick mice on day 16 after infection.3.4DI genomes are replicated by the infectious homologous virus and interfere with the production of infectious virus when a critical ratio of DI genomes: infectious genomes is reached. This suggests that there may be evolutionary pressure for the fixation of viral mutations that result in it no longer recognising, replicating or being inhibited by 244 DI RNA. Such resistance has been reported to occur in cell cultures persistently infected by VSV or Sindbis virus 3 ffu) in an in vivo protection experiment with 244 DI virus and immune competent mice. Data in To test this possibility we isolated infectious virus from the lungs of severely ill SCID mice at 16 days after inoculation with active DI virus + A/WSN . Virus w4Intranasal inoculation with 244 DI influenza virus completely protected SCID mice from rapid onset acute respiratory disease caused by A/WSN over the period that control groups became severely ill and died. Protected mice appeared completely normal showing no sign of disease or weight loss. This demonstrated that adaptive immune responses, in which SCID mice are totally deficient, are not needed for 244 DI virus-mediated protection. The main correlates of protection from clinical disease and weight loss in mice inoculated with active DI virus + A/WSN compared with control receiving inactivated DI virus + A/WSN are (a) reduction in the amount of infectious virus in the lungs of mice on day 2 (83-fold), day 4 (27-fold) and day 6 (10-fold), (b) reduction in genomic RNAs 1 and 7 in the lung on day 4, (c) larger amounts of 244 DI RNA in the lung on days 2 and 4, and (d) absence of lung consolidation. It appears therefore that the key events necessary to maintain animal wellbeing occur early in infection, with the main protective action of DI virus taking place at 2 and 4 days after infection or earlier. Protection correlated with high amounts of lung DI RNA and low amounts of lung infectivity. Despite the relatively high virus load in the lungs of protected mice, they appeared to be clinically normal at this time, gaining weight, and exhibiting no lung consolidation.6 ffu in the lung, with consolidation commencing 1–2 days later. In contrast, mice treated with DI virus attained similar levels of infectivity and significant consolidation on day 8, but significant weight loss and clinical signs were not apparent for another 3 days. However, once initiated the course of disease in the acute and late onset disease groups was indistinguishable. We have not seen any relapse in many hundreds of wild-type mice, with no known immune defect, protected with 244 DI virus from various influenza A viruses, and this includes observing most mice for 7 weeks and some for 6 months after infection (authors’ unpublished data). Lung consolidation in SCID mice infected with an influenza A virus is described as plum coloured areas on the lung surface (as we found), which microscopically presents as a proliferative pneumonia, comprising a massive multifocal to coalescing proliferative bronchitis, bronchiolitis, and alveolitis, marked proliferation of type II pneumocytes, and hyperplastic and hypertrophic columnar epithelium lining the airways A summary of the main features of the delayed onset disease in SCID mice given the lower dose (1.2 μg) of active 244 DI virus + A/WSN and the acute disease in SCID mice given the same amount of inactivated 244 DI virus + A/WSN is shown in + and CD4+ CTL responses in the lungs of H-2k mice infected with A/WSN or A/WSN + inactivated DI virus. These mice all died. CTL responses were diminished in mice inoculated with A/WSN + DI virus and these all survived + and CD4+ cells with specific antibody was needed to abolish lung consolidation and for mice infected with A/WSN or A/WSN + inactivated DI virus to survive + and CD4+ cells were inoculated with A/WSN + DI virus, lung infectivity was cleared, presumably with the assistance of local, T cell-independent, virus-specific antibody. These mice produced a haemagglutinin (HA)-specific antibody that was highly unusual as it was not neutralizing but, when adoptively transferred, protected naïve animals from A/WSN Previous work using wild-type mice, A/WSN challenge virus, and non-cloned DI WSN virus showed that there were MHC-restricted virus-specific CD8Analysis of RNA taken at day 16 from the lungs of sick SCID mice that had received active 244 DI virus + A/WSN showed that the sequence, and thus the properties, of the 244 RNA had not changed. Infectious A/WSN isolated from the same group of mice was also unchanged in sensitivity to interference by 244 DI virus in subsequent tests in immune competent mice in vivo. This lack of acquired resistance to DI virus contrasts with the appearance of oseltamivir-resistant mutants in influenza virus-infected SCID mice From days 4 to 8 there were small increases in the amounts of infectious virus, genomic RNAs and 244 DI RNA, with all showing a modest peak on day 8, and this build up appears to presage overt late onset disease. The interactive dynamics of infectious virus, genomic RNAs and 244 DI RNA during the initial acute disease/protection phase are difficult to reconcile with the conventional dogma that protection is mediated by the DI RNA competing for replication with cognate full-length segment 1, and thus reducing the amount of infectious virus produced. In fact, we see that on days 2, 4 and 6 after infection, infectivity is lower in the active DI group  than in the inactivated DI group as expected, but on day 2 both groups had the same level of segment 1. Segment 1 was reduced in the DI group only on day 4 (by 12-fold). In addition to this quantitative disparity, there was no preferential reduction in the cognate segment 1, as segment 7 was reduced in parallel (on day 4 by 5-fold). An intriguing feature of this work was the constant ratio of viral segment 1 RNA: 244 RNA, a segment 1 DI RNA. We saw no evidence of competition for replication between the DI and its cognate full-length RNA segment in the lung. However, we do not know if these data are affected by any asynchronicity of infection of cells by infectious and DI virus, or by heterogeneity of cells in the lung. There is no doubt that DI RNA is being replicated as the amount of DI RNA in lungs of mice inoculated only with DI virus declined by over 100-fold during the experiment. Data show that in the lung segment 1 RNA levels increase faster than lung 244 DI RNA levels and this may explain why there is disease breakthrough. The lowest recorded ratio of segment 1: DI RNA (1.3-fold) occurred on day 2 post infection, with the maximum ratio on day 12 (32-fold). Again there is no preferential difference as the maximum ratio of segment 7: 244 RNA was also on day 12.Several mechanisms have been proposed for the mode of action of 244 DI virus in vivo including interference with the production of homologous virus via competition between DI and full-length genomes, stimulation of adaptive immune responses, or activation of innate immune responses. The simplest explanation for the disparity between the lung infectious virus load and lung viral genomic RNA is that DI RNA is competing not at the level of RNA replication but at the level of assembly or packaging of virion RNA into new virions. The packaging of influenza virus genome RNA into virus particles is a very specific process with the virus selectively packaging one copy of each of the 8 segments