We present the near complete virus genome sequences with phylogenetic and network analyses of potential transmission networks of a total of 18 Australian cases of human parechovirus type 3 (HPeV3) infection in infants in the period from 2012–2015. Overall the results support our previous finding that the Australian outbreak strain/lineage is a result of a major recombination event that took place between March 2012 and November 2013 followed by further virus evolution and possibly recombination. While the nonstructural coding region of unknown provenance appears to evolve significantly both at the nucleotide and amino acid level, the capsid encoding region derived from the Yamagata 2011 lineage of HPeV3 appears to be very stable, particularly at the amino acid level. The phylogenetic and network analyses performed support a temporal evolution from the first Australian recombinant virus sequence from November 2013 to March/April 2014, onto the 2015 outbreak. The 2015 outbreak samples fall into two separate clusters with a possible common ancestor between March/April 2014 and September 2015, with each cluster further evolving in the period from September to November/December 2015. We now extend our previous study to include the near complete virus genome sequences and the phylogenetic and transmission network analyses of 12 of the Geelong 2015 cases and another 6 Australian cases from 2012–2015 for a total of 18 HPeV3 infant cases in Australia. Thus, our study provides valuable, near complete genome HPeV3 sequences and explores the potential value of phylogenetic and network analyses for better understanding HPeV3 transmission and evolution over time.The picornaviruses are small, single-stranded positive sense RNA viruses causing disease in animals and humans and include the human parechoviruses (HPeV). Like other picornaviruses, HPeV evolve rapidly due to nucleotide substitutions and recombination events, generating strains associated with changed infectivity, virulence or host range. The first detected outbreak of HPeV type 3 (HPeV3) in infants in Australia occurred in and around Sydney in New South Wales in late 2013, with subsequent infection in other parts of Australia in the first part of 2014. A second outbreak occurred across Victoria and Southern Australia in the second half of 2015. As described previouslyEnterovirus, but reclassified in 1998/99 into their own genus based on nucleotide and biological features including a lack of host cell protein synthesis shut-off during replication6. The RNA genome of HPeV is approximately 7300-7400 nucleotides with 5′ and 3′ untranslated regions (UTR) at each end of a single long open reading frame (ORF)9. The single ORF encodes a polyprotein including the P1 structural/capsid and the P2 and P3 nonstructural proteins. The capsid proteins of HPeV consist of only 3 proteins, VP0, VP3 and VP1, as VP0, in contrast to some other picornaviruses, is not proteolytically cleaved into VP4 and VP210. Some of the HPeV types have an arginine-glycine-aspartic acid (RGD) motif near the carboxy-terminus of VP1, thought to be the receptor-binding site likely binding to host cell integrins12. However, this RGD motif is not present in HPeV3, HPeV7-16, the related rodent/zoonotic virus Ljungan virus or in some strains within HPeV types normally encoding the RGD motif, for example for HPeV416. Like other picornaviruses, recombination of HPeV often involves breakpoints at the very end of VP1 and thus may remove or add the RGD motif, or alternatively occur downstream in the nonstructural coding region21.HPeV type 1 (HPeV1) and 2 were initially classified as echoviruses 22 and 23 in the genus 23. HPeV3 appears to cause more severe disease in young infants compared to the other HPeV types and is the most common type identified in cerebrospinal fluid (CSF) samples from young infants with CNS infection and/or sepsis-like presentation22. Evaluation by cranial magnetic resonance imaging (MRI) or ultrasound of a group of 10 infants with HPeV3 CNS infection showed abnormal periventricular white matter in all infants and for a number of the infants, clinical follow-up revealed sequelae including cerebral palsy, epilepsy, learning disabilities, and visual impairment25. Further cases like this including reports of autopsy cases of two infants that died with active HPeV3 infection and severe CNS disease are referenced in ref. 22.HPeV infections are prevalent in humans and most often cause only asymptomatic/subclinical or mild gastrointestinal and/or respiratory disease. More severe disease including central nervous system (CNS) dysfunction may be seen in particular in infants and one relatively large study reported mortality among young infants as high as 6%33. The reason for this is not known and as these viruses are easily transmitted by the oral-faecal and respiratory routes30, it may be related to exposure and immunity within families e.g. outbreaks occurring when there is both an older sibling lacking prior HPeV3 exposure plus waning or no maternally derived HPeV3 passive immunity in the young infant34. Studies from Japan have indicated that antibodies to HPeV3 are only present in 15% of children from 7 months to 1 year of age, but that seropositivity increases to 45% in 2–3 year old and to 85% in 4–6 year old children35. Due to these factors, virus testing results and virus sequences available for analyses are likely to only represent a minority of HPeV3 infections. Therefore, transmission network analyses will help elucidate part of a much larger transmission network, i.e. will represent sequences sampled from severe disease in young infants among a much larger population of infected older children and adults with mild or no clinical disease and thus not sampled and tested.HPeV3 was first isolated in 1999 and reported in the literature in 2004 and subsequent surveillance suggests a two to three-year cycle between outbreaks36. However, it appears that more recent strains have spread worldwide only within the last 20–30 years37, and during this time, may have evolved into being more virulent. One such example may be the Yamagata 2011 lineage of HPeV3 shown to cause severe disease in young children and myalgia in adults40. We have previously shown that the first reported outbreaks of HPeV3 in infants in Australia in 2013/14 and 2015, respectively, were caused by a novel recombinant HPeV3 with the capsid encoding region closely related to the Yamagata 2011 lineage, while the nonstructural encoding region derived from an as yet unknown/unpublished HPeV(s)1.Molecular clock analyses have estimated that the parechoviruses have existed for approximately 400 years with HPeV3 and HPeV7 diverging approximately 150 years ago1 that the major recombination event took place between March 2012 and November 2013 and we now show evidence of further temporal virus evolution and possibly recombination. While the nonstructural coding region appears to be evolving both at the nucleotide and amino acid level, the capsid encoding region appears to be very stable, particularly at the amino acid level. This apparent stability of the virus capsid may indicate that development of a vaccine or antibody treatment to avoid severe disease in infants may be feasible. It should be noted that the ancestor of the nonstructural coding region of the Australian recombinant HPeV3 is still not known and further sequencing and publishing/depositing of results from cases worldwide should be a priority to further the understanding of generation, evolution, spread and possibly even host range of new/recombinant strains of HPeV3.We now present the near complete virus genome sequences with phylogenetic and network analyses of potential transmission from a total of 12 of the Geelong 2015 cases as well as from another 6 positive Australian cases. These include an early Australian sample from 2012 obtained by retrospective testing, samples from Sydney from 2013 and 2015 in addition to two samples from Adelaide and one from Darwin from 2014 making a total of 18 cases of HPeV3 in infants in Australia. Overall the results support our previous finding1, thus not fully covered by our Ampliseq panels), to 2.3 million mapped reads with an average coverage of 67000 for sample CS-HP-16012. Apart from samples CS-HP-16018 mentioned above and CS-HP-16003 for which we only had very limited sample material left from our previous study, samples averaged 0.5–1.5 million reads and an average coverage of 14000–50000. This allowed us to assemble high quality near complete virus genome consensus sequences from each sample apart from sample CS-HP-16018, where high quality sequences were obtained for the first approximately 5000 nucleotides only. For that sequence we have chosen to only report and include in our analyses the capsid encoding region from nucleotide 701–3013. All sequences generated have been deposited in GenBank under accession numbers KY556659-KY556676.Next generation sequencing (NGS) was performed on RNA extracted from the clinical samples Table  as well in vivo virus, which is useful information to have if a virus isolate is used for animal trials or in vitro studies.We first compared sequences obtained from different sample types from the same patient . These two clusters are represented by GenBank Accession number AB759207 in one of the two clusters and AB759204 and AB759205 in another. We inadvertently based our initial NGS panel on AB7592071 and this particular panel coincidentally resulted in high quality sequence for sample CS-HP-16018 up to approximately nucleotide 5000, consistent with this sample, from March 2012, being of full Yamagata 2011 lineage, but really matched most closely to the AB759204/AB759205 cluster/recombinant and not the AB759207 cluster/recombinant. Nevertheless, based on BLAST comparison to all sequences available in the NCBI databases, the Yamagata 2011 lineage(s) of viruses are the closest related to our Australian HPeV3 outbreak viruses in the region up to approximately nucleotide 3100 (the capsid encoding region) and consequently are included in our initial phylogenetic analysis. In any event, GARD analysis of the full sequence and the coding region from nucleotide 701 to 7231 of our Australian HPeV3 virus sequences, (excluding CS-HP-16018 as mentioned above), indicated possible sites of recombination at nucleotide 997 and nucleotide 3025 or 3550. The level of significance only reached p = 0.01–0.05 and likely reflects the impact of slight differences in evolutionary rates among the regions derived from previous recombination event(s)1. The sequences found to have differences in topologies of segments in the GARD analysis were samples CS-HP-16003, 16006, 16007, 16010, 16014, 16017 and 16020, the potential importance of this is addressed later in the discussion. Nevertheless, in our phylogenetic analyses described below, we looked at the regions both separately and as full-length sequences to see an effect, if any, of a prior site of recombination. Sequences were divided into the capsid encoding region (nucleotides 701–3013); the nonstructural proteins encoding region (nucleotides 3014–7231); the full protein encoding region/ORF (nucleotides 701–7231); or finally, the full-length sequences from nucleotide 1–7334. The dataset from nucleotides 701–3013 included either the 17 sequences from the Australian samples containing the novel recombinant strain or another set with 21 sequences containing these 17 Australian recombinant virus samples and in addition sample CS-HP-16018, an Australian sample from 2012 with a fully Yamagata 2011 lineage virus, as well as the 3 closest related Yamagata 2011 virus sequences obtained from NCBI . For the remaining datasets we only included the 17 Australian samples containing the novel recombinant HPeV3, as the region downstream of the capsid coding region is likely to be derived from a previous recombination event/s and consequently will have a different evolutionary history than the fully Yamagata 2011 lineage viruses1.As a first step in our analyses of the sequences obtained, we screened for recombinants among the sequenced viruses using GARD  analysis. We discovered in our previous study that the Australian HPeV3 outbreak virus is a recombinant, with the sequence up to approximately nucleotide 3115 derived from a Yamagata 2011-like virus, whereas the downstream sequence is derived from recombination(s) with an as yet unknown/unpublished HPeV/sThe transition/transversion bias, nonsynonymous (dN)/synonymous (dS) nucleotide substitutions per site ratio (dN/dS), indication of positive or negative selection of sites and number of variable nucleotide and amino acid sites in the datasets are shown in Table The next step was to do phylogenetic inference of the sequence datasets by Maximum Likelihood (ML) phylogenetic analyses using the MEGA 6 software. Phylogenetic trees are shown in Figs Overall the ML phylogenetic analyses show that the capsid region of the Australian recombinant HPeV3 virus sequences are related to each other and more distantly to the Yamagata 2011 virus sequences. The Australian virus sequence from 2012 (sample NAS18/CS-HP-16018) has the closest relation to the Yamagata 2011 virus while the other Australian virus sequences from 2013/14 are more related to the Yamagata 2011 sequences than the Australian virus sequences from 2015. This latter group partitioned into two clusters; cluster 1 with CSF samples CS-HP-16003, 16004, 16005, 16006, 16007, faecal samples CS-HP-16010, 16017, 16020 and nasal sample CS-HP-16014 and a cluster 2 with CSF samples CS-HP-16001, 16008, faecal sample CS-HP-16012 and nasal sample CS-HP-16016 falling separately Fig. . It shouPhylogenetic transmission network construction was then done using the program Network v5. It should be emphasized that our virus sequences all came from severely ill infants and likely only represent a fraction of infected individuals. Consequently, the aim was not to look at direct transmission between these cases, but rather to look for an indication of a potential transmission network likely containing a large number of unsampled infections among older children and adults . Similar to the ML phylogenetic analysis shown above, we did this for the individual regions, for the full ORF and for the full sequence. Similar to what is mentioned above, we found the potential transmission networks generated to be very similar and again suggesting that the closest ancestor for the capsid region of the recombinant Australian 2013/14/15 virus sequences was AB759204 of the Yamagata 2011 virus lineage. Interestingly, while the Australian 2012 “non-recombinant” virus sequence (sample NAS18/CS-HP-16018) clearly was closely related to the Yamagata 2011 virus sequences, it may not be the direct ancestor of the subsequent Australian outbreaks that may be caused by separate introduction(s) Fig.  [21 sequ). Not unexpectedly, considering the anticipated large number of unsampled infected individuals and the likely wide travel network involved, in particular in relation to the holiday season up to December, we could not detect any spatial connection between either the 2015 clusters, or the earlier Australian virus sequences. However, for the 2015 virus sequences it is interesting to note that those with the highest number of nucleotide differences and located most distant in the transmission networks are the faecal sample CS-HP-16020 taken in Sydney and the CSF CS-HP-16001, CS-HP-16003, CS-HP-16008 and nasal CS-HP-16014, CS-HP-16016 samples taken from infants treated at University Hospital Geelong which, except for CSF sample CS-HP-16008, have home addresses located outside or even at some distance from Geelong  collection described previously1. An additional 3 positive samples selected from VIDRL’s stored collection of HPeV3 positive samples from Australia provided a total of 21 clinical samples from 18 cases of HPeV3 in infants in Australia; see Table An outbreak of HPeV3 was identified in infants in the Geelong area of Victoria, Australia during August-December 2015 and a total of 16 cases were identified at the University Hospital Geelong as described previously1.Virus isolates from the following 6 samples were also available for analysis: CS-HP-16006 faecal and nasal swab samples, CS-HP-16004 faecal sample, CS-HP-16018 nasal sample, CS-HP-16019 CSF sample and CS-HP-16020 faecal sample. These virus samples were cultured on monolayers of Vero cells as described previously1.All samples were stored at −80C and RNA extracted and converted into cDNA as described previouslyThe studies described here were performed in accordance with relevant guidelines and regulations and was provided with ethical exemption by the Barwon Health Human Research Ethics Committee.1, we initially constructed a composite reference genome from related HPeV3 sequences available in NCBI  and used this initial reference , we subsequently updated this initial reference sequence (updated reference available as GenBank accession number KY020128) and used that to design an updated Ion Ampliseq Panel, referred to as panel 2. Based on additional NGS runs, we further updated the reference sequence and designed a third Ion Ampliseq Panel, here referred to as panel 3. Details of panel 2 and 3 can be seen online , Geelong, Victoria, Australia. For the NGS results described here we did a total of 5 Ion Torrent S5 runs with a total of 6 Ion 530 Chips generating a total of approximately 25 billion nucleotides.Extracted RNA converted to cDNA from all samples were PCR-amplified with the Ion AmpliSeq Library Kit 2.0 and Ion AmpliSeq Panel 1, 2 or 3. The panels comprise 37 or 38 overlapping primer sets split into 2 pools per sample and amplified with the 5X Ion AmpliSeq HiFi Master Mix. The details of our NGS protocol has been described in detail previously41. IGV calculates the consensus sequence for the NGS results for each sample as described by Cavener42; if the frequency of a single nucleotide at a specific position is greater than 50% and greater than twice the number of the second most frequent nucleotide it is assigned as the consensus nucleotide or if the sum of the frequencies of two nucleotides is greater than 75% (but neither meet the criteria for a single nucleotide assignment) they are assigned as co-consensus nucleotides. If no single nucleotide or pair of nucleotides meets the criteria, IGV assign an ‘N’. In the few occasions where our consensus sequences contained co-consensus nucleotides at a given position, we manually inspected the results and assigned a single nucleotide when a given nucleotide was present in 60% or more of the reads. The final assembled virus genome sequences were based on the NGS results indicated above, but also included nucleotides 1–29 and 7292–7334 (the very 5′- and 3′end sequences). These did not derive directly from the NGS sequencing, but are assumed based on: (i) the high efficiency of the end-primers used; (ii) comparison to other closely related sequences, and (iii) the fact that the rest of the 5′- and 3′-UTR sequences obtained were highly conserved as indicated in the results.Sequences obtained from the Ion Torrent S5 were analysed based on each sample having a unique barcode. Raw sequences were analysed using the Ion Reporter Software contained within the Ion S5 Instrument using either of the 3 reference genomes or various HPeV reference genomes from NCBI. Obtained reads were then visualised and analysed using the Integrative Genomics Viewer (IGV)44 and the HyPhy package46 available on the Datamonkey webserver and using the HKY85 substitution model as suggested by using the automatic Model Selection Tool available on the site. The GARD technique identifies evidence for recombination breakpoints by searching multiple sequence alignments for phylogenetic incongruences.Screening for recombinants among the sequenced viruses were done using GARD  analyses48 and aligned using Clustal-W49. Maximum Likelihood phylogenetic analyses were conducted using the MEGA 6 software50 and the Tamura-Nei model51 . The robustness of different nodes was assessed by bootstrap analysis using 1000 replicates. To understand the chains of transmission between obtained samples, sequences were used to construct phylogenetic (transmission) networks using the Median Joining method implemented in the program Network v5 (http//www.fluxus-engineering.com) developed to reconstruct all possible shortest, least complex phylogenetic trees  and depict this as a network. The parameter epsilon was set at the same value as the weight of characters used to calculate the genetic distances  as described previously52. As the virus sequences available for this analysis most likely only represent a minority proportion of HPeV3 infections occurring in the total transmission network, i.e. only represent sequences sampled from severe disease in young infants among a much larger population of infected older children and adults with mild or no clinical disease and thus not sampled, connections in the transmission network are not an indication of direct contact transmission between individual cases. Rather, it should be viewed as cases linked by chains of transmission that include an unknown number of mild or undiagnosed infections, yet still able to indicate temporal evolution and chains of transmission.To understand phylogenetic relationships among the HPeV3 sequences, they were compared to the NCBI database using nucleotide BLAST 52.Transition/transversion bias was estimated using Maximum Likelihood (ML) and Maximum Composite Likelihood (MCL) analysis available in MEGA 6. Site-specific selection pressures were measured as nonsynonymous (dN) -synonymous (dS) nucleotide substitutions per site and estimated using the single-likelihood ancestor counting (SLAC), fixed-effects likelihood (FEL), internal fixed-effects likelihood (IFEL), and random effects likelihood (REL) methods available at the Datamonkey online version of the HyPhy package. All analyses utilized the HKY85 nucleotide substitution model, which was tested as the best fitting model for the data sets, and employed input Neighbor Joining phylogenetic trees. A cut-off p-value to classify a site as positively or negatively selected was set at 0.01 or 0.05 for SLAC, FEL, and IFEL methods and the cut-off value for the Bayes factor in the REL method was set at 100 to reflect a positive or negative selection at a given site as described previouslyAll sequences generated have been deposited in GenBank under accession numbers KY556659-KY556676. Other datasets generated or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Table S1


Zika virus infection during pregnancy can cause serious birth defects, including microcephaly and brain abnormalities  brain abnormalities or microcephaly; 2) NTDs; 3) eye abnormalities without mention of a brain abnormality included in the two previously defined categories; and 4) other consequences of central nervous system (CNS) dysfunction, specifically joint contractures and congenital sensorineural deafness without mention of brain or eye abnormalities included in another category. Because the evidence linking NTDs and congenital Zika virus infection is weak, prevalence estimates per 1,000 live births were calculated both overall and excluding NTDs for each quarter in 2016; CIs were calculated using Poisson regression  , includiThe prevalence of reported birth defects cases potentially related to Zika virus infection increased in jurisdictions with confirmed local transmission, from 2.8 per 1,000 live births (182 cases) during the first half of 2016 to 3.0 per 1,000 live births (211 cases) during the second half . In “higher” Zika prevalence jurisdictions, the monitored birth defects prevalence was 3.0 per 1,000 live births in both the first (753 cases) and second (775 cases) halves of 2016. In “lower” prevalence jurisdictions, the monitored birth defects prevalence declined significantly from 3.4 per 1,000 live births (549 cases) during the first half of 2016 to 3.0 (492 cases) per 1,000 live births during the second half  .When NTDs were excluded, the prevalence of birth defects potentially related to Zika virus infection in jurisdictions with local Zika transmission increased 21%, from 2.0 per 1,000 live births (CI = 1.7–2.4) to 2.4 (CI = 2.1–2.8) . This inLeveraging existing birth defects surveillance systems permitted rapid implementation of surveillance for birth defects potentially related to Zika virus infection early during the U.S. Zika virus outbreak. The prevalence of birth defects strongly linked to Zika virus infection increased significantly in areas with local Zika virus transmission . This finding underscores the importance of surveillance for birth defects potentially related to Zika virus infection and the need for continued monitoring in areas at risk for Zika transmission and exposure. An increase in birth defects potentially related to Zika was only observed in jurisdictions with local Zika virus transmission, and this difference was significant when NTDs were excluded. Brain and eye abnormalities and consequences of CNS dysfunction have been most consistently described in cases of congenital Zika infection, whereas the evidence supporting a possible association between NTDs and Zika virus infection during pregnancy is weak  was similar to a previously published baseline prevalence of birth defects potentially related to Zika virus infection from 2013–14  . Established birth defects surveillance systems can adapt to monitor other emerging pregnancy, infant, and newborn outcomes of concern beyond structural birth defects, including functional problems such as hearing loss, and can provide additional clinical information through standardized data collection and clinical review. Finally, birth defects surveillance systems can provide an important mechanism for facilitating timely access to services among infants with birth defects and serve as a resource for assessing subsequent health and developmental outcomes among these children. The unique contributions of ongoing birth defects surveillance and the U.S. Zika Pregnancy and Infant Registry are both critical to optimally monitoring pregnant women and infants from the threat of Zika virus infection and implementing appropriate prevention efforts  potentially related to Zika virus infection in a birth cohort of nearly 1 million births in 2016. A significant increase in birth defects strongly related to Zika virus during the second half of 2016 compared with the first half was observed in jurisdictions with local Zika virus transmission. Only a small percentage of birth defects potentially related to Zika had laboratory evidence of Zika virus infection, and most were not tested for Zika virus.Whereas the U.S. Zika Pregnancy and Infant Registry monitors women with laboratory evidence of possible Zika virus infection during pregnancy and their congenitally exposed infants, population-based birth defects surveillance systems make a unique contribution by identifying and monitoring all cases of these birth defects regardless of exposure or laboratory testing or results. Continued surveillance for birth defects potentially related to Zika virus infection is important because most pregnancies affected by Zika virus ended in 2017. These data will help communities plan for needed resources to care for affected patients and families and can serve as a foundation for linking and evaluating health and developmental outcomes of affected children.


In 1947, in a small forest in Uganda, near the capital, Kampala, a filterable transmissible agent was found in a sentinel rhesus macaque with a high fever. The agent was later identified as a virus and named Zika after the forest in which it was found. For the next 60 years, the virus was largely ignored by scientists because very few human cases were identified. However, in the summer of 2015, in several cities in eastern Brazil, a Zika epidemic was identified concurrently with a markedly increased number of infant microcephaly cases in maternity wards. Before long, Zika virus infection during pregnancy was identified as the cause of microcephaly, the virus was confirmed to be spreading among >40 countries in the Americas, and numerous public health agencies were faced with an emergency about how best to protect a generation of children conceived during the Zika outbreak.The journey taken by Zika virus from Africa, across the Pacific, to the World Health Organization–declared Public Health Emergency of International Concern in February 2016 is chronicled by New York Times writer Donald G. McNeil, Jr., in Zika: The Emerging Epidemic . The booMcNeil’s book is thorough in its timeline and excellent at describing the political history of public health management for Zika. This history includes the blaming of an insecticide for the initial cases of Guillain-Barré syndrome in French Polynesia in 2013, the US Centers for Disease Control and Prevention’s initial handling of evidence of a sexual transmission route for Zika virus, and the controversy of whether to recommend delaying pregnancy in Zika-endemic areas where travel advisories were already in place. McNeil presents convincing arguments at the end of the book for why the recommendations to delay pregnancy should have been stronger, clearer, and more prompt. The book also catalogs a range of alternative hypotheses that emerged in the media before a scientific consensus formed that Zika did in fact cause microcephaly.The book contains some poignant individual stories that humanize the toll of the epidemics in the Pacific and the Americas. McNeil correctly calls attention to Guillain-Barré syndrome, which typically does not generate headlines the way that a photo of a microcephalic infant does. In some instances, anecdotes about newspaper editors and public officials draw attention away from the victims of the epidemic.Toxoplasma, risk for death from dengue virus), and academic readers would be wise to follow their noses to the source material, much of which McNeil lists by chapter at the end of the book. The book is very readable, as expected from a New York Times writer, and it successfully presents a current-events summary of the 2015–2016 Zika epidemic and what the world’s public health systems did and did not do to minimize the risk for those in its path.The book sometimes strays into a narrative of epidemiologist or journalist as hero, a convention that should be avoided. It contains a small number of scientific inaccuracies (e.g., biological classification of


We describe a strain of Lassa virus representing a putative new lineage that was isolated from a cluster of human infections with an epidemiologic link to Togo. This finding extends the known range of Lassa virus to Togo. TThe Lassa virus infections in the index case-patient, secondary case-patient 1, and secondary case-patient 2 were confirmed by laboratory investigations at Bernhard Nocht Institute ; Centers for Disease Control and Prevention ; and Philipps University , respectively. The viruses from all 3 patients were isolated in Vero E6 cell culture in the respective Biosafety Level 4 laboratories. Full-length virus sequences were generated directly from clinical specimens, from the isolates, or both using next-generation sequencing technology in combination with Sanger sequencing  . The seqThe virtually identical viruses from the 3 patients confirmed the transmission chains suggested by the epidemiologic data. Only the virus from secondary case-patient 2 showed differences in coding regions—a deletion of 3 nt and a nucleotide exchange in the polymerase (L) gene—from the viruses in the other 2 case-patients. These differences were confirmed by sequencing the virus in the clinical specimens. Differences among the 3 strains in the highly structured intergenic regions might represent artifacts created by the difficulty in sequencing these regions.https://www.beast2.org/) with nucleotide sequences of full-length nucleoprotein (NP), glycoprotein precursor (GPC), and L and Z genes of the Togo strain in conjunction with representative sequences of Lassa virus and other Old World arenaviruses. The most stable reconstruction was obtained for the L gene with the Togo strain being placed in sister relationship with lineage II  (The phylogeny was inferred using BEAST2 (s >0.97) . In the s >0.97) . The phys >0.97)  Figure 1s >0.97)  Figure 2The long branch  separating the Togo strain from known lineages suggests that it represents a new lineage. Because Lassa virus lineages were originally established on the basis of uncorrected sequence distances  Table 1.In conclusion, sequencing Lassa virus from a cluster of imported infections, with the index case-patient originating from Togo, reveals a new lineage of Lassa virus in West Africa. It seems to be related to lineage II or lineages I/VI, which are all circulating in Nigeria.Additional methods used to detect a new Lassa virus strain, Togo, 2016; uncorrected pairwise amino acid distances among Old World arenaviruses; phylogeny of the Lassa virus Togo strain using Z gene sequences; and analysis of potential recombination and/or reassortment in its evolution.


Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3–12.5 kb divided into 3–4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ophioviridae, which is available at http://www.ictv.global/report/ophioviridae.The  CP, coat protein; RdRp, RNA-dependent RNA polymerase.Particles are non-enveloped, naked filamentous nucleocapsids forming either circles (open form) or pseudo-linear duplex (collapsed form) , Fig. 1.Citrus psorosis virus [Ranunculus white mottle virus, Freesia sneak virus and Blueberry mosaic associated virus have three RNAs  whereas members of the species Mirafiori lettuce big-vein virus and Lettuce ring necrosis virus have a fourth RNA (RNA4) [Tulip mild mottle mosaic virus.The genome of ophioviruses consists of three or four individually encapsidated RNA segments . Membersis virus , RanuncVirions encapsidate both the minus- and positive-sense RNAs, but a larger amount of RNA of negative polarity is detected. As virions appear circularized, the presence of panhandle structures has been suggested. RNA1 contains two ORFs in the viral complementary strand (vcRNA) encoding the 22–25K protein and the RdRp. The 24K protein of citrus psorosis virus is involved in the misprocessing of miRNA and RNA silencing suppressor activity. vcRNA2 encodes the cell-to-cell movement protein (MP), which also has RNA silencing suppressor activity. Mirafiori lettuce big-vein virus has a second putative ORF in vRNA2 that encodes a protein of unknown function. The coat protein (CP) is encoded by vcRNA3. A fourth genomic RNA has been reported for Mirafiori lettuce big-vein virus and lettuce ring necrosis virus, encoding putative proteins of unknown function.Ophiovirus is the only genus in the family, including seven species. CP amino acid sequence alignments show 31–52 % identity between isolates of different species, and 92–100 % identity within a species. The CPs of Mirafiori lettuce big-vein virus and tulip mild mottle mosaic virus  share about 80 % amino acid sequence identity, thus warranting an ophiovirus species demarcation criterion of <85 % CP amino acid sequence identity. The RdRp contains the SDD sequence in motif C, a signature for segmented negative-stranded RNA viruses. Phylogenetic reconstructions using sequences of the conserved core module from RdRps of ophioviruses and representative negative-stranded RNA viruses reinforce their separation as a monophyletic group. Citrus psorosis virus is present worldwide, transmitted by vegetative propagation of the host, and natural dispersion has also been observed. No vector is known for ranunculus white mottle virus and blueberry mosaic associated virus. Olpidium virulentus and Olpidium brassicae fungi transmit Mirafiori lettuce big-vein virus, tulip mild mottle mosaic virus and lettuce ring necrosis virus; freesia sneak virus is presumably transmitted by a member of the genus Olpidium. Ranunculus white mottle virus has been reported in Italy, France and Germany [ Germany . Mirafio Germany  has been Germany .www.ictv.global/report/ophioviridae.Full ICTV Online (10th) Report:


Background: Antibodies to microbes, or to autoantigens, are important markers of disease. Antibody detection (serology) can reveal both past and recent infections. There is a great need for development of rational ways of detecting and quantifying antibodies, both for humans and animals. Traditionally, serology using synthetic antigens covers linear epitopes using up to 30 amino acid peptides. Methods: We here report that peptides of 100 amino acids or longer (“megapeptides”), designed and synthesized for optimal serological performance, can successfully be used as detection antigens in a suspension multiplex immunoassay (SMIA). Megapeptides can quickly be created just from pathogen sequences. A combination of rational sequencing and bioinformatic routines for definition of diagnostically-relevant antigens can, thus, rapidly yield efficient serological diagnostic tools for an emerging infectious pathogen. Results: We designed megapeptides using bioinformatics and viral genome sequences. These long peptides were tested as antigens for the presence of antibodies in human serum to the filo-, herpes-, and polyoma virus families in a multiplex microarray system. All of these virus families contain recently discovered or emerging infectious viruses. Conclusion: Long synthetic peptides can be useful as serological diagnostic antigens, serving as biomarkers, in suspension microarrays. The frequent appearance of novel pathogens ,2,3,4,5,There are a significant number of severe infections that can be difficult to diagnose without appropriate serological information, which calls for diagnostic arrays covering many differential diagnoses. The increased travel between continents increases the need for geographically-unrestricted diagnostic panels.Moreover, the frequent appearance of novel pathogens emphasizes the need for an ability to generically detect, and rapidly evolve, diagnostic techniques that can efficiently respond to such challenges. Antigens for such serological diagnostic systems optimally need to be prepared without any delay. Novel pathogens are now often identified through rapid sequencing ,11,12. IIn the following, we give examples of the bioinformatic selection and experimental evaluations of diagnostically-useful large synthetic antigens. The diagnostic antigens were obtained from sequencing of targeted pathogens and they were produced in a form suitable for SMIA.2-PEG6-His6-PEG6 at their amino terminus, where PEG6 is hexaethylene glycol and His6 is hexahistidine (enabling coupling control via anti-His antibodies). The peptides were synthesized under optimized conditions for long peptides . Briefly, solid phase synthesis was done on an automated multiple-peptide synthesizer , using coupling times and conditions optimized for high coupling yield. For amino-terminal protection, the base-labile FluorenylMethylOxyCarbonyl chloride (FMOC) group was used. In a few cases, megapeptides were subsequently analyzed by reverse-phase high-performance liquid chromatography (HPLC) on a C18 column. They yielded several peaks, indicating non-homogeneity (unpublished data). Lyophilized peptides were dissolved in dimethyl sulfoxide (DMSO) .The peptide sequences are detailed in The epitope prediction tools at the Immune Epitope Database  and 3D sIn the Ebola study, control antigens were recombinant glycosylated E proteins of the Sudan (SEBOV), Zaire Ebola (ZEBOV), Reston, and Bundibugyo strains of Ebola virus, and Marburg virus. Control sera were rabbit anti-Bundibugyo, anti-SEBOV, and anti-ZEBOV glycoproteins, respectively. They were all purchased from Integrated BioTherapeutics Bioservices, Gaithersburg, MD, USA. A convalescent serum from a patient diagnosed with an uncertain filovirus infection was obtained with informed consent. Anonymous blood donor sera from the Uppsala Academic Hospital were used as negative controls. They were all used with informed consent according to the Swedish Biobank law which allows diagnostic patient samples to be used for similar purposes as the original sampling purpose.In the HHV7 and JCV studies, we used 82 and 131 blood donor sera  which contained HHV7 and JCV antibody reactivity.7 beads per ml. We have found the optimal quantity of antigen for coupling of 200 μL of Magplex microsphere suspension to be 5–10 μg of recombinant protein antigen and 50 μg of synthetic peptide antigens. However, it is possible to save reagents by using smaller amounts and volumes. All volumes in the recipe should then be decreased proportionally. After the coupling of antigen with beads, beads were incubated with 0.5 mL of PBS containing 0.05% (v/v) Tween 20 and 50 mM Tris (PBST) in the dark for 15 min on a rocking mixer at room temperature. The purpose of which was to block unreacted carboxyl groups with primary amines. The beads were then washed once with 0.5 mL StabilGuard  using a magnetic separator. The bead pellet was finally resuspended in 400 µL StabilGuard. This created a bead mixture consisting of 6250 beads/µL. The coupled beads were stored at 4 °C in the dark. We found that coupled beads could be stored for at least a month (unpublished data). Prolonged storage at 4 °C can create problems due to antigen degradation, bacterial, or fungal growth. The beads with different numbers can be combined to make a serological panel for use in a multiplex system which theoretically could have a maximal multiplexity of 200 at a time. However, this becomes expensive due to antigen and bead costs. In our work, we have stayed at multiplexities below 50.Luminex carboxylated differentially-color-marked magnetic microspheres  were used for the coupling of antigens. Coupling was performed according to the protocol provided by the manufacturer. See the sample protocol for two-step carbodiimide coupling of protein to magnetic beads . A detaiv/v) Tween 20, 50 mM Tris and 2% (v/v) Prionex  (PBSTP) was added to wells of a round bottom 96-well microtiter plate  excluding the blank and controls. Fifty microliters of a vortexed and sonicated bead mixture consisting of 25 beads/µL suspended in PBSTP was then added to each well. The plate was then incubated in the dark with gentle rotation for 1 h at 37 °C. Post incubation, the wells were washed with PBS using a magnetic plate separator . After that, the beads were resuspended in 50 µL of PBSTP and 50 µL of biotinylated-protein G  at the concentration of (4 µg/mL of PBSTP) in each well. The plate was then placed for second incubation for 30 min at 37 °C in the dark with rotation. The wells were washed again with PBS and the beads were resuspended in 50 µL of PBSTP followed by the addition of 50 µL of streptavidin-phycoerythrin (SA-PhE)  at the concentration of (4 µg/mL in PBSTP) in each well. The plate was placed for the final incubation for 15 min at 37 °C in the dark with rotation. The beads were washed once with PBS before they were resuspended in 100 µL of PBS and analyzed in a Luminex-200  instrument according to the instruction from the manufacturer. A minimum of 100 events for each bead number was set to read and the median value was obtained for the analysis of the data. All samples were analyzed in duplicate and average readings calculated. To detect any antibody binding to the beads themselves, a naked non-peptide-containing (“blank”) bead was included. A control His6 bead was also included in the SMIA reaction. The His6 tag allowed monitoring of coupling of the megapeptides to the magnetic beads using anti-hexahistidine antibodies . We did not observe false positive reactions due to anti-His6 in the sera tested for this paper. One negative control, where PBSTP instead of serum was added, was also used in all experiments. The work flow is presented in Fifty microliters of serum diluted 1/20 in phosphate buffered saline , pH 7.4, containing 0.05%  megapeptide derived from the extracellular portion of the surface glycoprotein of Zaire Ebola virus would function as an antigen in SMIA. We compared the reactivity with positive control, patient, and negative control serum, of the megapeptide and a glycosylated recombinant protein from the same viral strain and from related filoviruses .All 40 negative control sera (Swedish blood donors) were negative in the test (unpublished data). The positive rabbit control serum reacted as strongly with the megapeptide as with a recombinant glycoprotein from the same virus A. DilutiBetaherpesvirinae). We strive to optimize herpesvirus serology and the mapping of optimal gB antigens for HHV7 is part of this. Betaherpesvirinae remains to be established.Human herpesvirus 7 (HHV7) is ubiquitous and is the least known of the nine human herpesviruses. It has mostly been associated with mild disease . HoweverAnother group of cross-reactive viral antigens are the capsid proteins (VP1) of human polyomaviruses . Many neWe developed new rapid and rational ways to produce antigens for microarrays, both what we call “megapeptides”, extraordinarily long synthetic peptides, in essence synthetic proteins (described in this paper), and by simple production of recombinant proteins .By using megapeptides we are coupling the generation of sequences, which can be derived from next-generation sequencing, to bioinformatics for an optimal selection of sequences for diagnostic antigens. We assumed that although long synthetic peptides are imperfect compared to recombinant proteins of the same length, they are good enough for many purposes. If the coupling efficiency typically is 99% per amino acid, a 100 aa megapeptide is likely to contain at least one error. In a few control experiments we found that megapeptides gave broad peaks in HPLC, indicating inhomogeneity (unpublished data). However, the assumption was that the majority of the molecules would contain enough of correct sequence to be able to mimic also conformational epitopes. The results presented in this paper indicate that this assumption was correct.Currently, it takes around 70 h to generate an assembled sequence from a sample using Illumina technology. Then, it takes at least 5 h to identify any microbial sequence from this run; in total, around four days. Megapeptides likely to be useful as antigens are then selected and ordered. Peptide synthesis of a 200 aa takes 300 h (13 days). They must then be transported and coupled to SMIA beads (one day). After 18 days, i.e., less than three weeks, a multiplex serology with a reasonable chance of detecting antibodies to the emerging microbe could be in place. Here, we give examples of the utility of megapeptides in suspension multiplex immunoassay systems we currently work with. These are from Filoviruses (Ebola), Herpesvirus 7, and Polyomavirus JC.Before the antigens can be created, diagnostically-useful peptides must be selected. Unless the location of highly antigenic epitopes is known or highly likely from analogy with related microbes, it is recommendable to synthesize several, e.g., five different megapeptides. The selection of peptide sequences can be from highly-antigenic surface antigens, predicted epitopes, see e.g., the Immune Epitope Database , or by kThe megapeptides tested here were smaller than the complete protein counterparts. The Zaire Ebola glycoprotein (accession nr AHX24667) is 676 amino acids long. HHV7 gB (accession number YP_073779) is 822 amino acids long. JCV VP1 (accession number NP_043511.1) is 354 amino acids long. It is natural that they cannot mimic the antigenicity of the whole molecule. Long recombinant proteins can cover more of the antigenicity. However, megapeptides are simpler to produce and are often cheaper than recombinant proteins on a weight basis. In this paper we could, therefore, use a higher molar concentration of megapeptide than of recombinant protein. From numerous titration experiments with other peptides and megapeptides (unpublished data) we assume that the given conditions for megapeptides are within 50%–100% of the saturation limit of the beads. Like in any immunoassay, the final antigen concentration to be used in an assay ready for routine use must be found by checkerboard titration against suitable antisera. This was not done for this work, whose purpose was proof of principle.SMIA has several inherent advantages as a serological diagnostic and surveillance system. First, it can combine the antigenicities of several antigens for each targeted microbe. This enables automatic differentiation of true from false positive results, like for a Western or line blot, in a simple test suited for primary testing. The use of several antigens for a single microbe can also avoid false negative results due to target antigen variation, unequal timing of immune responses to different antigens during an infection, or genetic differences in immune response to individual antigens. It is also suitable for syndromic or microbial family-based serological panels. The megapeptide is one of several antigen types which can contribute to these applications.In this report, we show results with megapeptides of lengths between 102 and 142 amino acids from viruses of three expanding virus families. In the case of JC polyoma virus VP1 and Human Herpesvirus 7 gB, overlapping megapeptides were used. In the case of Ebola virus, much of the extracellular portion of the surface glycoprotein (the portion most exposed to the immune system) was synthesized. It is likely that, in the future, with further refinement, megapeptides can contribute to improved and quickly-implemented serological diagnosis of these, and other, viruses.We conclude that long synthetic peptides can be useful as antigens in microarrays for antibody detection. They can be created relatively simply and quickly just from sequence information.


The presentation of peptide-MHCII complexes (pMHCIIs) for surveillance by T cells is a well-known immunological concept in vertebrates, yet the conformational dynamics of antigen exchange remain elusive. By combining NMR-detected H/D exchange with Markov modelling analysis of an aggregate of 275 microseconds molecular dynamics simulations, we reveal that a stable pMHCII spontaneously samples intermediate conformations relevant for peptide exchange. More specifically, we observe two major peptide exchange pathways: the kinetic stability of a pMHCII's ground state defines its propensity for intrinsic peptide exchange, while the population of a rare, intermediate conformation correlates with the propensity of the HLA-DM-catalysed pathway. Helix-destabilizing mutants designed based on our model shift the exchange behaviour towards the HLA-DM-catalysed pathway and further allow us to conceptualize how allelic variation can shape an individual's MHC restricted immune response. MHCII proteins bind and present both foreign and self-antigens to potentially activate CD4+ T cells via cognate T cell receptors (TCRs) during the adaptive immune response. Here, the authors combine NMR-detected H/D exchange with Markov modelling analysis to shed light on the dynamics of MHCII peptide exchange. Professional antigen-presenting cells express a subset of different alleles of heterodimeric MHCII proteins on their surface. These MHCII proteins are capable of binding and presenting both foreign and self-antigens to potentially activate CD4+ T cells via cognate T cell receptors as part of the adaptive immune response. The structural hallmarks of antigen binding by MHCII molecules are well understood at the phenomenological level: Optimal occupation of the four main pockets  in the MHCII-binding groove by peptide side-chains and concomitant formation of a conserved set of hydrogen bonds mainly between peptide backbone and MHCII side-chains are necessary and sufficient to guarantee high-affinity binding of certain epitopes and their subsequent surveillance by T cells1245791011Antigen-specific recognition of peptide-major histocompatibility class II (MHCII) complexes (pMHCIIs) by CD4P1, P4, P, P9 in t1 pocket region required for DM binding of a pMHCII . The expected advantage of choosing this reference is an increased stability of the pMHCII as the tryptophan side-chain optimally fills the P1 pocket that dominates the DR1-binding groove, but cannot be accommodated by P9 and thus prevents peptide reorientationw complex is markedly more resistant to exchange against a fluorescein-labelled haemagglutinin (HA)-derived peptide than DR1 loaded with wild-type CLIP (DR1/C), as detected by an FP-based peptide-exchange assay  measurements that report the dynamical exposure of amide protons to the solvent. HDX protection factors were derived from exponentially-fitted (EX2 regime) intensity decays of backbone amide signals in a series of heteronuclear single quantum coherence (HSQC) spectra  simulations. Over 160 simulation trajectories adding up to about 90 microseconds of total simulation time were collected—and thus two to three orders of magnitude more sampling than in previous simulation studies9101124 C79-R93 , consist C79-R93 , consistW predominantly exists in a ground state, but the N-terminal end of the peptide-binding groove including the DM-binding site in the α1-domain as well as the β1-domain α-helix spontaneously samples low-populated conformations. Despite a well-occupied P1 pocket the outward-flipping of the αW43 side-chain appears to be a rare feature of both the ground state and the low-populated conformations.Taken together, the kinetically stable pMHCII DR1/C2 site, which have been shown to contribute remarkably to pMHCII stability13W is a kinetically destabilized complex that displays a clear reduction in thermal stability of 19 °C , while binding of DR1/CW to DM was hardly detectable  value of only 0.432 Å  that tightly binds DM. This biochemical phenotype is not explained by the ground-state structure as detectable by crystallography and NMR.Thus, even in the context of an optimal PW could not be explained by its ground-state structure, we wondered whether its structural ensemble comprises excited metastable states that account for accelerated peptide-exchange and increased DM binding.As the biochemical phenotype of DR1-βN82A/CW and DR1/CW is given by a comparison of 1H-15N TROSY-HSQC peak intensities: Signal-to-noise levels of the DR1-βN82A/CW β1 domain are reduced to 48% as compared with DR1/Cw , and one where the β1 domain α-helix is largely unfolded at residues βQ70-R93   . In ordeW in the DM-catalysed peptide exchange pathway.Taken together, the βN82A mutation destabilizes the β1 domain α-helix fold, which contributes to an overall increase of the mobility in the entire region surrounding the peptide N terminus. This destabilization shifts the conformational ensemble towards the low-populated state that shares features of the DM-bound conformationW is its high and DM-independent peptide-exchange rate  and additionally aimed at stabilizing the helix by replacing the same residues by leucine, which has a higher propensity for helix formation due to hydrophobic contacts (DR1-βG84LβE87L) .W) or two (DR1-βG84PβE87P/CW) leads to complexes with gradually increased binding to DM, whereas DR1-βG84LβE87L/CW shows a similarly negligible response to DM as DR1/CW  that is characterized by low affinity for DM, but exerts DM-dependent peptide exchange, with the kinetically destabilized mutant DR1-βN82A/Cw. This mutant, in contrast, showed strong DM-binding albeit with DM-independent peptide exchange. Based on aggregate results we are now able to propose a detailed mechanistic model of MHCII peptide exchange  for 48 h at 37 °C. Soluble monomeric pMHCII complexes and peptide-free MHCIIs were finally obtained by applying the proteins and loading reactions onto a size exclusion chromatography column and buffer exchanged to PBS, pH 5.8. DM was expressed and purified using the baculovirus-insect cell expression system  as previously describedHLA-DR proteins were produced as described previously49306-318-fluorescein isothiocyanate (HA-FITC) on 1 μM DR1/peptide complexes was followed by fluorescence polarization in the absence or presence of 250 nM DM (if not stated otherwise). The reactions were set up in 40 μl in triplicates in PBS buffer, pH 5.8 at 37 °C (W experiments were performed at 25 °C (306-318-FITC complexes was monitored in the presence and absence of 20 μM competitor peptide (HA306-318) and 150 nM DM in PB/Citrate buffer, pH 5.5 at 37 °C as previously describedLoading of 200 nM HAat 37 °C  and 4b. at 25 °C . Peptide−1 in a Biacore 3,000 device . Non-specific binding of the reference flow cells was subtracted from the DM-coupled flow cells. DR1/peptide complexes were injected for 300 s into DM-immobilized or control flow cells followed by 300 s buffer injection and 450 s of 50 μM HA peptide. The chip was regenerated by injecting high-affinity peptides for the respective HLA-DR alleles in the flow cell at concentrations ranging from 20 to 100 μM.In all, 400–500 RU of biotinylated DM was coupled to streptavidin , as previously described−1 monomeric pMHCII protein was mixed with 5 × Sypro Orange (Life Technology). While the temperature was increased for 2 °C min−1, the emission was detected at 575 nm in lifetime after exciting the dye at 490 nm. Fluorescence intensity was plotted versus the temperature and a sigmoidal function was fitted to determine the midpoint temperature of the unfolding reaction (Tm). Stability measurements were performed in PBS buffer, pH 5.8.In order to determine the thermal stability, 0.2–0.5 mg mlw and DR1-βN82A/CW by standard three-dimensional experiments in combination with transferring assignments from DR1/C spectraNMR spectra were acquired on a Bruker AV700 MHz spectrometer equipped with a 5 mm triple-resonance cryoprobe. Backbone assignments of approximately 90% α- and around 80% β-chain resonances were obtained for DR1/C2O with 35–100 μM pMHCII. Spectra were processed with Topspin (Bruker) and analysed with CcpNmr AnalysisAll NMR measurements were performed at 310 K in PBS buffer pH 5.8 containing 10% D15Nα- and/or 15Nβ-labelled. DR1-βN82A samples used for assignments were additionally 13C labelled. NOESY-HSQC spectra with 80 ms mixing time were acquired for 2H- 15Nα/β-labelled DR1-βN82A/Cw with specific labelling of 1H-15N-Ala/Trp in the α and 1H-15N- Trp in the β-chain.MHCII complexes were partly deuterated α and fully deuterated β and Chemical shift perturbation was calculated according to 1H-15N-TROSY-HSQC spectra of 50 μM 15N β-labelled DR1/Cw , 50 μM DR1-βN82A/Cw , 60 μM 15N α-labelled DR1/Cw  and 35 μM 15N α-labelled DR1/Cw  measured at 37 °C for CcpNMR Analysis was used to determine the average spectral noise levels from peak-free regions. For signal to noise level calculation peak-heights were determined from Protected hydrogen atoms can exchange against deuterium in a transient energy state of a protein region, typically involving H-bond breakage kobs is the observed exchange rate, kint the intrinsic exchange, which has to be experimentally determined , and P−1 is the amide protection factor. Based on the knowledge of Kex, the energy difference between the open and closed state is calculated using the following equation:where Gop/cl refers to the energy difference between the open and the closed state, R to the universal gas constant  and T to the absolute temperature in Kelvin.where Δ15Nα- and/or 15Nβ-labelled DR1/CW complexes were concentrated to 80–100 μM in PBS buffer, pH 5.7, 10% D2O, 0.02% NaN3. Reference HSQC spectra were acquired with 1,024 × 96 complex data points and 16 scans at 37 °C. Afterwards, these DR1/peptide complexes were shock frozen and lyophilized overnight. Immediately after re-solubilization in 100% D2O a set of 90 HSQC experiments were acquired with a measurement time of ∼35 min per HSQC. At least 20 data points of the HSQC data set was used to plot S/N ratios of NH peaks against the experiment time. NH groups exchanging to an undetectable level within the dead time of the HSQC measurement (∼35 min) were considered to have half-lives (t1/2) of <15 min, while NH groups not showing adequate signal decay after 60 h were classified as highly stable, with a t1/2 >4,000 min and peaks with overlapping maxima were mostly excluded from the analysis. For peaks, which showed a clear decay within the experiment time (∼60 h), an exponential decay function of first order was fitted to the data according to the EX2-limitKex1− was obtained by normalizing the observed exchange rates for the intrinsic exchange rates .Partly deuterated α and fully deuterated β W and 124 DR1-βN82A/CW residues displayed t1/2 of <15 min, 87 DR1/CW and 72 DR1-βN82A/CW residues displayed t1/2 >4,000 min, 85 DR1/CW and 64 DR1-βN82A/CW residues could be fitted.All together, 99 DR1/CEscherichia coli. To obtain DR1/CW the peptide was loaded a posteriori using a 10- to 20-fold molar excess of peptide in the presence of 1 mM AdEtOH2  PEG3350, 0.1 M BisTrisPropane, pH 8.5.Both proteins were crystallized using commercial sparse matrix screens. DR1 was purified and refolded from OH2 ref.  as molecW, the protein was purified and refolded from E. coli and gel filtrated using a Superdex 200 column equilibrated in 20 mM MES, 50 mM NaCl, pH 6.4. Protein was concentrated and mixed with an equimolar amount of DM purified from S2 cells in the same buffer. The final protein concentration was 10 mg ml−1. Crystals were grown by vapour diffusion in sitting drops in 0.2 M Na nitrate, 20% (w/v) PEG 3350, 0.1 M BisTrisPropane, pH 7.5.For DR1-βN82A/CIn both cases, crystals were harvested and flash frozen in the crystallization condition supplemented with 20% glycerol. Data from these crystals were collected at beamline 14.1 at Helmholtz-Zentrum Berlin.In both cases, data sets were processed using the XDS packageW, no DM could be identified in the electron-density. Both structures were deposited at the Protein Data Bank with accession codes 4X5W and 4X5X.Despite the presence of equimolar amounts of DM in the crystallization conditions for the crystals of DR1-βN82A/CW (4X5W) crystal structures were analysed. pKa values of titratable residues for each structure were estimated with the program propKaW and DR1-βN82A/CW were modelled atomically clash-free by substituting the βAsn82 (CLIP Met107) side-chain with the one of Ala (Trp) resulting in four different starting structures. The starting structures for 162 DR1-βT41A/CW simulations were generated from the MSM of the DR1/CW simulations by uniformly sampling in the kmeans-clustered (k=6≈ sqrt(no. of microstates in the largest connected MSM set)) space of the first three MSM eigenvectors. Each structure was aligned onto its principal axes and solvated with a 150 mM l−1 NaCl using VMDThe DR1/C (3PDO) and DR1/C−1 Langevin damping, 1 atm Berendsen pressure and 800 fs relaxation time, and with harmonic, positional restraints on all protein heavy atoms . For the first 4ns in steps of 0.1 ns, these restraints were gradually reduced to zero, while the simulation temperature was gradually increased from 10 K to 310 K. For the remaining MD simulations, the pressure control was turned off , and the Langevin damping was set to 0.1 ps−1. Each of these remaining simulations, that is, of the six replicates for each DR1/CW and DR1-βN82A/CW, we performed for about 1 μs. Each final restart point of these simulations was then replicated again (27 times), and simulated on the Titan supercomputer for another 550 ns each. Each of the 162 DR1-βN82A/CW simulations was run on an in-house graphics-processing unit (GPU) cluster for 200–1,100 ns. The resulting MD simulation data set of 486 individual simulations with 275 μs total aggregated simulation time was then used for analysis.Simulations were run using ACEMD−1 Å−2 and 2 kcal mol−1 Å−4, respectively) were carefully chosen, so that with a minimal bias, the minimal distances between pMHCII and each of its periodic images would not fluctuate on average over all three box dimensions by more than 0.8 Å during short MD test simulations (<50 ns). In the course of our MD simulations, these minimal distances were closely monitored not to be smaller than two times 9 Å, the electrostatic cutoff.Throughout all our MD simulations, we harmonically restrained the center of mass and the inertia tensor's off-diagonal elements of pMHCII's heavy atoms to zero. We implemented these restraints as an ACEMD plugin, which is available upon request. This strategy allowed us to perform our MD simulations with the periodic boundary conditions more efficiently, as the principle components of this intertia tensor had been aligned in the starting structures (see ‘MD set-up'). This gain in efficiency (∼50% speed-up for pMHCII) is due to the fact that an additional box size  is not necessary to accommodate for the rotational tumbling of pMHCII under periodic boundary conditions, as has been studied previously64www.pyemma.org)W as well as the DR1-βN82A/CW, and DR1-αT41A/CW mutants were transformed and discretized in the same way in order to arrive at models at which the effect of the mutant can be consistently compared: In particular, the MSMs for DR1/CW and DR1-βN82A/CW were computed from the combined discretized simulation set of DR1/CW and DR1-βN82A/CW, whereas the MSM for DR1-αT41A/CW was determined from the combined discretized simulation set of DR1/CW, DR1-βN82A/CW and DR1-αT41A/CW. To this end, all pairwise Cα-coordinates of residues within 15 Å of βN82 in a simulated 3PDO conformation , βR13-βL27 (not βN19), βR71-βR93 ) were used as input coordinates to a time-lagged independent component analysis6967MSMs2447666793 not βR, βn82) wPairwise interactions and their significant differences between pairs of metastable states were detected with a modification of the system-wide Pairwise Interaction Analyser75W (4X5W) and DR1-βN82A/CW (4X5X) have been deposited with the Protein Data Bank with accession codes 4X5W and 4X5X, respectively. The data that support the findings of this study are available from the corresponding authors upon request.The crystal structures of DR1/CHow to cite this article: Wieczorek, M. et al. MHC class II complexes sample intermediate states along the peptide exchange pathway. Nat. Commun.7, 13224 doi: 10.1038/ncomms13224 (2016).Supplementary Figures 1 - 10 and Supplementary Tables 1 - 4Molecular movie illustrating the intrinsic, partial peptide dissociation pathway in the ground state MS3. The movie was extracted from a βN82A simulation. The molecular representation was chosen as in Figs. 1e, 3d, 4g. The representative simulations are filmed here at a frame rate of 10ns/1sec, and are smoothed with an averaging window of 11ns.1-chain α-helix is followed by flipping out of the αW43 sidechain. The movie was extracted from a WT simulation. The molecular representation was chosen as in Figs. 1e, 3d, 4g. The representative simulations are filmed here at a frame rate of 10ns/1sec, and are smoothed with an averaging window of 11ns.Molecular movie illustrating a possible transition pathway from MS3 to the transition state candidate MS1, in which a destabilization of the β1-chain α-helix. The movie was extracted from a βN82A simulation. The molecular representation was chosen as in Figs. 1e, 3d, 4g. The representative simulations are filmed here at a frame rate of 10ns/1sec, and are smoothed with an averaging window of 11ns.Molecular movie illustrating a possible transition pathway from MS3 to the transition state candidate MS1, in which flipping out of the αW43 sidechain is followed by destabilization of the β


Cryptosporidium is a leading cause of pediatric diarrhea in resource-limited settings; yet, few studies report the health care costs or societal impacts of this protozoan parasite. Our study examined direct and indirect costs associated with symptomatic cryptosporidiosis in infants younger than 12 months in Kenya, Peru and Bangladesh. Inputs to the economic burden model, such as disease incidence, population size, health care seeking behaviour, hospital costs, travel costs, were extracted from peer-reviewed literature, government documents, and internationally validated statistical tools for each country. Indirect losses  were also estimated. Our findings suggest that direct treatment costs per symptomatic cryptosporidiosis episode were highest in Kenya ($59.01), followed by Peru ($23.32), and Bangladesh ($7.62). The total annual economic impacts for the 0–11 month cohorts were highest in Peru , followed by Kenya  and Bangladesh . For all scenarios, indirect societal costs far outweighed direct treatment costs. These results highlight the critical need for innovative improvements to current prevention, diagnostic and treatment strategies available in resource poor settings, as well as the need for solutions that span multiple disciplines including food and water safety, sanitation and livestock production. Cryptosporidium spp. oocysts shed in the stool of infected people or animals, and commonly experience mild to severe diarrhea, which may last up to one month [Cryptosporidiosis is a leading cause of moderate-to-severe diarrhea (MSD) in infants from resource poor regions, and particularly, where sanitation and drinking water infrastructure are lacking –3. Indivne month . Childrene month ,4. Cryptne month –10.Cryptosporidium spp. oocysts in stool samples do not yet exist, leading to treatment courses that focus on symptoms rather than pathogen reduction [Cryptosporidium has a low infectious dose, can survive long periods of time under a variety of environmental conditions, and is resistant to household disinfectants, such as chlorine [In recent years, two multiyear studies conducted in 12 countries across three continents investigated the etiologies of pediatric diarrhea  and the Interactions of Malnutrition & Enteric Infections: Consequences for Child Health and Development project (MAL-ED) . Althougeduction . Definiteduction . This inchlorine . Altogetchlorine .The economic costs of cryptosporidiosis, incurred both by infected individuals and by health care systems, have not been well characterized in the peer-reviewed literature. One cost-of-illness analysis conducted after a waterborne outbreak in Milwaukee, USA highlighted the relative importance of productivity losses (64.6M USD) over medical treatments (31.7M USD), and a wide range in costs between treating mild versus severe cases  . In the Cryptosporidium surveillance, health care costs, and health care seeking behaviour for each of these countries, and then ranked them within continent according to the amount of available data. We selected one country per continent in order to gain regional representation from areas that differed in relevant factors including healthcare capacity, pathogen distribution, demographics, economics, and healthcare seeking behaviour. Suitability for inclusion in our study was primarily based on two criteria: 1) adequate data, and 2) low-to-moderate cryptosporidiosis incidence, as reported by GEMS or MAL-ED.Together, GEMS and MAL-ED reported incidence of pediatric cryptosporidiosis in study sites in 12 low-resource countries in Sub-Saharan Africa, South America and South Asia. We searched PubMed and Google Scholar for literature reporting We used both a healthcare and societal approach to measure the economic burden of cryptosporidiosis for infants aged 0–11 months in Kenya, Peru and Bangladesh. To assess the burden from a societal perspective, we considered direct medical, direct non-medical and indirect costs. Direct medical cost estimates included health practitioner fees, diagnostic tests and drug costs, including ORS. Direct non-medical costs included transportation to seek care. We considered all types of healthcare costs including out-of-pockets expenditures, government payments, as well as private and public health insurance. Indirect costs included caregiver income loss, lost lifetime earnings due to morbidity , and mortality. All costs were converted using purchasing power parity and presented in 2016 international dollars. We discounted all future costs at a rate of 3% per year as recommended by the WHO and used a life-long time horizon [We conducted one-way sensitivity analyses on up to 10 key variables per country. Variables were selected either because they had the potential to substantially impact overall costs or because there was a high degree of uncertainty in the parameter estimate. We varied the discount rate between zero and five percent. As well, we included a scenario analysis where we varied all growth faltering parameters simultaneously. Furthermore, we conducted best and worst case scenario analysis to determine the projected highest and lowest economic impact of pediatric cryptosporidiosis for each country under our current assumptions.Cryptosporidium in Kenya and Bangladesh were derived from GEMS [Cryptosporidium, we combined these parameters with the proportion of MSD to LSD cases to calculate the total number of diarrhea cases attributable to Cryptosporidium for the 0–11 month age group (We obtained age-specific population size estimates from the most recent in-country census for Peru 2007), Bangladesh (2011), and Kenya (2009) and inflated them to 2016 values using World Bank population growth estimates . The num007, Bangge group .Cryptosporidium and the frequency of diarrhea episodes per child per year reported by MAL-ED [For Peru, we calculated the number of age-specific cases using the percentage of diarrheal cases attributable to y MAL-ED , as wellMedical costs were calculated using country-specific estimates of caregiver health care seeking behaviour for cases of diarrhea. To estimate health care seeking behaviour for Kenya and Bangladesh we used Health Utilization and Attitudes Surveys (HUAS) that were completed prior to the GEMS study. The HUAS qualify and quantify the health sectors accessed by caregivers of childhood diarrhea cases that seek health care  ,34. For We included three different types of indirect costs: (1) caregiver income loss costs; (2) mortality costs; and (3) growth faltering costs. Caregiver costs were extracted from in-country household interviews ,28,36,41Where:LE = life expectancy in a given countryn = value between 15 (year earning begins) and life expectancy (year earnings end)Cryptosporidium infection would not experience catch up growth, and that permanent stunting would impact future potential earning. Our estimates of these factors were based on a longitudinal birth cohort of children in Bangladesh monitored for growth faltering and cryptosporidiosis [Cryptosporidium infection (i.e. 8.8%). We assumed that only children categorized as severely stunted (Height-for-age- adjusted z-score (HAZ) ≤-3) would remain stunted into adulthood (i.e. 23.8% of the cohort). Country-specific income loss due to growth faltering was calculated as follows:Finally, we assumed that a fraction of infants with acute growth faltering attributable to ridiosis . To deteWhere:LE = life expectancy in a given countryn = value between 15 (year earning begins) and life expectancy (year earnings end)Overall, Peru had the highest incidence of cryptosporidiosis in children in the first year of life (0.091 cases/person-year), followed by Kenya (0.084 cases/person-year) and Bangladesh (0.0093 cases/person-year); however, Bangladesh had the largest cohort size for that age group . Kenya had the highest number of cryptosporidiosis cases, despite having less than half the number of infants in the 0–11 month cohort compared to Bangladesh. In the first year of life, mortality attributable to cryptosporidiosis was highest in Kenya , followed by Bangladesh  and lowest in Peru . Health care seeking behaviour also differed by country with variations in the type of care sought , and the percentage of caregivers who accessed each type of care for their children.Our evaluation of health care costs incurred by cryptosporidiosis patients suggests that the direct cost per case varied from $23.32 (range $9.97-$60.84) and $7.62 (range $3.45-$4.12) in Peru and Bangladesh to $59.01 (range $12.5-$454.80) in Kenya . This diCryptosporidium infections were lower in Kenya ($24.2M) and Bangladesh ($5.5M). Indirect costs contributed the largest portion of economic burden caused by pediatric cryptosporidiosis, accounting for 80.9%%, 97.1% and 97.6% of costs in Kenya, Peru and Bangladesh respectively. Growth faltering was responsible for the greatest impact on overall costs in all three countries. Of the direct costs, inpatient costs in Kenya had the greatest economic impact; however, in Peru and Bangladesh outpatient care was estimated to be the principal direct cost.When all types of costs were considered, Peru had the highest total annual economic burden due to cryptosporidiosis in infants aged 0–11 months ($28.5M), even though it had the smallest cohort population and the lowest mortality rate. Annual costs for symptomatic Cryptosporidium infection and the frequency of faltered growth persisting into adulthood , followed by Peru ($23.32) and Bangladesh ($7.62). The higher direct costs observed in Kenya are mainly attributable to higher outpatient and inpatient utilization rates in Kenya, relative to Bangladesh or Peru. GEMS reported mean costs per diarrheal episode as 1.82 USD in Bangladesh [We estimated that direct costs per ngladesh  and 6.24ngladesh . This dingladesh . Furtherngladesh . The ovengladesh .Cryptosporidium is a zoonotic protozoan, with the potential to inflict losses on other industries, such as cattle production. Approximately 26 Cryptosporidium species are currently recognized, most of which exhibit high host specificity and generally cross the species barrier only when they encounter immune compromised individuals. Humans are the primary definitive host for C. hominis, but are also infected by C. parvum, which is a zoonotic species infective to numerous nonhuman hosts. Molecular analysis of a subset of GEMS participants with MSD identified C. hominis as the most common Cryptosporidium species, indicating that human-to-human transmission was a greater cause of pediatric infection than zoonotic transmission [C. hominis versus other species [Our economic evaluation focused on human cases of cryptosporidiosis; however, smission . Other ssmission , Peru [4smission , and Bansmission , also re species ,46. TogeOur analysis aimed to quantify and describe country-specific practices related to seeking health care, diagnosing, and treating children with cryptosporidiosis symptoms. It is important to note that many of the reported norms did not align with practices endorsed by the medical community, and that this paper does not report health care costs in settings where all children receive evidence-based care. In Peru, for example, antibiotics are commonly prescribed to treat diarrhea cases in lieu of diagnostic tests paired with pathogen specific treatment. Only 31% of Peruvian children under five with diarrhea receive ORS, and nitazoxanide use is uncommon . In BangA critical gap currently exists in our understanding of the societal, pathological, and economic impacts of cryptosporidiosis in high-risk groups and geographic locales. We limited our evaluation to one high-risk age cohort in each of the three target countries because important limitations currently exist in data related to disease incidence, social determinants of health, health care seeking behaviour, cognitive delay and long-term sequelae . Pediatric cryptosporidiosis is a complex illness with risk factors that relate to the immune status of hosts, to species differences in the parasite, and to social determinants of health that impact exposure and recovery . In particular, it remains unclear whether short-term stunting observed in children younger than five persists into adulthood, and whether other sequelae associated with diarrhea  are also associated with cryptosporidiosis ,6,8,10. Cryptosporidium infection a challenging problem. Successful mitigation will likely require a multi-sectoral One Health type approach, integrating input from experts in water, sanitation, and hygiene (WASH), food safety, livestock production, education, and family health  [Other differences relating to demographics were not captured by our analysis, including disparities between rural and urban care seekers, and willingness to seek care for male versus female children. In the absence of more comprehensive data, our model used cryptosporidiosis incidence estimates established for rural districts in Kenya, Peru, and Bangladesh in combination with care seeking and cost information established for both urban and rural areas. Improvements to available data are needed in order to better characterize human, veterinary, and societal costs of cryptosporidiosis. Furthermore, the complexity of disease transmission along with poor treatment options and the complex interaction of this disease with other health compromising conditions  make mitigation of trition) . In partS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


We performed an in vitro dolutegravir resistance selection experiment by using a breakthrough method. First, MT4 cells were infected with human immunodeficiency virus type 1 (HIV-1) Lai. After integration into the host cell genome, cells were washed to remove unbound virus and 500 nM dolutegravir was added to the cell medium. This high concentration of the drug was maintained throughout selection. At day 80, we detected a virus highly resistant to dolutegravir, raltegravir, and elvitegravir that remained susceptible to zidovudine. Sequencing of the virus showed no mutations in the integrase gene but highlighted the emergence of five mutations, all located in the nef region, of which four were clustered in the 3′ polypurine tract (PPT). Mutations selected in vitro by dolutegravir, located outside the integrase gene, can confer a high level of resistance to all integrase inhibitors. Thus, HIV-1 can use an alternative mechanism to develop resistance to integrase inhibitors by selecting mutations in the 3′ PPT region. Further studies are required to determine to what extent these mutations may explain virological failure during integrase inhibitor therapy.Resistance to the integrase strand transfer inhibitors raltegravir and elvitegravir is often due to well-identified mutations in the integrase gene. However, the situation is less clear for patients who fail dolutegravir treatment. Furthermore, most  in vitro and involve the integrase gene. Most mutations selected by raltegravir, elvitegravir, or dolutegravir exposure are located inside the catalytic site of the integrase gene, but mutations outside the catalytic site of the integrase gene have also been selected with dolutegravir. Following in vitro selection with dolutegravir, we report, for the first time, a virus with selected mutations outside the HIV-1 integrase gene that confer resistance to all integrase inhibitors currently used to treat patients, such as raltegravir, elvitegravir, and dolutegravir. Our observation may explain why some viruses responsible for virological failure in patients treated with dolutegravir did not show mutations in the integrase gene.Integrase strand transfer inhibitors (INSTIs) are increasingly used both as first-line drugs and in rescue therapy because of their low toxicity and high efficacy in both treatment-naive and treatment-experienced patients. Until now, resistance mutations selected by INSTI exposure have either been described in patients or selected  After its entry into the host cell, the HIV-1 RNA genome is converted into double-stranded DNA. This step, carried out by the reverse transcriptase, requires a different primer for the synthesis of each DNA strand; the first is a host-derived tRNA primer used to copy the viral RNA into a minus strand DNA, resulting in an RNA-DNA duplex, of which the RNA is degraded by RNase H, and the second is a 3′ polypurine tract (PPT) used as a primer for plus strand DNA synthesis . SpecifiAfter synthesis of the full-length linear viral DNA, it is integrated into the host cell chromatin through the action of the viral integrase (IN) enzyme, which catalyzes two reactions , 4. WithIN is an important target for the treatment of HIV infection because of its central role in HIV-1 replication. In 2007, IN was the last viral enzyme to emerge as a target for inhibitors to block HIV-1 replication. Integrase strand transfer inhibitors (INSTIs) are small molecules that bind to the active site of IN in the PIC context, causing it to disengage from the 3′ end of the viral DNA . CrystalIn vitro strategies using conventional resistance selection, including a gradual DTG concentration increase, showed that DTG has a resistance profile markedly distinct from those of RAL and EVG. Several mutations of nonpolymorphic residues such as S153, R263, and G118 have been described, but all were reported to confer low-level in vitro DTG phenotype changes  without DTG to allow integration of the virus. Twenty-four hours later, cells were washed twice with phosphate-buffered saline to remove unbound virus and suspended in fresh medium in the presence of 500 nM DTG, approximately 170 times the 50% effective concentration (EC50) of DTG. At the same time, the virus was also cultured without DTG. This method, using a strand transfer inhibitor at a high concentration, has already been used for HIV-2 to select resistance to EVG , comparing it to that of the wild-type Lai virus.50 of each inhibitor . The 9053 virus lost approximately 50% of its infectivity relative to the wild-type Lai virus but showed a high level of resistance to all three INSTIs tested, with fold changes (FCs) of >270 for DTG, >80 for RAL, and >160 for EVG   . Finally0, 2 μM) .nef gene, where there were several modifications clustered in the 3′ PPT region of the 9053 virus. One mutation was located six nucleotides upstream of the 3′ PPT , and three mutations and one deletion were detected inside the GGGGGG 3′ PPT pattern . The resulting sequence was A9053C and 5′AAAAGAAAAG(G9069C)(G9070A)G(G9072T)(G9073deleted). Deletion of one nucleotide in the 3′ PPT involved a change in the open reading frame of the nef gene that generates a stop codon located 4 amino acids downstream of the deletion. Consequently, the nef protein would be shortened from 206 to 99 amino acids.The resistance of the 9053 virus to the INSTIs in the absence of mutations in the IN gene led us to explore the entire genome  of the 9053 and Lai viruses cultured in parallel without DTG and compared their sequences with those of the wild-type Lai virus. There were no differences between the two genomes except in the We retrospectively sequenced the 3′ PPT region of samples taken throughout the passages under DTG selection . The firWe cloned amplified DNA obtained at days 55 and 65 . The cloWe tested the ability of the 9053 virus to infect new cells and replicate in the presence of DTG . The wil50 of 80 nM) than the wild-type virus was (EC50 of 3.5 nM), with an FC of 23   .in vitro and in vivo, have shown that mutations conferring resistance to INSTIs are selected within the catalytic site of IN  process, resulting in linear DNA that is no longer fully compatible with integration, explaining the decrease in infectivity. This nonconventional linear DNA could impair DTG binding, explaining the resistance to DTG. In addition, the deletion of one nucleotide induced an interruption in the reading frame of the ogenesis –26. Alth culture . ResultsAlthough we do not have an explanation for the mechanism used by the virus to escape DTG, this virus is clearly resistant to all currently used INSTIs. Indeed, phenotypic analysis showed a high FC in resistance to INSTIs, especially DTG (>270), whereas the virus is still susceptible to AZT, thus behaving normally with respect to inhibitors of targets other than IN. In addition, introduction of all of the mutations detected in the 9053 virus into the pNL43 backbone was sufficient to establish DTG resistance . We have thus demonstrated, for the first time, that selection of mutations outside the IN gene could be involved in INSTI resistance. Already, resistance mutations located outside the catalytic site of the enzyme, at positions 262 and 263, are specific to DTG, and similarly, it has been shown that mutations involved in protease inhibitor resistance can be selected outside the target gene, in the gag substrate cleavage sites , 29.Discrepancies between the FCs (23 for pNL43 versus 270 for the 9053 virus) can be explained by the use of two different backbones (Lai versus pNL43). It would be necessary to use a plasmid closer to the Lai sequence for which the corresponding virus would have similar infectivity to compare the impact of the mutations identified.in vitro under DTG, all located in the IN gene, showed relatively low resistance to DTG when introduced into the pNL43 backbone, such as S153Y , R263K , and G118R   –18. EvenFC, 10) –. NeverthFC, 10) –. We haveFC, 10) –. OverallIn addition, the 9053 virus is still infectious, as shown by its ability to infect new cells, even if replication was quantitatively slowed by the presence of DTG, at least in the first days of infection. The production of p24 antigen by the 9053 virus was not due to the subpopulation of wild-type virus used for the initial infection, still present in the supernatants of the resistant virus, because the wild-type virus was completely impaired by DTG. Our data clearly show that this virus was able to replicate at a high DTG concentration.We highlight a new mechanism of HIV-1 replication in the presence of DTG. As no mutation in the IN gene was selected, DTG can still bind to the active site of IN in the same way as the wild-type virus, which is completely inhibited by 100 nM DTG. There are two possibilities that could explain the replication of the mutant; i.e., (i) the virus is not integrated into cellular DNA and replication involves only unintegrated viral DNA  or (ii) in vitro selection of this virus, which is resistant to DTG by an unprecedented mechanism, was based on a DTG concentration of 500 nM. This is significantly higher than the 50% inhibitory concentration of DTG (2.7 nM) (The (2.7 nM)  but on t(2.7 nM) . Understin vitro identification of a novel mechanism of DTG resistance involving mutations outside the IN gene warrants further studies to determine the extent to which mutations within the 3′ PPT could explain virological failures of DTG therapy in the absence of selected mutations in the IN gene.DTG is now widely used in the treatment of patients infected with viruses either with or without INSTI resistance mutations. The 2.293T and HeLa P4 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated FBS . MT4 cells were cultured in RPMI with 10% FBS. All cells were maintained at 37°C in 5% COThe HIV-1 pNL43 plasmid carrying all of the mutations detected in the 3′ PPT, as well as pNL43ΔNef (with the G deletion at bp 9073), was obtained by site-directed mutagenesis with the QuikChange II XL site-directed mutagenesis kit (Agilent) in accordance with the manufacturer’s instructions. Virus was prepared with Lipofectamine reagent (Thermo Fisher) by transfecting 293T cells. Forty-eight hours posttransfection, the viral supernatant was harvested and frozen at −80°C. The titer of the supernatant was determined by quantifying HIV-1 p24 antigen with the VIDAS instrument (BioMérieux).d‐galactopyranoside substrate by using the absorbance at 575 nm. Briefly, cells were infected in triplicate in 96-well plates with wild-type or mutant virus  containing increasing concentrations of the inhibitor DTG, RAL, EVG (0 to 800 nM), or AZT (0 to 51 µM). Phenotypic susceptibility to DTG was evaluated by the Agence Nationale de Recherche sur le Sida (ANRS) PBMC method as previously described  or containing DTG at a final concentration of 100 or 500 nM for 2 h and then infected with the 9053 Lai virus from supernatant day 80 or wild-type Lai virus, both at a level of 30 ng of p24 antigen /10(1−23)/RP(991−1021); gag, FP(734−754)/RP(2294−2315); protease, FP(2071−2100)/RP(2705−2735); reverse transcriptase, FP(2480−2499)/RP(3514−3546) and FP(3208−3236)/RP(4038−4069); RNase H, FP(3696−3715)/RP(4380−4399); IN, FP(4007−4026)/RP(5251−5270); vif, FP(4899−4922)/RP(5765−5786); vpr, FP(5431−5453)/RP(5970−5988); tat-rev-vpu, FP(5611−5639)/RP(6423−6443) and FP(7634−7653)/RP(8285−8304); gp120, FP(5833−5857)/RP(6553−6580) and FP(6423−6443)/RP(7831−7849); gp41, FP(7634−7653)/RP(9010−9038); nef-3′ LTR, FP(8688−8713)/RP(9607−9632). Purified RT-PCR products were directly sequenced for bulk genotyping by using a cycle sequencing reaction with the BigDye Terminator kit (Applied Biosystems). Furthermore, some of the purified RT-PCR products were cloned with the TOPO TA Cloning kit (Invitrogen) and 31 clones were reamplified by PCR for sequencing. All of the sequences obtained were analyzed with the CodonCode Aligner software.The RT-PCR assay was performed with the Transcriptor One-Step RT-PCR kit (Roche Life Science). Several genes were studied with the different primer sets  listed here and numbered relative to the HxB2 sequence: 5′ LTR, FP


Because of the global spread of Zika virus, accurate and high-throughput diagnostic immunoassays are needed. We compared the sensitivity and specificity of 5 commercially available Zika virus serologic assays to the recommended protocol of Zika virus IgM-capture ELISA and plaque-reduction neutralization tests. Most commercial immunoassays showed low sensitivity, which can be increased. Flaviviridae, genus Flavivirus, that was originally discovered in 1947 in Uganda  and chikungunya virus, in acute-phase clinical specimens  for arbovirus diagnostic testing. All samples were obtained from Canadian travelers who visited areas with known Zika virus transmission and consulted their physicians after symptoms consistent with Zika virus infection developed upon return.We obtained deidentified samples from 75 patients. Thirty samples were from patients with serologically confirmed Zika virus infections; 10 from patients with confirmed Zika virus infections identified by 2-target real-time reverse transcription PCR (RT-PCR); 10 from patients with suspected Zika virus infections, which were subsequently identified as DENV infections; and 25 acute-phase samples from flavivirus-negative persons tested by Zika virus RT-PCR and MAC-ELISA. Primary Zika virus diagnostic testing for all samples was conducted by using an in-house CDC-based MAC-ELISA and subsequent confirmation of Zika virus infection by cross-PRNTs for Zika virus and DENV, or molecular assays as described  and 3 MAC-ELISAs: Zika Virus Detect ; Ab213327 ; and NovaLisa ZVM0790 . On the basis of preliminary testing, we also tested the Euroimmun IgM ELISA in parallel with the Euroimmun conventional Zika virus IgG ELISA (EI 2668-9601 G). Both Euroimmun assays use recombinant Zika virus nonstructural protein 1 as the antigen; the InBios Zika Virus Detect uses a recombinant Zika virus envelope glycoprotein as the positive antigen, an unspecified cross-reactive control, and reference cell antigens; and the Novatec and Abcam ELISAs use an unspecified Zika virus antigen.Most tests evaluated provided algorithms that resulted in positive, negative, or equivocal results. However, the InBios kits account for antigenics associated with secondary flavivirus infections and reports results as Zika virus positive, possible Zika virus positive, or presumptive other flavivirus positive or negative on the basis of calculations of optical density ratios obtained from a sample with the 3 different antigens. Two independent laboratory technicians blindly evaluated the 5 assays by using the panel outlined, according to the manufacturer’s instructions. Comparisons and performance calculations were conducted by the Quality Control Office of the National Microbiology Laboratory.>90%) for confirmed DENV-positive samples , the IgM assays of Euroimmun, Abcam, and Novatec demonstrated sensitivities of 37%, 57%, and 65%, respectively. When we combined results of the Euroimmun IgM and IgG ELISAs, sensitivity increased to 82%. The InBios Zika Virus Detect IgM assay correctly identified all confirmed Zika IgM-positive samples identified by the recommended diagnostic assays, resulting in a sensitivity of 100%. The InBios ELISA also detected IgM in 50% of samples that were positive for Zika virus by RT-PCR, whereas the other assays did not detect IgM in most of these samples .The low sensitivity of most immunoassays evaluated could be improved by testing a repeat sample collected a few weeks after the initial specimen, although this sampling is not always practical, particularly if resources are limited. When performed in combination, the Euroimmun Zika Virus IgM and IgG ELISAs provide improved sensitivity. However, interpretation of recent versus past infections could be problematic, particularly when IgM results are negative and IgG results are positive. On the basis of our findings, the InBios Zika Virus Detect MAC-ELISA provides diagnostic results comparable to the CDC-based in-house MAC-ELISA for specimens collected from patients with primary flavivirus exposures . A needed follow-up to our study will be further evaluation of IgM detection by commercial ELISAs involving cases of secondary flavivirus exposures or previous immunization to related viruses, such as yellow fever virus.