West Nile virus  is the cause of the most widespread arthropod-borne viral disease in the world and the largest outbreak of neuroinvasive disease ever observed. Mosquito-borne outbreaks are influenced by intrinsic  and extrinsic  factors that affect virus activity and mosquito biology in complex ways. The concept of vectorial capacity integrates these factors to address interactions of the virus with the arthropod host, leading to a clearer understanding of their complex interrelationships, how they affect transmission of vector-borne disease, and how they impact human health. Vertebrate factors including host competence, population dynamics, and immune status also affect transmission dynamics. The complexity of these interactions are further exacerbated by the fact that not only can divergent hosts differentially alter the virus, but the virus also can affect both vertebrate and invertebrate hosts in ways that significantly alter patterns of virus transmission. This chapter concentrates on selected components of the virus-vector-vertebrate interrelationship, focusing specifically on how interactions between vector, virus, and environment shape the patterns and intensity of WNV transmission.  West Nile virus  was first detected in the Americas in 1999, the result of a single point introduction into the New York City area [ity area , followeity area ,4,5,6. Tity area  as has bity area . ExtrinsWNV has been successful in persisting in the environment of North America, partly a consequence of its generalist nature, but also likely a consequence of its ability to adapt readily to new hosts. Like all RNA viruses, WNV has the ability to mutate rapidly due to its error-prone RNA dependent RNA polymerase, rapid rate of replication, and replication to high titers. This chapter will delve into intrinsic and extrinsic factors, focusing on vector-virus-environment interactions that impact the intensity of WNV transmission. In evaluating the importance of each factor, it is critical to do so in the context of vectorial capacity (VC), a measure of a mosquito population’s capacity to transmit an infectious agent to a new susceptible population. Calculation of VC is founded on the basic formula:based on . A numbebased on ,11). HerCulex pipiens L. and Culex restuans Theobald may be responsible for up to 80% of human WNV infections in the northeastern USA [Cx. restuans populations peak in the spring and early summer [Cx. pipiens populations which peak later, and the former may be the critical vector of WNV early in the transmission season [Cx. restuans occurs from California to North Carolina, from southern Canada to Honduras; locally adapted populations may differ in epidemiologically significant traits [Recently available molecular tools have facilitated advancement of our knowledge of medically important mosquito vector genetics, feeding habits, and infection prevalence. Species distribution and population density suggest that tern USA . Cx. resy summer ,14,15, un season . Cx. rest traits . Cx. pipiens complex includes Cx. pipiens, Cx. quinquefasciatus Say, Cx. australicus Dobrotworsky & Drummond, and Cx. globocoxitus Dobrotworsky, mosquito species that have a worldwide distribution  is uillett, ,33. Unlindscapes  and is rndscapes , in whicndscapes ,37. Cx. is noted ,39, whicFollowing ingestion of an infectious blood meal by a mosquito, the virus must overcome barriers to transmission including the midgut infection barrier , midgut Cx. tarsalis calclulated an EIP of 109 degree-days for WNV [Cx. pipiens demonstrated an accelerating effect with increasing temperatures, suggesting degree day models are likely to underestimate the effects of temperature [Cx. pipiens and the possible interaction between mosquito genetics and local environmental factors was demonstrated in NYS [Cx. pipiens populations between counties in NYS, demonstrating that vector competence is not a static intrinsic trait of a particular mosquito population, but instead a dynamic trait governed by the intersection of multiple variables.The vector competence of colonized and field populations of mosquitoes for WNV has been a focus of research since WNV was first identified in the USA. It has been found to vary among mosquito species and genera ,47,48,49 for WNV , yet subperature . Even moperature ,55,56,57perature , and mayperature . Spatio-d in NYS . These sCx. pipiens, Cx. quinquefasciatus, and Cx. restuans, Cx. restuans are most likely to be affected by temperature increases, with increased sensitivity to temperature rises over 16 °C as evidenced by adult and immature mortality  and Ae. triseriatus [Cx. pipens and WNV, demonstrating that survivorship was directly proportional to sucrose intake, but also that mosquitoes with lower nutrient reserves as a result of less concentrated sucrose meals were more likely to orally transmit virus [The impact of larval nutrition on development and vector competence is virus- and vector-specific. Studies conducted with measured . These rer virus , but difseriatus . The effit virus .The interaction between rainfall and mosquito populations is a dynamic one, and also species-specific. Decreased precipitation has been associated with increased WNV infection prevalence in horses, birds and humans ,74,75. ACx. tarsalis tend to be more generalist feeders than the more ornithophilic Cx. pipens complex mosquitoes [Cx. pipiens, Cx. quinquefasciatus, and Cx. tarsalis from birds early in the transmission season to humans later [Cx. pipiens mosquitoes may also be an important contributor to host feeding preference. Northern European populations of Cx.pipiens are pure, whereas USA populations generally contain varying levels of Cx.molestus signature, a characteristic which may correlate to geographical differences in the propensity to feed on mammals and the likelihood of spillover to humans [Mosquitoes may be generalists or opportunists in feeding, or they may be specialists with a strong preference for a single host species. Primary WNV vectors may feed on a range of avian and mammalian hosts with variable levels of WNV competence , therefosquitoes ,79,80,81ns later ,83. Geneo humans ,84. Geneo humans . Cx. tarsalis with WNV demonstrated decreased fecundity and increased blood feeding rates in infected mosquitoes which, as has been shown with Plasmodium-mosquito interactions [Cx. pipiens demonstrated that WNV infection is not associated with alterations to life-history traits, showing species specificity, yet that resistance to infection may be associated with decreased longevity [Dengue virus , suggestype WNV . These dtype WNV ,92,93, atype WNV , yet they glands  and apopy glands , suggestRecent studies have also advanced our understanding of the complexity of interactions between invading arboviruses and mosquito immune defenses . It is nCell fusing agent virus [Kamiti River virus [Culex flavivirus  have been identified in recent years, many in primary vectors of WNV. These include nt virus , Kamiti er virus , Culex fs , Lammi mi virus , Calbertdo virus , and Palus . The prus  are among the most susceptible avian families to WN disease [i.e., R0) in the enzootic cycle partly by removing individuals who would have become immune. This increase in enzootic R0 might be expected to result in more human infections [The enzootic transmission cycle is comprised predominantly by ornithophilic Culex species and passeriform birds. West Nile virus causes neurologic disease in some avian species, particularly passeriforms, while other species demonstrate variable levels of morbidity and mortality as demonstrated in laboratory studies  and in s disease ,131, but disease . A seros disease , and 50% disease . Nonethefections .Ciconia ciconia) in Israel were identified during migration within two days of arrival at a stopover site [Migratory birds are thought to have been the principal agent for the spread of WNV in North America and globally. Infectious viremias were detected in birds during fall migration in 2002 and 2003 . In addiver site . Sciurus carolinensis) [Tamias striatus), and eastern cottontail rabbits (Sylvilagus floridanus), which develop viremia sufficient for infecting some mosquito species, but it is not clear whether the infected mosquitoes are competent, i.e., will transmit virus. These studies are well summarized elsewhere and therefore will not be discussed further [Although the main enzootic vectors, Culex species mosquitoes, are ornithophilic, it has been noted that late in the transmission season they become more opportunistic and largely switch to feeding on mammals ,136,137.inensis)  eastern  further .Japanese encephalitis virus, Murray Valley encephalitis virus, Usutu virus, and St. Louis encephalitis virus (SLEV), among others. WNV has historically been classified into at least two phylogenetically distinct lineages. Lineage 1 WNV, which is associated with West Nile fever in humans and CNS infection in approximately 1% of cases [Cx. pipiens in the Czech Republic in 1997 [Aedes rossicus Dolbescin, Gorickaja and Mitrofanova [et al. [WNV, first isolated in 1937 from a febrile patient in Uganda , is a meof cases , is the of cases . Lineageof cases . Althougof cases ,145 and of cases . Rabensb in 1997  and subsrofanova . Initialrofanova . Furtherrofanova ,152,153. [et al.  initiallSequence analyses following the WNV U.S. invasion are consistent with a single point introduction with a strain most closely related to a 1998 Israeli strain  which haCx. pipiens [Cx. quinquefasciatus [Cx. pipiens but not Cx. quinquefasciatus, consistent with species-specific differences in competence [. In addition, significant temporal decreases in intrahost diversity have been identified [Cx. pipiens, which utilized passage of virus-positive salivary secretions, demonstrate that a combination of selection and intrahost bottlenecks may purge diversity, yet this does not necessarily preclude WNV from adapting further to mosquito hosts [The commencement of infection in the mosquito mesenteron (midgut) is likely constrained to a limited number of cells in the posterior midgut epethelia ,160 and  pipiens  and Cx. asciatus , additiompetence . In addientified ,162, parentified  in conceentified . Consistentified ,166,167,entified . This geentified  and therentified , and mayentified ,172. Desto hosts . Although more extensive purifying selection within avian hosts serves to decrease WNV intrahost diversity ,167, genCulex populations, seasonal genetic bottlenecks are also likely, particularly in temperate areas in which mosquitoes must overwinter. Diapausing Cx. pipiens and Cx. tarsalis, likely infected via vertical transmission [As the intensity of WNV transmission cycles in concert with smission ,175,176,smission ,178. Thesmission . Althougsmission . Given dCx. pipiens salivary secretions [Cx. pipiens bodies may result in a loss of fitness in chicks, yet, interestingly, passage and adaptation to chicks may simultaneously increase WNV fitness in mosquitoes [Although recent phylogenetic analyses have revealed some instances of positive selection , and adacretions . In contsquitoes . These dChikungunya virus [Venezuelan equine encephalitis virus [in vivo remain(s) uncharacterized, studies with WNV-infected Cx. quinquefasciatus suggest that complementation could act to maintain deletion mutants [Ae. aegypti [in vivo exists and could contribute significantly to WNV swarm dynamics, adaptation, and evolvability. Theory predicts that under high mutation rates in which selection cannot outpace mutation, RNA virus evolution should favor mutational robustness to avoid accumulation of deleterious mutations . Such roya virus  and Veneis virus . An evolis virus , and mayis virus . Althoug mutants , as has  aegypti , demonstPatterns and intensity of WNV transmission are governed by complex and dynamic interactions between vector, vertebrate, virus and environment. The capacity to characterize the role of extrinsic and intrinsic factors that determine the outcomes of these interactions is complicated by the fact that effects may rarely act independently. Instead, as has been shown with DENV ,192, it


There are four principal arboviruses in mainland China. Two kinds of them are mosquito-borne viruses, namely Japanese encephalitis virus and dengue virus, which lead to Japanese encephalitis, and dengue fever/dengue hemorrhagic fever respectively; the other two are tick-borne viruses, namely tick-borne encephalitis virus and Crimean-Congo hemorrhagic fever virus , which contribute to tick-borne encephalitis and Xinjiang hemorrhagic fever respectively. With exception of these four main arboviruses, many other mosquito-associated viruses have been isolated and identified in recent years. These newly isolated and identified mosquito-associated viruses are probably responsible for human and animal infections and diseases. The purpose of this review is to describe the newly isolated mosquito-associated viruses in mainland China which belong to five viral families, including their virological properties, phylogenetic relationships, serological evidence, as well as to appeal the public health concentration worldwide. Arboviruses comprise a group of viruses that reproduce in sensitive blood-sucking arthropods . There aSince the 1950 s, four kinds of arbovirus-related diseases have been confirmed to be endemic in China, namely Japanese encephalitis (JE), dengue fever (DEN), tick-borne encephalitis (TBE) and Crimean-Congo hemorrhagic fever (CCHF)  -10. The This article focuses on the mosquito-associated viruses isolated from variety of arthropods, humans and animals that have been identified in mainland China in recent years, which belong to 5 viral families, and a lot of them are important zoonotic pathogens Figure .Culex spp. mosquitoes collected from corrals in Xinjiang, China, in the summer of 2006 by Lu et al. YUOV is a newly identified member of genus oviridae . It was articles . The vir species . In receBrevidensoviruses within the family Parvoviridae was firstly isolated from Culex pipiens pallens in Liaoning province. Additional strains of CppDNV were subsequently isolated from various provinces of China , Culex pipiens quinquefasciatus (two strains), Culex tritaeniorhynchus (three strains), Anopheles sinensis (one strain), unclassified Culex mosquitoes (six strains) and other miscellaneous mosquitoes (two strains) from the Liaoning, Yunnan, Xinjiang and Guizhou provinces in China demonstrated a nucleotide homology of more than 98%. This confirmed that the virus was a newly identified member of the species Aedes aegypti densovirus (AaeDNV) along with the previously identified Aedes albopictus densovirus  [During an investigation of arboviruses in China, a new kind of virus with the same biological characteristics and a genome sequence very similar to the viruses belonging to the genus na Table . The viralDNV-2) .Culex pipiens pallens, Culex modestus, Culex tritaeniorhynchus, Aedes (Och.) detritus, Aedes vexans, Aedes dorsalis, Aedes albopictus, Anopheles philippinensis, Anopheles sinensis and Armigeres obturbans. Of this growing list of arboviruses, most are mosquito borne. The ability of such viruses to spread between geographic regions through movements of people, animals and goods means that addressing them clinically is of great public health importance. Given the increasingly frequent international exchanges with China, the risk for further introduction and exportation of the mosquito-borne viruses via human activity and transportation of goods becomes greater, so enhanced monitoring and long-term surveillance of these viruses are of great public health importance both in China and internationally.In the last two decades, 12 species of viruses belonging to five families have been newly identified in mainland China. Four  are pathogenic to humans and livestock. Four  are initially isolated and identified in China and are considered to be potential threats to humans and livestock. One (CHIKV) is an imported virus, and no autochthonous cases have been reported in mainland China to date. One (GETV) is known as a pathogen in horses and pigs. Two (KDV and CppDNV) are associated with insects and are not pathogenic to humans. These species of viruses have been isolated from ten mosquito species in four genera, including The authors declare that they have no competing interests.HL and XYG contributed equally to the analysis and interpretation of the results, and involved in drafting the manuscript. Both of them have read and approved the final version of the manuscript. Dr. GDL contributed to conception and design of the manuscript, and involved in drafting and revising the manuscript as well as gave final approval of the version to be published.


To the Editor: In April 2009, the outbreak of influenza A pandemic (H1N1) 2009 virus was reported. Subsequently, the disease spread throughout the world, and the pandemic alert level was raised to level 6 in June by the World Health Organization. Pandemic (H1N1) 2009 virus infection spread to Thailand and is now found throughout Thailand. Similar to the effects of other viruses, pandemic (H1N1) 2009 virus may cause neurologic complications. Associated neurologic symptoms were first reported from Dallas, Texas, USA: 4 children experienced unexplained seizures or had an alteration of consciousness level that was associated with this virus , and areflexia developed. His motor weakness (grades III/V) began in both legs and then involved both arms and hands. Other neurologic examinations showed limitation of extraocular movement in all directions, normal pupil size and light reflex, and facial diplegia. A lumbar puncture was performed, and cerebrospinal fluid (CSF) contained neither leukocytes nor erythrocytes, with a protein level of 19.5 mg/dL.On day 3 after the patient’s admission, acute respiratory failure developed. A nasopharyngeal aspirate specimen was positive for pandemic (H1N1) 2009 virus by PCR. The patient received oseltamivir, zanamivir, and ventilator support. His chest radiograph showed diffuse alveolar infiltration. On day 10, his motor weakness worsened to grade 0, and his consciousness level was diminished to a drowsy state.A computed tomography scan of the brain showed diffuse white matter lesions . RepeateMycoplasma pneumoniae. A CSF antigen test was negative, and CSF culture was negative for bacteria. Meropenem was given to treat ventilator-associated pneumonia, which was caused by β-lactam–resistant Klebsiella pneumoniae. After a month of treatment, the patient regained consciousness, his motor strength improved considerably, and he was able to be gradually removed from the ventilator. After 3 months, he was discharged with self-assisted status.Other laboratory tests showed mild transaminitis and negative results for syphilis testing and for serologic tests for HIV, hepatitis B virus, hepatitis C virus, Japanese encephalitis virus, herpes simplex virus, and Our report shows neurologic manifestations associated with pandemic (H1N1) 2009 virus infection in an adult. The manifestation of progressive quadriplegia with diffuse sensory loss is compatible with a polyneuropathy. The neurologic signs developed 2 days after the respiratory tract signs.Although a diagnosis of Guillain-Barré syndrome was considered initially, according to the National Institute of Neurologic Disorders and Stroke criteria  2009 virus infection. Acute disseminated encephalomyelitis is a condition that might occur within 30 days after an infectious process (


Cotton leaf curl Gezira virus (CLCuGV), the begomovirus associated with CLCuD in Africa, was identified. However, the cognate African betasatellite (Cotton leaf curl Gezira betasatellite) was not found. Instead, two Asian betasatellites, the CLCuD-associated Cotton leaf curl Multan betasatellite (CLCuMB) and Chilli leaf curl betasatellite (ChLCB) were identified. Inoculation of the experimental plant species Nicotiana benthamiana showed that CLCuGV was competent to maintain both CLCuMB and ChLCB. Interestingly, the enations typical of CLCuD were only induced by CLCuGV in the presence of CLCuMB. Also in infections involving both CLCuMB and ChLCB the enations typical of CLCuMB were less evident. This is the first time an African begomovirus has been identified on the Indian sub-continent, highlight the growing threat of begomoviruses and particularly the threat of CLCuD causing viruses to cotton cultivation in the rest of the world.Cotton leaf curl disease (CLCuD) is a severe disease of cotton that occurs in Africa and Pakistan/northwestern India. The disease is caused by begomoviruses in association with specific betasatellites that differ between Africa and Asia. During survey of symptomatic cotton in Sindh (southern Pakistan)  Gossypium barbadense, and then from Sudan in 1924 and subsequently from Tanzania in 1926 Bemisia tabaci. Losses to cotton cultivation in the Sudan were ultimately overcome by the introduction of resistant cotton varieties. In Pakistan CLCuD was a minor, sporadic problem until 1986, when a plot of a newly introduced cotton variety (G. hirsutum) became infected. In subsequent years it became epidemic spreading to almost all cotton growing regions of Pakistan and into adjoining areas of India. Losses to the economy of Pakistan during this period were enormous; estimated at US$5 billion between 1992 and 1997. During the late 1990s losses were gradually reduced, returning Pakistan's cotton output to above pre-epidemic levels, by the introduction of CLCuD-resistant cotton varieties. Unfortunately, in 2001, all previously resistant cotton varieties began to show the typical symptoms of CLCuD, indicating that the resistance had been overcome st Century. For both southern Asia Begomovirus (family Geminiviridae) in association with a newly identified class of satellites, the betasatellites Cotton leaf curl disease (CLCuD) is a serious disorder of cultivated cotton that is characterised by greening , leaf curling, vein darkening, vein swelling, enations and production of cup-shapped leaf-like structures on the undersides of leaves. The disease has been reported from a number of countries across Africa and from southern Asia, specifically Pakistan and northwestern India. In Africa CLCuD was first reported 1912 from Nigeria, affecting Geminiviridae encompasses four genera  and viruses are assigned to these based on genome organization, host range and insect vector Begomovirus are the most numerous and economically the most destructive. They are transmitted by a single species of whitefly (Bemisia tabaci) and have genomes that consist of either two components (known as DNA-A and DNA-B) or a single component (homologous to the DNA-A component of the bipartite viruses). Each component is 2.6–2.8 kb in size, replicates in the nucleus and is transcribed bidirectionally from a non-coding region which also contains a hairpin structure (which within the loop contains the sequence TAATATTAC known as the nonanucleotide motif) that forms part of the origin of replication. All begomoviruses native to the New World have bipartite genomes. In the Old World, although a few bipartite begomoviruses have been identified, the vast majority are monopartite and most of these associate with a class of ssDNA satellites known as betasatellites as well as satellite like molecules known as alphasatellites.Geminiviruses are insect-transmitted viruses with single-stranded (ss)DNA genomes which are encapsidated in characteristic twinned icosahedral particles from which they derive their name. The family Satellites are defined as viruses or nucleic acids that depend on a helper virus for their replication but lack extensive nucleotide sequence homology to the helper virus and are dispensable for its proliferation Cotton leaf curl Gezira virus [CLCuGV]) and a disease specific betasatellite (Cotton leaf curl Gezira betasatellite [CLCuG]); the presence of an alphasatellite has thus far not been investigated The begomovirus-betasatellite complexes that cause CLCuD in cotton in Asia and Africa are distinct. The complex in Pakistan and India during the 1990s was shown to consists of multiple begomovirus species (often occurring as multiple infectionsmore than one virus per plant) supporting a disease specific betasatellite (Cotton leaf curl Multan betasatellite [CLCuMB]) as well as an alphasatellite Cotton leaf curl Kokhran virus , one from each plant, were determined and are available in the nucleotide sequence database under the accession numbers FR751142 to FR751146  and four in the complementary-sense ; the positions and coding capacities of these are indicated in Hollyhock leaf crumple virus (HoLCrV), another Malvaceae-infecting begomovirus from Africa, the levels of identity were between 81.9 and 83.4%. To all other begomoviruses in the database the levels of sequence identity were less than 77%. These results indicate that the virus identified in association with CLCuD-affected cotton in Sindh Province is CLCuGV. CLCuGV is a typical African begomovirus, distinct from those that occur on the sub-Continent, and is the first such virus identified in Pakistan.An initial comparison to the sequences available in the databases using BLAST (NCBI) showed the clones from Pakistan to have the highest levels of sequence identity to CLCuGV. Subsequent sequence alignment using MegAlign  showed them to have between 87 and 98.3% nucleotide sequence identity with CLCuGV sequences available in the databases (37 of which are available at this time). To In an effort to try and identify the possible origins of the CLCuGV identified in southern Pakistan, all available CLCuGV sequences were aligned and used in a phylogenetic analysis . At thisMalvaceae-infecting betasatellite identified on the continent. It is for this reason that efforts were made to identify CLCuGB in the cotton plants in which CLCuGV was found. Despite extensive efforts, that included Southern blotting probing with a CLCuGB probe and RFLP mapping RCA products (results not shown), only two species of betasatellite were identified and both are of Asian origin. The presence of CLCuMB was shown by Southern blotting with a CLCuMB probe and PCR amplification with CLCuMB βC1 gene-specific primers  isolated from cotton with all betasatellites in the databases showed it to have the highest sequence identity (84.9 to 100%) to isolates of Chilli leaf curl betasatellite (ChLCB) but only low levels of identity (<77%) to all other betasatellites  indicating that it is an isolate of ChLCB , which has its origins in the Middle East, but has since spread globally, including to the New World, North Africa and much of Asia, but does not occur in either Pakistan or India Tomato leaf curl Palampur virus having been reported from Iran, Pakistan and India Squash leaf curl China virus has been introduced from China into both Pakistan and India Bean yellow dwarf virus (BeYDV), a mastrevirus that was first reported from southern Africa Althea rosea). As well as indicating that the “Egypt” strain is a threat to cultivated cotton, this provides a possible way for CLCuGV to be transported by peoplein infected ornamental plants.The begomoviruses occurring in the Middle East and the Indian sub-Continent are distinct. This is likely due to the geographical hurdle to dispersal posed by the Sulaiman mountain range, which runs down the eastern edge of Balochistan, separating Pakistan from the Iranian plateau. Similarly, the begomoviruses of China and the sub-Continent are distinct, most likely due to the barrier posed by the Himalayan mountain range. This has led to the unusual situation with the monopartite begomovirus Potato virus X vector Only two betasatellites are known to be associated with CLCuD, CLCuMB in Asia and CLCuGB in Africa N. benthamiana suggest that ChLCB may modulate symptoms, since in the presence of both satellites the vein darkening, vein swelling and enation symptoms were more pronounced and occurred more towards the leaf margins than in infections of CLCuGV with either betasatellite alone.Although CLCuGB was not identified, the presence of the Asian CLCuD associated betasatellite CLCuMB was shown. This is the first documented case of CLCuD being caused by interaction between an African virus and an Asian betasatellite. Additionally a non-malvaceous betasatellite, ChLCB, was shown to be present in plants containing CLCuGV and CLCuMB. Recently ChLCB has been encountered increasingly in cotton having been shown to be present in an unusual collection of cotton species in Multan Malvaceae (including cotton), in Iran indicates that genetic exchange between even distantly related viruses can occur The encroachment of the African CLCuD-associated begomovirus CLCuGV into cultivated cotton in Pakistan is a worrying development. Southern Asia, and particularly Pakistan, already had a large genetic diversity of begomovirues capable of infecting cotton. It is likely this diversity, in both cotton and other plant species, added to the rapidity with which resistance to CLCuD in cotton was broken after it was introduced in the late 1990s. The possible effects of the added genetic diversity of an African virus remain to be determined. However, the evidence of recombination between CLCuGV and TYLCV, a distantly related virus that is not known to infect plants of the family B. tabaci occurs. In the United States cotton is affected by a bipartite begomovirus, Cotton leaf crumple virus  using ϕ29 DNA polymerase was used to amplify all circular DNA molecules in DNA samples BamHI yielded approx 2.8 kb fragment and this was ligated into the pTZ57R vector . No restriction enzymes yielding approx 1.4 kb fragments could be identified, so potential betasatellites were PCR-amplified from RCA amplified product with the universal primers Beta01/Beta02 Concatameric RCA product was digested independently with various restriction endonucleases to identify enzymes yielded fragments of begomovirus genome (∼2.8 kb) or half genome size (∼1.4 kb). The Basic Local Alignment Search Tool  was used to compare sequences with begomovirus and betasatellite sequences in the database. Sequence alignments of full length clones with the various references sequences were constructed using CLUSTAL X Agrobacterium-mediated inoculation. An ∼437 bp BamHI-PstI fragment of the begomovirus clone NT1 was ligated in the binary vector pGreen0029 BamHI insert of NT1 was ligated into BamHI restricted pGNT0.15 to yield a 1.15-mer construct of the viral genome (pGNT1.15). Similarly, to generate a 1.6-mer of the betasatellite clone NGVB, a fragment of ∼859 bp was released by digestion with KpnI and EcoRI and cloned into pGreen0029 to yield pGNGB0.6. The full-length betasatellite was released from pTZ57R/T by digestion with KpnI and ligated into KpnI restricted pGNGB0.6 to yield pGNGB1.6. Both constructs were electroporated into Agrobacterium tumefaciens strain GV3101 and inoculated to N. benthamiana plants as previously described Partial direct repeat constructs of virus and satellite clones were produced for Figure S1Detection of Cotton leaf curl Multan betasatellite (CLCuMB) in CLCuD affected cotton plants from Sindh. Southern blot analysis of nucleic acids extracted from field collected cotton plants probed for the presence of CLCuMB (A). Samples were extracted from a healthy, glasshouse grown, cotton plant and from three of the five cotton plants in which CLCuGV was detected (lanes 2–4). The blot was probed with the βC1 gene of CLCuMB. PCR-mediated detection of CLCuMB using primers specific for the CLCuMB βC1 gene (B). The template DNA included in the PCR reactions were extracted from a healthy, glasshouse grown, cotton plant (lane 1), from an N. benthamiana plant experimentally infected with CLCuMV and CLCuMB (lane 2) and from the 5 plants with CLCuD symptoms originating from Sindh in which CLCuGV was detected . PCR products were run on a 1% agarose gel and stained with ethidium bromide. A size marker was run in lane M.(TIF)Click here for additional data file.Table S1Genes encode by CLCuGV clones isolated from Pakistan. * The Rep gene of NT31 contains a frame shift mutation due to the insertion of an A (with respect to the other isolates) at coordinate 2179. The data in the table is for a reconstructed Rep gene product.(DOCX)Click here for additional data file.


Picorna-, Papilloma-, and Anelloviridae as well as known viruses from the families Astro-, Corona-, Parvo-, and Hepeviridae were identified in different ferret cohorts. Ferret kobu- and hepatitis E virus were mainly present in human household ferrets, whereas coronaviruses were found both in household as well as farm ferrets. Our studies illuminate the viral diversity found in ferrets and provide tools to prescreen for newly identified viruses that potentially could influence disease outcome of experimental virus infections in ferrets.Ferrets are widely used as a small animal model for a number of viral infections, including influenza A virus and SARS coronavirus. To further analyze the microbiological status of ferrets, their fecal viral flora was studied using a metagenomics approach. Novel viruses from the families  Infectious viral diseases, both emerging and re-emerging, pose a continuous health threat and disease burden to humans. Many important human pathogens are zoonotic or originated as zoonoses before adapting to humans Mustela putorius furo) can carry bacteria and parasites such as Campylobacter, Giardia, and Cryptosporidium in their intestinal tract and potentially spread them to people Relatively few studies have been conducted on diseases of non-domestic carnivores, especially regarding diseases of small carnivores (e.g. mustelids). Ferrets (Mustela putorius furo) in the Netherlands and Sweden and stored at −80°C (n = 12). The second cohort consisted of rectal swabs from ferrets (Sweden) that were used in an experimental procedure that consisted of 3 weeks of immunosuppression n = 12). The third cohort were rectal swabs from household ferrets (the Netherlands) with (n = 3) or without (n = 12) diarrhea gathered in 2010. This study was carried out in strict accordance with European guidelines  and Dutch legislation . In compliance with relevant laws and institutional guidelines, no ethical approval is required for most of the samples in this study, because live animals were not primarily and purposefully sampled to obtain material for scientific purposes, but for health care purposes, such as surveillance of viruses that could cause underlying ferret disease. Some of the samples from the ferrets in this study were obtained under ethical approval by the independent animal experimentation ethical review committee of the Netherlands Vaccine Institute (permit number 20110286) in compliance with relevant laws and institutional guidelines. All efforts were made to minimize animal suffering and the owners of the ferrets gave permission for their animals to be used in this study.Rectal swabs were collected from 39 ferrets  was performed as described previously and by the manufacturer 5′-CCTCCAGCTCCGCTGGCTCAA-3′), VS732 (5′-CAATCCTAGGGTGCAGTCCTT- 3′), and VS731 (5′-FAM- CCACTCTCTCTGTGGCACTTT-TAMRA- 3′) that target the 3D polymerase/3′UTR genome region of ferret kobuvirus (KF006985) and primers and probe VS733 (5′-CTGCTCCTCAATTAACAGGCT-3′), VS735 (5′-CACACTCCTGCATCCATTAGT- 3′), and VS734 (5′-FAM-TGAGATCATTGCATGACAATGT-TAMRA- 3′) that target the 3D genome region of ferret parechovirus (KF006989) with TaqMan® Fast Virus 1-Step Master Mix (Applied Biosystems) according to instructions of the manufacturer. Similarly, a diagnostic ferret hepatitis E virus real time RT-PCR was used, using primers and probe VS739 (5′- AAGATGCGTTTTGTTCTCTTGCT-3′), VS740 (5′- CCGGACGCCCTCCTGYA- 3′), and VS741 (5′-FAM- TGCTCGCCGCCCCAATGTACC-TAMRA- 3′) that target the ORF2/3 genome region of ferret hepatitis E virus (JN998606). A diagnostic ferret coronavirus real time RT-PCR was used with primers and probe as described previously (22).Sequence information obtained from the large scale molecular virus screening using next-generation sequencing was used to design specific primers for amplification of the papillomavirus genome MpPV1 using AmpliTaq Gold DNA polymerase (Roche), according to instructions of the manufacturer to confirm and/or extend 454-sequence reads. Primer sequences are available on request. Products were sequenced as described previously http://www.cbs.dtu.dk/services/NetPicoRNA/) or predicted based on alignments with other picornaviruses. The similarity between different picornaviruses was measured using Simplot version 3.5.1 with the Kimura-2 parameter, a transition/transversion (Ts/Tv) ratio of 3.0 and window and step sizes of 600 and 20 nucleotides, respectively Multiple alignments were created using ClustalX (2.0.10) The genomes of identified viruses described in detail here were deposited in GenBank under the following accession numbers: ferret kobuviruses MpKoV38, KF006985; MpKoV32, KF006987; MpKoV39, KF006986; ferret parechovirus (MpPeV1), KF006989; ferret papillomavirus (MpPV1), KF006988; ferret anellovirus (MpfTTV1), KF006990.Mustela putorius furo). More than 253,000 trimmed sequence reads were generated and the sequences from each animal were assembled de novo. Sequences were classified into eukaryotic viruses, phages, bacteria, and eukaryotes based on the taxonomic origin of the best-hit sequence. Many of the identified sequences were of eukaryotic or bacterial origin, and a large proportion of the total reads did not have any significant hits for nucleotide or amino acid sequences in GenBank in agreement with viral metagenomic studies of feces from bats, turkeys, rodents, pine marten, and humans Large scale molecular virus screening was performed on 39 rectal swabs from ferrets  and do not represent genuine enteric infections in the ferrets. Eukaryotic viruses with relatively high homology (85–100% on the amino acid level) to ferret coronavirus (Coronaviridae), ferret hepatitis E virus (Hepeviridae), and Aleutian mink disease virus (family Parvoviridae), were detected  were obtained from ferrets 37 and 26 respectively with ∼95% identity on the nucleotide level to murine astrovirus STL1 that was recently described (Anelloviridae), picobirnavirus (family Picobirnaviridae), kobu- and parechovirus (family Picornaviridae), and papillomavirus (family Papillomaviridae) were identified  Canis lupus familiaris) Kobuvirus is a viral genus belonging to the family A near-complete kobuvirus genome containing the complete coding region was obtained by 454-sequencing from a rectal swab from ferret 38. In addition, two partial polyprotein sequences were obtained from rectal swabs of household ferrets 32 and 39. The ferret kobuvirus (MpKoV) from ferret 38 encodes a 2,482-amino-acid polyprotein flanked on each side by untranslated regions (UTRs) . PolyproThe genetic relationship of MpKoV38 with other known kobuviruses was assessed by determining the pairwise distance to the corresponding genome regions of other picornaviruses . MpKoV38To obtain insight in the prevalence of ferret kobuvirus in ferrets, a ferret kobuvirus-specific real time PCR targeting the 3D/3′NTR region of the kobuvirus genome was performed on the total set of 39 rectal samples. Besides samples 32, 38, and 39, in which ferret kobuvirus was identified, three other rectal samples were positive as well, resulting in an overall prevalence of ∼15% ; Fig. 1.Picornaviridae. The genus is composed of two species, human parechovirus and Ljungan virus from bank voles. Parechoviruses are small, non-enveloped viruses with a single stranded positive sense RNA genome of ∼7.5 kb. The genome encodes a single polyprotein that is cleaved by virus-encoded proteases into the structural and nonstructural proteins. Human parechoviruses are widely spread infectious pathogens that are associated with mild gastroenteritis, respiratory disease, flaccid paralysis, encephalitis, and myocarditis in young children Parechovirus is a viral genus in the family Picornavirus Study Group of ICTV determined that members of a species of the parechovirus genus share greater than 70% amino acid identity in the polyprotein, and share a natural host range and genome organization. Members of a picornavirus genus should normally share phylogenetically related P1, P2 and P3 genome regions, each sharing >40%, >40% and >50% amino acid identity, respectively Picornaviridae family.A near-complete parechovirus genome containing the complete coding region was obtained by 454-sequencing from a rectal swab from household ferret 36 without diarrhea. The ferret parechovirus from ferret 36 (MpPeV1) encodes a 2,207-amino-acid polyprotein flanked on each side by untranslated regions (UTRs) . CleavagTo obtain insight in the prevalence of MpPeV1 in ferrets, a MpPeV1-specific real time PCR targeting the 3D region of the parechovirus genome was performed on the total set of rectal samples from 39 ferrets. Besides the sample in which MpPeV1 was identified, no samples were positive .Papillomaviruses (PVs) are a highly diverse family of viruses with double stranded circular DNA genomes of ∼8 kb in size. They infect a wide variety of mammals, as well as birds and reptiles, and are highly species specific. Some papillomaviruses cause benign or malignant epithelial tumors of the skin and mucous membranes in their natural hosts Taupapillomavirus  was typical for papillomaviruses, consisting of a long control region (LCR), and early  and late regions  . Whole gomavirus . Papilloomavirus , as MpPVTo obtain insight in the prevalence of MpPV1 in ferrets, a MpPV1-specific real time PCR targeting the L1 region of the papillomavirus genome was performed on the total set of 39 rectal samples. Besides the sample in which MpPV1 was identified, no samples were positive for MpPV1 .Mustela putorius furo torque teno virus 1. This sequence encodes for a partial open reading frame 1 (ORF1) as determined by alignment with ORF1 amino acid sequences from representative torque teno viruses. A taxonomic proposal submitted to the International Committee on the Taxonomy of Viruses (ICTV) by the Anelloviridae-Circoviridae Study Group proposes that genera in the family Anelloviridae are defined as having >56% divergence in the nucleotide sequence of ORF1 Xitorquevirus.The partial anellovirus genome sequence from sample 21 was designated MpfTTV1 for Mustela putorius furo).Previously, virus discovery in animals has focused on pathogenic infections or on animals that on the basis of relevance to (re-)emerging viruses are thought to represent a key risk host for emerging virus-associated disease in humans Sequences closely related to known viral sequences were identified, with homology to ferret coronavirus, ferret hepatitis E virus, Aleutian mink disease virus, different avian viruses and murine astrovirus STL1 Picornaviridae that currently comprises 17 genera and many more different virus species We report on the first identification of a kobuvirus, parechovirus, papillomavirus and anellovirus in ferrets. The kobu- and parechovirus belong to the family Picornaviridae based on the observed sequence diversity. Interestingly, the P3 genome region of MpPeV1, encoding the nonstructural proteins NS3A–3D, is not more conserved than the P1 genome region, encoding the structural capsid proteins, when compared to the corresponding regions of human parechoviruses 1 and 2. This has been observed for Ljungan virus as well by us and others The ferret parechovirus is the third parechovirus species identified to date. It is highly divergent from human parechovirus and Ljungan virus and may even constitute a new genus in the family Xitorquevirus, in analogy to the classification of torque teno viruses in nine genera named Alpha-, Beta-, Gamma-, Delta-, Epsilon-, Eta-, Iota-, Theta-, and Zetatorquevirus, and the proposed five genera Kappa-, Lambda-, Mu-, Nu-, and XitorquevirusLike many other mammals, it seems that ferrets also harbor anelloviruses. The ferret anellovirus MpfTTV1 is most closely related to the recently identified anellovirus from pine marten Ferrets are carnivores that are significantly affected by a number of human pathogens and hence are widely used as a small animal model for viral infections Table S1GenBank accession numbers of representative parecho-, papilloma-, and anellovirus strains.(DOC)Click here for additional data file.


Phloem transport of plant viruses is an essential step in the setting-up of a complete infection of a host plant. After an initial replication step in the first cells, viruses spread from cell-to-cell through mesophyll cells, until they reach the vasculature where they rapidly move to distant sites in order to establish the infection of the whole plant. This last step is referred to as systemic transport, or long-distance movement, and involves virus crossings through several cellular barriers: bundle sheath, vascular parenchyma, and companion cells for virus loading into sieve elements (SE). Viruses are then passively transported within the source-to-sink flow of photoassimilates and are unloaded from SE into sink tissues. However, the molecular mechanisms governing virus long-distance movement are far from being understood. While most viruses seem to move systemically as virus particles, some viruses are transported in SE as viral ribonucleoprotein complexes (RNP). The nature of the cellular and viral factors constituting these RNPs is still poorly known. The topic of this review will mainly focus on the host and viral factors that facilitate or restrict virus long-distance movement. Plant viruses are obligate intracellular parasites living exclusively in the symplast of their hosts. Virus accumulation at high levels throughout the whole plant is a necessary condition for virus survival. Massive titer of virions may facilitate virus transmission from one plant to another, whatever the mode of propagation: by seeds or pollen, by graftings, by mechanical wounds, or by vectors. Viruses are dependent on their hosts to complete their life cycle in the plant, i.e., replication, encapsidation, cell-to-cell movement, and long-distance transport. Therefore, multiple compatible interactions have to be established between viral proteins or virions and cellular factors. The plant reacts to these invaders by developing various strategies to restrict, or even better, eradicate the pathogens. On their side, viruses counteract these defense mechanisms by different ways. The result of this arm race leads to a complete resistance of the plant, if the virus cannot overcome the plant defenses, or to a systemic infection, eventually ending with the host death, if the viral counter defenses are efficient enough to bypass the plant protection system. A wide range of intermediate situations between plant immunity and death can be encountered, which highlights the complexity of interactions that may take place between the virus and the plant.Virus entry into plant cells, mostly epidermal, and mesophyll, is followed by virion disassembly and genome translation/replication in inoculated tissues. Then, viral transport complexes move from cell-to-cell and on-going replication takes place in the newly infected cells Figure . This shPhloem cells structure and composition reflect their high functional specialization in transporting molecules from source to sink tissues. SE are enucleated cells, modified by selective degradation of organelles, interconnected by wide sieve pores, and forming a low-resistance cellular conduit for elaborated sap flux  infection and patterns of photoassimilate distribution in sink organs, indicating that virus movement can be mapped very accurately onto the orthostichy. However, spatial and kinetic analyses of long-distance movement of some viruses revealed that the direction and speed of movement may be different than those of photoassimilates. For instance, Melon necrotic spot virus (MNSV) is first transported in melon plants from cotyledons to the roots through the external phloem before being carried to the shoot apex through the internal phloem  subunits assembly, and RNP complexes, in which the viral genome is associated with viral and/or cellular proteins. As described in more details in the following section, the requirement of a functional CP for systemic movement is common but not universal. Although this occurrence is usually associated with the need to produce virions, the CP can also be required to form RNP complexes. The nature of the complexes involved in long-distance transport of different viral species is described thereafter, emphasizing the central role of the CP  was shown to move simultaneously in the form of RNP complexes and virions, the three RNAs of this multipartite virus being transported in different forms  is another example for which systemic movement of each of the three genomic RNA may occur in different forms, and may involve constitution of RNP complexes with cellular factors. Gopinath and Kao  for example, moves in N. benthamiana and in its natural host, P. vulgaris, in a CP-dependent manner, most probably virions, but this virus can also be transported in beans, although less efficiently, as CP-independent RNP complexes , another Begomovirus  lacking the CP encoded by RNA2    that lack a CP-encoding gene and move naturally in the form of RNP complexes. These complexes are formed by the association of the viral RNA genome with the viral protein encoded by ORF3 and the host nuclear fibrillarin  forms a loop on the surface of the virion reinforces this hypothesis  require MPs for long-distance movement , which is the MP of TSWV, is essential for systemic movement  while other viruses  are dependent on the CP in addition to the TGB1 protein, for both cell-to-cell and long-distance movement  proteins, encoded by three partially overlapping ORFs in nine genera within the TGB1 is a multifunctional protein that has, among others, the property to bind single stranded RNA (ssRNA) and form RNP complexes  was shown to contain targeting sequences for the nucleolus and cajal bodies (CB) , interaction with fibrillarin may represent a more general mode of action promoting viral systemic trafficking. Similar in vitro interactions between PMTV TGB1 and fibrillarin were reported  regarding long-distance transport of its three genomic RNAs. All of them require the TGB1 to move systemically. While two of the genomic RNAs can spread in the absence of the CP, the third RNA encoding the CP needs the minor capsid protein (CP-RT) for phloem transport  in tobacco , Nicandra physaloides  VPg that abolishes its interaction with the cellular protein PVIP  was indeed sufficient to restore virus systemic spread. As 6K2-N is located on the cytoplasmic side of the membrane  is one of the tail components that targets the cell periphery and PD  which functions in RNA replication and in polyprotein processing , three non-conserved genes corresponding to the p33, p18, and p13 proteins can be deleted without affecting the ability of the virus to systemically infect the more susceptible citrus trees . Mutations in the CP gene that disrupt virion formation inhibit systemic transport  virions reduce or completely inhibit virus systemic movement, depending on the hosts . This protein of about 74 kDa is a C-terminally extended form of the CP produced by occasional suppression of the CP termination codon. It gets processed by an unknown mechanism into a 54 kDa protein (RT*), which is found incorporated into virions. CP, RT, and RT* are involved in virus long-distance transport  able to target cognate RNA sequences. These siRNA are generated from double-stranded RNA (dsRNA) by Dicer-like enzymes (DCL) and then recruited by RNA-induced silencing complexes (RISC) containing an ARGONAUTE (AGO) effector protein. siRNA guide the sequence-specific cleavage by AGO1 of homologous targets  is essential for long-distance spread in spinach and pepper plants, while it is dispensable for systemic infection of N. benthamiana and N. clevelandii, suggesting that P19 displays an essential host-dependent role in systemic movement  promoted virus exit from vascular tissues into the surrounding cells and the subsequent systemic infection of the upper leaves  was unable to systemically infect cucumber plants although it accumulated in inoculated cotyledons  and Papaya ringspot virus (PRSV) was essential to inhibit RNA silencing  in tobacco plants could be complemented in transgenic plants expressing the 5′ terminal region of the TEV genome (containing the HC-Pro coding sequence), but not in plants transformed with a mutated form of TEV HC-Pro . By monitoring the propagation of several TCV CP mutants in such plants they observed that only mutants bearing a functional silencing suppression activity could invade the vasculature of systemic leaves. Moreover, in this genetic background, all viral mutants unable to form particles remained restricted to the vascular tissues of upper leaves. These observations suggested the existence of two barriers that could block the systemic spread of TCV. The first barrier would be at the entry point into the vascular bundles and could be overcome by the CP, even if the protein is deficient for encapsidation. The second barrier corresponding to the exit from the vascular bundles of systemically infected leaves would be dependent on virus assembly. The apparent discrepancy between these data and those presented by Deleris et al.  is a multifunctional protein involved in virus assembly, but also in suppression of RNA silencing and in induction of R gene-mediated resistance . Mérai et al.  showed tClosteroviridae displays complex and diversified coding capacities. Among the 10 proteins encoded by BYV, two were reported to be enhancers of replication and involved in long-distance movement, the L-Pro and the p21 proteins. Only the latter exhibited silencing suppression activity , p14, was shown to be essential for virus long-distance movement in Beta macrocarpa  sequence resemble protein-binding sites on rRNAs  RNA-3 for systemic spread in B. macrocarpa  in which entry of Turnip vein clearing virus (TVCV) into vascular tissue is inhibited , another tobamovirus, is restrained in the A. thaliana mutant , a cell wall protein of tobacco required for cell-to-cell movement of TMV, may also assist virus long-distance transport  as a bait. Repression of IP-L expression by virus-induced gene silencing (VIGS) led to a delay in virus accumulation in non-inoculated leaves. This suggests that a high expression level of IP-L is important for efficient ToMV systemic infection  with the capacity to regulate gene expression through histone modifications  and OBERON2 (OBE2) respectively, which were described as having redundant functions in the establishment and/or maintenance of the shoot and root apical meristems  and RTM3-likes genes  is unable to move systemically in fibrillarin-silenced plants, while viral accumulation in inoculated leaves remains unaffected  systemic movement was reduced in tobacco plants treated specifically with low but not with high concentrations of the heavy metal cadmium. Using cDNA library subtraction experiments, a glycine-rich protein (GRP), which expression is specifically induced by low cadmium level, was identified and named cadmium-ion-induced GRP protein (cdiGRP). This vascular protein is localized in the cell wall of SE and CC. It contains an amino-terminal secretion signal, an internal glycine-rich domain and a carboxy-proximal cysteine-rich domain which could be responsible for protein cell wall targeting. Antisense expression of the cdiGRP cDNA in tobacco plants reduced cdiGRP mRNA accumulation in cadmium-treated plants, and allowed systemic movement of TVCV. Conversely, over-expression of cdiGRP reduces TVCV systemic movement by preventing the exit of virions from vascular bundles, which reinforces the role of cdiGRP in restricting virus long-distance trafficking. The blocking capacity of cdiGRP may be explained by callose deposition in the cell wall of phloem cells observed after cadmium treatment, or after constitutive expression of cdiGRP.Experiments performed by Ueki and Citovsky  showed tA. thaliana Col-0 populations identified several mutants allowing long-distance movement of TEV in an ecotype that normally restricts the virus to inoculated leaves. The identified RTM (for Restricted TEV Movement) resistance genes are dominant and effective against several potyviruses  and the SAR  may overlap the effect on SA-resistance. Lewsey and Carr  into the phloem tissue, virus movement inside SE, and virus unloading (exit) into cells of the sink tissue. Each of these steps represents a potential barrier for virus trafficking and several examples of viruses blocked at one stage or another were described in this review. Whether the crossing of such cell interfaces induces bottlenecks in a virus population constitutes a new challenge for the future because data on this issue are still sparse. The first quantitative analysis of a virus population in the vasculature has recently being conducted on CaMV using measures of virus titer in aphids as a reflection of virus load in the sap  and even structural motifs on viral RNA are implicated in virus long-distance transport. It becomes also clearer that in addition to the predominant virions, RNP complexes constitute an important form of long-distance spread and that several forms of viral devices can even co-exist, in the same host. Understanding whether there is a specialization of these different forms, either in the time course of infection or for the crossing of the various cell borders or even in some specific environments  will be a serious challenge. Plant physiology and virology are associated disciplines that should provide reciprocal feedbacks for the understanding of transport processes in phloem. In particular, a better knowledge of the structure and function of the various cell types composing the vascular tissues and the specialized PD at each cell interface would be greatly beneficial for virology. In addition, a deeper characterization of plant defense responses  induced during virus systemic movement is necessary to decipher their molecular mechanisms and their connexions with the viral life cycle. Finally, although many RSS with apparently very diverse modes of action were identified for most viruses, their precise involvement in virus systemic spread remains an essential issue. Among others, the question of how and where viral RSSs interfere with the movement of the extremely abundant siRNA is puzzling.An emerging field of research that appeared very recently concerns the size of virus population moving in the SE and able to invade the distant organs from the vasculature. Regulation of the virus population through the phloem involves very tightly regulated barriers that are essential check-points for plant development (Dinant and Lemoine, Finally from an agronomical point of view, identification of plant proteins required for viral systemic movement can potentially generate new sources of virus resistance in crops. Selecting from natural populations or by genetic engineering plants deficient for cellular proteins required in viral cycle is an efficient strategy to develop recessive resistance genes against viruses (Maule et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Poxviridae.A wildlife hospital and rehabilitation center in northwestern United States received several big brown bats with necrosuppurative osteomyelitis in multiple joints. Wing and joint tissues were positive by PCR for poxvirus. Thin-section electron microscopy showed poxvirus particles within A-type inclusions. Phylogenetic comparison supports establishment of a new genus of  Bats have several physiologic, cellular, and natural history characteristics that may make them particularly suited to their role as reservoir hosts (Chordopoxviridae is a subfamily of Poxviridae that contains large double-stranded DNA viruses that replicate in the cellular cytoplasm and are known to infect a wide range of vertebrates. Many of these viruses cause zoonotic disease in humans. Although poxviruses are known to have incorporated host genes into their genomes to subvert the host immune system (–Eptesicus fuscus) were brought to a wildlife hospital and rehabilitation center  during late spring or summer because they could not fly. All but 1 of the bats had >1 visibly swollen and occasionally contused joints involving the long bones of the legs and wings; 1 had contusions of the oral commissures. During 2009–2011, six  adult big brown bats  for further investigation. Histologic examination showed severe fibrino-suppurative and necrotizing tenosynovitis and osteoarthritis that involved the long bones and occasionally facial flat bones and joints with occasional localized vasculitis. No bacterial or fungal agents were seen by light microscopy of specimens stained with hematoxylin and eosin, Giemsa, Warthin-Starry, Brown and Brenn, or Gomori methenamine silver stains or in a Wright-Giemsa–stained cytologic preparation of a joint aspirate. Cultures for aerobic and anaerobic bacteria, and cultures of the joint from 1 bat for mycoplasma showed negative results.Thin-section electron microscopy of synovial tissue extracted from a wax histoblock showed poxvirus particles in inflammatory cells , panel Al-glutamine, and penicillin/streptomycin. Growth medium from a T25 flask containing green monkey kidney epithelial cells (BSC40) was removed and the virus mixture added. The flask was incubated for 1 hour at 56°C, after which 6 mL of RPMI 1640 medium was added to each flask. Cells and supernatant were harvested after 95% of the monolayer was infected. Material was taken from the wing and joint of an affected bat for real-time PCR testing and cell culture isolation. Wing and joint material was positive by real-time PCR for a poxvirus with low genomic G + C content  was briefly applied to the grid and blotted. The grid contents were visualized by using a Tecnai BioTwin electron microscope  operating at 120 kV. Digital images were captured by using a 2K × 2K camera (Advanced Microscopy Techniques). Poxvirus particles were identified in cell culture supernatant , panel Bwww.illumina.com/technology/sequencing_technology.ilmn). DNA sequences from 7 open reading frames  were extracted on the basis of sequence similarity. Data were deposited in GenBank under accession nos. KC181855–KC181861. Open reading frames were translated into amino acid sequences and aligned by using the ClustalW alignment option in Geneious version 6.0.5 (www.geneious.com/). The tree search was conducted by using MrBayes in Geneious 6.0.5 under default settings and Amsacta moorei (red hairy caterpillar) entomopoxvirus (Moyer) was used as the outgroup with a burn in of 10%.Genome sequencing produced data that were used to construct a phylogenetic tree . Virus D,Historically, osteomyelitis with arthritis has been reported in smallpox patients (osteomyelitis variolosa) and in smallpox vaccine recipients (vaccinia osteomyelitis), but did not occur frequently (–Chordopoxvirus; its nearest neighbor is Cotia virus, an as yet unclassified chordopoxvirus first isolated from sentinel suckling mice in a state reserve in Cotia County, São Paulo State, Brazil, in 1961 (Results of at least 3 investigations that involved detection of viral DNA detection in bat guano have been published; 2 involved bats from North America and 1 involved bats in China (Chiropoxvirus. Further efforts should be undertaken to determine whether Cotia virus should be included in this genus. The bat-derived virus requires a new species designation for which we propose Eptesipox virus because of its isolation from an Eptesicus fuscus bat specimen.Although the 2 viruses are nearest neighbors, levels of shared nucleotide and amino acid identity between them suggest they should likely be considered separate genera (


Lignin is often the most difficult portion of plant biomass to degrade, with fungi generally thought to dominate during late stage decomposition. Lignin in feedstock plant material represents a barrier to more efficient plant biomass conversion and can also hinder enzymatic access to cellulose, which is critical for biofuels production. Tropical rain forest soils in Puerto Rico are characterized by frequent anoxic conditions and fluctuating redox, suggesting the presence of lignin-degrading organisms and mechanisms that are different from known fungal decomposers and oxygen-dependent enzyme activities. We explored microbial lignin-degraders by burying bio-traps containing lignin-amended and unamended biosep beads in the soil for 1, 4, 13 and 30 weeks. At each time point, phenol oxidase and peroxidase enzyme activity was found to be elevated in the lignin-amended versus the unamended beads, while cellulolytic enzyme activities were significantly depressed in lignin-amended beads. Quantitative PCR of bacterial communities showed more bacterial colonization in the lignin-amended compared to the unamended beads after one and four weeks, suggesting that the lignin supported increased bacterial abundance. The microbial community was analyzed by small subunit 16S ribosomal RNA genes using microarray (PhyloChip) and by high-throughput amplicon pyrosequencing based on universal primers targeting bacterial, archaeal, and eukaryotic communities. Community trends were significantly affected by time and the presence of lignin on the beads. Lignin-amended beads have higher relative abundances of representatives from the phyla Actinobacteria, Firmicutes, Acidobacteria and Proteobacteria compared to unamended beads. This study suggests that in low and fluctuating redox soils, bacteria could play a role in anaerobic lignin decomposition. Panicum virgatum) could potentially provide energy via biofuels if a system for unlocking this energy were devised that was robust, efficient and inexpensive There is a strong impetus both nationally and internationally for devising new, non-fossil based fuels that are generated in a sustainable way with minimum greenhouse gas production p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, are synthesized in different ratios and combinations depending upon the type of plant, and so conferring its structural characteristics. The best understood mechanism for breaking open the rings in the lignin phenols belongs to fungi, specifically via oxygen free radical attached by the enzymes dioxygenases Streptomyces viridosporusKocuria and StaphylococcusFlavobacterium meningosepticumLignin is a complex heteropolymer linked to cellulose, giving plants structural integrity. The deconstruction of lignin and its dissociation from cellulose presents a challenge for soil microbes and biofuels engineers alike. The repeating units of phenolic monomers, Plant litter quality is a key controller of decomposition rates in soils, and lignin and the lignin∶N ratio play a particularly important role in late stage decomposition Humid tropical forest soils house an immense and unexplored microbial diversity We employed Bio-trap® samplers , where pure low-sulfonate alkali lignin  was trapped within Bio-Sep® (University of Tulsa) beads to create the lignin-amended traps, and unamended beads were used as controls. The traps contain about 200 g Bio-Sep® beads, 3 mm diameter balls composed of 75% powdered activated carbon and 25% DuPont aramid polymer, resulting in a matrix that has 75% porosity 2O2. We also performed cellulase enzyme assays using the fluorogenic detection molecule methylumbylliferyl (MUB): MUB-beta-d-glucopyranoside for beta-glucosidase (EC 3.2.1.21); MUB-cellobioside for cellobiohydrolase (EC 3.2.1.91); MUB-beta-xylopyranoside for beta-xylosidase (EC 3.2.1.37); MUB-N-acetyl-beta-glucosaminidase for chitinase (EC 3.2.1.30). Rates are the mean of sixteen technical replicates of amount of substrate evolved per unit time per gram bead.Enzyme assays were performed on beads fresh from the field at the 1, 4, and 30 weeks. For enzyme assays, 3–4 g beads were added to 50 mM acetate buffer solution pH 5.5, mixed by stirring for 2 min, then the buffer extract was analyzed for enzyme activity. We performed oxidative enzyme assays using a colorimentric method for phenol oxidase (EC 1.10.3.2) L-dihydroxyphenylalanine (L-DOPA), and peroxidase (EC 1.11.1.7) DOPA plus 0.3% HDNA from the beads was extracted using a modified CTAB extraction method as previously described 5′-aaactYaaaKgaattgacgg-3′) and 1392R (5′-acgggcggtgtgtRc-3′) were used to amplify the V8 variable region of the 16S rRNA gene from bacteria and archaea as well as the 18S rRNA gene in eukarya http://pyrotagger.jgi-psf.org/), which filters by removing low-quality sequences from the set based on the qual file, trims using a 225 bp sequence length threshold, dereplicates, clusters at the OTU level based on 97% identity, then classifies The same four biological replicates were sequenced for small subunit (SSU) rRNA genes using high-throughput amplicon pyrosequencing. The universal primers 926F  of total bacterial cells demonstrated significantly more bacteria associated with the lignin-amended beads compared to the unamended beads in the beginning and end of the experiment . There wMicrobial communities showed a significant separation of the lignin-amended bead community from the unamended bead community analyzed using amplicon pyrosequencing ; the sloThe PhyloChip is designed based on a well-supported phylogenetic tree of Archaea and Bacteria A summary of the taxa that are significantly different in the lignin-amended beads compared to unamended beads, broken down by time point, show differences in populations with lignin amendment and a community succession over time . ExaminiTo identify taxa enriched in the lignin-amended beads we did a more detailed analysis on the microbial communities identified at the first time point after one week of incubation (T1), chosen because these samples had significantly higher phenol oxidase activities compared to the control beads; significantly lower cellulase enzyme activities; significantly lower richness and significantly higher numbers of bacteria as determined by Q-PCR. Based on PhyloChip results we performed a one-tailed, paired t-test for each identified taxon, and found a total of 39 taxa were significantly different between the lignin-amended beads and the controls, of which 38 were enriched on the lignin-amended beads . These tCaulobacter intermedius and Brevundimonas diminuta, and these taxa are known catalase producers. Caulobacter crescentis is an obligate aerobe that produces catalase likely as protection from oxidative stress in late-stationary phase in culture Rhodomicrobium is an Alphaproteobacteria in the family Rhizobiales that was detected by the pyrosequencing analysis, and also a known purple non-sulfur bacterium. While taxa in this genus are able to link iron reduction and denitrification to photosynthesis Escherichia spp. observed as lignin-degrading from the guts of wood-boring beetles Streptomyces viridosporus and Rhodococcus spp. demonstrated to have lignin degrading activity This study demonstrates that the lignin-amended biosep beads are an effective method for trapping soil populations with the specific capability of decomposing lignin. Substantial phenol oxidase and peroxidase accompanied by depressed carbohydrate-active enzyme activity and low microbial community richness after one week suggests the capture of a fairly specialized group of microorganisms adapted to the lignin-amended bead environment. There were a number of taxa that were dominant early on in the experiment and more abundant in lignin-amended than unamended bead communities, which presumably play a role in lignin decomposition in the soil. Bacteria known to break down lignin are concentrated in the Alphaproteobacteria, Gammaproteobacteria, and Actinomycetes Enterobacteriaceae has been observed to out-compete obligate anaerobes under similar conditions Flavobacterium meningosepticum was isolated from soil and shown to not demonstrate catalase activity, though it has the ability to grow on phenolic, model lignin compounds as sole C and energy source Frequent episodes of soil anoxia have been observed in these soils and are known to affect the microbial community Kocuria (Actinobacteria) and Staphylococcus (Firmicutes) were previously described as phenol-degraders in soil Fungi are generally considered the main microbial decomposers of plant material There was a strong effect of time on the microbial community structure and function  suggestiWhile the pyrosequencing and PhyloChip microarray microbial community profiles agreed well with each other, there were some differences at the species-level identification of lignin bead-associated microbial taxa due to the fact that these two methods assay microbial communities in very different ways. Though both begin with PCR amplification, the primers used are tailored to each method; for pyrosequencing, the universal primers are designed to capture as much of the bacteria, archaea, and eukaryota in any environmental sample The lignin-baited biosep beads appear to be efficient bio-traps for capturing lignin-degrading microbial populations, baited with commercial alkali lignin and tested with L-DOPA, phenolic model lignin compounds which bear structural similarities to carbon compounds found in the environment like humics, lignin breakdown products and contaminants. The aromatic compounds benzoate, phenylpropionate and phenylacetate are produced as natural by-products in the anaerobic rhizosphere of rice field soil The data taken together suggest that the lignin had an adverse effect on all but a specific subset of the microbial community, and this select population is likely able to enzymatically access and assimilate carbon derived from the lignin. Phylogenetic analysis also demonstrated a significant increase in the diversity and clustering of the community on lignin-amended beads compared to unamended beads, suggesting that the lignin either directly created an chemical environment unfavorable to all but a small population of bacteria, or the taxa initially able to utilize the lignin had a competitive advantage and out competed later immigrant populations. The molecular mechanisms of this largely anaerobic lignin-degrading population are of interest and under investigation.Figure S1Photographs of bio-traps. These images show (A) Bio-Sep beads and (B) bio-traps made of slotted PVC to hold the beads.(TIF)Click here for additional data file.Figure S2Gas concentrations in field chambers. Oxygen concentration (A) and nitrous oxide, carbon dioxide, and methane (B) measured in the oxygen chambers in the field during the course of the experiment. The asterisks (*) show the times when samples were taken.(TIF)Click here for additional data file.Figure S3Comparison of taxa detected by amplicon pyrosequencing in lignin-amended compared to unamended beads. The correlations are shown with the R-square values, equations for linear fit as well as significance values. They are all significantly correlated, which would indicate no differences between lignin and no-lignin bead communities, however, from the R-square values it seems that there are some differences.(TIF)Click here for additional data file.Figure S4Phylogenetic relatedness of PhyloChip microbial communities. (A) Report of community relatedness by net relatedness index (NRI) or nearest taxon index (NTI). (B) Community relatedness is plotted as a function of community richness for PhyloChip microbial community analysis. The community analysis program phylocom was used to generate estimates of phylogenetic clustering in microbial communities using the net relatedness index (NRI), which is a measure of tree-wide phylogenetic dispersion, and nearest taxon index (NTI), which is a measure of branch-tip phylogenetic dispersion.(TIF)Click here for additional data file.Figure S5Shannon's diversity index for microbial communitiesby SSU rRNA pyrosequencing. The data are displayed as a box-and-whiskers plot.(TIF)Click here for additional data file.Figure S6Richness of PhyloChip and pyrosequencing microbial communities. Box plots display richness detected in lignin-amended and unamended biosep beads over time. For PhyloChip and pyrotag community richness, there was no significant trend between lignin-amended and unamended beads, though there was a significant effect of time.(TIF)Click here for additional data file.Table S1Enzyme activity rates measured on fresh beads.(PDF)Click here for additional data file.Table S2PhyloChip richness of taxa from the lignin-amended and unamended beads.(PDF)Click here for additional data file.Table S3PhyloChip richness of taxa with significantly higher relative abundance in lignin-amended beads compared to unamended beads.(PDF)Click here for additional data file.Table S4Phylogenetic classification at the phylum level of bacterial taxa identified by SSU rRNA amplicon pyrosequencing.(PDF)Click here for additional data file.Table S5Phylogenetic classification of identified SSU rRNA sequence tags (pyrosequencing) based on phylum level.(PDF)Click here for additional data file.Table S6Taxonomy and nearest neighbor of 38 taxa significanlty enriched on lignin beads by PhyloChip.(PDF)Click here for additional data file.


The main antivirals employed to combat seasonal and pandemic influenza are oseltamivir and zanamivir which act by inhibiting the virus-encoded neuraminidase. These have to be deployed close to the time of infection and antiviral resistance to the more widely used oseltamivir has arisen relatively rapidly. Defective interfering (DI) influenza virus is a natural antiviral that works in a different way to oseltamivir and zanamivir, and a cloned version  has proved effective in preclinical studies in mice. The active principle is the DI RNA, and this is thought to interact with all influenza A viruses by inhibiting RNA virus synthesis and packaging of the cognate virion RNA into nascent DI virus particles. We have compared the ability of DI virus and oseltamivir to protect ferrets from intranasal 2009 pandemic influenza virus A/California/04/09 . Ferrets were treated with a single 2 μg intranasal dose of 244 DI RNA delivered as 244/PR8 virus, or a total of 25 mg/kg body weight of oseltamivir given as 10 oral doses over 5 days. Both DI virus and oseltamivir reduced day 2 infectivity and the influx of cells into nasal fluids, and permitted the development of adaptive immunity. However DI virus, but not oseltamivir, significantly reduced weight loss, facilitated better weight gain, reduced respiratory disease, and reduced infectivity on days 4 and 6. 244 DI RNA was amplified by A/Cal by >25,000-fold, consistent with the amelioration of clinical disease. Treatment with DI virus did not delay clearance or cause persistence of infectious virus or DI RNA. Thus in this system DI virus was overall more effective than oseltamivir in combatting pandemic A/California/04/09. As a result their replication is dependent on complementation by a genetically compatible infectious helper virus to provide any missing function. All DI genomes retain sequences that allow them to be packaged and replicated. The resulting DI virus particle is usually indistinguishable from that of the infectious virus. In cell culture DI viruses are not only defective but also interfering, the DI genome being the structure responsible for this property. Thus, under appropriate conditions, the presence of the DI genome reduces the amount of infectious progeny virus produced . Some, bterferon . DI viruterferon .Influenza A DI viruses were the first to be described  and haveWe have shown that non-cloned and cloned DI influenza A viruses protect mice from lethal infection caused by influenza A virus . Only a Influenza viruses usually have to be adapted to grow in mice whereas the ferret is highly susceptible to new human isolates . BecauseIn addition to vaccines  antiviraOseltamivir was preclinically tested in ferrets and these animals are the preferred model for studying study new viruses and investigating oseltamivir-resistant strains . Thus we22.1Mustela putorius furo), 3–4 months of age, weighing 860–1367 g (mean 1082 g), were obtained from Highgate Farm, UK. They were seronegative for antibodies to A/Cal as determined by haemagglutination-inhibition. Ferrets were separated into 4 groups each comprising five animals: groups were treated intranasally with +300 μg active 244 DI virus and infected with A/Cal 2 h later, treated with oseltamivir by oral gavage (see below) and infected with A/Cal 2 h later, infected with A/Cal, and inoculated with saline. An identifier chip  was inserted subcutaneously into the scruff of each animal. Ferrets receiving 244 DI virus (see below) were sedated by isoflurane inhalation before intranasal delivery of 500 μl (250 μl per nostril) of a single dose of 2 μg of 244 RNA in 300 μg of carrier virus. Ferrets receiving oseltamivir treatment were given 2.5 mg/kg bodyweight administered by oral gavage twice-daily every twelve hours (5 mg/kg bodyweight/day) over a period of five days as used by others . Solutions were prepared on the day of challenge and the titre of the virus confirmed by infectivity assay. Control groups were infected with virus or inoculated with saline. Rectal temperatures were measured daily. Ferrets were monitored twice-daily post-challenge throughout the course of the study for clinical signs of influenza infection . Clinical signs were scored as follows: loss of activity scored 0 for normal activity levels, 1 for reduced activity, and 2 if inactive; nasal discharge scored 0 for no discharge and 1 for a discharge; sneezing scored 0 for no sneezing, and 1 for sneezing; appetite was scored 0 for no loss of appetite, and 1 for loss of appetite. Nasal washes were collected from each ferret following ketamine and xylazine sedation (as above) at days 1–6 and then at days 8, 10 12 and 14 post-challenge. For each nasal wash, 2 ml of PBS were instilled by small multiple volumes into each nasal cavity with expectorate collected into a beaker. The study was terminated at 14 days post-challenge.Ferret work was conducted according to UK Home Office legislation and was approved by the local ethical committee. Thirty outbred male ferrets . This is referred to as ‘active DI virus’. The UV inactivation target is viral RNA, and UV has little effect on the DI RNA because of its small target size, 395 nt compared with 13,600 nt for infectious virus. The absence of infectivity after UV-irradiation was checked by infectivity assay , and quantitative real time PCR performed to quantitate virion-sense RNA using an ABI prism 7000. The following primers and probe were used: 244 1F (5′ CTCTTTGCCCAGAATGAGGAAT 3′), 244 1R (5′ CATAATCAAGAAGTACACATCAGGAAGAC 3′) and probe (5′ FAM-CCCTCAGTCTTCTCC 3′). Primers were synthesized by Invitrogen, and the probes by ABI. Reverse transcriptase reactions (10 μl) were performed using 6 μl extracted RNA, RevertAid reverse transcriptase and random hexamer (Fermentas) according to the manufacturer’s instructions. cDNA (1 μl) was used in 20 μl of PCR reaction. A virion-sense 244 RNA standard was made by subcloning PCR products of full length 244 RNA in pGEMT-easy vector (Promega). RNA was transcribed using the T7 polymerase , the mix was digested with DNase I, and RNA purified by electro-elution. After ethanol precipitation, RNA was resuspended into RNase-free water and quantitated on a Nanodrop 1000 . Standard curves were generated by performing 10-fold serial dilutions of known RNA copy numbers with each dilution assayed in duplicate. The reaction was conducted at 50 °C for 2 min, 95 °C for 10 min, then 40 cycles of 94 °C for 15 s followed by 60 °C for 1 min.2.4Nasal washes from each ferret were titrated for A/Cal infectivity in a focus-forming assay using MDCK cells in 96-well plates in triplicate . After i2.5Before assay sera were treated with receptor destroying enzyme (RDE II (SEIKEN), Cosmos Biological) overnight at 37 °C to remove non-specific inhibitors of haemagglutination and then incubated at 56 °C for 30 min to destroy the enzyme. Serial 2-fold dilutions of serum were incubated with 4 HAU of A/Cal for 1 h at ambient temperature before adding chicken red blood cells . The HI titre is expressed as the reciprocal of the dilution of serum that causes 50% inhibition of agglutination, and is interpolated between full agglutination and no agglutination.33.1p < 0.05). The data also show a single treatment of infected ferrets with 244 DI virus resulted in a greater overall weight gain that was seen with the infected control animals (p < 0.05). This indicates that while the treated animals experienced a transient weight loss on day 3 . This was due to one ferret in the control A/Cal-infected group that recorded a non-elevated temperature, as omission of this data point gave p values of 0.02 and 0.04 for treatment with 244 DI virus or oseltamivir, respectively (p = 0.32).ectively b. There 3.3p = 0.009) reduced the score compared with the infected A/Cal control group by 1.7-fold  over the 14-day duration of the study showed that treatment with 244 DI virus significantly , a highly significant difference . Thus amelioration of infection by 244 DI virus appeared to improve rather than diminish the antibody response to the A/Cal haemagglutinin.There was a strong HI antibody response to A/Cal/04/09 (H1N1) in sera taken at 14 days after infection whether groups had been treated with 244 DI virus, oseltamivir or were untreated . The tit4In this study we compared the effects of treatment with DI virus and oseltamivir on the course of disease and virus load resulting from infection with 2009 pandemic influenza A virus  in the ferret. Data summarized in Oseltamivir treatment was also beneficial although it did not cause any significant decrease in weight loss or respiratory disease when compared to the infected animals that were not given any other treatment. Oseltamivir reduced virus load on day 2, but the virus load in oseltamivir-treated animals was more than 100-fold greater than the virus control on day 6. This differential appears consistent with a viral rebound observed following the cessation of oseltamivir treatment in people infected with pandemic 2009 virus . We alsoAll ferrets developed A/Cal-specific, serum HI antibody, but there was significantly less in the oseltamivir-treated infected group than in the DI virus treated infected group. In addition to the signs and symptoms described above ferrets were monitored in the morning and the afternoon for loss of appetite, appearance of diarrhoea, and reduction in activity. None of these was seen in any group.We conclude that treatment of ferrets with 244 DI virus ameliorates clinical disease and virus load resulting from infection with pandemic A/California/04/09 (H1N1) more effectively than did treatment with oseltamivir. DI virus did not cause virus to become persistent or delay clearance, and the presence of high levels of HI antibody in DI-treated, A/Cal-infected animals suggest that ferrets would be able to resist a repeat dose of virus.