Influenza A virus is a leading causative pathogen of human acute respiratory infection. Recently, the co-circulation of pandemic (H1N1) 2009 and seasonal H3N2 viruses was reported, and sporadic cases with reassortant avian H7N9 virus are continually reported in China. We aimed to establish a multiplex one-step real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay to simultaneously detect and discriminate FluA subtypes, including human seasonal H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus, in one reaction tube.Clinical samples, including throat swabs and sputum, were collected from the patients with influenza-like illness (ILIs). Total viral RNA from each sample or viral culture was extracted, and the specific detection of FluA virus and its subtypes was performed using a multiplex rRT-PCR assay.−2 50% tissue culture infective dose (TCID50) per reaction or 4.8 × 101 copies per reaction for each virus of the three viruses. For simultaneously detecting the three viruses, the LOD was 1.8 × 10−2 TCID50 per reaction or 1.6 × 10 copies per reaction for testing the total FluA virus RNA and 5.6 × 10−2 TCID50 per reaction or 5.1 × 10 copies per reaction for the H3, H1, and H7 genes in one reaction tube. The multiplex assay specifically detected these viruses, and no cross-reaction with other pathogens was found. Moreover, the assay had reliable clinical sensitivity (100%) and valuable clinical specificity (>95%). The detection of FluA with the matrix (M) gene contributed to the further determination of these subtypes, and the Rnase P gene (RP) was considered an internal control to favourably evaluate the quality of the clinical samples.The limitation of detection (LOD) of the multiplex assay was 5.4 × 10These findings indicate that the multiplex assay can simultaneously detect and discriminate FluA subtypes with reliable sensitivity and specificity, which is required for the early clinical diagnosis and viral surveillance of patients with FluA infection. Influenza A (Flu A) virus is the most widespread pathogen causing human respiratory infections, which is historically associated with annual seasonal epidemics and influenza pandemics worldwide, such as those outbreaks in 1918, 1957, 1968, and 2009  2009 virus and the human H3N2 virus was reported, and patients infected with reassortant avian H7N9 virus have continuously increased in China  gene of 85 FluA viruses, and the primers and probes of the human H3 gene of the human H3N2 virus, the human H1 gene of the pandemic (H1N1) 2009, and the H7 gene of the human with reassortant avian H7N9 virus infection were designed based on the highly conserved regions of the HA genes , which are available in the Global Initiative on Sharing Avian Influenza Data (GISAID) and GenBank. The RP gene was used as an internal control to monitor the quality of the clinical specimen. The new primers and probes listed in Table A total of 523 clinical specimens from the patients with ILIs, including 436 throat swabs and 87 sputum specimens, were obtained from March 2014 to November 2015 at the Infection Department, the First Affiliated Hospital, College of Medicine, Zhejiang University. All specimens were kept in viral transport medium supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) at −80 °C. The sample from each patient was divided into three tubes. The first was for viral detection, the second was for viral culture, and the last was stored. Informed consent was obtained from all individuals enrolled in this study, and the Ethics Committee of our hospital approved this study.Madin-Darby canine kidney (MDCK) cells were used for isolating the FluA virus by standard methods in a biosafety level-2 (BSL-2) laboratory for human H3N2 virus and pandemic (H1N1) 2009, and a BSL-3 laboratory was used for the reassortant avian H7N9 virus. Each fresh sample was inoculated with MDCK cells. When the cytopathogenic effect (CPE) of the FluA virus infected cells was observed, the cultured cells were harvested and clarified by low speed centrifugation (8000 g/min). The harvested virus was detected by a PCR assay and certified by a sequence assay. The total viral RNA of the individual sample or the viral culture was extracted in 50 μl of the elution buffer using the QIAamp Viral RNA Mini Kit  according to the manufacturer’s protocol, and the total DNA of the pathogens was extracted in 100 μl of the elution buffer using the QIAamp DNA Mini Kit  according to the manufacturer’s protocol. The samples were stored at −70 °C.® RT-PCR reagent . The final optimised 50 μl reaction mixture consisted of 5 μl of RNA, 25 μl of 2× One Step RT-PCR Buffer, 1 μl of TaKaRa EX Taq HS (5 U/μl), 1 μl of PrimeScript RT Enzyme Mix, 1 μl of forward primer (40 μM), 1 μl of reverse primer (40 μM), 0.5 μl of probe (20 μM) for each virus, 0.8 μl of RP forward primer (40 μM), 0.8 μl of RP reverse primer (40 μM), 0.4 μl of RP probe (20 μM), and 6 μl of RNase-free water. The thermal cycling parameters were as follows: cDNA synthesis at 42 °C for 30 min; denaturation at 95 °C for 5 min; and 40 cycles at 95 °C for 10 s; and 60 °C for 45 s. The fluorescence was recorded at 60 °C on an ABI 7500 real-time PCR system .The multiplex one-step rRT-PCR assay was performed by a one-step PrimeScript50). The sensitivity of the multiplex assay was determined by testing tenfold serial dilutions of the human H3N2 virus, the pandemic (H1N1) 2009 or reassortant avian H7N9 virus, or a mixture of the three viruses. Additionally, the sensitivity of the multiplex assay was also evaluated using tenfold serial dilutions of the RNA transcripts that were transcribed in vitro by a recombinant pGEM®-Easy plasmid  and the RiboMAXTM Large Scale RNA Production System-SP6  according to the manufacturer’s protocol, as described previously  at high concentrations . First, the three viruses and their mixtures were tested by the multiplex assay. Second, each nucleic acid (or their mixtures) of the 18 pathogens was tested by the multiplex assay. Third, the multiplex assay was used to test one or the three viruses, including one (or their mixtures) of the 18 pathogens. All of the pathogens were tested in triplicate by three independent runs using the multiplex assay and the rRT-PCR assay released by WHO and our previous study .The clinical sensitivity and specificity were calculated using 523 clinical specimens that were blindly detected and compared using the multiplex assay, the referenced methods and tissue culture. For the positive, suspected or different results, the partially amplified DNA products of the positive sample were cloned into the pMD18T vector  that was transformed into The M gene and the HA gene sequences of 26 seasonal H3N2, 27 pandemic (H1N1) 2009, and 32 reassortant avian H7N9 viruses available from GISAID and GenBank were aligned and compared, respectively. A conserved region was selected to design the primer and probe sets for the M gene from these FluA subtypes, and a conserved region was selected for the H3, H1 and H7 genes. These primers-probe sets were used to test the RNA of the corresponding FluA subtype at primer concentrations of 0.2, 0.4, 0.8, 1.6, and 3.2 μM and probe concentrations ranging from 0.2 to 3.2 μM in a 50 μl reaction volume. The optimal concentration of the primers and the probe sets was 0.4 and 0.2 μM, respectively, for the M, H1, H3, H7, and RP genes (data not shown).The final optimised 50 μl reaction mixture consisted of 5 μl of RNA, 25 μl of 2× One Step RT-PCR Buffer, 1 μl of TaKaRa EX Taq HS (5 U/μl), 1 μl of PrimeScript RT Enzyme Mix, 1 μl of forward primer (40 μM) and 1 μl of reverse primer (40 μM), 0.5 μl of probe (20 μM) for each virus, 0.8 μl of RP forward primer (40 μM), 0.8 μl of RP reverse primer (40 μM), 0.4 μl of RP probe (20 μM), and 6 μl of RNase-free water.3 50% tissue culture infective dose (TCID50)/ml or 4.8 × 106 copies/ml. The limitation of detection (LOD) was 5.4 × 10−2 TCID50 per reaction or 4.8 × 10 copies per reaction for each virus subtype.The sensitivities and specificities of the multiplex rRT-PCR assay as well as those of the referenced rRT-PCR methods were evaluated by analysing the serial dilutions of the three viruses and their related RNA transcripts. In our study, the original concentration of each of the three subtypes of FluA viruses  was 5.4 × 103 50% TCID50/ml, and the original mixture of the RNA transcripts for each FluA subtype was 1.6 × 106 copies/ml. The LOD of the multiplex assay for simultaneous detecting the three mixed viruses was approximately 1.8 × 10−2 TCID50 per reaction or 1.6 × 10 copies per reaction for total FluA virus and 5.6 × 10−2 TCID50 per reaction or 5.1 × 10 copies per reaction for the H3, H1, and H7 genes in one reaction tube.For the three mixed virus specimens, the final titer of each virus in 200 μl of the mixed virus specimens was 1.8 × 104 TCID50, copies, or cells/ml), excluding the FluA gene in this study  or among the panel comprising the 18 other common respiratory pathogens at high concentrations  2009, 88 human H3N2 and 9 reassortant avian H7N9 samples. Compared with the cell culture, the analyses of the 523 samples by the multiplex rRT-PCR assay and referenced methods in parallel showed that 144 specimens were positive for FluA, including 36 specimens with pandemic (H1N1) 2009 virus, 101 specimens with H3N2 virus, and 9 specimens with reassortant avian H7N9 virus  and neuraminidase (NA)  2009 virus and the human H3N2 virus infections are reported worldwide, and sporadic cases of reassortant avian H7N9 virus infection are continuously documented in China  should be included, as certain drugs may affect the result of the detection and the tissue culture. Finally, the clinical sample population was relatively small in this study.In conclusion, a sensitive and specific multiplex one-step rRT-PCR assay was developed for the early detection of most influenza A viruses and subtypes, including pandemic (H1N1) 2009, H3N2, and reassortant avian H7N9 virus, in clinical samples from patients with ILIs. This multiplex assay will provide a rapid and easy method for clinical diagnostics and viral surveillance of influenza A and its subtypes.


In early 2015, an outbreak of an acute exanthematous illness with dengue-like symptoms occurred in northeastern Brazil. By the end of the same year, an unexpected increase in the number of cases of microcephaly was observed in the region. The microcephaly outbreak cause was unknown and rumors pointing to various potential causes arose. Since we were unaware at the time if this scenario would attract the interest of the broader scientific community, due to the neglected regions associated and as often happens with many others health conditions related to infectious diseases in Latin America. This coupled with the fact that diagnostic testing for Zika virus was not available, prompted us to design a study that could demonstrate the correlation between the development of an exanthematous illness with Zika-like symptoms during pregnancy and the delivery of a newborn with congenital microcephaly.Aedes Aegypti and the enhancement of the Zika virus infection by dengue antibodies, respectively. The limitations of this study are: (a) participants recall bias, (b) absence of laboratory test results for Zika virus and other arboviruses and (c) incomplete test results for other pathogens that could lead to microcephaly.Mothers who experienced symptoms associated with the Zika virus during pregnancy had 10 times higher odds of delivering newborns with congenital microcephaly when compared with mothers who did not exhibit Zika-like symptoms. Thus, the acute exanthematous illness outbreak could be associated with the congenital microcephaly outbreak. We could not distinguish which virus caused the acute exanthematous illness in the study subjects , but these results could help to reduce the misquided speculation in regards to the cause of the microcephaly and could have expedited public health policies intended for controlling the mosquito vector. In addition to the lower head circumference, microcephalic neonates also had lower thoracic circumference, lower height and lower weight compared to non-microcephalic babies suggesting intrauterine growth restriction. Additionally, we found borderline association between mothers classified as homemakers and, who had past dengue infections with microcephaly. Prior contraction of dengue virus seems to play a role in the risk for the condition reflecting the domestication of the The study protocol was registered at ClinicalTrial.gov under the identifier NCT02741882. Registered on April 13th, 2016The online version of this article (doi:10.1186/s13104-017-2796-1) contains supplementary material, which is available to authorized users. In early 2015, an outbreak of an acute exanthematous illness with dengue-like symptoms occurred in northeastern Brazil .In late 2015, an unexpected increase in the number of microcephaly notifications was observed in the northeastern Brazil . The cauAs with other past outbreaks in Latin American, we did not know if the microcephaly increase would attract the interest of local and international scientific communities. Moreover, at the time, a diagnostic test that could be used to identify past exposure to Zika virus was not available . Thus, wFortunately, the World Health Organization declared the microcephaly outbreak a Public Health Emergency of International concern in 2016 , therebyAs a result of all this, the impact of this study was minimized slightly due to additional, available significant data that utilized definitive diagnostic testing for Zika. However, we did find an association between the studied exposure (suspected Zika virus disease) and the outcome . These findings provide valuable insights that could be useful in assisting health care providers to estimate the risk of microcephaly by assessing clinical signs and symptoms experienced by the mother during the pregnancy which, until now, remains a major challenge. Identification of Zika virus can be difficult due to the virus’s ability to cross-react with dengue virus and other flaviviruses tests and/or when sufficient diagnostic tests for Zika are not available as is often the case in poor underserved regions.We carried out a retrospective 1:2 matched case–control study among parturients admitted at the public maternity hospital, “Nossa Senhora de Lourdes”, located in Aracaju, in the state of Sergipe, on the northeast coast of Brazil. The aim of the study was to identify a correlation between the development of an acute exanthematous illness displaying Zika-like symptoms and pregnancies which resulted in the delivery of a new baby born with congenital microcephaly.From September 1st, 2015 to January 5th, 2016, the maternity hospital reported to Brazil’s ministry of healthy that 64 newborn babies were delivered with probable congenital microcephaly. The mothers of those babies were eligible to be analyzed as cases. Mothers from the same maternity whom delivered newborns without the condition were eligible to be a part of the control group.Maternal/neonate data was obtained from the medical records. The exclusion criteria were: neonates with head circumference in the normal range for the gestational age according to WHO guidelines , mother Of the 64 neonates born with probable congenital microcephaly, 21 had head circumferences in the normal range and one was diagnosis with Seckel syndrome. Two mothers had tested positive for syphilis (VDRL) and one for toxoplasmosis (IgM) during pregnancy. Three neonates/mothers had medical records that were substantially incomplete. All of them were excluded. For the included subjects, data for syphilis and human immunodeficiency virus were complete. However, data for toxoplasmosis, cytomegalovirus and rubella were partially complete , epidemiological week of the delivery (±4 weeks) and gestational age at birth (±2 weeks). The rationale for the matching was to compare women who had pregnancies in nearby places, during the same period of the year and of similar durations Fig. . The majhttp://www.lee.dante.br) considering a matched case–control design, two controls per case, α = 0.05 (two sided) and β = 0.8. According to the sample size calculation, 12 cases and 24 controls were required (12 trios of one case and two controls).For sample size calculation, we considered that 38% of the individuals exposed to Zika virus (IgM positive)  and a muFrom March 15th, 2016 to June 5th, 2016, two pediatricians applied telephone questionnaires to mothers in order to obtain information about the acute signs and symptoms experienced during pregnancy, along with other relevant information . Statistical analyses were performed using SAS software (version 9.4) and GraphPad Prism Software (version 6.0).The variables retrieved from medical records, and included in the questionnaire are presented in Table Results demonstrated that mothers who delivered neonates with congenital microcephaly were more likely to have experienced rash (mainly maculopapular), fever, arthralgia, periarticular edema, headache and fatigue/malaise compared with controls. In addition to the lower head circumference, microcephalic neonates also had lower thoracic circumference, lower heights and lower weights compared to non-microcephalic babies  cases versus 3 out of 38 (7.9%) controls met the study’s case definition. Therefore, the odds ratio for suspected Zika virus during pregnancy and subsequently giving birth to a neonate with congenital microcephaly was 9.28 . Mother occupation and past infection by dengue virus showed borderline significance in the univariate analysis . After the adjustment of these variables, the odds ratio remained significant  , fever, arthralgia, periarticular edema, headache and fatigue/malaise]. Taken together, they are similar to the suspected Zika-virus case definition considered for the study. These findings suggest that the risk of congenital microcephaly could be predicted by analyzing these clinical signs and symptoms. Because Zika virus diagnosis is not readily available, as is often the case in poor underdeveloped regions, or can be a significant challenge: (a) the RT-qPCR assay has a limited detection window because the virus is present for 11–17 days in the blood ; (b) then humans .On the other hand, as the present study was designed to locate the association between the suspected Zika virus during pregnancy and the delivery of a newborn with congenital microcephaly, further research with more statistical power is needed to draw definitive conclusions for the other signs, symptoms and exposures described in Table Because Zika, dengue and chikungunya viruses cause diseases with similar clinical symptoms, and they have circulated in northeastern Brazil in 2015, we could not distinguish precisely which one caused the acute exanthematous illness in the study subjects. However, evidence suggests that Zika virus was the likeliest etiology for the exanthematous illness outbreak. A report including 77 samples from patients with acute exanthematous illness collected in Tuparetama, Pernambuco, during the 2015 outbreak revealed that Zika virus was present in 40.2%, dengue virus in 11.7% and chikungunya virus 1.2% and coinfection of Zika and dengue viruses was also reported in 2.6% . AdditioIn conclusion, the objective of the study was reached; the acute exanthematous illness outbreak was associated with the congenital microcephaly outbreak. This knowledge could have helped to limit some of the misquided speculation and could have expedited public health policies more effectively targeting the mosquito vector. A deeper understanding of the specific microcephaly cause would be a next step.The limitations of the present study were: (a) participants recall bias, (b) absence of laboratory test results for Zika virus and other arboviruses and (c) incomplete test results for other pathogens that could lead to microcephaly. Regarding the participants recall bias, the use of “blind” surveys is considered the most effective device to reduce potential bias. Neither the mothers or the interviewers knew who was a member of the case or control groups . The absAdditional file 1: Table S1. Pregnancy data regarding signs and simptoms suggestive of zika virus infection for the 19 cases (or their neonates) included in the study.Additional file 2. Questionnaire.


We present an optimised metagenomics method for detection and characterisation of all virus types including single and double stranded DNA/RNA and enveloped and non-enveloped viruses. Initial evaluation included both spiked and non-spiked bird faecal samples as well as non-spiked human faecal samples. From the non-spiked bird samples  we detected 21 viruses, and we also present a summary of a few viruses detected in human faecal samples. We then present a detailed analysis of selected virus sequences in the avian samples that were somewhat similar to known viruses, and had good quality (Q20 or higher) and quantity of next-generation sequencing reads, and was of interest from a virological point of view, for example, avian coronavirus and avian paramyxovirus 6. Some of these viruses were closely related to known viruses while others were more distantly related with 70% or less identity to currently known/sequenced viruses. Besides detecting viruses, the technique also allowed the characterisation of host mitochondrial DNA present and thus identifying host species, while ribosomal RNA sequences provided insight into the “ribosomal activity microbiome”; of gut parasites; and of food eaten such as plants or insects, which we correlated to non-avian host associated viruses. The objectives of the physical preparation of samples for metagenomic sequencing of viruses are (i) to eliminate as many nucleic acids from the host and other elements like bacteria, fungi, parasites and, (ii) to ensure that as much of the virus nucleic acids are retained during the entire process and (iii) to generate good quality NGS reads.Detection and characterisation of viruses by metagenomics (VM) is a relatively new technique that takes advantage of the sensitivity of next-generation sequencing (NGS) while being broadly non-specific for any particular virus present in a given sample. Virus communities (viromes) are structurally and functionally diverse and distinct to habitats in hosts and environments, and prior to VM there was limited understanding of the virome present in human and non-human hosts. Now with the growing use of VM, information on eukaryotic viruses, prokaryotic viruses and even viruses that infect other viruses are increasing10. Among such proposed protocols, one designated NetoVIR by Conceição-Neto, N et al.6 was optimised on mock communities and later tested on actual and complex biological samples by the same research group13. The latter testing was performed without spiking the biological samples with known viruses, which would have served as a useful positive control to validate the protocol. As the overall aim of our project was to detect and characterise viruses in faecal samples from wild birds in Australia, we initially evaluated and compared the existing protocol (NetoVIR), including some additional modifications, and determined the optimum procedure for robust virus detection and characterisation in spiked and non-spiked bird faecal samples. Furthermore, we adopted the protocol downstream of the nucleic acid purification for library preparation and sequencing on the Ion Torrent platform. Using the optimised method, we identified viruses present in selected faecal samples from a Muscovy duck (designated as MUD) and a group of Pacific black ducks (designated as MAD) and from selected human faecal samples to show the broad applicability of the method. Finally, we demonstrate that additional information can be extracted from the sequencing reads, e.g. derived from mitochondrial DNA and ribosomal RNA present in the samples, allowing identification of host species, parasites present, food eaten and faecal bacterial populations present.Different approaches have been applied based on, for example, the sample type and the sequencing platforms usedSemi-quantitative assays for all marker viruses (see Table 14) was not retained during the virus enrichment process.The spiked MUD faecal sample was subjected to all the enrichment variations  and one virus from MUD (segment 1 of Hubei chryso-like virus 1) faecal samples are given in Table Paramyxoviridae, with individual reads being 95–98% identical to AB759118 (APMV-6 red-necked stint-Japan-2008)15. We assembled several areas of good coverage including 459 nucleotides of the polymerase gene (coverage 9–192), 1839 nucleotides covering the complete hemagglutinin-neuraminidase (HN) gene (coverage 5–103) and 651 nucleotides of the fusion (F) gene (coverage 2–8). For the polymerase gene, the similarity was 447/459 (97.3%) and 444/459 (96.7%) to AB759118 (APMV-6 red-necked stint-Japan-2008)15 and GQ406232 16, but only around 75% similar to the other known lineage of APMV-6, the so-called Hong Kong 1977 lineage17 . At the amino acid level, the 357 amino acids of the NSP12 RdRp gene had 353/357 amino acids (98.8%) and 326–346/357 amino acids (91.3–96.9%) identity to KM454473 and to other related gammacoronaviruses, respectively. Thus, a gammacoronavirus was detected in the Pacific black ducks which is somewhat related to a duck gammacoronavirus from China with a similarity at the nucleotide level of more than 96% in the NSP12 RdRp gene, but only 89–92% in the Spike gene.For the NSP12 RdRp gene, our sequence had 1030/1071 nucleotides (96.1%) match to KM454473 and 886–917/1071 nucleotides (82.7–95.6%) match to other related gammacoronaviruses. For the Spike gene, the similarity for the area of 524 nucleotides was 470/524 nucleotides (89.6%) and 365–381/524 nucleotides (69.6–72.7%) and for the area of 469 nucleotides, the similarity was 431/469 nucleotides (91.8%) and 336–343/469 nucleotides (71.6–73.1%) when aligned to KM454473 and other related gammacoronaviruses, respectively 19. A 201-nucleotide segment of the spike (S) gene was assembled. This had 81.5% (164/201) similarity to wigeon deltacoronavirus and 69.6–74.1% (140–149/201) similarity to the next nearest deltacoronaviruses  had high-quality reads (Q20 or higher) with individual reads being 82–89% identical to JQ065048; a wigeon deltacoronavirus isolate from Hong Kong collected in 2008Two large contigs of 14822 and 8322 nucleotides and two smaller contigs spanning parts of the deltacoronavirus Orf1ab replicase gene and the S1-S2 (Spike) and E, M and NS6 genes were obtained from the assemblerSPAdes analysis, and the NGS reads mapped to these contigs using the TMAP assembler to determine the coverage depth. From further analysis, we initially selected an area of 1131 nucleotides (coverage 290–1700) from the Spike gene and 2121 nucleotides (coverage 39–1266) from the Orf1ab gene which overlaps with NSP7, NSP8, NSP9 and NSP10. These sequences were then analysed using MEGA and aligned with ClustalW by codons. For the selected Spike gene segment alignment, the deltacoronavirus sequence had a 738/1131 nucleotides (65.2%) and 695–710/1131 nucleotides (61.4–62.7%) match to JQ065048 and other related deltacoronaviruses, respectively. At the amino acid level, this segment had a 277/376 amino acids (73.7%), and 227–248/376 amino acids (61.5–66.0%) match to JQ065048 and other related deltacoronaviruses, respectively , dividing areas into open reading frames . For all selected areas, the Wigeon coronavirus JQ065058 was the closest related sequence, and the percentage identities to the Wigeon and other related deltacoronavirus sequences at the nucleotide and amino acid levels are shown in Table Adenoviridae and approximately 75–86% identical to goose adenovirus 4 (JF510462) from Hungary20 and duck adenovirus 2 (KR135164) from China21 was found in the MAD faecal sample. The assembled sequences were analysed further including a 279 nucleotides area of the IVa2 protein coding region , a 177 nucleotides area of the IU34_gp11 gene  and a 114 nucleotides area of the IU34_gp21 gene . We found the similarity of assembled sequences to be 229/279 nucleotides (82%) match to the GoA4 and 210/279 nucleotides (75.26%) match to the DuA2 [Fig. [Figures A dsDNA virus belonging to the family uA2 Fig. , 99/114 rus 4 JF5462 from irus 2 KR5164 fromParvoviridae was identified in the MAD faecal sample. This virus was found to be related to a duck Adeno-associated virus (KX583629) from China21 with individual reads being 84–93% identical to KX583629. Several regions of this virus including 94-nucleotides area of the rep protein , 279-nucleotides area of the capsid protein  and a 142-nucleotides area of the capsid protein  were assembled. We matched these regions to KX583629 and other adeno-associated viruses respectively. We found the 94-nucleotides area of rep protein had 88/94 nucleotides (93.6%), the 279-nucleotides area of the capsid protein had 250/279 nucleotides (89.6%), and the 124-nucleotides area of the capsid protein had 120/142 nucleotides (84.5%) match to the adeno-associated virus from China. Thus, in our analysis, we found the adeno-associated virus from the Pacific black ducks from Australia to be somewhat related to the duck adeno-associated virus from China21. Representative phylogenetic trees generated using the consensus sequences are shown in Figures A ssDNA virus belonging to the family us KX58369 from Ch22 and Rotavirus G pigeon segment 1 and 8 (KC876010 and KC876006 respectively) isolated from China23. Rotavirus G is a segmented, dsRNA virus belonging to the Reoviridae family. For this virus, good coverage of 3 or higher with a mapping quality threshold of 20 or higher was not obtained. To the best of our knowledge, these sequences are the first assembled reads of a Rotavirus G from Australia. Hence, to undergo a phylogenetic analysis of the virus, the mapping quality threshold was dropped to 10 or higher to get coverage of 3 and above. Three regions each from segment 1, 3/4 and 8 with individual areas being a 230-nucleotides area of the VP1 protein of segment 1 (coverage 3–14), a 162-nucleotides area of the VP3 protein of segment 3/4 (coverage 3–7) and a 149-nucleotides area of the NSP2 protein of segment 8 (coverage 3–6) were analysed. The 230-nucleotides area of the VP1 protein had a 186/230 nucleotides (80.8%) match to segment 1 of KC876010 Rotavirus G pigeon from China, the 162 nucleotides area of the VP3 protein had a 142/162 nucleotides (87.6%) match to segment 3/4 for Rotavirus G chicken from Germany and the 149-nucleotides area of the NSP2 protein had a 130/149 nucleotides (87.2%) match to segment 8 of Rotavirus G pigeon from China [Figures The faecal sample from the Pacific black ducks had good quality individual reads (Q32 or higher) with 80–89% identity to Rotavirus G chicken segment 2 (NC021580) and 4 (JQ920005)  isolated from Germany1 and recently also identified in mosquitoes from Western Australia24 were found in the Muscovy duck (MUD) sample. We assembled several areas of good quality reads (Q20 or higher) for segment 1 including a 1496-nucleotides area of the polymerase gene (coverage 4–58) with 1494/1496 nucleotides (99.8%) match to segment 1 of Hubei chryso-like virus 1  from Western Australia24 and 1458/1496 nucleotides (97.4%) match to segment 1 of the Hubei chryso-like virus 1 from China1 (KX882962)  and a 275-nucleotides area (coverage 5–30) and one region from the membrane protein encoding region of 456-nucleotides area (coverage 3–19) were analysed. The polymerase sequences were found to have a 529/531 nucleotides (99.6%), and a 271/275 nucleotides (98.5%) match to the Culex Negev-like virus 3 from Western Australia (MF176277). The membrane protein encoding region had a 451/456 nucleotides (98.9%) match to the Culex Negev-like virus 3 from Western Australia (MF176277). Our sequences were also found to be somewhat closely related to Biggievirus  as evident from the phylogenetic tree given in Figures The MUD sample had good quality reads (Q32 or higher) that were assembled and found to be closely related to Culex Negev-like virus 3 which is a ssRNA virus detected in mosquitoes from Western Australia (MF176277)1 and not closely related to any other hitherto sequenced viruses, our sequences only resemble that from China. From the polymerase gene of the virus, partial gene sequences that were assembled from the NGS dataset including a 240-nucleotides area , a 184-nucleotides area  and a 117-nucleotides area  were analysed. We found that the assembled sequences of the polymerase gene had a 227/240 nucleotides (94.5%) match, a 161/184 nucleotides (87.5%) match and a 111/117 nucleotides (94.8%) match to the Hubei reo-like virus 7 (KX884635) isolated from China [Figures From the MUD faecal sample, virus sequences with similarity to the Hubei reo-like virus 7, an unclassified dsRNA virus was assembled. We were able to generate good quality reads (Q20 or higher) distributed throughout the genome. As this virus is a relatively newly characterised virus from mosquitoes in China25, 270/277 (97.5%) to enterobacteria phage ECGD1 (KU522583) and between 210–223/277 (75.8–80.5%) similar to other Escherichia and Salmonella phages  identical to enterobacteria phage phi92 (FR775895)To demonstrate that the optimised virus enrichment and NGS sequencing method can be utilised on other samples, we analysed human stool samples collected from 3 children aged 3 months (sample ST4), 18 months (ST5) and 4 years (ST6). The same protocol was followed (as for the duck samples), however, the analysis of NGS reads was restricted  to virus sequences that only included human single-stranded (ss) DNA viruses (32 human ssDNA virus species selected from NCBI on 7 Aug 2017), as well as known picornavirus reference sequences (180 picornavirus reference sequences downloaded from NCBI on 4 Aug 2017) in the NCBI databases.26, respectively. Sample ST6 had a single high-quality read  with 198/208 (95.1%) nucleotide match to the hypothetical capsid coding region of MF118166 human faecal virus Tarto27. Representative phylogenetic trees are presented in Figures 2.Samples ST4 and ST5 had good quality reads (Q10 or higher and coverage of 10–15) to a 116 nucleotide region of the 5′ untranslated region of torque teno mini virus. ST4 had a 113/116 (97.4%) and 111/116 (95.6%) match to KM259873 torque teno mini virus ALA22, and KT163899 torque teno virus isolate P13–4, respectively; and ST5 had 112/116 (96.8%) and 110/116 (94.8%) match to KT163899 torque teno virus isolate P13-4 and KM259873 torque teno mini virus ALA2228, EU681176 cardiovirus D/VI2229/200429 and JN652233 Saffold virus strain S19, respectively. This sample, ST6, also had good reads (Q10 and above and coverage of 3–18) with 296/422 (70.1%), 405/422 (95.9%) and 403/422 (95.4%) nucleotide match to the VP3 region and 150/261 (57.4%), 250/261 (95.7%) and 251/261 (96.1%) nucleotide match to the VP1 region of AB747252 Saffold virus isolate Pak-3290, EU681176 cardiovirus D/VI2229/2004 and JN652233 Saffold virus strain S19, respectively. Taken together this indicates that a Saffold/cardiovirus is present in sample ST6 although recombination among strains appears to be part of its ancestry. The representative phylogenetic trees are shown in Supplementary material Sample ST4 had reasonable quality reads (Q8 or higher and coverage of 5–6) with 157/163 (96.3%) nucleotide match to KY983589 human rhinovirus C11 strain SC3107. Sample ST6 had good quality reads (Q10 or higher and a coverage of 2–4) with 204/212 (96.2%), 185/212 (87.2%) and 184/212 (86.7%) nucleotide match to the 2 C region of AB747252 Saffold virus isolate Pak-3290From the contigs, a few larger contigs were selected that by BLASTN alignment contained sequences with a high degree of identity to human viruses or known bacteriophages. Contigs and closely related sequences in the NCBI databases were then used to map back the NGS reads using the TMAP assembler. Assembled BAM files were then analysed using IGV and areas with reasonable coverage and a Q value at 20 or higher selected for further analysis of consensus sequences by using BLASTN and BLASTX.30 and 880/909 nucleotides match (96.8%) to U53923, the VP4 gene from a bovine rotavirus isolated in the USA31 more than 20 years ago. It should be noted that the rotavirus sequence in our sample was almost certainly from the vaccine virus although at one position the GU565088 sequence in NCBI reportedly has a G nucleotide while our sequence had 12 reads (55%) with a G in this position and 10 reads with an A indicating a mixed nucleotide at this position albeit with a slight G nucleotide majority. Given that the RotaTeq vaccine is used in infants in Australia from 2 months of age, and the high genetic similarity to the vaccine virus VP4 gene, it is most likely that the detected rotavirus was a vaccine virus.From sample ST4 a sequence of 909 nucleotides in length was assembled with a coverage of 3–52 reads and a close nucleotide match to rotavirus sequences in the NCBI databases, e.g. 908–909/909 nucleotides match (99.9–100%) to GU565088, the VP4 gene of a reassortant rotavirus vaccine strain (USA RotaTeq)32.From sample ST5 we were able to assemble and map a sequence of 7044 nucleotides in length with coverage of 2–189 and a nucleotide match to norovirus sequences in the NCBI databases. It was found to have 7027/7044 nucleotides match (99.7%) to MG002634 and MG002632 Norovirus from Queensland, Australia in 2017 while showing 6743–6746/7044 nucleotides match (95.7%) to JX459901 and JX459904 (and covering 94.4% of the full norovirus genome), a GII norovirus from New South Wales, Australia in 2011We also did phylogenetic inferences of these sequence datasets by Maximum Likelihood (ML) phylogenetic analyses using the MEGA 6/7 software, and the representative phylogenetic trees are shown in Supplementary material In addition to virus enrichment, the optimised method also, (i) preserves a minor amount of host mitochondria/mitochondrial DNA, and, (ii) enriches for other small stable structures resistant or partially resistant to nucleases, such as, e.g. ribosomes. Thus, further scrutiny of the obtained NGS reads outside looking for viruses, can provide valuable information.Cairina moschata) there were good quality reads with a coverage of 23–66 and a 114/114 (100%) nucleotides match to the Cairina moschata mitochondrial COX1 gene (FJ808630)33 and only 104/114 (91.2%) to Anas platyrhynchos COX1 gene (KT803699) while the samples from the Pacific black ducks (coverage of 67–192) had a 114/114 (100%) nucleotides match to Anas platyrhynchos COX1 gene (KT803699) and only 104/114 (91.2%) to the Cairina moschata COX1 gene (FJ808630). For the mitochondrial cytochrome B gene (coverage 16–114), the samples from the Muscovy duck (Cairina moschata) had a 996/996 (100%) match to Cairina moschata (EU755254)34 but only around 89% match to Anas platyrhynchos while the samples from the Pacific black ducks had a 1012/1012 (100%) match to Anas platyrhynchos cytochrome B gene (KJ883269)35 and only around 88% to Cairina moschata. Consequently, the results “mined” from the NGS results supported the species of the birds sampled noting that Pacific black ducks (Anas superciliosa) are genotypically similar to the mallard (Anas platyrhynchos) or the bird that we sampled could be a cross-breed36.In the faecal samples from the Muscovy duck  and 176/177 ((99.4%) nucleotides match to Culex (quinquefasciatus) partial 28 S RNA (KY087523), while no such matches could be found in the Pacific black ducks sample that in contrast had many reads mapping to the 28 S RNA of tapeworms and other cestodes with, e.g. 167/167 (100%) nucleotides matches. Other reads could also be mapped to the 28 S ribosomal RNA of plants such as, e.g. triticum, sorghum, Oryza and mays. The finding of a low number of reads mapping to the ribosomal RNA of Culex mosquitoes is consistent with the abundant finding of Culex mosquito viruses in the Cairina moschata (MUD) sample and as no reads could be mapped to the Culex mitochondrial COX1 gene, indicate that the mosquito-associated viruses detected in the Cairina moschata faecal sample came from mosquitoes being eaten and going through the intestinal tract  rather than from potential mosquito contamination of the faecal sample before collection.Consensus reads from the The analysis of the NGS data using the ThermoFisher Ion Reporter microbiome software provided insight into the active bacterial communities present in collected faecal samples although did not necessarily correlate with results obtained by a conventional DNA extraction 16SrRNA microbiome diversity profiling (data not shown). Furthermore, an overall analysis of repeated samples indicated that while detection of viruses was very robust over time, the “ribosomal activity microbiome” is susceptible to freeze-thawing with the ratio of reads mapped to Gram-negative bacteria (particularly phylum Bacteroidetes) decreasing when compared to Gram-positive bacteria (particularly phylum Firmicutes).38. The use of marker viruses representing the different virus types enabled us to evaluate and optimise a metagenomics method suitable for detecting and characterising viruses from complex biological samples, in this study faecal samples.The optimum protocol for the metagenomics detection and characterisation of viruses from a given sample should allow detection of all types of viruses, including single and double stranded RNA/DNA, circular, linear or segmented genome viruses; enveloped and non-enveloped viruses; and small and large viruses present. To establish such a protocol for the enrichment, detection and characterisation of a maximum number of viruses in complex biological samples such as faecal samples, we used a method previously described by others on a mock community called NetoVIR as the starting point and tested different enrichment techniques using various biophysical methods6 itself, also produced good quality NGS reads for the viruses, although in lower amounts compared to variation D. Careful homogenization was significant for the homogeneous distribution of virus particles in the samples. Initial centrifugation and filtration using the 0.8 µm PES filter as described in NetoVIR removed larger particles while virus particles and some small particles such as ribosomes and some mitochondria were not filtered out and were subsequently spun down and concentrated using ultracentrifugation and thus enriching the sample. Harsh nuclease treatment ensured the reduction of free nucleic acids and other less protected nucleic acids. Finally, the nucleic acid extraction method did allow the extraction of genomes of both DNA and RNA viruses from the sample.The main aim was to minimise host and non-virus RNA/DNA and maintain as much virus RNA/DNA as possible to optimise subsequent random amplification of nucleic acids followed by NGS detection and characterisation. The method which maintained a maximum number of viruses and obtained high-quality and a higher number of NGS reads were found to be variation D; equivalent to the NetoVIR method with an added ultracentrifugation step to further concentrate and enrich for particles resistant to nucleases. However, variation C, or NetoVIR6 the authors used chloroform, correlate well with how the mock communities reacted to these enrichment techniques.Although detergent treatment may enhance enrichment of certain viruses, it was found to be disadvantageous for some enveloped viruses and thus not incorporated in the final protocol. We assume that the detergent disrupted the structure of the enveloped, non-inner capsid containing virus particles causing the nuclease to digest the virus nucleic acids. Filtration using 0.45 µm filters might also have negatively affected the virus enrichment process, as we were not able to produce many good reads for the marker viruses BTV, BVDV and IBV. Both our filtration results using 0.45 µm filtration and the detergent results, although in the NetoVIR study6, we used the SeqPlex RNA amplification kit instead of the Whole Transcriptome Amplification Kit 2  for cDNA synthesis, amplification and primer removal. While library preparation and NGS was carried out using Nextera XT DNA Library Preparation kit (Illumina) and HiSeq™ 2500 platform (Illumina) in NetoVIR6, we used Ion Plus Fragment Library Kit (Thermo Fisher Scientific) and Ion Torrent S5XL System (Thermo Fisher Scientific). However, a recent study has shown that the selection of different sequencing platforms and corresponding library preparation methods have a minimal impact on viral metagenomes39.Unlike NetoVIRWhile we detected and characterised 9 avian host associated viruses in the MAD faecal sample, it is noteworthy that no avian viruses were detected in the MUD faecal sample. We speculate that this is because of the age of the bird (a mature older bird of unknown age as compared to the MAD samples being from juveniles) and it, despite being a wild bird living outside, was from an urban-like environment with less interaction with many other wild birds.40. The virus seems to be non-pathogenic to chickens but can cause mild respiratory disease and a drop in egg production in turkeys41. While only distantly related to the Hong Kong lineage of APMV6 mentioned above, the APMV6 detected in the MAD faecal sample is around 95–98% identical to another lineage of APMV6 isolated from Japan15 and Italy16 in the year 2008 and 2007, respectively. Interestingly, the detection of the genetically similar APMV6 in Japan was from a red-necked stint , a migratory bird species which breeds in the Arctic, but migrates in the non-breeding season as far south as New Zealand and Southern Australia including the location from which the APMV6 from this study was collected. The fusion protein of the paramyxoviruses directs the membrane fusion42 and paramyxoviruses where the fusion protein cleavage site does not possess the furin recognition site (RXK/RR-F) are usually limited to the respiratory tract, and hence the sequence at this site contributes to the virulence of these viruses43. The sequences of the fusion protein in our APMV6 from the MAD faecal sample has an additional basic amino acid around the fusion protein cleavage site (REPR-L) characteristic for the Japan/Italy 2007/2008 lineage as compared to the Hong Kong lineage of APMV-617 and hence any further changes at this site could potentially make the virus highly virulent.Avian paramyxovirus 6 (APMV6) was first isolated in 1977 from a healthy duck at a domestic duck farm in Hong Kong and considered as the prototype strain for the entire serotypeCoronaviridae, have been previously detected in wild birds in other parts of the world44. Avian coronaviruses frequently cause severe infections in poultry in many countries45. Coronaviruses have also been associated with inter-species spill over with recent examples being Severe acute respiratory syndrome coronavirus and Middle Eastern respiratory syndrome coronavirus. The gammacoronavirus detected in the MAD faecal sample was more closely related to the duck gammacoronavirus from China in 201418 than to the other gammacoronavirus sequences available. While we observed more than 96% identity in the Orf1ab polyprotein gene, the Spike gene identity was only 89–92% to the duck gammacoronavirus. For the deltacoronavirus detected in the MAD sample, the closest relative was the wigeon deltacoronavirus from Hong Kong collected in 200819. The Spike protein of the coronaviruses mediates the entry of the virus particles into cells, and mutations in or recombination of this region are important for host species specificity and potential cross-species transmissibility of the virus43. These are the first wild bird coronaviruses from Australia to have their spike genes sequenced and further analysis to understand the relationship between wild bird coronaviruses is needed. A detailed study of the prevalence of coronaviruses among Australian wild birds (including the MAD sample) using PCR amplification of a small fragment of the Orf1ab gene with a detailed description and phylogenetic analysis of a small fragment of around 300 nucleotides is described elsewhere46. However, while that study did detect both gamma and deltacoronaviruses, the PCRs included were not able to detect coinfections (i.e. birds having both a gamma and a deltacoronavirus) while it is worth mentioning that such coinfection can be readily detected using our metagenomics method as evident from the NGS data generated in the present study.Gammacoronaviruses and deltacoronaviruses both from the family 20. Avian adenoviruses have great variability in their pathogenesis and virulence. The goose adenovirus 4 has been associated with hepatitis and hydropericardium20. The virus related to adeno-associated (parvo) virus from the Pacific black ducks was somewhat related to a duck adeno-associated virus from China in 2015. It is currently unclear whether the adeno-associated (parvo) virus detected in the MAD sample can replicate without helper virus or not, however, we consider it likely that the adenovirus (related to the DuA2 and GoA4 mentioned above) detected in the same sample, may act as a helper virus for enhanced replication as adeno-associated parvoviruses often need a helper virus for its replication47. As both viruses are present simultaneously and possibly enhance replication of the parvovirus present, this co-infection may be associated with disease in the host although this is currently speculative and will require further studies before any conclusion can be reached as to its significance in regards to the health of infected birds.In the MAD faecal sample, we also characterised two DNA viruses, one a virus related to DuA2 and/or GoA4 virus and another virus related to an adeno-associated (parvo) virus. The consensus sequences of the adenovirus that we assembled were related (75–86%) to both the DuA2 and GoA4 virus. The DuA2 (KR135164) was characterised in China in the year 2014 and found to be closely related to the GoA4 (JF510462) isolated from Hungary48. Although infection with this particular serotype has not been considered fatal, infection from another rotavirus serotype such as Rotavirus A can be lethal to the birds in extreme cases50. Even though rotavirus infections in avian species are associated with diarrhoea, cross-species transmission of the avian rotaviruses is usually very rare49. To the best of our knowledge, our sequences are the first assembled reads of a Rotavirus G from Australia and from a duck sample, which could explain why we had to drop the mapping quality threshold to 10 to get a coverage of 3 and above for the generation of consensus sequences. Nevertheless, even then, we were only able to generate consensus sequences for only segments 1, 3/4 and 8. We believe the sequences for one segment that matched to segment 4 of the NCBI reference (JQ920005) isolated from Germany22 is in reality segment 3 as it encodes for the VP3 protein of the rotavirus, which universally is accepted to be part of segment 3. Nevertheless, it appears that the rotavirus detected in the MAD sample is a new rotavirus and may have included reassortment in its evolutionary history as we were only able to map 3 of the gene segments and they, in turn, mapped most closely to different reference sequences.Rotavirus G has also been previously found in birdsPicornaviridae. The avian viruses in this virus family have been associated with different diseases such as duck viral hepatitis, turkey viral hepatitis and avian encephalomyelitis51. Avian encephalomyelitis virus is a highly infective virus to bird species such as chickens, partridge, turkey, quail, guineafowl and pheasants. They can severely affect the neuro-system and has high mortality rates49. However, the pathogenicity of avian calicivirus is still unclear although it has been isolated from chickens and turkeys having various diseases like the runting and stunting syndrome and enteric diseases53. The actual pathogenicity, if any, of the related viruses here detected at very low levels in the MAD sample is currently unknown.Other avian host associated viruses we identified in the Pacific black ducks sample (MAD) are related to avian megrivirus, avian encephalomyelitis virus and avian calicivirus. Among them, the avian megrivirus and avian encephalomyelitis virus belong to the virus family Culex mosquito in the sample supports this speculation. Ngewotan virus (Nam Dinh like virus), Hubei chryso-like virus 1, Culex Negev-like virus 3 (Biggie/Goutanap virus-like) and a virus related to Hubei reo-like virus 7 detected and characterised in the MUD sample are recently identified in mosquitoes24. All the mosquito viruses, except for the virus related to Hubei reo-like virus 7 (which was first characterised in China1) are nearly 100% identical to the viruses detected in Western Australia24, approximately 3000 km from Geelong, Victoria where the MUD faecal sample was collected. These viruses being RNA viruses, which can have high mutation rates due to the low polymerase fidelity54, a nearly 100% identity is rather interesting. The mode of travel of these viruses could be from birds that have eaten the infected mosquitoes and travel long distances and then could be depositing these viruses in their faeces. We also cannot rule out that the infected mosquitoes may be taken from one region to another by human means such as aircrafts, trains, vehicles or by the wind. Independent of mode, the close identity of these mosquito viruses from distant locations indicate that such viruses may be easily dispersed over great distances.The non-avian host associated viruses that were characterised in both MUD and MAD faecal samples were likely part of the food eaten by the ducks. For example, for the mosquito viruses detected in our MUD faecal sample, our detection of partial 28 S RNA of 55 and viruses related to Hubei picorna-like virus 19 and Hubei picorna-like virus 51, here detected and characterised in the MAD sample, were recently identified in leech and dragonfly, respectively1. Presence of plant viruses such as a virus related to black-grass cryptic like virus 2 and a virus related to Hordeum vulgare endornavirus is also likely to be from the food eaten. Only a single long good quality read with high similarity to the Israeli acute paralysis virus was detected. This virus may cause devastating infection in bees (colony collapse) when associated with varroa mite infestations. This virus has previously been detected in Australian bees, however, as Australia is free of varroa mites, is not thought to cause any disease in bees here57. Nevertheless, the finding in the MUD sample likely suggests that it could be from a bee eaten by the Muscovy duck.Virus related to invertebrate iridescent virus 30 has previously been found in mothsPicornaviridae, Adenoviridae, Parvoviridae, Partitiviridae, Endornaviridae, Myoviridae, Iridoviridae and Calciviridae were only around 70–80% similar to the closet sequences in the database  and Culex Negev-like virus 3 very recently described in Western Australia, none of the top matched sequences were to similar strains or related viruses from Australia. Hence to the best of our knowledge we are the first to provide virus sequences from Australia for avian paramyxovirus 6, avian adeno-associated parvovirus and avian calicivirus.Birds may serve as hosts, carriers or transporters of parasites and pathogensThe optimised virus enrichment metagenomics method presented here was able to not only provide information on viruses within the gut of a bird, but also identify the species of the bird, indicate dietary preferences and identify bacterial populations present from a fresh faecal sample. It is therefore a non-invasive technique that could have application to ecological and wildlife health surveys, and can be applied to many different species, including humans. Furthermore, the technique could be applied to different biological samples including saliva and tissue samples (data not shown) and be of use to medical and veterinary diagnostics.59, and as the optimised method allows ribosomes to be enriched together with the virus particles, the ribosomal profile of the faecal microbiome can also be investigated. These results provide a indication of the “ribosomal activity microbiome”, i.e. the microbiome of active bacteria in the final deposited feces as active bacteria have a much higher number of ribosomes per bacteria (up to 70000 ribosomes or more per bacteria) than resting or dead bacteria (7000 or fewer ribosomes per bacteria)61. Although the results based on this method cannot necessarily be directly compared to microbial assessment by 16 S DNA diversity profiling, understanding of the active bacteria may be important62. The decrease that was observed in the Bacteroidetes to Firmicutes ratio with repeated freeze-thaw cycles may also be useful to assess the prior freeze-thaw/storage history of a sample.The “ribosomal activity microbiome” that was revealed using the optimised method may provide valuable information about the composition of live and active bacteria in faecal samples. Ribosomal RNA is partially protected from nuclease activityFinally, we also would like to mention, that in addition to the bird and human faecal samples described here, the optimized method has been tested on dog faecal and saliva samples and on pig lung/tracheal swab samples with good results (data not shown) indicating that the method may have broad applicability for different sample types and species.Fresh faecal samples from one Muscovy duck (MUD) and a pooled sample from six juvenile Pacific black ducks (MAD) were collected in November and December 2016 from south-eastern Victoria, Australia (Geelong and Connewarre), respectively. These samples were stored at −80 °C within 1–3 hours of its collections and until its processing. Bird sample collection was approved under Deakin University’s Animal Ethics Committee project number B43–2016 and Department of Environment, Land, Water and Planning permit number 1008206.Marker viruses for spiking samples (Table Human stool samples collected from 3 children aged 3 months (sample ST4), 18 months (ST5) and 4 years (ST6) from Geelong were also used to test the optimised method. The study involving these samples here were performed in accordance with relevant guidelines and regulations and was deemed negligible or low risk by the Barwon Health Human Research Ethics Committee and therefore exempt from full committee review (project no: 17.119).The MUD faecal sample was selected for spiking with the marker viruses. A non-spiked MUD sample acted as control for the optimisation of the best protocol for metagenomics of viruses. All faecal samples were then processed according to the optimised protocol without any spiking. The detailed steps of the final optimised protocol are given in the supplementary material . However, the steps in the protocol are briefly described below.We initially optimised real-time PCR assays for the marker viruses to detect and quantify them during the virome detection and characterisation protocol development. Virus-specific quantitative assays were either developed or retrieved from the literature  and ultracentrifugation  and also tested filtration using 0.45 µm filters (variation E & F) as described, but not recommended as optimum on their mock community, by the authors of NetoVIR6. This enabled us to evaluate, compare and develop an optimised protocol for virus community analysis from a biological sample. The combination of biophysical enrichment techniques that maintained the maximum number and levels of marker viruses was selected to proceed for cDNA synthesis, amplification, library preparation and NGS. The method which then gave the maximum number and obtained high-quality NGS reads of the marker viruses was then selected as the final method to conduct metagenomics of project samples for the detection of viruses.Removing other impurities such as host cells, bacteria, food particles and free nucleic acids from the faecal samples and enriching for virus particles followed by nucleic acid extraction is the first step in metagenomics of viruses. For this purpose, we tested different enrichment techniques using various biophysical methodsout Fig.  on the sA 1:10 dilution of the bird and human faecal samples were prepared using sterile phosphate buffered saline (PBS). The samples were vortexed thoroughly prior to downstream processing.A volume of 2 mL of the faecal samples were taken for variations involving ultracentrifugation, while for other variations only 500 µL was taken to maximise handling efficiency and protect virus nucleic acid during extraction. Homogenization was carried out using TissueLyser II homogenizer (Qiagen) at 25 Hz for 2 min.A volume of 2 mL of the faecal samples were taken for homogenization. After homogenization, the sample was divided into two, one tube containing 1725µL which was processed according to Variation D and another containing 250 µL which was processed according to Variation C.To the homogenised sample of variation A and B, 0.25% (vol/vol) Nonidet P-40 detergent was added. It was centrifuged at 10,000 rpm for 30 min at 4 °C, and the supernatant was collected and processed downstream.The samples were centrifuged at 17,000 g for 3 min at room temperature, and the supernatant was collected for downstream processing.The samples were filtered through 0.8 µm polyethersulfone (PES) spin filters at 17,000 g for 1 min or until all of the sample had passed through the filter. The filtrate was taken and processed downstream.During method optimisation stage, the spike MUD faecal sample under variation E and F were then filtered through 0.45 µm PES syringe filters, and the filtrate processed downstream.The samples of variation A, D and F were then ultracentrifuged using a Beckman Coulter Airfuge Ultracentrifuge at 178,000 g for 1 hour (30 psi for 1 hour) at room temperature. The supernatant was discarded, and the pellet was suspended in 130 µL of sterile 1XPBS.To 130 µL of the sample, 7 µL of 20 × buffer (1 M Tris (pH8), 100 mM calcium chloride, 30 mM magnesium chloride), 2 µL of benzonase nuclease  and 1 µL of micrococcal nuclease (New England Biolabs) was added. After mixing gently, the sample was incubated at 37 °C for 2 h. The nuclease reaction was stopped by adding 3 µL of 500 mM ethylenediaminetetraacetic acid (EDTA).Virus nucleic acids were extracted using the QIAamp Viral RNA Mini Kit (Qiagen), importantly without adding any carrier RNA. Both DNA and RNA virus nucleic acids were extracted using this kit. The quantity of the isolated nucleic acids was determined using Nanodrop and in selected cases a Bioanalyzer, however, the quantity of extracted nucleic acids was as expected found to be very low after the enrichment and nuclease treatment process.Virus nucleic acids extracted from the spiked MUD faecal sample that undergone variations C, D, E and F were processed downstream as these methods showed presence or detection of all spiked marker viruses. Other samples were also processed as described below. Extracted virus nucleic acids were subjected to cDNA synthesis, amplification and primer removal using SeqPlex RNA Amplification Kit (Sigma) from all the samples according to the kit instructions. An initial RNA denaturation step of 95 °C for 3 min followed by placing the sample in −20 °C cold ethanol was carried out just before cDNA synthesis to ensure the denaturation of the nucleic acids of any double stranded RNA viruses. The quantity and quality of the amplified product were checked using the Bioanalyzer.Library preparation was carried out using Ion Plus Fragment Library Kit (Thermo Fisher Scientific), Ion Xpress Barcode Adapters 1–96 Kit (Thermo Fisher Scientific), Agencourt AMPure XP kit (Beckman Coulter) and Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific). A volume of 20 µL of the cleaned product was used for end repair using the end repair enzyme in Ion Plus Fragment Library Kit. The sample was then purified using Agencourt AMPure XP kit at a 1.8X sample to bead ratio and eluted in 25 µL of Low TE buffer. Following elution, barcoded libraries were prepared using Ion Plus Fragment Library Kit and Ion Xpress Barcode Adapters 1–96 Kit as per the kit instructions. A 1.5X sample to bead ratio of the Agencourt AMPure Kit was used to purify the library. Then it was eluted in 20 µL of Low TE buffer. Quantification of unamplified libraries was performed as per the protocol from Ion Library TaqMan™ Quantitation Kit using QuantStudio™ 6 Flex Real-Time PCR System. Based on the concentration estimated, the dilution that resulted in a concentration of ~100 pM was then calculated and made. Libraries were then pooled prior to loading onto Ion 530 Chips using the Ion Chef Instrument. Following template preparation, the chips were run on the Ion Torrent S5XL System (Thermo Fisher Scientific) as per company protocols. NGS and associated reactions were performed at the Geelong Centre for Emerging Infectious Diseases (GCEID), Geelong, Victoria, Australia. We ran NGS for the spiked MUD faecal sample for the variations C to F only once and generated around 3.4 million reads. Approximately 4 million reads were generated for the non-spiked MUD faecal samples on the first run during which it partly acted as a control, and 7 million reads generated for the MAD faecal sample. For further analysis, the MUD and MAD faecal samples were rerun, and about 9.5 and 7.1 million reads were generated respectively. However, NGS for the human samples were run only once as it was a demonstration of the optimised protocol only and about 7.1 million reads were generated.−10. Any virus sequences which matched either plasmid or ribosomal sequences were removed, and the remaining virus sequences used to create a reference sequence virus BLAST database. The BLAST package version 2.6.0 from NCBI was used to create the local databases and perform the database queries.Although the NGS data provided an insight into the virome of the bird faecal samples, we narrowed our focus to virus sequences that were somewhat similar to already known viruses, in most cases had good quality and quantity NGS reads, could serve as examples of the utility of the method, and/or were of interest from a virological point of view. All available virus sequences were downloaded from the NCBI GenBank genetic sequence database (May 2017), and a local BLAST database created. Bacterial plasmid sequences were downloaded from the NCBI Reference Sequence Database (RefSeq) repository and 16 S, 18 S, 23 S 25 S, 5 S and 5.8 S ribosomal sequences were downloaded from GenBank. These were used to create plasmid and ribosomal BLAST databases respectively. Virus sequences from the NCBI RefSeq database were downloaded and queried against the plasmid and ribosomal RNA databases using a megablast query and an e-value cut-off of 1 × 10−3. A BLASTN query against the virus reference sequence database was performed with an e-value cut-off of 1 × 10−10. A TBLASTX query against the virus reference sequence database was performed with an e-value cut-off of 1 × 10−10. BLAST query results files were converted into spreadsheet files, sorted by virus matches, and a list of potential virus targets created for each sample. The list was then manually inspected to identify viruses of interest.A BLASTN query was performed on the reads from each sample against the initial virus database with an e-value cut-off score of 1 × 10Mapping of sequence reads was performed against reference genomes of the viruses of interest using TMAP plugin on the Ion Torrent server. The full or partial consensus sequences of the viruses were obtained from TMAP using Integrative Genomics Viewer software (IGV) . Consensus sequences were generated from mapped sequences with a mapping quality score of 20 or higher and a coverage depth of at least 3.63. All the analysed sequences of the viruses found in MUD, MAD and human faecal samples are submitted to the NCBI GenBank database under the accession numbers MG991677-MG991679; MH000403-MH000428; MH013331-MH013339; MH028875-MH028887; MH085089-MH085101; MH107865- MH107867 and MH188072-MH188081.AssemblerSPAdes 5.6.0 plugin on the Ion Torrent Server was used to generate contigs from the sequence reads of each sample. The contigs were queried against the virus reference sequence database using BLASTN and TBLASTX to identify virus sequences as described above. Contigs were then used as references in TMAP and trimmed to regions with a mapping quality of 20 or higher and a coverage depth of at least 3. The contigs of deltacoronavirus from the MAD faecal sample and selected viruses from the human samples were then selected for more in-depth analysis using MEGA software63 as previously described in our laboratory65. To understand phylogenetic relationships among the virus sequences, they were compared to the NCBI database using nucleotide BLAST 67 and aligned using Clustal-W68 and some cases using Clustal-W (Codons) or Muscle using the MEGA 6 or 7 software69. Then the sequences were manually trimmed to put them in frame. Maximum Likelihood phylogenetic analyses were conducted using the MEGA 6 or 7 software69, and the best model for generating a phylogenetic tree was determined (data not shown). The robustness of different nodes was assessed by bootstrap analysis using 1000 replicates for nucleotide alignments and 500 replicates for amino acid alignments. The distance between the branches based on the number of nucleotide differences was calculated using the same software.Among the identified viruses, 10 viruses which provided good quality and coverage of the NGS reads mapped to NCBI virus genomes as references and also covered the different virus genome types  were selected for more detailed analysis. For these viruses, NGS reads were further analysed using MEGA softwareReads were uploaded and analysed on the ThermoFisher Ion Reporter microbiome software, and around 0.2–4.6% of all reads (depending on the sample and specific settings of the software) mapped to bacterial 16 S RNA sequences in the curated MicroSEQ® 16 S reference library v2013.1 and curated Greengenes v13.5 databases available in the Ion Reporter software. This gave a good insight into the active bacterial communities present in collected faecal samples.All sequences analysed have been deposited in NCBI GenBank under accession numbers MG991677-MG991679; MH000403-MH000428; MH013331-MH013339; MH028875-MH028887; MH085089-MH085101; MH107865- MH107867 and MH188072-MH188081. Other datasets generated or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Material


Yellow fever virus RNA is usually detected in blood of infected humans. We detected virus RNA in urine and semen samples from a convalescent patient. A complete virus genome was sequenced for an isolate from a urine sample. This virus had a South American I genotype and unique synapomorphic changes. Flavivirus and causes yellow fever in humans, characterized by fever, prostration, and hepatic, renal, and myocardial complications that lead to death in 20%–50% of cases (Yellow fever virus (YFV) is a member of the genus Despite availability of an effective vaccine, >200,000 cases of yellow fever and >30,000 deaths occur per year , headache, body pain, prostration, vomiting, dizziness, anorexia, dark stools, dark yellow urine, and bitterness in the mouth.3 ).The patient was admitted to a public hospital in Januária on January 9. An ELISA for nonstructural protein 1 (NS1) of dengue virus showed a negative result. The patient also had severe thrombocytopenia . High fever, gastrointestinal symptoms (vomiting and diarrhea), weakness, adynamia, and generalized myalgia were also observed. The patient had a weight loss of 4 kg over 8 days. Serum and urine samples were obtained  by qRT-PCR. We also performed a qualitative IgM-capture ELISA with a specific virus antigen and obtained positive results  . We tested samples for YFV RNA by using a real-time quantitiative reverse transcription PCR (qRT-PCR) and primers specific for YFV  and semen  samples were positive for YFV RNA. To evaluate infectivity, we tested a urine sample obtained on this date .On January 27, we obtained serum, urine, and semen samples and tested them by using qRT-PCR. Urine . Most of these changes were located in the NS5 (RNA-dependent RNA polymerase) gene, which plays a major role in virus replication. Changes in the NS5 gene have been associated with differences in viral replication, immune response, and protein–protein interactions during virus replication.Our results suggest that semen can be a useful clinical material for diagnosis of yellow fever and indicate the need for testing urine and semen samples from patients with advanced disease. Such testing could improve diagnostics, reduce false-negative results, and strengthen the reliability of epidemiologic data during ongoing and future outbreaks.Additional information on yellow fever virus in urine and semen of convalescent patient, Brazil.


Feed contaminated with feces from infected pigs is believed to be a potential route of transmission of porcine delta coronavirus (PDCoV). The objective of this study was to determine if the addition of commercial feed additives  to swine feed can be an effective strategy to inactive PDCoV.P < 0.05) for all the additives except for Amasil . Feed additives that contained phosphoric acid, citric acid, or fumaric acid were the most effective in reducing virus survival, although none of the additives completely inactivated the virus by 10- days post-inoculation.Six commercial feed acids , salt, and sugar were evaluated. The acids were added at the recommended concentrations to 5 g aliquots of complete feed, which were also inoculated with 1 mL of PDCoV and incubated for 0, 7, 14, 21, 28, and 35 days. In another experiment, double the recommended concentrations of these additives were also added to the feed samples and incubated for 0, 1, 3, 7, and 10 days. All samples were stored at room temperature (~25 °C) followed by removal of aliquots at 0, 7, 14, 21, 28, and 35 days. Any surviving virus was eluted in a buffer solution and then titrated in swine testicular cells. Feed samples without any additive were used as controls. Both Weibull and log-linear kinetic models were used to analyze virus survival curves. The presence of a tail in the virus inactivation curves indicated deviations from the linear behavior and hence, the Weibull model was chosen for characterizing the inactivation responses due to the better fit. At recommended concentrations, delta values (days to decrease virus concentration by 1 log) ranged from 0.62–1.72 days, but there were no differences on virus survival among feed samples with or without additives at the manufacturers recommended concentrations. Doubling the concentration of the additives reduced the delta value to ≤ 0.28 days  may be utilized as a strategy to decrease risk of PDCoV in feed, specially, commercial feed acidifiers at double the recommended concentrations reduced PDCoV survival in complete feed during storage at room temperature. However, none of these additives completely inactivated the virus. There are three enteric coronaviruses that can cause gastrointestinal illness in young pigs e.g., transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine delta coronavirus (PDCoV) . TransmiSalmonella spp. [Commercial swine feed is often fortified with various additives, including acidifiers such as organic and/or inorganic acids to control bacterial and mold growth in feed, increase growth performance of animals, improve nutrient digestibility, and control harmful bacteria in the animal gut . Acidifilla spp. , 5. Nurslla spp. . Acidifilla spp. . This st2 and were observed for the appearance of virus-induced cytopathic effects (CPE) for up to 6 days post-infection. The infected cells were subjected to 3 freeze-thaw cycles (−80 °C/25 °C) followed by centrifugation at 2500 × g for 15 min at 4 °C. The supernatant was collected, aliquoted, and stored at −80 °C until use.The strain of PDCoV was obtained from the National Veterinary Services Laboratory . Stock virus was propagated in swine testicular cells. The cells were grown in Minimum Essential Medium with Earle’s salts supplemented with L-glutamine , 8% fetal bovine serum , 50 μg/mL gentamicin (Mediatech), 150 μg/mL neomycin sulfate , 1.5 μg/mL fungizone (Sigma), and 455 μg/mL streptomycin (Sigma). The maintenance medium included 5 μg/mL of trypsin  and the same antibiotics as previously described. Cells inoculated with the virus were incubated at 37 °C under 5% CO2 for up to 6 days and examined daily under an inverted microscope for the appearance of CPE. The highest dilution showing CPE was considered the end point. Virus titers were calculated as Tissue Culture Infectious Dose TCID50/mL by the Karber method [Serial 10-fold dilutions of all samples were prepared in maintenance medium followed by inoculation in monolayers of swine testicular cells contained in 96-well microtiter plates  using 100 μL/well and 3 wells per dilution. Inoculated cells were incubated at 37 °C under 5% COThe CGI Enhance ground commercial starter feed used in this experiment was obtained from VitaPlus . This feed is designed for feeding pigs from 5–10 days post-weaning and does not contain any animal derived by-products. The feed was confirmed to be negative for PDCoV by real time reverse transcription-polymerase chain reaction (RT-PCR). A sample of the feed was submitted to Minnesota Valley Testing Laboratories , where dry matter , ether extract (method 2003.05), crude protein , crude fiber (method 920.39), and ash (method 942.05) were analyzed following standard procedures . The cheSix commercial feed acidifiers, UltraAcid P, , Activate DA , Acid Booster , Kemgest , Luprosil , and Amasil  were evaluated when added at their manufacturers’ recommended concentrations Table . In addi5 TCID50/mL) was added to all vials. The control treatment consisted of vials containing feed and virus but no feed additive. The samples were thoroughly mixed using a vortex mixer and stored at room temperature (~25 °C). An individual vial served as the experimental unit, and one vial from each set was removed at 0, 7, 14, 21, 28, and 35 days to determine the degree of virus inactivation. In the experiment involving double the recommended concentrations of additives, samples were removed and evaluated for virus inactivation at 0, 1, 3, 7, and 10 days. Different time points were selected to account for greater virus inactivation in the early stages of inoculation. To determine the amount of virus inactivation at each time point, the surviving virus in each vial was eluted by adding 10 mL of 3% beef extract-0.05 M glycine solution at pH 7.2. After thorough mixing by vortexing, the vials were centrifuged at 2500 × g for 15 min. Serial 10-fold dilutions of the supernatants (eluates) were inoculated in swine testicular cells as previously described for virus titration. The amount of surviving virus was calculated and compared with that in control vials (no additive) and was expressed as log10 TCID50/mL. All treatments were applied and analyzed in triplicate.Forty-eight aliquots of feed  were placed in plastic scintillation vials and the recommended concentrations of each feed additive were added. There were a total of 8 observations at each of the 6-time point for each of the 9 dietary combinations (control and 8 additives). Another set of 40 aliquots of feed were used at double of the recommended concentrations of the additives, for a total of 8 replications per each of the 5-time points and 9 dietary combinations Table . SubsequFifty mL of distilled water was added to 5 g of feed contained in a 100 mL glass flask. The feed suspension was stirred at room temperature for 2 h using a magnetic stirrer. The pH was measured using a pH probe  at 0, 15, 30, 60, and 120 min. The final pH value was calculated as the average of the values at different time intervals. The average pH for feed was 5.82 ± 0.02 and this value was used to compare the pH values after the addition of feed additives.50/mL) were analyzed by using GInaFIT software, a freeware add-on for Microsoft Excel  [N is the amount of surviving virus after treatment, N0 is the initial virus titer, k is the kinetic parameter (day−1), and t is the treatment time (d). The kinetic parameter k is usually expressed as D, which is also known as ‘decimal reduction time’  and was calculated as:Inactivation kinetics data  . The traond, WA) :1\documeN is the surviving virus titer after treatment, N0 is the initial virus titer, δ is the time (min or days) of first logarithm decline in virus titer, and n is the shape parameter. The n value provides an indication of the shape of the response curve. If n > 1, the curve is convex (it forms a shoulder-shaped response), if n < 1, the curve is concave (it forms a tail-shaped response), and if n = 1, the curve is a straight line and can be described by a linear model.The Weibull distribution function has been used to describe non-linear inactivation patterns of different microorganisms after thermal and non-thermal processing. Assuming that the temperature resistance of the virus is governed by a Weibull distribution, Mafart et al.  develope2 defined as follows:m is the number of observations, j is the number of model parameters, and SSQ is the sum of squares.Three replicates per treatment were used to determine how well the model fit the experimental data by calculating the Adj. RP < 0.05.The effect of different additives on the kinetic parameters and survival of virus was assessed by using a mixed model  that included the effect of additives and time as fixed effects and replicate/batch as random effects. Each vial was considered as the experimental unit. Data were analyzed for outliers and the presence of a normal distribution using the UNIVARIATE procedure of SAS that calls for calculations of sample moments, measurements of location and variability, standard deviation, test for normality, robust estimates on scale, missing values among others. The LSMEANS statement in SAS was used to calculate treatment means adjusted for model effects, while Tukey’s test was used to determine differences among treatments. For this study, significance was considered when 2 values from the log-linear and Weibull models. The Adj. R2 values for the log-linear model (0.48–0.57) were less than those obtained for the Weibull model (0.86–0.93), indicating that the Weibull model provide a better fit of the experimental data  for all additives, except for sucrose and formic acid Table . UltraAcThe pH of the complete feed without addition of acidifiers was greater than pH of the same complete feed with the addition of Luprosil, Activate DA, KEMGEST, Acid Booster, and Amasil. The pH of the complete feed with addition of UltraAcid P was not different from that of the complete feed. There was no correlation between the pH values of the diet with the addition of acidifiers and the inactivation kinetics of PDCoV  was present in UltraAcid P and KEMGEST, which suggests that this acid may be potentially responsible for inactivation of PDCoV. Phosphoric acid has been shown to inactivate pathogens such as Salmonella spp. on stainless steel surfaces, but there are no data available on inactivation of viruses in animal feed [The current experiment focused on determining inactivation kinetics of commercial additives available to the United States feed industry, and did not focus on evaluating the specific active ingredients present in these additives that may inactivate PDCoV. However, based on the description and order of the active ingredients listed for each commercial additive, it appears that some form of phosphoric acid  than 17 days observed for PEDV . CompariAddition of salt, but not sugar, to the control diet caused a decrease in delta values for inactivation of PDCoV. This observation is in agreement with inactivation of PEDV in complete swine feed, where adding both salt and sugar increased inactivation of PEDV . LikewisUsing feed acidifiers could be an effective strategy to decrease the concentration of PDCoV in swine feed, but double the manufacturer’s recommended concentration was required to observe an effect. Using feed acidifiers could be an effective strategy to decrease the concentration of PDCoV in swine feed, but double the manufacturer’s recommended concentration was required to observe and effect. In spite the observed results on inactivation of PDCoV more experiments are needed to demonstrate the effectiveness of these treatments as means of preventing PDCoV transmission in feed on more applied settings. None of the treatments applied in this experiment were completely effective in inactivating PDCoV. Therefore, the strategy proposed in this research should be used in combination with other virus inactivation procedures within the processing and distribution steps for swine feed rather than a single kill step for virus inactivation.


INK4/ARF locus encodes the key tumor suppressors, p15INK4b, p16INK4a and ARF, that together regulate the pRb and p53 major tumor suppressive networks. Given the importance of the products of the INK4/ARF locus in tumor suppression, transcription from the locus is tightly governed. Over the last decade, intense study to elucidate the regulation of this locus exposed that the genes encoded in the INK4/ARF locus are controlled both independently as well as in a coordinated manner.The INK/ARF locus is positioned in close proximity to the p15INK4b gene called Regulatory Domain (RD). Interaction of the CDC6 protein with the RD triggers recruitment of histone deacetylases, which increase methylation of histone H3 on lysine 9 (H3K9) and in turn cause heterochromatinization of the p15INK4b, p16INK4a and ARF promoters leading to their silencing. In this context, we recently reported that the E3 ubiquitin ligase and transcription cofactor E6AP  acts as a positive regulator of the INK4/ARF locus [INK4a-low expression endure the worst overall survival. This correlation predicts the prognostic value of this expression signature.A known regulatory site responsible for the coordinated silencing of all three genes in the CDC6 gene, as we demonstrated that the levels of other E2F1 gene targets were also altered in E6AP-deficient cells. In accord with these findings, we recently published an independent report showing that in prostate cancer cells, E6AP reduces the ability of E2F1 to bind to p27 promoter and to induce its transcription [A key finding from our work is that the ability of E6AP to modulate E2F1 transcriptional activity is not limited to the INK4/ARF locus is the long non-coding RNA (lncRNA) ANRIL (antisense non-coding RNA in the INK4 locus). ANRIL gene is transcribed in the antisense orientation relative to the INK4/ARF gene cluster. ANRIL promotes the epigenetic repression of its neighbor tumor suppressors in the INK4/ARF locus by physically interacting with the Polycomb proteins Suz12 and Cbx7, which in turn recruit the Polycomb repressive complex 1 and 2 to the locus, leading to histone modification and silencing of the locus [A particularly intriguing regulator of the expression of the tumour suppressors encoded in the he locus .ANRIL to cancer development has attracted much attention in recent years, and ANRIL oncogenic role is now solidly established in a variety of carcinomas. This is the case for NSCLC, where multiple recent papers convincingly demonstrate that high ANRIL levels correlate with poor prognosis . In thi by E2F1 , 6. Thisf cancer .et al. raises another potential regulatory mechanism of the INK4/ARF locus that is relevant in NSCLC, and opens new potential avenues for identification of novel pathways in NSCLC pathogenesis. An interesting future frontier will be to integrate all these pathways involved in the regulation of the INK4/ARF products to design better treatment strategies to ultimately improve outcomes of cancer patients.In summary, our paper by Gamell