We performed gene sequencing to identify mutations that may be responsible for the observed phenotype. The LP generally replicated faster than the SP and the difference in growth rate was more pronounced in Bunyamwera virus isolates. Ngari virus isolates were more conserved with few point mutations compared to Bunyamwera virus isolates which displayed mutations in all three genome segments but majority were silent mutations. Contrary to expectation, the SP of Bunyamwera virus killed suckling mice significantly earlier than the LP. The LP attenuation may probably be due to a non-synonymous substitution (T858I) that mapped within the active site of the L protein. In this study, we identify natural mutations whose exact role in growth and pathogenesis need to be determined through site directed mutagenesis studies.Biological phenotypes of tri-segmented arboviruses display characteristics that map to mutation/s in the S, M or L segments of the genome. Plaque variants have been characterized for other viruses displaying varied phenotypes including attenuation in growth and/or pathogenesis. In order to characterize variants of Bunyamwera and Ngari viruses, we isolated individual plaque size variants; small plaque (SP) and large plaque (LP) and determined  Orthobunyavirus genus of the Bunyaviridae family of arboviruses. The virus is associated with febrile illness with headache, arthralgia, rash and infrequent central nervous system involvement Orthobunyavirus genus are known to cause human disease, they were previously not associated with hemorrhagic manifestations. However, Ngari virus has been implicated in recent outbreaks of hemorrhagic fevers in Kenya and Somalia Bunyamwera virus is the prototype virus of the Bunyaviridae family, the Bunyamwera virus genome consists of three negative-sense RNA segments that employ a variety of coding strategies leading to generation of a limited set of structural and non-structural proteins Bunyaviridae family are likely underestimated because of the lack of detection tools arising partly from their high level of diversity, limited phenotypic and genetic characterization and segmented nature of their genome.Like other viruses within the Bunyaviridae, including Puumala virus in which the large plaque (LP) grows to higher titers than the small plaque (SP) and the parental wild type (WT) virus in vivo and in vitro has also been observed for the SP of West Nile virus Orthobunyaviruses are mostly isolated and amplified in interferon defective African green monkey kidney epithelial Vero cell line that may result in mutations yielding substrains that are phenotypically different from the parental wild type virus The study protocol (number SSC 2677) was approved by the Animal Care and Use Committee of the Kenya Medical Research Institute and by the Animal Ethics Committee of the University of Pretoria (Protocol number H012-13). All animal experiments were carried out in accordance with the regulations and guidelines of the Kenya Medical Research Institute and University of Pretoria Animal Ethics Committees.The sites in Kenya and vector species from which the 5 virus isolates used in the study were obtained is summarized in 2 incubator at 37°C overnight before use. The cells were used when they attained 75–90% confluence. Ten-fold dilutions of the virus isolates were prepared in maintenance media. Media was carefully aspirated from the wells using sterile transfer pipettes and 100 µl of the appropriate viral dilution added to each of duplicate wells of 6-well plates with gentle rocking to evenly distribute the virus. Plates were incubated at 37°C for 1 hour after which media was carefully aspirated and 3 ml of 1.25% methylcellulose solution gently added to each plate. Plates were placed in a CO2 humidified incubator and incubated for 5 days. Development of plaques was monitored by visualization under an inverted microscope. To facilitate visualization of plaques, methylcellulose solution was carefully aspirated using transfer pipette followed by fixation in 10% formaldehyde after which plates were stained with crystal violet solution.Vero cells were seeded on 6 well plates and incubated in a humidified CO9 PFU/ml were diluted in maintenance media to approximately 10 PFU/ml. Confluent Vero cells in 24-well plates were infected with 100 µl of diluted virus per well. After adsorption for 1 h at 37°C, the cells were overlaid with 1.25% methylcellulose. Five days later, the methylcellulose medium was carefully aspirated and sterile pasture pipettes used to pick plaques from wells with single plaques and placed in 500 µl of maintenance media. The plaque phenotypes were then propagated on Vero cells and the procedure repeated twice more, without intermediate amplification, for each of the plaque isolates. The purified isolates were then amplified by propagation on confluent Vero cells in flasks and then frozen at −80°C until use.Bunyamwera and Ngari virus isolates (previously passaged 3 times on Vero cells) with a titer of 1×10The viral isolates including the parental WT, i.e. mixture of SP and LP, were used to infect 90% confluent monolayers of Vero cells at a multiplicity of infection of 0.01 and incubated for one hour to allow virus adsorption. Infected monolayers were washed twice with sterile PBS and overlaid with maintenance medium and incubated at 37°C. An aliquot of tissue culture fluid (0.5 ml) was collected every 12 hours for the first 2 days and once on day 3 of infection, mixed 1∶10 with maintenance media and frozen at −80°C until use. Daily samples were titrated by plaque assay as described above. The statistical package R  For RNA extraction, the MagNA Pure LC RNA Isolation Kit I (Roche Diagnostics) was used. Complementary DNA (cDNA) was synthesized using Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science) with Random hexamers followed by PCR using Phusion High-Fidelity PCR Kit  and appropriate primers. Primers for each segment were either designed based on sequences of Bunyamwera, Batai and Ngari viruses available in GenBank or obtained from previous publications www.mbio.ncsu.edu/BioEdit/BioEdit.html), USA for both the reads from the forward and reverse primers. Sequences obtained were compared to those in the gene bank using the Basic Local Alignment Search tool (BLAST) http://www.ncbi.nlm.nih.gov/blast/Blast) to identify similar sequences. The clean sequences of each segment of each phenotype were aligned against the corresponding segment sequences of the wild type virus isolate using Bioedit. Nucleotide and amino acid similarity and diversity between the virus phenotypes were computed in MEGA v5.20 Sequencing was performed using different sets of primers for the S, M and L segments as defined above using Big Dye V3.1 kit (Applied biosystems) and injection on a 3500XL genetic analyser . The sequences obtained were cleaned and edited using Bioedit software  and 6 week old mice. Mice were inoculated intraperitoneally with 100 µl of 10Plaque titration of Ngari and Bunyamwera viruses yielded plaques of two significantly distinct phenotypes, large plaques (LP) (Range: 0.88–1.21 mm) and small plaques (SP) (Range: 0.47–0.66 mm) . Each plIn general, the LP phenotype of both Bunyamwera virus isolates grew at a faster rate and to a significantly higher titer (p = 0.009) than the SP phenotype by day 3 of infection . The BunFor Ngari virus isolates , the difComparison of the nucleotide and amino acid sequences of low passage Ngari virus isolates revealed little or no divergence within the S segments . The N aFor the Bunyamwera virus isolates, there were more transversions than transitions resulting in several non-synonymous codons.We selected the WT and plaque phenotypes of isolates GSA/S2/11232 and TND/S7/19801 for the mice pathogenesis experiments. Six-week old mice were not susceptible to infection by either virus isolate. However, newborn mice were susceptible to infection with both virus isolates and displayed clinical symptoms such as hind limb paralysis, tremors, disorientation and mortality beginning 2–3 days post inoculation. By day 4 post-infection, mice inoculated with isolate GSA/S4/11232SP had a 50% probability of survival compared to the LP phenotype that had approximately 70% survival probability . The difin vivo growth in Vero cells and evaluate pathogenesis of the viral phenotypes in Swiss Albino mice. Difference in growth was more pronounced in Bunyamwera than Ngari virus isolates. This may be explained by the more mutations observed for the former possibly due to the extra passages which the SP and LP phenotypes underwent compared to the WT. In contrast, Ngari virus phenotypes had fewer mutations despite undergoing extra passages than the WT during the purification and amplification processes. As expected, the LP phenotypes grew to a higher titer than the SP phenotypes for both Bunyamwera and Ngari virus isolates. Previous studies of other viruses have correlated plaque size to replication rate with the LP phenotypes displaying faster replication rate than the SP phenotype in vitro and in vivo on the assumption that LP phenotypes produce larger foci of cell destruction Orthobunyavirus genus have mapped such differences to the L segment In the current study we evaluate the genetic diversity of plaque purified phenotypes of Bunyamwera and Ngari virus isolates by gene sequencing. We also determine the rate of It is interesting that all the nucleotide substitutions on the M segment while resulting in non-synonymous amino acid changes, involved substitution of amino acids with similar properties, thus, a significant difference in protein function would be unexpected. The M segment substitutions resulted in exchange of positively charged amino acids, glutamic acid for lysine. However, two mutations in the L segment of the LP phenotype involved substitution of amino acids with different properties which are likely to alter the functionality of the L protein. Thus for isolate GSA/S4/11232, the LP attenuated pathogenesis may be mapped to any of the 2 non-synonymous mutations on the L segment. The T858I mutation resulting in amino acid substitution of a polar for a non-polar amino acid, occurring within the predicted catalytic site of the L protein (AA 597-1330) seems the most plausible cause of the observed attenuation in pathogenesis. Mutation within the catalytic site of the L protein has been demonstrated to abolish polymerase activity in a previous study With regard to isolate TND/S1/19801, the SP phenotype, which was attenuated in mice but genetically similar to the WT virus, it is likely that this phenotype was present in a higher quantity in the WT virus, which is a mixture of both LP and SP phenotypes, and could have preferentially been sequenced. However, in the mice pathogenesis experiment, it is likely that the LP phenotype in the WT grew at a faster rate as expected and resulted in earlier death of mice compared to the SP phenotype. However, we did not isolate the infecting virus from mice to confirm this observation. Another limitation was the use of interferon defective Vero cells for the one step growth curve analysis which may have limited our comparison with mice pathogenesis as the GSA/S4/11232 SP phenotype may have been better at counteracting the interferon response. Additionally, we did not sequence the non-coding regions of the genomic segments which have been documented to play a role in virus growth and pathogenesis In summary, we have identified a mutation in the L segment of Bunyamwera virus isolate GSA/S4/11232 LP phenotype which may be associated with decreased pathogenesis in suckling mice and virus replication in Vero cells. In addition, we have identified other natural mutations whose role in viral growth and pathogenesis should be determined. Site directed mutagenesis studies may clarify the exact mutation involved in the observed phenotypic changes.Table S1Primers used in sequencing of Kenyan Bunyamwera and Ngari virus isolates. Primers for each segment were either designed based on conserved regions of sequences of Bunyamwera, Batai and Ngari viruses available in GenBank or obtained from previous publications.(DOCX)Click here for additional data file.


Morbillivirus genus, including measles virus, canine distemper virus and peste des petits ruminants virus. Here, we describe the role of PVRL4 in morbillivirus pathogenesis and its promising use in cancer therapies. This discovery establishes a new paradigm for the spread of virus from lymphocytes to airway epithelial cells and its subsequent release into the environment. Measles virus vaccine strains have emerged as a promising oncolytic platform for cancer therapy in the last ten years. Given that PVRL4 is a well-known tumor-associated marker for several adenocarcinoma , the measles virus could potentially be used to specifically target, infect and destroy cancers expressing PVRL4.PVRL4 (nectin-4) was recently identified as the epithelial receptor for members of the  Morbillivirus, belongs to the Paramyxoviridae family in the order, Mononegavirales. The genome is a negative-sense, non-segmented, single-stranded RNA, and its structure is conserved with the nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), attachment (H) and large (L) genes present in all morbilliviruses [Morbilliviruses, which include measles virus (MeV), canine distemper virus (CDV) and peste des petits ruminants virus (PPRV), are among the most widespread and highly contagious pathogens in their respective hosts. Lapses in vaccination have produced frequent outbreaks of measles in recent years. Although measles virus infects human and non-human primates, humans are the only reservoir for MeV . On the iviruses . MeV, CDiviruses ,8,9,10. iviruses . As welliviruses ,15,16,17This review highlights recent advances on the role of PVRL4 (nectin-4) during morbillivirus infections. Specifically, the function of PVRL4 during morbillivirus entry and exit, and, in particular, the role the variable (V) domain of PVRL4 plays in the recognition of morbillivirus attachment receptors, is discussed. Finally, the oncolytic potential of morbilliviruses in targeting PVRL4 as a tumor marker on adenocarcinomas is proposed. Most morbilliviruses have established lymphotropism and epitheliotropism that are receptor-dependent  18,19,2,24,25. Iin vivo [Expression and distribution of cellular morbillivirus receptors in a host is particularly important, because the interaction of a virus with its cellular receptor links virus tropism and pathogenesis . High lein vivo . Moreovein vivo ,58,59,60in vivo . Similarin vivo . Taken tNevertheless, morbillivirus pathogenesis cannot be explained by the presence of SLAM and PVRL4 alone. Small ruminants infected with PPRV often exhibit lesions in the intestine that express relatively low levels of SLAM and PVRL4 , 61, 62.cis-, followed by trans-interactions with PVRL4 on adjacent cells, while PVRL4-PVRL1 interactions only appear in trans on adjacent cells [The characterization of virus-receptor interactions is particularly important to better understand virus entry and offers new opportunities for developing antiviral therapies. PVRL4 is a member of the nectin family of adhesion molecules, which belongs to the immunoglobulin (Ig) superfamily, comprised of nectin-1, -2, -3, -4 and the prototypic poliovirus receptor (PVR) ,65,66. Pnt cells ,72,73. Ain vitro [PVRL4 was biochemically shown to support binding to MeV H through its V domain, leading to virus entry . This isin vitro . This FPin vitro ,19,76  and late  stages of infections. Finally, many adenocarcinomas express PVRL4 on their cell surfaces, making them obvious targets for oncolytic and immune therapy based upon recombinant morbilliviruses.


Zika virus, which was first discovered in 1947, has become a global threat to human health as it is rapidly spreading through Latin America, the Caribbean, the US and Asia, after causing a large outbreak in the Northeast region of Brazil in 2015. There is ample evidence to support that Zika virus is associated with neurological complications such as microcephaly. The review aims to provide an overview on the complex issues involved in the emergence of Zika virus’s neurological disorders and to discuss possible explanations of Zika virus introduction and dissemination in Brazil. We also suggest national and global strategies to adequately respond to the Zika virus emergence.We provide an analytical evaluation of the main issues related to the Zika outbreak in Brazil, based on available scientific literature, including government documents, and on epidemiological information from national surveillance databases.Aedes aegypti might be the contributing factors for such an outbreak in Brazil. The Zika virus emergence emphasized the importance of cross-disciplinary perspective. Besides the scientific-based vector control strategies, it is important to understand the nature of the evolutionary processes involved in the viral evolution in complex ecosystems and to have social and anthropological knowledge on the conditions related to the spread of the disease in order to properly respond to the spread of the Zika virus.The studies on the clinical manifestations of the Zika virus infection coupled with the epidemiological surveillance information in Brazil have provided significant evidence that the Zika virus is associated with neurological disorders such as microcephaly and Guillain-Barré syndrome. Based on phylogenetic and molecular analysis, the hypothesis regarding the introduction of Zika virus in the country is that it took place following international events in 2013 and 2014, when many foreign visitors could have brought Zika virus into Brazil. The immunologically naïve status of populations in the Americas, previous infection with dengue virus, and the increased activity of The experiences of Brazil have demonstrated the significance of multi-disciplinary approach in response to new and resurgent arboviral diseases and provided important lessons that could be applied to other developing countries. Zika virus was first discovered in Uganda in 1947, and the first human case was documented in 1952 . The EmeThis paper will examine the complex issues involved in the emergence of Zika virus and discuss hypotheses regarding its introduction and dissemination in Brazil. Additionally, the paper also aims to provide insights into the eco-social determinants of Zika virus emergence and ways of integrating diverse conceptual frameworks into a transdisciplinary perspective. We hope that the lessons learned from the Brazilian experience can provide valuable insights for dealing with the emergence of new infectious diseases in other developing countries.Flavivirus genus and is mainly transmitted through the bite of infected Aedes mosquitoes (https://commons.wikimedia.org/wiki/File:Aedes_aegypti_feeding.jpg), which are also vectors for chikungunya, dengue, and yellow fever [Zika virus is an arbovirus in the ow fever .The symptoms of Zika virus infection are mild, similar to those of dengue, including fever, rash, joint pain, muscle pain and/or conjuntivitis and in most cases hospitalization is not required . HoweverTwo lineages of the Zika virus exist: the African lineage and the Asian lineage . The virSevere fetal birth defects such as microcephaly have been found in infants born to women infected with Zika virus, according to PAHO guidelines and National Surveillance Protocol in Brazil ,13. The Subsequently, several scientific studies have been conducted regarding Zika virus infection-related neurological disorders in newborns . In one Additionally, an increasing number of Zika virus infection-related cases of Guillain-Barré syndrome have been observed in Brazil. Although significant under-reporting may be expected to take place because Guillain-Barré syndrome is not a notifiable disease, information from Brazilian hospitals indicates an increase of 29.8% in hospitalizations from 2014 to 2015, from 1,439 to 1,868 cases; of note, some hospitals, such as Antonio Pedro Hospital in Rio de Janeiro, have reported an unusual increase in the number of cases .Aedes albopictus mosquitoes that are widespread in Latin and Central America, the US, and other regions not yet affected by Zika virus [Culex, a tropical domestic mosquito widespread in Brazil, might be able to transmit Zika virus since some arboviral infections are transmitted by several species of Culex [The pathogenesis and the mechanisms by which the Zika virus exerts its potentially severe effects on the brain of newborns and adults are still unknown. In Brazil, complete information regarding the number of pregnant women infected by Zika virus who gave birth to babies with microcephaly is lacking due to significant under-reporting and limited access to health care in the poorest regions. Although studies have suggested that Zika virus infection in the first trimester of pregnancy is related to more severe cases of microcephaly in newborns, a recent groundbreaking study in Brazil has found cases of Zika virus-associated severe microcephaly in all stages of gestation, including cases in the second and third trimester of pregnancy . We do nka virus . Furtherof Culex . This poAdditionally, other routes of transmission in addition to mosquito bites should be further studied. It is very likely that Zika virus can be spread through blood transfusions, and suspected cases in Brazil are under investigation. Sexual transmission has been confirmed by the US CDC as a route of transmission, although we do not know the transmission rate ,22. AlthAedes mosquitoes tend to concentrate. In 2014, the World Cup could have introduced Zika virus to Brazil, since many foreigners gathered in stadiums located in multiple states in Brazil. Finally, teams of rowers from French Polynesia, where a Zika virus outbreak occurred in 2013 and 2014, attended a canoeing championship in Rio de Janeiro in 2014. Concerns have been raised that the Olympic Games may lead to the introduction of new viruses into the country and to the possible global dissemination of Zika virus and other infectious diseases, despite significant governmental efforts to minimize these risks.A recent phylogenetic and molecular analysis by Brazilian researchers with international collaboration showed a single introduction of Zika virus into the Americas, estimated between May and December 2013 which indicates that the virus had been circulating in Brazil earlier than the first Zika virus infection case reported in 2015 ,25. ThisAedes aegypti mosquito activity. Additionally, it is important to note that during the large outbreak in Brazil, as in French Polynesia, the symptoms of Zika virus were reported in the context of a concurrent dengue epidemic [Aedes mosquitoes, produce more serious symptoms, such as higher fever and worse pain.Several explanations have been proposed regarding why the Zika virus emerged and caused an outbreak in Brazil. One possible explanation is the immunologically naive status of populations in the Americas compared to the populations of African and Asian countries where Zika virus has been circulating for decades and widespread immunity most likely exists against the virus ,25. A seepidemic . It is aWolbachia bacteria as a method of biologically controlling Aedes mosquitoes [For several political and institutional reasons, related to the decentralization of responsibilities in the National Health System and the elimination of federal structures in charge of vector control, such as the Superintendency of the Public Campaigns of Brazil - SUCAM in 1990, routine vector control has not been treated as a high priority in Brazil, although successful efforts have been made in some states and municipalities. In this context, emergency strategies are now under evaluation. In order to deal with Zika virus infection, several novel vector control strategies have been recently proposed and are now under the scrutiny of the scientific community and policy makers. One strategy is to modify the genes of mosquitoes to produce faulty offspring and reduce the quantity of larvae. This strategy has already been approved by the National Biosafety Commission in Brazil and applied by a British company, Oxitec, in some Brazilian cities. Another strategy is the sterile insect technology developed by the United Nations International Atomic Energy Agency, in which laboratory-bred male mosquitoes are exposed to nuclear radiation in order to make their sperm sterile before being released into the wild to mate with female mosquitoes. In this way, the mosquito population could be reduced in a few months. Another promising strategy to control Zika virus transmission by mosquitoes is using squitoes . NeverthThe Zika virus outbreak in Brazil revealed the need to further develop two important areas of knowledge in controlling and managing this arbovirus. The first crucial area of knowledge is the ecosystem. Having a comprehensive ecosystem-focused perspective is crucial in addressing vector-borne diseases with complex dynamic cycles involving vertebrates and blood-feeding vectors, such as mosquitoes and ticks. The importance of an ecological perspective is often missed in public health and epidemiological surveillance, and the absence of this perspective might explain previous failures in dealing with unexpected pandemics. The complex conditions affecting the evolution, introduction, mutations, variations, and adaptations of arboviruses in new ecological niches should be understood from this perspective -30. In pSocial and anthropological issues are another important area. Although genetic, immunological, and environmental conditions are important, understanding the social, economic, and anthropological aspects of the vulnerable population is also necessary because a pathogen requires a receptive population in order to cause disease. The specific social issues contributing to the Zika virus outbreak include rapid urbanization in conditions of extreme poverty, leading to intense deforestation that favors the contact of populations with unknown vectors and pathogens, as well as the intensification of international travel and population mobility. In addition, social vulnerabilities to new disease are not only a consequence of socioeconomic conditions, but also of the social behaviors related to risk and disease perceptions. It is therefore important to understand cultural and behavioral barriers when dealing with the emergence of new diseases. The importance of such barriers is demonstrated, for example, by the vulnerability of web users in Brazil to an erroneous rumor disseminated online that attributed Zika virus infection-related disease in newborns to vaccinations in pregnant women. Such barriers are also reflected by the social leniency of policy makers and the population overall regarding vector control measures, despite the availability of widespread information about this issue. Some studies have examined why access to information is not sufficient to change perceptions and behaviors. These studies have indicated that understanding the emotional component of attitudes is a crucial factor that could bring about effective access to behavior-changing information, and that an adequate understanding of emotions is required in the dissemination of new information ,32. ThesOther social and organizational issues in Brazil should be addressed in response to the Zika virus. Concerning health policy, the overall quality of National Health System services should be improved to deal with outbreaks of Zika virus. Although Brazil has a well-established universal health care system, it has been severely underfunded and undermined by the rapid increase of low-quality private health plans. Additionally, despite the existence of a well-established epidemiological surveillance system in Brazil, surveillance programs should be strengthened and refined in order to improve the quality of indicators and the ability to respond to unexpected occurrences of new and resurgent diseases.Aedes mosquitoes and to Zika as well as dengue and chikungunya, the poorest tend to be more vulnerable, and nearly 94% of Zika virus-related microcephaly cases have been reported in the economically deprived northeast region of Brazil. The majority of the Brazilian population lives in poor areas without adequate housing and sanitation, meaning that policies for better housing, sanitation, and garbage collection should be urgently implemented.Another issue is social policy concerning poverty and inequality. Although nearly everyone in Brazil is now exposed to The specific nature of the disease caused by Zika virus also highlights certain ethical issues. Abortion in Brazil is illegal; therefore, when effective diagnostic kits for Zika virus infection are developed, Brazilian society will have to come to a consensus regarding what to do when a pregnant woman tests positive for Zika infection. Women’s movements and organizations advocating for reproductive rights may be expected to play a crucial role in this process.Aedes in their houses as a response to the Zika virus outbreak, this campaign has not been effective because it has not been able to address transmission outside the home, which is conditioned by the urban and environmental determinants of transmission. Novel Zika virus control methods that incorporate environmental, urbanization, social, sanitation, and vector control policies with broader perspectives are urgently needed.Moreover, environmental policies are important in light of the dramatic global environmental and climate changes related to accelerated deforestation, urbanization, and global warming; these changes affect Brazilian ecosystems and have favored the proliferation of vectors and the emergence of new infectious diseases. Although the Brazilian government has put in place an extensive national campaign to mobilize the population to avoid the proliferation of Innovations and technological development policies focused on the development of new products are a major aspect of dealing with the Zika virus outbreak in Brazil. Currently, Brazilian institutions, despite very limited resources for research and development, are working on developing new and innovative diagnostic tools for the Zika virus, and it is expected that a new diagnostic tool produced by Bio-Manguinhos/Fiocruz will be available soon, after registration with the Federal Government Agency. This Bio-Manguinhos product is an ‘on-the-spot’ rapid test kit for measuring immunoglobulin M and immunoglobulin G.The experiences of Brazil with Zika virus underscore the conclusion that the conceptual approach to this new disease should shift from a traditional tropical neglected disease framework to an emerging and resurgent neglected disease perspective. Emerging and resurgent neglected diseases such as Zika virus are no longer confined to tropical regions and the most economically disadvantaged areas of the globe, but threaten other countries, including developed nations such as the US and European countries. Vector-borne viral diseases therefore deserve more public health attention, and an effective eco-social approach is needed in order to anticipate risks and provide interventions before the emergence and global spread of new and resurgent diseases. The experiences of Brazil have demonstrated that a cross-disciplinary approach supporting a social ecosystem health perspective  is cruci


An outbreak of Getah virus infection occurred among racehorses in Japan during September and October 2014. Of 49 febrile horses tested by reverse transcription PCR, 25 were positive for Getah virus. Viruses detected in 2014 were phylogenetically different from the virus isolated in Japan in 1978. Alphavirus, family Togaviridae) is a mosquito-borne virus that was first isolated in Malaysia in 1955 from Culex spp. mosquitoes  is available to prevent Getah virus infection and is mainly administered to thoroughbred racehorses registered by the Japan Racing Association. It is recommended that this vaccine be administered twice in the first year of registration (mainly 2-year-old horses) and then annually as a booster before each mosquito season. This vaccine contains the MI-110 strain isolated from a febrile horse during the outbreak in 1978.In mid-September 2014, the number of febrile horses began to increase at the Miho Training Center of the Japan Racing Association in Ibaraki Prefecture; we identified Getah virus infection among these horses. The outbreak of Getah virus infection in 1978 occurred at this facility. We summarize the epidemiologic features and molecular characterization of the epidemic virus.Approximately 2,000 horses are stabled at the facility in which this outbreak occurred. In September and October 2014, a total of 36 and 39 horses, respectively, became febrile. The numbers of pyretic horses in September and October during 2009–2013 were 16.0 ± 4.4 and 17.6 ± 1.7 (mean number ± SD), respectively.>4-fold increase in the antibody titer between paired serum samples. Serum collected from horses vaccinated after August 1 were not used in this study because the neutralization test cannot distinguish an increase in antibodies induced by natural infection from that induced by vaccination.EDTA-treated blood samples and nasal swab samples from 49 and 48 pyretic horses, respectively, were collected during September 25–November 2. Paired serum samples were collected from 19 febrile horses during the acute (September 15–October 12) and convalescent (2–4 weeks later) phases. Nasal swab samples were suspended in 2.5 mL of transport medium (The NSP1 and capsid genes in the RT-PCR–positive samples (strain designation Miho-2014) and MI-110 strain were amplified by using previously described primer pairs (>4-fold increase) to the MI-110 strain of Getah virus  and capsid (552 bp) genes were completely identical among the 10 Getah viruses detected in 2014. The nucleic acid sequence identities between the Getah virus detected in 2014 and MI-110 were 98.7% for the NSP1 gene and 99.1% for the capsid gene. Phylogenetic analyses were performed with the nucleic acid sequences of the Getah virus NSP1 and capsid genes, including those of isolates from horses, mosquitoes, and pigs , and shoThe first outbreak of Getah virus infection among racehorses occurred in September–November 1978, and another outbreak in 2014 occurred around the same period at the same facility. In 1978, of 1,903 stabled horses, 722 were affected (


A recent study by Fennessey et al. (Retrovirology 12:49,  Retrovirology the construction of the SIVmac239Opt clone with all four positions repaired, which could form an improved virus reagent for specific studies, e.g. when using a low multiplicity of infection to challenge macaques [SIVmac239 is a frequently used prototype virus in HIV-1 transmission, pathogenesis and vaccination studies in the SIV-macaque model. Early studies identified four suboptimal and therefore genetically unstable nucleotides in the molecular clone encoding this virus [The same PBS mutation has repeatedly popped up in literature in a diversity of HIV–SIV studies, e.g. virus mutation-reversion studies , 5. Its Lys3 as primer for reverse transcription by mutating the complementary 18-nucleotide PBS in the HIV-1 genome [Lys3 primer (PBSLys3) and this segment is among the best conserved parts of the retroviral genome. However, we reported a single nucleotide polymorphism in around 5 % of natural HIV–SIV isolates and argued for the infrequent use of a variant tRNA primer [Lys5 molecules that encode this PBS-mutation (PBSLys5) were identified when the sequence of the human genome became available [Lys3 initiated reverse transcription at PBSLys3 in 81 of 90 virus particles analysed, but the variant tRNALys5 was used in the other 9 particles (10 %) [Lys5 priming on PBSLys3 and tRNALys3 priming on PBSLys5), a heteroduplex PBS is generated with the plus-strand sequence copied from the tRNA primer and the minus-strand copied from the viral RNA. It cannot be excluded that this duplex is a target for mismatch repair as described in [Lys3 or PBSLys5 at a 50–50 ratio. Thus, 10 % tRNALys5 priming would result in 5 % PBSLys5-containing proviral genomes, which is strikingly similar to the frequency in natural HIV–SIV isolates and the 3 % frequency that we reported in HIV-1 evolution studies [We and others probed the exclusive usage of tRNA1 genome –13. The 1 genome , 14, 15.vailable . We usedribed in . Howeverribed in . In thisLys5 HIV-1, Fennessey et al. demonstrate that approximately 50 % of the proviruses contain a (homoduplex) PBSLys5 sequence. This result can be explained by the use of tRNALys5 for initiation of reverse transcription. The other 50 % of the proviruses contained either a PBSLys3/PBSLys5 heteroduplex (40 %) or the reverted PBSLys3 sequence (10 %), both resulting from tRNALys3 annealing . Thus, tRNALys3 and tRNALys5 anneal with similar efficiency to PBSLys5 (~50 %), whereas tRNALys3 binds with higher frequency than tRNALys5 to PBSLys3 . As a consequence, the PBSLys5 variant percentage will gradually decrease further upon subsequent rounds of virus replication and cell division, while the PBSLys3 fraction will increase. No matter the PBS composition of the starting clone, HIV–SIV will soon replicate as a mixture of roughly 95 % PBSLys3 and 5 % PBSLys5 variants, which are no stable entities but rather interchange continuously, but maintaining this fixed 95–5 ratio. In other words, this PBS-variation is driven by the tRNA primer usage and occurs independently of regular virus evolution, that is generation of new mutations (mostly by RT errors during the reverse transcription process) and selection of beneficial changes.In their single genome analysis of proviruses upon infection with a PBSLys3- and tRNALys5-using viruses for several reasons. These tRNA molecules may have a different intracellular concentration, which is supported by a recent deep sequencing study that revealed 1966 tRNALys3 sequence reads versus 53 tRNALys5 reads, yielding a 37:1 ratio ([Lys5 virus to the PBSLys3 sequence is caused by frequent tRNALys3 annealing at PBSLys5 and should not be interpreted as a large fitness impact. In other words, the special nature of the PBS-tRNA axis means that it cannot be compared to other genome segments in evolutionary terms and in fitness calculations. These insights also explain the ‘surprising’ result of rapid reversion in the absence of continuous virus replication [We realize that there may be a fitness difference between tRNA1 ratio ( and unpu1 ratio (, 19. Howlication . No contLys5 annealing. Such premature termination of reverse transcription—before the mismatching nucleotide in the tRNA primer or viral PBS is copied, which corresponds to maximally 7 tRNA nucleotides or 10 PBS nucleotides—seems unlikely anyway as the remaining complementarity between the PBS copy and the tRNA copy will not likely allow an efficient second strand transfer during reverse transcription.There is no need to implement a DNA repair action to explain the rapid reversion as this occurs by proviral DNA strand segregation upon cell division. There is also no need to introduce premature termination of reverse transcription to explain progeny proviruses with a mutant PBS, as this is caused by tRNAWe focused exclusively on the PBS mutation that was corrected in SIVmac239Opt. Rapid reversion of this mutation upon SIVmac239 replication was mistakenly interpreted as indicating a major impact on virus replication. The other three correcting mutations in SIVmac239Opt represent more regular positions in the viral genome and reversion of these mutations in long-term SIVmac239 cultures may indeed correlate with an fitness impact.


These include not only the known oligomannosyl antigens but also previously unrecognized tri-antennary or multi-valent GlcNAc-terminating N-glycan epitopes (Tri/m-Gn). These findings highlight the potential of N-glycan cryptic sugar moieties as conserved targets for broad-spectrum virus neutralization and suggest the GNA-model of glycan-binding warrants focused investigation.Identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the key steps toward development of vaccines against emerging viral pathogens. Owing to genomic and somatic diversities among viral species, identifying protein targets for broad-spectrum virus neutralization is highly challenging even for the same virus, such as HIV-1. However, viruses rely on host glycosylation machineries to synthesize and express glycans and, thereby, may display common carbohydrate moieties. Thus, exploring glycan-binding profiles of broad-spectrum virus-neutralizing agents may provide key information to uncover the carbohydrate-based virus-neutralizing epitopes. In this study, we characterized two broadly HIV-neutralizing agents, human monoclonal antibody 2G12 and Galanthus nivalis lectin (GNA), for their viral targeting activities. Although these agents were known to be specific for oligomannosyl antigens, they differ strikingly in virus-binding activities. The former is HIV-1 specific; the latter is broadly reactive and is able to neutralize viruses of distinct phylogenetic origins, such as HIV-1, severe acute respiratory syndrome coronavirus (SARS-CoV), and human cytomegalovirus (HCMV). In carbohydrate microarray analyses, we explored the molecular basis underlying the striking differences in the spectrum of anti-virus activities of the two probes. Unlike 2G12, which is strictly specific for the high-density Man Developing broad-range virus-neutralizing antibodies (bnAbs) requires identifying the immunological targets that are conserved among the targeted viruses and are suitable for antibody targeting. Discovery of oligomannosyl moieties as targets of bnAbs against HIV-1 has stimulated substantial interest in carbohydrate moieties as vaccine candidates ,9,10,11.Like HIV-1, virtually all human viruses decorate their virions with carbohydrate moieties. Thus, the potential of viral carbohydrates as immunological targets for other viral pathogens warrants exploration. One intriguing question is whether human viruses of distinct phylogenetic origins, such as HIV-1 and SARS-CoV, may display conserved glycan targets that are suitable for broad virus neutralization. Availability of bnAbs targeting such conserved glycans is clearly important for improving a biodefensive public health response to unexpected viral epidemics.In response to the 2003 SARS epidemic, our team introduced carbohydrate microarrays to study SARS-CoV-elicited anti-glycan antibodies . In esseet al. determined the glycan profiles of the spike protein produced by monkey Vero-E6 cells n-KLH (M9_2G12). The two Man9-KLH conjugates display Man9 moieties in two defined cluster configurations. M9_2G12 is highly specific for 2G12.(3)Ribonuclease B (RB) with Man5-6GlcNAc2Asn (M5-6) as the main glycans. (4)Phosphomannan (P-Man), a yeast polysaccharide. Both M5-6-RB and P-Man are known to be positive with GNA; the former but not the latter also binds to Con A. Using these microarrays, we characterized GNA, PHA-L, and 2G12 for their epitope-binding profiles. Results are summarized in To support exploration of the potential GNA glyco-epitopes in this study, we produced a set of comprehensive antigen microarrays, which include a large-panel of carbohydrates, lipids/liposomes, and protein antigens . Key com4, which stands for [(Man9GlcNAc2Asn)4]n-KLH (M9_2G12) conjugates, and M5-6-RB. The two control probes spotted are KLH and E. coli K1 polysaccharide. Microarray datasets corresponding to blue bars) versus those of local backgrounds surrounding the antigen microarrays (red bars).4, respectively, which is expected. However, GNA highly and selectively binds to a number of N-glycan cryptic sugar moieties, including Man9, (M9)4, M5-6, and AGOR. GNA-binding of AGOR is a novel observation. Given that GNA had no binding to ASOR (Tri/m-II), which only differs from AGOR (Tri/m-Gn) by having the terminal galactoses. This result demonstrates, therefore, that GNA is most likely specific for the Tri/m-Gn-glyco-epitopes of AGOR with exposed terminal GlcNAc moieties.i.e., GNA Ag/Bg, or 2G12 Ag/Bg. To assist a comparative analysis of binding profiles between GNA and 2G12, datasets were plotted in the same scale in the y-axis. The positive detections labeled with numbers in the graphs are AGOR (5), Man9 (9), (M9)4 (11), M5-6-RB (15), and P-Man in two dilutions (17 and 18). The whole datasets for these graphs are listed in In In this microarray analysis, GNA and 2G12 show minimal or no cross-relativities with irrelevant antigens but illustrate strikingly different glycan-binding profiles. 2G12 is “mono-specific” for Man9-clusters (9 and 11) as expected. By contrast, GNA is “pauci reactive” with a number of glycan targets. Its binding to the spotted oligomannose antigens  and P-Man (17 and 18) can be attributed to the known GNA-specificity for recognition of the shared Manα1,3Man and/or Manα1,6Man moieties. However, GNA-binding to AGOR, but not to ASOR or OR, indicates that GNA also specifically recognize the Tri/m-Gn-glyco-determinants displayed by AGOR. Supporting to this prediction is the fact that AGOR, ASOR, and OR are negative to other anti-mannose agents, including lectin Con A, mAbs 2G12, TM10, and polyclonal antisera that recognize oligomannosyl moieties of varies cluster configurations ,35. MoleA high-precision microarray robot  was used to spot antigen preparations onto glass slides pre-coated with nitrocellulose polymer  as described . The ant3 at room temperature (RT) for 30 min. They were then incubated at RT with 2G12 , biotinylated PHA-L (PHA-LBI), or GNABI  at an indicated titration in 1% (w/v) BSA in PBS containing 0.05% (w/v) NaN3 and 0.05% (v/v) Tween 20. The secondary antibodies or streptavidin conjugates applied for microarray staining are specified in the figure legends. The stained slides were rinsed five times with PBS with 0.05% (v/v) Tween 20, air-dried at room temperature, and then scanned for fluorescent signals using a ScanArray5000A Microarray Scanner  following the manufacturer’s manual.Immediately before use, the printed microarrays were rinsed with PBS, pH 7.4, with 0.05% (v/v) Tween 20 and then blocked by incubating the slides in 1% (w/v) bovine serum albumin (BSA) in PBS containing 0.05% (w/v) NaNi.e., GNA Ag/Bg, or 2G12 Ag/Bg. The whole microarray datasets for PHA-L, GNA, and 2G12 were presented in Fluorescence intensity values for each array spot and its background were calculated using ScanArray Express software . SAS Institute’s JMP-Genomics software package  was used for further microarray data processing and statistical analysis. In Two HIV-1 gp120 preparations, Bal-gp120-Man9 and Bal-gp120, were produced in HEK293 cells as previously described ,26. Bal-in vivo. Thus, effort must also be made to establish GNA-like potent and broadly virus-neutralizing antibodies, especially humanized or fully human mAbs that are readily applicable in the front-line biodefense against emerging viral pathogens.In this pilot study, we examined whether human viruses of distinct phylogenetic origins may express common carbohydrate moieties. Using carbohydrate microarrays and ELISA-based viral glycan-profiling analysis, we characterized two broadly HIV-neutralizing agents, human monoclonal antibody 2G12 and lectin GNA. Although these agents were known to target oligomannosyl antigens, they differ strikingly in the spectrum of viruses they effectively neutralize. The former is solely HIV-specific; the latter is broadly reactive with human viruses, including HIV-1, SARS-CoV, and HCMV, that are phylogenetically and pathogenically distinct. Our carbohydrate microarray analyses demonstrate the two probes differ strikingly in glycan-targeting specificities and the spectrum of glyco-epitopes they recognize. Although 2G12 is strictly specific for the high-density Man9 clusters that decorate the HIV envelope spike, GNA recognizes a number of N-glycan cryptic sugar moieties. These include oligomannoses and the previously unrecognized Tri/m-Gn-glyco-determinants. The latter appear to be the potent natural ligands of GNA. Molecular mechanisms underlying the GNA-model of “pauci reactive” glycan-binding and broad virus-neutralization warrant further investigation. Owing to the potential immunogenic activity as a plant-derived lectin, GNA is unlikely suitable for anti-virus therapy


Influenza viruses exist as a large group of closely related viral genomes, also called quasispecies. The composition of this influenza viral quasispecies can be determined by an accurate and sensitive sequencing technique and data analysis pipeline. We compared the suitability of two benchtop next-generation sequencers for whole genome influenza A quasispecies analysis: the Illumina MiSeq sequencing-by-synthesis and the Ion Torrent PGM semiconductor sequencing technique.in vitro. We also optimized an RT-PCR protocol to obtain uniform coverage of all eight genomic RNA segments. The sequencing reads obtained with both sequencers could successfully be assembled de novo into the segmented influenza virus genome. After mapping of the reads to the reference genome, we found that the detection limit for reliable recognition of variants in the viral genome required a frequency of 0.5% or higher. This threshold exceeds the background error rate resulting from the RT-PCR reaction and the sequencing method. Most of the variants in the PR8 virus genome were present in hemagglutinin, and these mutations were detected by both sequencers.We first compared the accuracy and sensitivity of both sequencers using plasmid DNA and different ratios of wild type and mutant plasmid. Illumina MiSeq sequencing reads were one and a half times more accurate than those of the Ion Torrent PGM. The majority of sequencing errors were substitutions on the Illumina MiSeq and insertions and deletions, mostly in homopolymer regions, on the Ion Torrent PGM. To evaluate the suitability of the two techniques for determining the genome diversity of influenza A virus, we generated plasmid-derived PR8 virus and grew this virus Our approach underlines the power and limitations of two commonly used next-generation sequencers for the analysis of influenza virus gene diversity. We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time. The data analysis pipeline that we propose here will also help to standardize variant calling in small RNA genomes based on next-generation sequencing data. Viruses outnumber all other known life forms on earth. Furthermore, viruses in general and RNA viruses in particular have a huge genetic diversity, which is the driving force of their evolutionary success. Viral genomic diversity is well captured in the term ‘quasispecies’. The term ‘quasispecies theory’ was first introduced by Manfred Eigen as a theoretical model to study molecular evolution by mutation and selection in self-reproducing macromolecules ,2. LaterOrthomyxoviridae family, which is characterized by enveloped viruses that have a segmented, single-stranded, negative sense RNA genome [−5) because the viral RNA-dependent RNA polymerase lacks 3′-5′-exonuclease activity and therefore has no proof-reading function [Influenza is an acute and highly contagious viral disease of the respiratory tract in humans. It is caused by influenza A and B viruses and occasionally by influenza C virus. These viruses represent three of the five genera of the A genome . Replicafunction ,13. Muta−5 per nucleotide per infectious cycle. Sequence analysis of multiple clones of cDNA fragments derived from one or more gene segments has also been used to study sequence variation of influenza virus derived from clinical samples [i.e. reverse transcription and PCR amplification, and the NGS method itself is also an error-prone process [The mutation rate of influenza A viruses has been traditionally determined by sequencing different cDNA clones obtained from multiple plaques descending from a plaque-purified influenza A virus . In othe samples ,16. In a samples ,18. Howe samples -21. Howe process .i.e. the number of times the genome is sequenced). However, its segmented RNA genome makes it technically challenging to obtain full genome coverage. Stoichiometric RT-PCR amplification of each of the eight genomic RNA segments is difficult, in particular when starting from ex vivo samples such as nasal swabs or bronchoalveolar lavage from experimentally infected animals. NGS studies of influenza virus reported to date did not start from the amplification of all eight full-length genomic segments in sufficient amounts in a single reaction, and homogeneous coverage across all eight segments was not always obtained [NGS enables sequencing of multiple gigabases of DNA in a single run; the output size depends on the sequencing instrument . Consequobtained -29.in vitro and mouse experiments. In addition, PR8 virus is used as a donor to generate egg-grown reassortant viruses for seasonal influenza vaccine production. Importantly, we also took advantage of the available plasmid-based reverse genetics system for PR8 virus because it is a genetically stable equivalent of the virus [Here, we compared the suitability of two NGS methods to determine the influenza A virus quasispecies diversity. We deep-sequenced A/Puerto Rico/8/34 (PR8) influenza virus, which is used extensively in many research laboratories for he virus . We compOur aim was twofold: (1) to compare the performance of two high-throughput sequencing instruments; (2) to determine the complexity of an influenza A virus quasispecies (to count the number of nucleotide variants present in a swarm of genomes of that virus). We selected the Illumina MiSeq and the Ion Torrent PGM sequencing platforms because the accuracy of single nucleotide polymorphism (SNP) identification of these two popular NGS platforms is unclear. A study by Quail and colleagues concluded that the overall SNP calling rate is slightly higher for the data generated by Ion Torrent PGM than for Illumina MiSeq data , whereasWe first compared the accuracy and sensitivity of these two sequencers. We used plasmid DNA to compare the accuracy of the sequencing output because it is genetically very stable. We also generated a plasmid with two tracer mutations, which allowed us to prepare mixtures with different, defined amounts of wild type and mutant plasmid before sequence analysis, in order to determine the sensitivity of the sequencers for picking out the occurrence of the introduced SNPs. For this comparison, we chose plasmids that also allowed us to generate PR8 virus with or without the introduced tracer mutations ,31.We generated a mutated version of plasmid pHW197-M (pHW197-Mmut). This mutant has two silent mutations in the influenza virus M1 open reading frame (ORF) that served as tracers when mixing pHW197-Mmut and pHW197-M plasmids at different ratios. Because we intended to perform such mixing experiments with both plasmids and viruses generated from these plasmids, we carefully selected two silent mutations that most likely would not affect virus fitness. We chose these mutations based on their prevalence in human H1N1 virus isolates see . We selede novo sequence assembly. In addition, longer read lengths increase the likelihood that one can conclude whether mutations observed in a genomic segment are linked or not. The two point mutations that we introduced in the M gene segment are 291 nucleotides apart. Therefore, to confirm the presence of these two mutations on the same DNA molecule, read lengths after processing should be at least 291 nucleotides long. Such a length should be obtained when using the Ion Torrent PGM 400 base-pair sequencing kits. The length distribution of the sequencing reads of the plasmid samples generated by both sequencers is shown in black in Figure Assuming an equal error rate per base, longer read lengths are preferred for the de novo sequence assembly. Therefore, we performed a quality control using the CLC Genomics Workbench software; we also propose a NGS data analysis pipeline that is generally applicable  can be confidently counted, because low quality reads could lead to over-interpretation of the number of mutations. In addition, high quality reads will lead to a higher accuracy of e Figure . First, The per-base quality distribution on both sequencers, using the plasmid samples as template, is shown in Figure To evaluate the accuracies of both sequencers, the processed reads were mapped to the plasmid reference sequence Table . The perFor both sequencers, we observed a striking fluctuation in coverage depth (times a nucleotide is sequenced plotted against the position in the genome) Figure . The larSince the plasmid reference sequence is known, we were confident that any mismatching nucleotide variant could be reported as a sequencing error. The error rate per read position was 0.08% ± 0.01% for the Illumina MiSeq and 0.12% ± 0.01% for the Ion Torrent PGM. The error rate increases slightly with the read length for both sequencers, with a pronounced rise at the end of the reads on the Ion Torrent PGM (data not shown). For the Illumina MiSeq, substitutions are the dominant error type with A-to-C and T-to-G being the most prevalent Figure A, which i.e. above 0.16% for the Illumina MiSeq and above 0.24% for the Ion Torrent PGM. Since we are dealing with proportions very close to zero, the proportion of variants that could be miscalled at this threshold was estimated using the Agresti-Coull interval as an approximate binomial confidence interval [in silico filtering parameters. We set the forward/reverse balance between 0.25 and 0.75, meaning that the minimum ratio between the number of forward and reverse reads that support the surmised variant should be at least 0.25. In addition, a nucleotide variant should be counted at least 10 times independently and should have an average Phred score of at least 20  when it was higher than twice the sequencing error background, interval . Settinginterval ,38,39). based on )  and then sequenced the mixtures on both platforms. On both sequencers, the calculated frequency of pHW197-M or pHW197-Mmut based on the output data closely resembled the used ratios Table . Neverthin vitro  to evaluate the consistency of the two NGS methods. In addition, wild type and mutant viruses were mixed at a ratio of 99:1 before RNA isolation to compare the accuracy of the two NGS sequencing methods to resolve this ratio. Finally, we also wanted to quantify the number of differences, if any, between the plasmid encoded influenza virus information and the in vitro cultured virus samples. This quantification would reflect the baseline quasispecies diversity, in the absence of exogenous selection pressure.To compare the efficacy of the sequencers for detecting mutations in an influenza A virus sample, we generated influenza virus starting from eight plasmids, including pHW197-M or pHW197-Mmut. This resulted in wild type PR8 and mutant PR8 (PR8mut), respectively, the latter carrying two silent mutations in the M1 ORF . These mutations did not seem to affect viral fitness because PR8 and PR8mut replicated equally well GCGAAAGCAGG, match in bold). By lowering the concentration of the CommonUni12G primer, we could avoid this extra band and obtained the eight amplicons of the expected size  samples. The viral RT-PCR products were purified and subjected to NGS on the Illumina MiSeq and the Ion Torrent PGM platforms. Before assembly, the reads were processed in silico as described above for the plasmid-derived sequences  resulted in sufficient full-length coverage of the entire influenza genome , were used to prepare RT-PCR products that were subsequently sequenced on both platforms  Figure A. All obin vitro grown PR8 and PR8mut virus. Sequencing each sample in duplicate and simultaneously on the same machine also allowed us to determine and compare the intrinsic variability of the two platforms. The number and types of nucleotide variants that were retained after applying the variant filter are presented in Table Next, we determined the number of variants at each nucleotide position in the virus-derived sequences, which would reflect the quasispecies diversity of The average difference between the frequencies of a variant in PR8 sequencing duplicates was only 0.17% ± 0.12% for the Illumina MiSeq and 0.16% ± 0.18% for the Ion Torrent PGM, again indicating that both sequencing platforms provide reproducible output Table . To deteAlmost all mutations detected in the wild type and/or mutant PR8 quasispecies are also present in H1N1 viral sequences retrieved from the Influenza Research Database and/or Genbank. The exceptions are indicated with a number sign (#) in Tables Taken together, these results show that both the wild type and mutant PR8 virus behave as a fairly heterogeneous virus populations even in the absence of external selection pressure.in silico processing of the sequencing data that allows identification and frequency determination of nucleotide variants in the influenza A virus  has become increasingly valuable to study virus diversity. NGS instruments have a very high sequencing capacity and therefore allow a very high coverage of the relatively small genome of RNA viruses. NGS analysis is thus in principle well suited for determining the genetic heterogeneity of RNA viruses. Unfortunately, in many research articles on viral quasispecies diversity there is little information on how the raw data were processed. Furthermore, the performance of different commercially available NGS platforms for quasispecies analysis has not been evaluated. Here, we compared the quality of the sequencing output obtained on the Illumina MiSeq and Ion Torrent PGM benchtop sequencers. We also propose an analysis pipeline for s Figure . This anTo determine the influenza genome diversity by NGS technology, different hurdles have to be overcome. First, it is technically challenging to obtain high quality full-length RT-PCR products that cover the complete segmented RNA genome of influenza viruses. We optimized an RT-PCR protocol with primers based on the conserved 3′ (Uni12) and 5′ ends (Uni13) of the eight genome segments ,43,71. Cin silico to retain only high quality reads. However, the available software and filtering parameters vary and are not always clearly described in the literature, making comparison of results very difficult. To reduce false positive variant calls introduced by the sequencing method, we applied specific trimming, filtering and variant calling parameters in the CLC Genomics Workbench software. We first applied this bioinformatics analysis pipeline to sequencing reads derived from plasmid DNA samples. We removed adaptor contamination, ambiguous nucleotides and trimmed low quality bases at the end of the reads by applying a Phred score of 20. Then, we excluded reads shorter than 50 bases to avoid unspecific mapping of these short reads. Trimming eliminated relatively more bases from the Ion Torrent PGM, meaning that the base quality of sequencing reads from the Ion Torrent PGM is lower than that from the Illumina MiSeq. In other words, the potential advantage of longer read lengths obtained with the Ion Torrent machine was cancelled by their relatively low quality. Together this resulted in a higher relative loss of bases for the Ion Torrent PGM data than for the Illumina MiSeq data (21.01% versus 14.01% respectively). Furthermore, the Phred score distribution across the reads, a measure of the intrinsic sequencing quality, was higher for the Illumina MiSeq data than for the Ion Torrent PGM data, resulting in a lower error rate. After this quality control, the sequencing reads were mapped to the reference sequence, resulting in a higher percentage of mapped reads for the Illumina MiSeq. The total mapping error rate of the Illumina MiSeq (mainly nucleotide substitutions) was lower than that of the Ion Torrent PGM (mainly indels). This finding is in agreement with Loman and colleagues [A second hurdle is to distinguish between mutations that truly represent the viral genome diversity from errors introduced by RT-PCR amplification and the NGS chemistry. The first step is to filter the output sequence data lleagues ,73.We then applied the analysis pipeline outlined in Figure de novo assembled PR8 and PR8mut sequences correspond very well to the plasmid-derived reference genome. We detected 19 mutations in PR8 and 29 mutations (including the two tracer mutations) in PR8mut with a frequency of 0.5% or higher. When a variant was present at low frequency (< 15%), the Illumina MiSeq detected it with significantly higher frequency than the Ion Torrent PGM. Most of the detected mutations were transitions and appeared with a frequency below 5%. However, three mutations in HA and one in NP of PR8, as well as four mutations in HA and one in NS of PR8mut, were present at a frequency > 10% (based on Illumina MiSeq data)  and Asn460Ser (10 − 15%) (Asn117Asp and Asn117Ser for H3 numbering of HA2). Based on pyrosequencing of the HA segment, the Asn460Asp mutation has been observed in 12.2% in PR8 virus grown on MDCK cells [in vivo but mutating it to alanine was tolerated in vitro [We focused on the mutations detected by both sequencers with a frequency > 5% and on the mutations that appeared in both wild type and mutant PR8 viruses. There are three such mutations in the HA head domain of PR8 and four in the HA head domain of PR8mut, and all of them are part of or close to the antigenic sites Figure A. The shCK cells . In addiCK cells . Both PRin vitro . In wildin vitro . The Glnin vitro . Remarkain vitro . Howeverin silico quality control, the two sequencers produced reads of comparable lengths. The Illumina MiSeq had a higher intrinsic sequencing quality than the Ion Torrent PGM, presumably because detecting incorporated bases based on a coupled fluorescent dye (Illumina) gives less noise than a change in pH caused by release of a proton after incorporation of a base (Ion Torrent). However, the Ion Torrent PGM had a lower false-positive rate for detecting SNPs. Another interesting observation is the lower coverage of the ends of the viral segments on the Illumina MiSeq due to the transposase-based fragmentation. Nextera transposase-based fragment library preparation is convenient and fast but results in low coverage of segment termini. We also noticed some sequence bias of this transposase-based fragmentation approach  is clearly advantageous to the Illumina MiSeq (about 3 days). In contrast, when analyzing many viral samples at high coverage, the greater output of the Illumina MiSeq is an important advantage.The proposed RT-PCR protocol and subsequent analysis pipeline for influenza viruses is widely applicable, in silico pipeline for selecting high quality reads obtained by NGS platforms. This pipeline is also more widely applicable. Due to the lower total error rate and the higher sequencing quality of the reads, we conclude that the Illumina MiSeq platform is more suited than the Ion Torrent PGM for detecting variant sequences, whereas the Ion Torrent platform has a shorter turnaround time. In addition, we found that the detection limit for reliable recognition of variants in the viral genome required a frequency of 0.5% or higher.Our study underlines the power and limitations of two commonly used next-generation sequencers for the analysis of influenza gene diversity. We propose an 2.MDCK and HEK293T cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, non-essential amino acids, 2 mM L-glutamine, 0.4 mM sodium pyruvate, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37°C in 5% COE. coli DH5α. Plasmid DNA was isolated with the Plasmid Midi Kit (Qiagen) according to the manufacturer’s instructions. The resulting air-dried pellet was dissolved in 50 μl of sterile ultrapure water. The presence of the introduced mutations in pHW197-Mmut was confirmed by restriction analysis and Sanger sequencing on a capillary sequencer .Reverse genetics plasmids for PR8 virus were kindly provided by Dr. Robert G. Webster  . We intr5 HEK293T and 2 × 105 MDCK cells in a 6-well plate). After 30 h, L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma) was added to a final concentration of 2 μg/ml. After 72 h, the culture medium was collected and the presence of virus was confirmed by hemagglutination of chicken red blood cells. Reverse genetics-generated PR8 and PR8mut viruses were plaque-purified on MDCK cells as follows. Confluent MDCK cells in a six-well plate were infected with a serial dilution series of virus. After 1 h, an overlay of low melting agarose  in serum-free cell culture medium containing 2 μg/ml TPCK-treated trypsin (Sigma) was added. After 56 h, cytopathic effect was checked, agar overlaying viral plaques were selected with a pipette tip, and virus was allowed to diffuse from the agar for 24 h at 4°C in serum-free medium. Afterwards, virus derived from one plaque was amplified on MDCK cells in serum-free cell culture medium in the presence of 2 μg/ml TPCK-treated trypsin (Sigma). After 96 h, the culture medium was collected, and cell debris was removed by centrifugation for 10 min at 2500 g at 4°C, and the virus was pelleted from the supernatants by overnight centrifugation at 16,000 g at 4°C. The pellet was dissolved in sterile 20% glycerol in PBS, aliquoted and stored at −80°C. The infectious titer of the obtained PR8 and PR8mut virus stocks was determined by plaque assay on MDCK cells, on three different aliquots each performed in triplicate. The presence of the introduced mutations in the M segment of PR8mut was confirmed by segment-7-specific RT-PCR followed by purification from 1% agarose gel  and conventional Sanger sequencing of the amplified PCR fragment.To generate recombinant wild type PR8 virus and PR8 virus with the two tracer mutations in the M gene (PR8mut), 1 μg of pHW191-PB2, pHW192-PB1, pHW193-PA, pHW194-HA, pHW195-NP, pHW196-NA and pHW198-NS, together with 1 μg of pHW197-M (wild type PR8) or pHW197-Mmut (PR8mut) was transfected using calcium phosphate co-precipitation into a HEK293T-MDCK cell co-culture in Opti-MEM  with a two-fold dilution series of the virus (made in serum-free cell culture medium containing 0.1% BSA) in 500 μl medium. After 1 h incubation at 37°C, an overlay of 500 μl of 1.6% Avicel RC-591 (FMC Biopolymer) in serum-free medium with 4 μg/ml TPCK-treated trypsin (Sigma) was added. After incubation at 37°C for 48 h, the overlay was removed and the cells were fixed with 4% paraformaldehyde and permeabilized with 20 mM glycine and 0.5% (v/v) Triton X-100. Plaques were stained with an anti-M2e IgG1 mouse monoclonal antibody  followed by a secondary anti-mouse IgG horseradish peroxidase (HRP)-linked antibody . After washing, TrueBlue peroxidase substrate (KPL) was used to visualize the plaques.MDCK cells were seeded in complete DMEM in 12-well plates at 3 × 107 PFU of stock virus in serum-free cell culture medium with 0.1% BSA was combined with 400 μl lysis-binding buffer and mixed by vortexing. The mixture was loaded on a two-layered glass fiber column. After binding to the column and washing, the RNA was eluted in 50 μl elution buffer .RNA was isolated with the High Pure RNA Isolation Kit (Roche) according to the manufacturer’s instructions, excluding the DNase I digestion step. In brief, a 200-μl sample containing 1 x 10Primers used for cDNA synthesis and PCR were designed based on the 5′ and 3′ conserved ends of the influenza A genomic segments and contain an additional sequence of 20 nucleotides at their 5′ end necessary for PCR amplification -43,79. cWe used 0.5 ng of purified plasmid or RT-PCR sample and the Nextera XT DNA Sample Preparation Kit (Illumina) according to the manufacturer’s instructions to generate multiplexed paired-end sequencing libraries. Sequencing libraries were generated in duplicate, meaning that from each plasmid or RT-PCR sample two libraries were prepared in parallel and sequenced on the same Illumina MiSeq sequencing chip. In brief, DNA samples were fragmented and tagged with adapters by Nextera XT transposase. These adaptor ligated DNA fragments were amplified by a limited-cycle PCR program (12 cycles) to add the barcodes and sequences required for subsequent cluster formation. The resulting fragments were purified and simultaneously size-selected by using 0.6× AMpure beads. Fragments were analyzed on a High Sensitivity DNA Chip on the Bioanalyzer (Agilent Technologies) before loading on the sequencing chip. The fragment lengths showed a negatively skewed distribution with a peak at approximately 700–1000 bases. From the optimized RT-PCR products, also 500 ng was sheared with an M220 focused-ultrasonicator (Covaris) set to obtain peak fragment lengths of 300–400 bp. Next, the NEBNext Ultra DNA Library Preparation kit (New England Biolabs) was used to repair the ends and to add Illumina MiSeq-compatible barcode adapters to 100 ng of fragmented DNA. The resulting fragments were size-selected using Agencourt AMPure XP bead sizing (Beckman Coulter). Afterwards, indexes were added in a limited-cycle PCR (10 cycles), followed by purification on Agencourt AMpure XP beads. Fragments were analyzed on a High Sensitivity DNA Chip on the Bioanalyzer (Agilent Technologies) before loading on the sequencing chip. Equimolar amounts of normalized libraries were combined and diluted 25-fold in hybridization buffer. The multiplex sample was heat denatured for 2 min at 96°C before loading on the MiSeq chip. After the 2×250 bp MiSeq paired-end sequencing run, the data were base called and reads with the same barcode were collected and assigned to a sample on the instrument, which generated Illumina FASTQ files (Phred +64 encoding). These files were imported in the CLC Genomics Workbench software . During import in CLC Genomics Workbench, the uncallable ends of the MiSeq reads (B in input file) were automatically trimmed and the failed reads  were removed.Samples for sequence analysis were generated in duplicate, meaning that from each plasmid or RT-PCR sample two libraries were prepared in parallel for sequencing on the same Ion Torrent PGM 318 sequencing chip. From each plasmid or RT-PCR product, 100 ng was sheared with an M220 focused-ultrasonicator (Covaris) set to obtain peak fragment lengths of 400–500 bp. After shearing, blunt ends were created using the end repair enzyme from the Ion Plus Fragment Library kit (Life Technologies). Next, the fragments were ligated to Ion Torrent PGM-compatible barcode adapters. Since the adaptors are not 5′ phosphorylated, the nick repair polymerase in the kit repairs subsequently the nick on one strand at each ligation site, in order to minimize adaptor-dimer formation. We purified and simultaneously size-selected the adapter-ligated library using Agencourt AMPure XP bead sizing (Beckman Coulter). Fragments were analyzed on a High Sensitivity DNA Chip on the Bioanalyzer (Agilent Technologies); the fragment length peak was situated around 450 bp. Barcoded libraries were pooled in equimolar amounts. From the resulting diluted multiplexed library, 20 μl was loaded on an Ion OneTouch 2 instrument (Life Technologies) to perform emulsion PCR on Ion Sphere particles using the Ion PGM Template OT2 400 kit. We used the Ion PGM sequencing 400 kit (Life Technologies) to sequence templated ion sphere particles deposited in the Ion 318 chip v2 . The Ion Torrent Suite version 4.6 (Life Technologies) was used with the default parameters for base calling and assigning of the reads to a sample based on their barcode. The default settings in the Ion Torrent Suite already filter and trim the sequencing reads to some extent. These default trimming parameters are not stringent and remove only very low quality 3′ ends (mean Phred score of at least 15 in a base window of 30) and adaptor contamination. The resulting FASTQ files were imported into CLC Genomics Workbench for further analysis.de novo assembly. In addition, the processed reads were also aligned with the pHW197-M plasmid reference sequence or the influenza PR8 reference genome  using local alignment. For this, the following default penalties were used: match = +1, mismatch = −2, insertion/deletion = −3, filtering threshold: length fraction = 0.9 and similarity fraction = 0.8. Non-specific matches, defined as reads aligning to more than one position with an equally good score, were ignored. Sequence variants were called using all available sequencing data that covered each nucleotide at least 100 times and had a central base quality score of Q20 or greater. The A-to-G variant introduced by the primer at position 24 in the HA, NP, NA, M and NS segments was not taken into account during the influenza quasispecies variant analysis. All numerical data mentioned in the text are presented as averages with their standard deviations (± SD).CLC Genomics Workbench version 7.0.3  was used to analyze and process the sequencing reads of both the Ion Torrent PGM and the Illumina MiSeq. First, adaptor contamination was removed from the reads. Next, the sequencing reads were trimmed from both sides using the modified Mott trimming algorithm to reach a Q20 score, which means that the chance that a particular base in the sequence is called incorrectly by the sequencer is 1 in 100. Afterwards, all ambiguous (N) bases were trimmed from the reads. We also removed the reads with a read length below 50. For the Illumina MiSeq, the broken pairs resulting from trimming and filtering were also removed. The remaining reads were assembled using default settings for Sequence variants with the lowest proportion were considered as minor alleles. Analysis of minor allele proportions was performed by fitting a logistic regression model of the form logit(p) = constant + PLATFORM*VIRUS*CLASS + error, where p indicates the minor allele proportion, PLATFORM refers to the sequencing platform, VIRUS refers to virus population, and CLASS refers to class of loci having either low (< 15%) or high (> 15%) minor variant frequencies. Significance of the fixed PLATFORM, VIRUS and CLASS effects was assessed by an F-test. Significance of pair-wise comparisons between mean proportions was assessed by a Fisher’s protected least significance difference test. The logistic regression and assessment of significance was performed in Genstat v16.The output sequencing reads obtained on the Illumina MiSeq and Ion Torrent PGM were submitted to NCBI’s Sequence Read Archive and can be found under project numbers SRP052608 (plasmid samples) and SRP052225 .


Using a proprietary combination of fluorescent dyes that stain both nucleic acid and protein in a single 30 min step, rapid, reproducible, and cost-effective quantification of filovirus particles was demonstrated. Using a seed stock of Ebola virus variant Kikwit, the linear range of the instrument was determined to be 2.8E+06 to 1.0E+09 virus particles per mL with coefficient of variation ranging from 9.4% to 31.5% for samples tested in triplicate. VC particle counts for various filovirus stocks were within one log of TEM particle counts. A linear relationship was established between the plaque assay, qRT-PCR, and the VC. VC results significantly correlated with both plaque assay and qRT-PCR. These results demonstrated that the VC is an easy, fast, and consistent method to quantify filoviruses in stock preparations.Development and evaluation of medical countermeasures for diagnostics, vaccines, and therapeutics requires production of standardized, reproducible, and well characterized virus preparations. For filoviruses this includes plaque assay for quantitation of infectious virus, transmission electron microscopy (TEM) for morphology and quantitation of virus particles, and real-time reverse transcription PCR for quantitation of viral RNA (qRT-PCR). The ViroCyt Ebolavirus and Marburgvirus. There are at least five Ebolavirus species and a single Marburgvirus species [Filoviruses are the causative agents of severe hemorrhagic fever with high mortality rates in humans . They ar species . Filovir species ,4. One oThe most commonly used methods for filovirus quantification include the plaque assay and transmission electron microscopy (TEM). The gold standard for quantifying infectious viruses is the plaque assay, which provides a titer based on virus-infected cells producing a plaque. In theory, each plaque arises from a single infectious virion. Results provide a concentration of infectious virus particles, termed plaque-forming units (PFU/mL) . Plaque Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a relatively new technique that enumerates genetic material by comparing an individual sample to a standard RNA curve. Results are expressed as genomic equivalents per mL (GE/mL). While the genomic sequences of filoviruses are very similar, different species can be distinguished using primers and probes with unique sequences ,7. Strai® Virus Counter (VC) 2100  instrument is a reengineered non-sorting analytical flow cytometer developed specifically to directly quantify virus particles in solution independent of the virus species. A single universal staining step uses a proprietary stain that is a combination of two fluorescent dyes, one specific for envelope proteins and the other specific for nucleic acids  [The ViroCyttranded) ,10. Virutranded) . Like fi® Virus Counter 2100 instrument and suitability for filovirus particle quantitation, we compared the VC to TEM, using a number of different filovirus stocks propagated in Vero E-6  cells. In addition, we investigated the relationship between the different methods used to quantitate filoviruses, providing valuable insight into results derived from these various techniques.Infectious titer, genomic equivalents, and particle count are all quantitative measurements that must be accurate, reproducible, and robust for the results to be meaningful. Use of well characterized stocks in pivotal efficacy studies requires these assays to be standardized and validated. To evaluate the ViroCytAll filovirus seed stocks used were media (supernatant) harvested from infected Vero E-6 cells after visual evidence of cytopathic effect in at least 50% of the cells. Media contained 10% fetal bovine serum. After harvesting, the medium was clarified, aliquoted into single use vials, and immediately stored at −70°C. TEM conducted shortly after harvest revealed morphology typical of filoviruses with virus particles present in numerous forms, predominately straight filamentous shapes, but torus, “6,” and “V” shapes were also present. Lengths ranged from 500 nm to excess of 2500 nm. Particle diameters were not reported.A seed stock of Ebola virus (EBOV), Kikwit variant, was diluted beginning at 1:10 into Hank’s balanced salt solution buffer. Four serial ¼ log dilutions were prepared from the 1:10 in order to provide samples that lie within the linear range of the VC. Four additional serial 10-fold dilutions were prepared from the 1:10 dilution  for a total of nine dilutions (samples). The stock used to prepare diluted samples had an initial plaque assay titer of 1.78E+07 PFU/mL. The coded samples were tested with each quantitative method and used to examine reproducibility, linearity, and correlation between the various methods. Previously characterized stocks of EBOV, Kikwit variant, Sudan virus (SUDV), Gulu variant, and Marburg virus (MARV), Angola variant, were analyzed to compare the VC and TEM particle concentrations. et al., 2012. Briefly, six wells were inoculated with 200 μL of each dilution and plates rocked every 15 min to minimize drying out of the monolayer. After 1 h incubation, 2 mL of a 0.5% semi-solid agarose overlay was added to each well. Plates were incubated for seven days and cells stained with 2 mL of a secondary overlay that included 5% neutral red . Plaques in each well were counted 24 h post-staining. The titer for each sample was calculated from wells with countable plaques (≤150) and factoring in the dilution. Final concentration of the seed stock was established using results from all samples with plaque counts between 10 and 150.Each sample generated was diluted in a serial 10-fold series and tested in a viral plaque assay using confluent six-well plates of Vero E-6 cells, as described in ShurtleffR2 values for the standard curve. Using the Ct value for each sample, the GE/reaction was interpolated from the slope and y-intercept of the standard curve. The TRIzol dilution factor was then used to calculate the GE/mL for each sample. Final concentration of the seed stock was established using all sample results that fell within the linear range of the assay and factoring in the dilution of the sample.Each sample was tested in triplicate with an EBOV-specific qRT-PCR assay developed and validated in our laboratory ,12. BrieAn equal mixture of the sample  and 1:100 dilution of 100 nm polystyrene beads  were adsorbed to charged 200-mesh formvar/carbon-coated nickel grids (SPI Supplies), fixed using 2% cacodylate-buffered glutaraldehyde, and sterilized by exposure to vapors from 1% osmium tetroxide before being removed from biocontainment. Three grids were prepared for each sample. All viral particles and polystyrene beads were counted in at least 10 randomly chosen squares on each grid. The volume was calculated by dividing the number of beads counted by the known concentration per mL (2E+10). The bead solution: viral stock solution ratio was 1:1, so VP/mL was determined by dividing counted particles by the calculated volume of sample: Final virus particle concentration of the seed stock was established using results from samples with countable number of particles on all three grids and factoring in the dilution of the sample.The VC reagent kit (ViroCyt) was used following the manufacturer-recommended procedures. The instrument performance was validated prior to testing samples by running a non-biological positive control  and cleanliness control to verify the flow path was clean. Briefly, 300 μL of each sample was stained using 150 μL of Combo Dye solution, incubated in the dark at room temperature for 30 min, and analyzed in the VC. Samples were tested in triplicate with inter-sample washes and a cleanliness control run between each sample. Results were automatically analyzed by the instrument software and reported as VP/mL. A negative control sample was diluted and tested to establish the sample quantification limit . The negneg is the mean value of the negative control samples; σneg is the standard deviation of the negative control samples; and t99% is the t value for N-1 degrees of freedom at the 99% confidence level.XFinal virus particle concentration of the seed stock was established using all samples whose VP/mL counts were above the sample quantification limit and within the linear range of the instrument and factoring in the dilution of the sample.p-values). A p-value of <0.05 was considered statistically significant.Microsoft Excel 2007  was used for linear-regression analysis. GraphPad Prism v5 software  was used to perform Pearson correlation analysis to determine the correlation between each assay (correlation coefficients (r) and A total of nine dilutions (samples) were generated using an EBOV, Kikwit variant stock. Samples were coded and then tested using the various quantitative methods. Results are shown in R2 for the experimental plaque assay result was 0.9984 with a slope of 0.9633. Experimental titers correlated well to theoretical values . Using the 8 samples whose titers were established using plaque counts between 10 and 150, the concentration of the stock virus was determined to be 1.10E+07 PFU/mL, a difference of 0.21 logs from the original titer (1.78E+07 PFU/mL). The coefficient of variation (CV) for the first seven samples ranged from 7.4% to 22.3%, with 27.8% and 37.7% for the two most dilute samples.Every sample was quantified by plaque assay a. The nuR2 for the standard curve used to determine GE/mL was 0.999 with a slope of −3.48 and an intercept of 45.08 (data not shown). The lowest copy number quantified by the qRT-PCR corresponded to a 1:10,000 dilution of the stock virus . While detected, results from the 1:100,000 dilution fell outside the linear range of the assay (CV = 72.9%). Using seven samples whose results fell within the linear range of the assay, the R2 for the experimental results was 0.9961 with a slope of 1.006. Experimental titers correlated well to theoretical values . The GE/mL of this seed stock was determined to be 3.40E+10, a difference of 0.25 logs from the original GE/mL concentration (1.91E+10 GE/mL). CV ranged from 1.2% to 7.4% for those samples that fell within the linear range of the assay.The EBOV-specific qRT-PCR assay was linear between 1E+02 and 1E+09 GE of synthetic RNA per reaction b. R2 forR2 for the experimental results was 0.1438 with a slope of 0.36. Experimental titers did not correlate with theoretical values . Using only the most concentrated sample, the particle concentration was determined to be 4.01E+07 VP/mL for this stock, a difference of 0.82 logs from the original VP/mL concentration (2.67E+08 VP/mL). The CV for replicate samples was 121.6%.Three grids of each dilution (sample) were prepared and counted using TEM. Particle morphology was not included in this assessment. Virus particles were not detected in sample dilutions greater than 1:31.6 . Results from the TEM are shown in i.e., the instrument qualification limit). Three replicates of each dilution (sample) were tested in the VC. No virus particles were detected in sample dilutions greater than 1:100 . Results from the VC are shown in R2 value for the experimental results was 0.9731 with a slope of 1.5045. Experimental titers correlated well to theoretical values . Particle concentration was determined to be 5.5E+08 VP/mL for this stock, a difference of 0.44 logs from the original VP/mL concentration (1.5E+09 VP/mL). CV ranged from 9.4% to 31.5% for those samples that fell within the linear range of the instrument. Similar dilution analyses were done for a number of filoviruses using the VC, with all stocks yielding linear regression fits of R2 ≥ 0.95 and slopes between 1.24 and 1.58. The R2 values and slopes confirm a strong positive correlation and linear relationship between VC results and dilution values (data not shown).Uninfected Vero E-6 cell culture media was used to determine the background or sample quantification limit of the VC. Using a total of 15 values, we determined the sample quantification limit of the instrument to be 2.8E+06 VP/mL (data not shown). Sample VP/mL results equal to or below this value were reported as below the limit of detection of the instrument  and slopes of 0.96 for the plaque assay, 1.01 for the qRT-PCR, 1.50 for the VC, and 0.36 for TEM. As presented in p = 0.4649) (data not shown).The relationship between infectious virus, genomic material, and virus particles as measured by VC and TEM are shown in A summary of the initial quantitative assay results for the EBOV, Kikwit variant stock described above is shown in Current filovirus quantitation methods include plaque assay, qRT-PCR, and TEM. Virus quantitation-specific assays used by different laboratories vary depending on the size of the biocontainment space, user preferences, and specific needs. Filovirus plaque assays are the gold standard assay for quantitation. Plaque assay quantifies infectious virus and has been used for many years by filovirus researchers. The assay is biological in nature, depending on living cells and their ability to be infected and produce plaques. The assay measures infectious virus but the ratio of infectious virus particle to plaque is unknown. There are many steps that contribute to the accuracy and reproducibility of the results. Standardization and training with periodic assessment can ensure the results are always optimal. Repeatability, a component of precision, was defined in this study as the CV between replicates. The plaque assay CV ranged from 7.4% to 37.7%, with the most dilute samples exhibiting the highest values. Titers established for each of these samples were within 0.28 logs. Reproducibility, expressed in this study as the difference in titer between the stock concentration determined at the time of preparation and that achieved in this study, was very good with only a 0.21 log difference in titer .Quantitative RT-PCR measures genetic material (RNA) that is specific for the primers and probes used in the assay. qRT-PCR has been used to quantify many viruses to include influenza , human iVirus particle count by TEM quantifies the number of virions meeting specific criteria of size and shape consistent with filoviruses. Filovirus particle size is fairly well established but problematic since the virus is pleomorphic with varying proportions of long filaments, ‘6’-shaped forms, and circular configurations. Although nucleocapsid and particle diameters were consistent within and among species, the average particle length was 996 nm but varied up to 20,000 nm depending on the virus and cells used to propagate . ParticlTEM is faster than plaque assay and, like the qRT-PCR, takes only hours to complete. No virus-specific reagents are required. However, it is relatively expensive and few laboratories have the required expertise and instruments. TEM particle counts, although the current method of choice for enumeration of virus particles, have a relatively high limit of detection of 1E+07 VP/mL, which contributed to our inability to quantitate virus beyond the first dilution . DiffereThe VC is a rapid method, providing real-time results for multiple samples in less than 1 h. Cost per sample, run in triplicate, is approximately $15 ($4.75 per replicate). Training to use and care for the instrument is minimal, but use of the instrument for filoviruses requires high containment and poses aerosol hazards that must be carefully mitigated. The one-step assay, instrument, and software are easy to use and do not require a highly trained individual. Due to its universal stain, the assay can be used with a wide variety of viruses. The VC is not amenable to quantifying filovirus particles directly from samples with high levels of proteins that are found in clinical samples  or stock preparations with fetal bovine serum concentrations greater than 10%. In these sample types, an initial dilution is required to lower background levels. Analysis of these matrices is possible, but would require purification or some other method to remove exogenous protein. While we can easily conceptualize automated counting of homogeneous spherical viruses such as herpes simplex, baculovirus, and dengue . For ther = 0.9984, p < 0.05) and the qRT-PCR .We have demonstrated that there is a linear relationship between the plaque assay, qRT-PCR, and VC particle counts. If higher virus concentrations were tested, work described in other studies  suggestsApplicability of the VC to other filovirus stocks is illustrated in The VC assay is simple to conduct, training is minimal, and no virus-specific reagents are required. Due to the use of an instrument for analysis, quantification is non-subjective and requires <10 min per reaction. The instrument is relatively small and can be placed inside a standard biosafety cabinet; installation and operational qualifications have been established; and assay controls are available to verify the instrument is functional. While we do not claim that particle counts are substitutes for techniques that measure infectious virus or genomic material, we do feel that the use of the VC to quantitate virus particles in seed stocks is a viable alternative to the more challenging, less sensitive TEM method. At a minimum, the VC provides an additional tool for rapid, precise, reproducible, and cost-effective quantification of filovirus particles in stock preparations and could prove useful to monitor various steps during virus propagation or purification.