Gli-B1-encoded γ-gliadins and non-coding γ-gliadin DNA sequences for 15 different alleles of common wheat have been compared using seven tests: electrophoretic mobility (EM) and molecular weight (MW) of the encoded major γ-gliadin, restriction fragment length polymorphism patterns (RFLPs) (three different markers), Gli-B1-γ-gliadin-pseudogene known SNP markers (Single nucleotide polymorphisms) and sequencing the pseudogene GAG56B. It was discovered that encoded γ-gliadins, with contrasting EM, had similar MWs. However, seven allelic variants (designated from I to VII) differed among them in the other six tests: I , II , III (Gli-B1b), IV , V (Gli-B1h), VI (Gli-B1d) and VII (Gli-B1a). Allele Gli-B1c (variant VIII) was identical to the alleles from group IV in four of the tests. Some tests might show a fine difference between alleles belonging to the same variant. Our results attest in favor of the independent origin of at least seven variants at the Gli-B1 locus that might originate from deeply diverged genotypes of the donor(s) of the B genome in hexaploid wheat and therefore might be called “heteroallelic”. The donor’s particularities at the Gli-B1 locus might be conserved since that time and decisively contribute to the current high genetic diversity of common wheat.The  Triticum turgidum L.) and the hexaploid species (AABBDD) bread wheat (Triticum aestivum L.). Bread wheat represents roughly 90 to 95% of the total production; hence, any advances related to knowledge or breeding in this species will have a major worldwide impact.Wheat is globally one of the most important crops. Currently, with world production at 728.5 million tonnes and a sown area covering 214 million hectares in 2019, wheat includes a quarter of total cereal production [Wheat provides 20% of the daily protein and food calorie intakes worldwide and represents one of the major protein components of the human diet. In recent decades, there has been an unprecedented increase in knowledge of the chemistry, genetics and functionality of the main proteins of the wheat seed, gliadin and glutenin. For example, there are classifications of different alleles at the seed-storage-protein-encoding loci in their relation to dough quality. This knowledge helps the production of new genotypes with improved characteristics . However, the immense complexity of the bread wheat genome  and of the genetic loci encoding the synthesis of the gliadins and glutenins leaves many questions unanswered. Such as the fact the same protein alleles might have different relationships to dough quality. Such findings raise the question of whether these alleles, identified by protein analysis in unrelated cultivars, are indeed identical. Moreover, the degree of difference at the DNA level of allelic variants differing on the protein level at the same seed-storage-protein-encoding locus is unknown.A common wheat genotype produces more than 50 different polypeptides of gliadin (gliadins) ,4. The pGli-A1, Gli-B1, Gli-D1, Gli-A2, Gli-B2, Gli-D2) [Gli loci. Analysis of segregating progenies from crosses between wheat genotypes showed that any Gli allele encodes two or more gliadin polypeptides (a block of electrophoretic bands) inherited together as a Mendelian unit. Alleles at one Gli locus differ in the number and/or EM of the encoded gliadin polypeptides of different gliadin types [About 20–25 bands differing in electrophoretic mobility (EM) may be observed in a one-dimensional electrophoregram  of a single grain, encoded in common wheat by six major ( Gli-D2)  and seve Gli-D2)  Gli lociin types . In totain types .Gli-1 and Glu-3 loci mapped in the distal part of the short arms of the homoeologous group 1 chromosomes [Gli locus encoding several gliadin polypeptides may exceed 2000 Kb as suggested for the Gli-A2 locus [A fundamental characteristic of the seed-storage-protein-coding loci , is theomosomes ,13,14, bomosomes ,16. The A2 locus .Gli locus. The largest part of a Gli locus was shown to consist of non-coding DNA sequences represented mainly by different types of transposable elements [Gli locus may differ in the number and types of active genes, in the distance between them, in the quota of pseudogenes among all the gliadin sequences and, probably, in the presence of non-gliadin genes harbored within a given Gli locus. The polymorphism of an entire Gli locus could be further augmented by the presence of single nucleotide polymorphisms (SNP).Hence all expressed gliadin genes together occupy only a minor part of the DNA of a elements ,18,19, belements  for whicelements  and evenelements ,22,23. TGli locus are routinely identified by the use of APAGE of gliadin proteins, thereby revealing sets of transcribed genes within the locus. However, an allele identified by APAGE may differ, in different genotypes, in the non-coding part of the locus. Hence alleles identified by APAGE may only be considered as markers of corresponding allelic variants of Gli loci.Alleles at a Gli locus, including its non-coding sequences. Restriction fragment length polymorphism (RFLP) analysis may disclose a difference between the alleles in the distribution of gliadin-coding sequences (including pseudogenes) along the whole locus. Two sets of molecular probes were also made available for the analysis of gliadin-coding DNA sequences. Firstly, the GAG56B γ-gliadin pseudogene was detected with the gene-specific PCR primer pair GAG29/30, and one of the variants of the pseudogene sequence (p-aes) was proven to be a characteristic of common wheat T. aestivum [Gli-B1-γ-gliadin sequence (probable pseudogene). Australian common wheat cultivars tested with these markers gave one PCR product with one pair of primers of either 369 or 397 bp [There are few possibilities to compare allelic variants of an entire aestivum . Secondlr 397 bp .Gli- locus is a highly complex chromosomic fragment that includes several gliadin-encoding genes, pseudogenes and sequences unrelated to gliadins. Some allelic variants at the Gli loci (and at the Glu-3 loci tightly linked to Gli-1) have been related to dough quality, providing useful markers to conduct quality breeding. However, the same allele at the Gli locus identified by protein electrophoresis in different cultivars might apparently have different relationships to dough quality, suggesting that this allele might be represented by more than one variant and tampering with the usefulness of gliadins as molecular markers. In order to explore this question, in this work, we compared 15 allelic variants at the Gli-B1 locus of common wheat (identified as blocks of Gli-B1-encoded gliadin polypeptides in APAGE) by means of several approaches: firstly, EM and molecular weight (MW) of encoded γ-gliadins using two-dimensional electrophoresis; secondly, their RFLP patterns (three different restriction enzymes and a γ-gliadin-specific probe); thirdly, the length of the PCR sequence of the γ-gliadin pseudogene; and fourthly, the DNA sequence of another γ-gliadin pseudogene present within the Gli-B1 locus. It was shown that the alleles studied might be divided into several variants strongly differing between them in their coding, as well as non-coding, sequences. The simultaneous multiple differences (without intermediate forms) among versions of the Gli-B1 locus may be explained through the suggestion that these variants maintain the particularities of the original genotypes of the corresponding diploid donor(s) in hexaploid wheat.Thus, a Gli-B1 locus  have been revealed in the wheat germplasm studied [Gli-B1l, were selected for further analysis.Twenty-four alleles at the  studied , and 16 Examples of gliadin electrophoregrams (APAGE) of the common wheat cultivars carrying the alleles studied  and scheGli-B1n, q, s and h are commonly accompanied by the allele Gli-B5b of the Gli-B5 locus, tightly linked to Gli-B1 [Gli-B5b encodes a pair of faint ω-gliadins, while the allele Gli-B5a is null. Allelic variation at Gli-B5 was accepted as a polymorphism procured by the distal part of chromosome 1B, and ω-gliadins encoded by the Gli-B5b were considered as a part of the Gli-B1-encoded blocks [Alleles producing the slowest-moving γ-gliadin and alleles o Gli-B1 . The alld blocks .Gli-B1 locus of common wheat produces one γ-gliadin (the Gli-B1a encodes two γ-gliadins), two or more ω-gliadins and some bands in the β-zone of the APAGE electrophoregram  were revealed: Gli-B1i, m, k, o ; Gli-B1n, q, s (II); Gli-B1b (III); Gli-B1e, g (IV); Gli-B1h (V); Gli-B1d (VI); and Gli-B1a   . The allriant IV l,j,k, buGli-B1c produces a weaker stained γ-gliadin and is linked to Gli-B5b  is to analyze a heterozygous seed of a cross between two genotypes carrying the alleles under study. Additionally, there is an “internal marker” present in most of the genotypes, the Gli-A3-controlled ω-gliadin: allelic variants of this polypeptide show identical apparent MWs of about 41 kD [Gli-D1 locus  may be also used as an internal marker of MW (The best way to compare the apparent MWs of the ut 41 kD . The majer of MW .Gli-B1 locus (Gli-B1-encoded γ-gliadins with different (including contrasting) EM had similar MWs of about 41 kD. For example, the MWs of γ-gliadins of alleles of variants I, III, VI (The intriguing result of this approach seems to be a close similarity (identity) of the apparent MWs of γ-gliadins produced by any variant of the B1 locus : Gli-B1- III, VI a–c and I III, VI d.Gli-B1a, b, c, d, e, f, g, h, k, m, o, q and s each controlled γ-gliadin of similar MWs (Gli-B1v (differing slightly from the Gli-B1o in the EM of one ω-gliadin), found in Spanish landraces also produced the γ-gliadin with an MW identical to those of variant I  electrophoregrams published by different authors, as well as the results of our work, showed that alleles ilar MWs . A new aariant I . In duruAlthough SDS-electrophoresis is hardly sensitive enough to document some probable minor differences in the length of compared polypeptides . The tree of relationships among Gli-B1 alleles , Gli-B1n and q (+s) (II), Gli-B1b (III), Gli-B1e and g (+f) (IV), Gli-B1d (VI), and Gli-B1a (VII). Alleles of different variants do not share or share only a few identical Gli-B1-derived polymorphic DNA fragments recognized by the γ-gliadin-specific probe. Furthermore, the approach has permitted some fine differences to be revealed between alleles belonging to the same variant.The results obtained with the RFLP approach show considerable differences in DNA sequence (positions of restriction sites) along the Gli-B1c may represent one more variant (VIII), related to, but different from, alleles of group IV, as has already been suggested on the basis of APAGE data. The allele Gli-B1a seems to be related to variant IV (endonucleases RsaI and HaeIII).In accordance with the RFLP analysis, the allele Gli-B1l (the 1BL.1RS) and any other allele studied.There was no convincing identity of any RFLP band produced by genotypes carrying the allele Gli-B1 locus. Each cultivar tested produced a PCR sequence with either primer GliB1.1 or GliB1.2, whereas cultivars carrying the 1BL.1RS translocation did not produce any amplification [Gli-B1l) at the Gli-B1 locus were analyzed by this test (γ-Gliadin SNP markers (PCR primers GliB1.1 and GliB1.2) are specific for a pseudogene located inside the fication . In our his test ; Table 1Gli-B1 locus were divided into two groups, according to [Gli-B1i, k, m, o), II  and III (Gli-B1b). Another group (PCR product with the GliB1.2) was composed of all other alleles  (Gli-B1l) did not give a PCR product with any pair of primers.Firstly, all alleles at the rding to . One groe, f, g) . As expeSecondly, there was a polymorphism in the length of the PCR products  , zone A.Gli-B1b and four cultivars from four countries with the allele Gli-B1d produced PCR sequences of approximately 369 bp (GliB1.1) and 409 bp (GliB1.2), respectively  and allele Gli-B1c each produced an apparently identical PCR sequence of 397 bp ; Gli-B1n, q, s (II); Gli-B1b (III); Gli-B1e, g, f (IV), Gli-B1h (V); Gli-B1d (VI); and Gli-B1a (VII). The allele Gli-B1c belongs, in this test, to variant IV.Therefore, there are seven groups of alleles identified in accordance with the PLP. These groups are identical to those revealed by the APAGE, TDE and RFLP approaches: alleles GAG56B of the Gli-B1 locus were divided into two groups by DNA sequence [Gli-B1 locus were compared  occurring in different cultivars has an identical sequence for the γ-gliadin pseudogene GAG56B. For example, haplotype 2 was registered in two unrelated cultivars from two countries with allele Gli-B1n, haplotype 4 occurred in three cultivars with Gli-B1c, and haplotype 5 in three cultivars from three countries with Gli-B1h (Gli-B1-encoded γ-gliadin) and noncoding (γ-gliadin pseudogene) parts of the Gli-B1 locus. The allele Gli-B1c belongs, in this test, to variant IV.Firstly, in most cases, any allele at the  Gli-B1h . TherefoGAG56B were divided into seven deeply diverging groups similar to those revealed by other methods in our work: alleles Gli-B1i, k, m, o (variant I: haplotype 1); Gli-B1n, q, s (II: 2); Gli-B1b (III: 3); Gli-B1e, g, f, c (IV: 4); Gli-B1h (V: 5); Gli-B1d (VI: 6); and Gli-B1a (VII: 7)  . The sma(VII: 7) .Gli-B1m differed slightly from other alleles of variant I by the length of its SSR sequence : the allele sequence , Table 2GAG56B sequence differed among wheat genotypes: Gli-B1m , Gli-B1e (4 and 11) and Gli-B1f (4 and 10)  into the new variant of the same allele . Three variants of the allele Gli-B1m differed in the number of the CAA repeats , while the only substitution in position 348 distinguished haplotype 4 from 10 , harboring different haplotypes of the GAG56B pseudogene in different cultivars, always produces the same set (a block) of encoded gliadins (data not shown).Fourthly, there were three alleles (identified by APAGE) for which the  and 10) , Table 2 from 10 . Any allRsaI data  chromosomes present in a given genotype. The differences among heterohomologues are distributed along the chromosomes involved and are assumed to be due to differences in the non-coding portion of DNA sequences ,35. DeleGli-B1 locus of common wheat has been documented in our work. These variants could be designated, analogously to heterohomologous chromosomes, as “heteroallelic”, and alleles belonging to the same variant would be “homoallelic”.The existence of several quite different variants of the DNA sequence at the Gli locus, because restriction sites for a given endonuclease may occur at any location along the whole long gliadin locus, including expressed genes and non-coding sequences. Using the RFLP approach permitted several distinct variants of DNA sequences of the Gli-B1 locus to be documented, strongly differing (without intermediate forms) in their restriction site positions. Moreover, some fine differences between alleles belonging to the same variant were revealed.RFLP seems to be especially useful for the comparison of allelic versions of a GAG56B were used for more detailed analysis of two particular regions of the non-coding DNA inside the Gli-B1 locus. In general, analysis of coding and non-coding parts of 15 allelic variants of the Gli-B1 locus revealed at least seven “heteroallelic” variants of the Gli-B1 locus (I–VII) without intermediate forms between them. Two pairs of allelic variants  were more closely related than the others. Nevertheless, allele Gli-B1a , for example, differed from alleles of variant IV by the EM (APAGE) of the encoded γ-gliadin  and in PLP and GAG56B tests but differed from them in another RFLP test (HaeIII) and in APAGE. Therefore, we assume Gli-B1c is one more variant (VIII) related to group IV.The allele Glu-B1 locus (B genome) among 58 cultivars of tetraploid and hexaploid wheat. It was suggested that these variants were present in genotypes of the diploid donors of this genome [Earlier, six contrasting variants without intermediate forms were described for DNA sequences of the s genome .Gli-B1 locus might originate from different genotypes of the donor(s) of the B genome in hexaploid wheat, as suggested by [Gli-B1 locus of these donors. It is suggested that genotypes of the diploid donors of genomes A, B and D were already highly polymorphic before the origin of allopolyploids [The pathways of origin of hexaploid wheat through successive crosses between diploid donors of the A and B genomes, and later, between AB and the D genome, are known in detail . Naturalested by , and inhlyploids  and thatlyploids  might allyploids .Gli-B1 locus not only dominate in the germplasm studied but are also spatially structured around the world and probably adapted to regional eco-climatic environments [Gli-B1 locus of common wheat germplasm studied is based mainly on the differences between variants of genotypes inherited by hexaploid wheat from its diploid donor(s) of the B genome. The donor(s) supplied common wheat with its own genetic variation of the distal part of chromosome 1B, which is now observed as heteroallelic variants at the Gli-B1 locus. A considerable downturn in the level of genetic diversity of common wheat for this chromosome region would be caused only by the disappearance (impossible) of one of these variants.Analysis of the gliadin genotypes in about 1000 common wheat worldwide cultivars of the 20th century  known i than 20 , the disTriticum aestivum L.) cultivars used in this work were obtained from genetic and/or breeding laboratories in their countries of origin and studied earlier , plus several Spanish landraces. The widest possible, first, allelic variation at the Names of cultivars, country of origin, year of registration and spring/winter habit were taken from the CIMMYT GRES database .In the current work, some grain samples from local collections were analyzed with the aim of confirming their gliadin genotypes. To do this, an acid polyacrylamide gel electrophoresis (APAGE) routine procedure  was usedFor RFLP analysis, DNA was extracted from seven-day-old seedlings) using a modified CTAB procedure . DNA digDNA was extracted from dry single seeds using the CTAB method . DNA quaPCR amplification was performed in the thermocycler Analytik Jena  using GliB1.1 and GliB1.2 primers . ProductGAG56B was amplified with GAG29 and GAG30 primers [® version 5.0 sequence analysis software  and aligned with acn M13712 in order to detect polymorphisms. The information obtained was used to reconstruct GAG56B for each cultivar and to define haplotypes. DNA sequences were aligned using clustalW [γ-Gliadin gene  primers . PCR-ampclustalW . PhylogeGli-B1 locus based on the RFLP data were also designed by the Neighbor-joining method.The trees of the relationship between alleles at the


Cut-off diameters below which droplets will be practically free of contamination, are presented as a function of viral loading. The median diameters of virus laden polydisperse droplet distributions will be 1.5 to 20 times higher depending upon the geometric standard deviation. The studies have implications to risk assessment as well as residence time estimates of airborne infections in indoor environments. Additionally, it will be also helpful for performance evaluation of sanitization and control technologies to mitigate infection risks in workplaces.For rebooting economic activities in the ongoing COVID-19 pandemic scenario, it is important to pay detailed attention to infection transfer mechanisms during interaction of people in enclosed environments. Utmost concern is the possibility of aerosol mediated infection transfer, which is largely governed by the size distributions of virus laden droplets, termed as virusols in this work, ejected from humans. We expand on the well-known theory of Poisson fluctuations which acts as statistical barrier against formation of virusols. Analysis suggests that for viral loads < 2 × 10 The contagion of COVID-19 is identified as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)2. Recognized routes5 of virus transmission from an infected person are, (1) surface/contact transmission, (2) direct droplet transmission, and (3) aerosol transmission. It is presumed that the first two modes of transmission pose the greatest risk, and have formed the backbone of instituting of intervention measures and strategies, such as social distancing, lock down, sanitization, wearing of masks etc. The airborne transmission risk, or the aerosol risk received prominence following a publication by Morawska and Milton6, in which special attention was drawn to certain superspreading events such as choir practice, restaurant, etc. As a result, WHO7 and CDC8 issued scientific brief indicating that airborne transmission is possible under special circumstances like enclosed spaces with inadequate ventilation and prolonged exposure from a severely infected person. Nevertheless, there are many other critical data such as minimum infectious dose for SARS-CoV-2, relationship of disease severity with viral load, proportion of infections acquired through airborne transmission, etc. which need to be known to answer the significance of airborne transmission. The problem is particularly relevant when transfer by severe symptomatic or asymptomatic individuals is considered. In spite of masks which would suppress direct transmission due to sneezing or coughing, it is still possible that viruses from an asymptomatic person might escape into air space through uncontrolled leaks. Recent studies10 show that the speech droplets are also potential in the virus transmission and the very recent case of COVID-19 outbreak in an air-conditioned restaurant suggests that virus-laden aerosol droplets could have remained in air and travelled long distances before infecting the others9.The outbreak of Coronavirus disease (COVID-19) has spread to more than 200 countries in the world, causing global health emergency as the number of confirmed cases reached 45,25,497 including 3,07,395 deaths worldwide as of May 17, 202012 have appeared over the past decade as well as recently on airborne risk, and Tellier et al.5 provide an excellent review on the subject. It has been found that significant (42–63%) portion of droplets containing virus causing influenza are in the respirable size range14 and support the hypothesis that influenza could be transmitted by the airborne route. The potential for airborne risk has a strong implication for the post lock down rebooting of business and office activities. This is because, in enclosed and indoor environments, such as public transport, offices, work places and schools, even a possibility of leaks from ill-fitting masks will be perceived as posing a risk of high consequence and will form a deterrent to a minimal level of interpersonal interaction. This can only be countered by building confidence through the deployment of adequate mitigation/sanitization technologies to stall aerosol route of transmission. The size distribution of airborne contaminants plays a crucial role in their risk potential, inhalability, site of deposition in the respiratory tract, transport in air and removal characteristics by intervention technologies.Several publications15, consist of sub-micron droplets directly emitted due to respiratory activities and the droplet nuclei formed from the evaporation of super-micron droplets contain viruses of size (0.02–0.3) μm14. These droplets are formed through atomization process of respiratory fluids 18 having a wide range of viral load (102 to 1011) copies/mL21. The droplets contain soluble nonvolatile materials 22 up to about 0.71% mole fraction. A rough but reasonable estimate22 shows that the respiratory droplet’s initial diameter is reduced by one-half to form droplet nuclei. The droplets of sizes less than about 20 µm , which are of importance from airborne risk perspective would dry up within a few seconds to form nonvolatile residues of size approximately half the droplet size22. In a recent paper by Stadnytskyi et al.10, a factor of 1/3 has also been used for the dehydrated residue sizes. The SARS-CoV-2 virus particles of size (100–200) nm will be incorporated into these residues which will then vector them across the indoor air space.The expiratory activities  of infected human subjects generate aerosol droplets of different characteristics in terms of their size and initial speed. The airborne droplet with sizes varying from 0.05 to 500 μm25. Due to the discrete nature of the virus (RNA copies), statistical fluctuations become very important for viral incorporation into droplet-residue system during their formation in human ejecta26. The studies by Fuchs and Sutugin23 and Raabe24 showed that particles contained in droplets produced by atomizing suspensions, are distributed according to Poisson distribution which makes allowance for the probability of occurrence of blank droplets with no viral copy. Alonso et al.27, in their experimental study indicated that the viral loading is higher in bigger particles than in smaller ones. Fernandez et al.28 demonstrated that the probability of number of bacterial cells in an aerosol droplet increases with bacterial solution concentration, and the probability follows Poisson distribution. Zuo et al.29 found that the virus loading of droplets follows a power law with exponent > 3, and they showed that the virus laden droplet size distribution shift towards larger particles. Although the work of Shindle and Galily30 raised doubts on the Poissonian assumption through their spray drying experiments, the assumption is still widely used for want of an alternative formulation. In a very recent work, Madas et al.31 have used Poisson distribution to estimate the probability of an airborne particle carrying at least one virus copy for modeling the deposition distribution of the SARS-CoV-2 virus in the human airways. As a result of the fluctuations, significant part of the ejected droplets would dry up to form blank residues carrying no RNA copies, thereby becoming unviable and harmless from the point of view of infection transfer. This situation is at variance with the assumption of virus distribution in the aerosol as being “proportional to droplet volume”17.Another important aspect of size distribution relevant to airborne risk arises from the well-known theory of atomization of suspensions and radioactive aerosol activation mechanisms10 estimated that for viral load of 7 × 106 RNA copies/mL, less than 0.01% of 3 µm, 0.37% of 10 µm, 37% of 50 µm droplets (prior to dehydration) will carry one or more virus and the remaining fraction will not carry any virus. Although they did not mention the basis of their calculations, a quick comparison with the formula [Eq.  only when propensity parameter exceeds the order of unity. In most practical situations of interest to airborne infections, this would be unlikely, as may be seen below.From Eq. , it is s20. Table 33 is comprehensive and shows a broad range between (102–107) RNA copies/mL. There is a general conformity between all the reported data regarding the range, and an atypical high value of 1.3 × 1011 RNA copies/mL is reported by Pan et al.21 from sputum sample of a patient. The propensity parameter corresponding to ~ 108 RNA copies/mL will be close to unity for a droplet diameter of ~ 20 µm. One can expect that droplets below this size will be increasingly blank. The viral load in the infectious subjects varies over a wide range due to many factors and infection time34 concluded that severe cases have distinctly higher viral loading as compared to milder cases. Also, from Table 33. Broadly speaking, mild cases fall in the category median of < 2 × 105 RNA copies/mL as compared to the levels of > 106 RNA copies/mL for those with severe symptoms34. With this empirical understanding, we tentatively classify viral load into the following two categories:Mild-to-moderate cases: Severe cases:  In a few recent studies36, asymptomatic cases have been found to carry viral load similar to mild cases with one case of exceptionally high load . For all practical purposes, a susceptible person will be exposed to inhalation of dried droplets. In view of the fact that virusols > 10 µm are unlikely to reach the pulmonary region, and cause risks, they are not considered from the risk perspectives22. From the droplet perspective, this amounts to a cut-off diameter of 20 µm and it’s required to examine the virus carrying potential  of droplets lower than 20 µm.The results presented here consider the cases of airborne droplets prior to evaporation, just released due to expiratory processes such as coughing, speaking, etc. Due to inevitable evaporation process, the droplet diameters are reduced to ~ 50% of their original value10. Relationship between viral loading and severity of the disease is a matter of considerable practical value. For viral loads of less than 104 RNA copies/mL, expected for mostly mild-to-moderate cases33, the virusol fraction is less than 0.1% for droplets below 60 µm; i.e. more than 99.9% of the droplets below 60 µm will not be carrying any virus. As larger than 60 µm droplets are very unlikely to remain airborne for infecting via aerosol route, this simple analysis leads to a conclusion that mild-to-moderate cases are least likely to infect via aerosol route.Figure 5 RNA copies/mL, droplets less than 20 µm are unlikely to carry any viral load. Thus, airborne contamination is most likely to arise from severe patients only. The figures also show that even for ejections from infected subjects with high viral load, the droplets < 2 μm (prior to evaporative water loss) are unlikely to be contaminated and carry no risk. From Table −3 (for speaking). The corresponding virus laden fraction (virusols) are in the range of 2 × 10−5–4 × 10−4 even for severe cases with viral load of 108 RNA copies/mL. In real indoor scenario, this would amount to risk of inhaling less than one virus carrying droplet if a person stays for an hour in the room with the infected person. One can therefore restrict attention on large droplets only for aerosolized risks, and the remaining fraction will be just uncontaminated droplets.Figure −3 to 2.5 × 103 cm−3  or less ejected from human ejecta of infected persons are matter of potential concern from the hazard perspective of viral transmission by airborne route in confined environments. As we know, not all droplets carry viruses and this fact has bearing on future intervention technologies which operate on size control basis. An important “statistical barrier” brought about by Poisson fluctuations limits viral incorporation into droplets during their ejection from the infected subjects. The present analysis illustrates the impact of this reasoning on the formation of virus laden droplet systems, termed herein as virusols, their size distributions and practically useful cut-off values. By combining the available data from the current literature on viral loading in different patients with the recent observation on its relationship with disease severity, it is argued that formation of virusols, which will remain stable for certain length of time as well as which are inhalable by humans, (i.e. droplets less than 20 μm) is virtually inhibited in mild-to-moderate cases of patients. Virusol formation and consequent infection transfer could be important for explicitly severe cases, that too for droplet sizes above 2 µm (prior to evaporation). Hence, for an effective control measure using filtration based air cleaners, it may not be necessary to worry about ultrafine particle filtration. This somewhat relaxes the constraint on the filtration efficiency as relatively coarser filters will be efficient in capturing larger particles. As a result, flow resistances can be significantly lowered thereby enabling higher Clean Air Delivery Rates. Furthermore, the finding of a significant upward shift in virusol sizes, implies that their residence times in indoor spaces will be considerably lower than other droplets ejected from humans. This will greatly help in providing a realistic assessment of air borne infection transfers in indoor environments.


Despite the economic burden of psoriasis for patients and societies, scant information exists regarding the impact and burden of the disease in Argentina.The objective of this study was to estimate medical resource consumption and direct health care costs for patients with moderate/severe psoriasis in Buenos Aires, Argentina from the perspective of the payer.Adults with moderate/severe psoriasis (severity was defined as receiving systemic treatment), during January 2010–January 2014, aged 18 years and older, members of the Italian Hospital Medical Care Program with at least 18 months of follow-up were included. All data on hospitalizations, drug prescription, outpatient episodes, consultations, and investigations/tests in the 12 months before inclusion in the study were considered for the estimation of medical resource consumption and direct health care costs. First-quarter 2018 costs were obtained from the IHMCP and converted into US dollars (using the January 2018 exchange rate).A total of 791 patients were included. The mean age at diagnosis was 34 ± 12 years. Almost 65% of the patients had a dermatologist as their usual source of care, 43% had internists, and 14% had rheumatologists. The average yearly direct cost was US$ 5326 (95% CI: 4125–7896) per patient per year.The single center design and the retrospective nature are the main limitations.This is the first Argentine study that evaluated the costs of moderate/severe psoriasis by taking into consideration the direct medical costs of the disease. Psoriasis is a chronic inflammatory immune-mediated proliferative skin disorder that predominantly involves the skin, nails, and joints.2In 2014, Member States of the World Health Organization (WHO) recognized psoriasis as a serious non-communicable disease and adopted the resolution of the World Health Assembly (WHA), which calls for multilateral efforts to raise awareness and fight stigma. The worldwide prevalence of psoriasis is estimated to be approximately 1–3%.3Although psoriasis is not usually a life-threatening disease, several studies have shown that it has a significant impact on medical resource consumption in affected patients.5Despite the economic burden of psoriasis for the patient and for society as reported by several authors,2 and has a subtropical climate. It is located on the western bank of the Río de la Plata and has a population of 2,890,151.A retrospective, epidemiological, and observational study was conducted. The population studied included the members of the Italian Hospital Medical Care Program (IHMCP), a prepaid health maintenance organization. The IHMCP provides comprehensive medical and health services through two main hospitals and 24 medical offices to more than 164,456 members, primarily located in the urban areas within the autonomous city of Buenos Aires in Argentina. The city covers an area of 202 kmData on hospitalizations, drug prescription, out-patient episodes, consultations with general or specialist medical practitioners, and investigations/tests in the 18 months before the inclusion in the study were collected from electronic records of the included patients. Electronic records of IHMCP patients provide daily routine information on their diseases and therapies.Costs were calculated using the bottom-up approach and grouped by perspective. To obtain the annual cost per patient per year, annualized resources were multiplied by the irrespective unit costs. January 2018 unit costs were derived from the IHMCP. The unit costs in Argentine pesos were converted to US dollars using the January 2, 2018 exchange rate (US$1 = AR $19.23). Unit costs derived from the IHMCP were compared with those from the public sources of Argentina, including the National institute of Statistics and Surveys,p < 0.05). Stata v. 10.1 software  was used for the analysis.Patients’ demographic and disease characteristics, as well as resource utilization and costs, were analyzed using descriptive statistics . Confidence intervals (95%) were estimated by using non-parametric bootstrapping. A non-parametric statistical test (Wilcoxon–Mann–Whitney test) for independent samples was used to determine whether differences between comparisons performed were statistically significant .All costs obtained from resource utilization are presented in With the introduction of biologics over the past years, there has been a marked shift in treatment modalities that has resulted in a great need for the updating of costs.This study is consistent with previous cost-of-illness studies performed in Germany and some European countries.The present study has many limitations. First, the sample involves patients from only one center. Therefore, a larger sample that involves more centers would be necessary to generalize the results. The second limitation is the way the information was obtained . Nevertheless, considering that the patients from the IHMCP are seen solely in its own centers and that all medical records are registered, the possibility of bias is low. The third limitation is that indirect costs were not obtained.In Argentina, this is the first study designed to capture the economic burden of the disease. The important and novel information provided about the disease-related economic burden is the main contribution of this study to the existing literature for psoriasis both in Argentina and the region in terms of costs in a developing country. This information is a valuable tool for relevant and wise decision-making in the field by understanding the exact behavior of the disease in the local setting, and is supplementary to the use of information from Europe and North America, where both resource utilization and costs may be quite different from those observed in a developing country like Argentina.7In conclusion, the study provides important information about the economic burden of the disease in Argentina. Future research performed here as well as in other countries of the region, taking into consideration direct and indirect costs of the disease, will expand knowledge and improve management of the disease in the region, an issue of great importance.None declared.María Laura Galimberti: Approval of the final version of the manuscript; conception and planning of the study; elaboration and writing of the manuscript; obtaining, analyzing, and interpreting the data; effective participation in research orientation; intellectual participation in propaedeutic and/or therapeutic conduct of the cases studied; critical review of the literature; critical review of the manuscript.Aldana S. Vacas: Approval of the final version of the manuscript; obtaining, analyzing, and interpreting the data; critical review of the literature.Barbara A. Hernández: Approval of the final version of the manuscript; obtaining, analyzing, and interpreting the data; critical review of the literature.María L. Bollea Garlatti: Approval of the final version of the manuscript; obtaining, analyzing, and interpreting the data; critical review of the literature.María J. Cura: Approval of the final version of the manuscript; obtaining, analyzing, and interpreting the data; critical review of the literature.Ricardo L Galimberti: Approval of the final version of the manuscript; obtaining, analyzing, and interpreting the data; critical review of the literature.None declared.


Provirus was present in Ghanaian women (6/87), in 1/6 Pygmy populations and in African American men (4/45) and women (6/68), but not in any Caucasian women (0/109). Two BrC cell lines (HCC 70 and DT22) from African American women had Xq21.33. Env regions of the virus which differed by 2–3 SNPs did not alter the protein sequence of the virus. SNP at 5730 and 8529 were seen in all persons with provirus, while 54% had an additional SNP at 7596.Two Nigerian women and 2 Ghanaian women had additional unusual SNPs. Homozygosity was seen in (5/27) BrC and (2/22) control women. The genetic variation and homozygosity patterns suggested that there was gene conversion of this X chromosome associated virus. The suggestive finding in this preliminary data of possible increased prevalence of Xq21.33 provirus in post-menopausal Nigerian women with BrC should be clarified by a more statistically powered study sample to see if postmenopausal African and/or African American women carriers of Xq21.33 might show increased risk of BrC. The implication of finding such a link would be the development of antiretroviral drugs that might aid in preventing BrC in Xq21.33+ women.The human endogenous retroviruses HERV-K HML-2 have been considered a possible cause of human breast cancer (BrC). A HERV-K HML-2 fully intact provirus Xq21.33 was recently identified in some West African people. We used PCR technology to search for the Xq21.33 provirus in DNA from Nigerian women with BrC and controls. to see if Xq21.33 plays any role in predisposing to BrC. This provirus was detected in 27 of 216 (12.5%) women with BrC and in 22 of 219 (10.0%) controls. These results were not statistically significant. The prevalence of provirus in premenopausal control women 44 years or younger [18/157 (11.46%)} vs women with BrC [12/117 (10.26%)] showed no statistical difference. The prevalence of virus in postmenopausal control women > 45 yrs. was 7.4% (4/54) vs 15.31% (15/98) in postmenopausal women with BrC. These changes were not statistically significant at <.05, but the actual  Much is lacking in our understanding of the causes of human breast cancer (BrC). There is some evidence that a mouse mammary tumor virus (MMTV) may be associated with, and be a cause of some human BrC. Such a virus is often called human mammary tumor virus (HMTV). The biology of MMTV has been summarized by Dudley et al.  with mucenv gene, and type 2, which have a more complete env gene. With time, most of these proviruses developed lethal mutations and indels which prevented any subsequent activation of infectious virus in the host. In addition, as these viruses had identical long terminal repeats (LTRs) on both the 5′ and 3′ side of the virus at the time of integration, through homologous recombination during cell replication some of these integrated viruses deleted all internal genes but solo LTRs which have remained in many thousand genomic locations in the hominid genome.The human endogenous retroviruses HERV-K HML-2 have also been implicated as having some role in the pathogenesis of BrC as discussed below. These retroviruses began to enter the evolving primate genome over 35,000,000 years ago (MYA) through infection, integrating in the genome of oocyte or spermatocyte cells. After fertilization, offspring of these infected animals now contained a provirus in every cell. If animals survived this infection they could pass this provirus from generation to generation –14. ManyMuch work has been done recently to fully detail the total number of HERV-K HML-2 proviruses in the human genome. Most of these proviruses are in all humans, but some have arisen in limited areas of the world and in limited populations, occurring at different times in evolution with variable spread throughout the world. Proviruses are now classified by their location in the chromosomal arm of the human genome. It is estimated that 117 of these proviruses remain in the human genome with several thousands of solo LTRs , 14–20. gag, prot, pol and env genes , MDA MB157 (ATCC® HTB-24™) from medullary carcinoma, MDA B 468 from a pleural effusion, HCC1806 (ATCC® CRL-2335™) primary acantholytic squamous cell carcinoma, SUM 149PT from a xenograft of a transplanted primary explant of a human invasive infiltrating duct carcinoma and ZR7530 (ATCC® CRL-1504™) from metastatic ductal carcinoma. Cell lines were grown in media suggested by the company providing such lines. Other cell lines were established in our laboratory from tumor biopsies obtained from left over pathology samples. This material was obtained with informed consent with the donor remaining unknown but the pathology of the tumor known. This protocol which allowed cultivation of cells was approved by the University of Michigan Investigation Review Board (IRB). This included cell lines 09–1521, 12–1697, 12–135, 13–1357, 14–130, 15–107. Cell lines K151 and NSUH1 were established at North Shore University Hospital, (NSUH) Manhasset N.Y., from pleural effusions cells obtained with informed consent in a protocol approved by the NSUH IRB. These cells were grown in RPMI 1640 with penicillin and streptomycin and 10% heat inactivated calf serum.DNA was extracted from PBL and cell lines using the DNeasy blood and tissue kit™ and/or the Qiagen Gentra Purgene® assay. Saliva was collected in the Oragene OG-500 collection kits. DNA was extracted manually using the prepIT-L2P DNA Genotek .Women from Nigeria with BrC were clinically staged according to the American Joint Committee on Cancer system . ImmunohPCR to search for Xq21.33 was carried out by screening with the following primers:Coff Xq21.33 ACG CTG TTT TGT CCC TTT GA.1016 R CGTCGACTTGTCCTCAATGACCACGCT.584R TTTGCGTTGACTGAGCCATTACCG.12477 R TTG AGC AAC ATC TTG GAG CCT TGC.3456R ACT TGC CCA ATA TGC AGC CTT TCC TCC.6115R TCA ACT ATG GCC TGT CCT TGC GAA.5355F TCG GCT CAA AGA GCA GAG ATG GTT.F Flank TCAGTC TGA AGA CAA TGA CAT ACT T T.Taq PCR Kit (New England Bio Labs). Reactions were carried using 5ul of 5X Long Amp Taq reaction buffer + .75ul of 10uM DNTP, + 0.2 ul of 50uM forward and 0.2ul of 50uM reverse primer with addition of 1ul of Long Amp Taq DNA polymerase plus the addition of between 50 to 250 ng of sample DNA to a final volume of 25ul. In general, the reactions were carried out 94 °C initial denaturation for 30 s followed by 35 to 40 cycles of 94° for 15 to 30 s, 55° to 60° for 15 to 30 s and 65° for 2 to 5 min, and then final extension at 65° for 10 min. All positive samples to 1011R to Coff Xq21.33 were confirmed with at least 3 other primer sets.Approximately 200 ng of DNA was tested with a primer Coff Xq21.33 in the 5′ insertion site and any second primer  located in the Xq21.33 genome, and/or a 3′ insertion site primer, F Flank (R) to 5355F in the virus. PCR was carried out using the LongAmp® Homozygosity was determined by using primers at Xq21.33 and F Flank to obtain a PCR product of 420 bp in heterozygotes and no amplification product in homozygotes.env region of Xq21.33 was carried out by creating an amplified product using F Flank to 5355F using the protocol noted above. The amplified product was resolved from 0.8% agarose gels and bands of DNA of interest were cut from gels and purified using the QIAquick Gel extraction kit QIAGEN according to the method of the manufacturer. The purified product was sent for direct sequencing to the DNA sequencing core of the University of Michigan using primers in forward or reverse orientation ranging from 5350 to 9472.Genotyping of the 3′ Neighbor-Joining trees were generated using MEGA 7. The evolutionary history was inferred using the Neighbor-Joining method . The perhttp://www.stata.com/support/faqs/statistics/adjusted-means-after-anova.Differences in the presence of the Xq21.33 provirus between cases and controls were examined using Fisher exact tests All analyses were performed using Stata SE version 15.1 . A type I error (α) level of 0.05 was considered significant for testing the study’s hypotheses. Two hundred thirty-nine Nigerian women with BrC participated in this study. In 4 the DNA concentration was too low for adequate use in PCR. In addition, in 19 consecutive samples there was an interfering substance present which prevented PCR reactivity. These samples were amplified multiple times with different primers to get a signal, but they would not amplify. These patients are not included in this study. This left 216 women with breast cancer who became part of this study. There were 229 control women enrolled in the study. Of these, 10 had very poor DNA content. This left 219 control women as part of this study.gag and pol genes of the provirus. In addition, we screened positive samples for the env gene using 5359F and F Flank. Representative positive samples are shown in Fig. Proviral DNA was screened for in all samples first using the primer set 1016R and Coff Xq21.33. All positive samples were then screened with other primer sets including 1584R, 2477R, 3456R and 6115R in conjunction with Coff Xq21.33. This covered the LTR and the The Xq21.33 provirus was present in 27 of 216 (12.5%) Nigerian BrC cases compared to 22 of 219 (10.0%) controls These results were not statistically different.Table n = 563 age range 44–87) [n = 123 age range 40–56) [We used this age 45 or greater as presumed postmenopausal age as we have broken our data into age groups at 5 years intervals. In Nigeria the average age of menopause is 48.4 +/− 5 years (samples size e 44–87) , and in e 40–56) .n = 109) confirming previous observations reporting the specific incidence of Xq21.33 in in people with African ancestry [We next looked at DNA from patients in the United States who had BrC and other conditions Table . Of noteancestry . We alsoWe looked to see if there was homozygosity/hemizogocity for this virus in women with BrC and virus vs those without breast cancer and provirus. An illustrative PCR gel is shown in Fig. We were particularly interested in seeing if the pathology of BrC was different in women who had the virus and those who were negative. Extensive biomarkers were carried out on the Nigerian cohort of women as shown in Table We also screened known cell lines for the presence of Xq21.33 proviruses as shown in Table env region of the virus are shown in Table We did genomic sequencing in order to determine if there is variation in the Xq21.33 sequence in populations from West Africa. Mutations found from PCR product amplified from primers 5355F to F Flank giving a sequence spanning the env protein.Genetic sequences were compared to the known HERV-K HML2 sequences to understand the evolution of Xq21.33 and how 2 Nigerians and 2 Ghanaians had more than 3 SNPs present. All proviruses had the 5370 and 8529 mutation, which may have existed in the founder virus, while about 54.3% of proviruses had an additional mutation at 7596. All mutations preserved the integrity of the We performed a phylogenetic analysis to understand the evolution of HERV-K HML-2 sequences isolated in Nigerian and Ghanaian populations. A Neighbor-Joining tree shows that genetic mutations and possible gene conversion events occurred during HERV-K HML-2 Xq21.33 evolution Fig. . PlacingTo date studies of endogenous retroviruses have been trying to determine if any of these proviruses can be activated again to produce infectious virions which may genetically alter cells and lead to a disease process. Thus far no provirus has been convincingly associated with a disease. Great interest has been placed on seeing if any or some HERV-K HML-2 viruses can be linked to BrC, as these viruses are genetically distantly related to MMTV. Early studies of BrC cell lines revealed the presence of viral particles possibly related to HERV-K HML-2. The T47D cell line grown from a neoplastic pleural effusion was shown to produce viral particles when stimulated first with estradiol and then progesterone . At firsenv surface protein [nv protein on tumors of both American and Chinese patients, and the level of expression correlated with poor prognosis [env was necessary for metastasis and tumorigenesis of BrC cells [Attempts were made to determine if HERV-K HML-2 proteins might appear in BrC cell lines or tumors. Indeed NP9, a 9 kb protein expressed predominantly in the nucleus as a product of Type 1 HERV-K HML-2 proviruses, was described by Armbruester et al. . This pr protein . Eighty- protein . Antibod protein . Various protein . Similar protein . Human trognosis . Using Trognosis . SubsequrC cells .Other studies have looked to see if H2K proviruses are involved in transcription during transformation of mammary epithelial cell to neoplastic cells. This was accomplished by taking human mammary epithelial cells and immortalizing them through telomere maintenance by “hTert” (encoding human telomerase reverse transcription) over expression. These cells were then transformed with SV40 small and large T antigen, and then further transformed with a murine leukemia virus (MLV) base vector. This led to over expression of the BrC oncogene HRAS (V12) and/or ERB2 (Her2/neu). These cells were then looked at to see what mRNAs were used that belong to the HERV-K HML-2 group. Surprisingly, the HML2 transcriptome analyzed showed that most of transcription arose from 15 HML2 proviruses. The majority of transcription was in an antisense orientation and “from mostly older proviruses integrated within introns” . VirusesWith all of this analysis so far, it still remains unclear if there is a specific HERV-K HML-2 provirus associated with BrC and/or how activation of HERV-K HML-2 might be playing a role in formation of BrC. With the discovery of the new Xq21.33 provirus , which aIn Nigerian women the prevalence of this virus is higher (49/444 women 11.03%) than that detected by Wildschutte et al. (6.9%) . This diThis provirus has not spread as widely as younger proviruses K113 or K115, which have been reported to be present in 21.8 and 34.1% of African populations . For insenv region but might modify the biology of the provirus. Several samples have multiple SNPs which, on genetic analysis, suggests some form of gene conversion occurring in some Nigerians and some Ghanaians. The mutations we found cannot be explained by PCR artifact which would produce many random mutations, rather than those seen. This would suggest that Xq21.33 is in a hot spot of the genome, which may allow for some recombination with other active viruses, such as K113.These SNPs found in HERV-K HML-2 Xq21.33 do not create stop codons in the viral There was no difference in the prevalence of virus between those women with BrC and women without BrC indicating that this virus does not play an active role in breast carcinogenesis. In an unplanned analysis by menopausal state if one divides these patients into pre and post-menopausal groups, then virus is more prevalent in post-menopausal women, rising from 12/117 or 10.25% to 15/98 or 15.30% in women with BrC while falling from 18/157 or 11.4% to 4/54 or 7.4% in post-menopausal women without BrC. This difference in frequency of provirus in post-menopausal women is not statistically significant at the 0.05 level as the study was not powered to detect such small differences, should they be relevant to the process or carcinogenesis, but is closer to the 0.079 level. It is clear that our sample size is too small. Clearly an adequately powered study of post-menopausal women might be undertaken to explore a potential significant association of this virus in post-menopausal African and or African American women with BrC.We did see a higher prevalence of triple negative BrC, as has been reported earlier in patients from Africa. Our patients are relatively younger than patients with BrC in Europe and the Americas. This may be more due to the fact that the average life span of women living in Nigeria, according to the World Health Organization, is about 54.7 compared to that in the United States, Germany, Sweden and the United Kingdom, which is about 81.0, years of age. As health is improving in Nigeria the percentage of triple negative BrC may reduce, as older women are less likely to have triple negative cancers.env gene from the artificially constructed Phoenix virus, [env construct similarly binds to heparan sulfate [In order to understand the potential role of this virus even if our study, or a future larger study with more post-menopausal women, had shown a statistically relevant association of Xq21.33 in post-menopausal BrC, it would still be necessary to show that this provirus has activated and is now in some area in the BrC cell different from Xq21.33 and the known HERV-K HML-2 integration sites and in a genomic area where it might disrupt some cellular growth factor. Such a virus would also have to be able to infect breast tissue. Recently it has been shown that the HERV-K HML-2 x virus,  encoded x virus, . It was  sulfate . HeparinSince cells have 117 proviruses present, as well as over 2000 LTRs and over 1000 pericentromeric and centromeric HERV-K HML-2 s, all of which are almost 97–99% the same, using current platforms for sequencing to detect provirus would be very difficult as a newly integrated provirus would be obscured by such background noise. One would have to find sequences that are within a new area of the genome and or repetitive elements in the centromeric regions. Such a sequence would have to span the full LTR and extend into the sequence of the virus itself. Current sequencing provides only fragments of 300–400 bp, which are not of sufficient length, or number, to distinguish virus from background HERV-K genomes and LTRs. However, if virus did get to the breast and integrate in an area to induce an oncogenic change, virus would be amplified many fold by the clonal expansion induced by the cancer. This new insertion site in the cancerous breast tissue could only be detected by sequencing larger portions of the genome with new available technologies that give long sequence reads. Similar technology has been used in a study of large granular cell leukemia thought to be due to a HERV-K HML-2 virus. While this study could not confirm the presence of a HERV-K-HML-2 virus it does provide insight into how such technology can be used in cancerous cells .We searched for evidence that a recently discovered HERV K HML-2 virus Xq21.33 might play a role in predisposing women to BrC in those carrying this provirus. Our data describes some of the genetic variation in this provirus but our data could not support an association of Xq21.33 with breast cancer. However, it does suggest that there may be some role for this provirus in predisposing post-menopausal women carrying this virus to breast cancer which could be discovered if a more appropriate sample size of older patients was studied. Finding of such an association would be important as antiretroviral drugs might be useful in preventing BrC in such women.Additional file 1: Supplementary Fig. 1. Alignment of HK2 X21q33 sequences from populations of African ancestry.


C. x latifolia (Yu. Tanaka) Tanaka) naturally originated from a merger between a haploid ovule of lemon (C. x limon (L.) Burm) and a diploid pollen from a ‘Mexican’ lime (C. x aurantiifolia (Christm.) Swing). The very limited natural inter-varietal diversity and gametic sterility of C. latifolia requires a phylogenomic based reconstruction breeding strategy to insure its diversification. We developed a strategy based on interploid hybridization between diploid lemon and the doubled diploid ‘Giant Key’ lime. This lime is a doubled diploid of ‘Mexican’ lime, itself a natural interspecific F1 hybrid between C. medica L. and C. micrantha Wester. For an optimized breeding program, we analyzed the meiotic behavior of the allotetraploid lime, the genetic structure of its diploid gametes, the interspecific recombination between C. medica and C. micrantha, and constructed its genetic map. A population of 272 triploid hybrids was generated using ‘Giant Key’ lime as pollinator. One hundred fifty-eight SNPs diagnostic of C. micrantha, regularly distributed throughout the citrus genome were successfully developed and applied. The genetic structure of the diploid gametes was examined based on C. micrantha doses along the genome. The diploid gametes transmitted in average 91.17% of the parental interspecific C. medica/C. micrantha heterozygosity. Three chromosomes  showed disomic segregation with high preferential pairing values, while the remaining chromosomes showed an intermediate inheritance with a preferential disomic trend. A total of 131 SNPs were assigned to nine linkage groups to construct the genetic map. It spanned 272.8 cM with a low average recombination rate (0.99 cM Mb-1) and high synteny and colinearity with the reference clementine genome. Our results confirmed that an efficient reconstruction breeding strategy for ‘Tahiti’ lime is possible, based on interploid hybridization using a doubled diploid of C. aurantiifolia. The tetraploid parent should be selected for favorable agronomic traits and its genetic value should be efficiently inherited by the progeny thanks to transmission of the high level of parental heterozygosity. However, it would require developing numerous progeny to overcome the linkage drag caused by the limited interspecific recombination associated with the predominant disomic inheritance.The triploid ‘Tahiti’ lime ( Polyploidy, the state of an organism having more than two paired set of chromosomes, is a major component of angiosperm evolution  and polyTetraploid meiosis behavior and particularly the transmission of parental heterozygosity greatly depends on the origin of the polyploid. Two extreme models are generally considered, disomic in allotetraploids and tetrasomic in autotetraploids . In alloThe specificities of tetraploid meiosis require dedicated tools for genetic mapping. While several software tools are available to construct linkage maps of diploid species, including MapMaker , JoinMapCitrus species and related genera are diploid with a basic chromosome number x = 9 (Most er x = 9 . Howeverer x = 9 . Cameroner x = 9 . Triploier x = 9 . The reser x = 9 . Ploidy er x = 9 . Both seer x = 9  and inteer x = 9  have beeC. x aurantiifolia (Christm.) Swing), triploid (‘Tahiti’ lime type C. x latifolia (Yu. Tanaka) Tanaka and ‘Tanepao’ lime type), and tetraploid (‘Giant Key’ lime) natural germplasm (C. maxima (Burm.) Merr., C. medica L., C. micrantha Wester and C. reticulata Blanco), while ‘Mexican,’ ‘Tanepao,’ and ‘Giant Key’ types involved only C. medica and C. micrantha gene pools. ‘Mexican lime’ types resulted from direct hybridization between C. micrantha and C. medica  Burm) ovule by diploid pollen from C. x aurantiifolia  were selected from GBS data  to analyze the meiotic behavior of the tetraploid ‘Giant Key’ lime and its impact on the genetic structure of the diploid gamete population, (ii) to implement its genetic map, and (iii) to analyze the interspecific recombination between GBS data  and 131 C. micrantha (ICVN 0101115), ‘Poncire commun’ citron (ICVN 0100701), respectively representative of homozygous genotypes of the C. micrantha and alternative alleles, ‘Mexican’ lime (ICVN 0100140) as heterozygous control with equivalent doses of C. micrantha and alternative alleles (1/1), ‘Tahiti’ lime (ICVN 0100058), and ‘Persian’ lime (ICVN 0101046) as representatives of the C. latifolia triploid lime ideotype, ‘Tanepao’ (ICVN0100836) and ‘Coppenrhad’ (ICVN010838) lime as representative of C. aurantiifolia triploid limes. All these varieties came from the collection of the Inra-Cirad Citrus Biological Resource Center in San-Giuliano, Corsica, France.Diploid lemons  were fertilized with pollen of the tetraploid ‘Giant Key’ lime (ICVN 0100785). Among the seedlings, nucellar diploid plants  and triploid hybrids were identified by flow cytometry. The meiotic behavior was analyzed and the genetic map of the tetraploid ‘Giant key’ lime was constructed using 272 selected triploid hybrids. In addition to the genitors, some varieties/species were also included in the study as controls at the genotype calling step: Genetic analysis of polyploid populations requires an unambiguous estimation of allele doses for heterozygous genotypes. GBS analysis of polyploid citrus revealed the difficulty to infer allele doses at individual SNP polymorphisms . TherefoC. maxima x C. reticulata) x C. medica] x [C. micrantha x C. medica] with [C. micrantha x C. medica] parent being a doubled diploid (C. micrantha (M) have the following segregation AA x MMAA where A is the alternative allele to the M one. They are therefore perfect markers to analyze the segregation of the tetraploid ‘Giant Key’ lime, construct its genetic map and to analyze the interspecific recombination between C. micrantha and C. medica. One hundred and eighty-nine diagnostic SNPs of C. micrantha (DSNPs) were selected from those identified from a GBS analysis which aimed at deciphering the mosaic genomes of citrus fruits ([( diploid . Therefos fruits . These Dwww.lgcgenomics.com). The KASPar™ genotyping system is a competitive, allele-specific dual Förster Resonance Energy Transfer (FRET)-based assay. LGC Genomics extracted the DNA from the leaf samples, designed the primers based on the SNP locus-flanking sequences, and performed the genotyping. Details on the KASPar method are provided in . Under intermediate inheritance the marker segregation is directly dependent of the rates of preferential pairing and double reduction for the considered marker . Using t(1) for each marker, parental heterozygosity restitution (PHR) was computed as the percentage of heterozygous diploid gametes. The estimation of PHR for each chromosome is the average of the values for all the markers on the chromosome.(2) the tetrasomic parameter (τ) which defines the proportion of gametes resulting from random meiotic chromosomal pairing . A value(2) the double reduction parameter (β) indicating the frequency of double reductions relative to the total frequency of random meiotic associations . For fix2 test, first considering all the gametes and then only homozygous gametes. The markers displaying an excess of the C. micrantha allele relative to the Mendelian segregation were identified by calculating the difference between the observed and the theoretical (0.5) frequencies of the C. micrantha allele.Segregation distortion was evaluated through a χC. micrantha/C. medica heterozygosity, C. micrantha or C. medica homozygosity were visually analyzed using GGT 2.0 software  were previously identified  and the method proposed by After competitive, allele-specific PCR (KASPar methodology), genotype calling was performed for the 189 CC. limon and C. micrantha respectively homozygous for A and M alleles). ‘Mexican’ and ‘Giant Key’ limes had a heterozygous profile with a fluorescent signal for the two alleles and were used as the reference for equivalent A and M doses. Heterozygous triploid hybrids with a value of theta (θ) angle (angle between the x axis and the line joining the origin and the samples considered) higher than the one for ‘Mexican’/’Giant key’ lime samples were considered to have two M allele doses (AMM) while the one with a lower θ value was considered to have only one M dose (AAM). Therefore, cluster analysis enabled the identification of triploid hybrids (and corresponding diploid gametes) having zero, one, or two doses of M and allowed us to directly infer the genotype of the diploid gamete .One example is provided for marker S03_46198875 Figure 2C. micrantha diagnostic markers, the 158 selected markers displayed balanced heterozygosity for ‘Mexican’ and ‘Giant Key’ limes (AM and AAMM respectively) and homozygosity for lemons (AA). Among the 272 genotyped triploid hybrids, three were discarded due to a rate of missing data higher than 10%. Thus, a total of 158 SNPs and 269 hybrids were used in this work.Twenty-two SNPs failed genotyping and were removed from the analysis. The remaining 167 markers were filtered to less than 10% of missing data, which resulted in the removal of nine SNPs. As expected for C. micrantha or C. medica homozygosity along a chromosome. Full C. micrantha homozygosity was observed only for chromosomes 5 and 7 (respective rates of 0.37 and 0.74%). Full C. medica homozygosity concerned chromosomes 1 (2.97%), 3 (1.49%), 6 (1.12%), and 7 (0.74%).The average of PHR over the whole genome was high (91.2%). The minimum values observed for markers and diploid gametes were 73.31 and 61.54%, respectively Figure 3C. micrantha allele doses in the diploid gamete that generated the ‘Tahiti’ lime  SNPs displayed an excess of the C. micrantha allele. All the markers on chromosomes 1, 6, and 9 on the one hand, and those on chromosomes 5 and 7 on the other hand were exclusively in excess of C. medica and C. micrantha alleles, respectively . One hundred and ten (69.62%) SNPs displayed an excess of the ectively Table 2.The disomic and tetrasomic inheritance behaviors of each chromosome were computed Table 3 -1. It ranged from 0.21 to 1.54 cM Mb-1 for chromosomes 8 and 7 respectively , 25 markers and 19 hybrids with redundant information were discarded using the R package {polymapR}  algorithectively Table 4 Syntheny with the reference Clementine genome  was high-1 in average and ranged from 2.56 to 4.12 cM Mb-1 for the analyzed genome segments of chromosome 9 and 7 respectively  and homozygous in lemons. Efficient evaluation of allele doses with the method described by Among the 189 SNPs diagnostic of The analysis of the ‘Giant Key’ lime diploid gametes data revealed high preferential pairing values, highlighting a predominant disomic inheritance, especially for chromosomes 2, 8, and 9. The other chromosomes displayed a preferential pairing trend, and chromosome 5 had the lowest value (0.633). Such variations among chromosomes have already been described, for example in sugarcane  and autoPoncirus trifoliata cv ‘Pomeroy’ with PP values ranging from 0.53 to 0.91   are part of the phylogenomic structure of the C. reticulata + C. limon somatic tetraploid hybrids  , betweenon (64%) , or for on (64%) . Rouiss on (64%) . The dou hybrids , the ‘Noticulata , and thezygosity . It is tGBS data .In addition to preferential chromosome pairing, PHR transmission from the doubled-diploid parent is also affected by the double reduction (DR) rate, which varies according to the crossover distance between the locus and the spindle fiber . It is hC. medica and C. micrantha chromosomes probably drive the preferential pairing and intermediary preferential disomic inheritance observed for the tetraploid ‘Giant Key’ lime. The difference in PP rates between the chromosomes may be explained by variations of the extent of differentiation between the different sets of chromosomes as proposed by de novo sequencing projects of citrus species will provide decisive elements to understand the preferential pairing tendency and its variation between chromosomes.Both sequence divergence and structural variations between -1) and those of the tetraploid clementine (3.21 cM Mb-1). The tetrasomic ‘Giant Key’ lime map spanned only 272.8 cM. The notable reduction in the recombination rate for the tetraploid lime compared to the tetraploid clementine is hypothesized to be a consequence of the ‘Giant Key’ inheritance behavior, which is close to disomic in three chromosomes  and intermediate, with a disomic tendency, in the remaining ones, while the tetraploid clementine showed a mainly tetrasomic tendency  rather displayed fragments of C. reticulata/C. maxima heterozygosity  showed 1.7 to 3.4 times more overall crossing overs between the homologous A chromosomes than diploid AA lines. Moreover, the authors observed a dramatic change in the crossing over pattern with homogeneous distribution along the genome in the triploid, while the diploid displayed a classical marked decrease in the extent of its pericentromeric areas. Chromosome interference was also strongly reduced in the allotriploids. In our case, the ‘Giant Key’ lime is a doubled-diploid of an F1 interspecific hybrid and therefore there is no intraspecific polymorphism between homologous chromosomes, and intraspecific recombinations are untraceable. Our conclusions on a strong reduction of the observed recombination compared with the reference clementine genetic map . The segregation distortion had very little effect on marker ordering . TherefoC. latifolia is a highly productive variety with researched organoleptic and pomologic qualities, such as specific aromatic profile, acidity/sugar ratio, seedlessness, fruit size, and moderate peel thickness. It is the Citrus variety most tolerant to the Huanglongbing disease (the most devastating citrus disease caused by the bacteria Candidatus Liberibacter sp.). However, it displays high content of furanocoumarin potentially harmful for human health, its production period is limited and the genetic basis of this horticultural group is very low. The main medium term objective of this study is to design a reconstruction-breeding scheme for the sterile triploid ‘Tahiti’ lime ideotype. Several studies have shown that an important part of the actual phenotypic diversity of edible citrus should be related to the differentiation between the ancestral taxa before reticulation and introgression processes and that the structures of the phenotypic and genetic diversities are closely correlated. Such correlations with genetic structure were observed for morphological and pomological characters  ovule and a diploid pollen of a ‘Mexican’-like lime (C. aurantiifolia). Research on citrus phylogenomic structures by C. micrantha x C. medica hybridization event  were observed for three chromosomes  with relatively high rates of tetrasomic inheritance . All these results are fully compatible with the ‘Tahiti’ lime originating from an interploid cross, as already highlighted by Concerning the ‘Giant Key’ lime, the present study revealed an average PHR of 91.2%, close to that detected for the doubled-diploid ‘Mexican’ lime . InteresC. micrantha/C. medica interspecific origin such as ‘Mexican’ lime, C. macrophylla, C. excelsa, or C. aurata  was found along the nine chromosomes and the observed interspecific recombinations between the constitutive C. medica and C. micrantha genomes were strongly limited in comparison with the reference clementine genetic map. It was nevertheless sufficient to construct a genetic map of a tetraploid lime that revealed for the first time high synteny and colinearity with the clementine reference genome. The phylogenetic structure of the diploid gametes generated by the tetraploid lime parent reinforces the hypothesized interploid hybridization origin of the ‘Tahiti’ lime. Such a mechanism simplifies the implementation of extensive breeding reconstructing programs in which the high parental heterozygosity restored to the diploid gametes would lead to the creation of hybrids phenotypically close to the ‘Tahiti’ ideotype.One hundred and fifty-eight SNP competitive allele-specific PCR markers, diagnostics of All datasets generated for this study are included in the article/PO and YF designed the experiment. YF and JE developed the triploid progeny. DA and FC selected and developed the diagnostic SNP markers. DA and PO analyzed the data and wrote the manuscript.This work received financial support from the European Regional Development Fund under the framework PO FEDER-FSE Corse 2014-2020 number 247SAEUFEDER1A, project called Innov’Agrumes . We thank also the Collectivité de Corse for the grant of DA (number ARR-15.036680.SR).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.


The evidence of the economic burden of road traffic injuries (RTIs) in Nepal is limited. The most recent study, conducted in 2008, is now considered outdated because there has been a rapid increase in vehicle numbers and extensive road building over the last decade. This study estimated the current economic costs of RTIs in Nepal, including the direct costs, productivity costs, and valuation of pain, grief, and suffering. An incidence-based cost-of-illness analysis was conducted from a societal perspective, employing a bottom-up approach using secondary data. All costs incurred by the patients, their family members, and costs to society were estimated, with sensitivity analyses to consider uncertainty around the data estimates available. Productivity loss was valued using the human capital approach. The total costs of RTIs in 2017 were estimated at USD 122.88 million. Of these, the costs of productivity loss were USD 91.57 million (74.52%) and the pain, grief, and suffering costs were USD 18.31 million (14.90%). The direct non-medical costs were USD 11.50 million (9.36%) whereas the direct medical costs were USD 1.50 million (1.22%). The economic costs of RTIs increased by threefold since 2007 and are equivalent to 1.52% of the gross national product, indicating the growing national financial burden associated with preventable RTIs. Road traffic injuries (RTIs) are a key injury burden worldwide. According to the Global Burden of Diseases (GBD) data for Nepal, RTIs are the leading cause of death among people aged 15 to 49 years [RTIs cause not only premature deaths and disabilities but also considerable costs to victims and their families ,3. ThereThe evidence of the economic burden of RTIs in Nepal is limited. A few global-level studies estimated the economic burden of RTIs for Nepal ,9. A stuThese national-level studies are now considered outdated because, since 2008, there has been a rapid increase in the use of motor vehicles and extensive road-building programmes that have shifted the ranking of RTIs from the eighth to the seventh leading cause of death in Nepal . The numA small number of local studies have attempted to measure the costs of RTIs ,16,17. AA cost-of-illness (COI) analysis was adopted to estimate the economic burden of RTIs in Nepal. The COI analysis identifies and measures all the costs resulting from a specific injury to society ,19 and iThe annual incidence of RTIs was obtained from Nepal Police records . AccordiThis study included direct medical and direct non-medical costs, productivity losses, as well as pain, grief, and suffering costs, as recommended by the guidelines for estimating the cost of road crashes in low- and middle-income countries (LMICs) ,36. The The average cost of two-way transportation was obtained from a household survey on a sample of RTI casualties conducted by the Nepal Injury Research Centre (NIRC) in 2018. The average food cost per day (USD 2.39) was based on a consensus of the researchers working in the NIRC. The unit cost of vehicle damage and administrative costs were obtained from a previous costing study of RTI in Nepal . Due to Any unit costs from other years were first converted into 2017 values in Nepalese Rupees (NRs) adjusting for inflation  using thDirect medical costs included the costs of inpatient services, outpatient services, diagnostic services, medication costs, and doctors’ fees. It was assumed that serious injuries would require inpatient services while both serious and minor injuries would require outpatient services. This is because a seriously injured patient may need outpatient visits following hospital discharge for follow-up and recovery. Serious RTIs also require diagnostic tests following a hospital admission and this study included X-rays, blood and urine investigations, CT scan, and ECG, similar to other studies on the cost of RTIs ,23,46. IDirect non-medical costs related to RTIs included transportation costs, caregivers’ food costs, vehicle damage costs, and administration costs. As the food cost of people seriously injured was included within the inpatient service cost, it was not counted here to avoid double counting. Indirect costs regarding productivity loss were estimated as income lost as a result of short-term absenteeism from work, long-term disability, and premature death due to RTIs. Productivity costs resulting from long-term disability and premature death were estimated using the human capital approach (HCA). The HCA estimates the present discounted value of future earnings of an individual assigning monetary value using the wage rate . The aveThe costs of pain, grief and suffering were calculated by assigning 20.0% of the total cost of productivity loss in line with the previous costing study from Nepal . This apCost per inpatient bed-day, using the cost of tertiary  hospitals in Nepal i.e., USD 5.34  ;Doctors’ fee per patient in private hospitals (USD 6.22) and government hospitals (USD 1.44) ;Administrative costs   because The discount rate of 10.0% for premature death and long-term disability in line with the previous costing study conducted in Nepal ;Average amount of working years lost due to premature death considering the age at retirement as used by other studies ,20,24. TOne-way sensitivity analyses were conducted to deal with the uncertainty surrounding the data in the estimations  for thesThe sum of the direct medical and non-medical, productivity loss and pain, grief and suffering costs resulted in a total economic burden of approximately USD 122.88 million in 2017 . The totThe direct medical costs of RTIs in 2017 were approximately USD 1.50 million, as shown in The direct medical cost was most sensitive to the cost of the doctors’ fee . ApplyinThis study estimated the total costs of RTIs in Nepal in 2017 as USD 122.88 million. The findings of this study show that the economic burden of RTIs in Nepal increased by threefold (without inflation) over the past decade from USD 40.65 million in 2007  and is nTotal direct costs of RTIs in Nepal were estimated at USD 13.0 million, representing 10.58% of the total costs of RTIs; equivalent to USD 1036.45 per injured person. This finding is lower than the costs incurred by motorbike crash victims and their caregivers found in a study from Kathmandu in 2015 (USD 1224 per injured person) . The aveThe total indirect costs of RTIs  were estimated at USD 109.88 million, representing 89.42% of the total costs. The higher indirect costs compared to direct costs were similar to the study conducted in Iran  and threThe total economic costs of RTIs were approximately USD 122.88 million, which amounts to an estimated USD 4.45 for each of the 27.63 million people living in Nepal, and equivalent to 1.52% of the total Nepal GNP for 2017 (USD 8.06 billion) . This fiThe Nepal Road Safety Action Plan (which has not been promulgated through parliament) recommends a mandatory 10% of the total budget of road construction to be allocated for road safety interventions . In the The costs reported in this study are likely to be a conservative estimate of the economic burden due to some limitations. The data were obtained from different sources, and there is the possibility of each source having some reporting bias. For instance, the study used the road crashes data from the Nepal Police record that is known to be incomplete due to traffic crashes that are not reported to the police ,34,57. GNepal police officers at the scene of a crash make a judgement on the severity of an injury  for recording in the official RTI statistics. There are no formal definitions of serious or minor injury, and therefore the police officers make this decision based on personal opinion. This risks misclassification of injury severity and will lead to incomplete reporting of fatal injuries, since some patients with minor or severe injuries may later die from complications arising from the injury. In Nepal, 2384 deaths were recorded by the Nepal Police in 2017  equivaleThis study included multiple costs in the cost estimates, but some additional costs were not included or not available for the analysis. For example, the costs of emergency services, surgical services, prosthetic devices, physiotherapy, rehabilitation, follow-up visits, and the costs associated with readmission following an RTI were excluded in the analysis. In Nepal, there is currently a poorly developed emergency medical service, so national data are not available. A further limitation involved the use of the HCA to value productivity loss which has been criticised for overestimating the value of productivity loss as it assigns more value to premature deaths . Other aThese results will be of value in studies modelling the impact of RTIs in Nepal or when estimating the cost-benefit of interventions to prevent RTIs in Nepal. Future research could generate more valid and nationally representative estimates. Specifically, the collection of primary data on costs experienced by patients suffering RTIs would strengthen the data included in the analyses. In addition, a household survey with the RTI victims and their family members could estimate costs associated with minor RTIs not requiring hospital admission, providing data not available in this study.This study provides an up to date estimate of the economic burden of road crashes in Nepal that could be used to advocate for improved road safety in Nepal and be available for future cost-effectiveness studies of interventions to prevent RTIs in Nepal. The high economic burden of RTIs suggests a need for investment in evidence-based, cost-effective multi-sectoral interventions such as the enforcement of speed limits, enforcement of helmet use by motorcycle riders and pillion passengers, seatbelt usage by drivers ,64, and In conclusion, the study sought to include direct medical, direct non-medical, and productivity loss costs, as well as a valuation of pain, grief, and suffering due to RTIs in Nepal. This study estimated the total costs of RTIs in Nepal at USD 122.88 million in 2017. The total direct medical costs were estimated at USD 1.50 million and direct non-medical costs at USD 11.50 million. Similarly, the total costs of productivity loss were estimated at USD 91.57 million, and the cost of pain, grief, and suffering associated with RTIs was estimated to be USD 18.31 million. The economic costs of RTIs increased by threefold since 2007 and is equivalent to 1.52% of the 2017 GNP, indicating the growing national financial burden associated with preventable road injuries and deaths.


To test the hypotheses that emerging viruses are associated with neurological hospitalizations and that statistical models can be used to predict neurological sequelae from viral infections.GBS) in the state of Rio de Janeiro from 1997 to 2017. Increases in GBS from month to month were assessed using a Farrington test. In addition, a cross‐sectional study was conducted analyzing 50 adults hospitalized for inflammatory polyneuropathies from 2015 to 2017. The extent to which Zika virus symptoms explained GBS hospitalizations was evaluated using a calibration test.An ecological study was carried out to observe time trends in the number of hospitalizations with inflammatory polyneuropathy and Guillain‐Barré syndrome (P<0.001) in the incidence of GBS following the introduction of influenza A/H1N1 in 2009, dengue virus type 4 in 2013, and Zika virus in 2015. Of 50 patients hospitalized, 14 (28.0%) were diagnosed with arboviruses, 9 (18.0%) with other viruses, and the remainder with other causes of such neuropathies. Statistical models based on cases of emerging viruses accurately predicted neurological sequelae, such as GBS.There were significant increases (Farrington test, The introduction of novel viruses increases the incidence of inflammatory neuropathies. The introduction of novel viruses increases the incidence of inflammatory neuropathies in Brazil. GBS is characterized by ascendant, bilateral muscle weakness with depressed tendon reflexes.We compared a historical time series of inflammatory polyneuropathies, which included GBS, to time series of influenza, dengue virus, and Zika virus in the state of Rio de Janeiro, Brazil. Further, we analyzed medical charts of arbovirus infections that resulted in hospitalization with neurological disease. Our objectives were to explore whether epidemics of emerging viruses are associated with neurological hospitalizations in Brazil and whether models could be used to predict neurological sequelae from viral infections.2We carried out an ecological study in which we observed time trends in the number of hospitalizations with inflammatory polyneuropathy ICD‐10: G61) and Guillain‐Barré syndrome (G61.1) in the state of Rio de Janeiro reported to Brazil's hospitalization database  from 1997 to 2017 . We transcribed the physicians’ notes about clinical signs and symptoms reported by the patient. Based on this information, we used the classification system proposed by Martyn and Hughes3P<0.001): in 2009 (n=115 cases) contemporaneous with the introduction of H1N1From 1997 to 2017, 1593 cases of GBS were reported in the state of Rio de Janeiro . Analysis of monthly GBS cases determined that there were significant increases in GBS from 1 month to the next for 3 years . As noted above, due to the difficulty of distinguishing Zika virus from dengue virus, we also pooled the data into the category arboviruses. The pooled data were not significant for explaining GBS cases .There was seasonal variation in GBS incidence with 15% fewer cases during winter. Zika virus cases were a significant variable for explaining cases of GBS  of the 97 admissions for inflammatory polyneuropathies. These 80 admissions corresponded to 50 patient charts because some patients were admitted repeatedly. The most frequent causes of inflammatory polyneuropathy were arboviruses n=14, 28.0%), followed by other viruses (18.0%)  patient; one patient had chikungunya virus confirmed by serology and one patient was positive for dengue virus by laboratory exams. The other 11 (78.6%) patients were diagnosed with arbovirus infection based on clinical symptoms. Of the 14 patients, 8 (57.1%) presented with cutaneous rash, 7 (50.0%) with fever, and 3 21.4%) conjunctivitis . Adding dengue virus to the model failed to improve goodness of fit because the epidemics that occurred in the early 2000s were not accompanied by an increase in GBS, and the incidence of dengue virus was low in 2015–2016.Flavivirus species is complicated because commercial tests for dengue virus cross‐react to Zika virus antibodies and vice versa.Chart review indicated that suspected Zika virus cases were seldom confirmed by laboratory exams. To this extent, we can only report that patients had some type of arbovirus, but cannot determine whether it was chikungunya, dengue, or Zika virus. Differential diagnosis of The present study has several implications for planning the treatment of neurological complications of arboviruses. Five (55.6%) of the nine patients treated with intravenous immunoglobulin were transferred to a different hospital. On the other hand, none of the patients admitted to a neurology reference hospital were transferred. It appears that only reference hospitals had access to intravenous immunoglobulin, whereas the treatment was not available in other public hospitals. To fill this gap, there is a need for public health decision makers to assure the supply of intravenous immunoglobulin in the public health institutions network. This would likely reduce the need for transfers, which worsen GBS patient outcomes and are costly.A limitation of this study is the diagnosis of suspected arbovirus infections. Diagnosis was primarily based on clinical signs and symptoms, which could not be confirmed by laboratory diagnosis in 78.6% of cases. Further, as this study was hospital based, we would not have detected inflammatory polyneuropathies that did not result in hospitalization. In addition, since the analysis was limited to the state of Rio de Janeiro, which had a high incidence of Zika virus, it is unknown whether our conclusions apply to other regions where outbreaks were smaller.Predictive modeling is useful for forecasting future observations, explaining processes, and testing theories.JRA and TLF contributed to study design, data analysis, and manuscript writing. BBSL, OCP, and PCS contributed to study design. HLFP and RDM performed field work and chart review. JMCF and CCBP contributed significantly to the patient database and chart review. KNS contributed to manuscript writing. OJMN: contributed significantly to the patient database and manuscript writing.The authors have no conflicts of interest.Figure S1. Zika virus incidence in the state of Rio de Janeiro by health district.Figure S2. Clinical cases of dengue virus in the state of Rio de Janeiro, 2001–2017 according to SINAN.Table S1. Notation for the hhh4 time series model to predict the number of cases of GBS per health district in each month.Table S2. Formulation of the hhh4 model. αν, βν, γ, δ, αλ, βλ, ϕit e ψi were estimated from the data using the library SURVEILLANCE in R.Table S3. hhh4 model based on Zika virus cases in the state of Rio de Janeiro, 1997–2017. Values in bold indicate P<0.05.Table S4. hhh4 model based on Zika + dengue virus in the state of Rio de Janeiro, 1997–2017. Values in bold indicate P<0.05.Click here for additional data file.


We detected 3 lyssaviruses in insectivorous bats sampled in South Africa during 2003–2018. We used phylogenetic analysis to identify Duvenhage lyssavirus and a potentially new lyssavirus, provisionally named Matlo bat lyssavirus, that is related to West Caucasian bat virus. These new detections highlight that much about lyssaviruses remains unknown.  Lyssavirus genus, which belongs to the family Rhabdoviridae. Researchers have described 17 lyssavirus species, and a putative species is awaiting formal classification ; Lagos bat virus, which is associated with various species of frugivorous bats; Mokola virus, for which the reservoir host is unknown; Shimoni bat virus, which is associated with the striped leaf-nosed bat (Macronycteris vittatus); and Ikoma lyssavirus, for which the reservoir host is unknown , which is associated with insectivorous bats, specifically the Egyptian slit-faced bat , exsanguinated them by cardiac puncture, and then performed full necropsies. We identified bats on the basis of morphologic ; these sequences were distantly related to West Caucasian bat virus (WCBV). We collected these samples from Matlapitsi cave, Limpopo, in 2015 and Madimatle cave, Limpopo, in 2016 . These 2 novel sequences had a shared nucleotide identity of 99.2%. Compared with other lyssaviruses, they shared the highest nucleotide identity with WCBV (80.9%–81%) and the lowest with Ikoma lyssavirus (70.5%). The DUVV sequence was 91.6%–99.3%, similar to previously described DUVV sequences  from the Russian Caucasus in 2002 (Miniopterus bats in Kenya spurred speculation that WCBV, or a closely related virus, was circulating in Africa (>23 species in Africa, does not host known lyssaviruses in phylogroup I or II (We detected a novel lyssavirus, MBLV, that belongs to phylogroup III and is most closely related to a sequence of WCBV that was isolated from a common long-fingered bat (Models have demonstrated that WCBV can cause fatal encephalitis in animals; commercial human and veterinary vaccines do not offer significant protection (In summary, the mechanisms of lyssavirus maintenance in bats is still unknown and could be influenced by various environmental and ecologic factors (Additional information on lyssaviruses in insectivorous bats, South Africa, 2003–2018.