Arthrobacter ureafaciens neuraminidase. The distribution of the diffusion coefficient shows that 70% of SIV particles were entrapped, while the rest diffused freely in the mucus. Additionally, SIV penetrated the porcine mucus with time, reaching a depth of 65 µm at 30 min post virus addition, 2 fold of that at 2 min. Both the microscopic diffusion and macroscopic penetration were largely diminished by NAI, while were clearly increased by the effect of exogenous neuraminidase. Moreover, the exogenous neuraminidase sufficiently prevented the binding of SIV to mucus which was reversely enhanced by effect of NAI. These findings clearly show that the neuraminidase helps SIV move through the mucus, which is important for the virus to reach and infect epithelial cells and eventually become shed into the lumen of the respiratory tract.Swine influenza virus (SIV) has a strong tropism for pig respiratory mucosa, which consists of a mucus layer, epithelium, basement membrane and lamina propria. Sialic acids present on the epithelial surface have long been considered to be determinants of influenza virus tropism. However, mucus which is also rich in sialic acids may serve as the first barrier of selection. It was investigated how influenza virus interacts with the mucus to infect epithelial cells. Two techniques were applied to track SIV H1N1 in porcine mucus. The microscopic diffusion of SIV particles in the mucus was analyzed by single particle tracking (SPT), and the macroscopic penetration of SIV through mucus was studied by a virus in-capsule-mucus penetration system, followed by visualizing the translocation of the virions with time by immunofluorescence staining. Furthermore, the effects of neuraminidase on SIV getting through or binding to the mucus were studied by using zanamivir, a neuraminidase inhibitor (NAI), and  Pigs are naturally susceptible to three subtypes of influenza A viruses: H1N1, H3N2 and H1N2, all of which have a strong tropism for the pig respiratory tract mucosa et al.Mucus is a complex mixture of mucous glycoproteins (mucins), proteins, proteases and protease inhibitors, lipids and water et al.et al.in vitro model resembling the in vivo situation is needed and the interactions of influenza virus with natural mucus should be studied in depth. In the present study, we aimed to address the following questions:Despite this inhibitory function of the mucus, the virus is ultimately able to reach the susceptible epithelial cells. It has long been assumed that NA promotes virus access to target cells in the airway by mucus degradation. However, this concept is scarcely supported by experimental data. Cohen Is the virus entrapped or able to penetrate through the native respiratory mucus?Can viral neuraminidase use mucin sialic acids as substrates and catalyze the cleavage of them away?Is this cleavage sufficient to liberate the virions and allow them to penetrate through the mucus layer?To this purpose, we applied swine influenza virus to a model we previously set up using porcine respiratory mucus, pseudorabies virus (PRV) and single particle tracking (SPT) Tracheas were collected from 6-month-old pigs which were negative for swine influenza A viruses as shown by a Hemagglutination Inhibition (HI) test. Using of tracheas from euthanized animals was approved by the Ethical and Animal Welfare Committee of the Faculty of Veterinary Medicine of Ghent University. Two days before euthanization, the pigs were treated intramuscularly with ceftiofur  to clear the respiratory tract from possible bacterial infections. The tracheas were dissected and the mucus was gently scraped with a spoon, collected with a syringe, and the mucus samples were stored separately at −70°C until use. These samples were negative for neuraminidase determined by NA assay using fluorescent NA substrate (4-methylumbelliferyl-N-acetylneuraminic acid [MU-NANA]).Sambucus nigra lectin (SNA-I)  for 1 h, at room temperature. After 2 washings in phosphate buffered saline (PBS), the sections were incubated with Streptavidin-Texas Red  and FITC labeled Maackia amurensis lectin (MAA)  for 1 h, at room temperature. Afterwards, the sections were washed, and mounted in 90% glycerin containing 2.5% 1,4-diazobicyclo--octane (DABCO). Images of the fluorescence staining were acquired using a confocal microscope  and the fluorescence signal was analyzed with ImageJ. The coverage of either SA was calculated by ratio of fluorescence signal to the region of interest (ROI).Freshly collected mucus was filled in a gelatin capsule (2.3 cm × 0.8 cm), snap frozen in methocel (Fluka). Cryosections of 12 µm were made with a trimming interval of 400 µm between each section. The mucus sections were blocked in 1% (w/v) bovine serum albumin for 1 h, followed by incubation with biotin conjugated Madin Darby Canine Kidney (MDCK) cells were maintained in Minimum Essential Medium  supplemented with 10% fetal calf serum (Gibco), 100 µg/ml of streptomycin and 100 units/ml of penicillin (Invitrogen). The avian-like H1N1 swine influenza virus Sw/Belgium/1/98 was used at the third passage on MDCK cells. The virus was propagated in MDCK cells in MEM supplemented with 5 µg/ml trypsin (Gibco), 100 µg/ml of streptomycin and 100 units/ml of penicillin (Gibco).Confluent MDCK cells were inoculated with SIV at a multiplicity of infection (m.o.i) of 0.01 in MEM. Twenty hours post inoculation, the supernatant was harvested. The cellular debris was removed by ultracentrifugation at 7 000 ×g for 20 min at 4°C in a Type 35 rotor  and the suspension was clarified by filtration with a 0.45 µm filter (Millipore). Afterwards, the virus was pelleted at 75 000 ×g for 2 h at 4°C in a type 35 rotor. Following resuspension in PBS , the virus suspension was brought on a discontinuous OptiPrep (Sigma) gradient containing 10–30% (w/v) of iodixanol and centrifuged at 100 000 ×g for 3 h at 4°C in an SW41Ti rotor . The visible opalescent virus bands at the interfaces were harvested separately. The buffer was exchanged with HNE  buffer by the use of a 50 kDa filter device (Millipore).To verify the purity of the virus from each band, double staining was performed by using 3,3′-Dioctadecyloxacarbocyanine perchlorate (Dio) and hyperimmune swine serum directed to influenza Sw/Belgium/1/98 virus, followed by Texas Red-conjugated goat anti-swine IgG antibody. Hyperimmune swine serum was diluted (1∶50) and mixed with the virus suspension . After 2 h incubation on ice, Texas Red-conjugated goat anti-swine IgG secondary antibody  was added and incubated further on ice for 2 h. Afterwards, the resulting mixture containing virus, hyperimmune serum, and secondary antibody was equilibrated to room temperature. Dio solution (1 mM in DMSO) was mixed with virus suspension  by fierce vortexing, followed by incubation at room temperature for 20 min. The resulting suspension was ultracentrifuged at 100 000 ×g for 1.5 h in a type 35 rotor to remove the free antibodies and dyes. After resuspension in PBS, the virions were clarified by a further ultracentrifugation at 100 000 ×g for 1.5 h. The staining was analyzed with confocal microscope by randomly selecting 10 regions. The ratio of viral antigen positive particles (virions) versus Dio positive particles was calculated, which is referred to as the degree of viral purity.zeta potential) of the labeled and unlabeled virions were measured by dynamic light scattering and laser Doppler anemometry as previously described et al.The band containing the most purified virus was obtained and the buffer was exchanged with HNE buffer. After incubation with Dio solution as previously described, the virus suspension was filtered by the use of a Sephadex G-50 column  to remove unbound dye. The unlabeled virus that was eluted through the Sephadex G-50 column was used as negative control. Dio is a lipophilic dye which integrates into the lipid components of the viral envelope. To determine that the integration of the Dio dye into the viral envelope does not change the biophysical properties of the virus, the size and surface charge (8.5 TCID50/ml) or PRV (108.5 TCID50/ml) suspension or 100 nm PEGylated beads  were mixed with 100 µl of porcine tracheal respiratory mucus by gentle stirring. To determine if NA would affect SIV diffusion, the virus was added to the mucus with or without the presence of 0.02 µM zanamivir (Sigma) or 10 mU/ml Arthrobacter ureafaciens neuraminidase (Roche Applied Science). The mixture was placed in a custom made glass chamber. The samples were incubated at 37°C for 10 min on the microscope using a stage-top incubator  before SPT measurement. Movies were captured with the NIS Elements AR software (Nikon) at a temporal resolution of 33 ms for 5 s. The illumination time was 30 ms per frame. Trajectories of n ≥ 500 particles were analyzed for each experiment and three independent experiments were performed for each condition. Movies were analyzed with the Image Processing Software  Da) was calculated as a function of the time scale (t) for each particle. Analysis of the movies was performed with IPS. The centroids of individual particles were identified in each frame of a movie. Based on the positions of the centroids, the trajectories of the particles can be determined by a nearest neighbor algorithm. The apparent diffusion coefficient Da corresponding to the first time lag Δt was calculated according to the classical formula: Da  =  MSD/4ΔtThe trajectories of fluorescent viral particles in porcine tracheal respiratory mucus were recorded by a fast and sensitive electron-multiplying charge-coupled device (EMCCD) camera  mounted on an inverted epifluorescence microscope  equipped with a 100× oil-immersion objective . Tracking experiments were performed in press-to-seal silicone isolators . Our previous study shows that PRV was highly immobilized while 100 nm PGEylated beads were diffusive in porcine respiratory mucus, hence GFP-PRV was used as a negative control and the latter was used as a positive control 6.5 TCID50 purified SIV were brought in the form of 5 droplets onto the surface of the mucus. Immediately, 10 min and 30 min after virus addition, the capsules were snap frozen in methocel. Due to a delay of freezing process, the time point “immediately after addition” was designated as 2 min. To determine if the NA would influence the SIV penetration, the virus was added with or without the presence of 0.1 µM Zanamivir (Sigma) or 50 mU/ml Arthrobacter ureafaciens neuraminidase onto the mucus, followed by snap-freezing at 30 min post virus addition. Cryosections of 12 µm were made with a trimming interval of 400 µm between each section. Double immunofluorescence staining was performed using mouse anti-Muc5AC IgG1  and mouse anti-NP IgG2a  monoclonal antibodies, followed by Texas Red conjugated goat anti-mouse IgG1 and Alexa Fluor 488 conjugated goat anti-mouse IgG2a secondary antibodies (Invitrogen), respectively. Ten sections were made for each condition, then 10 images were taken, and finally translocations of the virions was measured, as shown in Freshly collected mucus sample (150 µl) was added to a gelatin capsule (2.3 cm × 0.8 cm) to create a mucus “layer” at the bottom  Triton X-100 for 10 min. Immunofluorescence staining was performed using a mouse IgG antibody to SIV NP, followed by Goat anti-mouse IgG antibody conjugated with FITC to visualize the virions. Fluorescence images were acquired on randomly selected regions with a confocal microscope, and the bound virions on the mucus were calculated with ImageJ.Mucus sections (12 µm) were made, and incubated with 30 µl suspension containing 10Five sections were examined for each mucus sample for the semi-quantification of α2,3- and α2,6-SA coverage in the mucus. For each section, 2 images were taken and thus 10 images were obtained for each sample. As shown in After ultracentrifugation over a discontinuous OptiPrep gradient containing 10% to 30% of iodixanol, three visible opalescent bands were collected, named Band 1, Band 2 and Band 3, respectively, from top to bottom . The purAfter incubation with 10 µM Dio dye at room temperature, followed by elution in a Sepharose G-50 column, the labeled and unlabeled SIV were analyzed for different characteristics. The results show that the hemagglutination activity and infectivity were not altered by labeling. The neuraminidase activity of Dio-labeled SIV was 91% of that of unlabeled SIV. Measured by dynamic light scattering and laser Doppler anemometry, the size and surface charge of the labeled virions were not significantly altered .The motion of SIV in porcine respiratory mucus was investigated and compared with the diffusion of PRV and 100 nm PEGylated beads. Trajectories of 8 steps were analyzed, from which a distribution of the apparent diffusion coefficients was obtained. Similar to our previous data, PRV was highly hindered in the porcine respiratory mucus, while the 100 nm PEGylated beads diffused freely. The distribution of diffusion coefficient clearly demonstrated that, compared to one immobile fraction for PRV or a mobile fraction for the 100 nm beads, SIV experienced two diffusion patterns in porcine respiratory mucus, with 70% of viral particles being trapped while the rest of particles moving rapidly . The aveThe depth of SIV penetration could be visualized by double immunofluorescence staining of the Muc5AC and SIV nucleoprotein (NP). The distance from the surface down to the deepest point of virus translocation was measured and designated as the depth of virus penetration . Three iArthrobacter ureafaciens neuraminidase. Approximately 55% of the mobile viral particles (with Da larger than 0.2 µm2/s) became stuck by the effect of zanamivir whereas the exogenous neuraminidase increased the mobile particles by approximately 15% , collectins, defensins, cathelicidins, histatins and surfactant proteins in vivo influenza infection study in pigs despite reduced clinical symptoms and virus shedding in vivo situation and the minimal benefits neuraminidase inhibitors may have.While vaccination remains the primary option for the prevention and control of influenza, anti-influenza virus drugs are considered as a complementary approach, as vaccine production may not be rapidly achieved. Our findings provide experimental evidence for the essential role of NA in influenza virus penetration through the respiratory mucus. Blocking the NA activities clearly suppressed the movement of virus in mucus , illustret al.The ability of an influenza virus passing through the mucus may serve as a determinant for influenza virus transmission in addition to efficient virus attachment, high potential of replication and low infectious dose required Since the majority of viral particles were incapable of penetrating through the mucus layer, why do influenza viruses invade the respiratory tract of the animals after all E. histolyticaVibrio choleraeHelicobacter pyloriProduction of enzymes that aid the virus movement through the mucus. Influenza virus binds to and uses sialic acid-containing molecules as receptors. It is because of this capability that influenza virus has evolved a second viral surface protein, neuraminidase, as a receptor-destroying enzyme that cleaves sialic acid, allowing the virus to be released after binding to sialic acid-containing molecules that do not lead to viral infection. This strategy is utilized as well by many other microbes, such as The use of abundant and ubiquitous molecules as receptors. Although there may be a risk of binding to decoy receptors, the use of abundant and ubiquitous molecules as receptors provides the apparent advantage to the virus for allowing infection of multiple cell types and species. This can result in a low minimal infectious dose for initial infection. Based on the data of diffusion and penetration, the effects of the mucus network that virions encounter are so extreme that only a part of the particles can escape and reach susceptible target cells ultimately. Thus the viruses which require lower minimal infectious doses for the same tissues may gain higher chance to establish an infection.Spreads via aerosol. The slow settle of aerosols in the air can cause prolonged contact of the virus with the respiratory tract which benefits the virus penetration through the mucus layer. Furthermore, aerosol droplets can travel much more efficiently to the lower respiratory tract and the mucociliary apparatus may need a longer time to transport and exclude the virions out of the respiratory tract, which increases the chance of these viruses to penetrate through the mucus layer and reach the target cells eventually.The issues on if SIV would be able to penetrate through the porcine respiratory mucus and if the neuraminidase would contribute to move the virus through the mucus layer have been addressed. However, the ability of the viral neuraminidase to cleave sialic acid from mucus has not been investigated due to technical limitation. The viscous property impedes the separation of the free sialic acids from the mucus even if they would have been cleaved by the viral neuraminidase. Investigating the role of influenza virus neuraminidase in the cleavage of sialic acid from mucus may shed some light on unravelling the mechanism of influenza pneumonia. Hence the effect of influenza virus neuraminidase on mucus needs to be studied.


Circoviridae may have neurologic tropism more commonly than previously anticipated.A fox circovirus was identified in serum samples from foxes with unexplained neurologic signs by using viral metagenomics. Fox circovirus nucleic acid was localized in histological lesions of the cerebrum by in situ hybridization. Viruses from the family  Circoviridae) are nonenveloped, single-stranded, circular DNA (≈2 kb) viruses  medical treatment. All procedures were performed in compliance with relevant laws and institutional guidelines. Following euthanasia, necropsies were performed according to standard procedures. Samples were stored in 10% neutral buffered formalin and embedded in paraffin, and 4 μm–thick sections were stained with hematoxylin and eosin and evaluated for the presence of histologic lesions.Toxoplasma gondii, and Neospora canium (data not shown). Serologic test results for canine distemper virus, rabies virus, N. canium, and tickborne encephalitis virus were negative, and Ziehl-Neelsen and Giemsa staining results for microsporidia were negative. Minor white matter involvement, the duration of animal survival, and the current absence of documented rabies cases in the United Kingdom eliminated rabies virus as the cause of the neurologic disorder.All foxes had similar histologic findings consisting of chronic multifocal or diffuse lymphoplasmacytic meningoencephalitis oriented on the forebrain with a predilection for cortical gray matter  Figure 1Serum samples from 6 of the foxes (VS7100001–6) were available for virus discovery studies. To perform the studies, we used a viral metagenomics approach with the 454 sequence platform  as described previously . More thA diagnostic real-time fox circovirus PCR was performed targeting the Rep-coding sequence on 32 serum samples from foxes with and without neurologic signs . Viral nOur findings indicate that circoviruses commonly cause systemic infections in wild foxes in the United Kingdom and can be detected in the brains of foxes with neurologic disease. It has been suggested that circoviruses are involved in a plethora of diseases in pigs, dogs, and birds (Circoviridae, were recently found in serum and cerebrospinal fluid of humans with paraplegia and acute infections of the central nervous system (Circoviridae may have neurologic tropism more commonly than previously anticipated. However, a causal link between circovirus infection and disease in humans and animals remains to be proven. Because the prevalence of circoviruses in foxes was relatively high and closely related circovirus species seem pathogenic for both dogs and foxes, additional surveillance is warranted to clarify the epidemiology and pathogenicity of circoviruses in foxes.Cycloviruses, which belong to a proposed new genus in the family Histopathologic features of brain tissues from foxes with possible circovirus-induced neurologic disease and relative abundance of broad taxonomic categories in metagenomic sequences obtained from fox serum samples.


Culex spp. mosquitoes. We analyze these data, in conjunction with a comprehensive collection of flavivirus genomes, to characterize flavivirus evolutionary and biogeographic history in unprecedented detail and breadth. Based on the presumed introduction of yellow fever virus into the Americas via the transatlantic slave trade, we extrapolated a timescale for a relevant subset of flaviviruses whose evolutionary history, shows that different Culex-spp. associated flaviviruses have been introduced from the Old World to the New World on at least five separate occasions, with 2 different sets of factors likely to have contributed to the dispersal of the different viruses. We also discuss the significance of programmed ribosomal frameshifting in a central region of the polyprotein open reading frame in some mosquito-associated flaviviruses.To generate the most diverse phylogenetic dataset for the flaviviruses to date, we determined the genomic sequences and phylogenetic relationships of 14 flaviviruses, of which 10 are primarily associated with  Flavivirus includes an unusually large number of taxonomically recognised species  with a global distribution. The genus also includes a large and increasing number of unclassified or “tentative” species. Pathogenic mosquito- and/or tick-borne flaviviruses cause a variety of clinical diseases in a wide range of vertebrate species. These disease syndromes include mild/severe febrile illness, “flu-like” syndromes with a rash, or in other cases severe encephalitis or haemorrhagic disease. Dengue with / without warning signs and severe dengue, is the most devastating arboviral disease in tropical and, increasingly, sub-tropical areas of the world (300 to 400 million cases each year) , Jutiapa virus [JUTV], Phnom Penh bat virus [PPBV] and Sokuluk virus [SOKV] were amplified in the mammalian cell-line BHK21 and Sitiawan virus [STWV] was amplified in Vero cell cultures. STWV virus was kindly provided by Dr. Yuji Kono as inactivated nucleic acid in RNA-Now lysis buffer.Viral RNA was either extracted using the BioRobot EZ1  or RNA-Now (Biogentex) using the manufacturer’s recommendations. Reverse transcription was carried out using Taqman Reverse transcription reagents (Applied Biosystems) under standard conditions with random hexamers as primers.PCR was targeted at the E, NS3 and NS5 gene-conserved regions using consensus degenerate primers ,26–28. SSpecific primers were designed from the 3 conserved region sequences defined above and long-range PCR was conducted to complete the sequencing of gap between the E to NS3 genes, and NS3 to NS5 genes, using the cMaster RTplusPCR system (Eppendorf) ,30.Culex-spp. associated flavivirus sequences . Semi-nested PCR with a second inner virus-specific primer was also used when necessary. The 3'-terminal region of the genome was obtained using a similar strategy with virus-specific forward primers in the NS5 region and a reverse degenerate primer in the 3’UTR (3’UTR-MOS: 5’-GGTCTCCWMTAACCTCTAG-3’).The 5’-terminal region of the genome sequence was obtained using an E-gene specific reverse primer and a forward degenerate primer in the 5’UTR designed using an alignment of available 5’ UTRs of Sequencing was conducted with the primers used for amplification, or with M13 primers after cloning in a pCR2 cloning vector (Invitrogen).Complete polyprotein open-reading frame (ORF) sequences, excluding the partial untranslated regions (UTR) regions obtained by this protocol were used for further analyses.Resequencing of seven of the eleven new complete polyprotein ORF sequences  was performed using the Ion PGM Sequencer   and a rade novo assembly.BCV, JUTV and PPBV sequences were obtained using the same NGS method and Flavivirus. Genbank accession numbers of sequences used for the analysis are noted after each virus abbreviation on http://www.ebi.ac.uk/Tools/) and refined manually, for comparison. Nucleotide alignments were then deduced using amino acid (AA) alignments as a template using the TranAlign software available via the EMBOSS server . The effect of removing regions of ambiguous alignment via the GBlocks algorithm , Yokose virus [YOKV] and SOKV which cluster within the s group”  and formVespertilio pipistrellus bats in Kyrgyzstan  was isolated from A. cinereus mosquitoes in Finland  from O. caspius in Spain  from A. vexans nipponii in Korea , Murray Valley encephalitis virus [MVEV] and Alfuy virus [ALFV], form a strongly supported sister group to these viruses, all sharing a common ancestor with CPCV. Notably, the 2 Australian viruses MVEV and ALFV share the same ecological niche and are considered to be a single species , with ALWithin the recognized Ntaya virus group , we haveDuring the preparation of this manuscript, the sequences of 5 strains of ITV were reported . The autTMUV, STWV and the recently described duck egg drop syndrome virus [DEDSV]  and layeNTAV was originally isolated in Uganda. During the preparation of this manuscript, the sequence of another strain of Ntaya virus was reported . The strCulex spp., mosquitoes.With determination of the ORF sequences of AROAV and NJLV, the sequences of all viruses recognised to date within the AROAV group are now known. Both AROAV and NJLV were isolated from sentinel hamsters in South America (Venezuela and Ecuador respectively). NJLV has also been isolated from Bussuquara virus [BSQV] and Iguape virus [IGUV] are 2 other representatives of this group and have been respectively isolated in sentinel monkeys and sentinel mice, in Brazil. As shown in Aedes vexans mosquitoes whereas KOKV was also isolated from Culex annulirostris [With the complete ORF determination of STRV in this study, all of the recognised viruses known to date in the KOKV group have now been sequenced. Interestingly, STRV has only been isolated from irostris , both inirostris . Additioirostris ,76.SL have been isolated and their sequence data are consistent with the concept that they should be classified as a fourth major group of flaviviruses. Moreover it was recently observed that the ISFVSL could be sub-divided into 2 sub-groups: Stegomyia (Aedes) associated viruses and Culex associated viruses based on all inferred phylogenies. With the addition of recent discoveries such as Nakiwogo virus (NAKV) isolated from Mansonia [Coquillettidia [SL group is becoming increasingly complex. Indeed the phylogeny now shows a potential third and/or fourth sub-group that includes NAKV and PCV. This is not surprising considering that the likely mosquito vectors of the two viruses, Coquillettidia and Mansonia spp., are considered by morphological data to be sister groups . Also, these viruses are significantly undersampled and more vector distribution studies and virus discovery would help to clarify their phylogenetic status.During the past decade, many new ISFVMansonia  and Palmlettidia  the ISFVCulex-spp. associated virus clades are rooted by Old World Aedes-spp. associated viruses.From a phylogeographic point of view, the evolutionary origins and dispersal patterns of many of the flaviviruses can be tentatively deduced by considering their association between phylogenetic clustering and geographic location, knowledge of historical anthropological patterns, host/vector associations and estimated times from the present, of divergence from a common ancestor. For example, all the To provide formal support for these observations, we performed a separate Bayesian phylogenetic analysis under a relaxed molecular clock model. A summary of this analysis is represented by the maximum clade credibility tree, which has strong branching support for most of the clades; all nodes had high posterior probabilities (> 0.95) except those labelled with a star (*). Time-calibration for the tree, was based on the principle that YFV was introduced from the Old World into the New World during the slave-trade ,44,45. AIn Estimated times of divergence that belong to the “current time”, i.e., the past 500 hundred years were authenticated against historical facts relating to the slave trade and introduction of yellow fever virus into the Americas .Similar estimates for divergence between OW ancestral DENV and NW strains (150 to 450 years ago) are also consistent with the concept of human introduction of these viruses into the NW via the Slave trade and other commercial exchanges . The virus was most likely introduced from North Africa [The first outbreak of WNV in North America occurred in August 1999 in New York  is found in Russia  and sincThere has been considerable discussion concerning the significance of these observations  and the Based on the phylogenetic and phylogeographic evidence, we deduce that mosquito-borne flaviviruses, corresponding to those identified at nodes A to E, F1–5 and G1–2 , originaSL  and NW rodent NKVSL . These estimate are less accurate, due to the relatively short term calibration date we have used for YFV as reference, and could be thousands or even more years in the past. All apparent virus migrations during this period are referred to as having occurred “before the slave trade”.On the other hand, the TMRCA estimates for the other 6 NW lineages suggest that these viruses potentially could have been introduced to the NW more than one thousand years before the initiation of historic commercial and slave trading across the Atlantic Ocean . The ILHV group emerged presumably in Africa and dispersed westward to the NW. On the other hand, the BAGV group remained in Africa, eventually emerging in Europe and the Middle East, whereas the TMUV related ancestral lineages emerged and dispersed eastward into Asia node D—.On the other hand, the JEV/USUV/ALFV/MVEV group shares a common ancestor with a TMRCA of about 2000–3000 years before the present, implying that an ancestral lineage dispersed eastwards out of Africa and appears to have dispersed to south East Asia before emerging as JEV and then dispersing widely, throughout Asia .SL viruses. In common with the arboviruses, there are distinct lineages in the OW and NW (coloured black and red respectively). The TMRCA of the OW NKV, Apoi virus (APOIV), which was isolated from rodents in Japan, pre-dates all the other recognised NKVSL, and the MRCA of this lineage diverged to produce descendant NKV and arthropod-borne virus lineages. This leaves open the possibility that the association of flaviviruses with tick and mosquito vectors may have been an acquired trait from a non-vectored ancestral virus [Nodes N and O in al virus . Nodes NArteriviridae), and GGA in many ISFVs besides insect nidoviruses of the family Mesoniviridae and some umbraviruses and dianthoviruses. The efficiency of frameshifting depends on the identity of the slippery site nucleotides but is typically less than 1% in the absence of additional stimulatory elements. Thus, most known instances of eukaryotic-1 frameshifting are stimulated  by the presence of a 3' stable RNA secondary structure, such as a pseudoknot or stem-loop, that is separated from the slippery heptanucleotide by a 'spacer' region of 5–9 nt.Many viruses harbour sequences that induce a proportion of translating ribosomes to shift-1 nt and continue translating in the new reading frame to produce a 'transframe' fusion protein . Where fSequence elements associated with-1 PRF were previously described in the JEV serogroup viruses for JEV, WNV, USUV, MVEV and ALFV ,89Fig. . In thesA nucleotide alignment of the putative stimulatory elements responsible for the-1 PRF is presented in Analysis of nucleotide conservation at synonymous sites within alignments of related flavivirus sequences has previously been successful in identifying additional coding ORFs overlapping internal regions of the polyprotein ORF and accessed via-1 PRF ,90. Due Most flaviviruses exhibit enhanced synonymous site conservation at the 5' end of the polyprotein ORF. This is presumably associated with non-coding elements such as functional RNA structures involved in replication and/or translation enhancement –96. SeveCx theileri flavivirus, and 253 to 257 codons in KRV, CFAV, HANKV, AEFV and Ochlerotatus caspius flavivirus. The frameshift site generally appears to be G_GAU_UUY (Y = U or C), with notable exceptions appearing to be G_UUU_UUU in NAKV and A_AAU_UUU_UUC  in PCV.In the ISFVs  ,90. HereA dramatic and statistically significant peak in synonymous site conservation in an internal region was observed in one other clade of flaviviruses—the WESSV-SEPV clade . This coA statistically significant peak in synonymous site conservation was also observed in the NS3-encoding region of TBEV and related flaviviruses . HoweverInternal frameshift sites and/or overlapping ORFs were not predicted using these methods for other flavivirus groups such as SLEV, YFV and DENV. It should be noted, however, that the synonymous site conservation analysis would not necessarily detect cases of frameshifting where there is not an overlapping ORF subject to purifying selection and where the frameshift stimulatory elements comprise just a few codons (e.g. primary sequence rather than secondary structure stimulators) and/or are located in the nascent peptide rather than the RNA sequence. Also these analyses do not yet provide statistically useful results for all flavivirus clades .As phylogenetic methods have developed and more complete genome (ORF) sequence data have become available, increasingly robust analyses and interpretations of the flaviviruses have become possible. For example, we now know that the African virus, Kadam virus (KADV), sits at the root of the TBEV lineages and KADV diverged from a common ancestor of the seabird tick-associated viruses .Culex-associated flaviviruses evolved from ancestral Aedes-associated viruses [Our phylogeny strongly supports the NS3 branching pattern of divergence defined previously ,11,25 in viruses ,12. Howe viruses , which iThese recently-discovered apparently non-pathogenic mosquito-associated viruses (LAMV and NOUV group) are phylogenetically closely-related to human pathogenic viruses, such as DENV, YFV, WNV, JEV. However, they do not appear to replicate in vertebrate cells ,52,57 a Within the MBFVs, three NKV-like viruses, ENTV, SOKV and YOKV, have not been associated with any known vector. Nevertheless, they are genetically closely related to the YFV and EHV groups, have only been found in the Old World, and are associated with migratory bats. YOKV and SOKV were both isolated in Asia but appear to have their ancestral roots in Africa, as do many of the related viruses in the EHV group. Thus, flaviviruses seem able to lose or acquire a variety of genetic traits as illustrated by acquisition of the requirement for a tick or mosquito vector and therefore becoming arboviruses, loss of ability to infect vertebrate cells, exemplified by LAMV and NOUV, loss of the requirement for vector transmission illustrated by the ENTV group and originally proposed by Mattingly in 1960  and suppSL group remains poorly described. Based on the phylogenetic and TMRCA data, viruses within this group have been introduced to the NW on at least two separate occasions and these introductions may have occurred thousands of years ago. Estimations for the time of divergence between these OW and NW NKVSL ranged between 4000 to 14000 years ago . The arrival of viruses in the NW could alternatively have occurred via the gradual dispersal of OW rodents and migratory bats. On the other hand, these transoceanic dispersions might have occurred after the development of trading between the OW and the NW.In contrast with the MBFV and TBFV groups, the NKVFlaviviridae. Moreover, they infect only invertebrate hosts and DNA forms of ISFV genomes are generated during infection of cell cultures [Flaviviridae. These viruses are also interesting because similar ISFVs have been isolated from mosquito species that inhabit different ecological niches, raising the question how might this occur? Possible mechanisms of virus transfer between mosquito species include the diffusion of viruses between larvae at sites shared by different mosquito species, or biting midges could become infected when taking blood meals from infected mosquitoes [Until about ten years ago, only one ISFV, cell fusing agent virus (CFAV), was recognised. Currently, at least 9 genetically distinguishable ISFVs have been isolated and their sequences determined. This group of mosquito- and potentially sandfly-borne viruses is an extremely genetically diverse group with a divergence of up to 61% at the amino acid level. This compares with the genetic distances between the three current genera in the family cultures . Integracultures ,102,103.squitoes –108 and It is recognised that YFV and DENV were transported frequently to the Americas from Africa on the ships that transported slaves across the Atlantic Ocean during the centuries of slave trading ,109–111.Our choice was to use an ‘independently confirmed’ historical hypothesis  and to eCulex-associated viruses that circulate in Europe, Asia, Australia and/or the Americas, appear to have their evolutionary roots in Africa. Given the OW diversity of flaviviruses and the comparatively lower number of NW viruses, this analysis attempts to formalise a parsimonious argument for an ‘Out-of-Africa’ history. Unless there is a matching, but largely unsampled flavivirus diversity in the NW, this remains the most plausible interpretation of their phylogenetic distribution.Our results demonstrate that such movement of arboviruses from Africa to the Americas is not unique to YFV and DENV. The ancestral history presented in Given that we rely on a single, relatively recent calibration to estimate old divergence times, we do not consider our estimates to be a precise historical record of flavivirus evolution, but it does allow us to distinguish 2 major temporal periods during which flaviviruses may have been introduced from the OW into the NW. All historical and phylogenetic data are consistent with the concept that there was a recent period of 400 to 500 years during which African viruses such as YFV and DENV were transported on slave and commercial ships across the Atlantic Ocean to the Americas, ,111,112 Based on published estimates for the times of divergence in the tree ,113,114 Culex mosquitoes in Peru might represent another example of potential virus introduction from the OW to the NW.If we consider the possibility of a more ancient introduction of viruses, i.e. between 4000 to 14000 years ago, we have to consider the possibility that ancestral viruses emerged in the OW and were then introduced into the NW many years before the slave trade period, possibly by birds, rodents, bats, arthropods and/or humans. The tree shows that introductions to the NW were multiple and independent. The first introduction is represented in Culex spp.-associated viruses contain members from both the OW and the NW. It has been proposed that in the past, several different insect-specific flaviviruses have been introduced independently into Latin America, from the OW, rather than a single virus having been introduced with subsequent divergence to generate the different viruses found in the Americas [Recent discoveries of viruses in the ISF group such as Culex flavivirus ,122,123 Americas ,124.SL Apoi virus, an OW virus isolated in Japan from Apodemus mice  roots all other NKVSL. MODV and JUTV were the first recognised rodent NKVSL to be introduced to the NW  whereas JUTV was isolated in South America. These viruses were possibly introduced into the NW via rodents, when the OW and NW land masses in the northern hemisphere were joined by ice.The NKVo the NW —node O aRio Bravo virus (RBV) and Montana myotis leukoencephalitis virus  during the “recent period”. This is almost certainly not an accurate estimation of the evolutionary dynamics that occurred during the “ancient period”, which in the case of the NKVSL are not vectored by arthropods.Although the uncertainty in the TMRCA is quite significant, there are clearly two periods of emergence, It was previously reported that members of the JEV serocomplex express a transframe fusion protein, NS1', via-1 PRF . The fraThe CPCV sequence also lacks the potential to form the canonical JEV-serogroup 3' RNA stable pseudoknot that is associated with highly efficient-1 PRF ; thus iThe significance of the NS1' protein is not yet fully understood but it has been reported that it plays a role in viral neuroinvasiveness and reduced neurovirulence ,130,131.et al., 2010); ILOV and DGV [Frameshifting at a very similar genomic location occurs in the ISFVs but results in the translation of a much longer overlapping ORF . Potenti and DGV ; and SEPIn conclusion, with the addition of 14 new flavivirus ORF sequences, the estimation of phylogenies via Bayesian methods, plus biogeographic and bioinformatic considerations, we have identified 11 likely introductions of mosquito-borne flaviviruses from the OW to the NW over two separate temporal periods. We have also demonstrated that similar introductions have occurred eastwards from Africa to Australia, again over two distinct time periods. Clearly there have been far more introductory movements of flaviviruses from one part of the world to another than we have described here. However, more data for a larger number of samples will be required before we can draw specific and more detailed conclusions. Finally, in the context of flavivirus evolution and dispersal, we have extended our current understanding of frameshifting amongst the flaviviruses.


However, its functional character in lung cancer remains largely unknown. In this study, we show that S100P increases cancer migration, invasion and metastasis in lung cancer cells. Ectopic expression of S100P increases migration, invasion and EMT in less invasive CL1-0 lung cancer cells. Conversely, knockdown of S100P suppressed migration and invasion, and caused a reversion of EMT in highly invasive lung cancer cells. These effects were transduced by increasing the interaction of S100P with integrin α7, which activated focal adhesion kinase (FAK) and AKT. Blocking FAK significantly decreased S100P-induced migration by decreasing Src and AKT activation, whereas inhibiting AKT reduced S100P upregulation on ZEB1 expression. Further study has indicated that S100P knockdown prevents the spread of highly metastatic human lung cancer in animal models. This study therefore suggests that S100P represents a critical activator of lung cancer metastasis. Detection and targeted treatment of S100P-expressing cancer is an attractive therapeutic strategy in treating lung cancer.S100P, a Ca2 Lung cancer is the one of the most commonly diagnosed malignancies, with a high mortality rate worldwide. Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers . SurgicaS100P, a member of the S100 family of small EF-hand calcium-binding proteins, exhibits different cell biologic functions extra- and intra-cellularly by direct targeting with specific proteins. Its expression is upregulated in some cancers, including colon, pancreatic and endometrial cancers, and it is associated with poor clinical outcomes and decreased chemosensitivity -6. RecepIntegrins, large and complex transmembrane glycoproteins belonging to adhesion receptors, modulate various cell functions upon ligand binding . Focal aThe present study demonstrates that S100P binds preferentially to integrin α7 with the activation of FAK and AKT signaling. Consequently, the metastatic characteristics of lung cancer cells are enhanced. Thus, inhibition of S100P may provide a novel therapeutic target for combating lung cancer metastasis.n = 5), as determined by immunofluorescence staining  of specimens . There wg Figure . Evaluatg Figure . These dTo explore the role of S100P in lung cancer migration and invasion, endogenous S100P was stably inhibited by shRNA plasmid transfection in the CL1-5 and A549 lung cancer cell lines. The qRT-PCR of fractionated samples was used to measure the decreased expression of S100P in CL1-5 and A549 cells. Levels of S100P mRNA transcript  were reduced by approximately 70% and 90%, respectively, when compared to control plasmid transfected CL1-5 and A549 cells (CL1-5-AS2 and A549-AS2) Figure .Silencing S100P resulted in MET supported by morphologic changes  Figure  and someWe further confirmed the role of S100P overexpression in lung cancer. After having transfected S100P cDNA into CL1-0 and A549 cells, ELISA analysis revealed that S100P cDNA transfection increased protein expression in CL1-0 and A549 cells, compared to pCMV plasmid cells Figure . The ectTranscriptional factors, including Snail, Slug and ZEB1 have been demonstrated to regulate EMT , we consTo investigate the role of ZEB1 on S100P-mediated cancer migration and EMT, we inhibited ZEB1 by siRNA transfection, then assessed the expression of EMT markers and cell migration. Transfection of CL1-0 cells with ZEB1 decreased the mRNA transcript of ZEB1 . SilenciConsistent with a role in modulating cell movement, the S100P knockdown CL1-5 and A549 cells exhibited decreased FAK , Src (Tyr 416), and AKT (Thr 308 and Ser 473) phosphorylation, without any obvious alteration in their unphosphorylated forms. In contrast, ectopic expression of S100P protein displayed increased FAK, Src, and AKT activation in CL1-0 and A549 cells Figure .To discern whether FAK and AKT activation initiated S100P-mediated cancer migration, FAK and AKT siRNA transfection were assayed on cancer migration in S100P overexpressing CL1-0 cells. An inductive effect on cell migration by S100P overexpression was decreased by either FAK or AKT siRNA transfection Figure  and 5D. Previous studies have shown that cell surface molecules, such as RAGE (receptor for advanced glycation end products) and integrins, are receptors for S100 family proteins , 8. We iTo investigate the candidate of S100P binding receptor, we pulled down S100P from the cell membrane of S100P overexpressing CL1-0 cells, then determined the protein prolife by nanoUPLC-MS/MS. Mass spectrometry analysis shows integrin α7 abounds in S100P protein precipitant of CL1-0 cell membranes. Coimmunoprecipitation data revealed that S100P was bound to integrin α7 Figure . The rolTo investigate the influence of S100P in lung cancer metastasis, S100P-knockdown CL1-5 was transplanted into mice by tail vein injection. Inhibition of S100P decreased the metastatic ability of CL1-5 in mice Figure . As deteIncreased S100P levels have been reported in liver, skin, endometrial, colon, and pancreatic cancers, and have been indirectly implicated in promoting carcinogenesis and cancer metastasis in animal models , 16-18. The occurrence and progression of cancer metastasis are complicated. First, tumor cells disassociate from the primary tumor site by losing cell-cell connection and switching to a mesenchymal phenotype, resulting in increased movement and invasiveness , 20. TheThis study demonstrates that overexpression of S100P in CL1-0 or A549 cells increases the ability for migration, invasion, and EMT. Conversely, in metastatic CL1-5 or A549 cells that express high levels of endogenous S100P, knockdown of S100P decreases migration and invasion abilities, causing MET. Furthermore, inhibition of S100P in CL1-5 decreases the metastatic recurrence of CL1-5 in mice. More importantly, the tumor regions of specimens from lung cancer patients express higher amounts of S100P when compared to matching normal regions. Results from cell-based studies, mouse models, and clinical patient specimens suggest that S100P is a critical mediator contributing to the development of lung cancer.ZEB1, zinc finger transcription factor, is essential for regulating EMT and cancer metastasis , 25. ZEBIn addition, knockdown of AKT prevents ZEB1 upregulation and EMT, showing that AKT activation is responsible for ZEB1 upregulation. Silencing of ZEB1 by siRNA transfection also decreases the switch of EMT induced by S100P overexpression. Such findings support the phenotypic transition of lung cancer by S100P mediated FAK-mediated activation of AKT and subsequent AKT-activated ZEB1 gene expression, thereby contributing to cell migration.Integrin family proteins, the family of heterodimeric transmembrane adhesion receptors, have been implicated in the pathogenesis of various malignances, including lung cancer , 30. TheIn conclusion, S100P interacts with integrin α7 to regulate lung cancer cell migration and invasion via the FAK/AKT-ZEB1 signaling pathway. This suggests that S100P plays a pivotal role in integrin-mediated cancer progression, and that the inhibition of S100P may be a promising target for diagnostic and therapeutic interventions against metastatic events.Primary antibodies against phospho-Tyr 397 FAK, total FAK, phospho-Ser 473 Akt, total Akt, phospho-Tyr 416 Src, total Src, vimentin, snail, slug, and ZEB1 were purchased from Cell Signaling Technology . Antibodies against anti-human S100P and claudin-3 , SM-actin , Fibronectin, N-cadherin and E-cadherin  were obtained from their respective companies. Primary antibodies against GAPDH, FAK, and AKT inhibitors were purchased from Millipore . S100P ELISA kit was obtained from CircuLex .Less invasive (CL1-0) and highly invasive (CL1-5) human lung adenocarcinoma cell lines were provided by Dr. Pan-Chyr Yang  , 38. TheKnockdown of S100P in CL1-5 and A549 was performed using a lentiviral expression system obtained from the National RNAi Core Facility . The lentiviruses were produced by co-transfecting HEK293T with pLKO-AS2, pLKO-AS3-S100P and two packaging plasmids (pCMVVDR8.91 and pMD.G). Stable clones were established by puromycin selection. ON-TARGET plus SMARTpool siRNA  transfection was used for ZEB1, AKT1 and FAK knockdownQuantitative reverse transcriptional polymerase chain reaction (qRT-PCR) was used to assess the efficacy of the S100P shRNA plasmid and various siRNA transfection. Overexpression of S100P in CL1-0 and A549 was achieved by transfecting S100P cDNA . Stable clones were established by G418 selection.Cells were harvested using TRIzol reagent , followed by RNA extraction according to the manufacturer's instructions. Equal amounts of RNA were used to synthesize first-strand cDNA. RT-PCR was performed using SYBR Green on a StepOnePlus Real-Time PCR System . Each PCR reaction mixture contained 200 nM of each primer, 5 μL 2×SYBR Green PCR Master Mix , 5 μl cDNA, and RNase-free water, for a total volume of 10 μL.−ΔΔCT method. S100P levels in Lung Cancer cDNA Array I (Origene) were also measured by PCR. The primer sets used were S100P: 5′- TACCAGGCTTCCTGCAGAGT-3′ and 5′- CACGTCTGCCTGTCACAAGT-3′; RAGE: 5′- CAGTGTGGCTCGTGTCCTT-3′ and 5′- CAGTGTGGCTCGTGTCCTT-3′; and GAPDH: 5′- GAGTCAACGGATTTGGTCGT-3′ and 5′- TTGATTTTGGAGGGATCTCG-3′.The PCR reaction was conducted with a denaturation step at 95°C for 20 sec, 40 cycles at 95°C for 3 sec, and 60°C for 30 sec. All PCRs were performed in triplicate and normalized to internal control GAPDH mRNA. Relative expressions were presented using the 2in vitro using CHEMICON QCM 24 well migration or invasion assay kits . Briefly, cells were seeded into each insert (pore side: 8 μm) of the 24-well plate and incubated at 37°C for 24 h. After using cotton swabs to remove the cells on the upper side of the insert membrane, the cells on the bottom side of the insert membrane were fixed with methanol for 15 min, followed by staining with crystal violet for 60 min. After washing with distilled water, the invaded cells on the membrane were counted in three random fields of view per trans-well. The images were captured via microscope. Alternatively, the migratory and invading cells was quantified by fluorescence dye staining, and the OD value read by a fluorescence plate reader at excitation/emission wavelengths of 485/530 nm. The migration of A549 cells was also assessed by Scratch wound-healing assay.Cell migration and invasion was analyzed The cells were lysed on ice for 30 min in M-PER solution. Cell lysate was centrifuged at 12,000 rpm for 15 min and the supernatant fraction was collected for immunoblot. Equivalent amounts of protein were resolved by SDS-PAGE (10-12%) and transferred to PVDF membranes. After blocking in 5% non-fat dry milk in Tris-buffered saline, the membranes were incubated overnight with the desired primary antibody. The membranes were then treated with the appropriate peroxidase-conjugated secondary antibody and the immuno-reactive proteins were detected by an enhanced chemi-luminescence kit (Millipore), according to the manufacturer's instructions. To quantify the immunoblot images on unsaturated bands, densitometric analysis was performed using AlphaEaseFC (Alpha Innotech).For Co-IP, cell lysates  were incubated with 2 μg of isotype or anti-DDK antibodies (Origene) overnight, and then with 10 μL protein A-agarose beads  for 2 h at 4°C. DDK and intergrin α7 were detected by incubating the blots with specific antibodies.Cell membrane protein was extracted by the Novagen proteoextract Transmembrane protein Extraction Kit (Millipore). Protein solution 10 μL (∼10 μg) was mixed well with 100 μL acetone and centrifuged at 13,000 rpm for 10 min. After centrifugation, the supernatant was discarded, and the protein residues kept and dried by evaporation. Protein residues were then re-dissolved in 18 μL 25 mM ammonium bicarbonate aqueous solution. Then 2 μL sequence-grade trypsin  was added and protein solutions were digested at 37°C for 16 h. After trypsin digestion, 2 μL tryptic peptide solution was injected into the nano LC system and detected by LTQ Orbitrap.The LC-MS/MS conditions were LTQ Orbitrap Discovery hybrid FTMS (Fourier Transform Mass Spectrometer)  at 30000 resolution, with a nanospray source in the positive ion mode. The nano UPLC system (nanoACQUITY UPLC) was manufactured by Waters . The desalting column  and analytical column  were also purchased from Waters. Mobile phase A was the aqueous solution of 0.1 % formic acid, and mobile phase B was the organic solvent (100% ACN containing 0.1% formic acid).After desalting by 0.1% formic acid for 3 min at a flow rate of 5 μL/min, analysts were separated at a flow rate of 300 nL/min by a 15 cm UPLC column. The gradient conditions were t: 0-1 min, hold B: 1%; t: 1-20 min, B 1-45%; t: 20-30 min, B 45-85%; t: 30-35 min, B 85%; t: 35-45 min, B 85-1%; and t: 45-60 min, B 1%.Peptide elute from the column was directed to the nano-spray source and the MS was operated in the positive ion mode. The MS/MS was acquired with a mass spectrometer operated in the data-dependent mode. Individual raw data files were processed with Mascot Distiller  software and then uploaded to the in-house Mascot server  for protein identification.Noncancerous and cancerous lung tissue specimens from human lung cancer patients were embedded in OCT and frozen in liquid nitrogen. Sections (5 μm) were fixed with 4% paraformaldehyde, permeabilized with Triton (0.25%), and stained with S100P antibody . After washing with PBS containing 0.1% Tween-20 (PBST), slides were incubated with Dylight 488-conjugated secondary antibodies , with or without DAPI, for 1 h at room temperature. The data were analyzed with a confocal laser-scanning microscope .2O2. The expressions of phospho-FAK, and -AKT antigen were demonstrated using mouse/rabbit anti human-p-FAK and p-AKT (dilution 1:100) antibody. The sections were incubated with the primary antibodies overnight at 4°C. The studied antigens were then visualized using biotinylated antibodies and streptavidin, conjugated with horseradish peroxidase. Diaminobenzidine Dako Cytomation  served as the substrate, and all of the sections were counterstained with hematoxylin.Control plasmid transfected CL1-5 and S100P-knockdown CL1-5 (clone 217) were transplanted into nude mice by tail vein injection. Animals were sacrificed on weeks 12 and the number of tumor nodules was recorded for analysis of lung cancer incidence. All immunohistochemical reactions were performed on 8-μm-thick paraffin sections. In brief, the sections were deparaffinized in xylene and rehydrated, then incubated in target retrieval solution (DAKO) in an autoclave for 8 min to retrieve the antigens. Endogenous peroxidase activity was blocked by 10 minutes of incubation with a 3% solution of Ht-test was used when comparing the means of two groups. Statistical significance was set at p < 0.05.A two-tailed Student's


Tobamovirus species. We present the first complete genome sequence of a tomato mottle mosaic virus isolate from the United States.Tomato mottle mosaic virus was recently reported from the United States following its original description from Mexico as a novel  Tobamovirus in the family Virgaviridae have rigid rod-shaped virions that are readily transmitted by plant-to-plant contact and mechanical inoculation and, in some cases, by seeds (Tobamovirus (http://www.ictvonline.org/virusTaxonomy.asp), which are distributed worldwide and include tobacco mosaic virus (TMV), the first virus of any type or host identified  was 99% identical to the Mexican ToMMV isolate sequence with 27 single nucleotide differences and one insertion (G at 6212). Of the 24 nucleotide differences within the predicted four open reading frames  typical of tobamoviruses, 15 were silent, whereas 9 led to an amino acid change. Partial Sanger sequences of this and a second ToMMV isolate from the United States confirmed the insertion at 6212.KP202857.The complete genome of the U.S. ToMMV isolate 10-100 was deposited in GenBank under the accession number


Parvoviridae, three novel members of the genus Amdoparvovirus have been described in the last 4 years, expanding this genus that had contained a single species since its discovery, Aleutian mink disease virus. The increasing number of molecular and epidemiological studies on these viruses around the world also highlights the growing interest in this genus. Some aspects of amdoparvoviruses have been well characterized, however, many other aspects still need to be elucidated and the most recent reviews on this topic are outdated. We provide here an up-to-date overview of what is known and what still needs to be investigated about these scientifically and clinically relevant animal viruses.Many new viruses have been discovered recently, thanks in part to the advent of next-generation sequencing technologies. Among the  Aleutian disease was originally observed in the USA during the late 1940s in Aleutian mink, a novel gunmetal gray variety similar in color to the Aleutian foxes . The disThe first scientific evidence of an infectious cause was the demonstration that suspensions of infected mink organs retained their infectivity after filtration, indicating the causative agent was likely viral . The virFifty years after the discovery of AMDV, three related viruses have been identified in other mammals. AMDV is, however, the best characterized virus in its genus and was the focus of the majority of the literature considered.Amdovirus within the family Parvoviridae, subfamily Parvovirinae, for many years. It has now been reclassified as Carnivore amdoparvovirus 1 within the genus Amdoparvovirus . RFFAV is more closely related to GFAV, with an identity of >80%  and AMDV-3, which is only 82–84% identical to other AMDVs and might represent a different species  and occupies an intermediate position between AMDV and GFAV . The left ORF encodes the non-structural proteins NS1, NS2, and NS3, and the right ORF encodes the capsid proteins VP1 and VP2 (Carnivore amdoparvovirus 1 and 2 (The monosense genome consists of two open reading frames (ORFs), flanked by untranslated regions . These fold into hairpin structures and mediate viral DNA replication by functioning as primers and allowing the attachment of host polymerases, and they are also essential for packaging , a mechanism adapted from the rolling circle replication used by some circular DNA viruses [reviewed in ]. ReplicAedes fitchii, which can feed on mink, can carry AMDV for up to 35 days after an infectious blood meal, and the virus may replicate in this vector indicating that vector-borne transmission may also be possible  can contain viruses and transmission via direct or indirect  contacts are both possible . Virionspossible .Carnivore amdoparvovirus 2 has been identified only in gray fox to date  and also other animals such as skunks, raccoons, bobcats, cats, dogs, mice, and humans . Carnivo to date , whereas to date  and RFFA to date .Aleutian mink disease virus affects farmed and free-living animals, where it propagates and evolves with different dynamics. Viruses identified in the two reservoirs and in different locations can be very similar, indicating exchange of strains between the wild and farms, and amongst different farms following the movement of animals or contaminated materials. Accidental escape or deliberate release of infected animals from farms, where intensive farming facilitates disease spread, makes farms a source of viruses for wild populations , 2012.Table 1). In Nova Scotia the virus is highly prevalent in wild mink  and in other wild animals (Table 1). It is also prevalent on farms (up to 70% in 2003), but efforts for implementing eradication strategies are reducing the number of affected animals .American mink were introduced into Europe during the 1920s and 1930s for farming purposes , and thiTable 1). Between 1980 and 2014 the seroprevalence in Finnish farms fluctuated between 3 and 60%, a recent report indicates that 80% of farms in Ireland have Aleutian disease, and the virus was also reported in German and Dutch farms . RFAV was identified in six farms in China, RFFAV in wild foxes in the Spanish Basque region, and GFAV in two animals in California (Our understanding of the epidemiology of the other amdoparvoviruses is much more limited (lifornia .Infection of mink by AMDV manifests in different ways depending on the age of the mink, with different pathology and cell-tropism in kits versus adults. In adults, the virus causes a persistent infection that leads to a progressive wasting syndrome, characterized by weight loss and anorexia associated with splenomegaly, lymphadenopathy, plasmacytosis, hypergammaglobulinemia, necrotizing arteritis, and proliferative glomerulonephritis . The plaIn kits the virus causes an acute infection that manifests as respiratory distress and fulminant interstitial pneumonia, which is due to a direct cytopathic effect in pneumocytes, and is mostly fatal within 3 weeks post-infection . TreatmeIn ferrets and skunks, AMDV causes a similar wasting syndrome with kidney impairment and glomerulonephritis . A respiAsymptomatic and non-persistent infections are common in both mink and ferrets, and in these individuals a lower antibody response and a transient viremia are observed . The morCarnivore amdoparvovirus 2 in gray fox included gait abnormalities, lymphadenopathy and acute muscle inflammation (Clinical signs reported for RFAV included anorexia, emaciation, growth retardation, thirst, chronic diarrhea, splenomegaly, enlarged lymph nodes, renal cortex congestion, and brittleness in raccoon dog and emaciation, growth retardation, pale swollen kidneys, and severe diarrhea in Arctic fox . Clinicaammation . ClinicaAmdoparvoviruses cause severe diseases in a wide range of animals, resulting in major economic losses for farmers and threatening wild animal populations. Continued research and an increased awareness of these scientifically and clinically relevant animal viruses will provide a better picture of their total impact as more information becomes available in the coming years and will likely lead to the discovery of additional new species and susceptible hosts.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.


We describe a case with a false-negative PCR-based analysis for JC virus in cerebrospinal fluid (CSF) in a patient with clinical and radiological findings suggestive of progressive multifocal leukoencephalopathy (PML) who was on chronic immunosuppressive therapy for rheumatoid arthritis. Our patient developed rapidly progressive global decline with clinical and radiographic findings suggestive of PML, but JC virus PCR in CSF was negative. The patient passed away 3 months from the onset of her neurological symptoms. Autopsy confirmed the diagnosis of PML with presence of JC-polyoma virus by immunohistochemical staining. This case highlights the potential of false-negative JC virus PCR in CSF when radiographic and clinical features are suggestive of “possible PML.” We review the plausible causes of potential false-negative CSF results and suggest that when the clinical presentation is suspicious for PML repeat CSF analysis utilizing ultrasensitive PCR assay and subsequent brain biopsy should be considered if CSF remains negative. Additionally, appropriate exclusion of other neurologic conditions is essential. Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disorder of the CNS caused by infection of the JC-polyoma virus. PML has classically been described in immune-compromised individuals, such as acquired immune deficiency syndrome (AIDS), but it also occurs in people on chronic immunosuppressive therapy for solid organ transplants, in people with hematological malignancies receiving antineoplastic therapy, and in individuals with autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, psoriasis, and systemic lupus erythematosus with or without immunosuppressive or immunomodulatory therapies that depress the immune response and allow JC virus reactivation –3. HowevSome of the reported immunosuppressive therapies described in association with PML include efalizumab, natalizumab, infliximab, rituximab, belatacept, fludarabine, glucocorticoids, cytotoxic chemotherapy, and various transplant drugs such as tacrolimus and mycophenolate –10. In sAmong patients with autoimmune diseases, but without HIV or malignancy, one study estimated PML incidence at 0.2/100,000 .The diagnosis of PML is suggested by clinical and neuroimaging features and further established by demonstrating the presence of JC virus DNA in the cerebrospinal fluid using polymerase chain reaction (PCR) , 3. AlthWe describe a case of PML with false-negative CSF JC virus PCR, thus reinforcing the importance of obtaining histological confirmation when clinicoradiological findings are suggestive of PML, even with initially normal CSF studies.A 75-year-old woman with history of rheumatoid arthritis maintained on long term methotrexate and adalimumab presented with a progressive left hemiplegia and global decline. Initial magnetic resonance imaging (MRI) of the brain without and with contrast Figures  demonstrThe patient continued to decline rapidly with confirmation on serial MRI Figures . She expOur patient's MRI showed typical findings of PML, including bilateral asymmetric nonenhancing multifocal areas of periventricular and subcortical white matter demyelination that did not conform to the typical cerebral arterial distributions, along with lack of mass effect and U-fibers involvement. Unlike many prior reported cases, where, in the setting of subtle MRI findings, JC virus CSF PCR was diagnostic , 9, 10, Plausible explanations for the false-negative JC virus analysis of CSF studies include inadequate choice of JC virus DNA PCR targets , low virWe suggest that when the clinical presentation is suspicious of PML, a negative initial CSF JC virus PCR result does not exclude such diagnosis. Therefore, consideration for repeat CSF analysis, utilizing ultrasensitive PCR assay, should be undertaken, and exclusion of other potential masquerading neurologic conditions is essential. These conditions include autoimmune and inflammatory conditions such as multiple sclerosis and acute disseminated encephalomyelitis (ADEM), infectious conditions such as HIV encephalitis and Varicella-Zoster virus encephalopathy, and other conditions such as mitochondrial encephalopathies, CNS vasculitis, and reversible posterior leukoencephalopathy , 15. If,


The World Health Organization has declared the recent leap in the number of microcephaly cases and their suspected association with Zika virus a public health emergency of international concern. Ana Bispo tells Andréia Azevedo Soares why Brazil should have some scientific answers in coming months. Q: When did you first become aware of this virus?A: I first heard of Zika at a meeting of an international network of Latin American and Iberian laboratories called ViroRed in November 2014 in Uruguay. My lab is a member of this network. Some colleagues from Europe, who also attended the meeting, were concerned about an outbreak of Zika virus in French Polynesia in 2013. They described how they had genetically sequenced Zika virus and gave participants samples of the viral ribonucleic acid (RNA), which can be used to detect the virus using a technique known as reverse transcription polymerase chain reaction or RT–PCR.Q: Could you tell us some of the history of Zika virus in Brazil?Aedes aegypti mosquito, was widespread. In Brazil an increase in microcephaly cases was observed in Pernambuco state, in the north-eastern part of the country, in suspected association with Zika virus. In French Polynesia cases of neurological damage and Guillain-Barré syndrome were reported. But Brazil was the first place where such a steep increase in microcephaly in babies had been observed and this increase appeared to be associated with Zika virus: almost 4000 cases of microcephaly were registered in Brazil between August and December 2015.A: In November and December 2014, physicians from different states of the north-east region observed an outbreak of dengue-like illness and in April 2015 researchers from the Federal University of Bahia confirmed that Zika virus was the cause. Since then, 21 states have reported cases of Zika virus in Brazil. In Rio de Janeiro, the first cases were confirmed in May 2015. So Zika virus arrived in Brazil and found a very susceptible population because people had not been exposed to it before and because the main vector, the  “We are following about 300 pregnant women."Q: Is your institution conducting case–control studies to solve the mystery of whether Zika virus is indeed a cause of microcephaly?A: Yes, Fiocruz in Rio de Janeiro has started a case–control study with pregnant women mainly focused on microcephaly with Zika virus. Overall, we are following about 300 pregnant women. A similar case–control study is also being conducted by colleagues at the Aggeu Magalhães Research Center, a branch of Fiocruz in Pernambuco state, where an exceptionally high number of microcephaly cases was reported last year.Q: Can you tell us more about your lab’s work on the suspected association between microcephaly and Zika virus?A: The first important finding was described by our team in November 2015, when we detected Zika virus genome in the amniotic fluid of two pregnant women from Paraíba state. Both women had reported a medical condition consistent with Zika virus infection. The ultrasound images of their fetuses were normal until a month after their probable infection with Zika virus, when the physician who examined them saw the first micro-calcifications of the brain. The physician confirmed that both fetuses had microcephaly and offered the women amniocentesis tests to rule out other genetic problems. Aware of the growing number of microcephaly cases, the physician called us to check whether we could detect Zika virus in their amniotic fluid. The results were positive in both cases. Our findings came about a week after the Brazilian government declared a health emergency due to the increase in microcephaly cases and so these new data were very important for guiding the investigations. Some days after we announced the amniotic fluid evidence, scientists in the state of Pará found Zika virus in samples from a baby with microcephaly that had died after birth and, recently, my team detected Zika virus in the placenta, liver, lung, umbilical cord and brain of a fetus that had been spontaneously aborted.Q: When will it be possible to confirm whether Zika virus infection is a cause of microcephaly?A: According to the data we have been collecting – and the new babies that will be born in the meantime – we may have concrete results in June or July.Q: Could something else have caused the leap in the number of cases of microcephaly?A: I doubt it. The majority of women who have delivered babies with microcephaly report that they have had a medical condition consistent with Zika, especially during the first trimester. It is hard to establish a causal link between Zika and microcephaly, because it is difficult to detect the virus when the baby is born. The Ministry of Health recommends that samples of umbilical-cord blood, placenta and urine should be collected. We do not have a test that is serologically specific enough to detect Zika virus antibodies in a baby with microcephaly. It’s the same with Guillain-Barré syndrome: when the symptoms appear  patients are no longer in the acute phase of Zika disease. But we still think that Zika is responsible because when you compare current numbers of Guillain-Barré cases with those before Zika arrived in Brazil, the figures are much greater.Q: Could the recent leap in microcephaly be in any way attributed to heightened awareness and increased surveillance?A: You can always argue that health professionals are much more aware of such birth defects and more vigilant in terms of identifying them and, of course, we have better diagnostics in general. But, still, this is a very large increase, so it is puzzling. That’s why more research is needed to shed light on these mysteries.“It will be easier to develop a vaccine for Zika than dengue.”Q: Brazilian scientists are trying to develop a vaccine for Zika. What are the main challenges?A: There are three groups of scientists in Brazil that are trying to develop a Zika vaccine. One from Bio-Manguinhos, which is part of Fiocruz, is working with GlaxoSmithKline to develop a dengue vaccine that can be used as a model for a Zika vaccine. Another group based in São Paulo, from the Butantan Institute, has started to work on a Zika vaccine with the US National Institutes of Health. And a group at the Evandro Chagas Institute is collaborating with the University of Texas Medical Branch. It will be easier to develop a vaccine for Zika than dengue because Zika has only one serotype while dengue has four. In addition, only two Zika lineages, an Asian and an African one, are described in the scientific literature.Q: Fiocruz has just announced the creation of a rapid test to diagnose Zika, dengue and chikungunya simultaneously. Why is it innovative?A: This is a ready-to-use multiplex PCR test that allows you to detect three viruses: dengue, chikungunya or Zika. The technicians only need to add the extracted RNA sample to the device containing three different reagents. It is called the Nucleic Acid Test for Dengue, Chikungunya and Zika viruses and is in the final phase of validation. We are hoping to complete the validation process by the end of this month. It is expected to cost about US$ 20 per test kit, including RNA extraction. The test has great potential for improving epidemiological vigilance because it shows which virus is circulating at a specific time in a given area. The test could also be particularly useful in the first days of infection, when the symptoms of Zika, chikungunya and dengue are so similar that it is difficult for doctors to distinguish between dengue and Zika, for example. The kits are also user-friendly, so they can be used by a technician with basic knowledge in real time PCR. If the final validation goes well, about 500 000 test kits could be available in the country by the end of the year. I proposed the test and my colleagues at Fiocruz’s Institute of Molecular Biology in Paraná state developed it.Q: Although the virus is normally spread by mosquitoes, there have been reports of possible spread through sex. How strong is the evidence?A: There is a report in the scientific literature of two cases of Zika that were sexually transmitted. Recently my team detected Zika virus in semen and urine from a patient with Zika virus infection 16 days after the symptoms of the disease appeared. Additional studies are being conducted to verify whether Zika virus can be sexually transmitted. It is difficult to collect samples of semen and vaginal fluids, but we must pursue this line of investigation. We must also look at possible infection with Zika virus via breast milk. Nevertheless, mothers are encouraged to continue breastfeeding their newborns as evidence suggests that the benefits outweigh any theoretical risks of infection.Q: How will the Zika outbreak evolve in the coming months ahead of the Olympic Games that are scheduled to take place in Brazil in August?Aedes shuts down for winter and so we did not have a big problem with dengue during the World Cup. The measures to control the vector will be as stringent as they were during the World Cup. There was huge investment in vector control, especially around football stadiums and other mass-gathering areas. The government is working hard on prevention so that visitors to Brazil – apart from pregnant women – need not worry.A: We foresee the same scenario that we had with dengue during the 2014 World Cup, which also took place in the winter. Mosquitoes that hibernate need warm weather to become active: the


Schmallenberg virus (SBV) emerged in Germany in 2011, spread rapidly across Europe, and almost disappeared in 2013. However, since late summer 2014, new cases have occurred in adult cattle. Full-genome analysis revealed some amino acid substitution differences from the first SBV sample. Viremia developed in experimentally infected sheep and cattle for 4–6 days. Schmallenberg virus (SBV), an insect-transmitted orthobunyavirus with a negative-stranded tripartite RNA genome, causes no or only mild nonspecific clinical signs for a few days in adult ruminants  , to confirm an SBV infection  and to fTo evaluate sequence variations among SBV variants circulating in Germany since 2011, the original SBV isolate (BH80/11) and viruses isolated in 2012 from the blood of viremic sheep (BH619/12) or cattle (D495/12-1 and BH652/12) were compared with 3 genomes obtained from the viruses in acutely infected adult cattle in 2014 . RNA was extracted by using the QIAamp Viral RNA Mini Kit  according to the manufacturer’s recommendations, and the open reading frames of all 3 genome segments  were sequenced as described (For the S segment (830-nt long), which encodes the nucleocapsid protein (N) and a nonstructural protein (NSs), sequence analysis revealed very high stability. The samples obtained from acutely infected animals in 2014, BH619/12 and BH652/12, were 100% identical to the original SBV strain BH80/11 from 2011. In the sample D495/12-1 from 2012, a single nucleotide was substituted  Figure. The M segment (4415-nt long) encodes 2 glycoproteins (Gn and Gc) and a nonstructural protein (NSm). It is the most variable genome segment of SBV and related viruses . In contTo investigate whether the detected sequence variations correlated with any change in pathogenicity, 5 female sheep and 1 female calf were subcutaneously inoculated with pools of up to 5 serum or whole blood samples from 1 of the holdings with new cases confirmed in 2014 . None of the animals showed fever or any other clinical sign. Blood samples were taken daily for the first 2 weeks after inoculation and analyzed by real-time reverse transcription PCR , which aSBV, first detected in 2011, circulated again in Germany in 2014. Virus genome with a high sequence identity to the first SBV sample was repeatedly detected in the blood of acutely infected adult cattle, and in experimentally infected animals, viremia developed that was identical to the original SBV isolate. The renewed virus circulation during the 2014 vector season was observed primarily in an area less affected in the 2 previous years. The missing or markedly reduced virus circulation led to a decline in herd seroprevalence caused by a missing infection of the young stock (Technical Appendix. Comparison of the small-, medium-, and large-segment sequences of Schmallenberg viruses isolated in 2012 from the blood of viremic ruminants or detected in 2014 in acutely infected adult cattle with the first SBV isolate from 2011, Germany.


Phoca vitulina). This epidemic caused high mortality in seals along the north-west coast of Europe and represented a potential risk for human health. To characterize the spectrum of lesions and to identify the target cells and viral distribution, findings in 16 harbor seals spontaneously infected with Seal/H10N7 are described. The seals had respiratory tract inflammation extending from the nasal cavity to bronchi associated with intralesional virus antigen in respiratory epithelial cells. Virus infection was restricted to the respiratory tract. The fatal outcome of the viral infection in seals was most likely caused by secondary bacterial infections. To investigate the pathogenic potential of H10N7 infection for humans, we inoculated the seal virus intratracheally into six ferrets and performed pathological and virological analyses at 3 and 7 days post inoculation. These experimentally inoculated ferrets displayed mild clinical signs, virus excretion from the pharynx and respiratory tract inflammation extending from bronchi to alveoli that was associated with virus antigen expression exclusively in the respiratory epithelium. Virus was isolated only from the respiratory tract. In conclusion, Seal/H10N7 infection in naturally infected harbor seals and experimentally infected ferrets shows that respiratory epithelial cells are the permissive cells for viral replication. Fatal outcome in seals was caused by secondary bacterial pneumonia similar to that in fatal human cases during influenza pandemics. Productive infection of ferrets indicates that seal/H10N7 may possess a zoonotic potential. This outbreak of LPAI from wild birds to seals demonstrates the risk of such occasions for mammals and thus humans.Avian influenza viruses sporadically cross the species barrier to mammals, including humans, in which they may cause epidemic disease. Recently such an epidemic occurred due to the emergence of avian influenza virus of the subtype H10N7  in harbor seals ( Phoca vitulina) resulting in increased mortalities along the coasts of Sweden, Denmark, Germany and The Netherlands ) until end-point titration in MDCK cells. At 3 and 7 dpi, three animals were euthanized by exsanguination. Necropsies were performed according to a standard protocol  actinomycetemcomitans and Bordetella bronchiseptica) were described, e.g. with H7N7 [S. equi ssp. zooepidemicus and B. bronchiseptica were isolated only from lungs of seals that died during phocine distemper or influenza epidemics [The most severe lesions in the harbor seals infected with Seal/H10N7 were suppurative or pyogranulomatous bronchopneumonia caused by co-infections with bacteria and/or lungworms that most likely were the cause of death. Similar lesions caused by bacterial or parasitic infections represent common causes of death particularly in young harbor seals from the North Sea . Howeverhia coli . The prehia coli . Also inith H7N7  or H3N8 pidemics –33, whicpidemics . The lunpidemics .Virus infection, cell tropism and associated lesions of Seal/H10N7 infection were evaluated using intratracheally inoculated ferrets as an animal model for human disease (reviewed by van den Brand ). They wInterestingly, the severity of the lesions in the lungs of the ferrets was comparable or even more severe at 7 dpi than at 3 dpi despite lacking virus antigen expression. This may be caused by a delayed inflammatory response due to influx of inflammatory cells, response to cell necrosis and cytokine production after infection, even when there was no virus present in cells anymore (as reviewed by van den Brand ).Sequence analyses indicated that Seal/H10N7 virus was most closely related to LPAIV , 5. HumaWhen comparing the histopathology of H10N7seal infection between the naturally infected harbor seals and the experimentally infected ferrets, it has to be considered that the time course of the natural infection is unknown. However, similar changes were seen in the lower respiratory tract including tracheitis, bronchitis, bronchial adenitis and to a lesser extent bronchiolitis. During the influenza outbreak there were also seals with a similar pattern of lesion that were negative for influenza A virus by PCR and immunohistochemistry (data not shown) possibly indicating virus clearance. When comparing cell type tropism, in both seals and ferrets tracheal, bronchial and bronchial glandular epithelial cells were positive for virus antigen. Virus attachment studies with LPAI viruses  showed moderate virus attachment to trachea and bronchi, moderate to scarce attachment to bronchioles and scarce attachment to alveoli of harbor seals , which iThe difference between the seals and the ferrets is the absence of lesions and virus antigen in the nose of the ferrets which may be in part related to the natural infection in harbor seals versus the intratracheal inoculation of the ferrets. Since the ferrets were inoculated intratracheally to evaluate the pulmonary lesions and virus titers, presence of virus in the nose was very inconsistent and therefore not conclusive for virus replication and lesions in the nose.Another difference is that the seals had virus antigen presence in occasional type I pneumocytes and the ferrets in few type I and II pneumocytes which may reflect a slight difference in tropism between the two species. The rarity of virus antigen presence in alveolar epithelial cells in the seals may reflect the low tropism of Seal/H10N7 for alveolar epithelial cells, it may be related to the time-related clearance after infection as was seen in ferrets with less cells positive for antigen at 7 dpi, or it may be related to masking due to bacterial co-infection, which was not seen in the experimentally infected ferrets. In one ferret with influenza virus in the olfactory bulb, contamination during necropsy from the adjacent virus-positive nasal cavity cannot be excluded.In both the naturally infected harbor seals and the experimentally infected ferrets, virus antigen was seen in bronchial glands with associated bronchial adenitis. Similar tropism for bronchial glands with subsequent lesions has been described for H1N1pandemic infection in ferrets and for seasonal human influenza virus H1N1 and H3N2 infections in humans . Also, vIn conclusion, Seal/H10N7 infection caused mild to moderate inflammation in the upper and lower respiratory tracts of naturally infected harbor seals together with concurrent bacterial infection that resulted in fatal pneumonia. Additionally, intratracheally infected ferrets had moderate inflammation in the lower respiratory tract, indicating that humans may potentially be infected and develop disease when infected with Seal/H10N7. This mass outbreak of H10N7 in seals demonstrates the ease of a bird-to-seal infection of a low pathogenic avian influenza virus such as this Seal/H10N7 from wild birds and thus be a potential risk for humans. In case of a future outbreak in harbor seals caused by Seal/H10N7 or a related virus, its zoonotic potential needs to be evaluated and taken into account.S1 Table(DOCX)Click here for additional data file.S1 FigThe body weight loss from 1 to 3 dpi was more severe than that from 4 to 7 dpi.(TIF)Click here for additional data file.