Four hydrophilic polymers in the form of beads – chitosan, alginate, alginate/polyvinyl alcohol (PVA), and chitosan-coated alginate – were used as supports for lipase immobilisation. Hydrogel beads were characterised by bead-size-distribution estimation, surface morphology studies, and polymer interactions assessment. Matrix performances – loading efficiency, immobilisation yield, enzyme activity, and stability retention – were evaluated and compared. Although the loading efficiency of the chitosan-coated Ca-alginate beads (79.8%) was inferior to that of the Ca-alginate (87%) and of the Ca-alginate/PVA beads (81.3%), their enzyme immobilisation yield (63.96%) was the most important. Moreover, lipase encapsulated in chitosan-coated Ca-alginate beads demonstrated better pH, thermal, and storage  stabilities. Immobilised lipase activity also increased in the order: alginate/PVA > chitosan > alginate > alginate/chitosan, and displayed a maximum at pH 8 and at temperatures of 45 °C  and 50 °C . Thus, chitosan-coated Ca-alginate beads could be considered as a suitable support for lipase immobilisation. Lipases (EC 3.1.1.3 triacylglycerol acylhydrolase), due to their versatility as biocatalysts, able to act at the aqueous and organic interface, and to alter the rate of a number of processes of biotechnological interest, such as triglyceride hydrolysis, esterification, interesterification, transesterification, alcoholysis, acidolysis, and aminolysis, have found a large application in food, detergent, pharmaceutical, leather, textile, cosmetic, paper, and biofuel production industries. NowadaysEnzymatic catalysis provides several advantages over the chemical one, such as efficiency due to the enzyme specificity and selectivity, reduced energy consumption because of the mild reaction conditions, and limited release of wastes. Additional benefits offer the enzyme immobilisation, resulting from the repeated use of the biocatalyst, its enhanced stability, the localisation of the interaction, and the effective control of the reaction parameters.The use of immobilised lipases in biodiesel production, the techniques applied for enzyme immobilisation, and the factors affecting the process are extensively reviewed by Stoytcheva et al. Lipase iThe objective of this work is to compare the performances of four hydrophilic polymers in the form of hydrogel beads – chitosan, Ca-alginate, Ca-alginate/PVA, and chitosan-coated Ca-alginate – as supports for lipase immobilisation for their possible application in biodiesel production. Enzyme immobilisation was performed by adsorption and by encapsulation. Polymer beads were characterised, and polymer performances, in terms of loading efficiency and immobilisation yield, enzyme activity, and stability retention, were evaluated.All of the used reagents – sodium alginate, chitosan (low molecular weight), polyvinyl alcohol, sodium hydroxide, calcium chloride, boric acid, acetic acid, and so on – were of analytical reagent grade.Trichoderma harzianum, as described in a previous work.[Lipase (EC 3.1.1.3) was produced in solid-state fermentation by ous work.−1) and ethanol (26% (v/v)) under gentle stirring. The mixture was allowed to remain overnight. Then, the beads were removed by decantation, washed with deionised water until reaching pH 8.0, and soaked in hexane for 1 h under agitation (120 rpm). After hexane excess removal, lipase immobilisation was performed by physical absorption, according to the methodology reported in the literature [Chitosan solution (4% (w/v)) was prepared by dissolving the appropriate amount of chitosan powder in 1% acetic acid. Chitosan beads of various shapes and sizes (3 × 5 mm average) were obtained applying the “syringe method”, i.e. the viscous chitosan solution was extruded (needle diameter 1 mm) dropwise in a coagulant bath containing NaOH (1 mol Lterature  by chito2 (0.1 mol L−1) were prepared by dissolving suitable amounts of the corresponding reagent in Tris-HCl buffer (0.05 mol L−1), pH 8.0. After that, 11.2 mg of lyophilised lipase dissolved in 2 mL of the same buffer was added to 8 mL of the alginate solution and the mixture was gently stirred for 30 min. The obtained lipase containing homogeneous alginate gel was extruded dropwise through a syringe (needle diameter 1 mm) to the gelling solution of CaCl2 under stirring. The beads were allowed to polymerise for 40 min. The derivatives were washed twice with 3 mL aliquots of Tris-HCl buffer and the beads were stored at 4 °C for later studies. A similar approach was followed for the preparation of alginate beads placebo. The residual solutions were collected to determine the efficiency of the immobilisation.Lipase encapsulation in alginate beads was carried out using established protocols. First, s2 aqueous solution (1.5% (w/v)) and 65 mL of chitosan solution (2% (w/v)) in acetic acid. This procedure led to the lipase immobilisation in chitosan-coated alginate beads.[Lipase encapsulation in alginate was accomplished as described above. Nevertheless, the lipase containing alginate gel was extruded dropwise (needle diameter 1 mm) into a gelling bath obtained by mixing 35 mL of CaClte beads. The bead−1) under stirring for 30 min. The mixture was extruded (needle diameter 1 mm) as drops into a saturated boric acid solution containing CaCl2 (2% (w/v)). The beads were stirred for 45 min to complete the solidification, rinsed with double-distilled water, and finally stored wet at 4 °C. The same procedure was used for placebo preparation. The residual solutions were collected for further analysis.The immobilisation was achieved according to the PVA-alginate-boric acid method. A homogeBead characterisation implicated: counting of the number of beads obtained in each batch, bead-size-distribution estimation, surface morphology studies, and polymer interaction assessment. The studies were performed using an optical microscope VistaVision and a Fourier transform infrared spectroscopy (FTIR) spectrophotometer WQF FT-IR 520-A.The polymer loading efficiency and the enzyme immobilisation yield were the two parameters selected to evaluate the enzyme immobilisation performances of the used hydrophilic polymers.C1 and C2 are the initial and the residual protein concentrations, respectively, and V1 and V2 are the initial volume of the enzyme solution and the total volume of the residual solutions, respectively. The initial protein concentration C1 of the enzyme solutions and the concentration C2 of the protein remaining in the decanted and filtered solutions and washings were determined according to the Bradford method.[The loading efficiency was defined as the per cent of the immobilised enzyme, and was calculated using the following expression:d method. Bovine sAIm is the specific activity of the immobilised enzyme and AF is the specific activity of the free enzyme.The immobilisation yield, i.e. the specific activity ratio of immobilised lipase to free lipase, was calculated as follows:p-nitrophenyl butyrate (p-NPB) as a lipase substrate. The reaction mixture consisted of 3.9 mL of 0.05 mol L−1 Tris-HCl buffer and 0.1 mL of 0.05 mol L−1p-NPB. The hydrolysis was achieved for 25 min under stirring (120 rpm). Samples with a volume of 100 μL were withdrawn every 5 min, diluted up to 4 mL, and the released p-nitrophenol by the reaction was monitored at 410 nm. Control tests were carried out using the placebo preparations. The enzyme activity was calculated by the initial rate of the enzyme reaction (the slope of the absorbance plot) and the p-nitrophenol molar absorption coefficient. The lipase unit was defined as the amount of enzyme that liberates 1 μmol of p-nitrophenol per minute under the assay conditions. Enzyme activity was assessed under various experimental conditions, namely pH and temperature.The activity of the free and the equivalent amounts of immobilised lipase was determined spectrophotometrically, using p-−1 Tris-HCl, pH 8.0) incubated at 60 °C for 2 h followed by rapid cooling in ice water to reach 25 °C. The pH stability was evaluated by measuring the enzyme activity at pH 8 and 25 °C after sample incubation for 1 h in acetate or 50 mmol L−1 Tris-HCl buffers, pH 5.0, 6.0, and 7.0 for 1 h at the same temperature.The thermal and pH enzyme stabilities were estimated by measuring the enzyme activity after thermal treatment, and pH change. The thermal treatment involved sample buffered solution  during a month. Between the measurements, the samples were stored wet at 4 °C. The residual activity was expressed as a per cent of the initial activity.The number of the obtained polymer beads by applying various methods was approximately 250 per batch. Out of each batch, 25 beads were used for bead-size-distribution estimation. The diameter of each bead was measured in different angles employing an optical microscope (magnification 4x), and the average bead size was calculated. The obtained results are presented in The surface morphology of the beads was studied using optical microscopy (magnification 10x). The observations demonstrated that the Ca-alginate beads, the hybrid Ca-alginate/PVA beads, and the chitosan-coated Ca-alginate beads have a spherical shape, slightly rough surface, compact structure, and elastic properties. In contrast, the obtained chitosan beads were irregularly shaped, with ovoid form and soft deformable texture. The corresponding photos are presented in −1 are characteristics for the C–O–C stretching attributed to the saccharide alginate structure, and for the asymmetrical stretching of the –COO– groups. The chitosan spectrum shows the characteristic absorption bands of C = O and NH groups at 1633 and 1028 cm−1, respectively. The infrared (IR) spectrum of PVA evidences the adsorption bands at 1086 and 1423 cm−1, assigned to the C–O stretch. The FTIR spectra of the hybrid alginate/PVA beads and the chitosan-coated alginate beads demonstrate similar spectral features to those of the individual polymers. Therefore, FTIR analysis confirms the absence of strong chemical interactions between the polymers, leading to functional group modification.The possible interactions between the polymers forming the hybrid alginate/PVA beads and the chitosan-coated alginate beads were investigated by FTIR spectrometry. The recorded spectra are shown in As mentioned above, the enzyme immobilisation performances of the selected polymers were quantitatively evaluated by calculating their loading efficiency and immobilisation yield. These two parameters were defined as described in Equations (1) and (2). The data presented in p-NPB hydrolysis. It is noteworthy that the activity of the free lipase increased with enzyme concentration in the studied concentration range (0.05–0.65 mg mL−1) only up to 0.20 mg mL−1. Then, the lipase activity decreased (Lipase activity evaluation was performed measuring the initial rate of the enzyme-catalysed ecreased . A similThe results, graphically presented in It was found that the applied method of immobilisation and the nature of the selected support do not affect the pH optimum. Such results were expected, taking into consideration the mild immobilisation conditions and the specific characteristics of the immobilisation applied procedures. Nevertheless, at pH 10 as an example, the free lipase retained only 34% of its optimal activity measured at pH 8, while the retention of the activity of the immobilised lipase was higher, and varied from 42% to 46% of its optimal activity.The effect of the temperature on the activity of the immobilised lipase was investigated in the temperature range 30–60 °C, and at pH 8 corresponding to the pH optimum . Lipase All the carried out experiments demonstrated that enzyme activity retention also depends on the nature of the applied support and increases in the range: alginate/PVA > chitosan > alginate > alginate/chitosan. Similar results were obtained estimating the pH and the thermal stability of the immobilised lipase  and 9.FLipase encapsulated in chitosan-coated Ca-alginate beads conserved 42.08% of its activity after thermal treatment and 99.59% of its activity after incubation at pH 5. Since the process of encapsulation is mostly based on the electrostatic interaction between the negatively charged alginate acid groups and the positively charged chitosan amino groups, it results in the formation of a reinforced gel. Obviously, the restriction of the contact of the enzyme with the environment due to the gel reinforcement favours the retention of its stability and activity.The evaluation of the storage stability of the immobilised lipase demonstrated that it increases in the range: alginate/PVA > chitosan > alginate > alginate/chitosan , such asThe comparison of the performances of the four hydrophilic polymers – Ca-alginate/PVA, chitosan, Ca-alginate, and chitosan-coated Ca-alginate – demonstrated that the chitosan-coated Ca-alginate beads could be considered as a promising matrix for lipase immobilisation. Even if the loading efficiency of the chitosan-coated Ca-alginate beads was inferior to this of the Ca-alginate beads, they demonstrated better performance characteristics in terms of enzyme immobilisation yield, pH, thermal, and storage stability, and enzyme activity retention compared to the other selected polymers. The presented studies may be of potential value for the production of esters in the reactions of transesterification using immobilised lipases.


Southern tomato virus (STV), a double-stranded RNA virus that affects tomato in China, was determined using small RNA deep sequencing. This Chinese isolate shares 99% sequence identity to other isolates from Mexico, France, Spain, and the United States. This is the first report of STV infecting tomatoes in Asia.The complete genome sequence of  Southern tomato virus (STV), which causes tomato mosaic and yellow stunting disorder, was first characterized for its presence in Mexico and the United States , Tomato yellow leaf curl virus (TYLCV), and Tomato chlorosis virus (ToCV) were also identified. With such a high incidence of mixed infection, the disease symptoms observed could not be attributed to STV alone. For STV, the complete genome in a single contig was obtained, and its sequence was verified by Sanger sequencing through genome walking using reverse-transcription PCR with five pairs of STV-specific primers. In the summer of 2012, tomato plants in a greenhouse near Shouguang, Shandong province, in eastern China, exhibited a high incidence of virus-like disease symptoms, with severe mosaic, epinasty, leaf curl, and yellow stunting disorder. To determine the causal agent(s), total RNA was prepared with TRIzol reagent on a pooled sample collected from eight diseased tomato plants. The small RNA deep-sequencing technology  was emplThe verified complete genome for the Chinese isolate STV_CN12 comprised 3,463 nucleotides (GenBank accession no. KT438549). The genome contained a 5′-proximal open reading frame (ORF) encoding a 378–amino acid (aa) coat protein (p42). A second ORF contained an RNA-dependent RNA polymerase , which was likely expressed via an a + 1 ribosomal frameshift, as predicted for other STV isolates (KT438549.The nucleotide sequences of STV_CN-12 have been deposited in GenBank under the accession number


We suggest seropositivity affects survival depending on trade-offs among antibody protection, immunosuppression and virus lethality. Negative effects of seropositivity might be greater on juveniles due to their immature immune system. Also, while RHDV directly affects survival through the hemorrhagic syndrome, MV lack of direct lethal effects means that interactions influencing survival are likely to be more complex. Multi-event modeling allowed us to quantify patterns of host–pathogen dynamics otherwise difficult to discern. Such an approach offers a promising tool to shed light on causative mechanisms.Host–pathogen epidemiological processes are often unclear due both to their complexity and over-simplistic approaches used to quantify them. We applied a multi-event capture–recapture procedure on two years of data from three rabbit populations to test hypotheses about the effects on survival of, and the dynamics of host immunity to, both myxoma virus and Rabbit Hemorrhagic Disease Virus (MV and RHDV). Although the populations shared the same climatic and management conditions, MV and RHDV dynamics varied greatly among them; MV and RHDV seroprevalences were positively related to density in one population, but RHDV seroprevalence was negatively related to density in another. In addition, ( IP and SM participated in the design of the study. CR, IP, SM and AB assisted in the field with animal handling and collected and processed the samples. AB conducted the serological analysis. SS carried out all statistical analysis. CR, SS and IP drafted the manuscript and SM and AB helped edit the manuscript. All authors read and approved the final manuscript.Sex and age population structures in each enclosure . Frequency of males and females, juveniles and adults, as resulting from captured individuals for each session capture in the three enclosures.Click here for fileProbabilistic framework of the multi-event analyses. Matrice structure for each parameter  is given and its notation explained.Click here for fileSurvival estimates with 95% CI for each enclosure . Survival estimates in the three enclosures as depending on the main effects found through model selection.Click here for fileSeroconversion estimates with 95% CI for each enclosure . Seroconversion estimates in the three enclosures as depending on the main effects found through model selection. No age effect was tested because juveniles remain juveniles only for a short time and no recapture from one individual in juvenile state exists.Click here for fileMyxoma virus and RHDV antibody prevalence in enclosure E1. Myxoma virus and Rabbit Hemorrhagic Disease Virus seroprevalences over the study period as proxied by Initial State in enclosure E1. Prevalence estimates refer to the probability one first-captured individual is seropositive. This probability is corrected for the specific session probability of capture of seronegatives and seropositives. Vertical bars represents 95% confidence intervals.Click here for fileMyxoma virus and RHDV antibody prevalence in enclosure E2. Myxoma virus and Rabbit Hemorrhagic Disease Virus seroprevalences over the study period as proxied by Initial State in enclosure E2. Prevalence estimates refer to the probability one first-captured individual is seropositive. This probability is corrected for the specific session probability of capture of seronegatives and seropositives. Vertical bars represent 95% confidence intervals.Click here for fileMyxoma virus and RHDV antibody prevalence in enclosure E3. Myxoma virus and Rabbit Hemorrhagic Disease Virus seroprevalences over the study period as proxied by Initial State in enclosure E3. Prevalence estimates refer to the probability one first-captured individual is seropositive. This probability is corrected for the specific session probability of capture of seronegatives and seropositives. Vertical bars represent 95% confidence intervals.Click here for file


Eight hour after infection, the naïve contact groups were housed with those inoculated with each of the viruses as a means to monitor contact transmission. Our results indicated that genetically diverse viruses circulate in LBMs in South China's Guangdong Province and that NDV from different birds have different tissue tropisms and host ranges when transmitted in different birds. We therefore propose the continuous epidemiological surveillance of LBMs to support the prevention of the spread of these viruses in different birds, especially chickens, and highlight the need for studies of the virus–host relationship.Although Newcastle disease virus (NDV) with high pathogenicity has frequently been isolated in poultry in China since 1948, the mode of its transmission among avian species remains largely unknown. Given that various wild bird species have been implicated as sources of transmission, in this study we genotypically and pathotypically characterized 23 NDV isolates collected from chickens, ducks, and pigeons in live bird markets (LBMs) in South China as part of an H7N9 surveillance program during December 2013–February 2014. To simulate the natural transmission of different kinds of animals in LBMs, we selected three representative NDVs—namely, GM, YF18, and GZ289—isolated from different birds to evaluate the pathogenicity and transmission of the indicated viruses in chickens, ducks, and pigeons. Furthermore, to investigate the replication and shedding of NDV in poultry, we inoculated the chickens, ducks, and pigeons with 10 Due to its high morbidity and mortality, Newcastle disease (ND) is considered to be the most significant and widespread infectious disease in commercial poultry—one that can cause severe economic losses in poultry, particularly chickens, and affect a range of other domestic species, including ducks and pigeons  genotypes when the sequences are isolated over time  behave similarly to non-virulent viruses according to ICPI tests in 1-day-old chickens; whereas the strains are highly pathogenic to pigeons, the serial passaging of PPMV-1 in chickens results in increased virulence  2013-0019.50) was calculated by using the Reed–Muench method  surveillance program during December 2013–February 2014 by the Key Laboratory of Zoonosis Prevention and Control of Guangdong, China. Oropharyngeal and cloacal swab specimens were collected from commercial chickens, ducks, and pigeons from LBMs, also in Guangdong Province. A total of 214 swab samples were collected in 1.5 mL centrifuge tubes containing 1.0 mL transport medium  with antibiotics  and shipped to South China Agricultural University. The oropharyngeal and cloacal swab samples were inoculated in 10-days-old specific-pathogen-free (SPF) embryonated chicken eggs, as previously described  and 27 commercially available domestic ducks (2-weeks-old Peking ducks) were purchased from a pigeon farm in Gaoming and a duck farm in Yunfu, respectively, and housed in isolators. All pigeons and ducks were confirmed to be serologically negative for ND by HI assays during a 1-day period before experimentation.To test pathogenicity and reproduce the natural conditions of NDV transmission in chickens, ducks, and pigeons, we selected three strains representative of and circulating in South China: genotype VII Chicken/Guangdong/GM/2014 (GM), genotype IX Duck/Guangdong/YF18/2014 (YF18), and genotype VI Pigeon/Guangdong/GZ289/2014 (GZ289), each a predominant chicken-origin, duck-origin, and pigeon-origin genotype, respectively. A total of 27 6-weeks-old SPF white Leghorn chickens were supplied by Guangdong Dahuanong and housed in isolator cages under negative pressure with food and water provided We previously determined the complete genome sequence of the GM strain from LBMs in Guangdong Province preserved in our laboratory  were inoculated with 106 EID50 of the indicated virus in a volume of 200 μL via intraocular and intranasal routes. Eight hour after infection, an additional three chickens, ducks, and pigeons were inoculated intranasally with 200 μL PBS and placed in physical contact—that is, in the same cage and sharing food and water ad libitum—with inoculated birds in order to monitor contact infection. We euthanized three inoculated birds  from each subgroup 3 days postinoculated (DPI) to test the virus replication from various tissues from the thymus, cecal tonsils, bursa of Fabricius, trachea, lung, brain, kidney, and spleen. Oropharyngeal and cloacal swabs from all infected and contacted birds were collected for the detection of viruses shedding at 3, 5, 7, 9, and 11 DPI and suspended in 1000 μL transport medium (50% glycerol in PBS) with antibiotics  for viral detection and titration in eggs. Virus titers were calculated as described previously . The complete genome sequences of the GM, YF18, and GZ289 strains obtained in the present study are available from Gen Bank under the accession numbers DQ486859, KR014814, and KR014815, respectively.Gen Bank accession numbers of the complete F genes of the 23 South China strains described in this study are designated in Table Gallus gallus), 38 ducks (Peking), and 27 pigeons (C. livia) had oropharyngeal and cloacal swab samples taken as part of an avian influenza (H7N9) surveillance program on 3 DPI and from the cloacal (5.29 log10EID50) on 3 DPI (Table 10EID50). But, it only could be shed from the cloaca within 3 DPI (4.92 log10EID50). GZ289 virus shedding was detected from oropharyngeal and cloacal swabs only at 3 DPI .GM, YF18, and GZ289 viruses shedding from the inoculated chickens were detected in oropharyngeal and cloacal swabs at 3, 5, 7, 9, and 11 DPI (Table 10EID50) and from the cloacal swabs (3.75–3.92 log10EID50) at 3 and 5 DPI . The naïve contact chickens housed with GZ289 virus still survived in 14 DPI, but none seroconverted . The mean virus titers in the thymus, cecal tonsils, bursa of Fabricius, trachea, brain, kidney, and spleen were 3.33, 2.67, 2.75, 1.92, 1.58, 3.58, and 2.83 log10EID50, respectively. Generally, the GM and GZ289 virus titers were lower than those of YF18 in ducks. Those two viruses replicated in some tested tissues, including those of the thymus, cecal tonsils, bursa of Fabricius, lung, kidney, and spleen, but not in those of the trachea or brain .In the inoculated ducks, the YF18 virus was replicated systemically in the tissues of the thymus, cecal tonsils, bursa of Fabricius, trachea, lung, brain, kidney, and spleen on 3 DPI Table . The YF1in Table . The GM 10EID50, respectively). YF18 virus shedding was detected in the oropharynx in inoculated ducks within 7 DPI, with virus titers from 1.58 to 2.42 log10EID50, and from the cloaca within 3 DPI, with virus titers of 1.75 log10EID50. Lastly, GZ289 virus shedding occurred with both oropharyngeal and cloacal swabs at 3 DPI .GM, YF18, and GZ289 viruses shedding from the inoculated ducks were detected in oropharyngeal and cloacal swabs at 3, 5, 7, 9, and 11 DPI (Table 10EID50), whereas cloacal swabs did not show any detectable virus during the period , whereas two pigeons in the contact group seroconverted with relatively high titers (6.0 log2). The HI titers of groups inoculated with the GM and YF18 virus were 8.3 log2, and 8.7 log2, respectively, although none of the three contact pigeons seroconverted . In the YF18 virus- inoculated group, the virus titers of the cloacal swabs were detectable only at 3 DPI (1.75 log10EID50); however, the virus titers of the oropharyngeal swabs could not be detected during our observation period .GM, YF18, and GZ289 viruses shedding from the inoculated pigeons were detected in oropharyngeal and cloacal swabs at 3, 5, 7, 9, and 11 DPI (Table 10EID50) and tested from cloacal swabs only at 7 and 9 DPI (1.58–1.67 log10EID50).To determine whether these three viruses could be horizontally transmitted among pigeons, 8 h after infection, three pigeons were inoculated with 200 μL PBS via the same routes as a naïve contact group placed with those inoculated with the GM, YF18, or GZ289 viruses. No pigeons died in the naïve contact subgroup placed with those exposed to the three selected viruses Table . No viruIn sum, the GZ289 virus could infect and transmit among pigeons by naïve contact. Though the GM and YF18 viruses could infect pigeons, they could not transmit among pigeons by naïve contact  and genotype I vaccine virus (V4), are still used at a large scale, most extensively for protecting poultry flocks from ND in South China . In all, the efficient replication, high seroconversion, and shedding of relatively high titers in naïve contact groups suggest that NDV isolated from different birds was transmitted to the naïve contact group.A correlation exists between antibody response and shedding after infection with virulent NDV in susceptible animals (Miller et al., Altogether, our results provide clear evidence that genetically diverse viruses circulate in LBMs in South China's Guangdong Province and illustrate that the three NDV strains isolated from different birds have varying levels of infectivity, pathogenicity, and transmission in chickens, ducks, and pigeons. Our findings thus emphasize the need for constant epidemiological studies in LBMs, in order to enhance active surveillance toward preventing the spread and evolution of these viruses.YK conceived the study and wrote the paper. BX and YK designed, performed, and analyzed all the experiments. RY provided technical assistance and prepared all the figures. YL, SF, YL, and TR designed the study and revised the manuscript. All authors reviewed the results and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the US, noroviruses are estimated to cause 21 million cases annually with economic losses reaching $2 billion. Outbreak investigations frequently implicate vomiting as a major transmission risk. However, little is known about the characteristics of vomiting as a symptom or the amount of virus present in emesis.5 and 3.9 x 104 genomic equivalent copies (GEC)/ml for GI and GII viruses, respectively (p = 0.02). Sample pH was correlated with GII.2 Snow Mountain virus detection.Emesis samples and symptomology data were obtained from previous norovirus human challenge studies with GI.1 Norwalk virus, GII.2 Snow Mountain virus, and a pilot study with GII.1 Hawaii virus. Viral titers in emesis were determined using strain-specific quantitative RT-PCR. In all four studies, vomiting was common with 40–100% of infected subjects vomiting at least once. However, only 45% of subjects with vomiting also had diarrhea. Most of the emesis samples had detectable virus and the mean viral titers were 8.0 x 108 GEC in emesis. Unlike shedding through stool, vomiting is more likely to result in significant environmental contamination, leading to transmission through fomites and airborne droplets. This quantitative data will be critical for risk assessment studies to further understand norovirus transmission and develop effective control measures. The correlation between sample pH and virus detection is consistent with a single site of virus replication in the small intestine and stomach contents becoming contaminated by intestinal reflux. Additionally, the frequency of vomiting without concurrent diarrhea suggests that epidemiology studies that enroll subjects based on the presence of diarrhea may be significantly underestimating the true burden of norovirus disease.Half of all subjects with symptomatic infection experienced vomiting and the average subject shed 1.7 x 10 Noroviruses are estimated to cause 21 million cases of acute gastroenteritis every year in the US . CurrentNoroviruses are transmitted through the fecal-oral route via consumption of contaminated food or water, contact with contaminated surfaces (fomites), and person-to-person –10. Outb4 gEq/ml [Despite the clear role of vomiting in transmission, there is very little data on viral loads in emesis. An investigation of norovirus outbreaks among rafters in the Grand Canyon detected GI norovirus in a vomitus sample . In samp4 gEq/ml . There iThe goal of this study is to provide quantitative data describing the frequency of vomiting and virus titers during norovirus infection. We analyzed vomiting data from two GI.1 Norwalk virus challenge studies, a GII.2 Snow Mountain virus challenge and a pilot study with GII.1 Hawaii virus. Virus titers were determined by RT-qPCR in the archived emesis samples from these studies. The availability of this data will facilitate accurate transmission risk estimation and provide an evidence base for the development of appropriate control measures, such as disinfection after a public vomiting event.The emesis samples for this study are archived specimens from three previously published human challenge studies  –24 and oEmesis and stool samples were weighed, aliquoted into sterile containers and frozen at -80°C. For emesis samples, weight was used as a proxy for total volume with 1 g equal to 1 ml. Samples collected within 15 min of each other were combined and treated as a single specimen occurring at the earlier recorded time.Infection was defined as detection of norovirus RNA in at least one stool specimen or ≥ 4-fold increase in anti-norovirus IgG in serum. Diarrhea was defined as ≥ 3 unformed stools or ≥400 g unformed stool in a 24 hr period. Viral gastroenteritis was defined as detection of norovirus RNA in stool, and diarrhea  or one or more vomiting episodes plus one of the following: abdominal cramps, nausea, fever , myalgia, chills, fatigue, or headache.All challenge studies were approved by the Emory University Institutional Review Board (studies 1 and 2) , 24 or tEmesis (50% vol/vol) and stool (20% vol/vol) suspensions were prepared in sterile, molecular-grade water. For emesis specimens, only the liquid phase of the specimen was used to maintain testing consistency. Virus particles were separated from organic debris by phase extraction with an equal volume of Vertrel XF . After incubating for 2 hours at 4°C, the sample was centrifuged for 10 minutes at 9400 x g. RNA was isolated from 140 μl of the aqueous phase using the QiaAmp Viral RNA mini kit  following the manufacturer’s instructions. Isolated RNA was stored at -20°C until testing.Viral titers were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR) using previously described strain-specific primers and probes and the OneStep RT-PCR kit  , 26. TheThe pH of completely thawed emesis samples was determined using pHydrion plastic pH strips, 0.0 to 6.0  and colorPHast pH strips, 5–10 .Comparisons between studies and between genogroups were assessed using the Student’s t-test. ANOVA was carried out in GraphPad Prism 6 .The samples used in this study were archived from previous norovirus human challenge trials . Of the Vomiting is very common in symptomatic norovirus infection. Among subjects infected with Norwalk virus, 70% of symptomatic subjects experienced at least one vomiting event . Similar5 GEC/ml vs. 1.6x105 GEC/ml, p = 0.36). Samples from subjects infected with Hawaii virus had lower mean virus titers but a higher frequency of positive samples. To assess total viral shedding in emesis, cumulative shedding was calculated by multiplying the sample virus titer by the sample volume in ml and summing the resulting value across all positive samples for a subject. Overall, the cumulative virus shedding per subject was high . The cumulative shedding titers for subjects infected with Norwalk or Snow Mountain viruses were similar, but the cumulative shedding during Hawaii virus infection was 2–3 logs lower than the other viruses (2.3x105). However, it should be noted that only one subject infected with Hawaii virus had sample volume data available.Most subjects who experienced vomiting had at least one sample with detectable norovirus . Of the The relationship between virus titers in emesis and stool from representative subjects is shown in The pH of stomach contents can vary based on diet, time of day and prolonged vomiting . To asseIt is clear from outbreak and challenge studies that vomiting is the signature symptom of norovirus infection, which is colloquially known as the “winter vomiting disease.” However, vomiting has been largely overlooked in the quantitative studies of norovirus disease. This is the first report of vomiting frequency and viral titers from human challenge studies with GI and GII noroviruses. In these challenge studies, vomiting was more prevalent than diarrhea as a symptom and norovirus-infected subjects shed large amounts of virus through vomiting. There was no difference in viral titers in emesis samples between subjects infected with GI.1 Norwalk and subjects infected with GII.2 Snow Mountain virus. , in cont2) and produce potentially infectious aerosols [Simulated vomiting events have shown that a single vomiting event can contaminate a large area (7.8 maerosols , 30. A raerosols . Thus, iThis is the first report of results from a human challenge study with GII.1 Hawaii virus. The viral titers in emesis and stool  were sevThe site of norovirus replication within the host is not known, but the virus has been shown to bind to duodenal tissue . The resMany outbreak and epidemiology studies of norovirus define a case based on the presence of diarrhea. However, in this study, nearly half of subjects who experienced vomiting did not have concurrent diarrhea . Our fin


Nordal et al compared recently in a Nordic population the JADAS based on CRP with JADAS based on ESR and concluded that these instruments correlated closely, indicating that both scores can be recommended for assessing disease activity in JIA.Recently, Juvenile Arthritis Disease Activity Score (JADAS) was found to be a valid instrument for assessment of disease activity. JADAS was developed with erythrocyte sedimentation rate (ESR) because C-reactive protein (CRP) values were not available in all databases used to validate the tool. Determine JADAS-CRP and compare its performance to the JADAS-ESR and to test the agreement of both scores on each disease activity category, in a Portuguese population with JIA.A National cohort of patients with JIA, registered in Rheumatic Diseases Portuguese Register, was selected. Patients were included in the study when all disease activity measures were available for JADAS-ESR and CRP calculation and one visit per patient was randomly selected. JADAS-CRP was adapted by replacing ESR with CRP as the inflammatory marker. CRP was truncated to a 0-10 scale, similar to the truncated ESR used in JADAS. JADAS 27-CRP was calculated similarly to JADAS 27-ESR as the simple linear sum of its four components. Pearson correlations and K statistics were used in analyses.p < 0,0001), though the correlation coefficient between CRP and ESR was only 0,335 . When comparing the JADAS-ESR and JADAS-CRP within each subtype of JIA, the strong correlation was maintained . The agreement between JADAS-ESR and CRP across the 4 activity states  assessed by K statistics was very good, showing 91.1% of the observations in agreement, K = 0.867 (95%CI 0.824-0.91).358 children included, 65.4% were female, mean disease duration 11.75 ± 9.03 years. 37,5% were persistent oligoarticular, 14,8% had extended oligoarticular, 14,2% were polyarticular rheumatoid factor negative, 8,4% polyarticular RF positive, 10,9% systemic, 9,8% enthesitis-related arthritis, 3,1% psoriatic arthritis and in 1,4% patients we could not have access to the subtype of JIA. The correlation coefficient was 0,967, In our study the JADAS27 based on CRP and ESR correlated closely, indicating that both measures can be used in clinical practice.None declared.


A limiting factor in these studies, however, is rapid virus evolution and resulting polymorphisms, which prevent reads from aligning readily to commonly used virus reference genomes, and, accordingly, make virus integration sites difficult to detect. Another confounding factor is host genomic instability as a result of virus insertions. To tackle these challenges and improve our capability to identify cryptic virus-host fusions, we present a new approach that detects Virus intEgration sites through iterative Reference SEquence customization (VERSE). To the best of our knowledge, VERSE is the first approach to improve detection through customizing reference genomes. Using 19 human tumors and cancer cell lines as test data, we demonstrated that VERSE substantially enhanced the sensitivity of virus integration site detection. VERSE is implemented in the open source package VirusFinder 2 that is available at The online version of this article (doi:10.1186/s13073-015-0126-6) contains supplementary material, which is available to authorized users. Pathogenic viruses pose significant threats to public health throughout the world ,2. With Virus insertions in host genomes typically cause host genomic instability -9, whichStimulated by the strong demand for NGS investigations of virus-host interactions, a large number of tools were developed in the past three years for virus/virome characterization -20, noveTo facilitate the rapidly growing number of studies on disease-associated viruses, here we present a new approach that detects Virus intEgration sites through Reference SEquence customization (VERSE). The rationale of VERSE is to use short reads to iteratively 'correct' reference genomes so as to create new 'personalized' reference genomes. Corrections made to the references improve read mapability, and, accordingly, detection sensitivity. VERSE is specifically designed with the diversity and scale of today’s NGS applications and computational efficiency in mind. It allows quick analysis of NGS data of various types: whole genome sequencing (WGS), whole transcriptome sequencing (RNA-seq), targeted sequencing (TS), and so on. VERSE is implemented in a publicly available software package, VirusFinder . In its We used WGS of 13 hepatocellular carcinomas (HCCs), RNA-seq of 4 HCC cell lines, and TS of 2 Merkel cell carcinomas to evaluate VERSE (Table Paired-end WGS (2 × 90 bp) of the 13 HCCs was performed on an Illumina HiSeq 2000 sequencer as described in . AverageWhole transcriptomes of the four HCC cell lines were subjected to sequencing library preparation using an Illumina TruSeq RNA Sample Preparation Kit as reported in the original publication . SequencFor the two Merkel cell carcinomas, virus genomes were captured from formalin-fixed, paraffin-embedded tissues and enriched using PCR-generated capture probes . Targetesimulate' in pIRS to generate paired-end sequencing reads . Additionally, to mimic real data, we inserted single nucleotide polymorphisms (SNPs), small insertions and deletions (indels), and structural variants (SVs) into the data. We let the frequency of SNPs be 10 times higher than that of indels and the frequency of SVs 10 times less than that of indels. The simulation data are freely available at [Besides the data from the real tumors and cancer cell lines, we also simulated WGS of human chromosome 1 using the profile-based Illumina pair-end Read Simulator (pIRS) . We plugFigure The purpose of the read subtraction step is to collect viral reads, that is, the reads characteristic of the viruses infecting a host. In this step, VERSE uses the alignment tool Bowtie 2  to map rIn this step, VERSE utilizes ICORN , a tool Customization of large genomes is time-consuming. For instance, to use short reads to modify the UCSC hg19, it can take ICORN over a week to complete at the Vanderbilt Advanced Computing Center for Research and Education (ACCRE) . To speechrVirus). Next, we use BWA [chrVirus. The host genomic regions that potentially harbor virus integration sites are then derived from the mapped positions of the reads of the SVs . Then, following the same procedure as step (b), VERSE runs ICORN to iteratively align reads to these pseudo-chromosomes, call SNPs and indels from read alignment, and then correct the reference with the called SNPs and indels.The final outputs of this step are therefore customized host genomic regions, each of which corresponds to a potential virus integration event. By modifying only these small regions, we are able to reduce the computation time from over a week on a large reference genome to a few hours.In this step, VERSE concatenates the host genomic regions recruited in the previous step with the consensus virus genome to create an analysis-ready reference genome. VERSE runs BWA to map the viral reads to this new reference and then utilizes CREST  to detecAs demonstrated above, VERSE combines two complementary tools, SVDetect  and CRESTo measure the confidence of a predicted position relative to the real virus integration site, and based on the output of CREST, VERSE categorizes a prediction into one of two classes: (a) high confidence - if there are sufficient soft-clipped reads to support an integration locus so that CREST is able to detect it; and (b) low confidence - CREST fails to detect it for the lack of high-quality soft-clipped reads.For a high-confidence prediction, CREST’s output is used directly as a putative virus integration site. For a low-confidence one, however, VERSE predicts its position based on the soft-clipped reads that cover it. In particular, VERSE derives the boundaries of the region that potentially harbor the integration site from the output of SVDetect. Next, VERSE sorts the loci within the boundaries in the descending order of the number of soft-clipped reads that are aligned to them. The one covered with the most soft-clipped reads is then used as an estimate of the real integration locus.To discard the possible false-positives, VERSE requires the distance between two adjacent low-confidence virus integration sites to be at least 10 bp. The drawback of the use of this stringent cutoff is that VERSE could mistakenly discard a real integration event if it is within 10 bp of another.The input of VERSE includes NGS reads (in FASTQ format) sequenced from a host, a reference host genome (in FASTA format), and a reference virus genome (FASTA format). The entire pipeline of VERSE, from the initial read subtraction step to virus integration detection to result classification and filtering, is fully automated. The output of each step is used automatically as the input for the next step of the pipeline.sensitivity_level to allow users to designate the number of iterations of reference genome customization. Because the majority of errors in a reference gnome can be corrected using one or two rounds of ICORN iteration, when evaluating VERSE on the human tumors and cell lines in the section below, we let sensitivity_level = 1 for simplicity and to save time. We encourage users to tune its value in their applications so as to adjust VERSE’s detection sensitivity.VERSE provides an argument flank_region_size, which defines the size of the flanking regions upstream and downstream of a genomic region under study. In our experiments presented in the section below, flank_region_size was set to the default value of 4,000. This means VERSE will search both the upstream 4,000 bp and downstream 4,000 bp regions flanking a genomic segment predicted by SVDetect to harbor a candidate virus integration site. By allowing VERSE to examine the flanking regions, we reduce the chance to miss virus insertion sites therein.Another input argument of VERSE is As mentioned above, the source code of VERSE is publicly available, through the open source software package VirusFinder 2 . As the In this section, we first evaluate VERSE’s capability to customize virus reference genomes. Then, we examine the effect of host genome customization and VERSE’s performance in identifying virus integration in human tumor genomes. The utility of VERSE is available through the VirusFinder 2 software.Although the viruses infecting the human tumors and cancer cell lines collected by us were reported by earlier studies, their consensus sequences are unknown to investigators. In order to estimate the resemblance of the VERSE-created consensus virus genomes with the intra-host virus populations, we ran VERSE on the simulation data.The mutated copy of the HPV-16 virus reference genome (GI:310698439) that was inserted in the simulation data harbors 75 SNPs and 9 indels. VERSE recruited the viral reads by aligning the raw simulated reads to the UCSC hg19 sequence. Then, with the HPV-16 reference genome as input, VERSE used the viral reads to customize the HPV-16 reference. After one round of ICORN iteration, 68 (91%) out of 75 SNPs and 6 (67%) out of 9 indels (relative to the assembly of short reads) were successfully characterized in the HPV-16 reference and corrected. This result demonstrates that VERSE could accurately identify the consensus mutations of the intra-host virus populations.Next, we compared the consensus virus genome produced by VERSE with the initial virus sequence that was inserted into the simulation data. The identity of the resulting consensus virus genome with the initial sequence is 99.9%, in comparison with 99.1% between the HPV-16 virus reference and the initial inserted sequence. This result indicates that starting from a commonly used virus reference genome, VERSE is able to generate a consensus virus genome that better represents the virus populations within a host.To show how well VERSE customizes host genomes, we randomly chose a WGS sample, 26 T, from the 13 HCCs  out of 2.4 billion reads were recruited as viral reads. In step (c) of the pipeline, VERSE used SVDetect to identify three genomic regions  as putative virus integration-harboring regions. It is easy to see that the real virus integration site, Chr18:107920, is located in the third region.Next, VERSE ran ICORN to iteratively customize the three genomic regions. In each round of ICORN customization, short reads were mapped against the three regions and then consensus SNPs and indels were identified from these regions for reference corrections. The coverage of the mapped reads at each modified base before and after correction was compared and the correction that reduced the coverage was rejected. We performed 6 rounds of customization on this sample. Table Figure With improved alignment, VERSE is able to recruit more reads  to detect virus integration sites. An in-depth discussion of VERSE’s performance in detecting virus integration sites is the topic of the next section.More than half of our samples used WGS, currently the most powerful sequencing technology. It provides the most comprehensive and unbiased characterization of genomic alterations  in genoIn a previous study , WGS samnormal mode .Table Additionally, Table Table Finally, it may be worth mentioning the computational efficiency of VERSE. With its well-designed pipeline, it takes VERSE on average <3 days (using 8 CPUs) to analyze a WGS sample on ACCRE  to detect virus integration sites. Using 19 human tumors and cancer cell lines as test data, we demonstrated that VERSE improved detection sensitivity substantially. VERSE has been implemented in our open source package VirusFinder 2 .VERSE requires the presence of both spanning and soft-clipped reads to nominate a virus integration event. Comparing with other tools that focus either on spanning reads or on split reads, this is a stringent requirement. Though effective and fast, a potential drawback of this design is that it may miss real virus integration events if there is no supportive soft-clipped reads. Further improvement is needed in order not to miss real virus integration sites. Another limitation of VERSE is that ICORN, the tool used in VERSE for customizing reference genomes, cannot process single-end reads. This limits the applications of VERSE to paired-end sequencing data at present.


Mesoniviridae  comprises of a group of positive-sense, single-stranded RNA ([+]ssRNA) viruses isolated from mosquitoes.The family Alphamesonivirus-1 which also includes Nam Dinh virus (NDiV) from Vietnam and Cavally virus (CavV) from Côte d’Ivoire. The eight other isolates were identified as variants of three new mesoniviruses, based on genome organization and pairwise evolutionary distances: Karang Sari virus (KSaV) from Java, Bontag Baru virus (BBaV) from Java and Kalimantan, and Kamphaeng Phet virus (KPhV) from Thailand. In comparison with NDiV, the three new mesoniviruses each contained a long insertion (180 – 588 nt) of unknown function in the 5’ region of ORF1a, which accounted for much of the difference in genome size. The insertions contained various short imperfect repeats and may have arisen by recombination or sequence duplication.Thirteen novel insect-specific virus isolates were obtained from mosquitoes collected in Indonesia, Thailand and the USA. By electron microscopy, the virions appeared as spherical particles with a diameter of ~50 nm. Their 20,129 nt to 20,777 nt genomes consist of positive-sense, single-stranded RNA with a poly-A tail. Four isolates from Houston, Texas, and one isolate from Java, Indonesia, were identified as variants of the species Mesoniviridae, order Nidovirales. Species demarcation criteria employed previously for mesoniviruses would place five of these isolates in the same species as NDiV and CavV (Alphamesonivirus-1) and the other eight isolates would represent three new mesonivirus species . The observed spatiotemporal distribution over widespread geographic regions and broad species host range in mosquitoes suggests that mesoniviruses may be common in mosquito populations worldwide.In summary, based on their genome organizations and phylogenetic relationships, thirteen new viruses were identified as members of the family  Mesoniviridae  comprises a group of positive-sense, single-stranded RNA ([+]ssRNA) insect viruses  tail . The phylogenies exhibited highly similar topologies and strongly indicated that the viruses identified in this study cluster within the previously identified insect-specific nidoviruses in the genus Mesonivirus   phylogenetic trees were constructed based on the amino acid alignments of ORF2a (unprocessed S protein) and a concatenated region of the highly conserved domains within ORF1ab . Based on this analysis, NDiV (including the Ngewontan and Houston strains) and CavV can be considered a single species, which has previously been designated at Alphamesonivirus-1. The PED between each of these strains was substantially less than the suggested cutoff of 0.032 for species demarcation  were completely conserved across all new mesoniviruses examined in this study.Mesoniviruses have been shown previously to express a 3’-nested set of polyadenylated sub-genomic mRNAs produced by copy-choice mediated leader-body fusion at sites located immediately upstream of alternative transcription regulatory sequence (TRS) elements which occur in the 5’-UTR and in the regions preceding ORF2a/ORF2b and ORF3a/ORF3b [http://bibiserv.techfak.uni-bielefeld.de/pknotsrg/) in the same region of the genome may facilitate the frame-shift [http://rna.naist.jp/ipknot) which predicts the consensus secondary structure from a multiple sequence alignment (by using integer programming to select the maximum expected accuracy (MEA) structure) [In nidoviruses, translation of ORF1b characteristically occurs through a ribosomal frame-shift at a ‘slippery’ sequence in the ORF1a/ORF1b overlap region to allow read-through synthesis of a polyprotein (pp1ab). This is usually facilitated by an RNA pseudoknot in the sequence immediately downstream of the frame-shift site. However, a previous analysis of the NDiV sequence failed to identify a predicted pseudoknot structure, suggesting that a stem-loop structure  . As showhttp://www.expasy.org) indicated that a frame-shift at this site would result in a double-membrane-spanning glycoprotein (p3ab) of approximately 30–33 kDa (depending on whether one or two N-glysosylation sites are occupied) with both the N-terminal and C-terminal domains located in the extracellular lumen  that could result in read-through by a -1 ribosomal frame-shift. Structural analyses using SignalP and TMHMM and NetNGlyc , other previously described mesoniviruses - HanaV MenoV, and NseV - would be assigned to three new species , and eight of the new isolates would represent three new species  [In this study we have identified 13 mesonivirus isolates and characterized their genome organization and phylogenetic relationships. Based on species demarcation criteria employed previously for mesoniviruses [Alphamesonivirus-1 illustrate the wide geographic distribution and mosquito host range of some mesoniviruses. The original four isolates of NDiV were made in northern and central Vietnam from Culex vishnui and Cx. tritaeniorhynchus mosquitoes collected indoors during a surveillance program for Japanese encephalitis virus (JEV) [Cx. vishnui mosquitoes in 1981. The four NDiV isolates from Houston, Texas (Houston strain) were made from Cx. quinquefasciatus and Aedes albopictus collected outdoors within the Houston metropolitan area during West Nile virus (WNV) surveillance in 2004 and 2010, respectively. Interestingly, all of these isolates were from mosquitoes captured in or near human dwellings and from species that feed on humans. The close similarity of isolates from Houston and Vietnam may suggest a recent translocation, possibly during the Vietnam conflict when Houston hosted a major air base for embarkation/disembarkation. Isolates of Kampaeng Phet, Botang Baru and Karang Sari viruses, which may be considered three new mesonivirus species, have a geographic distribution extending at least from central Thailand to Kalimantan and Java in Indonesia, and have been isolated from at least two species of Culex mosquitoes.The isolates of viruses assigned to the species us (JEV) . Our NDiCx. vishnui and Cx. tritaeniorhynchus are important vectors of JEV in Asia, and Cx. quinquefasciatus is the major vector of WNV in Houston. Recent experimental studies with Ae. aegypti mosquitoes infected with certain strains of the Wolbachia indicate that the presence of the symbiont bacterium interferes with dengue virus replication and decreases vector competence, possibly by upregulating or priming the mosquito’s innate immune system [Wolbachia-infected Ae. aegypti and chikungunya virus [Wolbachia-infected Cx. quinquefaciatus and WNV [Another consideration is the potential effect of mesonivirus infection on the susceptibility and vector competence of mosquitoes for viral pathogens of vertebrates. For example, both e system ,12. Simiya virus  and with and WNV . If a ba and WNV . This iset al. [Due to the continuous efforts of the virus discovery program of World Reference Center for Emerging Viruses and Arboviruses (WRCEVA), we have continued to isolate mesoniviruses from various insect vectors collected from widespread geographic locations  suggesting that these viruses are more common than previously thought. Zirkel et al.  suggesteThe detailed analysis and comparison of mesonivirus genome architecture conducted here has revealed some unexpected characteristics. Firstly, the presence of block insertions of up to 588 nt in the 5’ terminal quadrant of ORF1a of several mesoniviruses has not been reported previously. The function of this region is presently unknown in mesoniviruses and other nidoviruses and so the structural and functional consequences of these insertions, which contain various imperfect repeats is unclear. Although sharing similar genome architecture, nidoviruses vary greatly in genome size. A previous analysis of the evolution of nidovirus genomes concluded that genome expansion has occurred in a wave-like fashion in which the three major coding regions  expanded consecutively in a hierarchy that reflects the roles of their encoded proteins in the virus replication cycle . This imThe comparative analysis also revealed that the stem-loop structure which had previously been identified at the RFS site of NDiV is not conserved in the other mesoniviruses and so may not be responsible for activating the -1 ribosomal frame shift. Secondary structure predictions using IPknot on the aligned sequences downstream of the conserved ‘slippery’ sequence site revealed a conserved pseudoknot structure that conformed to all nucleotide substitutions. However, the predicted structure featured only four relatively short regions of complementarity and no estimations of minimum free energy for the represented structures are available through this algorithm. A possible ‘slippery’ sequence (CACUUUU) was also detected in the ORF3a/ORF3b overlap region but no conserved stem-loop or pseudoknot structure was predicted by IPknot in the downstream sequence. It is, therefore, unclear whether ORF3b is expressed by internal initiation or as a read-through extension of ORF3a, which would generate a double-membrane spanning protein. Functional analysis of each of the regions corresponding to the RFS in ORF1a/ORF1b and the putative RFS in ORF3a/ORF3b would help resolve the mechanisms of mesonivirus gene expression.In conclusion, we have identified and characterized several new mesoniviruses from mosquitoes of human medical importance sampled over time from widespread geographic regions. Several important questions related to their transmission, maintenance in insect hosts in nature, their potential impact of infection on the insect’s behavior, fertility, fecundity and survival, their evolution, their mechanisms of gene expression and their potential to be developed as biological control agents, warrant further investigation.All viruses used in this study were obtained from the WRCEVA at the University of Texas Medical Branch. Some were isolated by the authors (RBT and HG), during arbovirus field studies; the remainder were isolated by other investigators and sent to the WRCEVA for identification and further characterization. All isolations were originally made in mosquito cell cultures (C6/36 or AP-61). The proposed names, original sources and geographic origins and GenBank Accession numbers of the sequences obtained for the 13 viruses included in our study are listed below and in Table Culex vishnui mosquitoes collected on 11/26/1981 at Karang Sari, Cilacap  Indonesia. The initial isolation was made at the Naval Medical Research Unit #2 (NAMRU-2) in Jakarta.JKT-10701 was isolated from a pool of Culex vishnui collected at Bontag Baru, East Kalimantan, Indonesia in February 1981. Strains JKT-9876, JKT-9891 and JKT-9853 were also isolated at NAMRU-2 appear to be almost identical to isolate JKT-7774 in the phylogenetic tree. Since JKT-7774 was the first virus in this group to be isolated, it should be the prototype.JKT-7774 was isolated at NAMRU-2 from a pool of 50 Culex vishnui collected at Cilacap, Central Java, Indonesia on 10/13/1981.JKT-9876 was isolated at NAMRU-2 from a pool of Culex vishnui mosquitoes collected on 10/14/1981 at Karang Sari, Cilacap , Indonesia.JKT-9891 was isolated at NAMRU-2 from a pool of Culex tritaeniorhynchus collected at Cilacap, Central Java, Indonesia on 10/6/1981.JKT-9853 was isolated at NAMRU-2 from a pool of KP84-0192, KP84-0344 and KP84- 0156 were isolated from pools of mosquitoes (species not given) collected at Kamphaeng Phet, Thailand in 1984. Original isolations were made at the Armed Forces Research Institute for Medical Sciences, Bangkok.Culex vishnui mosquitoes collected at Ngewotan, Central Java, Indonesia on 11/17/1981.JKT-9982 was isolated at NAMRU-2 from a pool of Culex quinquefasciatus mosquitoes collected in the summer of 2004 as part of an arbovirus surveillance program in Houston (Harris County), Texas, USA.V3872 and V3982 – Both viruses were isolated at UTMB from pools of Aedes albopictus mosquitoes collected in June 2010 as part of the Harris County arbovirus surveillance program in Houston, Texas.TVP16740 and TVP16757 – These two viruses were also isolated at UTMB from pools of Ae. albopictus cells [Before sequencing, all virus stocks were grown in cultures of the C6/36 clone of us cells , obtaineThe following mesonivirus genome sequences were determined in this study: Karang Sari virus, strain JKT-10701 (KC807171); Bontang virus, strain JKT-9891 (KC807169); strain JKT-9853 (KC807167); strain JKT-9876 (KC807168); strain JKT-7774 (KC807166); Kamphang Phet virus, strain KP 84–0192 (KC807173); strain KP 84–0344 (KC807174); strain KP 84–0156 (KC807172); NDiV Ngewotan strain JKT-9982 (KC807170); NDiV Houston strain V3872 (KC807175), V3982 (KC807176), 16740 (KC807177), and 16757 (KC807178).2 were added. The monolayers were washed in 0.1 M cacodylate buffer, cells were scraped off and processed further as a pellet. The pellets were post-fixed in 1% OsO4 in 0.1 M cacodylate buffer pH 7.3 for 1 h, washed with distilled water and en bloc stained with 2% aqueous uranyl acetate for 20 min at 60°C. The pellets were dehydrated in ethanol, processed through propylene oxide and embedded in Poly/Bed 812 . Ultrathin sections were cut on Leica EM UC7 ultramicrotome , stained with lead citrate and examined in a Philips 201 transmission electron microscope at 60 kV.For ultrastructural analysis in ultrathin sections infected cells were fixed for at least 1 hr in a mixture of 2.5% formaldehyde prepared from paraformaldehyde powder, and 0.1% glutaraldehyde in 0.05 M cacodylate buffer pH 7.3 to which 0.03% picric acid and 0.03% CaClViral RNA (0.05-1.7 μg) was fragmented by incubation at 94°C for 8 min in 19.5 ul of fragmentation buffer (Illumina 15016648). First and second strand synthesis, adapter ligation and amplification of the library were performed using the Illumina TruSeq RNA Samplec Preparation kit under conditions prescribed by the manufacturer (Illumina). Samples were tracked using the “index tags” incorporated into the adapters as defined by the manufacturer.2 and post filter reads ranged from 148–178 million per lane. Base call conversion to sequence reads wasperformed using CASAVA-1.8.2. Virus sequences were edited and assembled using the SeqMan and NextGen modules of the DNAStar Lasergene 7 program . In certain cases, prefiltering of reads to remove host sequence enhanced the assembly process. Assembly was carried out using a fasta file of Aedes albopictus sequences to remove host DNA from the assembly thus reducing the number of contigs present.Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation using conditions recommended by the manufacturer. Paired end 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1000 using protocols defined by the manufacturer. Cluster density per lane was 645–980 k/mmThe compiled sequences had their relationship to other viruses determined by a BLASTX search. The open reading frames were determined using Enzyme X . Relationships to the other two insect-specific nidoviruses were determined using MacVector  using their DNA identity matrix software. The presence of conserved protein domains was determined using the SMART webserver ,18.Mesoniviridae along with a concatenated region of the highly conserved domains within ORF1ab . ORF2ab alignments were determined using the MUSCLE algorithm [A region corresponding to the full-length product encoded by ORF2a (unprocessed S proteins) was used to determine the relationships within the family lgorithm  as aminolgorithm , the WAGpro, RdRp and ZnHel1) of the thirteen new isolates described in this study and those of five previously described mesoniviruses  was performed using the ML method in the program TreePuzzle [Calculation of the PED between the highly conserved protein domains of ORF1ab , ORF2a (S) and ORF2b (N).Click here for fileA Clustal X multiple sequence alignment of mesonivirus pp1ab polyproteins illustrating region containing the block insertions (yellow shading) and various imperfectly repeated sequences that occur at the boundary and within the blocks of inserted sequence.Click here for fileA Clustal X multiple sequence alignment of the polypeptides encoded in ORF2a (S proteins) of the mesoniviruses. A predicted signal peptide in MenoV and predicted transmembrane domains in all mesoniviruses are shaded in aqua, predicted N-glycosylation sites are shaded in green, cysteine residues are shaded in yellow and the sites of proteolytic cleavage to generate glycoproteins S1 and S2 and the unidentified N-terminal fragment are shaded in purple.Click here for fileA Clustal X multiple alignment of the sequences of putative polypeptides encoded on ORF4 which occurs in the 3’-terminal regions of all mesoniviruses except MenoV. To emphasize the alignment, the KSaV ORF4 protein has been shown to commence at the next available methionine residue located 34 amino acids downstream of the predicted initiation codon.Click here for fileA Clustal X multiple sequence alignment of region immediately downstream of the ORF1a/ORF1b RFS that has been predicted previously to adopt a stem-loop structure in NDiV. The alignment illustrates sequence variations in nucleotides predicted in NDiV to be involved in base pairs that sustain the structure.Click here for file


An increasingly asked question is 'can we confidently link bats with emergingviruses?'. No, or not yet, is the qualified answer based on the evidence available.Although more than 200 viruses - some of them deadly zoonotic viruses - have beenisolated from or otherwise detected in bats, the supposed connections between bats,bat viruses and human diseases have been raised more on speculation than on evidencesupporting their direct or indirect roles in the epidemiology of diseases (except forrabies). However, we are convinced that the evidence points in that direction andthat at some point it will be proved that bats are competent hosts for at least a fewzoonotic viruses. In this review, we cover aspects of bat biology, ecology andevolution that might be relevant in medical investigations and we provide ahistorical synthesis of some disease outbreaks causally linked to bats. We provideevolutionary-based hypotheses to tentatively explain the viral transmission routethrough mammalian intermediate hosts and to explain the geographic concentration ofmost outbreaks, but both are no more than speculations that still require formalassessment. These characteristics and the ecosystemservices they provide set them apart from all other mammals. Other aspects of their naturalhistory, ecology, biology and evolution also distinguish them from other mammals, but nonehas been more emphasised recently than their potential link with human diseases,particularly those caused by zoonotic viruses , the second smallest mammal known] to 1 kgin some flying-foxes [Pteropus spp (Pteropodidae)], whose wingspans canreach 2 m , crevices and foliage as day roosts  and manyBat reproduction is similar to that of other mammals, but mating and duration ofbreeding season are strongly influenced by hibernation and migration. In hibernatingbats of various species, copulation generally occurs in the fall, either with ovulationand fertilisation occurring immediately, followed by a slow development of the fetusthroughout the winter and birth in the spring, or with sperm storage until spring, afterwhich ovulation, fertilisation and foetal development occurs. In migratory bats, matingis generally concentrated in the early spring, after they reach their summer roosts.These bats and several others that do not need to migrate or hibernate, but that live inareas with food availability varying seasonally, reproduce once a year (monoestrus),whereas others facing less marked seasonal variation or having food resources availableyear-round generally reproduce twice or more a year (polyestrus). The former pattern ischaracteristic of most insectivorous bats from temperate regions, whereas the latter iscommon in most frugivorous bats living in tropical regions. Gestation averages about twomonths and bats of most species produce a single young per litter, but a few producetwins and a very few produce three or four young at a time . Halpin Myotis brandtii (Vespertilionidae)] from Siberia wasrecaptured 41 years after the first capture ] form the largest warm-blooded non-humanvertebrate colony in the world.Bats of some species also form large aggregations and a few of them form the largestaggregations of mammals in the world. Old World fruit bats (Pteropodidae) of variousspecies constitute colonies of hundreds of thousands to millions of bats that aggregateon exposed tree branches . InBracAlthough bats are outnumbered by rodents in species richness, they are first amongmammals (and probably among vertebrates) in dietary diversity  which inEvolutionary history - Chiroptera is a middle Paleocene or early Eocenelineage of placentals ,the prevention of diseases and the expanded studies of bats for one reason or anotherlikely will provide more information about the bats themselves and, in the long run,help us protect bat populations from further decline.Summary of viruses from bats - More than 200 viruses have been isolatedfrom or detected in bats. Represented are bats of both suborders, 11 families and 37genera . Classical rabies virus (street rabies) is the prototype virus of a complex ofviruses that have been detected in bats, dogs and other mammals. This negative sense,single-stranded RNA virus has been classified in the virus order Mononegavirales, familyRhabdoviridae  and genus Lyssavirus .Canis lupus ], now domesticated asdogs (C. lupus familiaris). Even as far back as 2300 BC dog owners inthe Babylonian city of Eshnunna were fined for deaths caused by their dogs having bittenpeople. In 500 BC, the Greek philosopher Democritus described a case of canine rabies.In 400 BC, Aristotle wrote 'dogs suffer from the madness. This causes them to becomevery irritable and all animals they bite become diseased', In the first century, Romanwriter Cardanus described the saliva from a rabid dog as a 'virus' . In 1804, a German scientist, Georg Gottfried Zinke,demonstrated that rabies could be passed through saliva from rabid dogs.Rabies may be the oldest human infectious disease known. Its origin has been associatedwith wolves  in 1931 ] in Kyrghyzstan, Central Asia, Australian bat lyssavirus, from black, gray-headed , little red (Pteropus scapulatus) andspectacled (Pteropus conspicillatus) flying-foxes (Pteropodidae) andyellow-bellied pouched bats [Saccolaimus flaviventris], in Australia, Bokeloh virus from a Natterer's myotis [Myotisnattereri (Vespertilionidae)] in Germany, Duvenhage virus, from Schreibers'slong-fingered bat [Miniopterus schreibersii (Miniopteridae)] andEgyptian slit-faced bat [Nycteris thebaica (Nycteridae)] in SouthAfrica, European bat lyssavirus 1, from common serotines [Eptesicusserotinus (Vespertilionidae)], European bat lyssavirus 2, from Daubenton'sbats [Myotis daubentonii (Vespertilionidae)], Irkut virus, from greatertube-nosed bats [Murina leucogaster (Vespertilionidae)] in Russia andChina, Khujand virus, from a whiskered myotis [Myotis mystacinus(Vespertilionidae)] in Tajikistan, Lagos bat virus, first isolated from a Africanstraw-coloured fruit bat [Eidolon helvum (Pteropodidae)] in Nigeria,Lleida bat lyssavirus, from Schreibers's long-fingered bat in Spain, Shimoni bat virus,from a Commerson's leaf-nosed bat [Hipposideros commersoni(Hipposideridae)] in Kenya and West Caucasian bat virus, from a Schreibers'slong-fingered bat in Russia. Mokola virus, although not isolated from bats, also is amember of the rabies-virus related virus group and has been isolated from shrews[Crocidura spp ] and domestic cats inNigeria. Since the initial detections of these viruses, with the exception of Mokolavirus, most have been detected in bats of species other than those mentioned above.Among those mentioned above, only pteropodids feed on plants, whereas all others feed onanimals, including insects, other invertebrates or vertebrates. rom bats . AravanSevere rabies-like disease in humans has led to additional studies of flying-foxes,resulting in a greater understanding of the epidemiology and geographic distribution ofAustralian bat lyssavirus. Based on this evidence, Hendra, Nipah and other paramyxovirus diseases - Hendra and Nipahviruses are both highly pathogenic zoonotic paramyxoviruses  that have been detected in pteropodid bats within the last decades ,responsible for a 1997 zoonotic disease affecting pigs and humans in New South Wales,Australia. Antibodies capable of neutralising Menangle virus were detected inflying-foxes, providing provisional evidence of a bat origin for this virus; the viruslater was isolated from black flying-foxes . SamplesIn 1998 yet another paramyxovirus, this one named Nipah virus, was recognised as theetiologic agent of a fatal disease of humans and pigs in Malaysia and Singapore . By JunePteropushypomelanus) and in fruits partially eaten by them, confirming these bats asnatural hosts of the virus (Pteropuslylei (Pteropodidae)] in Cambodia ] and viaperson-to-person route. Nipah virus RNAs detected in Bangladesh are variable in theirsequences, suggesting multiple introductions via Indian flying-foxesPteropus giganteus (Pteropodiae), which migrate long distances andare found in the Maldives, India, Bangladesh, China, Nepal, Pakistan and Sri Lanka.In contrast to transmission of Nipah virus from bats elsewhere, transmission inBangladesh was found to be Mustela putorius furo ] anddomesticated guinea pigs [Cavia porcellus ], bothsusceptible to infection and disease with known henipaviruses, confirmed virusreplication and production of neutralising antibodies, but no clinical disease. Also,the major genetic difference between Cedar virus and Hendra and Nipah viruses lieswithin the coding strategy of the P gene, known to play an important role in evading thehost innate immune system. Preliminary studies indicated that Cedar virus infection ofhuman cells induces a more robust interferon-β response than does Hendra virus. Cedarvirus is one that might be studied to develop a human and livestock vaccine.RNA of Cedar virus, another henipavirus, was detected in urine of pteropodid bats inAustralia in 2009, but little is yet known about this virus . ChallenIntriguing evidence for infection with a henipavirus in African bats was presented bySevere acute respiratory syndrome (SARS) - The hundreds of human casenotifications of SARS in Guangdong Province, People's Republic of China in late 2002,then elsewhere in the world, moved bat virus recognition from unanticipated andoccasional to well-planned and more methodical. Thereafter the World Health Organization(WHO) put the entire world on alert. SARS cases soon were diagnosed in patients not onlyin Vietnam, but also in Hong Kong and Canada, and cases were diagnosed in health careworkers and household members who had cared for patients with the disease. Many of thecases were traced back through chains of transmission to a health care worker fromGuangdong Province who had visited Hong Kong, where he was hospitalised with pneumoniaand died. By late April 2003, more than 4,000 SARS cases and 250 SARS-related deathswere reported to the WHO from more than 25 countries. Most of these cases occurred afterexposure to SARS patients in health care or household settings.in nature had not been determined at that time,but knowing it was a CoV made the search easier.The WHO coordinated a massive international collaborative effort that included clinical,epidemiologic and laboratory investigations and simultaneously initiated efforts tocontrol the spread of the disease. Attempts to identify the causative agent of theoutbreak were successful during 2003, when laboratories in the United States of America(USA), Canada, Germany and Hong Kong isolated a novel CoV (SARS CoV) from SARS patients.Classified as a virus in the order Nidovirales, family Coronaviridae and genus CoV,unlike other human CoVs, this one can be isolated in Vero cells. SARS CoV RNA hasfrequently been detected in respiratory secretions and convalescent-phase serumspecimens from SARS patients contain antibodies that react with SARS CoV, altogetherproviding evidence that it was a newly recognised virus and associated with the disease.The source of the virus CoVs comprise a diverse group of large, enveloped, positive-stranded RNA viruses thatcause respiratory and enteric diseases in humans and other animals. Their genomes arethe largest  of any RNA virus known. A great deal is knownabout CoVs, nicely summarised by Paguma larvata ] andraccoon dogs [Nyctereutes procyonoides ] in livemarkets of wild animals in mainland China - were proposed, but were more confusing thanhelpful to our understanding of the origin and spread of the virus. Many possible natural history scenarios - among others, human infections originatingwith masked palm civets [Middle East respiratory syndrome (MERS) - MERS is caused by a CoVcalled MERS CoV. Cases of this disease were first reported from Saudi Arabia by ProMEDand formally published soon after  (Neoromiciacf. zuluensis (Vespertilionidae)] by Camelus dromedarius ] may be a primary source of this virus in nature (on after . MERS afnuridae) ] in Saudn nature  and expen nature . MERS CoMarburg and ebolavirus haemorrhagic fevers - Marburg and ebolaviruses -, genera Marburgvirus and Ebolavirus, respectively, the'filoviruses' - were discovered because they cause severe, often fatal, haemorrhagicdiseases in humans and other primates.Marburgvirus disease - In late summer of 1967, an haemorrhagic feveroutbreak was observed in laboratory workers in Serbia, at the time part of Yugoslaviaand in Germany. It was soon shown that the disease was transmitted from green monkeys[Chlorocebus sabaeus ] consigned fromUganda to Europe, but the origin of the disease was unknown, other than that it wascaused by a hitherto unrecognised virus, named Marburg virus for the city in Germanywhere the disease was first recognised. Another infection with this virus occurred in atraveller in Africa in 1975 and cases of Marburg haemorrhagic fever have been documentedwith some frequency in various parts of Africa since then. Although a great deal waslearned about this virus from pathologic and laboratory studies, its epidemiologyremained undetermined. Nonetheless, bits and pieces of evidence suggested that batsmight be associated with Marburg virus. It was not until 1999 that Rhinolophus eloquens(Rhinolophidae)] and a greater long-fingered bat [Miniopterus inflatus(Vespertilionidae)] captured in the Democratic Republic of the Congo and Ebolavirus diseases - In 1976, a series of severe and often fatalhaemorrhagic fevers occurred in southern Sudan. Almost immediately after those caseswere recognised, a similar disease was observed in humans in Zaire, now known as theDemocratic Republic of the Congo, nearly 1,000 km away. A virus, originally termed'Ebola virus', named after the Ebola River near the epidemic site in Zaire, was isolatedfrom patients in Zaire and partially characterised ]imported from a commercial source in the Philippines to a primate holding site inReston, Virginia, USA. No human illnesses among animal handlers were recognised ] in the Taï Forest ofCôte d'Ivoire resulted in an accidental infection and non-fatal illness of aninvestigator performing a necropsy on one of these primates. Virus isolation from thispatient was successful and the virus named Taï Forest ebolavirus. The cause of deaths ofthe members of the chimpanzee group was later shown to be infection with this virus.This was the first time that a human ebolavirus infection had been connected tonaturally-infected non-human primates in Africa , was detected during the investigation of bat die-offs in Cueva del Lloviuin Spain in 2002. Lloviu virus is genetically distinct from marburgviruses andebolaviruses and is the first filovirus detected in Europe that was not imported from anendemic area in Africa. Whereas infections of bats with marburgviruses and ebolavirusesdo not appear to be associated with disease in the bats, Lloviu virus was detected in adead Schreibers's long-fingered bat.Epomophorus wahlbergi (Pteropodidae)], little free-tailed bats[Chaerephon pumilus (Molossidae)] and Angolan free-tailed bats[Mops condylurus (Molossidae)] as potential hosts for Zaireebolavirus. In addition, from 2001 and 2003, Hypsignathus monstrosus),Franquet's epauletted fruit bats (Epomops franqueti) and littlecollared fruit bats (Myonycteris torquata). These RNA sequences werequite similar to those of the ebolavirus isolated from humans during the 1976 outbreakin Zaire. Other investigations led to antibody detections in bats of the same species.Survival of bats from experimental infections led Based on the work of Eptesicus fuscus(Vespertilionidae)] experimentally infected with Japanese encephalitis virus atvarious environmental temperatures and then tested them for both virus and neutralisingantibody. Viraemias were demonstrable in most of these bats within two-three days afterinfection and persisted for one-two weeks. However, not all viraemic bats producedantibody to the virus, a few others had equivocal and then delayed (nearly 2 months)antibody responses and some that had produced antibody soon after being infected nolonger had detectable antibody three months later.In addition to their other well-known uniquenesses, it may be asked whether bats haveunique responses to virus infections. That is, why (or how) do bats survive infectionswith viruses and other agents that are pathogenic for other vertebrates? Or do they?These questions have not been answered satisfactorily and data accumulated thus far havenot definitively answered them, but have led to speculation, much of which isfascinating. Serologic studies demonstrated that bats have antibody to many viruses, butit was not determined whether their antibody isotypes were similar to those of othermammals. Nearly 50 years ago, for example, Carolliaperspicillata (Phyllostomidae)], whereas little brown myotis [Myotislucifugus (Vespertilionidae)] have five IgG subclasses ] differs somewhat from that of In an effort to provide a foundation for understanding adaptations by bats that mightallow them to at least somewhat peacefully coexist with viruses that infect them, Other studies of bat genomes have indicated differences in genes associated with earlyimmune responses, which might allow virus to replicate in the absence of a primary andhearty immunological response. It is further reasonable to assume that this in turnmight allow at least low-level virus replication over an extended period. There is someevidence that these genes may be evolving more rapidly in bats than in vertebrates ofother taxa, which would suggest that the genes are co-evolving with the bats in responseto viral and other infections. An additional note of caution: when studying viruses frombats in order to understand their effects, it is clearly more appropriate to use bats ortheir cells in culture, rather than other hosts or cells from other hosts. That is,simply because a virus or other pathogen might interfere with or activate a gene productin one host, there is no certainty that it does the same in another host; thus theextrapolatory and speculatory nature of many available publications. A recent paper byHumans and most other mammals respond to infections by activating their immune systemsat the time they are infected, usually producing type I and type III interferons, whichare temporising responses activated prior to humoral antibody responses. The immunesystems of bats of at least one species (black fling-fox), on the contrary, appear to beperpetually switched on at a low level, thus allowing them to respond quickly to aninfectious agent .AlternaPseudogymnoascus destructans , maybring about a severe immune reconstitution inflammatory syndrome . Because bats represent such a largeproportion of mammals (about 20%) and are so diversified in their biology, habitats andnatural history, it seems reasonable to assume that they have many hundreds moreviruses, just as do other diversified groups of life forms. A few hypotheses have beenraised to explain so many viruses in bats ( in bats .HoweverArtibeus lituratus) and little yellow-shoulderedbats (Sturnira lilium) had circulating virus levels high enough toinfect culicid mosquitoes  that may serve as vectors for the virus,whereas great spear-nosed bats [Phyllostomus hastatus (Phyllostomidae)]did not develop sufficient viraemia to infect subsequently feeding mosquitoes  also figure among the mammals in which viruses apparently circulate. Except forhumans, these mammals are from groups that have evolved within the superorderLaurasiatheria. Horses are representatives of the order Perissodactyla, pigs and camelsare in the order Artiodactyla and ferrets, palm civets and raccoon dogs are in the orderCarnivora; all are members of the superorder Laurasiatheria.Although bats have been distinct from other mammals for more than 52 million years,bats, carnivores perissodactyls and artiodactyls share part of their evolutionaryhistory that is not shared with other mammals and their ancestors may have co-evolvedwith ancient lineages of certain viruses. This suggests that they may sharephysiological characteristics that could facilitate circulation of these viruses. The taxonomic and geographic distribution of bats whose viruses have potentially jumpedto humans is also noteworthy. Except for rabies, all episodes are in the Old World andpteropodid, hipposiderid and rhinolophid bats - representatives of families in thesuborder Yinpterochiroptera - have been mostly involved . To dateFuture investigations - We still know comparatively little about theimmunological and physiological systems of wild mammals of different orders. With batsbeing potential reservoirs of a long list of viruses ] are potential models for research in South America.They are widely distributed on the continent, adapt well to human constructions and feedon insects  that have continental distributions as potential models to testenzootic (or endemic) viruses. As an example, Pallas's mastiff bats [ insects , making In addition, fieldwork is necessary to continually searching for new pathogens and tounderstand the mechanisms underlying the dynamics of zoonotic diseases. It is importantto design field studies to understand the role of different biotic and abiotic factorsaffecting bat populations and pathogen circulation in bats and how these factors mayfavour spillovers to humans .Joining expertise from bat biologists and medical investigators, this scenario canquickly move forward to a new one in which the role of bats in the circulation ofzoonotic viruses and other pathogenic agents will be at least minimally understood.After understanding the role of bats  in the maintenance andcirculation of pathogens and the mechanisms underlying the emergence of zoonoticdiseases, wildlife biologists and epidemiologists should work together to developappropriate management plans to control virus circulation and minimise risks of humaninfection without causing significant biases against specific animal populations. Wecannot ignore the potential role of bats in the maintenance, circulation andtransmission of pathogens to humans and we cannot ignore the important ecosystemservices provided by these animals. Thus, the only possible approach is to developresponsible research to avoid the obstacles and keep safe both human and batpopulations. In addition, wildlife biologists and medical investigators should worktogether to provide expertise to wildlife epidemiologists. This training could beprovided by graduate programs in public health.Finally, can we confidently link bats with emerging viruses? No, or not yet, is thequalified answer based on the evidence available. Only integrative and organised fieldand laboratory research, using ecological and epidemiological approaches conducted bybat biologists and medical researchers, will provide a useful and satisfactorysolution.