Previously unknown or unexpected pathogens may be responsible for that proportion of respiratory diseases in which a causative agent cannot be identified. The application of broad-spectrum, sequence independent virus discovery techniques may be useful to reduce this proportion and widen our knowledge about respiratory pathogens. Thanks to the availability of high-throughput sequencing (HTS) technology, it became today possible to detect viruses which are present at a very low load, but the clinical relevance of those viruses must be investigated. In this study we used VIDISCA-454, a restriction enzyme based virus discovery method that utilizes Roche 454 HTS system, on a nasal swab collected from a subject with respiratory complaints. A γ-papillomavirus was detected (complete genome: 7142 bp) and its role in disease was investigated. Respiratory samples collected both during the acute phase of the illness and 2 weeks after full recovery contained the virus. The patient presented antibodies directed against the virus but there was no difference between IgG levels in blood samples collected during the acute phase and 2 weeks after full recovery. We therefore concluded that the detected γ-papillomavirus is unlikely to be the causative agent of the respiratory complaints and its presence in the nose of the patient is not related to the disease. Although HTS based virus discovery techniques proved their great potential as a tool to clarify the etiology of some infectious diseases, the obtained information must be subjected to cautious interpretations. This study underlines the crucial importance of performing careful investigations on viruses identified when applying sensitive virus discovery techniques, since the mere identification of a virus and its presence in a clinical sample are not satisfactory proofs to establish a causative link with a disease. Acute respiratory tract infections (ARTIs) can be caused by a wide variety of pathogens, among which viruses and bacteria are the main agents involved. Still, even with the sensitive diagnostic assays employed nowadays, a substantial amount of respiratory diseases cannot be attributed to any of the known commonly involved microorganisms .The existence of previously unknown or unexpected respiratory pathogens can be postulated as an explanation of this phenomenon. Furthermore, the pool of human respiratory viruses keeps growing because of the continuous introduction of “novel” pathogenic viruses from the animal reservoir . These vOne of these methods is VIDISCA-454, a restriction enzyme based virus discovery technique which was developed in our laboratory and utilizes Roche-454 as HTS method , 2012. TAmong the obtained sequence reads an unexpected virus was found, a papillomavirus. Papillomaviruses carry a circular double stranded DNA genome, are non-enveloped and are known for their capacity to induce warts and benign lesions of mucous membranes , and received antibiotics (amoxicillin). A chest X-ray was performed and the patient kept a diary to monitor the course of symptoms and their severity. Fourteen days after onset of symptoms the patient fully recovered. Four weeks after the first visit the patient underwent a check up visit and again specimens from each nostril and serum were collected (Visit 2: V2). The NPFS of the first and second visit were screened for the presence of known respiratory pathogens associated with ARTI: adenovirus, respiratory syncytial virus, human metapneumovirus, influenza A and B, parainfluenza viruses 1–4, human bocavirus, human coronaviruses , human rhinovirus, polyomaviruses WU and KI, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Bordetella pertussis, Legionella pneumophila, Streptococcus pneumoniae, and Haemophilus spp  during a visit to his local general practitioner where he reported symptoms of a respiratory tract infection. The onset of symptoms was 2 days before visiting his general practitioner. Symptoms included cough, phlegm production, shortness of breath, wheeze, coryza, fever, chest pain, muscle aching, and headache. During this first visit respiratory specimens  from each nostril  and serum were collected (Visit 1: V1). The patient was part of the GRACE study, a randomized antibiotics placebo-controlled double-blind trial (ilus spp . All diaEthics review committee of Cardiff and Southampton (UK) approved the study and written informed consent was provided by the participants .VIDISCA-454  was performed with 110 μl of sample resuspended in UTM as previously described . ObtaineThe γ-papillomavirus fragments identified after performing VIDISCA-454 were used as template for primer design and PCRs were performed using the Expand Long Template PCR system (Roche). The complete genome was obtained by sequencing two large amplified fragments which were overlapping on both sides for at least 400 nucleotides. Fragments were cloned into a XL-TOPO-kit (invitrogen), and sequenced . Primer sequences used in this study are available on request.D-thiogalactopyranoside (IPTG) for 5 h. Samples were collected every hour to monitor the L1-protein production. Attempts to purify the protein via Ni-NTA resin (Qiagen) failed, probably due to the formation of aggregates that shield the HIS-Tags; therefore the crude harvest was used to determine the reactivity of V1 and V2 serum on Western blot.The full late gene L1 (coding for the major capsid protein) was amplified with the Expand Long Template PCR system (Roche), cloned into pET100D expression vector (Invitrogen), and transformed into chemically competent Escherichia coli BL21-derived strain Rosetta 2 (Novagen). Overnight cultures of the transformed bacteria were inoculated into Luria broth medium, supplemented with 1% glucose, carbenicillin (10 μg/ml), and chloramphenicol (17.5 μg/ml). Cultures were grown to the exponential phase prior to induction with 0.5 mM isopropyl-β-Protein production on SDS-Page was visualized by coomassie staining and SDS-page gels were blotted onto PVDF (Millipore) membranes. The blots were incubated with 1:600 dilutions of either V1 or V2 serum, and with a 1:1000 dilution of a rabbit anti HIS-tag, followed by incubation with anti-human IRDye800CW  and anti-rabbit DyLight649 (1:5000). Signals were measured via the Odyssey infrared imaging system (LI-COR). The intensity of the signals was normalized with the anti-HIS signal to correct for difference in protein concentrations.′-CATACCCTAACGAAGAGGTAGCAGAC-3′, RTReverse-675–700: 5′-TGGCGGGCAACTGCCTTTATCTAG-3′, RTProbe-620–650: 5′ FAM- GGGCTGTATTCCGCCAACTGGTGAATATTGGGATG -TAMRA 3′.Real time PCRs were performed with the Platinum®; qPCR-kit (Invitrogen) on an ABI PRISM 7000 sequence detection system of Applied biosystems according to the manufacturer’s protocol, using the following primers and probe: RTForward-540–570: 5Screening was performed in duplicate on 54 respiratory samples collected from patients with respiratory complaints involved in the GRACE study . Among these, 10 sequences showed significant identity to HPVs. By genome walking the entire sequence of the virus (named HPV isolate A2619) was determined: the viral genome is 7142 bp in size, circular and 99% identical to the recently described HPV-KC5 isolate .Since the patient fully recovered after 14 days of illness, an increase of the specific antibody response to the responsible pathogen was expected, along with viral clearance. We examined the clearance of the virus with an HPV-A2619 strain specific real time PCR targeting the L1 gene. The virus was detected in the NPFS of both nostrils at V1, but also in the NPFS of both nostrils after the patient recovered (V2). The viral load in all samples was very low, between 2EFigure 1).Furthermore, western blot analysis was used to detect antibodies directed against the L1 capsid protein in serum samples collected at the same time as the 2 respiratory samples. A poor recognition of the protein was observed and no difference between V1 or V2 serum was noticed: the two samples had similar intensity ratios . Both patients came from the same city in the UK as the indexcase.Respiratory samples of 54 other patients from the GRACE study with respiratory tract infection were used for prevalence inference. Of these, 25 were from the same geographical area of the index patient and 39 from other European cities. Two patients (3.7%) were positive for HPV-A2619. In both patients also a low viral copy number was detected (between 2ENot much is known about γ-papillomaviruses (γ-HPVs). Occasionally γ-HPVs have been related to skin warts or keratotic lesions of immunosuppressed organ transplant recipients  is not an exception , 2012. TWith state of the art HTS based virus discovery techniques and the possibility to obtain such a huge number of sequences from a clinical sample, the identification of novel viruses became very effective, and nowadays the detection of low load viruses is not uncommon since minority nucleic acids can be efficiently sequenced . These mWith the development of HTS techniques the experimental time needed to discover novel viruses has dramatically reduced. However, targeted experiments and careful evaluation of all available data are essential to assess the relevance of every finding and to determine the role of every identified virus, since – as we show here – the mere identification of a virus is not sufficient to prove its involvement in a disease. This is especially true when dealing with body parts which are contiguous with the external environment – like the respiratory tract – and therefore more subjected to the presence of bystander or non-host-specificmicroorganisms.Marta Canuti and Martin Deijs performed the experiments, participated to study design and wrote the paper. Seyed M. Jazaeri Farsani, Melle Holwerda, Maarten F. Jebbink, and Michel de Vries performed the experiments. Saskia van Vugt, Curt Brugman, Theo Verheij, Christine Lammens, Herman Goossens, Katherine Loens, and Margareta Ieven provided the clinical samples and participated to the study design. Lia van der Hoek participated to the study design, supervised the study and helped with the manuscript preparation. All authors critically revised the manuscript and approved the final version.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.


Transmission via shared water implicates passerine birds as possible vectors for dissemination of this virus. Low pathogenicity avian influenza A(H7N9) virus has been detected in poultry since 2013, and the virus has caused >450 infections in humans. The mode of subtype H7N9 virus transmission between avian species remains largely unknown, but various wild birds have been implicated as a source of transmission. H7N9 virus was recently detected in a wild sparrow in Shanghai, China, and passerine birds, such as finches, which share space and resources with wild migratory birds, poultry, and humans, can be productively infected with the virus. We demonstrate that interspecies transmission of H7N9 virus occurs readily between society finches and bobwhite quail but only sporadically between finches and chickens. Inoculated finches are better able to infect naive poultry than the reverse. Transmission occurs through shared water but not through the airborne route. It is therefore conceivable that passerine birds may serve as vectors for dissemination of H7N9 virus to domestic poultry. Despite their avian genetic background, some H7N9 viruses have HA and polymerase protein mutations that confer a replication advantage in mammals  viruses emerged in eastern China . In addition, during surveillance in 2013, the influenza strain A/tree sparrow/Shanghai/01/2013 (H7N9) was identified in a tree sparrow  found at a site where migratory and local birds congregate (The polymerase acidic (PA) and polymerase basic 2 genes derived from A/Anhui/1/2013 (H7N9)–like virus are homologous to those from A/brambling/Beijing/16/2012 (H9N2) (Lonchura striata domestica), zebra finches (Taeniopygia guttata), sparrows (P. domesticus), and parakeets (Melopsittacus undulates) are susceptible to H7N9 virus and shed virus into water .For the experiments, we used A/Anhui/1/2013 (H7N9) (hereafter referred to as Anhui/1) from an index human patient (L. striata domestica) ; 5-week-old white leghorn hens  ; and 16-week-old bobwhite quail (Colinus virginianus) . The birds were quarantined for 1 week, and prechallenge swab samples were confirmed influenza virus–negative by egg isolation. Food and water were provided ad libitum. Animal experiments were approved by the St. Jude Children’s Research Hospital Animal Care and Use Committee and complied with all applicable US regulations.Study birds were of both sexes and included 3- to 6-month-old society finches (5 log10 50% egg infectious doses (EID50) of virus in 100 μL (finches) or 500 μL (poultry) of phosphate buffered saline. Oropharyngeal and cloacal swab samples were collected on days postinoculation (dpi) 2, 4, 6, 8, 10, and 13. Water samples (500 μL) were obtained 1–4 and 8 dpi. Samples were titrated in eggs  were housed in a 97 cm × 58 cm × 53 cm cage that contained a 30 cm × 41 cm × 41 cm cage housing finches (n = 4 or 5). This setup was used in duplicate for each experiment, and the data obtained from each set of cages were combined. Waterborne transmission was examined by sliding a water pan (15 cm × 25 cm) halfway into a notched hole in the finch cage , panel ANecropsies were performed on birds that died during the study. Trachea and/or lung and intestine samples were harvested  and homo>5 birds from each species for influenza A virus antibodies; all results were negative. On dpi 16, we collected blood samples from the surviving birds and tested them for H7N9 virus seroconversion by using the IDEXX AI MultiS-Screen Ab Test  according to the manufacturer’s instructions.Before beginning the experiments, we tested t-test in Excel  or GraphPad Prism v5 . Area under the curve (AUC) analysis for cumulative shedding was performed by using GraphPad Prism v5.Mean infectious titers were compared by using the 1-tailed Student 5 log10 EID50 units of virus and pairing the donor birds with the naive bird species (water contacts) (Waterborne virus transmission between finches and chickens was investigated by inoculating 1 species (donors) with 10ontacts) , panel AAll donor finches were productively infected with Anhui/1 or Ck/Rizhao and shed virus by the oropharyngeal route for 10–13 dpi . No viru>2.5 log10 EID50/mL of virus and/or when the bird shed during >2 consecutive time points. Under such criteria, waterborne transmission from finches to chickens was limited. Of 6 water-contact chickens paired with Anhui/1-donor finches, 2 shed <2 logs of virus for a single time point, which did not meet our transmission criteria .In the converse experiment, all donor chickens were productively infected with both viruses and shed virus an average of 10 days . Chicken10 EID50/mL) (10 EID50/mL), but the fourth finch had higher virus titers (peak titer 4.3 log10 EID50/mL) and shed virus for 7 days (4 sampling time points)  . Four wa points) . All wat10 EID50/mL). Water-contact quail were quickly infected and shed virus by 2 dpi. As in the finch–chicken experiments, society finches or bobwhite quail in this experiment were inoculated with Ck/Rizhao or Anhui/1 (donors) and shared water with naive birds. As in the other experiments, all donor finches in this experiment shed both viruses an average of 10 days . Virus wAll water-contact quail shed Anhui/1 by the oropharyngeal route , and peaIn the converse experiment, in which quail served as donors, all quail shed Anhui/1 and Ck/Rizhao by the oropharyngeal and cloacal routes for 13 and 10 days, respectively . DespiteThe birds in our experiments shared the same airspace, so we examined whether virus transmission occurred between the species via large droplet particles or smaller, fully aerosolized particles. Droplet transmission was less likely to occur because the animals were separated and droplet sources  were minimized through placement of separate water pans. Society finches (n = 2 or 3) were housed with chickens or bobwhite quail (n = 3) , panel BIn the previously described experiments, we observed interspecies transmission of H7N9 virus when birds shared water, but transmission did not occur when they shared airspace but not water. We hypothesized that this effect was primarily mediated by water contact. Because finches, chickens, and quail shed virus by the oropharyngeal route (often exclusively), transmission via water is possible and may occur during drinking and other events associated with water contact. To test this hypothesis, we sampled 500 μL of the water remaining in each pan on 4 different dpi and at 8 dpi .>4 of 5 sampled time points, virus was detected in all water pans, regardless of the inoculated bird species (10 EID50/mL for finch–chicken experiments and 3.8–4.5 log10 EID50/mL for finch–quail experiments (10 EID50/mL) (During  species . Peak vieriments . At 8 dpID50/mL) . TherefoOverall, 5 of 86 finches, 4 of 30 quail, and 0 of 30 chickens died during the experiments , 2. All 10 EID50/mL   , 2. An aID50/mL) , and hadID50/mL) . This de10 EID50/mL) and lung (4.5 log10 EID50/mL) at necropsy .Two of 6 naive water-contact quail in Anhui/1 experiments died at 15 dpi, and 1 of 6 naive water-contact quail in Ck/Rizhao experiments died 10 dpi . These qSeroconversion was tested by using IDEXX ELISA. Prechallenge serum and swab samples were negative for avian influenza antibodies and virus (data not shown), strongly suggesting the lack of prior exposure to influenza A virus .More than half of donor birds seroconverted: for Anhui/1-inoculated birds, finches 66%–77% (range for donor groups), chickens 50%, quail 100%; for Ck/Rizhao-inoculated birds, finches 50%–100%, chickens 83%, quail 100% . Among wNovel influenza A(H7N9) viruses emerged in China in 2013 and were first detected in humans with severe illness (,H7N9 viruses and viruses with genes homologous to those of H7N9 virus have been isolated from 2 passerine birds: bramblings and a tree sparrow (The isolation of LPAIVs from water has been reported (Airborne transmission of LPAIV among poultry has been demonstrated for multiple influenza subtypes, but Zhong et al. (We found little difference between the 2 viruses used in our study, except that replication of Ck/Rizhao was significantly better than that of Anhui/1 in donor chickens. Anhui/1 has several mutations conferring mammalian replication and receptor binding (Although the type of finches used in this study would not necessarily be expected to have poultry contact, other finch and sparrow species are peridomestic and susceptible to H7N9 virus; intermingling of these birds in nonsecured poultry operations like farms or live bird markets could facilitate transmission of H7N9 virus to poultry. Chickens and, in particular, quail could then act as an amplifying host, releasing large amounts of H7N9 virus into the environment, thereby posing a health risk to humans with direct contact. Bobwhite quail and Japanese quail have been shown to be highly susceptible to LPAIVs, including subtype H7N9, and to highly pathogenic avian influenza viruses (, We did not address interspecies transmission between birds and mammals. Passerine and psittacine birds shed H7N9 virus at levels lower than or nearly equal to those of domesticated poultry (In summary, in this follow-up of our study identifying small bird species as potential vectors of H7N9 virus (


In Portugal, entomological surveys to detect phleboviruses in their natural vectors have not been performed so far. Thus, the aims of the present study were to detect, isolate and characterize phleboviruses in sandfly populations of Portugal.Phlebotomus perniciosus pools. Direct sequencing of the amplicons showed that 2 samples exhibited 72 % nucleotide identity with Arbia virus, and two showed 96 % nucleotide identity with Massilia virus. The Arbia-like virus (named Alcube virus) was isolated in cell culture and complete genomic sequences of one Alcube and two Massila viruses were determined using next-generation sequencing technology. Phylogenetic analysis demonstrated that Alcube virus clustered with members of the Salehabad virus species complex. Within this clade, Alcube virus forms a monophyletic lineage with the Arbia, Salehabad and Adana viruses sharing a common ancestor. Arbia virus has been identified as the most closely related virus with 20-28 % nucleotide and 10-27 % amino acid divergences depending on the analysed segment.From May to October 2007–2008, 896 female sandflies were trapped in Arrábida region, located on the southwest coast of Portugal. Phlebovirus RNA was detected by using a pan-phlebovirus RT-PCR in 4 out of 34 Alcube virus in Ph. perniciosus and co-circulation of Massilia virus, in Arrábida region, southwest of Portugal. Further epidemiological investigations and surveillance for sandfly-borne phleboviruses in Portugal are needed to elucidate their medical importance.We have provided genetic evidence for the circulation of a novel phlebovirus species named  Leishmania, but they also act as vectors of several phleboviruses  that are associated with human illness (from transient febrile illness to severe neuroinvasive diseases) [Sandfly fever Naples virus species complex was known to circulate in Portugal after first being reported in 1985 when a tourist was infected in the Algarve region, in the south of the country [The world-wide impact of sandflies (Diptera: Psychodidae) as significant public health concern and vectors of several zoonotic diseases affecting humans is well known. The most widely distributed pathogens transmitted by sandflies are protozoan parasites of the genus iseases) –3. Basediseases) –10. In t country , 12. HowPhlebotomus perniciosus and subsequently grouped in pools of up to 60 specimens. Pools of sandflies were homogenized and suspended in Hank’s solution containing 7.5 % bovine albumin and antibiotics. Total RNA and DNA from homogenized sandfly pools were extracted using RTP® DNA/RNA Virus Mini Kit according to the instructions of the manufacturer . Extracted samples were analyzed for the presence of phlebovirus RNA by using a pan-phlebovirus RT-PCR published elsewhere [Ph. perniciosus trapped in two distinct trapping sites  and analyzed in an Illumina MiSeq run of 250-bp end reads. Complete genome assembly, sequence analysis, and multiple alignments were performed using Geneious v7.1.8 . The new virus is designated Alcube virus since the isolate was obtained from sandflies collected near a small river named Alcube. The complete genome sequence was determined and used to compare the genetic and phylogenetic relationships with other phleboviruses. Alcube virus exhibits the characteristic phleboviruses genome organization with L (encoding the RNA-dependent RNA polymerase), M , and the S segment  (GenBank acc. no. KR363190-KR363192). The complete sequence comprises 1,758 nt, 4,164 nt, and 6,405 nt for the S, M, and L segments, respectively. Genetic distances of the Alcube virus were compared with the members of the Salehabad virus antigenic complex and other representative phleboviruses  in Arrábida region, located on the southwest coast of Portugal. The virus was isolated and characterized using full-length genome sequence data. Phylogenies demonstrated that Alcube virus clustered with members of the Salehabad virus species complex within the genus Phlebovirus and forms with the Arbia, Salehabad and Adana viruses a distinct monophyletic lineage within this clade. In Turkey, high Adana virus seroprevalence rates in goats, sheep, and dogs were demonstrated [To date, Toscana virus , 12 fromnstrated . In contnstrated . In the nstrated , 16. TheAlcube virus in Ph. perniciosus and co-circulation of Massilia virus, a previously recognized phlebotomine-borne phlebovirus, in Arrábida region, southwest of Portugal.In conclusion, we have provided genetic evidence for the circulation of a novel phlebovirus species named


Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses.Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to  Influenza A viruses cause yearly epidemics in humans and are an important cause of worldwide morbidity and mortality . Occasioin vivo in mice. However, a clonal GFP positive virus was only obtained after plaque purification. Moreover, GFP was found to be unsuitable for live in vivo imaging, due to the low tissue penetration of the fluorescent signal and the high background. When the same strategy was employed to create an A/PR/8 strain expressing tRFP (TurboRFP), reporter expression was lost after two passages [in vitro when compared to the wild-type virus [et. al. [To date, both bioluminescent and fluorescent influenza A reporter viruses have been described –22. The passages . A/PR/8 pe virus . RecentlGaussia luciferase (gLUC) was introduced into the PB2 [in vitro, they were visualised in the lung during live in vivo imaging experiments. Alternatively, Tran and colleagues [in vitro and in vivo, was stable across five passages in MDCK cells and showed a clear signal in real-time in vivo imaging in mice [in vivo imaging [In order to address some of these limitations, others have generated bioluminescent influenza A reporter viruses. For example,  the PB2  and NA [ the PB2  gene seglleagues  inserted in mice . Recentl imaging . However imaging . Furtherin vivo imaging experiments.Here, we provide a strategy which was used to create five different reporter viruses, using enhanced GFP (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), gLUC and firefly luciferase (fLUC) in the same virus backbone. The levels of attenuation, reporter expression and stability of the reporter viruses were compared and this strategy was also used to generate pH1N1, HPAI H5N1 and H7N9 eGFP-expressing viruses. A selection of these viruses were then used for Reporter virus constructs were cloned using the PA gene segment of influenza A viruses A/PR/8/34 (H1N1), A/Netherlands/602/09 (pH1N1), A/Indonesia/5/05 (H5N1) and A/Anhui/1/13 (H7N9). Expression plasmids for H7N9 PB2, PB1 and PA were cloned into the pCAGGS plasmid, kindly provided by Dr. A. Garcia-Sastre .Influenza A virus reporter constructs were assembled using the pCAGGS plasmid as a shuttle vector. First, the 5’ untranslated region (UTR) and PA gene of the respective influenza A viruses was inserted into pCAGGS. The 17-aa 2A proteolytic site from foot and mouth disease virus (FMDV) , kindly et al. [et al. for plasmids pHL1102 and pHL1103, respectively [The packaging and promoter regions of various reporter constructs were further modified. The length of the initial PA packaging region was based on data described by de Wit et al.  and contet al. , 23 wereectively .Vibrio cholerae neuraminidase  and L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK) treated trypsin (10 μg/μl) was added. When no virus was obtained after the first passage in MDCK cells, a second passage was performed. In order to efficiently rescue the eGFP-expressing H7N9 virus, pCAGGS expression plasmids containing PB2, PB1 and PA were co-transfected. 293T supernatants were harvested by low-speed centrifugation and used to inoculate a confluent layer of MDCK cells and/or embryonated chicken eggs as described previously [To produce recombinant influenza A reporter viruses, 293T cells were transfected with 5 μg of each plasmid encoding the 7 gene segments of the A/PR/8, pH1N1, HPAI H5N1 or H7N9 virus and the PA reporter construct of interest using the calcium phosphate method as described previously . Cells weviously . The seq2 and the percentage of eGFP positive cells was determined by Fluorescent Activated Cell Sorting (FACS) on a FACS Canto .Minigenome assays using fLUC as a reporter were performed as described previously for PA constructs that contained fluorescent reporters . An eGFPMDCK cells were inoculated at a multiplicity of infection (MOI) of 0.1. Approximately 24 hours after inoculation, cells were washed once with PBS and lysed in hot lysis buffer (1% sodium dodecyl sulfate (SDS), 100 mM NaCl, 10 mM EDTA, 10 mM Tris-HCl pH 7.5). The samples were treated with 3x dissociation buffer  for 10 minutes at 96°C and loaded on a 12.5% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membrane by electroblotting. Blots were blocked overnight at 4°C in PBS containing 5% non-fat dry milk. The next day blots were washed three times with PBS 0.05% Tween-20. To detect the PA protein, a monoclonal mouse antibody was used in a 1/40 dilution . To detect M1 a monoclonal mouse antibody  was used in a 1/2000 dilution. Antibodies were diluted in PBS containing 5% non-fat dry milk. Blots were incubated for one hour at RT and washed three times with PBS 0.05% Tween-20. A secondary goat-anti-mouse HRP antibody  was then used in a 1/2000 dilution, in PBS containing 5% non-fat dry milk, for one hour at RT. After washing three times with PBS 0.05% Tween-20 blots were developed using ECL WB detection reagent  and imaged with a Chemidoc MP imaging system . Analysis was performed using Image Lab software (BioRad).Expression of eGFP was visualized using an Axiovert 25 IF microscope  followed by quantitative analysis using FACS. For iRFP, infected cells were visualized using a LSM-700 confocal microscope with filter set 32 (Zeiss) and quantitative analysis was done by FACS using the APC-Cy7 channel. Expression of fRFP was visualized using fluorescent microscopy with filter set 00 (Zeiss) and by FACS using the PerCP-Cy5-5-A channel.Gaussia luciferase flash assay kit  according to the manufacturer’s instructions. The Luciferase assay system  was used for fLUC, according to the manufacturer’s instructions. Luminescence was measured on an Infinite F200 reader  in flat 96-black well plates .The reporter activity of the gLUC virus was determined using a 50) in virus stocks was determined as described previously [μg/μl TPCK treated trypsin (Sigma). Infection medium consisted of EMEM  supplemented with 100 IU/ml penicillin (Lonza), 100 μg/μl streptomycin (Lonza), 2 mM glutamine (Lonza), 1.5 mg/ml sodiumbicarbonate , 10 mM Hepes (Cambrex) and non-essential amino acids .The fifty percent tissue culture infectious dose (TCIDeviously  using inMultistep replication kinetics were determined by inoculating a confluent monolayer of MDCK cells at a multiplicity of infection (MOI) of 0.001. One hour after inoculation, at time zero, cells were washed twice with PBS and fresh infection medium was added. At 0, 6, 12, 24, 48 and 72 hours after inoculation samples were taken and used to determine the virus titre, as described above.in vitro stability of the different reporter viruses, three individually rescued viruses were blindly passaged ten times in MDCK cells. MDCK cells were inoculated with 20 μl of virus supernatant from the previous MDCK passage in 3 ml of infection medium. Virus from each passage was used to inoculate a confluent monolayer of MDCK cells, using a 1/10 dilution to determine the presence of the reporter. Approximately 24 hours after inoculation, cells were used for FACS  or luciferase reporter assays (gLUC and fLUC) as described above.To test the Mice were housed and experiments were conducted in strict compliance with EU directive 86/609/EEC on animal testing and the Dutch Experiments on Animal Act, 1997. Each cage contained 6 mice with sawdust as bedding. Enrichment was offered in the form of paper tissues. Food and water were checked daily and the facility was dark/light for 12 hours. The protocol was approved by an independent animal experimentation ethical review committee “Stichting DEC consult” (Erasmus MC permit number EUR3385). Stichting DEC considers the application and pays careful attention to the effects of the intervention on the animal, its discomfort, and weighs this against the social and scientific benefit to humans or animals. The researcher is required to keep the effects of the intervention to a minimum, based on the three R’s . Animal welfare was monitored daily. All experiments with HPAI H5N1 viruses in mice were performed under DM-III conditions.2 and inoculated intranasally with 50 μl of 105 TCID50 of A/PR/8 reporter or wild-type viruses, 104 TCID50 of the HPAI 2UP_PA_eGFP_dPR(H5N1) or HPAI H5N1 wild-type virus or PBS for the mock-infected group. Each group consisted of 24 mice with six mice for each time-point. Before inoculation with gLUC, iRFP or fRFP viruses, the imaging region of interest was shaved to prevent the fur from quenching the signal [Female BALB/c mice  were randomly distributed into the relevant experimenal groups. Mice were anaesthetized with 3% isoflurane  in Oe signal . Mice wee signal .in vivo, mice were anaesthetized with 3% isoflurane in O2 and transferred to a custom made, transparent, airtight IVIS box with a black inlay . The box was filled with a maintenance concentration (3%) of isoflurane in O2 and transported to the IVIS Spectrum system  for in vivo imaging. For mice inoculated with the gLUC virus, 300 μg of coelenterazine-SOL in vivo substrate  was administered via the tail vein, prior to transfer to the IVIS box and subsequent imaging. Next, the mice were euthanized and the lungs were transferred to a 6-well equiglass cell culture plate  for imaging. Given the high turnover rate of the substrate, lungs from the gLUC virus-inoculated mice were injected with 100 μg of coelterazine-SOL substrate, via the trachea. Lungs were imaged using the IVIS spectrum; lungs from mice inoculated with fluorescent reporter viruses were additionally imaged using fluorescent microscopy.To visualise the reporter activity in vivo stability of the reporter viruses MDCK cells were inoculated with lung homogenates at a MOI of 0.1. At approximately 24 hours after inoculation cells were washed twice with PBS-1% FCS. Cells were resuspended in cytofix-cytoperm (BD biosciences) and incubated for 20 minutes on ice. Subsequently they were incubated for 1 hour, on ice, with a monoclonal mouse antibody against NP , diluted 1/50 in perm-wash buffer (BD Biosciences). Cells were washed once in perm-wash and incubated for 1 hour, on ice, with either polyclonal rabbit-anti-mouse FITC  or goat-anti-mouse APC (BD Biosciences), diluted 1/100 in perm-wash. Cells were washed once with perm-wash, resuspended in PBS and FACS was performed. Data was analysed with FlowJo, version Vx.07 .To test the At the relevant time points after inoculation mice were euthanized and lungs were inflated with 10% formalin and embedded in paraffin. Lungs were subsequently sectioned and immunohistochemistry (IHC) NP-staining was performed as described previously . To deteStatistical significance of virus titres was determined using the nonparametric Kruskal-Wallis test with a Dunns post-test where p<0.05 was considered significant. Statistical significance of bodyweight was determined using the two-way ANOVA with a Bonferroni post-test where p<0.05 was considered significant. All statistical analyses were performed using GraphPad Prism 5.00 for Windows .Confluent monolayers of MDCK cells, in a 5 cm poly-D lysine coated glass bottom culture dish , were inoculated with the 2UP_PA_eGFP_sPR virus at a MOI of 1. The plate was transferred to a temperature controled 37–2 digital mini-incubator  and imaged using the LSM-700 confocal microscope (Zeiss), using a 10x/0/45NA Plan-Apochromat objective. EGFP fluorescence was excited at 488nm and detected through a 500–550nm bandpass emission filter. Ten positions were selected and imaged every 30 minutes for 72 hours. At each position a 3x3 tilescan  was made, with a pixelsize of 625nm, resulting in an imaged area of 1.92x1.92nm. The sample was kept in focus through a reflection-based autofocus routine. The images were used to generate a movie using the Fiji ImageJ software package .MDCK cells were grown on sapphire discs that had been imprinted with a finder grid pattern according to the method described by McDonald et. al. . At 24 hTo optimize the strategy to make an influenza A reporter virus, different constructs were cloned using iRFP as a representative fluorophore . FirstlyDuplication of the packaging region (dPR) was essential to rescue iRFP reporter virus efficiently, whilst introduction of the 3UP mutation did not result in recombinant virus production. Although viruses that only contained the dPR had virus titres similar to the WT virus, no iRFP expression was detected. In contrast, the 2UP_PA_iRFP_dPR construct was the only one that resulted in recombinant virus expressing iRFP in MDCK cells, although the virus titres were lower than that of the wild-type. A reduction in virus titre was also observed when the 2UP mutation was introduced into the wild-type PA gene segment. Interestingly, none of the reporter viruses resulted in iRFP expression upon inoculation of MDCK cells when egg-grown viruses were used.The 2UP_PA_iRFP_dPR cloning strategy was subsequently used to insert a selection of different fluorescent and luminescent reporters into A/PR/8, including gLUC, eGFP, fRFP and fLUC .The PA-reporter plasmids, as shown in 10 lower virus titres when compared to the wild-type virus. To confirm that the 2UP mutation was necessary for improved reporter expression (as was observed for the 2UP_PA_iRFP_dPR virus), reporter expression levels were compared to those of viruses without the 2UP mutation  were imaged (n = 3) and virus titrations were performed (n = 3) at day 1, 3, 4 and 5 post-inoculation. All groups showed a reduction in bodyweight from day 2 onwards . The NP-positive cell population was selected and the percentage of (fluorescent) reporter expression was determined  virus was reduced by 1.5 log10 compared to wild-type HPAI H5N1 virus, and the HPAI 2UP_PA_eGFP_sPR(H5N1) virus had a significantly (p<0.01) lower virus titre of approximately 3.5 log10. The 2UP_PA_eGFP_dPR(H7N9) virus had a significantly (p<0.05) lower virus titre of approximately 2 log10 compared to the wild-type H7N9 virus. Strikingly, a difference between the various virus subtypes was observed. For the pH1N1 virus, the data resembled that of A/PR/8 , 2UP_PA_eGFP_sPR(H5N1) and 2UP_PA-eGFP_dPR(H7N9) viruses showed stable eGFP-expression up to passage 4. Together, these data demonstrate that the described strategy can be successfully applied to avian and human virus strains, although the pH1N1_GFP virus displayed limited in vitro stability.Next, the termined . The 2UPtermined  and 3B ain vivo characteristics of the HPAI 2UP_PA_eGFP_dPR(H5N1) reporter virus, BALB/c mice were inoculated intranasally with 50 μl of 104 TCID50 of HPAI 2UP_PA_eGFP_dPR(H5N1), HPAI H5N1 wild-type virus or PBS. As the sPR construct had a negative influence on the virus titre, the HPAI 2UP_PA_eGFP_dPR(H5N1) virus was selected for in vivo experiments. Bodyweight was monitored daily and if the loss in bodyweight exceeded ≥20%, mice were killed humanely. Both groups lost bodyweight from day 2 onwards and at day 2, 3 and 4 a significant difference was observed for the HPAI 2UP_PA_eGFP_dPR(H5N1) virus when compared to the HPAI H5N1 wild-type virus  virus is attenuated in vitro and in vivo, it still resulted in a detectable eGFP signal in mice and can thus be used for in vivo experiments.Nose and lung tissues were homogenised and used for virus titration on day 1, 3 and 4 post-inoculation. The HPAI 2UP_PA_eGFP_dPR(H5N1) virus replicated to significantly (p<0.05) lower virus titres both in the nose and lung compared to the wild-type HPAI H5N1 virus . Due to in vitro and in vivo. This strategy was then successfully applied to a set of relevant human virus isolates.Reporter viruses can be a valuable tool across a wide variety of research areas , 47, 48.in vitro, probably due to an increased production of non-infectious virus particles [in vitro and in vivo in the absence of the 2UP mutation [In this study, a 2UP mutation in the promoter region of the PA gene was necessary for successful reporter expression. Indeed, without this mutation only eGFP and gLUC were detectable in influenza A virus infected cells. A disadvantage of the 2UP mutation was that it resulted in lower virus titres (with the exception of the 2UP_PA_fRFP_dPR virus). When these mutations were introduced into the PA gene segment without a reporter, a similar decrease in virus titre was observed. This is consistent with the findings of Belicha-Villanueva and colleagues who showed that viruses with promoter mutations in the PB1 and PA gene segments of A/PR/8 displayed reduced replication articles . Interesmutation , 16–18. in vitro and in vivo. In contrast, viruses containing the relatively large fLUC had very low virus titres, were very unstable and showed a large variation in intensity if individually rescued viruses were compared. This is consistent with previous studies in alphavirus reporter systems [Another striking finding of the present study was that different reporters had a differential effect on virus attenuation and reporter activity. The selected reporters had a broad range in size with gLUC being the smallest 530 nt) and fLUC as the largest (1653 nt). Previous studies have shown that the coding capacity of the influenza virus gene segments is limited , 52. Thu0 nt and et. al. [et al. [in vivo imaging. This is in spite of the fact that the strategy described here was very similar to that of Tran and colleagues. This discrepancy may be due to study-dependent differences in the virus backbone, the characteristics of the selected reporter or interactions between the two. Indeed, this is consistent with the findings of the present study whereby the use of different reporters in the same virus backbone resulted in unpredictable stability and virus replication.Interestingly, when compared to the recent publications by Tran et. al.  and Karl [et al.  the PA-rin vivo imaging for any of the reporter viruses tested and all reporter viruses showed some level of in vivo attenuation. It remains possible that additional passaging of these viruses in mice would result in increased pathogenicity/reporter gene expression, as has recently been described by Fukuyama et. al. [in vivo imaging. Such optimisations may also ensure that a lower viral doses could be used to infect mice and the pathogenesis of mild influenza virus infections could be examined. This remains an important area for future studies.Unfortunately, a signal was not obtained during live  et. al. . Alterna et. al. , 55 may in vitro. This then affords the researcher the opportunity to analyse, in depth, the morphology of infected cells and the virus particles which they produce using techniques such as CLEM. Moreover, the detection limit of microscopy is such that even areas with very few infected cells can be identified and then further processed using EM. This may be important to detect the early stages of virus infection and identify the actual site of replication to determine the associated cellular changes.Despite these limitations, the fluorescent viruses created here could successfully be used to rapidly identify virus-infected cells This study also provided a cloning strategy that could be used to create eGFP-expressing reporter viruses derived entirely from pH1N1, HPAI H5N1 and H7N9. To date, the majority of studies have focused on the creation of reporter viruses in laboratory strains of influenza (such as A/WSN/33 and A/PR/8), although recently a bioluminescent pH1N1 virus  and a Vein vitro stability. The percentage of eGFP-expressing cells was low and the sPR viruses lost the ability to replicate at passage three for two out of three viruses. However, for HPAI H5N1 eGFP a lower virus titre was observed with the decreased PR and for H7N9 eGFP no virus was rescued with the decreased PR. Both HPAI H5N1 and H7N9 e-GFP expressing viruses stably expressed eGFP until passage four. Previously, the minimal regions for optimal packaging were obtained using A/WSN and A/PR/8 viruses [Interestingly, there were striking virus dependent differences in the influence of the length of the packaging region on virus titre, eGFP-expression and stability in the various reporter viruses. For the pH1N1 eGFP virus the length of the PR had no effect on virus titre, as was seen for A/PR/8 eGFP virus, although they performed poorly in terms of  viruses . Here, tin vivo experiments. However, the strategy developed here can clearly be used across a broad range of different reporter genes and virus backbones. This study thus provides a basis to continue to optimise influenza reporter viruses that can then be used to answer key questions in influenza A virus pathogenesis.From the experiments described in this paper it is clear that a there is no “one size fits all” strategy to produce influenza A reporter viruses. Attenuation levels and stability are unpredictable and have to be assessed thoroughly before proceeding to S1 FigRenilla) were calculated. Error bars indicate the standard error of the mean based on data from three replicates. The white and black bars represent two independent experiments. MDCK cells were inoculated at a MOI of 0.1 and ~24 hours post-inoculation cells were harvested and loaded on a 12.5% SDS-PAGE gel. Antibodies against PA and M1 were used to detect the levels of protein produced (C). Numbers represent the ratio between the level of PA and M1 protein expression.The PA reporter constructs were tested in a minigenome assay, using eGFP (A) or fLUC (B) minigenomes. (A). For the fLUC minigenome relative light units (fLUC divided by (EPS)Click here for additional data file.S2 FigThe influence of the 2UP mutation on the level of reporter expression was determined for eGFP (A), fRFP (B) and iRFP (C) expressing viruses. For each virus a negative control (yellow square), noUP (blue square) and 2UP (red square) was compared. Data are a representative of 3 independent experiments. This was also done for the gLUC and fLUC expressing viruses. (D) Three individually rescued viruses were used to determine the expression levels for the luminescent viruses. Data represent mean expression levels and error bars indicate standard error of the mean. A threshold of ~3x the background values was applied and is indicated with a dotted line.(EPS)Click here for additional data file.S3 FigA schematic representation of the 2UP_PA_eGFP_dPR gene segment showing the sequence analysis data from seven passage 10 MDCK-grown viruses. V1-V7 represent the different viruses that were sequenced. Vertical bars in the figure represent the nucleotide position of mutations or insertions (indicated by a red triangle). An asterisk shows if the mutation caused an amino acid change. Deletions are shown by horizontal bars and indicate the virus number, location of the deletion and the length of the deletion, which is shown in brackets.(EPS)Click here for additional data file.S4 FigA confluent monolayer of MDCK cells was inoculated with the 2UP_PA_eGFP_sPR virus at a MOI of 1. Time (hours post-inoculation) is depicted in the upper right corner.(MOV)Click here for additional data file.S5 FigOverview of a bright-field and eGFP image from the gridded sapphire disc. The dotted area represents the section that was used for further analysis by EM.(EPS)Click here for additional data file.


Sclerotinia sclerotiorum. In this study, we report a novel mycovirus S. sclerotiorum botybirnavirus 1 (SsBRV1) that was originally isolated from the hypovirulent strain SCH941 of S. sclerotiorum. SsBRV1 has rigid spherical virions that are ∼38 nm in diameter, and three double-stranded RNA (dsRNA) segments  were packaged in the virions. dsRNA1 encodes a cap-pol fusion protein, and dsRNA2 encodes a polyprotein with unknown functions but contributes to the formation of virus particles. The dsRNA3 is dispensable and may be a satellite-like RNA of SsBRV1. Although phylogenetic analysis of the RdRp domain demonstrated that SsBRV1 is related to Botrytis porri RNA virus 1 (BpRV1) and Ustilago maydis dsRNA virus-H1, the structure proteins of SsBRV1 do not have any significant sequence similarities with other known viral proteins with the exception of those of BpRV1. SsBRV1 carrying dsRNA3 seems to have no obvious effects on the colony morphology, but can significantly reduce the growth rate and virulence of S. sclerotiorum. These findings provide new insights into the virus taxonomy, virus evolution and the interactions between SsBRV1 and the fungal hosts.A variety of mycoviruses have been found in  Sclerotinia sclerotiorum  are widely distributed in fungal groups and typically contain RNA genomes. Mycoviruses with RNA genome includes positive single-stranded RNA (+ssRNA) virus and double-stranded RNA (dsRNA) virus. Meantime, an increasing number of novel mycoviruses have been reported and showed some unique molecular and biological attributes as well as taxonomic considerations ,b. Mycovrotiorum . Mycoviroviridae . A propo1 BpRV1; . BotybirR. necatrix megabirnavirus 1 (RnMBV1), Magnaporthe oryzae chrysovirus 1 (MoCV1), B. porri RNA virus 1 (BpRV1) and S. sclerotiorum partitivirus 1 (SsPV1), lead to seriously debilitating symptoms in their fungal hosts and show great potential to control the fungal diseases  plant in Yingjing County, Sichuan Province, PR China. Strains SCH941R6 and SCH941R7 were derived from the protoplast regeneration of strain SCH941. Strain Ep-1PNA367, a virus-free strain and a single-ascospore isolate derived from Ep-1PN (S. sclerotiorum isolates were cultured on potato dextrose agar (PDA) at 20–22∘C and stored on PDA slants at 4∘C.m Ep-1PN , shows nThe growth rate and virulence test of different strains were measured as the method described by 2, 10 mM DTT, 1 mM ATP, 20U RNase inhibitors, 25% PEG4000 (W/V), and 40 U T4 RNA ligase  and incubated at 4–8∘C for 18 h. The reaction mixture was then supplemented with 600 μl of DEPC-treated double distilled water and extracted using an equal amount of chloroform. The supernatant was collected, supplemented with an equal amount of isopropanol and 0.1 amount (V/V) of 3 M NaAc solution (pH 5.2), and precipitated at -20∘C for 30 min. After centrifugation, the precipitates were collected, washed with 70% ethanol (V/V) and re-dissolved in 16 μl of DEPC-treated double distilled water. The first cDNA synthesis was conducted in a reaction mixture containing 200 U RevertAidTM Reverse Transcriptase , 50 mM Tris-HCl (pH8.0), 50 mM KCl, 4 mM MgCl2, 10 mM DTT, 1 mM dNTPs, 20 U RiboLock RNase InhibitorTM , and distilled water in a final volume of 25 μl. The mixture was incubated at 42∘C for 1 h, 50∘C for 10 min, and 65∘C for 10 min. The cDNA was amplified using the PC2 primer (5′-p-CCGAATTCCCGGGATCC-3′) complementary to the oligonucleotide PC3-T7 loop and a sequence-specific primer corresponding to the 5′-and 3′-terminal sequences of the dsRNA. Sequencing was performed through the dideoxynucleotide termination method using a Big Dye Terminator Sequencing kit  and an ABI PRISM377-96 automated sequencer (BGI).Double-stranded RNA isolation, purification, cDNA cloning, and sequencing were performed as previously described by 1. Multiple sequence alignments were performed in the M-Coffee server2 and modified using the Clustal_X software by manually removing any gaps. The best-fit model (LG+I+G+F) of protein evolution was obtained using Akaike’s information criterion (AIC) and searched using the ProtTest server3  and re-centrifuged at 210,000g and 4∘C for 1.5 h. The fractions containing virus particles were carefully collected and re-suspended using 200 μl sodium phosphate buffer . The virus particles were stained with 2% (W/V) phosphotungstic acid (PTA) and observed using a transmission electron microscope . To identify the structural proteins of SsBRV1 virions, viral particles were purified from SsBRV1-infected strain Ep-1PNA367 carrying satellite RNA and virus-free Ep-1PNA367 was used as the negative control following the same method. polypeptide mass fingerprint-mass spectrum (PMF-MS) analysis of the structural proteins was performed following a previously described method  for 30 min. The RNAs were transferred to an Amersham Hybond-N+nylon membrane  by capillary action. Digoxigenin (Dig)-labeled DNA preparation, prehybridization, and hybridization were performed as the user manuual . The probe-RNA hybrids were detected using a CDP-Star kit . Probe 1 was amplified using the specific primers BRV1S1-F (5′-CCAGGGCAGTTGCTACAGTCAT-3′) and BRV1S1-R (5′-TTGCCGCAGAGGGGAATC-3′), probe 2 was amplified using the specific primers BRV1S2-F (5′-TCCCCGAGCAACTCAAAAACC-3′), and BRV1S2-R (3′-GCACGATTTAGTGCTGCGGTTT-3′), and probe 3 was amplified using the primers BRV1-p1-F (5′-GCAATAAAAAGCACAGCCGG-3′) and BRV1-p1-R (5′-TGTTGTGTTATTTGGTATGTTGATCG-3′).Figures 1A,B). Multiple dsRNA segments with sizes ranging from 6.6 to 1.6 kbp were detected from strain SCH941. To examine whether these dsRNA segments are responsible for the hypovirulence of strain SCH941, a protoplast regeneration technique was introduced. The results showed that some protoplast regenerants were cured, and exhibited a normal phenotype and strong virulence on detached leaves of rapeseed . dsRNA isolation revealed that two dsRNA segments with sizes of 6.6 and 1.6 kbp were extracted from those cured regenerants (such as SCH941R6 and R7), whereas the other dsRNA segments were eliminated . Thus, 6.6- and 1.6 kbp-dsRNA segments may not be the hypovirulence factors of strain SCH941. Agarose gel-electrophoresed analysis further revealed that the 6.6 kbp-dsRNA bands were actually composed of two dsRNA segments (temporarily named dsRNA1 and 2) with similar size . The 1.6 kbp-dsRNA segment was temporarily named dsRNA3 in the present research.Strain SCH941 exhibits an abnormal morphology and is incapable of causing lesions on detached leaves of rapeseed . Three dsRNA segments released from purified virus particles were similar in size to those isolated directly from mycelia of strain SCH941R6 . Furthermore, the yield of dsRNA3 from virus particles was lower than those of dsRNA1 and 2, which is consistent with that in mycelia . Virus particles purified from the virus-infected Ep-1PNA367 strain were subjected to SDS-PAGE gel-electrophoresed analysis. The results showed that three major protein bands with approximate sizes of 120 kDa (p120), 100 kDa (p100), and 80 kDa (p80) were detected . These proteins were not detected in the preparations from the virus-free isogenic fungal strain following the same method as that used for the virus-infected strain . A protein with a size of 90 kDa (p90) was detected in both the virus-free and virus-infected preparations, and was thus likely a host protein co-fractionated with the virus particles .Virus particles were successfully purified from the mycelia of strain SCH941R6. Transmission electron microscope observation showed that the purified virus particles had a spherical morphology with a rough surface and 38 nm in diameter . The GC content of SsBRV1 was 49.5%. The 5′-untranslated region  and 3′-UTR (60 bp) of dsRNA1 and dsRNA2 are strictly conserved with 95 and 85% sequence identities, respectively . Moreover, a Northern blot analysis was conducted to demonstrate the conservation of the 5′ UTRs . This result also demonstrated that the yield of dsRNA1 from viral particles was much lower than that of dsRNA2 . We also identified the possible secondary structures at the UTR and found one and three contiguous stem-loop structures were detected at the 5′ UTRs and 3′ UTRs, respectively .We have cloned all the full-length cDNA sequences of dsRNAs (1–3) from strain SCH941R6. The full-length cDNA sequences for dsRNA1 (GenBank Acc. KP774592) and dsRNA2 (GenBank Acc. KP774593) of SsBRV1 were 6,457 and 5,965 bp  at the C-terminal region, which has a low level of sequence similarity with the putative RdRps of some unclassified dsRNA viruses, i.e., B. porri RNA virus 1 (BpRV1), Spissistilus festinus virus 1 (SpFV1), Circulifer tenellus virus 1 (CiTV1), and members of Totiviridae and Chrysoviridae (Supplementary Table Only one large putative open reading frame (ORF1) was detected in dsRNA1 (nt 568-6343), and this ORF encodes a putative polyprotein of 1925 amino acids (aa’s) with a mass of approximate 217 kDa. This protein, named p1, contains a conserved RNA-dependent RNA polymerase (RdRp) domain , which encodes a large polyprotein with a mass of approximate 195 kDa. This polyprotein, named p2, shows a sequence similarity with the hypothetical protein encoded by the ORF2 of BpRV1. The regions (aa position 94-1009 and 1409-1730) of p2 were significantly similar to the corresponding regions (aa position 254-1148 and 1461-1761) of the BpRV1 ORF2-encoded polyprotein with 27 and 23% identifies, respectively  was detected in the p2 protein . Multiple alignment analysis suggested that some conserved amino acid residues of the GHBP domain were identified in the p2 protein . Multiple sequence alignment also showed that there is an existence of widely conserved amino acid residues in those two regions . This property was also found for the bipartite dsRNA virus BpRV1 with a lower level of sequence similarity  binding domain . Therefore, the dsRNA3 is not indispensable for virus SsBRV1, and we hypothesized it may be a satellite-like RNA (SatlRNA) and thus named it SatlRNA.The full-length cDNA of dsRNA3 is 1647 bp in length (GenBank Acc.KP774594), and its GC content is 46.9%. No major ORFs were found in dsRNA3, whereas one small ORF was deduced to encode a peptide with a mass of 13.7 kDa . However, sequence analysis revealed that no known proteins showed a significant similarity to dsRNA3 ORF-encoded proteins. Furthermore, the potential sequence relationship between dsRNA3 and dsRNA1 or dsRNA2 was not observed. The dsRNA3 can be lost during protoplasts transfection by SsBRV1 virions using PEG-mediated method . A maximum-likelihood phylogenetic tree was constructed based on the RdRp domain. The phylogenetic tree indicated that SsBRV1 clusters with BpRV1 and a totivirus Ustilago maydis dsRNA virus-H1 (UmV-H1) to form a separated evolutionary clade . BpRV1 is a bipartite dsRNA virus while UmV-H1 is a monopartite dsRNA virus  and beet leafhopper .Multiple alignment analysis revealed that the RdRp domain of SsBRV1 contains eight conserved motifs in the C-terminus of the ORF1-encoded protein, which is widely present in dsRNA viruses , and were individually cut and subjected to PMF-MS analysis in order to identify their corresponding genes. The results showed that p120, p100, and p80 generated a total of 45, 34, and 34 peptide fragments, respectively . These results suggested that the dsRNA3 segment was eliminated in some transfectants and may be a SatlRNA . All of the SsBRV1-related strains were subjected to biological assessment. There was no significant difference in the colony morphology between the virus-free strain Ep-1PNA367 and the transfectants, regardless of the presence of SatlRNA . However, in the presence of the SatlRNA, SsBRV1 can significantly reduce the virulence and growth rate of the transfectants . There was no significant difference in the virulence and growth rate between the transfectants and the virus-free strain Ep-1PNA367 when the SatlRNA is absent . Therefore, SsBRV1 carrying the SatlRNA can confer hypovirulence to the fungal host S. sclerotiorum.Virus particles of SsBRV1 were purified from strain SCH941R6 and were used to successfully transfect the protoplasts of the virus-free strain Ep-1PNA367. SsBRV1 transfectants were confirmed by agarose gel-electrophoresed dsRNA isolation and RT-PCR detection  and also has high pathogenicity on the detached leaves of rapeseed depending on the presence of the SatlRNA . Nevertheless, BpRV1 leads to seriously debilitating symptoms in its fungal host B. porri . Additional dsRNA segments (satellite or defective) may also be present in some bipartite dsRNA viruses , but occur in that of BpRV1  of ORF1 and ORF2 are not located in the strictly conserved 5′ UTR of SsBRV1 , while those of BpRV1 were p85, p80, and p70 with sizes of 85, 80, and 70 kDa, respectively . Obviously, differences between SsBRV1 and BpRV1 in the molecular features, virus particles and biological effects may be dependent on the different fungal hosts.Although three structural proteins were identified in both SsBRV1 and BpRV1, there were also some significant differences among these proteins. In SsBRV1, the three structural proteins were p120, p100, and p80 with sizes of 120, 100, and 80 kDa . Growth hormone (GH) is a member of a large class of evolutionarily related protein hormones that have been cloned from human, mouse, rat, cow, chicken, pig, horse, and many other species  in SsBRV1 ORF2-encoded protein, which was not detected in that of BpRV1  were encoded by ORF2 and ORF1, respectively. This finding prompts us a question that how these proteins are expressed. Because the peptides fragments from p100 include almost all of those from p80, p80 may be degraded from p100 or derived from p100 via a post-translational process resembling that of the related capsid proteins of the victorivirus HvV190S; . Most moHvV190S; . UmV-H1 HvV190S; . In someHvV190S; . It appeFigures 5). Similarly, this attribute is also present in the corresponding region of BpRV1, indicating some conserved roles in these novel fungal bipartite dsRNA viruses  further indicates the separate encapsidation of each dsRNA segment. Notably, smaller genome segment of birnaviruses and picobirnaviruses is responsible for viral replication, whereas larger genome segment encodes a polypeptide including the coat protein (Supplementary Figure There are obvious differences between SsBRV1 and the typical bipartite dsRNA viruses in genome size, the possible gene expression, structural protein components. In SsBRV1, there is a conserved region between ORF1 and ORF2 that shares significant similarity (Totiviridae and some unclassified dsRNA viruses, as well as the quadripartite dsRNA viruses quadriviruses and chrysoviruses (Figure 7B; R. necatrix (Totiviridae are not a monophyletic group and have diverse evolutionary lineages. It remains difficult to clarify the evolutionary history of these phylogenetically related monopartite and multipartite dsRNA viruses.However, SsBRV1 and BpRV1 have a close relationship with monopartite dsRNA viruses, i.e., members of necatrix . Liu et Birnaviridae, Picobirnaviridae and Partitiviridae, in terms of genome organization and molecular properties but was phylogenetically related to bipartite dsRNA virus BpRV1, monopartite dsRNA viruses and quadripartite dsRNA viruses. Three structural proteins were identified and involved in the formation of the virus particles. SsBRV1 virions can successfully transfect virus-free S. sclerotiorum strain and have limited impacts on the fungal hosts. A satellite-like dsRNA was closely related with the hypovirulence of SsBRV1-infected strains. SsBRV1 may be a new member of the virus family Botybirnaviridae.We have characterized a novel bisegmented dsRNA virus SsBRV1 that is distinct from the typical bipartite dsRNA viruses, i.e., members of 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.


Newcastle disease (ND) is an OIE listed disease caused by virulent avian paramyxovirus type 1 (APMV-1) strains, which is enzootic and causes large economic losses in the poultry sector. Genotype VII and genotype IX NDV viruses were the predominant circulating genotype in China, which may possibly be responsible for disease outbreaks in chicken flocks in recent years. While ducks and geese usually have exhibited inapparent infections.In the present study, we investigate the complete genome sequence, the clinicopathological characterization and transmission of two virulent Newcastle disease viruses, SS-10 and NH-10, isolated from domestic ducks in Southern China in 2010.(112)R-R-Q-K/R-R-F(117) at the fusion protein cleavage site. Animal experiment results showed that the SS-10 virus isolated from ducks was highly pathogenic for chickens and geese, but low pathogenic for ducks. It could be detected from spleen, lung, kidney, trachea, small intestine, bursa of fabricius, thymus, pancreas and cecal tonsils, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. Moreover, it could transmit to chickens, ducks and geese by naive contact. However, the NH-10 virus isolated from ducks could infect some chickens, ducks and geese, but only caused chickens to die. Additionally, it could transmit to the naive contact chickens, ducks, and geese.F, and the complete gene sequences based on phylogenetic analysis demonstrated that SS-10 (genotype VII) and NH-10 (genotype IX) belongs to class II. The deduced amino acid sequence was The two NDV isolates exhibited different biological properties with respect to pathogenicity and transmission in chickens, ducks and geese. Therefore, no species-preference exists for chicken, duck or goose viruses and more attention should be paid to the trans-species transmission of VII NDVs between ducks, geese and chickens for the control and eradication of ND.The online version of this article (doi:10.1186/1743-422X-11-147) contains supplementary material, which is available to authorized users. However, viruses with low levels of virulence had the sequence 112 G/E-K/R-Q-G/E-R116 at the C terminus of the F2 protein and leucine at residue 117 at the N terminus of the F1protein [Newcastle disease (ND) is caused by specified viruses of the avian paramyxovirus type I APMV-1) serotype of the genus Avulavirus belonging to the family Paramyxoviridae  serotype. Lentoge1protein –9.In China, Newcastle disease was first described in 1946  and, sinIn the present study, two NDVs isolated from outbreaks in domestic ducks in China during 2010 were genotypically and pathotypically characterized. The pathogenicity and transmission of SS-10 strain and NH-10 strain were evaluated in chickens, ducks and geese, which provided experimental reference data to the integrate control and prevention of ND in waterfowl.The initial biological characterizations of the two NDV isolates, including the MDT and ICPI, are presented in Table 112RRQKRF117, while strain NH-10 contained the 112RRQRRF117, which are a molecular characteristic of virulent NDV strains. The nucleotide sequence data were deposited into the GenBank database and the accession numbers were KF219497 (SS-10) and KF219498 (NH-10). The genome of the two isolates  are 15,192 nt in length, containing a six nucleotides insertion in the 5′-noncoding region (NCR) of the nucleoprotein gene between positions 1738 and 1743 and G + C content of 46.48%, 46.72%, respectively. Additionally, nucleotide sequences of the 3′ leader and 5′ trailer regions of the two NDV isolates which have identical to length, 55 nt or 114 nt, respectively. The two NDV isolates contain mRNA edit site (UUUUUCC↓C) in positions 484 nt of the P gene.The results of virulence tests, as determined by the MDT and ICPI tests, were for the most part, in accordance with those determined by the F0 protein proteolytic cleavage site motifs (residues 112 to 117). Strain SS-10 shared the cleavage site motif According to the phylogenetic tree of F gene nt 1 to 1662) and the complete genome 1 to 15192 nt) [192 nt [1 to 1662 Pathogenicity and transmission in chickens In order to investigate the pathogenicity of these three viruses in chickens, we inoculated intranasally with 106 EID50 of SS-10 or NH-10 viruses in a volume of 200 μl in SPF chickens, respectively. Then all the chickens were observed for 14 days after infection.10EID50 and 7.5 log10EID50, respectively  as a naive contact group, which was housed with those inoculated with the SS-10 or NH-10 viruses.10EID50 to 3.42 log10EID50 in oropharyngeal swabs and 1.75 log10EID5 to 3.17 log10EID50 in cloacal swabs . The mean virus titers in the spleen, lung, kidney, brain, trachea, small intestine, bursa of fabricius, pancreas and cecal tonsils were 3.58, 2.58, 2.17, 1.58, 2.00, 2.75, 3.83, 2.17 and 2.67 log10EID50, respectively. The NH-10 virus replicated in some tested organs, including heart, lung, spleen, kidney, trachea, small intestine, bursa of fabricius, thymus, pancreas and cecal tonsils, but not the brain (Table 10EID50).No ducks died during the observation period. All the ducks seroconverted in the SS-10 and NH-10 groups Table . The SS-10EID50 and 1.89 log10EID50, respectively. In the NH-10 group, the virus titers of oropharyngeal swabs could only be detected at 3 DPI (2.17 log10EID50). The virus titers of the cloacal swabs were from 1.58 log10EID50 to 2.17 log10EID50 within 7 DPI.SS-10 and NH-10 viruses shedding from the inoculated ducks were detected in oropharyngeal and cloacal swabs at 3, 5, 7, 9 and 11 DPI. The SS-10 virus could be detected in both oropharyngeal and cloacal swabs within 3 DPI Table . The meaTo study the horizontal transmission of these two viruses, three ducks were inoculated intranasally with 0.2 ml phosphate buffered saline (PBS) as a naive contact group housed with those inoculated with the viruses previously mentioned.10EID50 to 2.25 log10EID50 within 5 DPI in oropharyngeal swabs and 1.83 log10EID5 to 2.00 log10EID50 within 7 DPI in cloacal swabs, respectively (Table 10EID50). According to these, the shedding time and virus titers of SS-10 virus were longest and highest among the two viruses.The naive contact ducks, housed with the ducks inoculated SS-10 or NH-10, experienced no deaths during the test time. SS-10 viruses shedding of the naive contact group could be detected in the oropharyngeal and the cloacal swabs. The virus titers were from 1.75 logly Table . In the In the ducks study, these results indicate that SS-10 and NH-10 viruses could infect ducks and transmit between ducks by naive contact.Pathogenicity and transmission in geese To evaluate the pathogenicity of the two viruses in geese, we inoculated intranasally with 106 EID50 of each virus in a volume of 200 μl in geese, and then observed them for 14 days after infection.10EID50 and 5.75 log10EID50, respectively  as a naive contact group, which was housed with those inoculated with the two viruses described previously.10EID50 within 5 DPI , even though there were no clinical signs or gross lesions, indicating that ducks may carry duck-origin NDV strains without overtsigns of clinical disease and are likely to shed viruses via the oral or cloacal route. Secondly, duck-origin NDV strains passaged in ducks enhanced virulence (date not shown). Finally, the two NDV viruses exhibited highly pathogenic for chickens and geese, and replicated systemically in the inoculated chickens and geese. Therefore, outbreaks by highly pathogenic NDVs can occur by the introduction of benign viruses into waterfowl followed by serial passages under natural conditions, emphasizing the importance of the host environment in the process of NDV selection.Nowadays, vaccine strains Lasota, B1 and V4 are used widely in China to control the infection of NDV. However, virulent NDV strains are still isolated frequently in the vaccinated birds , which dTo sum up, it is an urgent to take stricter biosecurity measures in poultry breeding management, in particular to avoid mixing different types of poultry rearing in order to reduce cross-species transmission. Additionally, we also strengthen immunoprophylaxis and regular monitoring of ducks, geese and other waterfowl of the NDV geographic distribution. The present investigation provides important information on the epidemiology, diagnosis and control of NDV in South China and highlights the importance of supporting surveillance in developing countries for transboundary animal diseases.In 2010, two Newcastle disease viruses Duck/CH/GD/SS/10 (SS-10) and Duck/CH/GD/NH/10 (NH-10) were isolated from cloacal swabs of apparently healthy birds in live bird markets during 2010. All viruses were propagated in 10-day-old embryonated specific-pathogen-free (SPF) chicken eggs. All experiments were carried out in animal biosafety level 3 (ABSL-3) facilities. All animal procedures performed in this study were reviewed, approved, and supervised by the Guangdong Administration Committee of Laboratory Animals under the leadership of the Guangdong Association for Science and Technology, permit number SCXK(Guangdong)2008-0019.50) of two isolates were also determined with 10-day-old chick embryos and calculated by the method of Reed and Muench [The pathogenic potential for the two isolated viruses were evaluated using standard assay methods to determine the MDT in 10-day-old chick embryos and the ICPI in 1-day-old chicks . The 50%d Muench .Viral RNA was isolated from these two NDV-infected allantoic fluids of 10-day-old SPF chicken embryos, using an RNeasy mini kit  according to manufacturer’s recommendations and transcribed into cDNA with SuperScript III reverse transcriptase (Invitrogen). Amplification reactions were performed with the one-step RT-PCR kit . PCR was performed using fragment-specific primers. A set of 12 pairs of specific primers  according to the manufacturer’s instructions. Nucleotide sequence editing, analysis, the prediction of amino acid sequences and alignments were performed using the MegAlign program in the Lasergene package . The sequences of the two isolates were compared with 62 previously reported NDV sequences representative of different genotypes available in GenBank  as naïve contact group housed with those inoculated with the SS-10 or NH-10 viruses, respectively. All chickens were observed for clinical symptoms for 14 days. On 3-day post-inoculated (DPI), three inoculated chickens in each group were euthanized to test the virus replication in organs, including heart, liver, spleen, lung, kidney, brain, trachea, small intestine, bursa of fabricius, thymus, pancreas and cecal tonsils. Similar actions were performed on chickens that had died. Oropharyngeal and cloacal swabs were taken from chickens at 3, 5, 7, 9 and 11 DPI, and suspended in 1 ml PBS. All of the tissues and swabs were collected and titrated for virus infectivity in SPF chick embryos. Seroconversion of the surviving birds on 14 DPI was confirmed by hemagglutinin inhibition (HI) test.Six-week-old SPF White Leghorn chickens were purchased from Guangdong Dahuanong and housed in the isolator cages and randomly divided into 2 groups. Six chickens of the SS-10 group were inoculated intranasally with 106 EID50 of each virus in a 0.2 ml volume. The other three ducks were inoculated intranasally with 0.2 ml PBS as a naive contact group housed with those inoculated with the SS-10 or NH-10 viruses, respectively. Three inoculated ducks in each group were euthanized to test the virus replication in organs on 3 DPI, including heart, liver, spleen, lung, kidney, brain, trachea, small intestine, bursa of fabricius, thymus, pancreas and cecal tonsils, and the six remaining ducks were observed for clinical symptoms  for 14 days. Swabs from the oropharynx and cloaca were collected for detection of viruses shedding at 3, 5, 7, 9 and 11 DPI. All of the tissues and swabs were collected and titrated for virus infectivity in SPF chick embryos. Seroconversion of the surviving birds on 14 DPI was confirmed by HI test.Eighteen two-week-old healthy Peking ducks were purchased from a duck farm in Guangzhou and housed in the isolators and randomly divided into 2 groups. Peking ducks were confirmed serologically negative for Newcastle disease by hemagglutination inhibition (HI) assays. Six of nine ducks were inoculated intranasally with 106 EID50 of each virus in a 0.2 ml volume. Three geese were inoculated with 0.2 ml PBS as a naive contact group housed with those inoculated with the SS-10 or NH-10 viruses, respectively. On 3 DPI, three inoculated geese in each group were euthanized to test the virus replication in organs, including heart, liver, spleen, lung, kidney, brain, trachea, small intestine, bursa of fabricius, thymus, pancreas and cecal tonsils. Oropharyngeal and cloacal swab specimens were collected at 3, 5, 7, 9 and 11 DPI. All of the tissues and swabs were collected and titrated for virus infectivity in SPF chick embryos. Seroconversion of the surviving birds on 14 DPI was confirmed by HI test.Two groups of two-week-old healthy domestic geese (Qingyuan geese) were purchased from a goose farm in Guangzhou and housed in isolators. Geese were confirmed serologically negative for Newcastle disease by hemagglutination inhibition (HI) assays. Six geese of each group were inoculated intranasally with 10Additional file 1: Table S1: NDV isolates and their accession numbers used in phylogenetic analysis. (DOC 93 KB)


MAPIT is a three-arm randomized controlled trial assessing in-person Motivational Interviewing (MI) condition versus computerized (MAPIT) condition versus control. Our goal is to estimate the costs and cost-effectiveness for the study. We present preliminary findings on the startup costs and the implementation costs of the two intervention conditions, MI and MAPIT. To our knowledge, there is no published study of the cost-effectiveness of MI as a pretreatment intervention in a criminal justice setting. Similarly, there is no evidence to date on the cost-effectiveness of relevant web-based interventions in a criminal justice setting. An important contribution of this study is that it will address these significant gaps in the literature.The economic perspective—which determines whose costs are included—is that of the criminal justice system. The costs of the two interventions were assumed to be in addition to the control-arm costs. Implementation costs for both interventions were estimated by tracking the time interventionist staff screened, assessed, delivered, and supported each intervention. Interventionist staff kept timesheets on the time delivering and supporting each intervention. To estimate the study protocol cost per probationer in each intervention condition, a loaded hourly cost—including space and other relevant costs—was applied to that time. Costs incurred solely for research purposes were excluded.Three preliminary findings on the average time that interventionists spent are: the time per probationer on appointment reminders for both the MI and MAPIT arms was 113 minutes; the time overseeing delivery of an MAPIT session was 21 minutes; and the time delivering an MI session was 53 minutes. The costs per protocol of MAPIT and MI were $76 and $137, respectively.Preliminary results suggest that using a computerized approach (MAPIT) to deliver the intervention is cheaper than using in-person counselors (MI). However, MAPIT costs were higher than anticipated, in part because of the relatively large amount of time spent on appointment reminders and overseeing delivery of that intervention. Before drawing any conclusions for clinical practice, it is important to combine costs and outcome data in cost-effectiveness analyses, which is a planned next step.NCT01891656


Cobalamin and folate are especially important for women of childbearing age due to their ubiquitous role in fetal growth and development. Population-based data on cobalamin and folate status are lacking from Nepal, where diets are mostly vegetarian. The objectives of the study were to investigate cobalamin and folate intake and status, and to explore associations with socio-demographics, anthropometrics, anemia, and dietary habits. Following a random selection of geographical clusters, we collected blood samples from 500 non-pregnant women and 24-h dietary recalls and food frequency questionnaires from a subsample of 379 women. Twenty percent of the women did not consume any food containing cobalamin during the days recalled, and in 72% nutritional cobalamin intake was <1 μg/day. Eighty-four percent of the women had cobalamin intake lower than the estimated average requirement (EAR) (<2 μg/day). In contrast, only 12% of the women had a folate intake less than 100 μg per day, whereas 62% had intake between 100 and 320 μg. Low plasma cobalamin (<150 pmol/L) was found in 42% of the women, most of whom (88%) also had elevated levels of methylmalonic acid. Our results indicated a high prevalence of nutritional cobalamin deficiency, while folate deficiency was uncommon. Deficiencies of cobalamin vitamin B12) and folate among women of reproductive age have received increased interest not only due to associations with neural tube defects and poor cognitive development in offspring 2 and fol,4,5,6, bCobalamin is required for DNA synthesis and metabolism of folic acid, and is important for central nervous system function and most rapidly dividing cells, including maturation of red blood cells. Similarly, folate coenzymes are essential for methionine regeneration, the shuttle of one-carbon units, and RNA and DNA metabolism, as well as protein synthesis . In humaet al. found that 60% of pregnant Nepali women attending a hospital in Kathmandu Valley had biochemical signs of cobalamin deficiency , while only 7% had low serum folate levels (<4.5 nmol/L)  3.0), with 11% having a BMI of <18.5 kg/m2 and 13% a BMI of >25 kg/m2 indicating chronic energy deficiency and overweight, respectively (The mean age of the 500 enrolled women (97 from the carpet factory stratum) was 23 years and 34% were <20 years old. Fifty-two percent of the women lived in joint families, and the mean family size was seven. The mean BMI was 21.8 kg/mectively . Two thip = 0.02). Twenty percent of the women did not consume any cobalamin during the days recalled, and about two thirds (72%) consumed cobalamin <1 μg/day. Eighty-four percent of the women had cobalamin intakes less than the EAR (<2 μg/day) . The cobalamin intake varied according to age. Median intake was 0.25 μg  among women younger than 20, while it was 0.42 μg  in women ≥20 years ( μg/day) . The disBuffalo meat, milk, and eggs were the foods that contributed most to the cobalamin supply. Analysis of the 24-h dietary recalls showed that buffalo meat provided 58% of the cobalamin intake in this population and it was recorded in 18% of the recalls . Milk, sBased on the FFQs, 40% of the women reported buffalo meat consumption at least once a week, 51% reported intake of once a month or less, and 9% reported they had not eaten buffalo meat within the past six months. Sixty-three percent reported that they had tea with milk at least once a week; among them, 41% reported at least once daily. Twenty-two percent had tea with milk once a month or less, while 15% reported not having consumed tea with milk during the last six months. One third of the women reported consumption of eggs at least once per week, while 60% consumed eggs once a month or less, and 7% had not consumed any eggs during the last six months.p ≤ 0.0005), probably reflecting the seasonal availability of different green vegetables in the local community. Similar seasonal differences were also observed in the p-folate level . Among the 394 women who completed the FFQs, 81% reported consumption of different types of green leafy vegetables, and 27% reported consuming dried vegetables at least once a week within the past six months. Sixty-one percent of the women reported consumption of pulses, and 65% had wheat flour bread at least once a week during the past six months.The median intake of folate was 211 μg . Green leafy vegetables like spinach, mustard, radish leaves, and dry leafy vegetables contributed to 54% of the total folate intake. Rice was recorded in all recalls, while 40% of the recalls reported different seasonal green leafy vegetables. Pulses or legumes were recorded in 61% of the 24-h dietary recalls. Only 12% of the women had a folate intake less than 100 μg per day, whereas 62% had intake between 100 and 320 μg. We also estimated the proportion with low intake adjusting for within-subject variability. With this adjustment, 69% had an intake lower than 320 µg/day. Folate intake during the monsoon season (May–August) was low, with a mean intake of 199 μg (SD 129) as compared with 262 μg (SD 124) in other seasons , indicating most of the women with low p-cobalamin also had a higher MMA concentration. Spearman’s rank-order correlation showed a strong positive association between Hcy and MMA (r = 0.27), but no associations with cobalamin and folate levels (r = −0.01). The plasma MMA concentration was strongly and negatively associated with p-cobalamin (r = −0.37), but only weakly with the p-folate levels (r = −0.02).Four of the 500 women had missing MMA and Hcy results, while one had missing values for p-folate and cobalamin results. The mean (SD) of Hcy, MMA, cobalamin, and folate were 10.5 μmol/L (6.1), 0.62 μmol/L (0.58), 206.0 pmol/L (188.1), and 22.4 nmol/L (17.1), respectively. 82% had elevated MMA levels (>0.26 μmol/L), while 72% had Hcy >7.5 μmol/L . Hcy valp-cobalamin concentrations. P-folate was, however, significantly lower among these women, though marginally significant among women who reported consuming meat at least once a week. Plasma MMA was significantly higher among women younger than 20 and illiterate women. Surprisingly, none of the biochemical parameters of cobalamin and folate were significantly associated with anemia.Multiple linear regression analyses  indicatep-cobalamin level , and negatively associated with MMA . A similar relationship was also observed with the folate intake and p-folate. Neither cobalamin nor folate intake was significantly associated with Hcy.Intake of cobalamin adjusted for dietary energy, as determined by the 24-h dietary recall method, was strongly and positively correlated with the et al. [In this cross-sectional study of healthy young non-pregnant women from a semi-urban community, we found a high prevalence of cobalamin deficiency as defined by plasma cobalamin <150 pmol/L, but only a few women were folate deficient. The results from the dietary recalls support our findings, as the most frequently consumed foods contained high levels of folate but low amounts of cobalamin. There are limited data on cobalamin and folate status in representative samples from low- and middle-income countries. Moreover, few studies include both biochemical analyses and dietary recall assessment of vitamin deficiencies. The available data on biochemical markers of folate and cobalamin status among women are mainly based on elderly or pregnant women ,39 and net al.  but loweet al. .versus 76% consuming <2200 Kcal also indicate possibilities of underreporting food items. Or it could be partly because we did not require dietary recall during major local festival and celebrations. Using the Indian RDA, which is 1 μg/day for cobalamin and 200 μg/day for folate [We compared our dietary intake of cobalamin and folate with the dietary reference intake values from the WHO/FAO . As Nepar folate , 72% of r folate . HoweverThe poor cobalamin status in our population may partly be explained by the traditional predominantly vegetarian diet in Nepal. Among the 775 24-h dietary recalls, only 18% reported any meat intake, which indicates that even though the majority of women were not vegetarian, meat consumption is not regular. More than half of the study population reported that they had only consumed meat 1–3 times a month or less during the past six months. Consumption of meat  at least once a week was strongly associated with improved cobalamin status. This could be one of the reasons for our finding of relatively better cobalamin status among women from carpet factories, as 59% of them reported at least weekly meat consumption compared to 36% among the local resident women. Buffalo meat constitutes about two thirds of the total meat consumption and carpet workers, in contrast to the indigenous population, also consume pork meat . Non-dieFish is a rich source of cobalamin (2.32 μg per 100 g), but less than 1% of the recalls included any fish. Fish and yogurt, however, were two foods that were associated with improved cobalamin status among urban South Indian pregnant women . The cobFolic acid supplementation is recommended primarily to prevent neural tube defects and is effective if given during the periconceptional period . In NepaResults of blood samples and dietary intake recalls from representative women indicated a high prevalence of cobalamin deficiency, while folate deficiency was uncommon. It is important to investigate the potential negative effects of cobalamin deficiency in this population, and further research should focus on the health consequences of elevated plasma Hcy and impaired cobalamin status in Nepal. Prospective studies in representative populations identifying dietary and other attributable risks of cobalamin deficiencies and elevated Hcy are warranted.