Monilinia fructicola and Monilinia laxa are the most destructive fungal species infecting stone fruit (Prunus species). High-throughput cDNA sequencing of M. laxa and M. fructicola isolates collected from stone fruit orchards revealed that 14% of isolates were infected with one or more of three mycoviruses: Sclerotinia sclerotiorum hypovirus 2 , Fusarium poae virus 1 , and Botrytis virus F . Isolate M196 of M. fructicola was co-infected with all three viruses, and this isolate was studied further. Several methods were applied to cure M196 of one or more mycoviruses. Of these treatments, hyphal tip culture either alone or in combination with antibiotic treatment generated isogenic lines free of one or more mycoviruses. When isogenic fungal lines were cultured on nutrient agar medium in vitro, the triple mycovirus-infected parent isolate M196 grew 10% faster than any of the virus-cured isogenic lines. BVF had a slight inhibitory effect on growth, and FPV1 did not influence growth. Surprisingly, after inoculation to fruits of sweet cherry, there were no significance differences in disease progression between isogenic lines, suggesting that these mycoviruses did not influence the virulence of M. fructicola on a natural host. Mycoviruses are viruses that infect fungi. They have been identified from all major fungal phyla, namely the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota ,2. SinceCryphonectria parasitica, the causal agent of chestnut blight [Sclerotinia sclerotiorum [Rosellinia necatrix [The influence that mycoviruses have on the ecology of their hosts is not well studied. Some mycoviruses reduce the ability of the fungal host to cause disease in plants. These are known as hypovirulent mycoviruses, and they have potential as biological control agents. The most well-known are Cryphonectria hypoviruses 1 and 2 , which significantly decreased the virulence of the fungus t blight ,7. Otherrotiorum ,9 and whnecatrix . In contnecatrix ,12,13. Onecatrix ,14,15,16Monilinia fructicola and M. laxa were first recorded in Western Australian stone fruit production regions in 1997 [Monilinia species. Tsai, et al. [M. fructicola isolates infecting nectarine and peach orchards in New Zealand. Although not characterized genetically, the authors described virus-like particles resembling those of partitiviruses, totiviruses, tobraviruses and furoviruses. They identified no differences in host growth rates between isolates with and without virus infection. In this current study, we identified three mycoviruses infecting a collection of M. fructicola and M. laxa isolates, and undertook to determine if these viruses influenced growth rates of infected fungal cultures in vitro, and the virulence of the pathogen on a natural host. in 1997 ,18, and , et al.  identifiM. laxa and ten M. fructicola isolates were collected from symptomatic flowers, twig cankers and fruits from stone fruit orchards in Western Australia  aminomethane (Tris) pH8.0, 0.5 mM Ethylenediaminetetraacetic acid (EDTA) pH8.0, 1% Sodium dodecyl sulfate (SDS), 1% Polyvinylpolypyrrolidone (PVPP) and 450 µL phenol-chloroform (50:50) saturated with Tris-EDTA buffer (pH 8.0). The mixture was homogenized before being centrifuged at room temperature for 2 min. After that, 400 µl of the aqueous phase was transferred to a new tube containing 400 µl of phenol-chloroform, then mixed and centrifuged for 2 min. Then, 300 µL of the aqueous phase containing nucleic acids was removed to a new tube containing 58 µL of absolute ethanol to which 200 mg of cellulose powder  was added and mixed. After centrifuging at high speed for 1 min, the material was separated into two phases, the DNA-containing supernatant and the pellet containing RNA. The supernatant (250 µL) was transferred to a fresh tube and 20 µL 3M NaOAC pH5.2 and 625 µL absolute ethanol was added to precipitate DNA. The pellet containing RNA was washed three times with 750 µL application buffer . The RNA pellet was eluted in 450 µL elution buffer . The RNA solution (450 µL) was removed and precipitated after incubation at −20 °C in a new tube containing 1 mL absolute ethanol and 45 µL 3M NaOAC pH5.2 for RNA collection.M. fructicola isolate M196 by high-throughput sequencing.DNA was used to identify the fungal species based on the comparison of internal-transcribed spacer (ITS) region sequences of ribosomal RNA genes, as previously described . RNA wascDNA synthesis was carried out in 20 µL volume containing the following components: 4 µL 5× GoScript™ Buffer; 2 µL 0.1 mM DDT; 1 µL 10 mM deoxynucleotides; 1 µL GoScript™ reverse transcriptase ; 1 µL 10 mM Tris EDTA; 8 µL water; 1 µL RNA, and 1 µL random primer adaptor . The mixture was incubated at 25 °C for 10 min; 42 °C for 60 min; and 72 °C for 15 min. cDNA was amplified by PCR using primer 5′-CGTACAGTTAGCAGGC-3′, which annealed to the complement of the adaptor sequence added to cDNA molecules, in 20 µL volume of 10 µL 2× GoTaq® Green Master Mix ; 4 µL cDNA products; 5 µL water; 1 µL barcode primer. The reaction was carried out with an initial cycle of 5 min at 94 °C; 40 cycles of 10 min at 94 °C, 20 s at 45°C and 30 s at 72 °C, an extension cycle of 5 min at 72 °C, and a final cycle of 5 min at 37 °C. The library was purified, quantified, and paired-end sequenced on an Illumina MiSeq platform at the Australian Genome Research Facility.De novo assembly was carried out on the trimmed reads to form contigs >300 nt in length. Assembly parameters were word size of 40 and bubble size of 50. Contigs were compared to sequences lodged in GenBank  databases (https://blast.ncbi.nlm.nih.gov) using Blastn and Blastx [The sequences obtained were analyzed as previously described . Brieflyd Blastx  to identd Blastx . AnnotatSpecific primers  were desFour methods were tested alone or in combination to eliminate viruses from fungal isolates:(1). Cold treatment. Fungal mycelium was stored in 30% glycerol at −80 °C for two years before being recovered on V8 liquid medium.(2). Temperature shock. Fungal mycelium was stored in 30% glycerol at −80 °C for two years was heated to 30 °C for 30 s, incubated in liquid nitrogen for 45 s, then heated again to 30 °C for 45 s. Mycelium was then recovered on V8 liquid medium.(3). Hyphal tipping. Hyphal tips were harvested from the edges of rapidly-growing colonies, and sub-cultured to a fresh water agar plate.(4). Antibiotic treatment. Antibiotics were added singly to water agar when the autoclaved media had cooled to approximately 50 °C. Antibiotic concentrations used were 12.5 mg/L cycloheximide, 100 mg/L kanamycin, and 250 mg/L streptomycin. Hyphal tips were inoculated to plates and cultures incubated in the dark at 22 °C for six days before hyphal tips were harvested from it and the process repeated. Each line was treated this way five times before it was tested for the presence of all three mycoviruses by RT-PCR using species-specific primers. Species-specific primers were designed from high-throughput sequences .Virus-free lines were maintained in culture for up to six months before their virus-free status was reconfirmed by RT-PCR with species-specific primers. These virus-free lines were used for subsequent growth rate and virulence experiments.Prunus avium) of cultivar Bing. Inoculated fruits were incubated in 100% humidity in the dark at room temperature (17–20 °C) for seven days. Each fungal isolate was inoculated to 36 cherry fruits; each fruit was treated as a replication. At the end of the incubation period, the widest extent of the fungal lesion was measured using a compass and ruler. The entire experiment was repeated twice.Isolate M196 (triple virus-infected) and isogenic M196 lines free of at least one virus   were groFour virus-free and virus-infected isogenic lines  were grown on V8 agar plates at 22 °C for six days. Each isolate was inoculated into 36 V8 plates (three replications × 12 plates per replication). After six days, the maximum diameter of each colony was measured and differences in morphology recorded. The entire experiment was repeated twice.To determine the differences between treatments, a one-way analysis of variance (ANOVA) was done at a significance level of 0.05 using SPSS version 24.de novo assembly resulted in 37,146 contigs within the size ranges of 300 to 5385 nucleotides. Blastn and Blastx analysis of the contigs revealed 20 that shared nucleotide or amino acid identities with viral sequences. Where two or more contigs mapped to the same virus, gaps in the sequence were filled by RT-PCR so that the sequences of complete or almost complete genomes were determined  sequences were used to assign reads to samples. After screening for quality and trimming the barcode and primer sequences, termined . Three pola M196 . RT-PCR ola M196  with appM. fructicola isolate M196. The nucleotide and deduced amino acid sequences shared the greatest identities with the replicase gene of three previously described isolates of Sclerotinia sclerotiorum hypovirus 2 (SsHV2)  classified as a ssRNA virus. Alignment of the new sequence revealed that it shared 85–89% pairwise nucleotide and 93–95% amino acid identities with SsHV2 isolates 5427 from New Zealand (KF525367) [Sclerotinia sclerotiorum hypovirus 2. It is designated Sclerotinia sclerotiorum hypovirus 2 isolate Monilinia-TNS. SsHV2 was identified only in Monilinia M196.A contiguous virus-like sequence of 13,535 nucleotides was obtained from F525367) , isolateF525367) . AlthougThe sequence of SsHV2-Monilinia-TNS is estimated to represent 93% of the complete genome. The SsHV2-Monilinia-TNS genome comprises one large ORF, which is incomplete at the 5′end. The ORF extended from nucleotide 1–13,205 where it was terminated by an opal stop codon (UGA). The ORF is followed by a 3′ untranslated region (UTR) of 333 nucleotides, present from nucleotides 13,206 to 13,535. The conserved RdRp core motifs V and VI (S/TG x3 T x3 NS/T x22 GDD) (where x is any amino acid residue)  was presM. fructicola M196. The sequence shared the highest sequence identity with published isolates of Fusarium poae virus 1 (FpV1) , a double-stranded RNA virus. One segment (RNA1) encodes the replicase (RdRp) and the other (RNA2) encodes the coat protein. Comparison of the 2100 nucleotide sequence of RNA1 revealed that it shared 90% pairwise identity with RNA1 of two FpV1 isolates: A11 from Slovakia (AF047013) and 240374 from Japan (LC150606). The deduced amino acid sequence of the RdRp segment shared 92% identity with the RdRps of these two isolates. The ORF of RNA1 extended from nucleotide 54-2084 where it was terminated by an opal stop codon, encoding an RdRp-like protein of 689 amino acid residues with an estimated molecular weight of 79.9 kDa. The conserved core RdRp motifs V and VI [Two contigs representing the complete genomic segments of a bipartite virus were identified from V and VI  were preThe RNA2 segment, encoding the coat protein (CP) gene, was 2093 nucleotides in length. The single ORF encoding a protein deduced to be 654 amino acid residues in length, with an estimated mass of 72.6 kDa. Surprisingly, a partial RdRp-like motif (T x2 DS x27 GDD) was present with the CP ORF. The deduced amino acid sequence of ORF1 of RNA2 shared greatest identity (85–86%) with CPs of the two other FpV1 isolates, and the nucleotide identity was 83–84% with them. The 5′ UTR extended from 1 to 102 nt, and the 3′ UTR from 2017 to 2093 nt.Fusarium poae virus 1. The isolate from M196 was designated Fusarium poae virus 1 isolate Monilinia-TNS. FpV1-Monilinia-TNS RNA1 was assigned GenBank accession MH665658, and FpV1-Monilinia-TNS RNA2 assigned GenBank accession MH665659.The levels of identity of this new virus with isolates of FpV1 were above the species demarcation of partitiviruses recommended by the ICTV  of <40% M. fructicola M196. The deduced amino acid sequences of fragments each shared 90–95% identity to those of an isolate of Botrytis virus F (BVF) (accession NP068550) , a ssRNA virus. Together, the three fragments were estimated to represent about 30% of the BVF complete genome, designated Botrytis virus F isolate Monilinia-TNS. One of the fragments held the core RdRp motifs V and VI as SG x3 T x3 NT x21 GDD. The three contigs of BVF-Monilinia-TNS were assigned GenBank accessions MH665660, MH665661, and MH665662.Contigs representing partial genomic sequences of a virus were detected from M. laxa isolates and nine M. fructicola isolates were screened using SsHV2, FpV1 and BVF-specific primers , M84 (Fusarium poae virus 1 and Botrytis virus F), and M140 (Fusarium poae virus 1) (M. fructicola isolates tested held these viruses.Eighteen  primers . Three Mvirus 1) , but nonM. fructicola M196 of one or more of the three mycoviruses. It is unclear whether any of the antibiotics played a significant role in eliminating the viruses because tipping alone without antibiotics generated isogenic line M196-1 that was free of all three mycoviruses. Treatment with 12.5 mL/L cycloheximide eliminated SsHV2 from two isogenic lines, one of which, M196-4, was used in subsequent experiments. Treatment with kanamycin (100 mg/L), or with 250 mg/L streptomycin, eliminated both SsHV2 and BVF, but not the partitivirus FpV1  than lines lacking one or more viruses. After six days in culture, the mean diameter of colonies of isolate M196 was 73.8 mm, while virus-cured lines M196-1, M196-4, and M196-6 were less: 66.4, 62.9, and 66.0 mm, respectively (Triple-infected parent isolate M196 grew significantly faster (ectively .Where SsHV2 was absent but FpV1 and BVF were present (M196-4), colony growth was suppressed, suggesting that SsHV2 enhanced growth. There was no significant difference (P > 0.05) in growth between virus-free line M196-1 and FpV1-infected M196-6, indicating that FpV1 had no influence on growth. Where FpV1 and BVF co-occurred (M196-4), mycelial growth was suppressed compared to virus-free line M196-1. Although FpV1 alone had no apparent effect on growth, in combination with BVF it suppressed growth. This result could be interpreted as BVF alone suppressing growth and FpV1 remaining latent or as a synergistic suppressive influence of both viruses .p > 0.05) in the diameters of the resulting lesions. Although mean lesion size was lowest for M196 (8.7 mm), and virus-cured lines grew faster  (p > 0.05)  , they we > 0.05) . Mock inM. fructicola and M. laxa isolates tested were not widely infected with mycoviruses. Of the 18 M. laxa isolates and 10 M. fructicola isolates tested, only three M. laxa and one M. fructicola isolate were infected with one or more of three mycoviruses. This overall infection rate of 14% is far below that reported in the only other study of mycoviruses from Monilinia [M. fructicola isolates tested were infected with at least one mycovirus. This report was based on RNA profiles and visualization of virus-like particles, not by sequence analysis [The onilinia . That stanalysis . VisualiThe genome sequences of SsHV2 and FpV1 obtained using this approach were complete or almost complete, while that of BVF was partial. The incomplete genome may be a function of the low titer of BVF relative to the other two viruses, but it is possibly a function of differential RNA extraction efficiencies of the extraction procedure (cellulose-based) used.Monilinia fructicola isolate M196 was infected with three mycoviruses, all of which were originally identified from other host genera, and none had previously been identified from Australia. Their presence in Australia probably reflects anthropogenic international translocations of mycovirus-infected M. fructicola isolates infecting Prunus fruit and germplasm, although the other known hosts of these mycoviruses, Sclerotinia sclerotiorum, Fusarium poae and Botrytis cinerea, all occur in Australia and may carry these viruses. Notably, M. fructicola and other known fungal hosts of the three viruses identified all have international distribution, they are all serious plant pathogens, and they are all members of the family Sclerotiniaceae [iniaceae .S. sclerotiorum in China, New Zealand and the USA [Monilinia and not from Australia. Two groups have shown SsHV2 induces hypovirulence in its host. A study with isogenic lines of S. sclerotiorum infected with SsHV2 and an endornavirus showed that the presence of these mycoviruses reduced mycelial pigmentation and sclerotia formation in vitro. SsHV2 induced hypovirulence of S. sclerotiorum on lettuce and soybean, delaying production of sclerotia and reducing their numbers, but its influences on mycelial growth rate or virulence were not recorded [S. sclerotiorum on canola (Brassica napus) in China. In contrast, our studies with SsHV2 infecting a M. fructicola line (co-infected with with FpV-1 and BVF) did not indicate hypovirulence. Our studies suggest SsHV2 enhanced Monilinia mycelial growth in vitro, it did not visibly change pigmentation or conidia production, and it did not appear to influence virulence on cherry. Unfortunately, we were unable to generate a line containing only SsHV2 to confirm these findings.Until now, SsHV2 has been identified in  the USA ,25, but recorded  reportedFusarium poae, and its first report from Australia [F. poae [M. fructicola, FpV1 does not seem to induce abnormal morphology or changes to virulence, and this is a common observance for partitiviruses generally [Aspergillus fumigatus [Sclerotinia sclerotiorum after damaging cell organelles [This is the first report of the betapartitvirus FpV1 being identified in a host beyond ustralia . In F. p[F. poae  and hereumigatus , and Sclganelles .Botrytis virus F is the sole species in genus Mycoflexivirus, family Gammaflexiviridae, a group closely aligned to other fungus-infecting viruses in the Deltaflexiviridae and to plant-infecting viruses in the Alphaflexiviridae and Betaflexiviridae [Botrytis cinerea, on strawberries in New Zealand [M. fructicola growth in vitro. That BVF may negatively impact growth of M. fructicola is of potential interest, given that both B. cinerea and M. fructicola are important plant pathogens, together affecting over 200 plant species [iviridae . BVF was Zealand . It was  Zealand . Our res species ,36. The M. fructicola is not a trivial exercise, involving ‘curing’ parent fungal isolates of viruses to obtain isogenic lines [Aspergillus niger cultures infected with a number of virus-like particles on cycloheximide failed to eliminate any of the viruses [Investigation of the influence of mycoviruses on the growth of ic lines . Culturiic lines ; however viruses ,40. It wM. fructicola growth in vitro, but not affect virulence on fruit. In another study, growth of a chryso-like-virus-infected strain of Alternaria alternate in vitro was severely restricted, while virulence against fruit was enhanced [M. fructicola could metabolize nutrients more efficiently on the plate than could the virus-free isogenic line, but the mechanism for this is unknown.A limitation of our experiment is that conclusions of hypervirulence are based on linear colony growth measurements, not on measurements of total biomass, metabolism, or fecundity. It is unclear how the triple-infection by viruses might stimulate enhanced . The agaenhanced ,43,44. W


Bunyavirales, Flaviviridae, Reoviridae, Togaviridae). Further, ecological associations between bats and hematophagous viral vectors are characterized . Lastly, knowledge gaps related to hematophagous ectoparasites (bat bugs and bed bugs (Cimicidae) and bat flies (Nycteribiidae and Streblidae)), in addition to future directions for characterization of bat-vector-virus relationships are described.Bats are known to harbor and transmit many emerging and re-emerging viruses, many of which are extremely pathogenic in humans but do not cause overt pathology in their bat reservoir hosts: henipaviruses (Nipah and Hendra), filoviruses (Ebola and Marburg), and coronaviruses (SARS-CoV and MERS-CoV). Direct transmission cycles are often implicated in these outbreaks, with virus shed in bat feces, urine, and saliva. An additional mode of virus transmission between bats and humans requiring further exploration is the spread of disease via arthropod vectors. Despite the shared ecological niches that bats fill with many hematophagous arthropods  known to play a role in the transmission of medically important arboviruses, knowledge surrounding the potential for bats to act as reservoirs for arboviruses is limited. To this end, a comprehensive literature review was undertaken examining the current understanding and potential for bats to act as reservoirs for viruses transmitted by blood-feeding arthropods. Serosurveillance and viral isolation from either free-ranging or captive bats are described in relation to four arboviral groups ( Bats and the viruses they harbor have been of interest to the scientific community due to the unique association with some high consequence human pathogens in the absence of overt pathology. Virologic and serologic reports in the literature demonstrate the exposure of bats worldwide to arboviruses (arthropod-borne viruses) of medical and veterinary importance . HoweverRousettus aegyptiacus) as the reservoir for Marburg virus . Mu. Mu29].  sampled . Mosquiterfuscus . It is uinfected . Kading st virus . Therefost virus . Antibodst virus .Nycteribiidae and Streblidae), bat bugs and bed bugs (family Cimicidae), and ticks (families Argasidae and Ixodidae) [A number of hematophagous arthropods feed on bats, including bat flies (genera xodidae) ,171,172.xodidae) ,174,175.Cx. quinquefasciatus and Cx. tritaenhiorhynchus, the only blood-fed mosquitoes collected from the cave were Cx. quinquefasciatus, at least 20 of which had fed on Leschnault’s rousette (Rousettus leschenaulti) bats [Culiseta morsitans Theobald mosquitoes  were found to have fed on Eastern pipistrelle bats (Pipistrellus subflavus), but these blood meals comprised only 1% of the total blood meals identified from this mosquito species [Aedes (Stegomyia) species, but it is unclear how much investigation has been done in this area. Sixteen of 20 field-collected Ae. funereus mosquitoes (vector of RRV) had fed on Pteropus alecto bats [Coquillettidia (Cq.) fuscopennata  , Culex (Cx.) perfuscus Edwards , Cx. (Cx.) neavei Theobald , and Cx. (Cx.) decens group  are recognized as primary vectors of WNV, Sindbis, Babanki, and Usutu viruses, only for Babanki virus has additional field data been collected so far that support a potential role for bats in virus circulation (discussed above) [Kading and Schountz (2016) reviewed instances in the literature where mosquito blood meals have been identified as originating from bats . Informati) bats . Culiset species . No infocto bats . In Afri viruses ,178. Whid above) .Nycteribiidae, Streblidae) are highly host-specific obligate hematophagus ectoparasites of bats [Eucampsipoda africana) associated with Egyptian rousette bats [Strebla wiedemanni, Trichobius parasiticus) associated with common vampire bats (Desmodus rotundus) [Bartonella spp. bacteria have also been found infecting both fruit bats and the bat flies parasitizing them in Madagascar, providing a documented example of a pathogen-vector-bat association involving bat flies [Bat flies  . Bartoneat flies . More reCimicidae are found in close association with bat populations [Oeciacus vicarious) transmit Fort Morgan virus and Buggy Creek virus [Cimex insuetus and Strcticimex parvus) host the orthobunyavirus KKV which likely causes disease in humans [In addition to parasitizing humans by infesting their dwellings, bed bugs and other arthropods in family ulations . Some ciek virus ,184,185 ek virus . As prevn humans ,22, but Ornithodoros tadaridae) obtained from a palm tree colonized by Cuban bats [R. aegyptiacus) and soft-bodied ticks in caves, the argasid tick Ornithodoros faini was screened for Marburgvirus RNA but all pools were found to be negative [Macronyssidae, Dermanyssidae, and Spinturnicidae, though the significance of these arthropod’s role in virus transmission between bats and other vertebrates is largely unknown [Macronyssidae and Dermanyssidae have also been demonstrated to efficiently transmit arboviruses .Hypsignathus monstrosus and identified five viruses: one dicistrovirus , one nodavirus (family Nodaviridae), and two tombus-like viruses. They also detected a related tombus-like virus isolated from fig wasps and primitive crane flies co-habitating with bats, suggesting the ability of arthropods to host “bat-associated” viruses [A recent study explored viruses of potential arthropod-origin in the fruit bat  viruses . Tombus  viruses ,194,195. viruses ,197, andWith regard to experimental infections, one must take into account the method of inoculation , the inoculating titer, as well as whether or not vector bites were associated with the infection. In analyzing serologic results, one must take into account whether the assay is detecting neutralizing or non-neutralizing antibodies, and consider the dynamics of antibody decay which may impact detection sensitivity . ArthropImportantly, the intent of this review is not to vilify bats, but to analyze the existing literature and its support for bats as arboviral reservoirs, in addition to identifying areas for future study. Bats provide vital ecosystem services, such as arthropod suppression, pollination, and seed dispersal . Past deTo truly elucidate the role of bats as reservoirs for arboviruses, field surveillance studies documenting natural infection and transmission dynamics among vector and vertebrate species must be supplemented with experimental infections to characterize viremia profiles and infectiousness to vectors, virus-induced pathology, and immune kinetics following infection. With bats, these tasks are not trivial, and carry significant challenges in both the field and the lab. These challenges are evidenced by the few arboviruses for which there are substantial field and laboratory data involving the infection of bats. While many studies have presented serologic data indicative of past exposure of free-ranging bats to arboviruses of medical and veterinary importance, these studies should be followed up with laboratory assessments of reservoir host competence to shed light on the true epidemiological significance of the field data. Further, the detection of viral nucleic acid in free-ranging bats does not necessarily implicate the species as an arboviral reservoir. Rather, recovery and isolation of live virus at biologically relevant titers, and demonstrating the persistence of the pathogen in nature among connected populations of a potential reservoir species is more definitive. Unfortunately, few established bat colonies exist for use in vivo viral pathogenesis studies, limiting the achievability of these studies. Pteropus poliocephalus to recipient mosquitoes in addition to identification of RRV-seropositive bats trapped in Australia and Indonesia highlights the need for further investigation into the role of bats in the ecology of this disease [To fulfill the vertebrate reservoir host paradigm, in vivo infections must support findings in the field. The isolation of Marburg virus from Egyptian rousette bats in Uganda in addition to experimental infections demonstrating viremia and shedding in the absence of overt pathology support the role of this bat species as the reservoir for Marburg virus ,7,208. F disease ,149,157. disease . Serolog disease , but exp disease ,29. Addi


Streptococcus pneumoniae) colonization density has been associated with invasive pneumococcal disease, but factors that increase pneumococcal density are poorly understood. We evaluated pneumococcal densities in nasopharyngeal samples from asymptomatic young children from Peru and their association with subsequent acute respiratory illness (ARI). Total pneumococcal densities  during asymptomatic periods were significantly higher when a respiratory virus was detected versus when no virus was detected (p<0.001). In adjusted analyses, increased pneumococcal density was significantly associated with the risk for a subsequent ARI (p<0.001), whereas asymptomatic viral detection alone was associated with lower risk for subsequent ARI. These findings suggest that interactions between viruses and pneumococci in the nasopharynx during asymptomatic periods might have a role in onset of subsequent ARI. The mechanisms for these interactions, along with other potentially associated host and environmental factors, and their role in ARI pathogenesis and pneumococcal transmission require further elucidation.Increased nasopharyngeal pneumococcal ( Streptococcus pneumoniae (pneumococcus) is one of the most important bacterial causes of pneumonia among children and adults worldwide  study is a prospective cohort study conducted in the Province of San Marcos, Department of Cajamarca, located in the northern highlands of Peru. Malaria is not endemic in this high-altitude study community . The population is primarily rural with low income and limited access to healthcare services, as previously described  was introduced in the study region in late 2009 in a 2+1 schedule (2 primary doses at 3 and 5 months of age and a booster dose at 12 months). Very few children enrolled in 2009 had received any PCV7 doses at the time of enrollment. By the end of the study (September 2011), 62% of the study cohort had received >8 days apart from an ARI episode, to undergo real-time reverse transcription PCR (rRT-PCR) viral testing at Vanderbilt University for influenza virus , respiratory syncytial virus (RSV), human metapneumovirus (MPV), rhinovirus (HRV), adenovirus (AdV), and parainfluenza virus (PIV) types 1–3  by bacterial culture for pneumococcal identification and quantitative PCR (qPCR) for pneumococcal density determinations (10(x + 1) transformation to colonization density values, where x represents the measured density. We performed unadjusted group comparisons by using the Wilcoxon rank-sum test, Kruskal-Wallis test, or median test with Bonferroni adjustments, as appropriate. We used multivariable linear quantile mixed effects regression to model the median of log-transformed pneumococcal density against viral detection group, with random effects to account for correlation among multiple measurements collected from the same child. Other covariates included age at the time of sample collection, sex, recent exposure to antibiotics , pneumococcal vaccination status (receipt of >1 dose of PCV7), and calendar month. Because viral infections could lead to rhinorrhea, we did not directly include this variable in our main regression model, and rhinorrhea was considered as a potential variable in the causal pathway between viral infections and pneumococcal density changes. To examine the association of pneumococcal densities during asymptomatic periods with the risk for subsequent ARI, we conducted a survival analysis by using a frailty model and accounted for repeated measurements from individual children. Because severe ARI occurred uncommonly in this household surveillance study, we did not stratify the outcome variable (ARI) by severity (>8 days after the last day of ARI symptoms) through the earliest of the following: ARI, the collection of another nasopharyngeal sample, or 60 days after collection of the initial asymptomatic nasopharyngeal sample. These censoring criteria were implemented on the basis of our monthly nasopharyngeal collection strategy and the anticipated transient duration of colonization with an individual serotype in young children, typically 6–8 weeks or less (–https://www.stata.com) and R version 3.5.0 (https://www.r-project.org), including lqmm and survival packages.We transformed pneumococcal density values to stabilize their variance, as previously described (<3%), as previously reported  than in those in which a respiratory virus was not detected (286/492 [58%]) (p<0.001). Rhinorrhea was reported in 219/357 (61%) children in whom a virus was detected, compared with 227/492 (46%) in whom a virus was not detected (p<0.001). At least 1 respiratory virus was detected in 219/446 (49%) children with rhinorrhea, compared to 138/403 (34%) without rhinorrhea. Antibiotic exposure preceding collection of each asymptomatic nasopharyngeal sample was uncommon, occurring in only 12/849 (1%) samples (antibiotic exposure was unknown in 9/849 samples [1%]).Co-detection of >1 respiratory virus occurred in 48/849 (6%) samples. HRV was present in 40/48 (83%) co-detections, most commonly in combination with AdV, with or without an additional virus (36/40 [90%]). Colonization with pneumococcus was more common in samples in which 10-transformed pneumococcal densities were higher during asymptomatic periods when >1 respiratory virus was detected (median 4.95 [interquartile range (IQR) 3.11–6.35]) than when no respiratory viruses were detected . When densities were examined according to detection of specific viruses, densities differed significantly by viral group (p<0.001); the highest densities were observed in the HRV-only group. In Bonferroni-adjusted pairwise median comparisons, pneumococcal densities were significantly higher when HRV only  or viral co-detections  were present compared with samples that were virus-negative (3.35 [IQR 0–4.95]). In Bonferroni-adjusted median comparisons, pneumococcal density from samples with AdV-only infection (median 4.39 [IQR 3.15–6.08]) was not significantly different from that from samples with no respiratory viruses detected (p = 0.087).In unadjusted comparisons, log>1) virus was significantly associated with higher log10-transformed pneumococcal densities  was associated with lower incidence of subsequent ARI. Similar findings were observed when rhinorrhea was included in the model instead of viral detection . These findings suggest that asymptomatic viral infection during asymptomatic periods might present colonizing pneumococci with an opportunity to expand.Our findings demonstrate that viral detections during asymptomatic periods are associated with increases in nasopharyngeal pneumococcal colonization density, and further, that higher pneumococcal density during asymptomatic periods is associated with subsequent onset of an ARI. These findings expand upon earlier observations from our group and others that increases in nasopharyngeal pneumococcal colonization density are associated with symptomatic respiratory illness and pneumonia and with the detection of respiratory viruses during ARI periods . The study was conducted in a rural region in which the incidence of pneumonia is higher than national rates (In summary, we found that viral detections during asymptomatic periods are associated with increases in nasopharyngeal pneumococcal colonization density. Furthermore, we found that pneumococcal density, especially at high levels, is associated with subsequent development of ARI in young children in Peru. These findings suggest that interactions between viruses and pneumococci in the nasopharynx during asymptomatic periods might have a role in onset of subsequent ARI.Additional information about nasopharyngeal pneumococcal density during asymptomatic respiratory virus infection and risk for subsequent acute respiratory illness.


Nicotiana tabacum; family Solanaceae) is a minor crop, which is produced only for local consumption. In 2015, tobacco plants exhibiting severe downward leaf curling, leaf thickening, vein swelling, yellowing and stunting were identified in fields of tobacco in Suhar Al-Batina region, Oman. These symptoms are suggestive of begomovirus  infection.In Oman tobacco . Viral genomes were cloned from RCA products by restriction digestion and betasatellites were cloned by PCR amplification from RCA product, using universal primers. The sequences of full-length clones were obtained by Sanger sequencing and primer walking. Constructs for the infectivity of virus and betasatellite were produced and introduced into plants by Chilli leaf curl virus (ChiLCV) and the betasatellite to be a variant of Tomato leaf curl betasatellite (ToLCB). Both the virus and the betasatellite isolated from tobacco show the greatest levels of sequence identity to isolates of ChiLCV and ToLCB identified in other hosts in Oman. Additionally clones of ChiLCV and ToLCB were shown, by Agrobacterium-mediated inoculation, to be infectious to 3 Nicotiana species, including N. tabacum. In N. benthamiana the betasatellite was shown to change the upward leaf rolling symptoms to a severe downward leaf curl, as is typical for many monopartite begomoviruses with betasatellites.The full-length sequences of 3 begomovirus and 3 betasatellite clones, isolated from 3 plants, were obtained. Analysis of the full-length sequences determined showed the virus to be a variant of The leaf curl disease of tobacco in Oman was shown to be caused by ChiLCV and ToLCB. This is the first identification of ChiLCV with ToLCB infecting tobacco. The study shows that, despite the low diversity of begomoviruses and betasatellites in Oman, the extant viruses/betasatellites are able to fill the niches that present themselves. Begomovirus (family Geminiviridae) cause economically important diseases of many crops throughout tropical and subtropical regions. Begomoviruses have circular single-stranded (ss) DNA genomes, that are encapsidated in distinctive twinned icosahedral (geminate) particles and are transmitted exclusively by the whitefly Bemisia tabaci [Viruses of the genus a tabaci . The gena tabaci . The gena tabaci .Most monopartite begomoviruses are associated with a group of ssDNA satellites collectively known as betasatellites , 4. BetaTomato leaf curl Sudan virus and African cassava mosaic Zanzibar virus [Chilli leaf curl virus (ChiLCV) and Tomato leaf curl betasatellite (ToLCB [Tomato yellow leaf curl virus (TYLCV) and Watermelon chlorotic stunt virus [The first disease caused by a begomovirus in Oman was identified in 1993, although the causative viruses and satellites were not characterized until much later . Despitear virus , 8, begont virus , 11.Agrobacterium-mediated inoculation of the cloned virus and betasatellite were used to satisfy Koch’s postulates. The significance of the findings is discussed.The study described here has analysed the etiology of a leaf curl disease of tobacco recently identified for the first time in Oman. The results show that the disease is associated with ChiLCV and ToLCB. Additionally Total nucleic acid was extracted from leaf samples using a cetyltrimethylammonium bromide-based method  and keptBamHI resulted in ~ 2.7 kb fragments, which were eluted from agarose gels using a GeneJet Gel Extraction Kit (Thermo Fisher Scientific) and cloned in BamHI restricted pGEM-3zf (+) . Potentially full-length clones resulting from RCA (for begomovirus) and PCR (for betasatellite) were sequenced commercially using the primer-walking approach .Circular DNA molecules in nucleic acid samples were enriched using rolling circle amplification (RCA) as described earlier . Restrichttp://blast.ncbi.nlm.nih.gov/Blast.cgi). Open reading frames (ORFs) in sequences were identified using the ORF Finder program run on-line (https://www.ncbi.nlm.nih.gov/orffinder/). The Species Demarcation Tool (SDT), with the MUSCLE option, was used to calculate sequence identity values [Sequences were assembled using SeqMan, part of the Lasergene package of sequence analysis software . Related sequences available in the GenBank database were identified using the Basic Local Alignment Search Tool nucleotide (BLASTn)  run on-ly values . Pairwisy values . The evoy values .HindIII and SalI to release a fragment of ~ 1000 bp which was ligated into the binary vector pGreen0029 [HindIII, was ligated into the pGreen0029 clone containing the fragment, linearized using HindIII, to yield a partial direct repeat of the virus genome. A similar strategy was used to produce a construct for clone Tob44 in pGreen0029 using a ~ 425 bp KpnI-XbaI fragment and the full-length insert released using KpnI.Clone Tob11was digested with reen0029 . Then thAgrobacterium tumefaciens strain LBA4404. Agrobacterium inocula were prepared and inoculated to Nicotiana benthamiana, N. glutinosa and N. tabacum seedlings by agroinfiltration as described previously [Binary vector constructs were electroporated into eviously . Infiltr+ membrane (Amersham) and UV cross-linked. Probes were produced by PCR using a DIG-high prime kit (Roche Applied Science) and primers βC1F (5′-AGACCCGGGATGACGATCAGATATAATAACA-3′)/ βC1R (ACGTCGACTCACACACACACTTTCGTACA-3′) for betasatellite and (qPCRChV F (5′-AGTTCATATAGGGAAGGTTATGTGTA-3′) / qPCRChV R (5′- GGACAAGGAAGAACATCACACTATTC-3′) for virus. Membranes were hybridized and washed as described previously [Total genomic DNA (~ 10 μg) extracted from plant tissue was resolved on 1.5% agarose gels, transferred overnight by capillary action onto Hybond Neviously , 23 and Nicotiana tabacum) were observed with plants showing severe downward leaf curling, leaf thickening, vein swelling, mild yellowing and stunting , fields of tobacco , were collected, as well as a non-symptomatic plant from each field. A high molecular weight product was obtained by RCA with DNA extracted from all four symptomatic plants but not from the non-symptomatic plants (results not shown). Three potentially full-length clones , resulting from digestion of RCA product with BamHI, were obtained, one from each of three N. tabacum plants  to an isolate of ToLCB recently identified in watermelon in Oman . OveraNicotiana benthamiana, N. glutinosa and N. tabacum plants, either in the presence of absence of a construct for the associated ToLCB  and the symptoms showed a pronounced downward leaf curling. Similarly for N. tabacum inclusion of ToLCB in the inoculum led to a decrease in latent period (from 21 to 18 dpi) and an increase in symptom severity, with slightly more severe downward leaf curling, although this was less pronounced than for either N. benthamiana or N. glutinosa. However, for both singly inoculated plants and co-inoculated plants, the infectivity was significantly less to N. tabacum  than to either of the other Nicotiana species.Inoculation of N. benthamiana and N. glutinosa  and has been shown previously for infection of N. benthamiana with ChiLCV and the betasatellite Tomato leaf curl Bangladesh betasatellite (ToLCBDB) and another Oman isolate of ChiLCV with ToLCB [N. benthamiana, the results of Khan et al. [The symptoms induced by atellite . The chath ToLCB , 30. Altth ToLCB  showed Tn et al.  mirroredN. tabacum with cloned ChiLCV and ToLCB resemble those seen in field infected plants, quantitatively they do not; the symptoms in field-infected plants being more severe. There are a number of possible reasons for this. It is possible that the clones obtained from tobacco are not very pathogenic . Alternatively the conditions in the glasshouses where the experiments were conducted, required to keep whiteflies away from the experimental plants, may not be ideal for either tobacco or virus propagation. It is interesting to note that, in both experimentally inoculated plants and field collected plants there is a smear of fast migrating, small molecular weight material which hybridizes to the virus and betasatellite probes. This likely represents defective viral/betasatellite DNA replication products which may suggest that species of Nicotiana are not good hosts for ChiLCV and ToLCB. This idea is supported by the relatively low virus DNA levels which are present in N. tabacum. Nevertheless, the results show that the leaf curl disease of tobacco in Oman is caused by ChiLCV with ToLCB.Although qualitatively the symptoms induced by inoculation of The study here is the first identification of ChiLCV infecting tobacco, as well as the first identification of ToLCB in tobacco. This virus and betasatellite do not cause disease of tobacco in their regions of geographic origin. This finding is in-line with earlier studies showing that, despite the low diversity of begomoviruses and betasatellites in Oman in comparison to the sub-continent, the extant viruses/betasatellites are able to fill the niches that present themselves. Careful monitoring will be required to assess the threat of local viruses and satellites as agriculture in Oman expands and seeks to introduce new crops and crop varieties.Additional file 1:Table S1. Features of Chilli leaf curl virus and associated Tomato leaf curl betasatellite clones isolated from field infected tobacco.


The adsorption of Cd2+ by a S. cerevisiae–alginate system was tested either by batch or fixed-bed column experiments. The S. cerevisiae–alginate system was characterized using dynamic light scattering , size, hardness, scanning electron microscopy (SEM), and Fourier-transform infrared spectroscopy. Beads of the S. cerevisiae–alginate system showed a spherical–elliptical morphology, diameter of 1.62 ± 0.02 mm, 96% moisture, negative surface charge (−29.3 ± 2.57 mV), and texture stability during storage at 4 °C for 20 days. In batch conditions, the system adsorbed 4.3 µg of Cd2+/g of yeast–alginate beads, using a Cd2+ initial concentration of 5 mg/L. Adsorption capacity increased to 15.4 µg/g in a fixed-bed column system, removing 83% of total Cd2+. In conclusion, the yeast–alginate system is an efficient option for the removal of cadmium at low concentrations in drinking water.The aim of this study was to determine the Cd Long-term exposure to low concentrations of Cd2+ may imply chronic intoxication strongly associated with irreversible damage in the liver, kidneys, lungs, bones, and reproductive system [Cadmium . Also, these treatments can consume large quantities of chemical products or high amounts of energy [Also, freshwater bodies polluted with low concentrations of Cdf energy ,9. Thereration 1–0 mg/L. AThe use of any metal biosorbent is an environmentally friendly option. Moreover, metal biosorbents are cheaper than other conventional methods, and they are more versatile and diverse in terms of their molecular composition due to the variety of sources from which they can be obtained . BiosorpS. cerevisiae is widely used in food biotechnology processes, like bread, wine, and beer production. It is a GRAS organism. For this reason, efficient and low-cost processes were developed to produce large-scale yeast biomass. In addition, S. cerevisiae biomass can also be obtained as a high-volume brewing by-product. S. cerevisiae is a promising biosorbent that shows the capacity to remove heavy metals from aqueous solutions, such as lead, cadmium, chromium, copper, nickel, and zinc, even when inactive, and it shows good efficiencies in short contact times [The yeast ct times ,18,19.2+) and other divalent cations induce the covalent crosslinking of alginate; therefore, gel formation under simple and mild conditions makes it possible to immobilize eukaryotic cells [Saccharomyces cerevisiae could be a feasible option. This strategy would provide an eco-friendly Cd2+ removal system, mainly from water with low metal ion concentration, which may not be feasible using conventional methods. The aim of this study was to determine the Cd2+ removal capacity of a biosorbent system formed by S. cerevisiae immobilized in a calcium alginate gel matrix for its potential application in drinking water. The yeast–alginate system was also characterized using different techniques.However, after biosorption treatment, the cell–liquid separation may be a problem, adding a unit operation to the process. Therefore, the immobilization of yeast cells in a fixed matrix is an alternative for their application ,21. Algiic cells . In addiic cells . Therefoic cells . SaccharLow-viscosity sodium alginate (130–300 mPa) was purchased from BÜCHI . Cadmium standards were acquired from Sigma-Aldrich . All other chemicals were analytical grade from Sigma-Aldrich , unless otherwise specified.Saccharomyces cerevisiae  was obtained from three local markets from Hermosillo, Mexico. Yeast biomass was inactivated by heating in an autoclave for 30 min at 121 °C, then frozen at −40 °C and lyophilized for 48 h . The lyophilized cells were stored in a freezer at −40 °C until encapsulation.Dried commercial 2+ solution for atomic absorption of 1000 mg/L . Two working solutions were prepared by dilution with ultrapure water at concentrations of 5 and 10 mg/L.A stock solution of 100 mg/L was obtained from a standard CdS. cerevisiae’s encapsulation was carried out using an external ionic gelation technique with a semi-automatic Encapsulator B-395 equipped with a 750-µm nozzle . The vibration frequency was adjusted to 350 Hz to generate 350 droplets per second. Yeast biomass (1.5 g/L) was dispersed in a 1.5% sodium alginate solution. The mixture was dispensed into an inlet reservoir of an airflow-assisted nozzle system and applied dropwise  into a 0.4 M CaCl2 crosslinking solution under gentle stirring (70 rpm) with a magnetic bar. Synthetized beads were maintained in the crosslinking solution for 30 min to complete the hardening process, recovered with a micro-sieve, and washed three times with deionized water to remove CaCl2 excess. Produced beads (eight batches) were routinely stored at 4 °C for further experiments. Empty Ca-alginate beads (without yeast) were used as a control.S. cerevisiae–alginate system was characterized and analyzed based on particle size distribution, optical microscopy, microstructural analysis, surface charge, and moisture content.The obtained S. cerevisiae alginate beads were subjected to size analysis using an electronic digital caliper , with a diameter measurement of 150 beads. In addition, size distribution analyses were performed with an inverted microscope Zeiss Axio Vert A1 . The diameters of 20 arbitrarily chosen beads were measured using Zen 2 Lite software . Measurements for control and treatments were performed in duplicate.Wet S. cerevisiae was characterized with a Stemi DV4 stereomicroscope . Beads were placed in a petri dish containing deionized water and gently shaken for better separation. Optical images were obtained using a digital camera .Morphology of wet alginate beads loaded with S. cerevisiae–alginate system beads were obtained before and after incubation (15 min) in a water solution containing 5 mg/L Cd2+. Particle size of lyophilized alginate beads was determined by SEM microscopy.Microstructure of lyophilized alginate beads was characterized by scanning electron microscopy (SEM) using a JEOL-JSM-5900 LV microscope , operating at 10 kV electron acceleration and 100 mm of focal length. Samples were coated with a thin gold layer (60 A) prior to observation. Images of empty Ca-alginate control and The surface charge was obtained by triplicate measurements of zeta potential  using deionized water as a diluent. The moisture content of capsules was determined using the AOAC 4.1.06 method  in triplThe mechanical resistance of beads was determined in order to observe the performance of beads firmness during storage (for possible preservation). Firmness measurements were carried out at days zero, four, eight, 12, 16, and 20 of storage in refrigeration (5 °C) in deionized water. Calcium alginate beads without yeast were used as control. A texturometer  was used with a load cell of 5 kg and a semi-cylindrical acrylic probe of 18 mm of diameter and 39 mm of height. Beads were placed on a flat stainless-steel surface underneath the probe, removing water excess with a soft tissue. A compression of 40% was applied to each bead with a test speed of 0.1 mm/s. A force (gf) versus time (s) plot was obtained and the max force value (gf) was recorded as bead firmness. At least 15 measurements were performed for each treatment.2+ working solutions at pH 6.0 were individually mixed with 12 g of calcium alginate beads loaded with S. cerevisiae in 50-mL conical tubes. These conditions were chosen because they proved to be the most effective for the biosorption of cadmium by yeast [3 for the analysis of non-adsorbed residual Cd2+ by atomic absorption spectroscopy (AAS) . Controls included free S. cerevisiae cells, Cd2+ solutions without biosorbent, and empty Ca-alginate beads. Free cells of S. cerevisiae were separated from supernatant by centrifugation at 4500 rpm for 10 min at 15 °C for non-adsorbed residual Cd2+ determination.For batch biosorption experiments, 20 mL of 5 mg/L or 10 mg/L Cdby yeast . After 5t is the adsorption capacity at time t (µg/g), Ci is the initial concentration of Cd2+ (µg/mL), Ct is the Cd2+ concentration at time t (µg/mL), V is the volume of the solution (mL), and m is the mass of the adsorbent (g).Cadmium removal efficiency and adsorption capacity were calculated using the following equations:S. cerevisiae. The temperature of the column was maintained at 25 °C throughout all experiments. Fifteen milliliters of Cd2+ aqueous solution  was pumped through the column  in an up-flow mode with a peristaltic pump . After resident time, samples were collected, acidified, and analyzed for residual Cd2+ as described before. For desorption experiments, 50 mL of 25% HNO3 was applied  to the packed beads [2+ desorbed was determined by AAS from the filtered eluates. Following elution, beads were washed twice with 50 mL of deionized water.For fixed-bed column biosorption, a glass column  was packed with 12 g of calcium alginate beads loaded with ed beads . The amoS. cerevisiae–alginate system were recorded before and after Cd2+ adsorption. Empty Ca-alginate beads and free S. cerevisiae were used as controls. Samples were prepared as discs using potassium bromide (KBr). FTIR spectra were obtained in a NICOLET Protégé 460  spectrometer with WinFIRST version 2.10 software . The spectrum was recorded in the range of 4000 to 400 cm−1. Spectra were normalized at the 1400 cm−1 region using Origin Pro 9 software .FTIR spectra of the p ≤ 0.05). Minitab 17  statistical software and SigmaPlot 11  were used for data analysis and graphic representations, respectively.Results were expressed as means ± standard error. Values were treated by analysis of variance (ANOVA) and Tukey–Kramer test to determine significant differences between means  larger (1.62 ± 0.02 mm). These results are in agreement with earlier reports for microorganism encapsulation generated by semi-automated vibration technology [Empty Ca-alginate beads showed almost a spherical–elliptical morphology with a continuous smooth surface and a diameter of 1.51 ± 0.02 mm . Morpholchnology ,30.p > 0.05); however, a reduction in the size of lyophilized capsules loaded with yeast was observed, compared to the freshly synthesized ones (p ≤ 0.05). This was attributed to a lower polymer concentration inside the bead than the surface, related to cell incorporation, which led to a greater shrinkage after water loss by freeze-drying [SEM images of the lyophilized Ca-alginate beads, as well as their shapes and surfaces, are shown in e-drying .2+ produced a bead size reduction which was significant (p ≤ 0.05) in the empty ones  for beads loaded with S. cerevisiae (−26.9 to −32.0 mV) than for empty beads (−12.2 to −19.7 mV). The negative charge of empty Ca-alginate beads is mainly attributed to the carboxyl groups of β-d-mannuronic acid units of the polymeric alginate structure due to most of the α-l-guluronic acid units being crosslinked with calcium ions forming the hydrogel structure [S. cerevisiae is attributed to negatively charged functional groups, like phosphates and carboxylic groups, from cell-wall proteins and polysaccharides [The zeta potential of all alginate beads was negative  being grtructure . On the charides .2+ and, thus, its removal from aqueous solutions [S. cerevisiae. These results indicate that the Cd2+ cations formed a secondary shell around the anionic calcium alginate beads.A negative biosorbent system surface charge favors the electrostatic interaction with Cdolutions ,43. Cadmp > 0.05) between empty Ca-alginate beads and S. cerevisiae-loaded alginate beads. The general behavior in both beads was an apparent gradual decrease in alginate gel strength over storage time. This loss of strength was higher after eight days of storage in both types of beads , without significant changes in the subsequent days (p > 0.05). The observed diminution may be attributed to an ion exchange process between calcium and some residual cations in solution, like sodium and magnesium, and also to a slow dissociation of calcium to aqueous media [The effect of storage time on the gel force of alginate beads was studied over 20 days . Textureus media ,45,46.S. cerevisiae binds heavy-metal cations on its outer surface due to the presence of negatively charged phosphomannan, phosphate, and carboxyl groups [S. cerevisiae was less effective in removing cadmium in solution at both concentrations (5 mg/L and 10 mg/L) than its free form .The Cdng sites . At a co2+ removal efficiency of empty Ca-alginate beads exposed to aqueous solutions containing 5 mg/L Cd2+ was higher (63.5% ± 0.7%) than the removal efficiency of the yeast–alginate system (51.1% ± 1.8%). However, the removal efficiency of empty Ca-alginate beads decreased gradually when the contact time increased (2+ showed a similar (p > 0.05) removal efficiency to that of the S. cerevisiae–alginate system. However, the removal efficiency of empty Ca-alginate beads decreased to 46.2% ± 0.2% when they reached equilibrium at 60 min.At a contact time of 15 min, the Cdncreased , and the2+ concentration were 4.3 and 9.2 µg of Cd2+/g of capsules (at 15 min of aqueous solution contact time), respectively  adsorbed by the yeast–alginate system was 185.2 µg and corresponded to a removal efficiency of 82.3% ± 2.3%. This percentage was higher than the removal efficiency of encapsulated S. cerevisiae in the batch study (51.9% ± 0.7%) using the same concentration and contact time, and even higher than the Cd2+ removal efficiency of free S. cerevisiae (75.0% ± 0.1%). A possible explanation for the increase in Cd2+ efficiency when changing from batch to continuous conditions is the increase in the driving force of Cd2+ due to the constant flux of solution [Fixed-bed column experiments were performed to investigate the potential application of the yeast–alginate system in a continuous process. During the column testing, a flow rate of 1.5 mL and Cdsolution .2+/g of loaded yeast–alginate system beads, i.e., an increase of almost three-fold, with respect to batch experiments.The total adsorption capacity in fixed-bed columns was 15.4 µg of Cd3 allowed the recovery of 17.8% of total cadmium biosorbed by the S. cerevisiae–alginate system. In addition, a visible loss in the integrity of the capsules was observed after the contact with the eluent. The Cd2+ removal efficiency after HNO3 treatment was low compared with that reported by other authors (when using similar conditions to elute other toxic elements). For example, Ferraz et al. reported a Cr3+ recovery of 80% from a S. cerevisiae biosorbent system, while Chen and Wang observed a similar desorption efficiency for U6+ eluted from a yeast–alginate biosorbent system [3 was more effective for desorbing Cd2+ than other acids or bases [2+ from different biosorbents decreased significantly after a few cycles of regeneration due to the damage caused in the adsorbent [Cadmium elution with 25% HNOt system ,50. Stiror bases . Howeverdsorbent . Thus, iSaccharomyces cerevisiae . The bands at 1636.3 and 1542.1 cm−1 were attributed to amide I and amide II, respectively. Bands in the region from 1032.3 to 827.0 cm−1 were associated with carbohydrates, nucleic acids, and phosphate groups [The spectrum of free revisiae a showed e groups .−1 arose from the stretching of –OH groups. Very-low-intensity bands at 2919.3 cm−1 were attributed to stretching vibrations of aliphatic C–H. Observed bands between 1603.8 and 1415.8 cm−1 were attributed to asymmetric and symmetric stretching vibrations of COO−, respectively. The bands at 1100.0 and 935.0 cm−1 were attributed to the glycoside bonds in the alginate molecule (C–O–C stretching) [Saccharomyces cerevisiae.The spectrum of empty Ca-alginate beads showed all the characteristic peaks due to the alginate b. A broaetching) . The FTI2+ biosorption. 2+ adsorption. The decrease in peak vibration intensity in the region between 3000 and 3600 cm−1 indicated that Cd2+ interacts with –OH formed in alginate beads. Hydroxyl groups were also designated as binding sites for Cu2+ and Zn2+ on the surface of alginate beads [FTIR analysis was used to characterize changes of functional groups before and after Cdte beads .−1 in the Cd2+-loaded yeast–alginate system spectrum indicated that free –OH and –NH groups of immobilized S. cerevisiae probably bound with the metal.Changes in the intensity of 3367.3 to 3359.9 cm−1 could be assigned to the participation of aliphatic C–H, C=O asymmetric and symmetric stretching, phosphate groups, and C–O stretching from carbohydrates of yeast biomass groups in the biosorption process. Changes in the wavenumber intensity in the Cd2+-loaded yeast–alginate system (1598.1 cm−1) in comparison with the raw yeast–alginate system (1598.3 cm−1) emphasized the role of C=O stretching of S. cerevisiae carboxylates groups in metal uptake [In addition, alteration in wavenumbers at 2162.0, 1590.2, 1435.9, 1033.0, and 818.0 cml uptake .2+ biosorption properties of a yeast–alginate system were studied. A small gradual decrease in alginate gel strength over storage time was observed and attributed to an ion exchange process between calcium and some residual cations in solution, and also to a slow dissociation of calcium to aqueous media. However, the mechanical resistance of the yeast–alginate system was practically stable for 20 days. The FTIR attenuated total reflection (ATR) analysis of the yeast–alginate system before and after Cd2+ exposure showed that –OH formed in alginate beads and aliphatic C–H, C=O, phosphate groups, and C–O from carbohydrates of the yeast biomass groups participated in the biosorption process. The cadmium (Cd2+) removal efficiency and capacity using the S. cerevisiae–alginate system were higher in the fixed-bed column mode. This system allowed the removal of 83% of total Cd2+. Therefore, the S. cerevisiae–alginate system operated in the fixed-bed column mode has the potential to be used as an eco-friendly continuous water filter for Cd2+ biosorption.In this study, the Cd


Lyssavirus, and haematophagous bats are the principal reservoir; however, the virus has also been detected in non-haematophagous bats. The objective was to determine the rabies virus in non-haematophagous bats in the Colombian Caribbean region.Bats are an important ecological group within ecosystems. The rabies virus is a In 2017, a cross-sectional study was carried out with a base-risk sampling in twelve geographic zones of the Colombian Caribbean area that included the main ecosystems of two departments. 286 bats were captured, which were euthanized with a pharmacological treatment following the ethical protocols of animal experimentation. The taxonomic identification was done with dichotomous keys. The necropsy was carried out at the capture site, and brain samples were kept in liquid nitrogen. The extraction of the RNA was carried out from the frozen brains with Trizol™; a fragment of 914 bp of the glycoprotein G of the rabies virus was amplified with RT-PCR. The amplicons were sequenced with the Sanger method.Artibeus lituratus and Artibeus planirostris, amplicons were obtained and sequenced as the rabies virus.Twenty-three genera of bats were identified, and, in two frugivorous, This is the first evidence of natural infection of the rabies virus in frugivorous bats in the Colombian Caribbean area; this result is important for the surveillance and control of rabies. Lyssavirus and produces fatal acute encephalitis in humans [Rabies is a zoonotic disease that affects humans through saliva, bites or scratches . The natn humans . Rabies n humans ; once thn humans . Global n humans . In deven humans .Lyssavirus do not appear to cause disease in bats, suggesting co-evolution between the viruses and their hosts [Bats are an important ecological group in nature because of their ability to control insects, disperse seeds  and pollir hosts , 11; higir hosts  and repeir hosts . Bats cair hosts .Desmodus rotundus, Diaemus youngi and Diphylla ecaudata are reservoirs of the rabies virus and are distributed from the Tropic of Cancer to the Tropic of Capricorn [D. rotundus [The haematophagous bats apricorn . These srotundus .Tadarida brasiliensis [There are two cases reported of human rabies apparently transmitted by non-hematophagous bats. The first was reported in the USA in 1953  and the iliensis . In the Carollia perspicillata [molossus and Eptesicus brasilensis [On the other hand, according to the Colombian Agriculture Institute (ICA), there have been 184 cases of encephalitis ; the Naticillata . The secsilensis . In CordIn 2017, in two departments of the Colombian Caribbean region, a cross-sectional study was carried out with a base-risk sampling, for which 286 bats were captured. Based on the fact that, during 2014 and 2016 in the departments of Cordoba and Sucre, there were 146 cases of bovine rabies, according to the Colombian Agriculture Institute , 12 sampThe bats were captured using five mist nets for 4 h of sampling, which corresponded to 240 h/network. The bats were identified with dichotomous taxonomic keys based on morphometric parameters . The batThe RNA extraction was performed with Trizol™ (Invitrogen) from 286 samples of brain tissues; the aliquots were resuspended in 150 μL of nuclease-free water. The concentration of the RNA obtained with the NanoDrop 2000 equipment (Spectrophotometers™) was then quantified. The cDNA synthesis was done with the reverse transcriptase enzyme M-MLV (Invitrogen™) using random primers (Invitrogen™), following the recommendations of the manufacturers. Subsequently, a conventional PCR was carried out that amplified a fragment of the gen G with the primers Ga3222-4) (5′CGCTGCATTTTRTCARAGT3′) and (Gb4119-39) (5′GGAGGGCACCATTATTGGTMTC3′), which amplify a fragment of 914 bp [222-4 , where two sequences of 793 bp were obtained. These sequences were aligned with Clustal W, found in Mega7. For the phylogenetic reconstructions, 86 reference sequences available in the GenBank were used for typing by groups. The sequence of the vaccine strain was also included. The best evolution model was applied to explain the nucleotide diversity observed between the aligned sequences, and the phylogenetic reconstructions were done using Neighbor-Joining (NJ), Maximum Likelihood (ML) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA) using the software MEGA7.A. lituratus and A. planirostris, captured in Coloso, Department of Sucre . Four D. rotundus specimens were captured in this study, two in San Carlos, Cordoba  and two in Coloso (Sucre), both were negative for the rabies virus with the RT-PCR. The sequences detected in the frugivorous bats were recorded in the GenBank under the numbers MH763616 and MH763617. These two sequences were grouped within the clade D. rotundus or rabies virus of sylvatic origin , a high percentage of seropositivity (52.46%) was detected in an areas . Studiesan areas . It has an areas .D. rotundus; this bat is the principal vector, with high viremia and the ability to infect other animals. D. rotundus uses different animal species to obtain the blood, or by with high grooming social interactions [On the other hand, the phylogenetic analysis showed 304 variable sites of 793 analyzed sites, and a total of 203 parsimoniously informative sites were determined. The phylogenetic analyzes showed ten clades with an average distance of 14.3%. The sequences MH763616 and MH763617 were grouped with 100% branch support within the sequences that are directly related to the rabies virus isolated from ractions , 34 or bractions . The topDesmodus rotundus (AgV-3), Tadarida brasiliensis (AgV-4) and Lasiurus cinereus (AgV-6) [M. molossus, E. brasiliensis), in three dogs and one human [D. rotundus (AgV3). The circulation of the genetic variants of the rabies virus in bats and other mammals is important because it establishes the virus and determines the risks to public health. The rabies virus variants are a significant public health concern; thus, the entire rabies viruses are potentially infectious for human’s beings. Previous studies in Colombia showed that rabies virus transmitted in epizootic outbreaks, the viruses were closely genetic related. Hence, reservoirs can carry any infectious of rabies variant [In non-hematophagous bats from Sao Paulo (Brazil), three antigenic variants were identified , all previously identified in Latin America –38. Thes (AgV-6) . In Colone human . The seq variant , 41. The variant .The control of wild-origin zoonoses is limited by the insufficient knowledge on the biology of pathogens in the hosts, which coincides with pulses of viral excretion within bat populations produce densities changes and therefore contact rates . VirusesA. lituratus and A. planirostris, captured in the Colombian Caribbean region. It is necessary to expand to other areas of the Colombian Caribbean and compare with other regions like the Pacific area, to observe better the biology and behavior of these animals and their involvement in the transmission of rabies.This study presents the first molecular evidence of the natural infection of the rabies virus in frugivorous bats,


Invertebrate organisms include vectors of human viruses , model organisms (fruit fly), insect pollinators (honey bees and bumble bees), plant virus vectors (aphids), and commercially valuable aquatic species (oysters and shrimp) that play important roles in shaping ecosystems throughout the world. Like all organisms, invertebrates are infected by viruses and have, in turn, evolved strategies to limit virus infection. There are some fundamental similarities in host defense mechanisms, including the host recognition of non-self, pathogen-associated molecular patterns  that in turn stimulate the activation of host proteins, and expression of genes required to restrict virus replication, as well as unique aspects of specific host–virus interactions that are a result of co-evolution. Invertebrate antiviral defense mechanisms include canonical immune signaling cascades , heat shock responses, apoptosis, and dsRNA-triggered responses including the sequence-specific RNA interference mechanism and a less well characterized, non-sequence-specific dsRNA mediated response.Viruses features articles written by prominent researchers in the field of invertebrate immunology. The goal of this compilation is to better understand the similarities and differences in antiviral responses mounted by invertebrates, while recognizing that some of what is learned is unique to specific virus–host pairs. Interesting parallels can be drawn between invertebrate and vertebrate antiviral responses and, in some cases, homologous genes that play similar roles in a wide range of organisms have been identified. Together the articles in this issue serve as a foundation and inspiration for future studies aimed at further elucidating the molecular mechanisms of invertebrate antiviral defense.This special issue of Crassostrea giga), fruit flies (Drosophila melanogaster), mosquito species , honey bees (Apis mellifera), and bumblebees (Bombus spp.), as well as describe the influence of endosymbionts on virus replication in fruit flies and mosquitos at the cellular level [vago, in response to dsRNA is important in fruit flies and mosquitos, the predominant dsRNA-triggered antiviral mechanism is RNA interference (RNAi), which is a sequence-specific post-transcriptional silencing mechanism [Review articles led by Green and Speck, van Rij, Smagghe, Flenniken, Newton and Hardy, summarize the current understanding of antiviral responses in Pacific oysters , as compared to other innate immune pathways, virus-specific antiviral and pro-viral host-encoded genes, and the heat shock response [Drosophila miRNAs in antiviral defense against arboviruses [The most well-studied invertebrate antiviral response are those of Dipteran insects, specifically, us pairs . This syus pairs . Furtherus pairs . Sweversresponse . Whereasoviruses .Wolbachia, a maternally transmitted endosymbiotic bacterium, in mosquitoes, which would in turn lessen the number of mosquito to human transmission events [Wolbachia strains wMel and wAlbB limit mosquito reproduction and inhibit virus replication in mosquitoes  [Spodoptera frugiperda (Sf) 21 cells [Original research articles provide a glimpse into cutting-edge research in invertebrate immunology. Specifically, Adelman and Myles expand our understanding of the repertoire and functions, including proviral and antiviral roles, of C-type lectin domain containing proteins (CTLDcps) in s family ; Wang, Gsquitoes ; Traub-Cipalpis) ; van Oer21 cells ; and Mar21 cells .Together the articles in this issue serve as a strong foundation and inspiration for future studies aimed at elucidating the molecular mechanism of invertebrate antiviral defense.


Different types of viruses have different envelope proteins, and may have their shared or distinctive host-virus interactions which result in various post-infection effects in humans and animals. These effects often do not appear at once but take time to unfold. To characterize the virus-specific effects, we applied a Multivariate Polynomial Time-dependent Genetic Association (MPTGA) method, previously proposed for detecting differences in temporal gene expression traits, to test for the differences in mouse lung transcriptome response to infection of different subtypes of influenza A viruses.We compared two methods: the Multivariate Polynomial Time-dependent Genetic Association (MPTGA) method, and the conventional modified t-test, to study the virus-specific effects on mouse lung gene expression. Both methods found H3N2 to be the most different virus among the three viruses tested, with the largest number of genes with H3N2-specific effects. However, the MPTGA method demonstrated much higher power of detection, and the detected genes with virus-specific effects showed better biological relevance.Transcriptome response to virus infection is dynamic. MPTGA which leverages temporal gene expression traits showed increased power in detecting biologically relevant virus-specific effects comparing with conventional t-test method. Influenza viruses have various types and subtypes that differ in their morbidity, virulence and pathogenicity. Influenza A virus is the most virulent type and can be further divided into several subtypes . These sPrevious studies  have shoIn this paper, we investigated temporal mouse lung gene expression traits in response to infection with influenza A/California/04/09 (H1N1), A/Wyoming/03/03 (H3N2), and A/Vietnam/1203/04 (H5N1) HALo virus (GSE98527) and aimed to identify the virus-specific effects. To compare temporal gene expression traits, we proposed a multivariate polynomial temporal genetic association (MPTGA) method  previousTo characterize the cellular transcriptome response of mouse lung to infection of H1N1, H3N2, and H5N1 influenza virus, six to eight week-old female C57BL/6 mice were infected with influenza A/California/04/09 (H1N1), A/Wyoming/03/03 (H3N2), and A/Vietnam/ 1203/04 (H5N1) HALo virus in 3 experimental groups respectively. The Influenza A/Vietnam/ 1203/04 (H5N1) HALo mutant virus is an attenuated H5N1 virus generated from wild-type Influenza A/Vietnam/1203/04 (H5N1) virus m1×,I1= [1⋯m], U=  and V= .The detailed derivation is as follows. Define β.0’s gives the following linear system: αij=n0Q, bi=Q, β.0’s can be estimated by Taking derivative of β.’s can be derived accordingly.Similarly, define Next, taking derivative with respect to β’s and ρ, the log-likelihood can now be written as With the previously derived estimators of H1 with the null model H0 to test for virus-specific effects.Based on the MLE of the unknown parameters, a LRT v, we define j=0,1 as the indicator variable of a sample infected by virus v or other viruses. The null hypothesis H0 assumes that the temporal gene expression trait yj share the same trajectory pattern after infected by virus v and other viruses: H1, assuming that the temporal post-infection gene expression trait yj with virus v has a unique trajectory pattern distinct from the post-infection trajectories with other viruses: The MPTGA method can be applied to a broader setting that compares the temporal gene expression traits between two different conditions, whereas the original setting proposed by Lin et al.  is a speSimilarly, MPTGA can be applied to cases with three or more groups as outlined above.


Qiu et al. made a security analysis about the protocols of Chaudhry et al. and Kumari et al. in 2018, and they pointed out that there are many security weaknesses in the protocols. To improve the security, Qiu et al. proposed an advanced authentication scheme for Session Initiation Protocol on the basis of the previous protocols and claimed that their own protocol was very secure and practical. However, we demonstrate that the protocol of Qiu et al. has a serious mistake which causes their protocol cannot be executed normally. Beyond that, we also find out that their protocol cannot withstand insider attack and denial service attack. In order to remove these weaknesses, we propose an efficient provably secure mutual authentication scheme. Furthermore, our scheme provides security analysis with the help of Burrows-Abadi-Needham (BAN) logic. Compared with their protocol, ours has greater security and better performance. With the network of universal gradually, we often send messages, make phone calls and watch videos via the network, which brings great convenience to our lives. However, the network security issues become increasingly prominent. In order to enhance the security of the Session Initiation Protocol (SIP) in communication via the network, lots of scholars have proposed numerous solutions. The first authentication scheme about SIP on the basis of Hypertext Transport Protocol (HTP) authentication was proposed in 1999  proved tIn recent years, Zhang et al.  proposedWe will focus on the rest seven sections to describe our paper, section 2 reviews Qiu et al.’s scheme. In section 3 we pay attention to analyze the weaknesses about Qiu et al.’s scheme. Section 4 describes our proposed scheme. Next section 5 and section 6 mainly provide analysis and proof of security. Section 7 gives the performance comparison among our scheme and the relative schemes of other scholars. Finally, section 8 shows the conclusion of our scheme.In this section, we will briefly review Qiu et al.’ s scheme , which cBefore we show each phase, the notations which throughout this paper are introduced in U and server S do the following operations to finish registering.During the registration phase, user U⇒S:{Id,HId}:U registers to server S with his/her identity Id, password Pw and secret key Up. After that, U completes VPw = h(Pw‖Up), HId = h(Id‖VPw) and transmits {Id, HId} to sever S.A user Id and HId, S will compute N =HId⊕h(Sp) and store N into database.After receiving U completed the registration phase successfully and wants to access request to the server S, U and S should perform the following steps.If a user U→S:{B,X,T}:U accesses server S with his or her identity Id, password Pw and secret key Up. After that U calculates VPw = h(Pw‖Up), generates a long random number r and computes HId = h(Id‖VPw), W = h(Id‖Up), X = r⋅P, Y = r⋅Qs, B = W⊕h(HId‖Y) and T = h(B‖Y‖W). After completion of calculation, U sends {B,X,T} to server S.The user S→U:{E,Auths}:S receives message {B,X,T}, it will calculate HId = N⊕h(Sp), Y = Sp⋅X, W = B⊕h(HId‖Y) and check whether T* = h(B‖Y‖W) is equal to T. If not valid, the session will be terminated by S, otherwise server S generates a long random number r′ and calculates E = r′⋅Qs, sks = r′⋅Y, Auths = h(sks‖W‖Y). After that, S transmits information {E,Auths} to U.When server U→S:{Authu}:E,Auths}, U calculates sku = r⋅E,Auths′= h(sku‖W‖Y). Then U checks whether Auths′ = Auths. If valid, then U computes Authu = h(sku‖W‖Y‖E) and sends result Authu to S, else terminates the session.Upon getting {Authu}, S computes Authu′= h(sks‖W‖Y‖E). If Auth′u is not equal to Authu, S terminates the session. After that, the user U communicates with server S based on the common session key sk = sku = sks = r⋅r′⋅Qs.Once receiving the message {U, if he or she wants to change own password Pw for some reasons, the following steps will be performed.For a legitimate user U→S:{V,M}:U already picks a new password Pwnew and a new secret key Upnew, after that he or she inputs identity Id, password Pw and secret key Up. U computes V = h(sk‖h(Id‖h(Pw‖Up))) and M = h(Id‖sk)⊕h(Id‖h(Pwnew‖Upnew)), then U transmits message {V,M} to S.V,M}, S computes V* = h(sk‖N⊕h(Sp)) firstly, then S judges if the value of V* is equal to V. If holds, S will replace N with Nnew by computing Nnew = h(Sp)⊕h(Id‖sk)⊕M into database. If not, S fails to change password and exits the session.After obtaining message {U have poor experience [In this section, we analyze the weaknesses of Qiu et al.’ s scheme  carefullperience .N is stored into database alone. As is known to all, there should be some information like identity Id correspond to N in the database. Or else in login and authentication phase in their scheme, when S receives message from users, S cannot match corresponding N in database without the help of the corresponding information. So, the scheme of Qiu et al. is unable to carry out normally.In registration phase of Qiu et al.’s scheme, we notice that information S only knows information Id, HId and N which relates to U. HId is the most important secret data during the entire protocol execution process, so the server cannot store HId in the database but stores Id. We assume a semi honest server S0 has the ability of gaining and calculating the sensitive information in the sever. If Id corresponds to N, we notice that an adversary S0 can obtain {Id*, Up*, Pw*} of a legitimate user U* in login and authentication phase by off-line guessing attack, the specific steps are as follows.Perhaps Qiu et al. just forgot corresponding information, here we help them supply corresponding information on the basis of the scheme of Qiu et al. In registration, Step 1: According to the login and authentication process in the scheme of Qiu et al., S0 will get the values of HId* and W* at time of calculating HId* = N*⊕h(Sp) and W* = B*⊕h(HId*⊕Y*). Besides, because Id* corresponds to N*, S0 can get the corresponding value of Id*.Step 2: After getting user U's sensitive information {Id*, HId*, W*}, S0 can guess the value of Up* from the identity space by calculating W* = h(Id*||Up*). According to the same truth, S0 can guess the value of Pw* from the identity space by calculating HId* = h(Id*||VPw*) = h(Id*||h(Pw*||Up*)). Obviously, S0 can access {Id*, Up*, Pw*}.S0 has successfully accessed information {Id*, Up*, Pw*} of a legal user U*.Through the above steps, A can obtain some sensitive information in the database of server S. In Qiu et al.’s scheme, the adversary A can achieve insider attack by registering as a legitimate user. Firstly, he masquerades as a legitimate user in registration to input identity Id*, password Pw* and secret key Up*, then S will store the corresponding value N* into database and he can get the value N* form the database of sever S. After that A has already mastered the information {Id*,Up*,Pw*,N*}. On the basis of formulas HId = h(Id‖h(Pw‖Up)) and HId = N⊕h(Sp), A can get h(Sp) = N*⊕h(Id*‖h(Pw*‖Up*)). In addition, A can get other user’s N0 in the database, so he will get the user’s corresponding HId0 by formula HId0 = N0⊕h(Sp). In login and authentication phase, A can impersonate to be the user U0 to access sever S by corresponding N0 and HId0. The specific steps are as follows.In this part, we assume that a malicious insider adversary U0→S:{B0,X0,T0}:A chooses random numbers r0, W0 and computes X0 = r0⋅P, Y0 = r0⋅QsB0 = W0⊕h(HId0‖Y0) and T0 = h(B0‖Y0‖W0). After completion of calculation, U0 sends information {B0, X0, T0} to server S.Adversary S→U0: {E,Auths}:B0, X0, T0}, S computes HId0′ = N⊕h(Sp), Y0′ = Sp⋅X0 and W0′ = B0⊕h(HId0′‖Y0′). Obviously, the verification equation T0′ = h(B0‖Y0′‖W0′) is true. Next S generates random number r′ and computes E = r′⋅Qs, sks = r′⋅Y0′, Auths = h(sks‖W0′‖Y0′). After that, S transmits {E,Auths} to U0.Once getting message {U0→S:{Authu}:E,Auths}, U0 computes sku = r0⋅E, Authu = h(sku‖W0‖Y0‖E) and sends {Authu} to S.When getting {Authu}, S computes Authu′ = h(sks‖W0′‖Y0′‖E) and gets a conclusion that Auth′u is equal to Authu. Finally, the user U0 communicates with server S based on the common session key sk = sku = sks = r0⋅r′⋅QsAfter receiving the message {A can masquerade as an arbitrary legitimate user for entering server S by insider attack.We draw a conclusion that A is able to intercept message which is transmitted between U and S. At password update phase in Qiu et al.’s scheme, U sends the result values of V and M to S. The adversary A intercepts message {V, M} and forges M0 by generating random number, then A transmits {V, M0} to S. Apparently, A will pass verification of S by checking whether V is equal to V*. After that, S computes Nnew = h(Sp)⊕h(Id‖sk)⊕M0 and replaces N with Nnew in the database. Because of the falsify of Nnew, the user U will fail to pass verification in next login and authentication phase.We assume that an adversary U needs to remember identity Id, password Pw and secret key Up. It is a burden for users to keep in mind with three different data. If a user U wants to change password, according to the password update phase of Qiu et al., he or she has to bear in mind with a new password Pwnew and a new secret key Upnew. For the most users, it is hard to remember so much information. Therefore, the scheme of Qiu et al. has poor practicability.In term of Qiu et al.’s scheme, we find that a user In this section, in order to improve the security, we design an efficient provably secure mutual authentication scheme. Compared with the scheme of Qiu et al., our proposed scheme can resist various attacks and there is less pressure for users to remember. Our scheme consists of three phases: registration phase see , login aU, if he or she wants to access the system, the necessary step is to register with the server S by submitting identity ID and password PW. User U and server S will perform the following steps.For a legal user U generates a long random number r and computes VPw = h(PW‖ID‖r) and HId = h(ID⊕r). Then U transmits {VPw,HId} to S by a secure channel.S computes N = VPw⊕h(Sp‖HId) and R = h(Sp⊕VPw). Then S stores {HId,N} into database and issues a smart card which contains R to U through a secure channel.After receiving the data, U computes VR = h(PW⊕ID)⊕r and stores VR into the smart card.After receiving the smart card, U wants to acquire the service from server S, he or she should insert his or her smart card into card reader and enter his or her identity ID and password PW. U and S will perform the following steps.When a user U generates a random number ra, and computes r′ = VR⊕h(PW⊕ID), HId′ = h(ID⊕r′), VPw′ = h(PW‖ID‖r′), C = h(R⊕VPw′)⊕ra and Authu = h(ra⊕HId′⊕VPw′). Then U transmits {HId′,C,Authu} to the server S.The user S extracts N according to corresponding HId′ from the database and calculates VPw′′ = N⊕h(Sp‖HId′), R′ = h(Sp⊕VPw′′), ra′ = h(R′⊕VPw′′)⊕C. Then checks whether the value of Authu is equal to h(ra′⊕HId′⊕VPw′′). If holds, S picks a random number rb, computes sks = h(ra′‖rb‖HId′), D = h(R′‖VPw′′)⊕rb, Auths = h(sks‖D) and transmits {D, Auths} to U. Otherwise, S terminates the session.After receiving the message, U receives the message, U computes rb′ = h(R‖VPw′)⊕D, sku = h(ra‖rb′‖HId′), checks whether Auths is equal to h(sku‖D). If success, user U and server S share the same session key sks = h(ra′‖rb‖HId′) = sku = h(ra‖rb′‖HId′). If not, the smart card aborts the session.When U needs to change password PW for a number of reasons, he or she only needs to input identity ID, password PW and new password PWnew. User U and server S will perform the following steps.If a user U calculates r′ = VR⊕h(PW⊕ID), HId′ = h(ID⊕r′), VPw′ = h(PW‖ID‖r′), VPwnew = h(PWnew‖ID‖r′) and C1 = h(R⊕VPw′)⊕VPwnew. After generating a random number ra, U continue computing C2 = h(VPw′⊕VPwnew)⊕ra, Authu = h(ra⊕HId′⊕VPWnew). After that, U transmits {C1, C2, HId′, Authu} to S.The user S computes VPw′′ = N⊕h(Sp‖HId′), R′ = h(Sp⊕VPw′′), VPw′new = C1⊕h(R′⊕VPw′′) and ra′ = C2⊕h(VPw′′⊕VPw′new). Then S checks whether the value of Authu is equal to h(ra′⊕HId′⊕VPw′new). If not, S terminates this session, else continues to calculate N′ = VPw′new⊕h(Sp‖HId′) to replace N with N' in the database. Finally, S computes Rnew = h(Sp⊕VPw′new), D = h(R′‖VPw′new‖ra′)⊕Rnew and Auths = h(VPw′new⊕Rnew). After that, message {D, Authu} will be transmitted to U.Upon receiving the message, D, Auths}, U gets R'new by computing R′new = D⊕h(R‖VPwnew‖ra) and checks whether the value of Auths is equal to h(VPwnew⊕R′new). If success, S computes VR′ = h(PWnew⊕ID)⊕r′ and replaces {VR, R} with {VR', R'new}, else terminates this session.After receiving the message {A might perform various attacks [In this part, we demonstrate our scheme is secure, practical and can provide kinds of security requirements. We assume that an adversary  attacks –21. MoreA can obtain N and HId of a legitimate user that stored in database. Because computational formulas are HId = h(ID⊕r), N = VPw⊕h(Sp‖HId), VR = h(PW⊕ID)⊕r, R = h(Sp⊕VPw) and VPw = h(PW‖ID‖r). Without the random number r, A cannot get the sensitive information ID or PW and cannot compute important secret data such as R and VPw form known data. Therefore, our scheme can resist insider attack.Assume that an adversary Id directly but transmit HId which is computed by means of the formula HId = h(ID⊕r). Even if A can access the value of HId, A still cannot get the sensitive information Id. Because the formula contains a random number r which A does not know. Therefore, our proposed scheme provides user anonymity.In the public channel, we do not send ra and rb change in every login. For an adversary A, he can intercept information {HId', C, Authu} and replay this message. Obviously, A can pass the verification of the server S and will receive the corresponding message {D, Auths} form sever S. Because A does not have a knowledge of the correct values R, VPw and ra, he cannot compute rb′ = h(R‖VPw′)⊕D and sku = h(ra‖rb′‖HId′). Therefore, our scheme is security even under replay attack.In our proposed scheme, the random numbers A accesses exchanged information {HId', C, Authu, D, Auths} which is transmitted in a public channel [HId′ = h(ID⊕r′), A is unable to guess correct ID without r′. In addition, in the formulas VPw′ = h(PW‖ID‖r′), C = h(R⊕VPw′)⊕ra, A cannot guess PW correctly without the value ID and r'. Therefore, our scheme has an advantage of resisting off-line password guessing attack.In our scheme, if an adversary  channel ,23. BecaA steals the smart card, he is able to get the VR and R which are stored in the smart card. Because the formulas are VR = r′⊕h(PW⊕ID) and C = h(R⊕h(PW‖ID‖r′))⊕ra, without the knowledge of r’ and ra, A cannot guess the correct identity ID and password PW. If A wants to communicate with S, he needs to structure legitimate {HId', C, Authu}. Because of the formulas HId′ = h(ID⊕r′), C = h(R⊕h(PW‖ID‖r′))⊕ra, Authu = h(ra⊕HId′⊕h(PW‖ID‖r′)), without identity ID and password PW, A is unable to pass through the verification of server S. Therefore, our scheme can resist smart card lost attack.If an adversary A, in authentication phase, if he wants to masquerade as a legal user U and login in server S, he must forge a valid login message {HId', C, Authu}. It is impossible for A to forge valid login message without legitimate identity ID, password PW, VR and R. The same is true, if A wants to masquerade as the server S, he has to counterfeit message {D, Auths}. Without valid information N, A is unable to obtain {D, Auths} which can pass through verification of the user U. Furthermore, in password update phase, A is unable to forge valid C1, C2 and Authu to pass the authentication of server S. In the same way, A is unable to forge valid D and Auths to pass the verification of user U. Therefore, our scheme can resist impersonation attack.For an adversary A here, he could authorize and limitless access to the target. All security protections stand up to A for a specific period of time. Because Man-At-The-End attack has concrete form in certain circumstance, one of the defense details is as follows:Man-At-The-End attack  containsA could personate a legitimate user to register and access the sensitive value of N* from the database end. His own identity ID*, password PW* are known. According to formulas h(Sp‖HId) = VPw⊕N, VPw = h(PW‖ID‖r) and HId = h(ID⊕r). Without having knowledge about the value of random number r, he will take unpractical time cost to computer Sp, by formulas h(Sp‖h(ID*⊕r)) = h(PW*‖ID*‖r)⊕N*. Our scheme offer a defense and A is unable to obtain important information Sp.A could execute other forms and is hard to analyze, many protective devices have ability to against the attack, include software protection, hardware protection and digital asset protection, more details are reference no.24. Hence, our scheme can make a defense against Man-At-The-End attack.Although ID, PW, Sp and intercept message {HId', C, Authu} and {D, Auths}. For an adversary A, he wants to get sks = sku = h(ra‖rb‖HId′). But without the knowledge of the correct values VR and R, he cannot compute ra′ = h(R′⊕VPw′′)⊕C, rb′ = h(R‖VPw′)⊕D correctly. Finally, A still have no ability to get the session key to eavesdrop session. Hence, our scheme provides perfect forward secrecy.When the system crashes in one session, an adversary can acquire information Security model: Burrows–Abadi–Needham logic  is a set of rules for defining and analyzing information exchange protocols . SpecifiIn this section, we will demonstrate that our scheme is secure and practical by the Burrows-Abadi-Needham (BAN) logic. We list some essential BAN-Logic symbols and formulas as follows, In order to make our scheme practicable, we list some goals which need to be achieved.Goal 1. Goal 2. Goal 3. Goal 4. U and server S as follows.We show the idealized message form between user Msg 1. Msg 2. We transform some premises about authentication and login phase in our scheme.U|≡#(ra).Aspt 1. S|≡#(ra).Aspt 2. Aspt 3. Aspt 4. Aspt 5. Aspt 6. Aspt 7. Aspt 8. Aspt 9. Aspt 10. Aspt 11. Aspt 12. U and server S can set up the session key by combining with the above information.We prove that user (1) The proof of goal 1 and goal 2:S 1.On the basis of Msg 2, we get that S 2.U|≡#(ra). Owing to the nonce verification and the freshness rule, which is listed as follows.According to S 1 and Aspt 1, we know that S 3.In line with S 2, we could achieve: Goal 1 is realized apparently.S 4.sk = h(ra‖rb‖HId), we make a representation is as follows:According to So, we have achieved Goal 2.(2) The proof of goal 3 and goal 4:S 5.From Msg 1, we know that S 6.S|≡#(ra), owing to the nonce verification and the freshness rule, we obtain result what is as follows:On the basis of S 5 and Aspt 2, we know S 7.sk = h(ra‖rb‖HId), we are able to get:According to S 6 and Aspt 4 Goal 4 is accomplished.S 8.sk = h(ra‖rb‖HId), we have following.According to S 6 Distinctly, Goal 3 of our claim is achieved.U and S believe that the session key sk which is shared between U and S.According to the proof above, our goals are achieved. We hold a sufficient reason to believe that both Tpm, one hash function operation is Th. Other operations like generating a random number and exclusive-OR operation spend less time, which have little effect on performance comparison. So, we neglect the lightweight operations at this time. Before performance simulation test, we analysis performance of other protocols and ours in theory. So, we list The experience of users play an important role in protocol. During this part, we will show a performance comparison between our scheme and the other schemes see . Before Th. In other three protocols, execution time mainly lies on executing 3Th, 3Th and 2Th. At authentication phase, execution time of our scheme mainly depends on executing 15Th. Meanwhile, execution time of other three protocols mainly depends on executing 12Th +6Tpm, 7Th +6Tpm, 10Th +6Tpm. At password update phase, execution time of our scheme mainly lies on executing 19Th. And the other two protocols’ execution time mainly depends on executing 10Th and 8Th. The protocol of Chaudhry et al. lacks the phase of password update. In those four protocols, total execution time are 39 Th, 25 Th +6 Tpm, 10 Th +6 Tpm, 20 Th +6 Tpm respectively.From Tpm is many times as much as Th. Compared with other protocols, our scheme has a great advantage on computational costs in usual authentication phase and total phases.In those four protocols, we find that our scheme performance mainly bases on hash function and the protocols of Qiu et al. , ChaudhrWe perform simulant performance comparison under the same computer simulation environment and write programs according to the schemes strictly. In the experiment, we run one hundred times to get the average data. According to the In this paper, we review Qiu et al.’s protocol and find that it is vulnerable to some known attacks such as insider attack and denial service attack, then we review the scheme and carry on a strict security analysis about their scheme. Next, in order to solve these problems, we propose our more secure and more convenient scheme. Security analysis shows that our scheme can resist insider attack, off-line password guess attack and more, we give a sufficient reason. Then in security proof section, we adopt the BAN-logic to prove our scheme is secure and realizable. In the end, we make a performance comparison, the result shows that our scheme can be more suitable for users in SIP. Because under the same conditions, we can establish connections faster. In conclusion, compared to other protocols, our scheme is more security and practical.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 CodeThe VS code for analyzing the performance of the Zhang’s scheme.(ZIP)Click here for additional data file.S2 CodeThe VS code for analyzing the performance of the Qiu’s scheme.(ZIP)Click here for additional data file.S3 CodeThe VS code for analyzing the performance of the Kumari’s scheme.(ZIP)Click here for additional data file.S4 CodeThe VS code for analyzing the performance of the Chaudhry’s scheme.(ZIP)Click here for additional data file.