The epidemics of Ebola virus in West Africa and Zika virus in America highlight how viruses can explosively emerge into new territories. These epidemics also exposed how unprepared we are to handle infectious disease emergencies. This is also true when we consider hypothesized new clinical features of infection, such as the associations between Zika virus infection and severe neurological disease, including microcephaly and Guillain-Barré syndrome. On the surface, these pathologies appear to be new features of Zika virus infection, however, causal relationships have not yet been established. Decades of limited Zika virus research are making us scramble to determine the true drivers behind the epidemic, often at the expense of over-speculation without credible evidence. Here we review the literature and find no conclusive evidence at this time for significant biological differences between the American Zika virus strains and those circulating elsewhere. Rather, the epidemic scale in the Americas may be facilitated by an abnormally warm climate, dense human and mosquito populations, and previous exposure to other viruses. Severe disease associated with Zika virus may therefore not be a new trait for the virus, rather it may have been overlooked due to previously small outbreaks. Much of the recent panic regarding Zika virus has been about the Olympics in Brazil. We do not find any substantial evidence that the Olympics will result in a significant number of new Zika virus infections (~10 predicted) or that the Olympics will promote further epidemic spread over what is already expected. The Zika virus epidemic in the Americas is a serious situation and decisions based on solid scientific evidence - not hyped media speculations - are required for effective outbreak response. Fast forward to today and people are talking about canceling one of the world’s most watched events, the Olympics, due to the Zika virus epidemic in Brazil3. So, what happened? Did the virus change? Did we misinterpret its threat from the beginning? And will the Olympics this summer really exacerbate the current epidemic or provide new opportunities for Zika virus emergence? Zika virus research is now pouring in fast, but at times at the expense of fast-tracked studies and misinterpretation of results. As a result, there is significant confusion surrounding the Zika virus epidemic and many of the core questions need to be revisited.Zika virus, a name now synonymous with birth defects by many people, was not always a topic of public health debate. In fact, for 67 years, the virus was mostly ignored . That is because when Zika virus was discovered in 1947 it was not thought to cause severe enough disease in humans to warrant intense research“The Brazilian strain of Zika virus harms health in ways that science has not observed before”3 and“[Africa and Asia] have mostly avoided the post-2013 neurotrophic strains of the virus that are ravaging Brazil”2. Based on available evidence, however, it is too early to say whether this strain is in fact fundamentally different from other Zika virus strains. Only recently has Zika virus been associated with large outbreaks (since 2007 - Yap Island4) and severe disease such as microcephaly and Guillain-Barré syndrome (since 2013 - French Polynesia5). The epidemic in the Americas has proven to alarmingly increase these trends - 0.5 to 1.5 million suspected infections and~4,000 cases of microcephaly in Brazil alone6. What are the real reasons behind the severity of this epidemic? We will explore aspects of1) viral genetics that might alter transmission and pathogenicity in humans,2) the ecological conditions in the Americas,3) the potential impact of dengue virus on Zika virus-associated pathology, and4) how small sample sizes and under reporting may have skewed our previous assumptions of Zika virus and the disease it can cause. Using this knowledge, we will discuss whether a global event like the Olympics would really impact further Zika virus emergence and the expansion of the epidemic.It has been suggested that~1 mutation per genome replication)9. Therefore, all RNA viruses have the ability to evolve fast relative to most DNA-based organisms10, and Zika virus has evolved into at least two distinct lineages: African and Asian11. The viruses circulating in the Americas belong to the Asian lineage, which, to the best of our knowledge, originated in East Africa12. Comparing the genetics of the first discovered Zika virus strain from 1947  to the strain currently circulating in the Americas  reveals mutations in >1,100 nucleotide positions (~89% similarity), and confirms that yes, the viral genome is different. While the genetic makeup has changed - as is expected - the more important question, however, is whether this means that the currently circulating strain of Zika virus has a fundamentally different “behavior” ?Undoubtedly, yes, Zika virus circulating today is genetically different from the Zika virus of the past. A key aspect of Zika virus is that it has an RNA genome. Central features of RNA virus biology is that these viruses replicate, produce large population sizes, but do so with lots of errors  between Zika virus strains in Africa and the Americas? While studies have shown that competence of the suspected Zika virus vectors in the Americas,Aedes aegypti andAe. albopictus, can vary between mosquito populations13, the influence of Zika virus genetics has not been tested. There is, however, precedence for mosquito-borne viruses to adapt to local mosquitoes, increasing their epidemic potential. This happened with chikungunya virus during the Indian Ocean epidemic14 and West Nile virus during its invasion of the United States16. Hence there could be some yet-to-be discovered Zika virus mutations that facilitate faster transmission and increased rates of mosquito infection. A lot of work needs to be directed towards lab and field mosquitoes studies to actually determine if this has occurred and whether Zika virus could have adapted to enhance local transmission. At this point, however, there is no evidence that the Zika viruses in the Americas have adapted to the local conditions or can be transmitted any more efficiently than previous strains.Unfortunately, we are critically lacking comparative studies to directly address whether genetic changes in the virus are significantly contributing to the epidemic. For example, are there differences in mosquito vector competence (1) being transmitted during pregnancy or2) causing neuropathogenesis leading to complications such as microcephaly17 and Guillain-Barré syndrome5 than strains from Africa or other Asian strains? Several recent laboratory studies have shown that Zika virus can infect placental cells20, be vertically transmitted to offspring during pregnancy22, target and replicate in neuronal cells24, and cause birth defects25 . These studies, however, were conducted with a variety of Zika virus strains indicating that some of these phenotypes are common features of the virus, irrespective of the strain. In fact, the virus was first isolated in 1947 by injecting serum from a febrile rhesus macaque directly into a mouse brain1 and a subsequent paper published in 1971 showed that the same Zika virus strain could cause neurological disease in mice26. While these observations may not be that surprising - the mice were infected intracerebrally after all (as is common for these types of experiments) - these early experiments already demonstrated that Zika virus can replicate and cause pathology in neurons. Together, these studies suggest that vertical transmission and neuropathogenesis are not specific attributes of the Brazilian strain and perhaps Zika virus has always been capable of this.Perhaps even more controversial and urgent are the questions: is the Brazilian strain of Zika virus more capable ofe.g.,~30% in Nigeria during the early 1970s27). Perhaps disease associated with Zika virus was just overlooked in Africa because of the many other diseases such as malaria, acquired immune deficiency syndrome (AIDS), and tuberculosis that ravage the continent. In the Americas, a large number of Zika virus-naive people  are getting infected at the same time, which may reveal previously unknown clinical features of viral infection. Finally, genetic analysis of Zika virus strains has yet to discover any appreciable patterns associated with adaptation towards humans, vector species, or disease outcome28. This is not to say that it has not occurred, only that at this point in time our sampling is too insufficient to make any conclusions. Therefore, more experimental evidence is required before we can say whether Zika virus genetics or phenotype has changed in any significant way.So were those ancestral strains from the 1940’s to 1970’s in Africa reported to cause mild disease actually misunderstood? We know that people in some areas of Africa had high seroprevalence to Zika virus (31. Since chikungunya and Zika viruses share similar ecologies (humans andAe. aegypti), the current Zika virus outbreak should not be so surprising, given recent histories. Even the 2007 Yap Island outbreak gave us some indication of its potential - it is estimated that 73% of the population became infected with Zika virus4. A large outbreak in the Americas almost seemed inevitable, but why?Zika virus is not the first, nor likely the last mosquito-borne virus, to explosively emerge in the Americas. In 1999, West Nile virus was introduced into the New York area and quickly spread across the continent, killing thousands of people and millions of birds  without previous Zika virus immunity, and vectors (mosquitoes) capable of transmission. The climate may also have contributed to the scale and intensity of the epidemic; 2015 was the warmest year on record in the Americas38. This, however, could just be a consequence of numbers and reporting. Previous outbreaks may have missed these links because they were too small. In Brazil, the current estimate is that between 1–13% of pregnant women who become infected with Zika virus in their first trimester will deliver babies with microcephaly39. That is 8-650× the baseline microcephaly rate of 0.02–0.12% per live birth39. The largest previous Zika outbreak - which occurred in French Polynesia - had an estimated size of 30,000 infections based on serological evidence 40. Retrospective analysis of first trimester pregnancies indicated that about 1% of Zika virus infections resulted in babies born with microcephaly - a total of eight cases41. During the Yap Island outbreak, researchers estimated that about 5,000 of the 7,000 inhabitants over the age of three became infected4. Based on Yap State census reports42, roughly 200–300 women may have been pregnant during the outbreak, and only about ⅓ would have been in the first trimester. If the previous estimates were accurate during the Yap Island outbreak, then it may be possible that no babies were born with microcephaly just because there were not enough infected pregnant women for the chance occurrence. The current epidemic in the Americas, including Brazil, may therefore just seem more severe because there are more infected people to detect rare pathological complications such as microcephaly.The unfortunate surprise was the discovery of an association between severe neurological complications and Zika virus infection, especially among newborns43, however, redefined our perception of the virus. From 1996–1997, there were more than 500 clinical West Nile cases with a fatality rate approaching 10%, representing the largest mosquito-borne virus outbreak in Europe in more than a decade. Other outbreaks in urban areas soon followed, occurring in Russia44, Israel45, and the United States46, all of which included neurological disease in about 1% of the cases29. While genetic changes to the virus may account for some of the increase in severity47, most of it can be attributed to previously underestimating its severity due to small sample sizes. Many parallels can be made between what happened with West Nile virus and the sudden increase in Zika virus associated disease during its current emergence.Discovering new disease associated with a particular virus only after a large outbreak is not unique to Zika virus. In fact, we can learn from previous epidemics with different viruses, such as West Nile virus. Prior to the 1990’s, West Nile virus was only known to cause sporadic outbreaks with a few cases of severe neurological disease. An outbreak in Romania48, a~250% increase since 199049. One interesting hypothesis is that antibodies produced from a previous dengue virus infection may enhance subsequent Zika virus infection53. The proposed mechanism is that antibodies targeting dengue virus can bind to Zika virus during an active infection, but cannot always neutralize it. Instead, the bound antibodies can actually help Zika virus infect monocytes by attaching to the cell surface receptors (Fc gamma) and mediating efficient entry. This process of antibody-dependent enhancement is also known to occur between different serotypes of dengue virus and is a risk factor for severe dengue disease  the risk of further spread and2) personal risk to visitors. These are two very different questions, but often they get blurred together. Here we will discuss them separately. First, how many of the expected half a million visitors to Rio de Janeiro during the summer Olympics this August will become infected with Zika virus? Massadet al. predict that the numbers of individuals acquiring Zika virus during the Games is low - 1 in~30,000 to 100,000 people56. This translates to only 5 to 15 visitors during the 3-week games will locally acquire Zika virus. The same model was used to predict that 3 to 59 visitors would become infected with dengue virus during the 2014 World Cup57. The actual reported number was three, suggesting that such estimates are relatively accurate58. Another group estimated that 3 to 80 visitors will become infected with Zika virus during the Games59. Based on previous experience and scientific evidence, we might therefore only expect that about 10 people traveling to Brazil for the Games will get infected with Zika virus. Compared to the overall number of cases, that is an astonishingly low number.Now turning our attention from the biology and genetics of Zika virus, to the different risks associated with Zika virus and the Olympics. There are two main risks to consider:61. That alone will severely decrease the likelihood of exposure to Zika virus through fewer mosquito “bites”. The remaining risk is dependent upon the1) mosquito species,2) proportion of infected mosquitoes, and3) transmission rates upon blood feeding. A recent report helped to validate our assumption thatAe. aegypti is vectoring Zika virus in at least some parts of the Americas62, though other species may be involved63. The proportion ofAe. aegypti that are infected with Zika virus at any given time, however, is unclear.Ae. aegypti infection rates range from extremely low (unpublished data suggesting only a few Zika virus RNA-positive mosquitoes among thousands tested) to very high (5–17% near homes of suspected Zika patients62). If the infection rates are similar to chikungunya or dengue viruses, then we can expect that 1–2% ofAe. aegypti are infected with Zika virus65. Moreover, only a small portion (5–50%) of infected mosquitoes can actually transmit the virus13. So even if you get fed upon by hundreds of mosquitoes, odds are that you will not get exposed to Zika virus.One main reason behind the few predicted Zika infections of visitors is that August is winter in Brazil, so mosquito densities will have declined66. That is already 7–80× greater than the number of people predicted to become infected during the Games (~10 - see above). In reality, not enough people will get infected with Zika virus while visiting Brazil for three weeks to have a significant impact on the already expected viral spread. Unfortunately, Zika virus will spread, just as dengue, chikungunya, and West Nile viruses have done before. Holding the Olympic Games in Brazil will have no, or extremely limited, effect on this process.The world is interconnected. Zika virus and many other mosquito-borne viruses have already utilized this interconnectivity to travel great distances. Does a global event like the Olympics really enhance this problem? One estimate indicates that 100 to 400 people infected with Zika virus will enter Europe in 2016 due to normal travel from endemic regions1) have an environment that is inhospitable toAe. aegypti (or other susceptible mosquitoes) or2) have infrastructure to minimize the risks of mosquito exposure 67. Much of the tropical and subtropical regions of the world, however, have suitable environmental conditions to support Zika virus transmission67. Spread to many of these places is not concerning, because they already have autochthonous  transmission of Zika virus. The Centers for Disease Control and prevention (CDC) recently conducted an assessment of countries that could be at risk of Zika virus importation following the Olympics68. They suggest that Angola and China (among other countries) could be at risk because1) they are currently not reporting autochthonous Zika virus transmission,2) they likely have conditions to support transmission , and3) there is a high amount of expected air travel from Brazil. In short, it really depends on the home country of the traveler, what season it is, their economic status, whether they can protect themselves from mosquitoes, and many other variables. While a single traveler could be responsible for a new outbreak (as suggested for the Zika virus introduction into Brazil28), in all likelihood these events are extremely rare.To really understand the risks, let’s use an example. If a person is infected with Zika virus and returns to their home country, what are the real chances that the infected person could initiate local mosquito-borne transmission? The answer is largely dependent on the local conditions. Specifically, does the environment support enough competent mosquitoes that feed on humans to facilitate transmission? And will such transmission be sustained? Most of Europe, the United States, and other temperate regions cannot support local Zika virus transmission because they eitheret al.28 has often (wrongly) been used to suggest that it only takes one infected traveler to start an outbreak 2. That is not correct and was also not suggested by the authors. Instead, what the Fariaet al. data show, is that the chance of starting an outbreak is extremely low. If it had been high, we would have seen multiple introductions of Zika virus into Brazil (and elsewhere), due to travelers arriving from Zika endemic countries. We don’t see that, hence it likely takes many - not just one - infected travelers for the chance occurrence to start an outbreak.The ‘single introduction’ hypothesis put forward by Faria69. Other forms of human-to-human transmission scenarios also appear to exist70. Therefore, could sustained Zika virus transmission occur without mosquitoes and should this be a concern for further spread of the epidemic? Again, let’s use an example: an infected man returns home from the Games and has sex with his partner. There are numerous reports of Zika virus infection associated with sex with a man (or woman71) returning from an endemic region74. Therefore, in this scenario, there is immediate risk to his partner. Importantly, however, in each of these reports, Zika virus spread was limited to just those single contacts. Thus, sex and other modes of direct contact with an infectious individual is highly unlikely to lead to sustained transmission in a new population. It has also been estimated that the role of sexual transmission in Brazil is minimal compared to mosquitoes75, and without mosquitoes, transmission would dissipate. The single most compelling piece of evidence to support that Zika virus is primarily mosquito-borne is that it is only known to occur in regions withAe. aegypti67. Therefore, Zika virus is still considered to be primarily transmitted by mosquitoes and sexual transmission (or other yet-to-be-discovered human-to-human means of transmission) will likely not expand the expected range of the Zika virus epidemic.Direct human-to-human transmission is another possible route of Zika virus infection. These routes notably include transmission from mother to child during pregnancy and sexual transmission from a man to a woman1) There is currently no definitive evidence that the strain of Zika virus in Brazil has altered potential for transmission or pathogenicity in humans compared to the strains circulating in Africa and Asia .2) Major factors for the scope of the epidemic were likely large urban settings housing people without immunity and an abnormally warm climate leading to a large population of mosquitoes.3) Previous exposure to dengue virus could increase Zika virus disease severity, though such a connection is yet to be demonstrated as an important risk factor.4) The recent associations of some Zika virus infections with severe neurological conditions, such as microcephaly and Guillain-Barré syndrome, may be simply a reflection of sample sizes - large numbers of infections are often required to discover rare pathologies.Our rapidly expanding knowledge about Zika virus is starting to reveal important information about the current epidemic and suggests that we may have misjudged its epidemic potential for decades. We explored four key areas to demonstrate how the epidemic severity may be more related to the conditions in the Americas rather than new disease caused by Zika virus.1) whether it will enhance the epidemic spread and2) personally to people attending the games. The numbers of Olympic visitors expected to get infected with Zika virus in Brazil and travel home is far lower than the total numbers of these occurrences already expected to happen throughout the year. Therefore, the Olympics will not be a significant conduit for further epidemic spread. There is a personal risk of infection, though it is also predicted to be low. Obviously, pregnant women have the greatest risk as they could pass the virus to their developing fetus, with the possibility of causing severe neurological complications. Therefore each family needs to evaluate the consequences and likelihood of Zika virus infection to determine if they should travel to any region of the world with active Zika virus transmission. The Zika virus epidemic is a severe problem, but decisions should be based on scientific evidence79 and not fear-mongering2. These should be lessons to keep in mind when we argue about some other relatively unknown virus before the start of Tokyo Olympics in 2020.The risks regarding Zika virus and the Olympic Games in Brazil are In this interesting and well-executed review, Grubauch and Andersen 'navigate' the literature to provide a clean, well-driven and totally enjoyable text about the current situation generated for the Zika outbreak in the Americas. Based on the bibliography, they conclude there is no scientific evidence that the Brazilian Zika strain presents a higher pathogenicity compared to others circulating elsewhere. In addition, as was discussed in previous reports, they agreed with the role played by urban and weather factors to particularly enhance this outbreak.  The authors also believe that is totally necessary to confirm the connection between Dengue and Zika as a risk factor in disease severity. Finally, given the sample size of the recent outbreak, we could have under estimated rare diseases associated to this virus in the past.  The article itself is very nicely written and provides a very balanced viewpoint of Zika virus, which is something that has been lacking from several media sources. The authors have put significant thought into the attributes that affect the severity of the outbreak, but perhaps the most important statements concerning the evolution of the virus and the neurological phenotypes recently observed. With their calculations for the number of microcephaly cases that could have been detected on Yap Island (quite possibly none due to the size of the outbreak), they suggest that statistics and surveillance, rather than genetic differences, affect disease severity. Of course, additional research is necessary into understanding the severity of the Zika virus outbreak and its connections to microcephaly and Guillain-Barre syndrome and whether the genetic differences between the strains of Zika virus are or are not responsible for these newly observed phenotypes. The authors also expand on the details on how Zika virus could infect a traveler in Brazil and then induce an outbreak in their home country, which they estimate to have a much lower probability than popular media sources might suggest. Though the commentary surrounding the possibility of the Olympics enhancing Zika virus comes after the games, the thoughtful consideration of the risks supplies rational thinking that has been lacking, especially when juxtaposed with the hysteria prior to the games. The review superbly quells the hyperbole that has surrounded the Zika outbreak in the Americas. Regardless of this low possibility of traveler-associated transmission of Zika virus in new locales, regions with the proper conditions for Zika virus transmission should remain vigilant, and continued campaigns of mosquito control, especially given the breadth of viruses spread by mosquitoes . We would like to highlight that the authors didn’t cite any of their previous works, which surprised us in a positive way, showing the author’s will to deliver a clean and not biased opinion about the topic. Minor comments:El Niño. Even if wouldn’t be necessary for most of the readers.   The article is written for any kind of reader, which we found excellent, but in that case would be nice to clarify what is The paragraph that starts with “One main reason behind …August is winter in Brazil…”th in the list sovereign states and dependencies by area in the world. Particularly, the weather in Rio, still in august, can be warm enough for mosquitos. Last August the lower temperature registered was 71 F and the higher 78 F. Brazil is huge, is the he largest country in South America and in the Southern Hemisphere. Is placed 5We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In an attempt to justify the disastrous Iraq invasion in 2002, the then 13th Secretary of Defense of the USA, Donald Rumsfeld, stunned reporters when he uttered his (in)famous reflection on "known knowns",  "known unknowns" and "unknown unknowns" . In their interesting review on Zika epidemics, Grubauch and Andersen address the panic ensued by the overwhelmed number of cases in Latin America and elsewhere. They call the attention to several "known knowns" and "known unknowns" of the current Zika epidemics, although they do not shy away of pointing to possible "unknown unknowns". Grubauch and Andersen find no conclusive evidence in the literature for significant biological differences between the Latin American Zika virus strain and those circulating elsewhere. They argue that the epidemic scale in the Americas has been influenced by climate, humans and mosquitoes population densities and local prevalence of other viruses, in particular flaviviruses, like dengue and yellow fever. The severe neurological abnormalities associated with Zika virus would not be a new trait of the Latin America strain, but rather may have been overlooked due to previously small outbreaks. Every time the world is hit by an emergent or re-emergent pathogen with pandemic potential, panic ensues . This is understandable due to previous history of recurrent pandemics, like the medieval Black Death or the 1918 Spanish Flu, which killed millions of people in Europe and around the world . After the Pasteur-Koch germ theory was proposed, the previously "unknown unknowns"  could explain the panic. The recent panic observed when SARS, H5N1 and more recently MERS-CoV emergence, or the current Zika, should not be justified due to the prompt discovery of the ultimate cause and the possible control mechanisms. Unlike Londoners of the XVII century , who burnt witches and culled thousands of cats , we have the scientific tools and mechanisms to face the threat with the necessary rationality. In the case of Zika virus, however, many "known unknowns" still remain, like the true phenotypic repercussion of genotypic differences between the strains circulating in different parts of the world, differences in mosquito competences, the actual number of people that has already been infected, the competition by the vector of different viruses transmitted by the same aedes mosquitoes, vertical transmission of the virus in hosts and vectors, neuropathogenic potential of different strains, cross-immunity, antibody-enhancement by previous infection, just to mention a few. In addition, it is not well known the actual role of sexual transmission of Zika in triggering or maintaining an outbreak. As for the "unknown unknowns", just time will disclose.R0, of Zika is around 3, slightly higher than that of Dengue  and it is even possible to predict the likelihood of Zika being exported to unaffected regions of the world, either causing a single and self limited outbreak or establishing itself, depending on whether localis lesser or greater than 1. One important unknown currently being discussed in the literature is whether Zika will disappear from the affected regions and whether and when it will recur.  Mathematical models, very useful in the understanding and control of previous epidemics, have been widely applied in an attempt to describe and make prediction about the current Zika outbreak ). We now know that the Basic Reproduction Number, For the sake of completeness, two additional speculative unknowns are worth mentioning. The circulation of Zika virus and its potential interaction with dengue raises new concerns regarding vaccination strategies against the latter. The subtle trade-off justifying the recommendation of the vaccine might no longer hold. On the other hand, the patchy distribution of serious outcomes due to Zika has not been satisfactorily explained. Our navigation map cannot overlook the apparent clustering of cases of microcephaly reported in northeastern Brazilian states so far. We think that the many "known unknowns" related to Zika epidemic explored in this paper are worthwhile being published and we are sure that Grubauch and Andersen reflections could help bring science and rationality to the fore, soothing the perhaps unjustifiable panic. Althoug Grubauch and Anderson do not cross all the t's nor dot all the i's, they at least show us all the uncrossed t's and undotted i's of the current Zika "unknowns" epidemic. This definitively qualify their paper for publication. Finally, for the record: the World Health Organization officially declared - zero cases of Zika during the Olympic Games!We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Fantastic review! Thank you for taking the time and pointing out some of the issues that we did not get into, mainly because we were already >4000 words! There are so many unknowns of many varieties, we just ask for over-speculation to be tempered until the science can catch up. No new Zika virus infections during the Olympics is amazing news!


Workers Memorial Day, observed annually on April 28,Occupational injuries and illnesses also have economic costs. The societal cost of work-related fatalities, injuries, and illnesses was estimated at $250 billion in 2007, based on methods that focus on medical costs and productivity losses  and uses these data to inform the industry and other stakeholders about health and safety risks and to guide intervention activities.New data on fatal falls in the oil and gas industry are reported in this issue of


Picornavirales represents a diverse group of positive-stranded RNA viruses with small non-enveloped icosahedral virions. Recently, bats have been identified as an important reservoir of several highly pathogenic human viruses. Since many members of the Picornaviridae family cause a wide range of diseases in humans and animals, this study aimed to characterize members of the order Picornavirales in fruit bat populations located in the Southwest region of Cameroon. These bat populations are frequently in close contact with humans due to hunting, selling and eating practices, which provides ample opportunity for interspecies transmissions.The order Eidolon helvum and Epomophorus gambianus), were combined into 25 pools and analyzed using viral metagenomics. In total, Picornavirales reads were found in 19 pools, and (near) complete genomes of 11 picorna-like viruses were obtained from 7 of these pools. The picorna-like viruses possessed varied genomic organizations (monocistronic or dicistronic), and arrangements of gene cassettes. Some of the viruses belonged to established families, including the Picornaviridae, whereas others clustered distantly from known viruses and most likely represent novel genera and families. Phylogenetic and nucleotide composition analyses suggested that mammals were the likely host species of bat sapelovirus, bat kunsagivirus and bat crohivirus, whereas the remaining viruses  were most likely diet-derived.Fecal samples from 87 fruit bats  contains supplementary material, which is available to authorized users. Picornavirales consists of viruses with a monopartite or bipartite positive strand RNA genome of the families Dicistroviridae, Iflaviridae, Marnaviridae, Secoviridae, Picornaviridae, the genera Bacillarnavirus and Labyrnavirus as well as many other proposed unassigned species, all of which possess small non-enveloped icosahedral virions of approximately 30 nm in diameter with a pseudo T = 3 symmetry [Picornaviridae and Iflaviridae, the replication block is found in the C-terminal region of the polyprotein, while the structural proteins are in the N-terminal region, all encoded within a monocistronic genome (except for members of the dicistronic Dicipivirus genus of the family Picornaviridae). In others, such as the Marnaviridae and Dicistroviridae, the reverse order is true and can either be on a single (monocistronic) open reading frame (Marnaviridae) or on two separate (dicistronic) open reading frames (ORFs)  [Secoviridae contains viruses with either monopartite genomes with the replication block at the N-terminus or bipartite genomes [Picornaviridae have been implicated in human and other vertebrate diseases such as gastroenteritis, meningitis, encephalitis, paralysis (non-polio and polio-type), myocarditis, hepatitis, upper respiratory tract infections and diabetes [The order symmetry , 2. A ma genomes . Of all,diabetes , 6.Picornavirales have been discovered in stools of different animals: feline stool-associated RNA virus (fesavirus) from cats in an animal shelter [fish stool-associated RNA virus (fisavirus) in the intestinal content of a carp [porcine stool-associated RNA viruses (posaviruses) in the feces of healthy pigs [human stool-associated RNA viruses (husavirus) in stools of HIV positive and negative patients [Ascaris suum [Eidolon helvum fruit bat.Recently, members of the order  shelter ; fish stf a carp ; porcinethy pigs , 10 and patients . Moreoveris suum , suggestris suum . VirusesEidolon helvum and 2 from Epomophorus gambianus) and the pools were treated to enrich for viral particles using the NetoVIR protocol [Fecal samples were collected from fruit bats between December 2013 and May 2014 as previously described in [protocol . Sequencde novo assembled and contigs annotated, open reading frames (ORF) were identified and further analyzed for conserved motifs identification in the amino acid sequences using NCBI's conserved domain database (CDD) [Picornavirales. All sequences were deposited in GenBank under the accession numbers KX644936-KX644946. Raw reads were submitted to the NCBI’s Short Read Archive (SRA) under the project ID PRJNA344863 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA344863/).NGS reads were analyzed as described in Yinda et al. , 14. Brise (CDD) , Pfam [1se (CDD)  and Phyrse (CDD) . Nucleotse (CDD) . For physe (CDD) , 21 usinTo infer the host of the viruses we identified, we used NCA as described in . BrieflyPicornavirales  had the highest number of reads   flanked by at least a 393-nt long 5’ UTR and a 41-nt long 3’ UTR  is the second monocistronic picornavirus identified and its genome showed the same organization as the yet unclassified shrew crohivirus and the genus rus Fig.  in the fnt Table , althougnt Table . The 3D 10GxHxxG motif of 3C proteinase and the KDE, PSG, YGDD and FLKR motifs of RNA-dependent RNA polymerase) except DYSNWD of the RdRP that differs from sapelovirus A1 RdRp at position 3 (DYTNWD)  Table . ProposeIflaviridae tentatively named bat iflavirus. The genome is at least 10520 nt long and contains a 3179 aa ORF flanked by at least 28 nt and 273 nt at the 5’-UTR and 3’-UTR, respectively . Arrows in Fig. 16GxHxxG, KDY, DYSNFG, ASG, YGDD and FLKR, respectively  domains usually found only in bacteria [Bat posalivirus and fisalivirus possess a single large ORF with the replication proteins at the N-terminus and capsid proteins at C-terminus Fig. . The 5’-bacteria . The fisPicornavirales genomes with a dicistronic organization.Apart from the monocistronic picorna-like viruses above, we also identified five Cripavirus genus [Firstly, the bat cripavirus genome contained two predicted ORFs of 2919 nts (ORF1) and 3702 nts (ORF2) encoding replication proteins and structural proteins, respectively Fig. . There sus genus  and ther768XXGXGKT consensus sequence , a proteinase (1129–1317 aa) with a conserved GXCGX16GXH motif and an RdRP (1510–1710 aa) with conserved KDE, PSG, YGDD and FLKR motifs (Table The bat felisavirus is a dicistronic virus with a predicted ORF1 5638 nt) encoding functional proteins and ORF2 (3020 nt) encoding capsid proteins. Both ORFs are separated by a very short intergenic region (IGR) of 73 nt Fig. . The 5’-8 nt encofs Table . ORF2 shfs Table .Bacillarnavirus which presumably infect diatoms. The ORF1 encodes functional proteins, containing conserved domains of an RNA helicase (aa 432–541) with a GX4GKS Walker A motif and a RsdvnGiyidD Walker B motif of P-loop NTPases, a proteinase (aa 1030–1198) with a GLCMX13GXH motif also found in other bacillarnaviruses and an RdRP (aa 1430–1701) with KDE, PSG, YGDD and FLKR motifs  encodes two conserved motifs of rvh  while ORF2 (3061–8481 nt) was predicted to encode replication proteins: RNA helicase (634–743 aa), peptidase (2012–2290 aa) and an RdRP (1295–1785 aa). The 5’-UTR and 3’-UTR ends contain 352 and 155 nt, respectively.Surprisingly, two dicistronic picorna-like viruses with a 5’ ORF encoding capsid proteins and a 3’ ORF coding for the replication proteins were identified and provisionally named bat badiciviruses 1 and 2 Fig. . The batPicornavirales ; (iii) bat posalivirus and fisalivirus forming a clade of unassigned viruses comprising posaviruses 1 and 2 (JF713720 and JF713721), fisavirus 1 (KM434233), husavirus (KT215901-KT215903) and Pow Burn virus (KU754519); (iv) bat cripavirus, the only virus of the family Dicistroviridae described in this study formed a clade with cripavirus  and Himetobi P virus (AB017037) within the genus Cripavirus, family Dicistroviridae; (v) bat felisavirus, bat dicibavirus and bat badiciviruses 1 and 2 clustered to yet unassigned viruses. Bat felisavirus was closest to fesavirus 1 (KM017736) while bat badiciviruses 1 and 2 formed a cluster with Aphis glycines virus 1 (KF360262) and soybean-associated bicistronic virus (KM015260), with very low sequence identity even in the most conserved parts of the genome; (vi) finally, bat dicibavirus clustered with the diatom-specific Chaetoceros tenuissimus RNA virus type-II SS10-16 V (AB971661), Chaetoceros sp. RNA virus 2 Csp02RNAV01 (AB639040), marine RNA virus JP-A (EF198241), Asterionellopsis glacialis RNA virus (AB973945) and antarctic picorna-like virus 2 (KM259870), though clearly distant from these viruses (34-53% amino acid identity of 3Dpol).Phylogenetic analysis was performed using the nucleotide sequences of the proteinase-polymerase domains 3CD) of the eleven novel viruses, and 60 representative members of the order CD of theCripavirus and Aparavirus and bat cripavirus, (iv) monocistronic viruses with C-terminal capsid proteins , and (v) insect iflaviruses and bat iflavirus. Due to the lack of representative sequence data for comparison, bat badiciviruses 1 and 2 and the bat felisavirus are not shown.We further analyzed the phylogenetic relationships of the capsid proteins  we performed a linear discriminant analysis (LDA) in an attempt to identify the most likely host of the viruses described in this paper. Based on the predicted LDA model, the classification and posterior probabilities of the viruses described in this paper were inferred Fig. , Table 5Picornavirales. Six out of these eleven viruses are likely to represent members of yet unassigned virus families of this order whereas bat crohivirus, bat sapelovirus, bat kunsagivirus, bat iflavirus, and bat cripavirus, belong to the established families Picornaviridae, Iflaviridae and Dicistroviridae.Using the NetoVIR protocol for viral metagenomics, we could retrieve the (nearly) complete genomes of eleven diverse and highly divergent viruses belonging to the order Picornaviridae identified here were bat kunsagivirus, bat crohivirus and bat sapelovirus. The genome layout of bat kunsagivirus exhibits close similarity to that of roller kunsagivirus (species Kunsagivirus A) of the family Picornaviridae, but its deduced amino acid sequence exhibits significant differences suggesting classification into a novel species (proposed name “Kunsagivirus B”). The host of roller kunsagivirus was unclear as several rodent-related viruses were also detected in the same fecal sample, e.g. mosavirus, murine kobuvirus and Puumala virus (21). Therefore, a dietary origin of roller kunsagivirus could not be excluded. In line with this dietary hypothesis, bat kunsagivirus is predicted to have a mammalian host by NCA. Therefore, we speculate that fruit eating bats are most likely the host of the kunsagivirus identified in this study. Similarly, crohivirus , first isolated from a Zambian shrew [Sapelovirus A, Sapelovirus B and Avian sapelovirus [Picornaviruses of the family an shrew  is a yetelovirus . The novelovirus –40. TherIflaviridae. The latter virus infects the honey bee and together with Varroa destructor virus may be critical for colony mortality [Bat iflavirus is closely related to slow bee paralysis virus, belonging to the family ortality . All curortality .−03, the presence of these domains requires confirmation. Fisalivirus does not have this domain and is more fisavirus-like. Posaviruses 1 and 2, fisavirus 1, husavirus, bat posalivirus, and fisalivirus do not only have a similar gene arrangement but also cluster together in the 3CD phylogenetic tree  domains usually found only in bacteria . These pris suum , a nematris suum  postulatLaodelphax striatellus [. Furthermore, another cripavirus was recently described , possessing five rather than three VPg genes, in the guano from an insectivorous bat, Pipistrellus pipistrellus [Bat cripavirus is closely related to Himetobi P virus, an insect picorna-like virus isolated from the small brown planthopper, iatellus . Furtherstrellus . GeneticDicistroviridae but only part of one rhv conserved domain could be identified in a conserved domain search. Phylogenetic analyses of the pro-pol domains revealed clustering with the fesavirus 1  as closest relative [Bat felisavirus has a dicistronic genome with gene organization similar to viruses of the relative . There iPicornavirales. Both bat badiciviruses cluster with two insect viruses, Aphis glycines virus and soybean-associated bicistronic virus. This implies that the origin of this virus in bat stool remains obscure and is probably dietary. NCA analysis groups them within the insect cluster thereby indicating that they may be indeed insect viruses originating from the diet. Further, these viruses may also constitute a novel family in the order Picornavirales.Bat dicibavirus, and badiciviruses 1 and 2 are other virus with a dicistronic genome. In each case, the two ORFs are separated by an IGR of 196, 457 and 227 nt, respectively, and the ORF2 starts with a glycine codon, which is typical for IGR-IRES driven translation initiation. However, characteristic secondary structures were not yet identified. Phylogenetic analyses (Fig. Picornavirales. This point to the considerable genetic variability of picorna-like viruses found in Eidolon helvum fruit bats. Bat kunsagivirus, crohivirus, and sapelovirus are clearly picornaviruses and this is consistent with the growing number of picornaviruses detected in bats. Many picornavirus infections cause a wide range of symptoms depending on virus-specific and host-specific factors. Whether the affected bats were diseased, is unclear. The captured bats of the present study were apparently healthy without any obvious clinical symptoms. This, however, does not exclude the potential of these picornaviruses to induce diseases in their natural hosts or in other species after spillover infections.We were able to identify and characterize eleven novel and diverse RNA viruses of the order Picornaviridae family is made up of a vast amount of highly divergent human pathogens, zoonoses therefore are not unlikely. However, the potential of these viruses to infect humans remains to be investigated.Given that many severely pathogenic zoonotic viruses have been traced to originate from bats, the knowledge obtained in this study is valuable to rapidly trace potential future zoonotic infections from bats to humans. Furthermore, all bat samples analyzed here were collected from regions where people hunt, sell and eat bats, so coupled with the fact that the Iflaviridae and Dicistroviridae, respectively, and are likely of an insect dietary origin. Bat posalivirus, bat fisalivirus, bat felisavirus, bat dicibavirus and bat badiciviruses 1 and 2, are unassigned members of the order Picornavirales, and their natural hosts is currently unclear, and remains to be determined.Bat iflavirus and cripavirus are viruses of the family


Insects have presented human society with some of its greatest development challenges by spreading diseases, consuming crops and damaging infrastructure. Despite the massive human and financial toll of invasive insects, cost estimates of their impacts remain sporadic, spatially incomplete and of questionable quality. Here we compile a comprehensive database of economic costs of invasive insects. Taking all reported goods and service estimates, invasive insects cost a minimum of US$70.0 billion per year globally, while associated health costs exceed US$6.9 billion per year. Total costs rise as the number of estimate increases, although many of the worst costs have already been estimated . A lack of dedicated studies, especially for reproducible goods and service estimates, implies gross underestimation of global costs. Global warming as a consequence of climate change, rising human population densities and intensifying international trade will allow these costly insects to spread into new areas, but substantial savings could be achieved by increasing surveillance, containment and public awareness. Invasive insects impose many economic costs, for example by consuming crops and spreading disease. Here, Bradshaw et al. compile a database of the costs of invasive insects and conservatively estimate that the yearly global cost  is at least $70 billion for goods and services and $6.9 billion for human health. For millennia, insects have been responsible for spreading devastating infectious diseases in both humansA global challenge this century will be meeting the world's food requirements while maintaining economic productivity and conserving biodiversity. Globally, insect pests have been reported to reduce agricultural yields by 10–16% before harvest, and to consume a similar amount following harvestInsects are also among the most pervasive of invasive species. For example, 87% of the ∼2,500 non-native terrestrial invertebrates in Europe are insectsReliable global cost summaries therefore remain a major challenge. Indeed, there are currently only 86 insect species listed in the International Union for Conservation of Nature (IUCN) Global Invasive Species Databaseper se because of a lack of standard, objective criteria to assess the accuracy of published estimates; Methods). We categorized studies that did not meet these criteria as ‘irreproducible'. We further divided all costs into two main categories: ‘goods and services'  than we report here. Further, future costs are likely to increase as invasive insects expand their ranges in response to climate change, as well as to increasing human movements and international trade.We determined that invasive insects cost a minimum of US$70.0 billion per year globally for goods and services, of which US$25.2 billion per year comes from reproducible studies . There wCoptotermes formosanus estimated at >US$30.2 billion per year globally (Plutella xylostella as the most expensive (US$4.6 billion per year)Tetropium fuscum (US$4.5 billion per year in Canada), the gypsy moth Lymantria dispar (US$3.2 billion per year in North America) and the Asian long-horned beetle Anoplophora glabripennis  costs represent 84% of total health costs, followed by 15% for West Nile virus transmitted by Culex spp.  and 85% of the latter (n=99) we deemed reproducible. Ecosystem-regulating services, which have high economic value worldwideVespula wasps in New Zealand). That study showed that damages arising mainly from reduced pollination are comparable to the direct costs to goods and services  and are much higher than associated health costsThe estimated total global costs, even after attempting to correct for sampling bias, are therefore necessarily gross underestimates. We found only 86 (goods and services) and 117  estimates globally, of which only 55% of the former , of variable reproducibility and are likely to increase as the planet warms and international trade expands, we conclude that the costs of invasive insects to human society are underestimated and will escalate with time. The available data describe only the costliest insects of mainly industrial and/or biosecurity concern, and non-market costs are rarely estimated but see , even thEffective, early response and vigilant biosecurity are often cheaper (by up to 10 times for mosquito-borne disease4We began our review of the literature on the economic impacts of invasive insects using the ISI Web of Science database with a specific search string to identify relevant papers (see below). We then used the Web of Science's ‘refine' function to restrict the studies identified to the relevant fields, yielding 488 sources from 1911 to January 2014. We analysed each source to reject irrelevant papers and retained those containing economic estimates. We completed our database with 267 relevant studies up to December 2015 (including grey literature) opportunistically gathered. In total, we screened 737 sources, 470 of which were relevant to the economic impacts of invasive insects and from which 158 yielded useable economic estimates  and 2. WFor health costs, we limited the criteria for invasive vector-borne diseases and their related vector mosquitoes following Juliano and Lounibosa priori as having potentially important economic impacts. In addition, the search string included exclusion terms to reject irrelevant studies, for example, those related to medicine. We completed the search for citations in Google Scholar and internal government reports.We searched on Web of Science in February 2014 and extracted records from 1911 to January 2014. Our search string was composed of three elements: ‘invasive' AND ‘insects' and ‘economic impacts'. For each element we used a range of synonyms widely found in the literature. For example, for ‘invasive' we used invasi*, invader, alien, exotic, non-native, introduced, naturaliz*. For ‘insects', we also specified the names of a range of taxa that we identified Full search string: TS= AND TS= AND TS=(economi* OR monetary OR dollar*) NOT TS=(cancer* OR cardio* OR surg* OR carcin* OR engineer* OR operation OR medic* OR rotation OR ovar* OR polynom* OR purif* OR respirat* OR invasive technique).We made every effort to eliminate redundant amounts from the monetary values we used to estimate cost sums. First, we removed values that were obvious re-estimates of older values  versus our sum of the total costs  reported in the main text is only 10.1%, which suggest that even in the unlikely case of double counting, the bias is minimal, and well within the margin of error expected for a sum of median cost rates across the globe. It is essential to note that even if a species includes both extrapolations and actual values, it does not necessarily equate to double counting because often the different estimates apply to different regions of the insect's distribution or different economic components of their costs. However, this does not exclude the possibility of double counting within the irreproducible category, simply because we cannot verify how the estimates were derived to check for instances of potential double counting.It is possible that the impact rate of any invasive species will vary over time, with rates being initially low following original establishment, and then increasing as the species expands its range and possibly declining as hosts are eliminated or humans adapt to the invasion. Consequently, a simple sum of rates from many species that invaded at different points in time might not provide a practical measure of standardized costs. However, ascertaining the year of invasion of all species we examined was impossible or suspect, given a lack of monitoring data for many species. To examine the potential problem indirectly, we plotted the cost rates versus the applicable year  for the goods and services and human health estimates separately. The subsequent bivariate plots  do not rWe determined the reliability of the cost estimates given in each study by identifying the source of all the figures used to extrapolate regional costs. When monetary values were based on available calculation methodologies, traceable original references and clearly identified uncertainties, we deemed the resulting final costs to be ‘reproducible'. This reproducibility is not an assessment of quality or realism of the estimation; rather, it is a qualitative assessment of whether the initial values, assumptions and methodology applied to obtain the monetary value can be fully understood . Conversely, we defined as ‘irreproducible' any monetary values that could not be fully traced, clearly understood or justified. Thus, we deemed a monetary value to be irreproducible when it was not properly referenced, was not traceable, was derived from a potentially subjective source , did not have the full details of the calculations or did not provide a clear list of the underlying assumptions. We assessed reproducibility for every monetary value we found in the literature; hence, some values might be reproducible and others irreproducible in the same study .Supplementary Figures 1 - 9, Supplementary Table 1, Supplementary Note 1, Supplementary Methods and Supplementary ReferencesGoods and services costs. List of estimates (and references) for 'goods and services' costs due to invasive non-native insects. Human health costs. List of estimates (and references) for 'human health' costs due to invasive non-native insects. . Outbreak frequencies derived from Supplementary Data 3.Outbreak frequencies. National outbreak frequencies of disease epidemics arising from invasive insects. Years of epidemics of dengue, yellow fever, and chikungunya for 72 countries are shown to calculate outbreak frequencies (probabilities). Source: www.who.int/csr/don/archive/year/en/ (Microsoft Excel file).


It has been widely established that several virus infections are strictly correlated to the cancerogenesis and may induce, through their reactivation, severe damages during chemotherapy treatments. For example, the direct role that Hepatitis B virus (HBV) plays in the development of liver cancer is well known. Nevertheless, in other cases, as Hepatitis C virus (HCV) or Hepatitis Delta Virus (HDV) the direct role of viral replication in cancer progression is still controversial, especially in some areas, like Italy, where these infections are endemic.Aim of this project is to up date the epidemiological status of the incidence of HCC in Europe and HCV and HDV infection , with special attention to the changes during the time of route of transmission, and also investigate the interaction between viral reactivations and tumors.11, Giuseppe Corano Scheri1, Claudia Pinacchio1, Maura Statzu2, Arnolfo Petruzziello3Ivan Schietroma1Department of Public Health and Infectious Diseases, "Sapienza" University of Rome, Rome, Italy;2Department of Molecular Medicine, Laboratory of Virology, "Sapienza" University of Rome, Italy;3Virology and Molecular Biology Unit , Department of Pathology, Istituto Nazionale Tumori- IRCCS Fondazione G. Pascale, Naples Corresponding author: Giuseppe Corano Scherigiuseppe.coranoscheri@uniroma1.it ; +393896943773)EMAIL: a.petruzziello@istitutotumori.na.it# 1 Nicola Coppola - Section of Infectious Diseases, Department of Mental health and Public Medicine, University of Naples “Luigi Vanvitelli”, Italy.# 2 Carolina Scagnolari- Virology – Department of Molecular Medicine- University La Sapienza, Rome , Italy31, Ali Zalan1, Arnolfo Petruzziello3, Iftikhar ud din2, Fazle Haq2, Yousaf Hayat2Najeeb Ullah Khan1 Institute of Biotechnology and Genetic Engineering , The University of Agriculture Peshawar, Pakistan2 Department of Mathematics, Stats & Computer Science, The University of Agriculture Peshawar, Pakistan3 Virology and Molecular Biology Unit “V. Tridente”, Department of Pathology, Istituto Nazionale Tumori- IRCCS Fondazione G. Pascale, Naples  1Corresponding author: Najeeb Ullah KhanE-mails: najeebkhan@aup.edu.pk, naji_banni@yahoo.com# 1 Maria Lina Tornesello, PhD- Molecular Biology and Viral Oncology, IRCCS, Fondazione Pascale, Naples, Italy# 2 Sabrina Bimonte, Md- Division of Abdominal Surgical Oncology, Hepatobiliary Unit, National Cancer Institute , IRCCS Italia G.Pascale, Naples


Cotton leaf curl Gezira virus with associated DNA-satellites causes CLCuD. Quite recently, in the Old World (both Asia and Africa), bipartite begomoviruses have started appearing in the areas under cotton cultivation. Under such aggravated circumstances, it seems we are heading toward another epidemic of CLCuD in the Old World. Here we articulate the causes and potential emergence of the third epidemic of CLCuD in Asia. The current situation of CLCuD in Asia and Africa is also discussed.Cotton leaf curl disease (CLCuD), the most complex disease of cotton, is a major limiting biotic factor to worldwide cotton productivity. Several whitefly-transmitted monopartite begomoviruses causing CLCuD have been characterized and designated as CLCuD-associated begomoviruses. Despite of being reported over 100 years ago in Africa, CLCuD became economically pandemic causing massive losses to cotton production in Pakistan and India during past couple of decades. In Asia, cotton has faced two major epidemics during this period viz. “Multan epidemic” and “Burewala epidemic.” The “Multan epidemic” era was 1988–1999 after which the virus remained calm until 2002 when “Burewala epidemic” broke into the cotton fields in Indo-Pak subcontinent, till 2013–2014. However, both the epidemics were caused by monopartite begomovirus complex. Similarly in Africa,  Bemisia tabaci) and is caused by a complex of five single-stranded DNA viruses belonging to genus Begomovirus (family Geminiviridae) along with their DNA satellites, viz. betasatellite and alphasatellite (Cotton leaf curl disease (CLCuD) is a major threat to worldwide cotton productivity. Indo-Pak subcontinent is a paradigm where cotton production has been severely compromised in past few decades because of the onset of two CLCuD epidemics. India and Pakistan are third and fourth largest cotton producers in the world with 6.05 and 2.17 million ton annual production, respectively . The CLCatellite . UnderstCotton leaf curl Gezira virus (CLCuGeV) associated with Cotton leaf curl Gezira betasatellite (CLCuGeB) and Cotton leaf curl Gezira alphasatellite (CLCuGeA) has been predominantly the causative agent of CLCuD (Cotton yellow mosaic virus (CYMV) has been reported for the first time in cultivated cotton (Gossypium raimondii) from the west and central Africa (Gossypium hirsutum) favored the onset of this disease (Cotton leaf curl Multan virus (CLCuMuV)] kept on proliferating and burst into an epidemic in 1988 at Moza Kokhran, Multan, Pakistan. The country-wide spread of CLCuD most likely started from this vicinity and later moved across the borders into the northwestern states of India. In India, the first evidence of CLCuD was reported near border area Sri Ganganagar in 1989, which later spread as an epidemic in 1994 , which today are named: Cotton leaf curl Alabad virus (CLCuAlV), Cotton leaf curl Kokhran virus (CLCuKoV), and CLCuMuV  and Papaya leaf curl virus (PaLCuV) were also identified infecting cotton , named Cotton leaf curl Burewala virus [now known as CLCuKoV-Burewala strain (CLCuKoV-Bu)] in the year 2002–2003, broke this resistance and all the resistant germplasm became susceptible to this unique member of CLCuD (C2 gene in CLCuKoV-Bu was a key player in resistance breaking (C2 gene have been reported from India (Bur), which was a recombinant of CLCuMuB and Tomato leaf curl betasatellite . The significance of both these invaders was the breakdown of established resistance in all the CLCuD-resistant cotton germplasm. The reasons for the ultimate selection of these recombinants through the use of resistant cultivars are not yet fully understood. According to Bur may enhance the interaction between the betasatellite and the helper begomovirus.In the late 1990s, after the Multan epidemic era (1989–1999), the cotton productivity was reinstated by developing resistant cultivars in Pakistan. However, theatrical appearance of a natural recombinant species that lacks a of CLCuD . This waof CLCuD . It was breaking . Howeverom India . Anothere ToLCB;   (Tomato leaf curl New Delhi virus (ToLCNDV) (Okra enation leaf curl virus (OELCuV) (Mastrevirus Chickpea chlorotic dwarf virus (CpCDV)  in the infected plants . The ability of ToLCNDV to develop synergistic interactions with other begomoviruses has recently been shown to cause resistance breakdown in chilies   and TomaToLCNDV)  (SupplemToLCNDV) , in Paki(OELCuV)  and a Ma (CpCDV)  has furtd plants . The DNAd plants  and alsod plants . During d plants . ToLCNDVd plants . It has d plants  but alsod plants  and Eurod plants  and Itald plants . If we h chilies . CLCuAlV chilies . Thus, iFigure 1). Yet this similarity has been extended to the possibility of another epidemic in both continents because of the identification of bipartite begomovirus from the field cotton in Africa -cotton hybrids in Indo-Pak subcontinent. The cultivation of BT-crops not only affects the pest populations in an area but could also alter the weed flora in a particular region (Maize rough dwarf virus (MRDV) and Fijiviruses from maize in Spain (-4), which is comparable to many RNA viruses  between the disease severity and the disease incidence with respect to humidity and temperature was observed in all cotton growing areas of Pakistan . Moreover, positive correlations between and among disease incidence, humidity (0.15), and temperature (0.25) support our hypothesis. Concomitant to this, disease incidence data of 19 years (1997–2015) along with the “Burewala epidemic” were mapped on a bar graph. Likewise to the Burewala epidemic during the years 1999–2002 when the disease incidence was dramatically at its lowest incidence , similar disease scenario is prevailing in cotton growing areas of Pakistan  (2014–2015). Usually, during long hibernation/incubation periods, some pathogens remain cryptic while their infection continuously spreading . The present study demonstrates that an extensive SNPs diversity is associated with CABs genome and a highest SNPs diversity exists in CpCDV followed by ToLCNDV, CLCuGeV, and CLCuMuV, respectively, while least SNPs were evident in CLCuKoV-Bu genome . The occurrence of fewer SNPs in CLCuKoV-Bu may likely be due to recombination event from which this virus has emerged out. Further analysis revealed that >30% of the SNPs were non-synonymous, thus can have substantial role in the virus divergence and evolution.The correlation between various environmental factors and the vector-borne disease incidence has been a mechanistic determinant to assess the geographic spread of the disease risk . Such copreading . This stFigure 2B). Based upon our statistical modeling and the currently available data, we may infer that the Multan, Pakistan  may face a serious eruption of CLCuD with new arsenals, which may spread across the borders.Thus, we may speculate that today we come across a similar situation of early 2000s in terms of virus incubation period (Various strategies encompassing classical breeding and biotechnological techniques have been employed to control the CLCuD . All theWhitefly populations responsible for transmitting CLCuD appear to be favored by the humid and high temperature conditions in the Indo-Pak subcontinent, where cotton is a key crop. Due to expansion in the geographic range of CABs from South Asia to China, a dynamic change in CABs in South Asia is an emerging threat to cotton production . The keyMS conceived the idea, created the figures and drafted the first draft. ZI wrote part of the manuscript, collected meteorological and CLCuD incidence data, and helped in preparation of the first draft. MT helped in retrieving all the data and prepared the tables. SU performed all statistical analysis. Final draft was checked and approved by all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alzheimer’s disease (AD), the commonest cause of dementia, represents a significant cost to UK society. This analysis describes resource utilisation, costs and clinical outcomes in non-institutionalised patients with AD in the UK.The GERAS prospective observational study assessed societal costs associated with AD for patients and caregivers over 18 months, stratified according to baseline disease severity . All patients enrolled had an informal caregiver willing to participate in the study. Healthcare resource utilisation was measured using the Resource Utilization in Dementia instrument, and 18-month costs estimated by applying unit costs of services and products . Total societal costs were calculated using an opportunity cost approach.p < 0.001 for difference across all severities) and had a greater probability of death  at 18 months. Greater disease severity at baseline was also associated with concomitant increases in caregiver time and mean total societal costs. Total societal costs of £43,560 over 18 months were estimated for the MS/S group, versus £25,865 for the mild group and £30,905 for the moderate group (p < 0.001). Of these costs, over 50% were related to informal caregiver costs at each AD dementia severity level.Overall, 526 patients  were recruited from 24 UK centres. Mini-Mental State Examination (MMSE) scores deteriorated most markedly in the MS/S patient group, with declines of 3.6 points in the mild group, 3.5 points in the moderate group and 4.7 points in the MS/S group; between-group differences did not reach statistical significance. Patients with MS/S AD dementia at baseline were more likely to be institutionalised (Kaplan–Meier probability 28% versus 9% in patients with mild AD dementia; This study demonstrated a mean deterioration in MMSE score over 18 months in patients with AD. It also showed that AD is a costly disease, with costs increasing with disease severity, even when managed in the community: informal caregiver costs represented the main contributor to societal costs. Alzheimer’s disease (AD) is the commonest cause of dementia representing 60–80% of all cases of dementia within Europe . In the There have been several studies that have looked at the costs of dementia, most of which have used different methodology. Overall, and not surprisingly, there is general agreement that as the disease progresses the costs increase, especially once institutionalisation is required , 4. FurtLooking specifically at the UK, a recent analysis based on a model using estimates of prevalence, percentage of home versus care home usage, expenditure and quality of life, has estimated that the total cost of dementia to UK society is £26.3 billion per annum, representing an average annual cost of £32,250 per person .As the aforementioned studies lack robust, prospectively collected, local data, we performed an analysis of real-world UK data from the GERAS study. GERAS was a European prospective observational study that assessed the outcomes and societal costs associated with AD dementia for community-dwelling patients and their caregivers over at least 18 months. Patients were stratified according to their dementia severity at baseline into mild, moderate and moderately severe/severe disease groups, which broadly reflect the guidelines from NICE. The study design and baseline societal costs have been previously published . The objThe GERAS study was an 18-month, multicentre, prospective, non-interventional, observational cohort study conducted in three European countries: the UK, France and Germany  instrument) . Other oThe RUD instrument was used to measure healthcare resource utilisation by both patients and caregivers, and time spent on informal care by caregivers . The RUD assisting the patient with basic activities of daily living (ADL) such as eating, bathing and dressing; assisting with instrumental ADL such as housework and shopping; and supervising the patient as required to prevent dangerous events such as risk of fire, and walking into the road if alone.Resource use items collected included healthcare ; community care services ; changes to patient living accommodation ; caregiver working status ; and primary caregiver time spent caring for the patient. Caregiver time was calculated as the number of hours spent:Information relating to patient medications, out-of-pocket expenses and additional financial support received, such as whether the patient or caregiver receives money from the government or an insurance company, was also collected during the interview with the caregiver. Data were collected from patients and caregivers at the baseline visit, and then during routine care visits at 6, 12 and 18 months (+/− 6 weeks ).Eighteen-month costs were estimated by applying unit costs of services and products  to the health and social care resource use collected over the 18-month follow-up period. For resource-use items, full details of the unit costs applied and their sources have already been reported .Total societal costs were calculated using an opportunity cost approach taking into account productivity loss for working caregivers and lost leisure time for non-working caregivers. Costs were calculated for the month before each visit. Total societal costs were calculated by adding patient healthcare costs , patient social care costs  and caregiver informal care costs (including time spent giving care and missing work). The number of hours for basic and instrumental ADL were used to calculate caregiver time, with 24 hours per day availability for total caregiver time assumed, but time spent on supervision applied a zero cost value. The unit cost of caregiver time for working caregivers was considered to be the value of lost production time based on the national average wage per UK population ; for noThe following imputation rules were applied for missing data: for institutionalised patients, mean monthly costs from the last visit were used for the period until institutionalisation, then monthly costs for institutionalisation (standard monthly cost of care with zero other costs) were used from institutionalisation up to 18 months; for patients who died, last observation carried forward was used, such that costs from the last known visit were extrapolated up to the date of death (no costs after death were computed). For those patients with other reasons for discontinuation, multiple imputation regression method . In addiDemographics and baseline characteristics were summarised using descriptive statistics and were based on non-missing observations. In common with other clinical studies in AD –23, the The Kaplan–Meier method was used to describe the probabilities of institutionalisation and death, and differences across baseline AD dementia severity groups for these time-to-event endpoints were tested using the log-rank test. Descriptive statistics were used to describe baseline variables and discontinuations.Comparisons across baseline AD dementia severity groups were made using analysis of variance (ANOVA) for continuous variables or the Cochran–Mantel–Haenszel test for categorical variables.All analyses were performed using SAS version 9.2 .n = 94; 50.8% of discontinuations), death  or other reasons  e.g., lost to follow up, patient decision. Proportions of patients discontinuing increased with increasing baseline severity of AD dementia  were recruited from 24 centres across the UK; baseline patient characteristics are shown in Table p < 0.001), which was most marked in the moderately severe/severe patient group .Over the 18-month duration of the GERAS study, a statistically significant deterioration in mean MMSE scores was observed . Among patients assessed as having mild disease at baseline, 43.5% remained with mild disease, 36.7% transitioned from mild to moderate disease, 11.6% transitioned from mild to moderately severe/severe disease, and 8.2% had an MMSE score of >26. For those with moderate disease at baseline, 6.1% had scores indicating mild disease, 52.6% still had moderate disease and 41.2% progressed to moderately severe/severe disease. Among patients with moderately severe/severe disease at baseline, 2.9% had MMSE scores for moderate disease, but the majority (97.1%) remained with moderately severe/severe disease . Patients with moderately severe/severe AD dementia at baseline were more likely to be institutionalised at 18 months  than those with mild AD dementia (9.0%) at baseline . At 18 months, the probability of death in those with moderately severe/severe AD dementia at baseline was greater than in those with mild AD dementia (14.9% compared with 5.3%). The probability of death at 18 months overall was 8.2%  and with increases in any of its components: time spent on basic or instrumental ADL or supervision  of £43,560  in patients with moderately severe/severe AD dementia compared with £25,865  in those with mild and £30,905  in those with moderate AD dementia (p < 0.001), but patient healthcare costs were not significantly increased (p = 0.624). The results of the sensitivity analyses confirmed these base case findings (data not shown).As with total societal costs, there was a significant increase in caregiver informal care costs and patient social care costs associated with increases in AD dementia severity , although differences in MMSE score changes between the groups were not statistically significant. The probability of progression to institutionalisation or death increased in line with increases in baseline AD dementia severity.Historically, the deterioration in MMSE score over time has been described as non-linear , as suppIn our analysis, 8.2% of patients with mild AD dementia at baseline had an MMSE score >26 at 18 months, while 48.3% of patients demonstrated a decline in MMSE score, with 11.6% transitioning from the mild to the moderately severe/severe group. Although variability between individual patients is often observed , some ofOur study demonstrated that patients with more severe dementia had a higher rate of institutionalisation and were more likely to die than those with less severe AD dementia. This is not surprising, and is consistent with the findings of a study conducted in 779 dementia patients recruited at nine memory centres, which demonstrated that a shorter time to institutionalisation was associated with lower cognitive ability, lower functional ability and more neuropsychiatric symptoms at baseline . The greConsistent with baseline results , total sThe costs of Alzheimer’s disease to society observed in our study are substantial and were estimated to be approximately £26,000 for mild severity, £31,000 for moderate severity and £44,000 for the moderately severe/severe group per patient over an 18-month period. This translates to annual costs per patient of approximately £17,000 per year for mild, £21,000 for moderate and £29,000 for moderate/severe severity groups. These are comparable to the community figures calculated from the Alzheimer’s Society which have been rounded to annual rates per patient of £26,000 for mild, £43,000 for moderate and £55,000 for severe cases supporting the robustness of the data despite some methodological differences [Strengths of this study include that it was a large, prospective, observational study with a well-defined patient cohort, which collected longitudinal data over an 18-month period and assessed patients within the community across a range of disease severities. This study design would be expected to generate more reliable estimates of clinical outcomes, resource use and costs than either small cross-sectional studies , 30, 31 The study also has limitations. Given the recruitment of patients principally from secondary care sites, it is possible that the sample is not truly representative of the overall AD dementia patient population. In addition, caregivers and participants were required to be sufficiently fit to participate in the study, which may represent a selection bias across all severities. It can also be challenging to make comparisons between studies due to different cost estimation methods, making analysis and interpretation of cost and resource data more difficult. Moreover, the 18-month duration of this study is still relatively short for accurately determining correlations between AD dementia severity and rate of cognitive decline or the probability of institutionalisation or death.This observational study demonstrated a rate of deterioration in cognition of at least 3.5 points on the MMSE scale over the 18-month study period across all patient groups stratified according to baseline AD dementia severity. Increasing severity of disease was associated with increased patient institutionalisation and death, together with increased total societal costs. Our results show that even when managed within the community, AD dementia is a costly disease, with costs associated with informal caregivers representing the main contributing factor to societal costs. Given the greater costs associated with increasing severity of disease, any treatment that could help slow disease progression may have a significant impact on the costs of community-based care for patients with AD dementia.


Hepatitis E virus (HEV), an important agent of viral hepatitis worldwide, can cause severe courses of infection in pregnant women and immunosuppressed patients. To date, HEV infections can only be treated with ribavirin (RBV). Major drawbacks of this therapy are that RBV is not approved for administration to pregnant women and that the virus can acquire mutations, which render the intra-host population less sensitive or even resistant to RBV. One of the proposed modes of action of RBV is a direct mutagenic effect on viral genomes, inducing mismatches and subsequent nucleotide substitutions. These transition events can drive the already error-prone viral replication beyond an error threshold, causing viral population extinction. In contrast, the expanded heterogeneous viral population can facilitate selection of mutant viruses with enhanced replication fitness. Emergence of these mutant viruses can lead to therapeutic failure. Consequently, the onset of RBV treatment in chronically HEV-infected individuals can result in two divergent outcomes: viral extinction versus selection of fitness-enhanced viruses. Following an overview of RNA viruses treated with RBV in clinics and a summary of the different antiviral modes of action of this drug, we focus on the mutagenic effect of RBV on HEV intrahost populations, and how HEV is able to overcome lethal mutagenesis. Hepatitis E virus (HEV) was first described as novel agent responsible for enterically transmitted non-A, non-B hepatitis by Reyes and colleagues in 1991 .Orthohepevirus in the family of Hepeviridae [Hepeviridae Study Group of the International Committee on the Taxonomy of Viruses [HEV is a nonenveloped single-stranded RNA virus with a 7.2 kb genome of positive orientation. Three open reading frames (ORFs) encode for: (1) the nonstructural proteins (ORF1), comprising a methyltransferase, a papain-like cysteine protease, a helicase, and an RNA-dependent RNA polymerase (RdRp), connected by a Y-domain and a hypervariable region (HVR); (2) the capsid protein (ORF2); and 3) small proteins whose functions are not yet completely understood (ORF3) [ small previridae . Differe Viruses  and othe Viruses .An infection with HEV is usually self-limiting, causing arthralgia, flu-like myalgia, vomiting, and symptoms characteristic of hepatitis like jaundice and itching . ProgresRibavirin (RBV) is a broad-spectrum antiviral agent with numerous clinical applications against viral pathogens; it is currently the only treatment option for chronically infected HEV patients. Several publications have documented the emergence of single-nucleotide variants (SNVs) in viral genomes that cause either reduced RBV sensitivity or RBV resistance ,21,22,23In this article, we start with an overview of selected RNA viruses that are or have been clinically treated with RBV, and summarize this drug’s different antiviral modes of action. The second part focuses on the mutagenic effect of RBV on HEV intra-host populations and how HEV is able to overcome the lethal mutagenesis induced by this guanosine analog.Flaviviridae), dengue virus , respiratory syncytial virus , influenza A and B virus (Orthomyxoviridae), chikungunya virus , poliovirus (Picornaviridae), Hantaan virus (Bunyaviridae), and Lassa virus (Arenaviridae) [In 1972, RBV was described as a broad-spectrum antiviral against several DNA and RNA viruses . Since tviridae) ,29  ,31.Flaviviridae, Crance et al. investigated the in vitro antiviral properties of RBV against 11 flaviviruses including DENV, Japanese encephalitis virus (JEV), and yellow fever virus (YFV). Inhibition of virus replication was observed for all tested flaviviruses [Arena-, Bunya-, and Togaviridae, were demonstrated to be susceptible to inhibition by RBV (Picornaviridae: both foot-and-mouth disease virus (FMDV) [Studying multiple viruses from the family iviruses . Furtheriviruses ,34. Howeiviruses . Therefoiviruses . Addition by RBV . For exas (FMDV) ,40 and ps (FMDV)  were inhWhile displaying broad antiviral activity against a wide range of RNA viruses, clinical data on the application of RBV are still limited and restricted to only a few viruses. Initially, RBV was considered as a treatment option for influenza A and B virus infections. However, clinical trials showed inconclusive data; although some studies reported an improvement of symptoms of influenza virus infection, results were generally inconsistent ,43. Due Taken together, since its first description as an antiviral in 1972, RBV has been shown to be active against a broad range of RNA viruses. However, due to limited clinical trial data supporting its in vivo efficacy, clinical applications are currently limited to a minority of viruses.The broad antiviral effect of RBV against numerous RNA viruses suggests different modes of action for the molecule; indeed, several antiviral mechanisms have been described in the past ,52 and aAnother indirect mode of action for RBV is the inhibition of the cellular inosine monophosphate dehydrogenase (IMPDH), which was already proposed in 1973  Figure A. After The influence of RBV on the expression of IFN-stimulated genes (ISG) is controversial in the literature. Most studies, both in vivo and in vitro, come from the HCV and RSV fields. RBV is able to increase the antiviral effects of an IFN-based therapy and restore IFN-responsiveness in HCV-infected livers ,64,65,66O-methyltranferase at the N-terminus of the NS5 polymerase, termed NS5MTaseDV. NS5MTaseDV binds GTP and catalyzes the formation of a 5′ cap structure [DV, thereby blocking the synthesis of the 5′ cap [Some RNA viruses, as well as cellular mRNAs, harbor a 7-methylguanosine cap structure at the 5′ end . The RBVtructure . After Re 5′ cap . Likewise 5′ cap ,73. Howee 5′ cap ,73,74,75Another suggested mechanism is the direct impact of RBV treatment on the function of viral polymerases A,B. HereIn recent years, a mutagenic effect of RBV via its incorporation into newly synthesized RNA genomes, leading to viral extinction was described for several RNA viruses B. In conEver since the first reports by Sidwell et al. describing RBV as a broad-spectrum antiviral , there hIn summary, several mechanisms have been postulated for RBV activity. Among these, there are indirect, immunomodulatory mechanisms and effects on IMPDH. Furthermore, mechanisms on the virus itself were described by inhibition of the capping efficiency, the viral polymerase, and a mutagenic effect on newly synthesized RNA genomes.RNA viruses do not exist as a clonal population of genomes within the infected host, but rather diversify into a swarm of related but non-identical genome sequences . This he11 and 1012 new virions are produced in one infected individual per day [These viral populations are the product of very high replication rates found in RNA viruses, coupled with a lack of an RdRp proofreading function. For HCV, it is estimated that between 10 per day ,99. Esti per day . In coro per day ; for HCV per day ,104. Des per day .As expected, HEV also exists as a heterogeneous population within infected individuals ,106,107.HEV and most other RNA virus populations exist in close proximity to the so-called genomic error threshold, which defines a maximum error rate that still guarantees the maintenance and transmission of the genetic information of the master sequence ,84. A reAs a consequence of the emergence of RBV-resistant mutations and subsequent treatment failure, clinicians could draw back on combination therapies to overcome or avoid this phenomenon. Possible combinations are one mutagen and a conventional antiviral drug or using several RNA mutagens in combination or sequence as proposed by Perales and Domingo ,110,111.HEV is one of the pathogenic viruses that can currently only be treated with RBV as an off-label drug. IFN-α as an alternative therapy has been evaluated in small patient cohorts with limited success and considerable side effects ,113. In Recently, two independent studies were able to correlate RBV treatment failure with the emergence of novel single-nucleotide variations in the viral genome during treatment ,26. BothRBV has been shown to block HEV replication through a depletion of cellular GTP pools in cell culture model systems , in addiThe mutagenic effect of RBV-based therapy can have divergent effects on HEV populations, which may impact the therapy success. On the one hand, RBV increases the mutation rate in the viral genome, driving the population towards its extinction threshold. In contrast, the increased variability in the viral population can result in selection of variants with improved replication fitness which become dominant in the viral population and are associated with therapeutic failure. These advantageous variants could be (i) a downregulation of the replication machinery, thus preventing the accumulation of more mutations, as shown from in vitro data when reverse engineering the K1383N variant into HEV cell culture systems , and (iiHEV is a life-threatening infection when immunosuppressed individuals fail to achieve an SVR during RBV treatment. Currently, clinicians do not have alternative therapy regimens available. Recent studies have suggested that the heterogeneous viral population is able to acquire SNVs that decrease RBV sensitivity ,26. Thei


A 43-year-old Hispanic male without significant previous medical history was brought to emergency department for syncope following a blood draw to investigate a 40 lbs weight loss during the past 6 months associated with decreased appetite and progressive fatigue. The patient also reported a 1-month history of jaundice. On examination, he was hemodynamically stable and afebrile with pallor and diffuse jaundice but without skin rash or palpable purpura. Normal sensations and power in all extremities were evident on neurological exam. Presence of hemolytic anemia, schistocytosis, thrombocytopenia, and elevated lactate dehydrogenase (LDH) was suggestive of thrombotic thrombocytopenic purpura (TTP). However, presence of leukopenia, macrocytes, and an inadequate reticulocyte response to the degree of anemia served as initial clues to an alternative diagnosis. Two and one units of packed red blood cells were transfused on day 1 and day 3, respectively. In addition, one unit of platelets was transfused on day 2. Daily therapeutic plasma exchange (TPE) was initiated and continued until ADAMTS-13 result ruled out TTP. A low cobalamin (vitamin B12) level was evident at initial laboratory work-up and subsequent testing revealed positive intrinsic factor-blocking antibodies supporting a diagnosis of pernicious anemia with severe cobalamin deficiency. Hematological improvement was observed following vitamin B12 supplementation. The patient was discharged and markedly improved on day 9 with outpatient follow-up for cobalamin supplementation. Microangiopathic hemolytic anemia (MAHA) refers to any hemolytic anemia associated with fragmented red blood cells (schistocytes) and small vessel pathology. Thrombotic microangiopathy (TMA) includes heterogeneous disorders characterized by MAHA, thrombocytopenia, and organ damage due to microvascular occlusion .Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are two types of TMAs which may be fatal without prompt recognition and treatment. Thrombotic thrombocytopenic purpura (TTP), first described in 1924 . It can TTP results from immunoglobulin G (IgG) and autoantibodies to von Willebrand factor-cleaving protease (ADAMTS-13). ADAMTS-13 is required for degradation of the highly adhesive and reactive forms of large von Willebrand factor multimers which can be up to approximately 20000 kDa in size , 8. The TTP and other hemolytic microangiopathies are often severe with significant morbidity and mortality rates remaining as high as 10–20% in spite of TPE , 14. TheTMA has been reported in association with pregnancy –19, humaCobalamin (vitamin B12) deficiency induced TMA is a rare condition which closely resembles the clinical features of TTP such as thrombocytopenia, hemolytic anemia, and schistocytosis. This report presents a case of cobalamin deficiency induced TMA in a patient initially suspected of having TTP, with important implications for the work-up and treatment of patients with suspected TTP.A 43-year-old Hispanic male with no past significant medical history presented to the emergency department (ED) with syncope, progressive fatigue, decreased appetite, weight loss of 40 lbs over the past six months, and one-month history of jaundice. The patient denied any chest pain, dyspnea, fever, cough, tingling, numbness, headache, visual changes, focal weakness, abdominal pain, bleeding, leg swelling, rash, changes in urinary or bowel habits, or sick contacts or any history of anemia. Upon initial physical examination, the patient was afebrile with a blood pressure of 135/69 mmHg and tachycardia. The patient had pallor and diffuse jaundice without hepatosplenomegaly, skin rash, or palpable purpura. On neurological exam, the patient was alert and oriented to person, place, and time with normal sensation and muscle strength in all extremities. Examination of the remaining systems was unremarkable.Initial laboratory  investigOn days 1 and 3 of admission, the patient was transfused with two and one units of packed red blood cells, respectively. On day 2, the patient was transfused with one unit of platelets. The combination of a peripheral blood smear suggestive of MAHA and thrombocytopenia serves as an acceptable indication for the initiation of TPE , 33 and,Cobalamin deficiency was noted at admission laboratory evaluation but was not accepted as the underlying cause of patient's clinical condition. However, one dose of 1000 mcg of cyanocobalamin was administered intramuscularly on day 3. Additional TPE sessions were continued until day 5, while ADAMTS-13 report was pending. The platelet count remained in a range of 18–37 K/uL despite daily TPE. On day 6, a normal ADAMTS-13 report was reported and TTP was ruled out. Platelet count improved to 90 and 138 K/uL on days 8 and 9, respectively.Further investigations were also positive for intrinsic factor-blocking antibodies and an elevated gastrin level consistent with a diagnosis of pernicious anemia. The patient's condition improved markedly and the patient was discharged on day 9 with outpatient follow-up for vitamin B12 supplementation.Cobalamin deficiency induced TMA poses a diagnostic hurdle for clinicians encountering patients presenting with thrombocytopenia, hemolytic anemia, and schistocytosis. Although the differential diagnosis should always seek ruling out the most life-threatening conditions first, measurement of cobalamin and methylmalonic acid level to the current diagnostic panel for the evaluation of TTP can direct physicians to proper diagnosis and management . MoreoveCobalamin is vital for DNA synthesis and hematopoietic cell division. Its ingestion occurs in the protein-bound state and, thus, requires release by gastric acid and pancreatic protease. Cobalamin is most efficiently absorbed in the distal ileum bound to intrinsic factor, a protein produced by the gastric parietal cells. Passive diffusion alone contributes to absorption of only 1–5% of the daily intake of cobalamin . Cobalam clinical cobalamin deficiency as was seen in a Danish study which reported a lack of significant difference in the prevalence of cobalamin deficiency related symptoms in individuals with methylmalonic acid ≥0.4 μmol/L versus <0.4 μmol/L [ functional deficiency [Cobalamin derivatives serve as cofactors in two major cellular reactions. The generation of methionine from homocysteine requires cytoplasmic methylcobalamin and the conversion of methyl-malonyl-coenzyme A to succinyl-coenzyme A requires mitochondrial 5-deoxyadenosylcobalamin . Hence, 4 μmol/L . In renaficiency , 52. Theficiency –55. Therficiency . Moreoveficiency . The preficiency , 59.Cobalamin deficiency was traditionally thought to be a disease of elderly Caucasians but is now known to be common in multiple ethnic groups and ages . The cauThe prevalence of cobalamin deficiency is 3–40% depending on the definition and biochemical markers used as well as the population explored –66. PernTMA develops in about 2.5% of cobalamin deficiency cases , 74. TheNoël et al. compared clinical and biochemical parameters in seven patients with cobalamin deficiency induced TMA and seven patients with TTP . First, TTP is associated with significant mortality and demands early diagnosis and prompt initiation of TPE. The diagnosis of TTP can be supported by a deficient ADAMTS-13 activity level but the long turnaround times of ADAMTS-13 testing limit its utility and TTP largely remains a clinical diagnosis. Although fluorescence resonance energy transfer (FRETS) method for determining ADAMTS-13 activity is a rapid technique, it is not widely available. Alternative underlying causes, especially cobalamin deficiency, should be explored at presentation in all cases of suspected TTP. The 2012 British Journal of Hematology Guidelines recommends that serological testing for HIV, hepatitis B and hepatitis C, autoantibody screen, and pregnancy test, when appropriate, be performed at presentation for patients with suspected TTP, given the reported correlation between HIV infection, viral hepatitis, and thrombocytopenia . Given tCobalamin deficiency induced TMA is a rare condition but should be considered in all patients presenting with clinical and laboratory features of TTP. The presence of leukopenia, macrocytes, and a slightly elevated reticulocyte count are initial clues that raise suspicion for cobalamin deficiency induced TMA. The emergence of multiple case reports of cobalamin deficiency induced TMA warrants the addition of cobalamin and methylmalonic acid testing in all cases of suspected TTP. By adding cobalamin and methylmalonic acid testing to all TMA screens, the diagnostic delays in detecting and treating cobalamin deficiency as well as the potential TPE related iatrogenic harm may be prevented.