We investigated synanthropic small mammals in the Ethiopian Highlands as potential reservoirs for human pathogens and found that 2 rodent species, the Ethiopian white-footed mouse and Awash multimammate mouse, are carriers of novel Mobala virus strains. The white-footed mouse also carries a novel hantavirus, the second Murinae-associated hantavirus found in Africa. Mastomys and Arvicanthis are linked to human activity; these rodents are widespread throughout sub-Saharan Africa and are crop pests and zoonotic reservoirs for human pathogens. Histories of synanthropy are likely longest for rodents in areas of early human sedentism, making RNA virus richness in early centers of domestication such as the Ethiopian Highlands of particular interest.Most emerging infectious diseases of humans or domestic animals are zoonoses, and among emerging pathogens, RNA viruses are highly represented . The synBunyaviridae) are RNA viruses primarily carried by rodents and soricomorphs (shrews and moles), although 2 new species have recently been described in bats (Arenaviridae) are primarily rodent-borne RNA viruses. Members of both genera can cause life-threatening diseases in humans: arenaviruses cause hemorrhagic fevers in the Americas and Africa, and hantaviruses cause hemorrhagic fever with renal syndrome in Asia and Europe and hantavirus cardiopulmonary syndrome in the Americas. In Africa, only Lassa and Lujo arenaviruses are known to be highly pathogenic to humans. In contrast, hantaviruses have not yet been found to cause life-threatening human diseases in Africa, but hantavirus-specific antibodies have been found in human serum samples from several countries in Africa  and Mahbere Silassie , and in 1 lower-altitude locality, Aroresha , in the Tigray region of the Ethiopian Highlands. Kidney samples preserved in RNAlater reagent  and stored at –80°C were used for total RNA extraction by using the NucleoSpin RNA II Kit . Samples were pooled in pairs by locality and host species. RNA was reverse transcribed by using random hexamers as primers. Screening for arenaviruses was performed by using a pan–Old World arenavirus PCR targeting the large (L) gene  from Golgolnaele and 2 Awash multimammate mice (Mastomys awashensis) from Aroresha were positive for arenavirus RNA; 10 white-footed mice from the 2 highland localities  were positive for hantavirus RNA. Amplicons were purified and sequenced, and nucleotide sequences were aligned on the basis of the amino acid alignment. Phylogenetic analyses were performed on the nucleotide sequences by using a maximum-likelihood (ML) approach , with the Menekre virus, found in Hylomyscus sp. in Guinea  of representatives of Old World arenaviruses . The 3 s in 1983 . Howevern Guinea , as outgS. albipes mice, a Murinae species endemic to Ethiopia, they do not group with the Murinae-associated hantaviruses or with hantaviruses found in other African small mammals, such as bats (S. albipes mice are carrying a novel hantavirus. We propose the name Tigray virus for this virus because it was found in the Tigray region of Ethiopia. Additional genetic characterization, in particular of the small and medium segments, will be conducted to further clarify the evolutionary relationship of this virus within the hantavirus genus.After sequencing of the 10 hantavirus-positive samples, 4 distinct hantavirus sequences were obtained, 2 from Golgonaele and 2 from Mahbere Silassie.  as bats  or shrew as bats ,13. The Two rodent species living in close proximity to human settlements in Ethiopia are carriers of arenaviruses and hantaviruses. Recently, several new arenaviruses and hantaviruses have been described in small mammals in Africa, but no clear association with human diseases has been found ,9,11–13.M. awashensis and S. albipes. These rodents belong to the exclusively African Praomyini tribe (In conclusion, our screening of 201 small mammals led to the identification of 2 novel strains of Mobala arenavirus and a novel hantavirus in 2 rodent species found in Ethiopia, ni tribe , which h


WMSA volume, particularly in the centrum semiovale, on TEA MRI was a significant predictor of standardized language and cognitive scores at 2 years of age. Independent validation of our automated WMSA detection algorithm and school age follow-up are important next steps. The developmental significance of the frequently encountered white matter signal abnormality (WMSA) findings on MRI around term-equivalent age (TEA) in very preterm infants, remains in question. The use of conventional qualitative analysis methods is subjective, lacks sufficient reliability for producing accurate and reproducible WMSA diagnosis, and possibly contributes to suboptimal neurodevelopmental outcome prediction. The advantages of quantitative over qualitative diagnostic approaches have been widely acknowledged and demonstrated. The purpose of this study is to objectively and accurately quantify WMSA on TEA T2-weighted MRI in very preterm infants and to assess whether such quantifications predict 2-year language and cognitive developmental outcomes. To this end, we constructed a probabilistic brain atlas, exclusively for very preterm infants to embed tissue distributions . Guided with this atlas, we then developed a fully automated method for WMSA detection and quantification using T2-weighted images. Computer simulations and experiments using  Very preterm infants are at high risk for neurodevelopmental impairments. By school age, 30-50% of them exhibit cognitive impairment ,2. Many A few studies with neurodevelopmental follow-up have observed a significant negative association between WMSA and developmental quotient at 18 months corrected age ,6 and inThe limited progress in resolving the above question can partly be attributed to the use of conventional qualitative MRI readings. Such an approach remains subjective and lacks sufficient reliability for accurate and reproducible WMSA diagnosis  and may For T2-weighted sequences without fluid attenuation, the signal intensity distribution of WMSA greatly overlaps with that of cerebrospinal fluid (CSF), making the distinction between them difficult ,19. To oAtlas-guided analysis methods are usually characterized by how the atlases are generated and how the knowledge regarding atlases can be carried forward to target images. The atlas can be constructed using manual/semiautomatic/automatic segmentations of an individual or a group of individuals -27. Our  In this work, we constructed a probabilistic brain atlas, exclusively for very preterm infants and then guided with this atlas, we proposed a fully automated WMSA detection and quantification method using clinically available T2-weighted images. Computer simulations and experiments on clinical MRI data presented very high detection accuracy. Finally, in a prospective cohort of ELBW infants, we demonstrated a significant correlation between objectively quantified WMSA volumes and language and cognitive developmental scores on the third edition of the Bayley Scales of Infant and Toddler Development at two years of age. The Children’s Memorial Hermann Hospital (CMHH) and The University of Texas Medical School at Houston joint institutional review board (IRB) approved the study. Written informed consent for each infant was obtained prior to enrollment in the study. The consent was approved by IRB and signed by each subject's parent or guardian prior to enrollment and participation in the study.The study population was derived from a cohort of 50 ELBW infants without any major congenital anomalies, cared for in the NICU of Children’s Memorial Hermann Hospital from 2007 to 2009. Their mean (standard deviation [SD]) gestational age was 25.5 (1.6) with a range of 23 to 30.1 weeks; birth weight was 750.8 (143.1) grams with a range of 468 to 1000 grams; and the postmenstrual age at MRI was 38.7 (2.3) with a range of 34.1 to 43.9 weeks. Nineteen MRI scans were randomly selected for atlas construction from the cohort study. Their mean (SD) gestational age was 25.8 (1.8) with a range of 23.4 to 30 weeks; birth weight was 724.2 (148.4) grams with a range of 489 to 1000 grams; and post-menstrual age at MRI was 38.1 (2.1) weeks with range of 34.1 to 41.7 weeks. 2; the imaging matrix = 256×256 mm2; slice thickness = 2 mm. In this study, T2-weighted images (corresponds to TE2) were used to construct the atlas and perform the analysis. All infants were transported to the MRI scanner by an experienced neonatal transport nurse after feeding, swaddling and placement of silicone ear plugs  and Natus MiniMuffs  to facilitate natural sleep and attenuate MRI noise. Sedation was not used for any of the cases. All scans were completed under the supervision of a neonatal research nurse and a neonatologist experienced in neonatal MRI. All MRI scans were read by a neuroradiologist using a standardized reporting tool.All infants were imaged with a 3 T Philips scanner using a dual-echo fast-spin-echo sequence with use of an eight-channel SENSE-compatible phased array receive head coil. The imaging parameters used were: TE1 = 8.75ms; TE2 = 175 ms; TR = 10,000 ms; flip angle = 90°; FOV = 180×180 mmAll 19 ELBW infants randomly chosen for the atlas creation, had varying degrees of WMSA ranging from only in the frontal and/or occipital periventricular white matter up to signal abnormalities covering two-thirds of the cerebral WM. The procedures for atlas creation were similar to the general concept for creating an adult/neonatal group-averaged atlas: 1. Extrameningeal tissues were manually stripped and brain tissues  were manually parcellated using ANALYZE 8.1 software , as previously described in detail in  months corrected age). The cohort BSID-III language scores had a mean of 94.3 and a standard deviation of 14.0; cognitive scores had a mean of 88.7 and a standard deviation of 17.4. Correlational statistical analyses showed that BSID-III language and cognitive scores were significantly (ns of WM . WMSA atatlas should be weighted heavily; otherwise, one should consider weighing μspm heavily. Importantly, this approach likely contributed to our finding a significant association between WMSA volume and short-term standardized measures of language and cognitive development in ELBW infants.Accurate and reproducible quantification of WMSA is critical for determining its developmental significance and potentially identifying high-risk preterm infants that may benefit from neuroprotective and early intervention therapies. In this paper, we present a fully automated atlas-guided method to detect and quantify WMSA on conventional T2-weighted imaging. Atlas-guided anatomic information was incorporated with unified segmentation and the relative importance of both components was controlled by two parameters. We assigned equal weights to both components, recognizing that different weighting would likely produce different results. Intuitively, if the knowledge carried by an atlas can be precisely forwarded to a target, μSeveral studies have found that adult atlases do not form a suitable reference space for children and neonates, due to the considerable differences in size, geometrical proportions and brain tissue properties -39. HoweOur studies were performed on unsedated infants who typically fall asleep after being fed and swaddled. The ability to now use conventional T2-weighted over multiple echo weighted imaging helps reduce scan time, resulting in less motion artifacts and need for sedation. Use of parallel imaging techniques, as used in our study, is also helping greatly in this regard. These innovations will facilitate larger multicenter imaging studies in very preterm infants and aid in definitively establishing the role of advanced brain MRI in this vulnerable population. Once more cases can be added to our preterm atlas, additional refinements such as localization of WMSA will be necessary to fully understand the developmental significance of WM hyperintensities in periventricular crossroads regions versus subcortical or centrum semiovale regions ,13.In our cohort of very preterm infants, we demonstrated automatically detected WMSA volume on TEA MRI to be a significant predictor of cognitive and language development at 2 years of age. Prior efforts to correlate WMSA with cognitive outcomes may have failed due to the low reliability of diagnosing WMSA qualitatively ,14. Our The volume of WMSA in the centrum semiovale was the strongest predictor of Bayley scores, particularly language scores. This is consistent with findings from Krishnan et al. , who repin vivo very preterm data showed very high detection accuracy. Correlational analyses demonstrated that WMSA volume at TEA predicted short-term language and cognitive developmental outcomes. Our work will facilitate population-based studies to more accurately characterize WMSA’s long-term sequelae. In summary, we constructed an age-specific very preterm infant probabilistic atlas. Guided with this atlas, we then proposed a fully automated method for WMSA detection and quantification using clinically available T2-weighted images. Computer simulations and validation using


RNA was amplified based on isothermal amplification by Phi29 polymerase before hybridization. We were able to detect and characterize Nipah virus and Crimean–Congo haemorrhagic fever virus (CCHFV) in the brains of experimentally infected animals. CCHFV was finally used as a paradigm for epidemics because of recent outbreaks in Turkey, Kosovo and Iran. Viral variants present in human sera were characterized by BLASTN analysis. Sensitivity was estimated to be 105–106 PFU/mL of hybridized cDNA. Detection specificity was limited to viral sequences having ∼13–14% of global divergence with the tiled sequence, or stretches of ∼20 identical nucleotides. These results highlight the benefits of using the PathogenID v2.0 resequencing microarray to characterize geographical variants in the follow-up of haemorrhagic fever epidemics; to manage patients and protect communities; and in cases of bioterrorism.Several haemorrhagic fevers are caused by highly pathogenic viruses that must be handled in Biosafety level 4 (BSL–4) containment. These zoonotic infections have an important impact on public health and the development of a rapid and differential diagnosis in case of outbreak in risk areas represents a critical priority. We have demonstrated the potential of a DNA resequencing microarray (PathogenID v2.0) for this purpose. The microarray was first validated  Henipavirus, family Paramyxoviridae and haemorrhagic fever viruses in the families Arenaviridae, Filoviridae, Bunyaviridae and FlaviviridaeFlavivirus (family Flaviviridae) are arthropod-borne, as are those of the family Bunyaviridae, except for the genus Hantavirus which is rodent-borne or insectivore-borne Arenaviridae are also rodent-borne Henipavirus have bat reservoirs but may also infect humans through contact with infected horses or pigs FiloviridaeViruses recognized as highly pathogenic for humans must be manipulated in a Biosafety level 4 (BSL-4) laboratory. They include viruses associated with encephalitis and respiratory infections, such as recently emerged members of the genus athogenID v2.0, has been shown to be useful for rapid diagnosis during emerging viral infections, such as the 2009 influenza pandemic RhabdoviridaeDevelopment of vaccines to prevent infection by these emerging zoonotic viruses is limited and only ribavirin has been used as an efficacious treatment for several of them athogenID v2.0 microarray to detect highly pathogenic viruses. We first validated the microarray with in vitro samples by analysing supernatants from cells infected by prototype virus strains and variants belonging to five families of BSL-4 agents . We then evaluated its performance during a health emergency situation by testing human sera from CCHFV outbreaks in Turkey (2009), Kosovo (2001) and Iran (2009). CCHFV belongs to the genus Nairovirus, family Bunyaviridae and has the largest geographic distribution among haemorrhagic fever viruses Hyalomma, or indirectly through contact with infected livestock. Hospital environments are also vulnerable to inter-human transmissions Here, we used the PThis work includes a retrospective study on 12 human sera from clinical specimens submitted to France National-WHO-OIE Reference Centres for diagnosis during CCHF epidemics in Kosovo, Turkey and Iran.The collection of the remaining samples to be used for scientific purpose was declared to and approved by the Comité de Protection des Personnes, Ile-de-France I and the French Research Ministry (no. DC 2011-1471) according to French regulations.Animal experimental methods were approved by the Région Rhône Alpes Ethics Committee (France).Arenaviridae) and Sin Nombre virus  synthetic cDNA sequences  were used as templates for the amplification step.Viral strains and geographical variants  were culSera from 12 infected humans were collected during CCHFV outbreaks (2003–09) in the Balkans  and the Middle East .Saimiri sciureus) was experimentally infected intravenously with 103 PFU UM-MC1 Malaysian isolate of Nipah virus One non-human primate, a New World squirrel monkey (3 PFU) each in the BSL-4 animal-care facility in Lyon. Seven days after infection, mice were euthanized. Brain was collected, crushed in phosphate-buffered saline 1 × (1/10 weight/volume), and clarified by centrifugation for 15 min at 600 g before storage at −80°C.In another experiment, ten newborn Swiss mice were intracranially inoculated with 20 µL CCHFV  as previously described Extracted viral RNAs were reverse transcribed into cDNA using SuperScript III reverse transcriptase  then amplified by the whole transcriptome amplification (WTA) approach in the presence of random hexamer primers. An optimized protocol based on isothermal amplification by the Phi29 polymerase was applied to the QuantiTect Whole Transcriptome Kit (Qiagen) as previously described Quantitative RT-PCR and PCR amplifications of CCHFV sequences present in infected cell supernatants or human sera were performed in a Light-Cycler Instrument  athogenID v2.0 microarray is the second generation of a microarray developed through a collaboration between Affymetrix and Institut Pasteur The PPathogenID v2.0 microarray. The array was then washed and scanned according to instructions provided by Affymetrix. Results were analysed using GeneChip Operating Software version 4.0 (GCOS), GeneChip Sequence Analysis Software version 4.0 (GSEQ), and the ABACUS algorithm The entire microarray experimental procedure is summarized in The call rate value (the percentage of nucleotides identified by the microarray) obtained from each sample hybridized on the microarray was used to determine the degree of hybridization of that sample and to compare it with that of other samples. All the obtained sequences were exported into a FASTA-formatted file and then subjected to BLASTN analysis to identify viral variants.After scanning and analysis, all the chips were destroyed according to BSL-4 waste guidelines.PathogenID v2.0 microarray or for clinical investigation of the outbreaks, were sequenced directly. To analyse the CCHFV strains, classical, nested or semi-nested PCR were performed to amplify the region tiled on the microarray, e.g. the 531 bases of the L segment encoding the RNA-dependent RNA polymerase. Degenerate primer design and sequence analysis were performed using MacVector software . Primer position refers to the L genome segment of the prototype CCHFV strain (IbAr10200): fw2645 (5′-TGCTCWTTYATTGCCTGTGC-3′); rev3269 (5′-TNACACCRTTGGGGTGACA-3′); fw2576(5′-GGGAAAATAAGGACAGACCA-3′); rev3371 (5′-TCYGTTAAGCATTCATTRCT-3′). The PCR fragments were purified by ultrafiltration before sequencing . Sequencing was performed using a BigDye Terminator v1.1 cycle sequencing kit  and purified by ethanol precipitation. Sequence chromatograms from both strands were obtained on an automated sequence analyser ABI3730XL (Applied Biosystems) with the PCR primers. The percentage of sequence divergence was calculated for each sample by determining the number of mutations relative to the prototype sequence tiled on the microarray.All specimens used either for the validation steps of the BioNumerics software for Windows . The sequences used for this purpose were: (i) all the RNA-dependent RNA polymerase L sequences available in GenBank, (ii) the sequences obtained by direct sequencing and (iii) the sequences obtained from the microarray results.A phylogenetic analysis of CCHFV sequences was performed by the neighbour-joining method using PathogenID v2.0 resequencing microarray to detect and identify a number of different highly pathogenic viruses. This work was divided into two parts: (i) a validation step, in which we used supernatants from cultured cells infected with viral strains that matched the prototype probes tiled on the microarray and their variants, and (ii) an exploratory step, in which we used human sera from CCHFV outbreaks to evaluate the potential of the microarray to be used in public health emergencies.We used the high-density PathogenID v2.0 microarray could be used to correctly detect and identify different viruses present in a single sample designed to resemble a complex biological specimen that might occur in nature or the laboratory (i.e. screening a pool of samples). Hence, total RNA was extracted from the supernatants of cells that had been infected with a single viral strain. Then, pools of RNA from up to three different supernatants were made to resemble likely combinations that might coexist in the same geographical area or animal host . For Old World viruses of the family Arenaviridae, the tiled Lassa virus sequence  detected the homologous strain  and a variant from Guinea . In addition, even a divergent variant from Ivory Coast (AV) was significantly detected . Among the family Bunyaviridae, the Sin Nombre virus NM–R11, Hantaan virus 76-118, Rift Valley fever virus ZH548 and CCHFV IbAr10200 hybridized to their homologous sequences as expected . Moreover, the tiled Nigerian CCHFV IbAr10200 sequence also detected CCHFV variants from Mauritania, Turkey and China . Similarly, the tiled Dobrava-Belgrade virus sequence (DOBV/Ano-Poroia/Af19/1999) detected the variant 3970/87 from Slovenia  whereas the Seoul virus sequence detected the related Tchoupitoulas virus . For the family Filoviridae, the tiled Popp strain of Marburg marburgvirus was as efficient for detection of the Musoke strain  as for the homologous strain . The tiled Reston ebolavirus and Zaire ebolavirus sequences allowed detection of the homologous species  and an additional variant from Gabon 2001 . Among the family Flaviviridae, the tiled sequences from the Kyasanur Forest disease virus KFD P 9605 and tick-borne encephalitis virus Neudoerfl detected the heterologous Alkhurma virus  and Omsk haemorrhagic fever virus Balangul , respectively. Finally for the genus Henipavirus (family Paramyxoviridae) Nipah virus Malaysia and Hendra virus Australia were perfectly detected by the homologous sequence .The microarray detected and characterized each virus prototype to similar levels of sensitivity whether the viral RNA was tested alone or in a pool . SimilarPathogenID v2.0 resequencing microarray very efficiently detected: (i) prototype virus strains and the two synthetic probes with excellent call rates (>97%); (ii) variants with high call rates similar to those of prototype strains ; (iii) variants with moderate call rates . Variants with low call rates  were also detected, although less significantly. Finally, highly divergent variants were not detected by the microarray (data not shown).In summary, the We next evaluated the ability of the microarray to detect viruses in human serum samples that were collected during virus outbreaks. CCHFV was chosen as an example because this virus has emerged several times in recent years, particularly in Eastern Europe  and the Middle East. We used (i) sera from 12 CCHFV-positive patients from recent outbreaks in Turkey (2009), Kosovo (2001) and Iran (2009); and (ii) four CCHFV strains  grown in cell culture . We sequThe microarray clearly detected three out of the four CCHFV reference strains, the China strain being only poorly detected . It also allowed the geographical characterization of five out of the 12 CCHFV serum samples: two samples from Turkey and three from Kosovo, all belonging to the Eurasian cluster . The two1 maximum) the amount of specific cDNA after random priming. This indicated that reverse transcription did not substantially affect the amount of specific viral genetic material. In contrast, the WTA isothermal amplification of cDNA by Phi29 polymerase significantly increased this amount. The increase from the original amount of RNA in the sample to the cDNA after WTA was between 1.08 × 102 PFU/mL to 3.46 × 105 PFU/mL, with a mean increase of 3.72 × 104 PFU/mL. Interestingly, the lower amount of amplified cDNA detected by the microarray was 2.8 × 105 PFU/mL for the Kosovo sample 429 . On the other hand, the Turkey sample 090139 with 9.9 × 105 PFU/mL was not detected. As Kosovo and Turkey samples are equally divergent from the tiled sequence  were all well above the 105/106 PFU/mL detection limit (8 PFU/mL). Therefore, a degree of divergence of about 13.7–14.7% from the tiled sequence (Nigerian IbAr10200 strain) is the specificity detection limit of the microarray.Limited genetic material did not explain why the five samples from Iran were not detected, because the amounts of amplified cDNA hybridized on the microarray  and 70.8% (Kosovo 427), which were globally lower than those obtained from the infected cell supernatants (62.9–99.6%). Nevertheless, the BLASTN analysis allowed the geographical origin of the different isolates to be assessed with a precision dependent on the quality of the call rate. Sequences from samples having call rates >70% were precisely segregated into their specific sub-cluster in the phylogenetic tree along with sequences obtained by their direct sequencing . This is5 from the original RNA, 104 from the cDNA), which indicates that the complexity of genetic material of the sample did not impair WTA amplification. However, the call rate was lower than for supernatants of cells infected with the same CCHFV IbAr10200 strain (82.6% versus 99.6%), suggesting a higher background for hybridization  but generated a higher background for hybridization  compared with that obtained with cell supernatant infected with the same viral strain  and the Middle East.Among the molecular techniques used in clinical and field diagnosis, (RT)-PCR is considered a reference standard because of its versatility and rapid turnover. However, it may also be limited by pitfalls such as the genetic variability of the viral isolates or doubtful aetiology requiring the design of a battery of specific or degenerated primers, etc. Under these conditions, DNA microarray technology offers the advantage of performing a differential diagnosis in a single test. It has already proven effective for pathogen detection and epidemiological studies PathogenID v2.0 microarray might be competitive because it does not require (i) designing specific primers for all potential etiologic agents, (ii) setting up the corresponding PCR assays, and (iii) performing the sequence and bioinformatic analyses.Validation was performed using different types of samples  at different degrees of complexity and divergence from the tiled sequence. In single analyses containing multiple virus types, the microarray was able to identify specific viruses among pathogens that produce similar symptoms, and to discriminate between variants of different origins. This is crucial for clinical management of outbreaks that may involve viruses, bacteria or parasites Crimean–Congo haemorrhagic fever virus was chosen as a model infectious agent with which to test the microarray because it has a widespread geographic distribution 5 and 106 PFU/mL of hybridized cDNA per sample. As the mean amplification ratio from the original RNA to the cDNA after WTA was 3.72 × 104  and 1.75 × 104 (only considering serum samples), the detection limit of the microarray is between 101 and 102 equivalent PFU of original viral RNA per mL of serum. This compares favourably with the sensitivity limit of the quantitative RT-PCR method described by Wölfel et al. Using quantitative RT-PCR Saimiri sciureus infected intravenously with the neurotropic Nipah virus. In both cases, the amplification of the viral sequences was not affected by the complexity of brain genetic material but a higher background was observed during the hybridization step .Apart from human samples, the potential of the microarray was also preliminarily tested in animal organ material. It was able to detect viruses in brain samples from experimentally infected animals. This has been demonstrated not only in mouse brain that was intentionally infected intracranially for virus isolation, but also in moribund PathogenID v3.0, will include additional CCHFV sequences from the Middle East, Greece and Asian clusters, as well as geographical variants of families Filoviridae , Arenaviridae , Paramyxoviridae (Tioman virus) and Bunyaviridae (Prospect Hill virus). This improved covering of the sequence space will allow detection of new emerging viruses substantially divergent from the tiled sequences.Taking all results together, there is still room to enlarge the spectrum of pathogen detection by increasing the capacity of the microarray. This would allow not only the detection of all currently known isolates but also the discovery of new ones with reliable sequence information. To this purpose, the next-generation panvirological microarray,


TOC summary: Virus titers are useful for assessing which mosquito species may transmit virus. Culiseta melanura mosquitoes and avian hosts. Other mosquito species that feed opportunistically on mammals have been incriminated as bridge vectors to humans and horses. To evaluate the capacity of these mosquitoes to acquire, replicate, and potentially transmit EEEV, we estimated the infection prevalence and virus titers in mosquitoes collected in Connecticut, USA, by cell culture, plaque titration, and quantitative reverse transcription–PCR. Cs. melanura mosquitoes were the predominant source of EEEV (83 [68%] of 122 virus isolations) and the only species to support consistently high virus titers required for efficient transmission. Our findings suggest that Cs. melanura mosquitoes are primary enzootic and epidemic vectors of EEEV in this region, which may explain the relative paucity of human cases. This study emphasizes the need for evaluating virus titers from field-collected mosquitoes to help assess their role as vectors.Eastern equine encephalitis virus (EEEV) is maintained in an enzootic cycle involving  Togaviridae, genus Alphavirus) has reemerged in the northeastern United States and resulted in 26 human cases of infection and 9 deaths . Virus transmission has intensified throughout this region, spread to locales where it had not been previously detected, and extended north into New Hampshire and Maine, USA, and Nova Scotia, Canada  and passerine birds in freshwater swamps . Other mosquito species such as Aedes vexans, Coquillettidia perturbans, Ochlerotatus canadensis, and Oc. sollicitans have been implicated as epidemic/epizootic bridge vectors from viremic birds to horses and humans  values when the change in fluorescence increased above the baseline threshold value calculated by using IQ5 Optical System Software . Samples that failed to amplify or yielded Ct values >30 were reconfirmed by reisolation of EEEV in cell culture. Supernatants from virus cultures were also tested for EEEV by qRT-PCR or by conventional RT-PCR and by sequencing a portion of the nonstructural protein gene of EEEV as described (Mosquito pools that yielded infectious virus in cell culture were directly tested for EEEV by quantitative reverse transcription–PCR (qRT-PCR). RNA was extracted from mosquito pool homogenates by using spin columns and reagents in a viral RNA kit . A total of 2.5 µL of this preparation was added to a 25-µL qRT-PCR by using the TaqMan One-Step RT-PCR Kit  and primers/probe (9391/9459c/9414probe) . The relationship between Ct values and log10 virus titers were analyzed by regression analysis. Statistical significance was assigned at p<0.05 or by nonoverlapping 95% confidence intervals.The Pooled Infection Rate add-in for Excel  was used to calculate virus infection rates  and 95% confidence intervals (CIs) on the basis of maximum likelihood estimation (MLE) (Cs. melanura mosquitoes yielded the greatest number of EEEV isolations (n = 83) and was followed by Oc. canadensis (n = 10) and Ae. cinereus (n = 6) mosquitoes. Relatively few (<4) or no EEEV isolates were obtained from the remaining mosquito species. EEEV infection rates were higher in Cs. melanura mosquitoes  than in all other mosquito species tested except An. quadrimaculatus  and Ur. sapphirina  mosquitoes.In 2009, a total of 291,641 mosquitoes (35 species) were collected and processed as 16,909 pools for virus isolation. EEEV was isolated from 122 mosquito pools, which represented 14 species and 7 genera , obtainet values (<37) on the basis of criteria established by Lambert et al. (t 37.6–39.0), and 7 isolates failed to amplify after 50 amplification cycles. All mosquito pools that were negative or had Ct values >30 by qRT-PCR were retested in Vero cell culture and confirmed by reisolation of EEEV. Mean Ct values were lowest for Cs. melanura mosquitoes (Ct 22.3) and exceeded 30 for all other mosquito species  than all other mosquito species for which statistical comparisons were possible (p<0.01 by Mann-Whitney U test). Ae. cinereus and Oc. canadensis had the next highest virus titers , and mean titers ranged from <0.8 log10 PFU/mosquito pool to 1.69 log10 PFU/mosquito pool in the remaining species. The percentage of Cs. melanura mosquito pools with high virus titers (>3.0 log10 PFU/mosquito pool) was 88% compared with 0%–16.7% for the 13 other mosquito species , which indicated a predictive relationship between these 2 measures of virus concentration.The relationship between CCs. melanura was the only species in which titers developed that were associated with EEEV transmission, estimated to be 4–7 logs of virus in virus-transmitting mosquitoes in previous studies , suggest that this species could serve as an enzootic and epidemic/epizootic bridge vector of EEEV. We provide additional support for this hypothesis by estimating virus titers in field-collected mosquitoes, which enabled us to determine which infected mosquitoes could potentially transmit virus.Infrequent human and horse infections by EEEV may arise when Cs. melanura or Cs. morsitans mosquitoes, depending on the region. These 2 species comprised 46%–100% of all isolations from field-collected mosquitoes in published studies  in all Ae. vexans mosquito pools tested (n = 4).Oc. canadensis mosquitoes were the second major source of EEEV after Cs. melanura mosquitoes and accounted for 10 (8%) of 122 virus isolations in this study. EEEV has been detected in this species throughout the northeastern United States, including New Jersey, Rhode Island, New York, Massachusetts, and New Hampshire , which is consistent with observations in which 0 of 2 EEEV-positive pools of Oc. canadensis mosquitoes contained high titers of virus (>3.0 log10 PFU/mL) . This species may serve as a potential bridge vector on the basis of certain ecologic and behavioral criteria. Host-seeking females are abundant during June–October in many habitats throughout Connecticut  and are found near EEEV foci. Host-seeking females emerge as 1 generation that peaks in early July and then decrease sharply by mid-August when EEEV begins to amplify in Connecticut , which suggested a bridge role for this species. However, its low abundance in late August and September when EEE activity is greatest argues against its involvement as a primary epidemic vector in this region.We did not isolate EEEV from t values estimated directly from mosquito pools. On the basis of the fitted line estimated by regression analysis in the t<29.8 corresponded to virus titers >3.0 log10 PFU/mL. We used this titer as a threshold for classifying low or high virus titers in mosquito pools for purposes of comparison and to discern whether a virus infection was established and replication occurred in the mosquito vector. Competent vectors must support virus replication during the extrinsic incubation period to be able to transmit virus. However, quantitative data for the minimum virus titer necessary for transmission are limited or absent for most virus–vector systems. Despite these uncertainties, investigations showed that mosquitoes transmitted virus when their body titers exceeded 4–5 logs of virus for EEEV .Our results obtained by qRT-PCR corresponded to those estimated by plaque titration, which provided a basis for interpreting Ct<37). However, our ability to reisolate EEEV from another 14 mosquito pools with either equivocal or negative results indicates that Vero cell culture is a highly sensitive assay for detection of EEEV. Mosquito pools containing infectious virus should be reconfirmed and quantified by another independent method; qRT-PCR is currently the most convenient method to accomplish this test.Most of the pools that showed EEEV in cell culture also showed positive results by qRT-PCR . Our results were consistent with those of other studies (Cs. melanura was the only species that had consistently high titers of EEEV sufficient for transmission. This finding may help reconcile the paucity of symptomatic human and equine cases, despite frequent detection or isolation of virus from mammalophilic mosquitoes during episodes of virus amplification. These results should be verified in other regions, where involvement of other locally abundant mosquitoes is suspected during disease outbreaks. When information on virus titers in mosquitoes is considered, the number of candidate vectors may be reduced to a few key species that are capable of supporting virus transmission in nature.Our findings highlight the need to consider virus titer when interpreting virus isolation or PCR detection data for field-collected mosquitoes. Although we isolated EEEV from several mosquito species,


Duck enteritis virus (DEV) is the causative agent of duck viral enteritis, which causes an acute, contagious and lethal disease of many species of waterfowl within the order Anseriformes. In recent years, two laboratories have reported on the successful construction of DEV infectious clones in viral vectors to express exogenous genes. The clones obtained were either created with deletion of viral genes and based on highly virulent strains or were constructed using a traditional overlapping fosmid DNA system. Here, we report the construction of a full-length infectious clone of DEV vaccine strain that was cloned into a bacterial artificial chromosome (BAC).E. coli was introduced into the intergenic region between UL15B and UL18 of a DEV vaccine strain by homologous recombination in chicken embryoblasts (CEFs). Then, the full-length DEV clone pDEV-vac was obtained by electroporating circular viral replication intermediates containing the mini-F sequence into E. coli DH10B and identified by enzyme digestion and sequencing. The infectivity of the pDEV-vac was validated by DEV reconstitution from CEFs transfected with pDEV-vac. The reconstructed virus without mini-F vector sequence was also rescued by co-transfecting the Cre recombinase expression plasmid pCAGGS-NLS/Cre and pDEV-vac into CEF cultures. Finally, the in vitro growth properties and immunoprotection capacity in ducks of the reconstructed viruses were also determined and compared with the parental virus.A mini-F vector as a BAC that allows the maintenance of large circular DNA in in vitro growth properties of these reconstructions were very similar to parental DEV, and ducks immunized with these viruses acquired protection against virulent DEV challenge.The full genome of the DEV vaccine strain was successfully cloned into the BAC, and this BAC clone was infectious. The in vitro and in vivo. The generated infectious clone will greatly facilitate studies on the individual genes of DEV and applications in gene deletion or live vector vaccines.DEV vaccine virus was cloned as an infectious bacterial artificial chromosome maintaining full-length genome without any deletions or destruction of the viral coding sequence, and the viruses rescued from the DEV-BAC clone exhibited wild-type phenotypes both  Anseriformes) that is caused by duck enteritis virus (DEV). According to the most recent virus taxonomy reported in 2012 by the International Committee on Taxonomy of Viruses (ICTV), DEV  is classified into the genus Mardivirus and the subfamily Alphaherpesvirinae of Herpesviridae . The results are shown in Table xgpt and gfp genes, was excised by site-specific recombination using flanking loxP sites. This excision was achieved by co-transfecting the Cre recombinase expression plasmid pCAGGS-NLS/Cre and pDEV-vac into the CEF cultures. Following this step, a typical DEV CPE without green fluorescence appeared; then, the cloned virus without the BAC sequence, named rDEV-Cre, was obtained by limiting dilution and plaque purification. The correct excision of BAC sequences from the recombinant virus rDEV-Cre was confirmed by PCR and sequencing (data not shown).pDEV-vac DNA was transfected into CEFs by calcium phosphate precipitation to produce the recombinant virus rDEV-BAC. A typical cytopathic effect (CPE) with green fluorescence was observed in the cell monolayer 4 days later. To exclude the effect of additional BAC sequences on the virus, the BAC sequence, including the P = 0.241; P = 0.05; P = 0.485), and there was also no significant difference between rDEV and rDEV-BAC (P = 0.554) . The other is to transfect the transfer vector into cells first and to then follow with a virus infection (transfection-infection method). For cells with low transfection efficiency, the latter is more efficient. Primary chicken embryo fibroblasts are difficult to transfect, and low transfection efficiency limits homologous recombination of duck enteritis virus genome to transfer plasmids. In this study, we adapted the transfection of suspended CEFs using the calcium phosphate method to improve transfection efficiency.In animal experiments, viral DNA was detected using PCR of serum samples of all ducks immunized for 2 weeks without challenge with highly virulent DEV. The results indicated that viral DNA still existed in the blood of ducks in the immunized groups (data not shown). This finding demonstrates that those viruses were still active in ducks after being injected into the body for 2 weeks, and that viruses lies in body for a long time may be the prerequisite for live attenuated vaccines to express viral antigens that periodically stimulate the immune system to establish long-term immunoprotection.We established an infectious bacterial artificial chromosome clone of a widely used attenuated live vaccine strain in China. The results revealed that the rescued virus has similar replication and immunogenic characteristics as its parental strain. Based on this infectious clone, it would be possible to develop novel vaccines, including gene-deletion vaccines, or other marker vaccines for serological differentiation of DEV naturally infected from vaccinated animals. In addition, it would also be possible to use a viral vector to construct a bivalent or multivalent vaccine.All research was approved by the relevant committees at the Zhejiang Academy of Agriculture Sciences.The DEV vaccine virus  and the standard highly virulent CHv strain (CVCC AV1221) used in this study were provided by the China Institute of Veterinary Drugs Control  . Chickenxgpt and gfp genes was released as a Pac I fragment from plasmid pDS-pHAII, which was a gift from Dr. M. Messerle  with Vector NT1 Advance 9 software (Invitrogen). The regions of the sequenced pDEV-vac that were different from several reference nucleotide sequences were amplified by PCR and sequenced again using standard chain termination protocols. The ultimate sites of difference between pDEV-vac and the reference strain KF487736 were determined, and the corresponding sites in four viral  genomes were also determined by PCR and sequenced.n = 7) and housed in separate rooms. Four groups were separately inoculated intramuscularly (i.m.) with 0.5 mL of medium containing 1 × 105 TCID50 (50% tissue culture infection dose) of rDEV, rDEV-BAC, rDEV-Cre, and DEV-vac. The fifth group served as a negative control and was inoculated with medium without virus. Two weeks later, all groups were challenged with a 100-fold 50% duck lethal dose (DLD50) of a highly virulent DEV CHv strain i.m. Then, the signs of disease and death were observed within 2 weeks.A total of 35 20-day-old specific-pathogen-free (SPF) ducks  were used for these studies. The animals were randomly assigned to five groups (DEV: Duck enteritis virus; CEFs: Chicken embryo embryoblasts; BAC: Bacterial artificial chromosome; RFLPs: Restriction fragment length polymorphisms.The authors declare that they have no competing interests.LC planned and carried out virus recombinant, selection and identification of recombinant BAC clone, and rescue of reconstructed viruses, prepared figures, and drafted the manuscript. BY, JH and WY performed animal experiment. TY performed characterization of viruses in cells. ZN carried out viral detection and sequence alignment. XD constructed transfer vector for homologous recombination. And CZ gave suggestion for this research and helped with overall planning and drafting of the manuscript. All authors read and approved the final manuscript.


Immunocompromised individuals tend to suffer from influenza longer with more serious complications than otherwise healthy patients. Little is known about the impact of prolonged infection and the efficacy of antiviral therapy in these patients. Among all 189 influenza A virus infected immunocompromised patients admitted to ErasmusMC, 71 were hospitalized, since the start of the 2009 H1N1 pandemic. We identified 11 (15%) cases with prolonged 2009 pandemic virus replication (longer than 14 days), despite antiviral therapy. In 5 out of these 11 (45%) cases oseltamivir resistant H275Y viruses emerged. Given the inherent difficulties in studying antiviral efficacy in immunocompromised patients, we have infected immunocompromised ferrets with either wild-type, or oseltamivir-resistant (H275Y) 2009 pandemic virus. All ferrets showed prolonged virus shedding. In wild-type virus infected animals treated with oseltamivir, H275Y resistant variants emerged within a week after infection. Unexpectedly, oseltamivir therapy still proved to be partially protective in animals infected with resistant virus. Immunocompromised ferrets offer an attractive alternative to study efficacy of novel antiviral therapies. Immunocompromised patients, such as transplant recipients on immune suppressive therapy, are a substantial and gradually expanding patient group. Upon influenza virus infection, these patients clear the virus less efficiently and are more likely to develop severe pneumonia than immunocompetent individuals. Existing antiviral strategies are far from satisfactory for this patient group, as they show limited effectiveness with frequent emergence of antiviral resistance. For ethical and practical reasons antiviral efficacy studies are hard to conduct in these patients. Therefore, we developed an immunocompromised ferret, mimicking an immune suppressive regimen used for solid organ transplant recipients. Upon infection with 2009 pandemic influenza A/H1N1 virus these animals, like immunocompromised patients, develop severe respiratory disease with prolonged virus excretion. Interestingly, all immunocompromised ferrets on oseltamivir therapy excreted oseltamivir resistant viruses (H275Y) within one week after start of treatment. Furthermore, high dose oseltamivir therapy still proved to be partially effective against these oseltamivir resistant viruses. These immunocompromised ferrets provide a useful tool in the development of novel antiviral approaches for immunocompromised patients suffering from influenza. During the first 12 months of the 2009 influenza A/H1N1 virus (pH1N1) pandemic an estimated 284,000 patients died and hospitalization rates were considerably higher than for seasonal influenza It has been recognized that every winter season a significant number of immunocompromised patients are admitted to a hospital with influenza Unfortunately, present antiviral strategies are merely based on clinical trials conducted in otherwise healthy patients Although antiviral therapy has a documented positive effect on clinical outcome in immunocompromised patients We investigated the incidence of prolonged virus shedding and emergence of antiviral resistance by studying the course of infection of the immunocompromised patients infected with pH1N1 virus treated in our university hospital. These phenomena were studied in more detail in immunocompromised ferrets experimentally infected with pH1N1 virus, that closely mimic immunocompromised patients with influenza. These ferrets all showed prolonged virus shedding and emergence of antiviral resistance. Unexpectedly, the group of immunocompromised ferrets treated with an oseltamivir dose equivalent to a 450 mg dose (twice daily) in humans, had a higher survival rate than similarly untreated animals when infected with an oseltamivir resistant virus.We quantified prolonged virus replication and resistance development in immunocompromised patients retrospectively, who were infected between August 2009 and July 2012, and hospitalized in our tertiary hospital with influenza A virus. Among the 189 RT-qPCR confirmed influenza A virus infected patients , 71 (38%) patients were classified as being immunocompromised . These pmax) levels for MPA, the active form of MMF, were 65±30 µg/mL with trough (C12) levels becoming undetectable after 8 hours. Peak and trough tacrolimus levels were 86±30 ng/mL and 14±30 ng/mL respectively. We determined area under the curve (AUC0–12) values of 54±14 µg·h/mL for MPA and 438±265 ng·h/mL for tacrolimus. In humans, MPA AUC0–12 values between 30–60 µg·h/mL and tacrolimus trough levels between 5–15 ng·h/mL are proposed 1/2t) and high tacrolimus peak levels, we further optimized the ferret regime by administration of the cocktail every 12 hours containing half of the initial tacrolimus dose (0.5 mg/kg).We investigated whether the observations on prolonged virus replication and emergence of antiviral resistance in immunocompromised patients could be mimicked in ferrets receiving a cocktail of immunosuppressive drugs similar to that administered to SOT patients (combination of mycophenolate mofetil (MMF), tacrolimus and predisolone). First, pharmacokinetics were studied following oral administration of ferrets (n = 4) given 20 mg/kg MMF, 1 mg/kg tacrolimus and 8 mg/kg prednisolone . From thP<0.0001)  –D. As an<0.0001) –F. No si<0.0001) .The animals of groups 1, 2 and 3 had been inoculated with oseltamivir sensitive  and the animals in groups 4, 5 and 6 with oseltamivir resistant virus   and 3. OP<0.001). Such difference was not observed in the animals infected with oseltamivir resistant virus  was started 24 hours after infection and continued for 21 days  and S2C.nt virus .Emergence of oseltamivir resistance was studied in the animals infected with wild type virus  using an RT-PCR assay specifically detecting the H275Y oseltamivir resistance mutation  [40]. FrUnexpectedly, oseltamivir treatment appeared to have a positive effect on the proportion of surviving immunocompromised animals and animal body weight loss . WithoutP = 0.03), and not for the oseltamivir resistant virus infected animals .In addition, oseltamivir treatment appeared to have a protective effect on body weight loss of the immunocompromised animals . This trHere we show that prolonged influenza virus shedding and the emergence of oseltamivir resistance are two phenomena commonly observed in immunocompromised patients during antiviral therapy. As resistance development is low (19 out of 874 (2.2%)) treated influenza virus infected patients Since for both logistic and ethical reasons randomized studies are difficult to perform in often critically ill immunocompromised patients, we showed that prolonged influenza virus replication is also a common feature in immunocompromised ferrets. We observed an absence of a rise in body temperature, reduction of lymphocyte proliferation and follicle formation in ferret lymphoid tissues, which are also hallmarks in immunocompromised patients In the 2007/2008 H1N1 virus season an H275Y oseltamivir resistant mutant emerged, which had completely overtaken the circulating virus population by the end of 2008 max) from 3052 ng/ml in 4 hours to 833 ng/ml 12 hours after administration , a large  tertiary university hospital in the Netherlands, with an influenza A virus positive respiratory specimen taken between August 2009 through July 2012. Patients had a prolonged virus infection if the virus could still be detected after 14 days. Virological data, patient immune status and administration of antiviral therapy were obtained by reviewing medical records. Immunosuppression was defined as any of the following: receipt of treatment for any cancer, the use of any immunosuppressive medication to prevent transplant rejection or for management of pulmonary or autoimmune conditions, premature birth and below gestational age or a diagnosis of AIDS. Influenza A virus and the H275Y oseltamivir resistance mutation were detected by reverse transcriptase RT-PCR assays. These assays have been described previously Two biologically cloned pH1N1 influenza viruses were used in this study. Both viruses were isolated from an oseltamivir treated patient during the first wave of the pandemic in October 2009. Wild type influenza virus A/Netherlands/1715b/2009 (genbank ID code: CY065810) and H275Y mutant virus were isolated from the original quasispecies by co-cultivation of a respiratory sample in a Madin-Darby Canine Kidney (MDCK) cell culture in a single passage ad libidum with commercial food pellets and water. Approximately three to four weeks prior to the experiment a temperature logger  was placed in the peritoneal cavity of the animals. This device recorded the body temperature of the animals every 10 minutes. From day −8 to day −4, an average baseline temperature was recorded for each group of 6 animals.Animal were housed and experiments were conducted in strict compliance with European guidelines  and Dutch legislation . The protocol was approved by the independent animal experimentation ethical review committee from the Netherlands Vaccine Institute (permit number 200900201). All experiments were performed under animal bio-safety level 3 conditions. Animal welfare was observed on a daily basis, and all animal handling was performed under light anaesthesia using a mixture of ketamine and medetomidine to minimize animal suffering. After handling atipamezole was administered to antagonize the effect of medetomidine. All ferrets were eleven-month-old purpose-bred males (body weights between 1562 and 2362 g) and were seronegative for Aleutian disease virus and circulating influenza virus (sub) types A/H1N1, A/H3N2 and B virus. The animals were maintained in standard housing and were transferred to negatively pressured glove boxed on the day immunosuppressive therapy was started. They were provided food The following immunosuppressive drugs were used to suppress the immune system of ferrets: Mycophenolate mofetil (MMF)  powder for infusion, tacrolimus concentrate (5 mg/ml) for infusion  and prednisolone sodium phosphate (5 mg/ml) oral solution . All ferrets received an antibiotic prophylaxis of amoxicillin supplemented with 62.5 mg clavulanic acid (250/62.5 mg per 5 ml) oral suspension . Prodrug oseltamivir phosphate, used in the ferret experiments, was kindly provided by Hoffman-La Roche LtD. .Oseltamivir standards for mass spectrometry, oseltamivir phosphate (OS), oseltamivir-d3 (OS-d3), oseltamivir carboxylate (OSC) and oseltamivir carboxylate-d3 (OSC-d3) were purchased from Toronto Research Chemicals . Mycophenolic acid (MPA) standard and internal standards (MPAC) were purchased from Sigma Aldrich  and from Hoffman-La Roche LtD. respectively. The tacrolimus standards and internal standards (Ascomycin) were purchased from, respectively, Chromsystems and Sigma Aldrich. ULC/MS grade methanol and water containing 0.1% formic acid were obtained from Biosolve . Trichloro acetic acid (TCA) and formic acid  were obtained from Sigma Aldrich and both were from ACS reagent quality.A schematic of the ferret experiment is presented in 4 TCID50 of wild type  or mutant virus . Each day, pharyngeal and nasal swabs were collected just before administration of the drugs. Swabs were resuspended in 3 ml virus transport medium On day 0, three days after start of immunosuppressive therapy, ferrets were intratracheally infected with 1×102EDTA) samples, 5 µl of a 50% TCA solution (w/v) was added for plasma protein precipitation. Precipitated plasma proteins were removed by centrifugation for 10 minutes at 2000×g at ambient temperature. De-proteinized plasma samples (20 µl) were 2.5 times diluted with ultrapure water and 40 µl of the diluted samples were injected by an auto-sampler (kept at 4°C) into the liquid chromatography mass spectrometry (LC-MS) system. The calibration curves for OS and OSC showed a linear relationship between the SRM peak area of ratios between OS/OS-d3 and OSC/OSC-d3, respectively . The LLOQ and LOD were determined according to FDA guidelines and were, respectively, 2.5 and 1.0 ng/ml for both OS and OSC. The LC-MS system used was an 4000 API triple quadruple mass spectrometer containing a Turbo V electron spray ion source (ESI)  operating in the positive ionization mode using selected reaction monitoring (SRM) in combination with a Dionex Ultimate 3000 UHPLC system  using an Ascentis Express RP-C18 column  applying a gradient separation at 30°C.Oseltamivir and MMF plasma levels and whole blood tacrolimus levels were determined in a pharmacokinetic pilot study. For 4 days, four groups of ferrets (n = 4) received MMF, tacrolimus, or oseltamivir in combination with amoxicillin/clavulanic acid and prednisolone or as the complete cocktail. On day 4, blood was collected from these animals after 0, 10, 20, 30 minutes and 1, 2, 4, 5, 8 and 12 hours after administration of the drugs in order to determine MMF and tacrolimus levels, as described previously Samples for histological examination of the tonsils and tracheobronchial lymph nodes were taken to evaluate the immune status and were stored in 10% neutral-buffered formalin. Subsequently, these were routinely processed and embedded in paraffin wax, sectioned at 4 µm and stained with haematoxylin and eosin (HE) for examination by light microscopy.P values for comparison of influenza HI antibody titers in P≤0.05 was considered significant.Data are reported as mean ± standard error of the mean (s.e.m). The Figure S1a; b) and oseltamivir phosphate (c) for 4 days. On day 4, blood was collected after 0, 10, and 30 minutes and 1, 2, 4, 5, 8 and 12 hours after the final cocktail was administered. Plasma levels of the active form of mycophenolate mofetil (MMF), the metabolite mycophenolic acid (MPA) (a), whole blood tacrolimus levels (b), oseltamivir phosphate  and its metabolite oseltamivir carboxylate  plasma levels were determined by mass spectrometry. Area under the curve (AUC0–12), peak (Cmax) and trough (C12) levels and half-life (t1/2) values are presented in Mean steady state (day 4) pharmacokinetics of MMF, tacrolimus and oseltamivir in ferrets. Twice daily, four ferrets were given a cocktail of antibacterial prophylaxis, immune suppressive therapy ((TIF)Click here for additional data file.Figure S2a) and more distant viruses A/sw/NL/25/80 (b), A/IT/1443/76 (c), and A/New Jersey/08/76 (d). Individual data points represent antibody titers for each animal and horizontal bars represent the mean titer per group. Body temperature profiles show the absence of fever during the acute stage of infection in ferrets infected with wild type (e) or mutant (f) virus. Animals in this experiment were either immunocompetent (circles), immunocompromised (triangles) or immunocompromised and oseltamivir treated (squares). Data are mean ± s.e.m. Data used for Ferrets on immune-suppressive therapy show reduced antibody titers. Reduction of serum hemagglutination inhibiting (HI) antibody titers against pH1N1 A/NL/602/2009 virus ((TIF)Click here for additional data file.Figure S350/ml) were determined in nose and throat swabs daily taken from immunocompetent  and immunocompromised ferrets . The animals were infected either with wild type  or mutant virus . Ferrets in groups 3 and 6 were treated with oseltamivir (10 mg/kg twice daily) starting 24 hours after inoculation.Total number of animals positive for replication competent influenza virus in the upper respiratory tract. Influenza virus titers (TCID(TIF)Click here for additional data file.


Acanthamoeba and that are themselves infected by the virophage Sputnik. This study examines the evolutionary dynamics of this system, using mathematical models. While the models suggest that the virophage population will evolve to increasing degrees of giant virus inhibition, it further suggests that this renders the virophage population prone to extinction due to dynamic instabilities over wide parameter ranges. Implications and conditions required to avoid extinction are discussed. Another interesting result is that virophage presence can fundamentally alter the evolutionary course of the giant virus. While the giant virus is predicted to evolve toward increasing its basic reproductive ratio in the absence of the virophage, the opposite is true in its presence. Therefore, virophages can not only benefit the host population directly by inhibiting the giant viruses but also indirectly by causing giant viruses to evolve toward weaker phenotypes. Experimental tests for this model are suggested.Giant viruses contain large genomes, encode many proteins atypical for viruses, replicate in large viral factories, and tend to infect protists. The giant virus replication factories can in turn be infected by so called virophages, which are smaller viruses that negatively impact giant virus replication. An example is Mimiviruses that infect the protist  Acanthamoeba, and was shown to have characteristics that are atypical for the majority of viruses  was first discovered in the water of a cooling tower in the United Kingdom, infecting the protist Giant viruses and virophages are thought to be abundant in aquatic environments, infecting a variety of protists  that describes the average development of populations over time. These include the host rx(1−(x+y1+y12)/k). The intrinsic growth rate is given by r and the total amoeba population  cannot exceed the carrying capacity k. Contact between the primary virus and uninfected amoeba cells leads to infection with a rate β1. The primary virus can be released from two sources. Obviously, one source is cells infected with the primary virus alone, y1. An additional source is cells that contain both the primary virus and the virophage, although they are likely to release the primary virus at a reduced rate. This is expressed by the parameter f, which describes the degree of primary virus inhibition by the virophage  and can vary between zero and one. If f = 0, the primary virus cannot replicate at all in the presence of the virophage. If f = 1, the replication of the primary virus is not inhibited by the virophage. Amoeba infected with the primary virus only, y1, die with a rate a1 and become infected with virophage upon contact with a virophage-containing cell with a rate β2. Amoebae infected with both the primary virus and the virophage, y12, die with a rate a12. This death rate is determined both by the virophage and the primary virus. We assume that the primary virus contributes less to cell death in the presence compared with the absence of the virophage, due to inhibition of viral replication (parameter f). In addition, the virophage itself can cause cell death with a rate aph. Thus, the overall death rate of this cell population is given by a12 = aph + fa1. Note that while the reduced replication rate of the primary virus in virophage-infected cells is likely to be reflected in a reduced death rate of these cells, the death rate does not have to be decreased by the same factor, f, as written here. However, if the death rate of the infected cell is decreased by a different amount, expressed by an additional factor g, the results presented here do not change qualitatively. We do not track amoeba cells that are infected with the virophage only, as the virophage cannot replicate without the primary virus.The amoeba population is characterized by logistic, density-dependent growth, described by the term In order to address questions concerned with population extinction, we also consider a stochastic version of this model by applying the Gillespie algorithm to these ODEs  influences the approach to equilibrium  greatly stabilizes the dynamics, leading to significantly shorter damping times  while keeping the basic reproductive ratio of the primary virus constant. If the primary virus turnover is much larger than the turnover of infected cells , the dynamics are similar compared with the quasi–steady state assumption, that is, pronounced oscillations are observed and extinction occurs relatively fast , returning to the quasi–steady state assumption for the free virus populations. The model is now formulated as follows:Here, we examine the evolutionary dynamics of the virophage and concentrate in particular on the evolution of “virophage pathogenicity,” defined by the parameter 12 and z12. The two strains only differ in their pathogenicity, which is denoted by fy and fz. The death rate of these infected cells is thus given by a12 = aph + fya1 and α12 = αph + fza1 (note that the death rate of cells infected with the second strain is different and given by a greek letter). The basic reproductive ratio of virophage strain 1 is given by f = 0.Cells containing the primary virus can become infected by two virophage strains, and the respective virophage-infected cells are denoted by yf = 0 characterized by the most unstable dynamics. This can render populations prone to extinction, and these aspects were explored with stochastic Gillespie simulations of the ODEs. Figure As shown in the previous section, an increase in virophage pathogenicity can lead to more extensive population oscillations and longer damping times, with f = 0) depends on the model parameters and this is explored systematically in Figure y1, is of particular importance. If the virophage drives this population extinct, then it depletes its own targets for replication. High population levels of primary virus–infected cells, and thus persistence, are promoted by slow spread of the virophage, that is, by a slow virophage replication rate, β2, and a fast virophage-induced cell death, aph . The basic reproductive ratio of the first strain is the same as before, that is, The infection rate of the second strain primary virus is given by In the absence of the virophage, the primary virus strain with the larger basic reproductive ratio wins the competition, and thus evolution will maximize the basic reproductive ratio (subject to constraints that are not included in this model).β1 or γ1) it always wins the competition. The most obvious reason is that a faster replication rate increases the basic replicative fitness of the virus. In addition, however, a faster replication rate of the primary virus indirectly conveys a benefit by weakening the virophage. As shown in equilibrium expression γ1 > β1) and that it is also characterized by a higher death rate of infected cells (b1 > a1). Further assume that the increase in the death rate of infected cells is greater than the increase in the viral replication rate. In this case, the basic reproductive ratio of the second primary virus strain, R0 can win the competition. Interestingly, the strain with the smaller R0 can also win and exclude its competitor. Alternatively, coexistence of the two strains can be observed. The dependence of the outcomes on parameters is explored in Figure γ1 >> β1. If R0 excludes the strain with the higher R0. This is shown in Figure R0 because it lessens the impact of the virophage. In other words, the presence of the virophage can lead to evolution toward reduced replicative fitness of the primary virus.The situation is more complex in the presence of the virophage. If a strain is characterized only by a higher replication rate  such that the dynamics become unstable enough to cause extinction in stochastic settings. It is possible that there are constraints that limit the degree to which the virophage can inhibit primary virus replication, thus preventing the virophage to evolve to a state that would leave the system prone to extinction. It is currently not known whether such constraints exist, but it is feasible from a biological point of view, for example, if there is a trade-off between the ability of the virophage to inhibit the primary virus and its ability to reproduce in the viral factories maintained by the primary virus.Beyond the basic dynamics, some interesting evolutionary insights emerged. While the virophage is expected to evolve toward higher levels of primary virus inhibition, this can lead to more oscillatory dynamics which can result in extinction of the virophage and also the primary virus. For pathogenic virophages, persistence is only possible for relatively slow virophage replication kinetics, which is again not favored by evolution. This theoretical result brings up the question whether the presence of a virophage in food chains is transient and eventually destined to go extinct as a result of virophage evolution itself. Data, however, argue against this notion. Sputnik appears to have a long evolutionary history with Mimi-virus . Even within the perfect mixing assumption, uncertainties remain in the model because the same process can be formulated in different ways. For example, consider the infection term, which is generally given by Acanthamoeba population is infected with both the mimivirus and the virophage at different initial concentrations. Nonlinear least squares fitting procedures can be used to see how well the system can describe the data, and to estimate parameters in the context of one set of initial conditions. By changing the initial concentrations of cells and viruses, the parameterized model should then successfully predict subsequent experiments. Once the model has been validated/revised, more complex experiments can be performed to address specific model predictions discussed in this study. For example, conditions can be altered such that growth and death parameters are changed, or the cells and viruses could be manipulated to have the same effect. The influence of these parameter manipulations on the stability of the dynamics should be observed. This would provide an important piece of information to advance our understanding of the evolutionary dynamics discussed here. This in turn will be important for a better understanding of the microbial ecology and the evolutionary dynamics of aquatic and marine systems.The basic model validity can be tested by very simple in vitro experiments where a host


Work during the past few decades has provided a good understanding of alterations that occur in viruses when they interact with host cells. This is generally possible because of the relative simplicity of virus systems, as compared to the more complex cellular systems. There has also been advancement made in delineating host genomic responses to virus infection because of the availability of gene arrays for diverse organisms. However, work that seeks to equally comprehend host responses to virus infection at the protein level (proteomics) has lagged behind because of technical challenges and the great complexity of the cellular proteome.This series consists of six review articles, three original research papers and a single commentary that all focus on current research activities in the field of proteomics of virus-infected host cells, all designed to begin to fill these important gaps in our knowledge. The articles span quantitative proteomic analyses of host protein alterations induced upon virus infection of a range of host cells: archaeal, insect, and various animal systems. Several articles present results and reviews of new discovery-based functional assays to assess broad enzymatic groups within these experimental systems to complement the quantitative proteomics assays.The review article by Enrique Santamaria and co-workers provides an overview of mass spectrometry (MS)-based proteomics and how this method has been useful for delineating some of the interactions between herpes simplex virus type 1 (HSV-1) and the host cells , with an emphasis on interactions between the virus and T cells  to study virus-host interactions (Shahiduzzaman and Coombs, The review by Monteiro and co-workers discusses some of the applications of proteomic analyses of baculoviruses (Monteiro et al., Most of the original research and review articles presented above focus on individual virus-host model systems. The final review article by Zheng and colleagues provides a holistic overview of current approaches being undertaken to better define and functionally validate responses elicited within the host proteome by virus infection (Zheng et al., In the first of three original research papers within this volume, Jiang and colleagues label HeLa cells with either normal isotopic amino acids or heavy forms of arginine and lysine in the SILAC approach (Jiang et al., The research article by Milev and co-workers describes characterization of Staufen 1, a host protein that binds double-stranded RNA and that is associated with HIV-derived ribonucleoprotein complexes (Milev et al., The final article by Maaty and colleagues provides a global analysis of host responses to virus infection in an archaeal system (Maaty et al., In summary, these articles present overviews and ongoing original research in one of the “frontier” areas of virology—a better understanding of the complexity of various host omic responses to virus infection, with some examples of proteomic and functional genomic approaches.


The growing global demand for seafood together with the limited capacity of the wild-capture sector to meet this demand has seen the aquaculture industry continue to grow around the world. A vast array of aquatic animal species is farmed in high density in freshwater, brackish and marine systems where they are exposed to new environments and potentially new diseases. On-farm stresses may compromise their ability to combat infection, and farming practices facilitate rapid transmission of disease. Viral pathogens, whether they have been established for decades or whether they are newly emerging as disease threats, are particularly challenging since there are few, if any, efficacious treatments, and the development of effective viral vaccines for delivery in aquatic systems remains elusive. Here, we review a few of the more significant viral pathogens of finfish, including aquabirnaviruses and infectious hematopoietic necrosis virus which have been known since the first half of the 20th century, and more recent viral pathogens, for example betanodaviruses, that have emerged as aquaculture has undergone a dramatic expansion in the past few decades. Even in the modern molecular era virus isolation remains an important tool for research and diagnosis. Thus, over the past decade, through the use of cell culture and molecular approaches our knowledge of the biology of fish viruses has grown exponentially. Coincidentally, fish farming has expanded globally with an increase not only in absolute production (kilotonnes/year) but also in the number of fish species being cultured in both freshwater and marine systems. The increase in aquaculture operations world-wide has provided new opportunities for the transmission of aquatic viruses and the occurrence of viral diseases remains a significant limiting factor for aquaculture production and for the sustainability of biodiversity in the natural environment. Here we provide an overview of some of the significant viral pathogens affecting finfish species.While early studies pre-1950s) of fish pathology indicated the presence of ‘filterable agents’, 950s of f viruses ,3. Over in situ hybridization and various immunodiagnostic techniques [Diagnostic techniques for viral diseases of fish will not be discussed since these have become relatively standard and include pathological and histopathological examination, virus isolation on cell culture (where cell culture systems exist), molecular techniques including conventional and real-time polymerase chain reactions, chniques .2.Aquabirnavirus is the largest and most diverse of the three genera within the family Birnaviridae—non-enveloped viruses with a bi-segmented, double-stranded RNA genome. The type species, infectious pancreatic necrosis virus (IPNV), was the first fish virus isolated in cell culture [Seriola quinqueradiata) in Japan [Aquabirnavirus genus makes classification down to the species level difficult. culture  and unti culture . The vir culture . Since t culture ,8. An exin Japan . Since tin Japan . This diIPN was first recognized as an acute contagious disease of young salmonid fry in the freshwater phase of production that could cause up to 100% mortality. More recently, disease associated with significant mortality has emerged in the post-smolt, seawater stages ,12. AquaTo date, 4 serogroups, A, B, C, D ,13,14 haAquabirnavirus particles are non-enveloped icosahedrons, 60 nm in diameter , containInfectious pancreatic necrosis in its acute form can cause up to 100% mortality in young salmonids and remains one of the most significant diseases of major concern to the salmonid aquaculture industry. Of further significance, the majority of fish that survive a disease outbreak become sub-clinical carriers—fish that are persistently infected with no clinical signs. These sub-clinical carriers are a source of horizontal transmission, shedding virus in their feces particularly under stressful conditions such as spawning.Mortality rates associated with disease outbreaks can be quite variable (5–100%) and it is probable that various host, viral and environmental factors influence the severity of the outbreak . While sAs with all viral diseases of finfish, avoidance is an important control strategy through the use of good bio-security measures that have been developed over the decades and are now well-established . However3.Betanodavirus [Nodaviridae and is the etiological agent of viral nervous necrosis . The disease was first reported in barramundi  farmed in Australia [Oplegnathus fasciatus [Scopthalmus maximus [Dicentrarchus labrax [Epinephalus akaara [Pseudocaranx dentex [odavirus  is one oustralia ,30, Japaasciatus  followed maximus , Europeas labrax , redspots akaara  and strix dentex . The disx dentex  but resex dentex  and sincThe virus infects a large range of host species—at least 40 species of marine and freshwater fish world-wide —and the Viral nervous necrosis has a wide geographical distribution and includes south and east Asia, Oceania, Mediterranean Europe and Tunisia, UK, Norway and North America. To date, the virus has not been reported from South America.Virions of nervous necrosis virus (NNV) are small (25–30 nm in diameter), spherical and non-enveloped  with a gCurrent research is focused on vaccination as a potential means of control. While protection has been demonstrated in various host species using a number of different vaccination schemes –53, a co4.Salmo salar). The disease initially reported in Norway [in vitro and obtain purified virus, research accelerated and the virus is now well-characterised [The orthomyxovirus, infectious salmon anemia virus (ISAV), is the causative agent of infectious salmon anemia (ISA), a disease of sea-farmed Atlantic salmon  from which virus was isolated (5 fish positive out of 203 tested). In addition, ISAV was isolated in cell culture inoculated with one of 24 tissue pools (5 fish/pool) from cod (Gadus morhua) sampled from a well-boat that contained ISA-diseased salmon.While fish species other than Atlantic salmon are susceptible to infection with ISAV, only Atlantic salmon develop ISA disease following infection with ISAV. Of interest, a survey of wild fish undertaken following the 1998 outbreak in farmed salmon in Scotland  revealedClupea harengus) can be infected after immersion in ISAV-contaminated water [Oncorhynchus mykiss) by co-habitation with ISAV-infected salmon [S. trutta), Arctic char  and rainbow trout [O. kisutch) in Chile [Experimental infections have demonstrated that herring  and sleeping disease virus (SDV) has been clarified. At least six subtypes of SAV (Pancreas disease (PD) has been recognized in farmed Atlantic salmon since the 1970s but the infectious nature of the disease was not proven until a toga-like virus was isolated two decades later from an outbreak in Ireland  and, subs of SAV , based oTypical alphaviruses are transmitted by arthropods or insects, usually mosquitoes . While aca. 60 nm diameter) viruses with a ssRNA genome (11–12 kb in size) the coding sequences of which are organized into two large, non-overlapping open reading frames [Alphaviruses are enveloped, spherical  is one of three rhabdoviruses of finfish listed by the OIE , the other two being viral hemorrhagic septicemia virus (VHSV) and spring viremia of carp virus (SVCV). It is the causative agent of infectious hematopoietic necrosis (IHN) which affects most salmonid fish species . First dRhabdoviridae and the genome consists of a single molecule of negative-sense ssRNA (ca. 11 kb) encoding six proteins: a nucleoprotein, a phosphoprotein, a matrix protein, a glycoprotein, a non-virion protein and a polymerase [Novirhabdovirus which also contains VHSV.The virus has the typical bullet shape of members of the lymerase ,114. IHNOncorhynchus, until in 1984 when an outbreak in Atlantic salmon  occurred for the first time [Acipenser transmortanus) demonstrated this species susceptibility to IHNV and suggested that it represented a potential virus vector [Paralichthys olivaceus) and ayu  in Japan [Novirhabdovirus [Originally, the host range was thought to be restricted to species within the genus rst time . Since trst time , severals vector . Of furtin Japan , has beebdovirus .The clinical signs and histopathology have been well-documented . Risk faDue to the severe losses caused by IHN  there ha7.Ranavirus in the family Iridoviridae; other genera in the family include Iridovirus, Chloriridovirus, Lymphocystivirus and Megalocytivirus. There are six viral species within the genus . Species can be differentiated via RFLP profiles, virus protein profiles, DNA sequence analysis and host specificity, however none can be differentiated via classical antigen-antibody interactions  [Epizootic hematopoietic necrosis virus (EHNV) is a member of the genus e ELISA) .EHNV is a large icosahedral virus, approximately 175 nm  with a dPerca fluviatilis) in Victoria, Australia. It was the first virus isolated from freshwater finfish in Australia [Oncorhynchus mykiss ) [Macquaria australasica), silver perch (Bidyanus bidyanus), mosquito fish (Gambusia affinis) and mountain galaxias  are susceptible to infection and associated mortality. Other host species including Murray cod (Maccullochella peeli), golden perch (Macquaria ambigua), Australian bass (Macquaria novemaculeata), Macquarie perch, silver perch and Atlantic salmon  were also shown to be susceptible following intraperitoneal injection with varying degrees of mortality [EHNV was first isolated in 1985 from fatalities of juvenile redfin perch ) . To datealbaum)) . Laboratortality .The disease associated with EHNV is termed epizootic hematopoietic necrosis (EHN). The name implies the pathology of the disease includes necrosis of hematopoietic tissues. Specifically, EHNV causes multifocal necrosis of the spleen and renal hematopoietic tissue as well as the liver. Microscopically, there is a distinctive feature of infected cells—basophilic intracytoplasmic inclusion bodies—these structures are assembly sites of the virus where, by electron microscopy, paracrystalline arrays can be observed. Other lesions include hyperplasia and multifocal necrosis of gill epithelial cells, necrosis of atrial trabeculae and gastrointestinal epithelial cells, focal pancreatic necrosis, necrotic circulating hematopoietic cells and degenerate vascular endothelial cells in many organs ,139–141.Within Australia the geographical range is limited to south-east Australia . EHNV is endemic in these areas and is now only infrequently reported. Early work by Langdon  demonstr7.Novirhabdovirus (Family: Rhabdoviridae) and is the causative agent of viral hemorrhagic septicemia—the most serious disease of farmed rainbow trout in Europe [Salmonidae, Esocidae, Clupidae, Gadidae, Pleuronectidae, to name just a few [Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus of the genus n Europe . Well-ren Europe ,143 untin Europe ,145. SinViruses is devoted to the emergence of the North American genotype IVb which has caused recent significant mortality episodes in a range of fish species inhabiting the Great Lakes of North America [Currently, four genotypes (I-IV) and several sub-genotypes of VHSV have been identified using modern molecular tools . Basical America . Therefo America ,147,148.It is recognized that the existence of various strains of VHSV, the large host range, and the differences in pathogenicity of these strains in the various host species presents difficulties for disease control programs. Nevertheless, it is significant that eradication of VHS post-disease outbreaks in the UK  and NorwIt was recognized early on that survivors of VHS outbreaks developed resistance to re-infection  and so r