After assembly in the cytosol, some Vaccinia virus particles go through a complex process that leads to virus egress and eventually cell-to-cell transmission. Intracellular particles are fully infectious, and therefore virus mutants lacking essential functions in the exit pathway are unable to form plaques but can multiply intracellularly. We isolated virus mutants in which two of the genes required for virus spread (F13L and A27L) were deleted independently or concurrently. The phenotypes of the mutant viruses were consistent with the need of A27L and F13L for intercellular virus transmission, the effect of the ΔA27L mutation being more severe than that of ΔF13L. Despite their defect in spread, ΔA27L mutant viruses could be expanded by infecting cell cultures at high multiplicity of infection, followed by the release of virions from infected cells by physical means. We developed a novel system for the isolation of recombinant Vaccinia virus in which selection is efficiently achieved by recovering plaque formation capacity after re-introduction of A27L into a ΔA27L virus. This system allowed the insertion of foreign DNA into the viral genome without the use of additional genetic markers. Furthermore, starting with a double mutant (ΔA27L-ΔF13L) virus, A27L selection was used in conjunction with F13L selection to mediate simultaneous dual insertions in the viral genome. This selection system facilitates combined expression of multiple foreign proteins from a single recombinant virus. Therefore, improvements of the methods to modify the viral genome are of interest.Vaccinia virus (VV), the best characterized member of the Poxviridae family, has been extensively used as an expression vector. Additionally, there is intense interest for its use as the backbone for recombinant vaccines, and as an oncolytic agent. Genetic modifications are usually introduced in the viral genome before use, with the exception of its original application as the smallpox vaccine and early oncolytic trials. In addition, some applications require the construction of multiple modifications of the viral genome or co-expression of several proteins6, the standard technique to obtain recombinant Poxviruses relies on homologous recombination between transfected plasmids and the viral genome replicating in the cytoplasm. This infection/transfection procedure generates only a small fraction of recombinant virus in the progeny population. In this regard, selective markers are commonly used to remove parental virus from the mixture to facilitate the isolation of recombinant virus7. One of the selection systems for VV is based on plaque size8, and it involves the use of the F13L gene as a selective genetic marker. The F13L gene is required for virus envelopment and therefore F13L-deficiency leads to very low virus propagation, resulting in a tiny-plaque phenotype10. Thus, re-introduction of the F13L gene in a F13L-deleted virus, with the concomitant plaque recovery, can be used as a genetic selection method.Since the introduction of the first methods to insert genes in the VV genome11, membrane fusion13, intracellular virus transport14 and the formation of enveloped virions15. Given the critical role for A27L in these processes, it is not surprising that inactivation or certain modifications of the A27L gene result in viruses with severely diminished plaque sizes17.The VV A27L gene encodes a 14 kDa protein that participates in virus attachmentTo expand the applications of the plaque-based VV selection system, here we explored the use of A27L as a selectable genetic marker, and developed a virus/plasmid pair to mediate the insertion of foreign DNA into the VV genome. We demonstrate that the ensuing system is reliable and highly efficient for the isolation of recombinant VV expressing foreign proteins. Furthermore, we show that A27L selection can be used in conjunction with the F13L system to simultaneously drive dual gene insertion into the viral genome.18. To allow direct monitoring of the deletion process, the TagGFP2 and TagBFP genes (which are derived from distinct fluorescent proteins and do not share significant homology) were placed at both sides of the A27L gene. A synthetic promoter was only included upstream of the GFP gene, in such a way that recombination between the LoxP sites can be detected as a switch from green to blue fluorescence  would be cumbersome. Therefore, to facilitate this procedure, we devised a strategy , thereby indicating that the codon changes introduced into A27L did not disturb A27L function to a significant extent. In a second step, A27L gene deletion was accomplished by repeatedly passaging vLg-A27-LbL′ in a cell line expressing Cre recombinase under the control of a Tet-inducible promoter. The emergence of BFPopy Fig. . The inc19.Using a similar procedure, a double F13L-A27L deletion mutant virus (v-ΔA27-ΔF13) was isolated, starting from virus mutant vRB12, which contains a deletion of the F13L gene16, the virus plaques produced by v-ΔA27 were tiny, even after prolonged incubation times. Indeed, they were slightly smaller than those of v-ΔF13L . Analysis of the progeny virus obtained from the infection/transfection experiment indicated that recombination products were present at a frequency of about 10−2, and that the single cross-over recombinant viruses accounted for 84% of the large viral plaques. Plaque isolation allowed us to isolate the final, stable, double cross-over recombinant virus using established procedures20. The high frequency of recombinant virus may indicate a degree of enrichment in the transfected culture, due to the deficiency of the parental virus in spread.The defective phenotype of v-ΔA27 suggested that restoration of the A27L gene might serve as the basis for an efficient genetic selection system. To test this notion, we designed a plasmid to mediate the insertion of foreign DNA downstream of the A27L gene Fig. . The traIsolation of recombinant virus was also feasible after transfection of linear PCR products that included the recombination flanks. However, transfection of linear molecules resulted in a significant decrease in the recombinant virus titre with respect to the circular plasmid. Both with plasmids and with linear DNA, A27 selection outperformed the previously described F13 selection system Fig. .Overall, the A27 cloning system demonstrated high efficiency, required no selective media or special cell lines, and allowed rapid enrichment of recombinant virus. Furthermore, it offered several advantages over similar current selection methods, including the small size of the A27L gene, the higher rate of recombinant virus obtained, and the smaller plaque phenotype of the parental virus. In addition to these benefits, direct monitoring of the double/single cross-over nature of the viruses forming large plaques was straightforward due to the presence of the BFP gene in the parental virus.21. However, as the plasmids pA.S-Scarlet and pRB-TagGFP2 are both derived from pGEM plasmid, they share significant backbone homology, which may result in unwanted recombination events. Taking this into account, we performed parallel transfections in which linear PCR fragments lacking the plasmid vector sequences were used instead of complete plasmids. After transfection of plasmids, viruses forming large-plaques were detected in the viral progeny, indicating successful insertion of the A27L and F13L genes. Large plaques displayed a diversity of colours under the fluorescence microscope , and followed the progression of fluorescent plaques by microscopy Fig. . As expe−4). Most large plaques displayed fluorescence, thereby hindering the direct isolation of the non-fluorescent double recombinant. From this mixed population, the double recombinant could be isolated by serial plaque isolation starting from a fluorescent single recombinant. As an alternative, we carried out serial passages of the initial virus population to enrich for A27L+ F13L+ viruses. We then determined the amount of different virus recombinants after each infection round  prevents the occurrence of large plaques as the result of spontaneous reversions. Although based on similar concepts, the A27L system offers several advantages over the classical F13L version. First, the A27L gene is smaller than F13L, and thus the plasmids for insertion in the A27L locus are easier to manipulate. Also, the efficiency of the A27L system was higher see Fig. , which mThe A27L/F13L system allows for straightforward insertion of two DNA segments in distant genomic loci. Notably, the only practical difference between the respective A27L and F13L selection protocols is the starting virus, since the same selection principle (i.e. plaque formation/virus cell-to-cell virus spread) is applied for the isolation of the double recombinant virus.VV has considerable insertion capacity, thereby allowing the simultaneous expression of multiple genes. The availability of multiple insertion sites will facilitate the construction of complex recombinant viruses for various applications.BSC-1 cells (ATCC CCL-26) were grown in Eagle’s Minimal Essential Medium (EMEM) supplemented with 0.1 mg/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine (Lonza) and 5% foetal bovine serum (FBS).Flp-In T-Rex 293 cells (Invitrogen) were maintained in DMEM supplemented with 0.1 mg/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine (BioWhittaker), 7% foetal bovine serum (FBS), 300 µg/ml Zeocin and 10 µg/ml blasticidin.The 293-Cre cell line, inducibly expressing Cre recombinase, was derived from Flp-In-293 cells by Flp recombinase-mediated insertion of plasmid pcDNA-FRT-Cre using the Flp-In T-Rex system (Invitrogen) following manufacturer’s instructions. 293-Cre cells were grown in DMEM supplemented with 7% foetal bovine serum (FBS) containing 0.1 mg/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine (BioWhittaker), 10 µg/ml blasticidin and 100 µg/ml hygromycin. Routinely, for induction of Cre expression, cells were incubated with medium containing 1 µg/ml tetracycline for 18–24 h before infection. After virus adsorption, incubation was continued in medium containing 2% FBS and 1 µg/ml tetracycline.23 flanked by LoxP and Lox 2272 sites between the XhoI and HindIII sites of pLg-A27-L.A segment containing an A27L gene modified by synonymous substitutions and flanked by LoxP (ATAACTTCGTATA GCATACAT TATACGAAGTTAT) and Lox2272 (ATAACTTCGTATA GGATACTT TATACGAAGTTAT) sequences, in addition to ∼400 bp A27L recombination flanks, was synthesized by GenScript Corp and inserted into plasmid pUC57-simple, to generate pL-A27-L. A cassette comprising a synthetic early/late promoter, a LoxP sequence and the gene TagGFP2 sequence was obtained by digestion of pRB-E/L-LgL  with XhoI and BamHI. This cassette was then inserted between SalI/BglII sites of pL-A27-L to generate pLg-A27-L. Finally, plasmid pLg-A27-LbL′ was obtained by inserting the TagBFP gene21 obtained from plasmid pmScarlet-i_NES_C1, a gift from Dorus Gadella  by PCR amplification with oligonucleotides Scarlet5′  and Scarlet3′ . The PCR fragment containing the Scarlet coding sequence was digested with EcoRI and HindIII and inserted between the corresponding sites of pRB21, thus placing the mScarlet-I gene downstream of the synthetic early/late promoter in pRB218. The resulting plasmid, pRB-mScarlet-i-NES, is referred to herein as pRB-Scarlet.pRB-Scarlet was obtained by insertion of the gene coding for mScarlet25, was obtained by synthesis of a DNA fragment and subsequent cloning in plasmid pUC57 (done by Genscript Corp). Subsequently, pA-LE-Scarlet-NES was obtained by insertion of the gene coding for mScarlet-i from pRB-Scarlet into the EcoRI/BamHI sites of pA-LE-R_R . Then, plasmid pA.S-TagGFP2 was obtained by substituting the promoter-Scarlet gene cassette by the synthetic E/L promoter-TagGFP2 gene cassette derived from pRB-TagGFP2 (Supplementary Info). Finally, plasmid pA.S was obtained by inserting the synthetic E/L promoter-multiple cloning site region from pRB21 between the XhoI and BamHI sites in pA.S-TagGFP2.A plasmid was designed to mediate the reinsertion of the A27L gene, together with foreign sequences, into the A27L locus. This plasmid (pA-LE-R_R), containing A27L recombination flanks (566 and 551 bp), the A27L gene and an early/late promoter26 was synthesized by GenScript Corp and inserted between the EcoRI and HindIII sites in pRB21 plasmid8.The sequence for nanoLuc27 using the same sites to generate pTIH-Luc. Finally, pA.S-Luc was generated by inserting the luciferase coding sequence, obtained by EcoRI / BamHI digestion of pTIH-Luc, and insertion between the corresponding sites in pA.S plasmid.The Firefly luciferase coding sequence was obtained from plasmid pTM1-Luc (unpublished) by NcoI and XhoI digestion and was cloned in the pTIH plasmid28  and inserted into pRB21 to generate pRB-Cre. Subsequently, a DNA segment containing the Cre gene was amplified by PCR with oligonucleotides Cre5′ HindIII Fw (5′-CCCAAGCTTACCATGTCCAACCTGCTGACTGTGCAC-3′) and HF3400 (5′ CGTTCTAAAGCTAGTGCTATATCTCCC-3′ corresponding to a sequence in the F13L right recombination flank of pRB21). This PCR fragment was digested with HindIII and inserted into the unique HindIII site of plasmid pcDNA5/FRT/TO (Invitrogen). The final plasmid construct was used to generate a 293T-derived cell line inducibly expressing Cre, following the instructions of the Flp-In T-Rex system (Invitrogen). The resulting inducible cell line was termed 293-Cre.A codon-optimized version of the Cre gene was derived from plasmid pDIRE29 as follows: a PCR product corresponding to the F13L left recombination flank was amplified from VV DNA using oligonucleotides VVLL  and VVRR . This PCR product was digested with SphI and XhoI and inserted between the corresponding sites in pRB-rsGFP to generate plasmid prsGFP. Since the SphI-XhoI fragment removed spans the left flank and the F13L gene, insertion of the left F13L flank resulted in the net removal of the F13L gene.The F13L gene was removed from plasmid pRB-rsGFPBSC-1 cells in 6-well plates were infected with VV strain WR at a MOI of 0.05 plaque forming units (pfu) per cell and subsequently transfected with 2 µg of prsGFP complexed with Fugene HD (Promega). Infection was allowed to proceed for 3 days, and the progeny virus was plated in BSC-1 cells. Large GFP(+) plaques (simple recombinants) were isolated twice by plaque picking. In an ensuing plaque assay, several tiny, green fluorescent plaques were isolated and expanded in BSC-1 monolayers in 24-well plates. A culture well devoid of large plaques was selected for further amplification. Finally, the virus -termed v-∆F13g was characterized by western blot using antibody against F13 and by positive rescue of the normal plaque phenotype following re-insertion of the F13L gene.30 or vΔF13g at a MOI of 1 pfu/cell and transfected with plasmid pLg-A27-LbL′ using FuGeneHD (Promega). After 2 days, cells were disrupted by three cycles of freeze-thawing, and the released virus was used to infect fresh BSC-1 cell monolayers. Virus plaques showing green fluorescence were selected and isolated by plaque picking. After five consecutive rounds of plaque isolation, virus was expanded and titrated. Viruses obtained by insertion of this construct in WR, vRB12 and vΔF13g were termed vLg-A27-LbL′, v∆F13-Lg-A27-LbL′ and v-L-A27-LbL′-∆F13g respectively, and contained an A27L gene flanked by LoxP recombination sites. To promote deletion of A27L by Cre-mediated recombination, these viruses were passaged in 293-Cre cells pre-incubated for 18 h with medium containing tetracycline. For serial passaging, viruses in cultures were recovered after 24 h of infection and used as the inoculum for the following passage. Finally, to eliminate any residual A27L(+) virus, BSC-1 cells were infected with serial dilutions of the last passage. After 72 h the wells were observed by fluorescence microscopy, and the mutated A27L-deficient virus was recovered from a dilution that showed blue fluorescence but was devoid of GFP fluorescence. The final virus stocks were amplified from these infected cultures and termed v-∆A27, v-∆A27-∆F13 or v-∆A27-∆F13g when derived from VV WR, vRB12 or vΔF13g, respectively.BSC-1 cells in 6-well plates were infected with virus WR, vRB1220 using circular (plasmids) or linear (PCR-generated) molecules. Linear DNA fragments including the desired recombination flanks were PCR-amplified from plasmids containing flanks for the A27L or F13L genomic loci. For amplification of A27L constructs, PCR was performed with oligonucleotides pALE FI Fw (5′-TCCGCGCACATTTCCCCGAAAAGTG-3′) and pALE FD Rv (5′-GCCAAGCTCGCTTGCACGCCTCTGC-3′). For amplification of F13L constructs, PCR was performed with oligonucleotides pRB FI Fw (5′-GACTCACTATAGGGCGAATTGGGCC-3′) and pRB FD Rv (5′-AGGTGACACTATAGAATACTCAAGC-3′).Virus recombinants were isolated using standard protocols7 was followed. Briefly, BSC-1 cells in 6-well plates were infected at an MOI of 0.05 pfu/cell with a VV mutant deficient in plaque formation (viruses deleted in A27L or F13L). After a 1-h adsorption period, cells were transfected with DNA (2 µg of plasmid or 1 µg of PCR fragment) using FuGeneHD, following the manufacturer’s instructions. After 2–3 days, cells were harvested and recombinant viruses were isolated by at least three consecutive rounds of plaque purification. Alternatively, where indicated, serial passages were carried out in BSC-1 monolayers in T75 culture flasks.To generate recombinant virus, the previously described protocol for F13L selectionCells grown on 6-well plates were infected for 48 h with viruses expressing a fluorescent protein . After this time, cells were washed twice with PBS and fixed by the addition of ice-cold 4% paraformaldehyde for 12 min. All subsequent incubations were carried out at room temperature. Cells were permeabilised by a 15-min incubation in PBS containing 0.1% Triton X-100 and then treated with PBS containing 0.1 M glycine for 5 min. They were then incubated with primary antibodies diluted in PBS-20% FCS for 30 min, followed by incubation with secondary antibodies diluted 1:400 in PBS-20% FCS. The following antibodies were used: rat monoclonal antibody anti-B5 diluted 1:100; and anti-rat IgG-Alexa Fluor 594 (Invitrogen). Finally, cells were washed extensively with PBS, mounted with FluorSave reagent , and observed by fluorescence microscopy.Supplementary Information


The Zika virus (ZIKV) was overlooked as a potential threat for more than half a century. It was discovered first in a Rhesus monkey in 1947 in the Zika Forest of Uganda, hence its name, but not until its emergence in 2007 did the flavivirus grab the attention of researchers . HoweverAedes aegypti, also can be transmitted through sexual contact as well as through blood transfusions [Pathogenicity on the population level is being analyzed to understand, prevent, and treat this emerging disease. By October 2016, ZIKV outbreaks were being reported in more than 60 countries, with 1 million cases alone in Brazil . These fsfusions , 4. The sfusions .Ways to combat the outbreak include practical considerations. One of the main emphases needs to be low-cost and rapid field detection, especially in low-resource settings where assays such as real-time reverse transcription polymerase chain reaction (RT-PCR) require specialized laboratories and skilled professionals . RT-PCRsIsothermic nucleic acid amplification tests (NAATs) show promise, proving successful even with low loads of ZIKV. This is important as ZIKV RNA is frequently not detectable in the blood until several days after symptom manifestation, with one recent study finding the mean time for viremia detection to be 10.4 days, with a range from 0 to 53 days . A new aWith the currently recommended RT-PCR for detecting ZIKV being limited and despite new assays and detection methods being discovered, biosensors and nanotechnology may become the new standard. Biosensors are selective for a specific analyte, even in a sample containing contaminants . This coNanotechnology may already be providing some insights into the cross-reaction of antibodies in flaviviruses. One study utilized the antibody to antigen relationship, focusing specifically on an anti-Zika IgG monoclonal antibody (Z-ab) by using functionalized nanoparticles (MRNPs) with Z-ab . The stuOn the molecular level, understanding the virus—including structure, replication, and transmission—is essential. New research points to the binding and entry stage as vital to ZIKV propagation . In the Vaccine development currently underway focuses on neutralizing antibodies, utilizing nucleic acid vaccines, inactivated virions, and live-attenuated ZIKV in order to stimulate neutralizing antibodies against proteins present on the surface of the virion . Some ofWhile there is no current vaccine or treatment, researchers are making advancements in understanding the virus by studying transmission and molecular structure of the virus to impede the progression of this global health threat. With numbers continuously changing as to how many are infected and new research data covering varied aspects of molecular structure, diagnostic tools, binding properties, and more, it is hard to stay abreast of current research. The unchanging factor is that resources and research are needed to learn about the virus and how to control the spread and effects of this disease.In the meantime, the top priority in prevention needs to be protection and/or testing for sexually active women of child-bearing age that could become or are infected. Testing for the virus could also prove beneficial for women living in endemic or infected areas that are considering having children. Men and women both should be aware of possible infection, however, as more evidence emerges that ZIKV is able to be transmitted sexually. Precautions when traveling or if living in an infected region are important, with mosquito repellent being a basic but effective means of prevention. The main precaution is limiting mosquito bites by means of pesticides or exposure, with public awareness in infected regions essential. Taking measures to either avoid times outdoors when mosquitoes are active, such as dusk, and/or wear protective clothing or repellent is prudent. Using screens for open windows and doors is also a practical prevention policy.


Mirtazapine has recently emerged as a promising agent for the treatment of progressive multifocal leukoencephalopathy (PML). While there is no Class I evidence for its use, numerous case reports have illustrated mirtazapine’s efficacy. True to its name, PML is known to occur mostly in the white matter of the brain as its causative agent, John Cunningham virus (JC virus), has a predilection for infecting glial cells. The virus replicates vigorously in oligodendrocytes and causes lysis of the glial cell culminating in demyelination. Therefore, gray matter involvement is rare. Mirtazapine’s 5HT2A receptor blocking capacity is presumed to hinder JC virus’ entry into glial cells. We report a case of a patient with human immunodeficiency virus (HIV) with predominantly gray matter lesions from JC virus reactivation. This case is the first reported case of gray matter PML in an Arabic patient who responded favorably to mirtazapine therapy. John Cunningham virus (JC virus) is notorious for being highly lethal. Non-archetype JC virus is harmful to brain cells. Archetypal JC virus is ubiquitous in distribution; its worldwide seroprevalence is approximately 80% . JC viruThe virulence of JC virus is established when extensive deletions and duplications in the nucleotide sequences within the promoter/enhancer region of the genome occur . The resNevertheless, wherever it affects in the brain, JC virus leads to disastrous consequences. In the era before highly active antiretroviral therapy, the prognosis for patients with progressive multifocal leukoencephalopathy (PML) was grave; death occurred in approximately 95% of patients within four to six months of diagnosis . Those wA 37-year-old Yemeni male patient was brought to the hospital by his coworker after the patient had been disoriented for several days. A report from another medical center indicated the patient was diagnosed with tuberculosis (TB). According to this report, he had presented to the other facility one month previous with fever, productive cough, and disorientation. He was advised to start anti-TB therapy (ATT). However, the lack of collateral history made it impossible to confirm whether he had been receiving ATT. At presentation to our center, he was drowsy but arousable to strong verbal commands. He uttered a few incomprehensible sounds, and he obeyed spoken commands only occasionally. He appeared to have mild weakness in his left arm. His cranial nerve examination showed no abnormalities. The results of his systemic examination were unremarkable: we noted no skin rash, indications of intravenous drug use, evidence of peripheral stigmata of infective endocarditis, or lymphadenopathy. On subsequent days, he sustained several attacks of generalized tonic-clonic seizures. Given that he was febrile with evidence of meningismus, he underwent lumbar tap.InvestigationsHis cerebrospinal fluid (CSF) was light yellowish with 373 cells (73% monomorphic) with glucose at 2.4 g/dL, protein at 620 g/dL. The CSF was positive for JC virus DNA with a viral load of 2800 copies/mL. His serology for human immunodeficiency virus (HIV) was positive with a CD4 count of 135 /µL and an HIV viral load of 179,795 copies/mL. Test results for hepatitis B virus and sputum acid-fast bacilli were negative. CSF polymerase chain reaction results for TB were negative, so was the culture for TB in the CSF sample. However, the report from the peripheral center had shown positive TB culture in CSF. Computed tomography (CT) of the patient’s brain revealed left basal ganglia hypodensity and left periventricular white matter hypodensity. Magnetic resonance imaging (MRI) showed bilaterally scattered patchy confluent subcortical and periventricular foci confirmed on T2-weighted images (T2WI) and fluid-attenuated inversion recovery (FLAIR) images  along with the ATT. Given the reported cases of successful treatment with an anti-5HT2A antagonist, we treated the patient with mirtazapine along with standard cART and ATT.OutcomeThe patient remained clinically stable with no apparent worsening of his neurological deficit. He became more alert and could reply to one or two questions coherently. He sustained no further seizures. He developed rigidity in all limbs and bradyphrenia. He also became hypophonic and apathetic. A second MRI after six weeks of therapy revealed remarkable improvement Figure . After sPML is a demyelinating disease caused by JC virus, a polyomavirus that affects CNS white matter. This disease was first noted by Astrom et al. in 1958 based on the characteristic histopathological features of demyelination, bizarre astrocytes, and enlarged oligodendroglia nuclei . JC viruThere is currently no specific treatment available for this catastrophic illness. However, the pragmatic approach targets the basic mechanism of action of the virus. This includes immune response modulator agents that inhibit JC virus entry into the glial cells and inhibit JC virus DNA replication . CombineOur case presents a favorable response to mirtazapine in an HIV-related, predominantly gray matter affected PML. Nevertheless, as this case had quite a few atypical features without a histopathological confirmation of PML therefore, we can not completely rule out having other less likely differentials for the lesions seen in MRI brain and for the rapid response to combination treatment . In other words, it may not be incorrect to presume that there was concomitant CNS TB and restoration of ATT use may have influenced the quick resolution of the brain lesions. However, as CSF TB culture was negative at our center, we were hesitant to make the diagnosis of TB. Instead, in our patient, JC virus DNA was detected by PCR in the CSF, the presence of which is generally considered highly specific for the diagnosis of PML. Therefore, we feel it was most likely a case of predominantly gray matter involved PML in an HIV positive patient.This case represents the first report of PML in the gray matter of an Arab patient with HIV who responded well to mirtazapine treatment in conjuction with cART.


Rhopalosiphum maidis and R. padi. We found that pre-inhabitation by R. padi on plants facilitates the subsequent foraging of conspecifics and R. maidis. Without the virus, the occurrence of facilitation is asymmetric because it depends on the order of species arrival. However, with virus we found facilitation irrespective of the order of species arrival. Furthermore, the virus also boosted the fecundity of both aphids. Analyses of nutrient content of virus-free and virus-infected plants show significant increases of essential amino acids, sterols, and carbohydrates. Such nutrient increases appear to underlie the facilitative interactions and fecundity of aphids on virus-infected plants. Our experiments demonstrate that the virus dramatically increases the food consumption and fecundity of aphids through intra and interspecific trophic facilitation, resulting in processes that could affect community organization.Pathogens and other parasites can have profound effects on biological communities and ecosystems. Here we explore how two strains of a plant virus – Barley Yellow Dwarf Virus, BYDV – influence the foraging performance and fecundity of two aphid species:  However, our understanding remains limited about the ways in which parasites influence key ecological processes such as community assembly and organization3. Plant parasites such as viruses might be expected to have large effects on such processes, as plants lie at the center of most community interaction webs and thus mediate direct and indirect interactions among a wide range of organisms4. For example, many studies have shown how viruses affect the outcome of competitive interactions7. However, few studies have examined how viruses affect positive intra and interspecific interactions such as facilitation among phytophagous vector species and its implications for ecological communities10. Facilitation is any direct or indirect interaction among two or more organisms that increases the fitness of one or more organisms without negatively affecting the other(s)11. Phytophagous insect communities associated with grasses provide good experimental systems for the investigation of such questions as they exhibit complex interactions among species that include competition for space and nutrients, that are often mediated by the host plants.Understanding the outcome of interspecific interactions among species with similar ecological requirements is one of ecology’s persisting challenges. Work emerging over the past two decades indicates that parasites can alter the outcome of intra and interspecific interactions17. In the case of aphids, for example, small species such as Rhopaloshipum padi are first to colonize grasses , while others like the larger R. maidis may follow20. Studies that have investigated the effects of viruses on the feeding behavior of insect vectors have focused exclusively on intraspecific effects22 and thus, the consequences of pre-inhabitation and virus infection with respect to interspecific aphid interactions are largely unexplored. For example, foraging activity of a pioneer species like R. padi might affect the food supply and feeding rates of late-coming species such as R. maidis in ways that could be negative (competition), neutral, or positive (facilitation).Previous work has established that the order and timing of species colonization can influence the structure and dynamics of communities through facilitation in different types of organisms ranging from bacteria to vertebratesR. padi and R. maidis – and two virus strains of Barley yellow dwarf virus – BYDV-PAV and BYDV-RMV– affect feeding and fecundity. These virus strains are transmitted by aphids in a species-specific manner: Rhopalosiphum maidis transmits BYDV-RMV and R. padi transmits both BYDV-PAV23. The viruses are transported in the accessory salivary glands of aphids and can be transmitted into plants during feeding for weeks25. Our null hypothesis was that pre-inhabitation by an aphid species on a host plant and the presence of viruses had no effect on the food consumption and fecundity of a succeeding aphid species. Alternatively, if the chronological order of species arrival and the virus matter, outcomes could range from competition to facilitation, potentially including scenarios in which interactions among species are asymmetric . We tested these hypotheses using a combination of field and laboratory experiments that manipulated the presence of virus strains and the order of aphid species arrival to examine (1) the time it takes an aphid to start feeding, (2) the feeding duration, and (3) aphid fecundity. We also examined (4) how the nutritional quality of host plants was affected by aphid foraging and plant virus strains. Our results show that the plant both strains of the virus significantly increase the feeding rates and fecundity of aphids, resulting in niche expansion and changes in the organization of the plant-virus-insect community.Here we examine how the order of colonization by two aphid species – R. padi and R. maidis were kept under greenhouse conditions at 20 ± 2 °C and ambient photoperiod. All aphids used in experiments were kept in small colonies (~ten aphids per plant) to avoid stress on individuals associated with high population densities. Spring wheat plants (Triticum aestivum L.) were grown from individual seeds planted in a tray of greenhouse cones (4 cm diameter x 21 cm long) with a standard soil substrate with macro- and micro-nutrients . Plants were watered twice a day and kept in a growth chamber at 22 ± 1 °C with a 16 h photoperiod (430 µmol m−2 s−1), 8 h dark period at (20 ± 1 °C), and a relative humidity of 60%. Virus infection of host plants. To infect plants, we fed aphids on oat leaves (Avena strigosa Schreb) infected with BYDV-PAV and BYDV-RMV during 2–3 days. Then, we exposed healthy wheat plants to the viruliferous aphids for a period of ten days to allow virus inoculation (ten aphids per plant). Infection on each wheat plant was confirmed after 12 days using a double-antibody sandwich, enzyme-linked immunosorbent assay (DAS-ELISA) . The virus titer was estimated following the protocol described in Jimenez-Martinez et al.26 (Table Our laboratory stock colonies of 26 Table .R. padi transmits BYDV-PAV and R. maidis transmits BYDV-RMV). Levels of pre-inhabitation were: pre-inhabited by the same aphid species (intraspecific), pre-inhabited by the other aphid species (interspecific), and control (not pre-inhabited). Levels of virus infection were: host plant infected with BYDV-PAV, host plant infected with BYDV-RMV, intraspecific control (pre-inhabited by the same species but no virus), and interspecific control (pre-inhabited by the other species and no virus). We did not include a double-negative control  because virus inoculation only occurs through aphid foraging. The experimental unit was an individual spring wheat plant (Triticum aestivum L.) not previously used in any experiment, and not used again in any other experiment . There was a total of six replicates – six different plants – for each unique combination of pre-inhabitation and possible virus-aphid treatments.We used a factorial experimental design to test the hypothesis that pre-inhabitation and viruses affect the foraging behavior of late-coming aphid species. Experimental factors (independent variables) were pre-inhabitation with three levels, and virus infection with three levels. Virus-infection factors cannot be combined between the aphid species because virus transmission is species specific  and allowed them to feed for a period of 12 days removing their offspring daily. We chose this duration because a successful virus-inoculation can require aphids feeding on the plant up to 10–12 days. For the interspecific pre-inhabitation treatments, we did the same, but with viruliferous aphids of the other species.N = 6 for all treatment combinations) we placed a single adult aphid connected to a channel of a Giga-8 Direct Current-EPG  for a period of eight hours. The Giga-8 DC-EPG system has two electronic components, a voltage source and an input resistor connected to each other. The substrate electrode (output wire) is placed in the soil where the plant is rooted. The other electrode  is attached to the insect’s dorsum with a small drop of silver glue – a conductive adhesive27. The circuit is completed when the insect’s mouth parts enter the plant and the current flows from the voltage source to the plant and through the insect, the input resistor, and back to the voltage source. The voltage changes resulting from this “biological resistance” are monitored by a computer that graphs and records the values of the insect-plant resistor. Wave forms generated by voltage changes are associated with biological processes involved in feeding behavior such as plant cuticular surface probing, salivation, time to reach the phloem, duration of ingestion, among others28. Our EPG recordings started one hour after the aphid was placed on a horizontally oriented leaf, and continued for eight hours. We analyzed the electrical waves associated to each behavior using software (Stylet +a & d version 1.2) developed by EPG Systems . For more details on virus inoculation see Supplementary section We measured two key parameters of aphid foraging as response variables in all treatments: the time taken by aphids to reach the wheat plant’s phloem, and the duration of ingestions. Both responses were measured using electrical potential graphing (EPG). In each trial  having two windows (3 cm × 5 cm) made of Lumite fabric . Each combination of pre-inhabitation and virus treatments had 15 replicate wheat plants in both the lab and the field experiment. As a response variable in both experiments we recorded the total number of offspring produced for 30 days.Here we also used a factorial design in two experiments – one in the laboratory and one in field conditions – to test the hypothesis that pre-inhabitation and viruses affect aphid species’ fecundity for any late-coming aphid species. Experimental factors (independent variables) were exactly as in experiment 1 for both the laboratory and field experiments. However, this time to create pre-inhabitation treatments we exposed each 3-week old wheat plant to 10 adult aphids (virus-free or viruliferous depending on treatment) for 10 days before removing them (the offspring were removed every day). Next, both in the lab and the field, we placed a 1R. padi or R. maidis, as there were no aphid foraging treatments following pre-inhabitation by the same or the other species of aphid.To test the hypothesis that aphid inhabitation and virus infection increase the nutritional quality of host plants, we modified the same general design used in experiments 1 & 2 in order to compare the nutritional content of wheat plants never exposed to aphids or viruses (controls) with plants infected with viruses and inhabited with our aphid species (treatments). To perform this experiment, we grew 360 wheat plants in four trays in the laboratory at a density of 90 plants/tray. From these trays, we assigned 32 randomly chosen plants from across all trays to each unique experimental factor combination of aphid inhabitation and virus presence . Note that in this experiment the aphid treatments of interest are not “pre-inhabited” but just “inhabited” by R. padi, R. maidis, negative control) and virus treatments . After 10 days of exposure to treatments, we combined all the leaves from eight wheat plants from each treatment to produce one sample for nutrient analysis. Leaves were preserved in liquid nitrogen at −80 °C immediately after harvesting. Thus, although there were 32 independent experimental plants from each treatment, the final replication per treatment was four because of the biomass requirement of chemical analyses. In each sample, we measured the concentration of simple sugars (sucrose and glucose), total sterol content, and amino acids  using gas chromatography mass spectrometry or liquid chromatography mass spectrometry analysis  was caged in the aforementioned way, and exposed to 10 adult aphids for ten days according to species  of the treatments (pre-inhabited or pre-inhabited with virus) against their respective controls  were significant.The data from the experiments were analyzed using a Multivariate Analysis of Variance (MANOVA) and Wilks’ lambda. Significant differences in MANOVA were followed by individual analyses of the response parameters  per aphid species, by pre-inhabitation type (intra and interspecific), and by type of virus presentFor the data from experiment 2, we compared the fecundities among treatments using one-way ANOVAs for each aphid species in each location , followed by post-hoc Tukey-Kramer HSD mean comparisons.For experiment 3 we used a completely randomized design to compare the effect of viruses on the nutritional quality of wheat plants. Differences in sugars, total sterols, and amino acids were tested using one-way ANOVA followed by Dunnett’s test comparing treatments against the control plants . All analyses were performed in Python programming language (version 3.5.2). The data that support the findings of this study are available on AEKOS data repository (Data Cite DOI 10.4227/05/5a555d6cc6165).F18,98 = 117.26, P ≤ 0.0001). Rhopalosiphum maidis reached the phloem significantly faster on plants previously foraged by conspecifics , but we did not detect any significant effect on the duration of ingestion  and increased the time of ingestion by 57% (t = 13.13, df = 10, P < 0.0001; Fig. R. maidis with BYDV-RMV significantly decreased the time taken by R. padi to reach the phloem by 56% (t = 8.75, df = 10, P < 0.0001; Fig. t = 3.98, df = 10, P = 0.0026; Fig. We found that the presence of virus strains altered the feeding behavior of both aphid species, pre-inhabitation of R. padi and R. maidis were able to colonize and reproduce on virus-free plants that were pre-inhabited (MANOVA: Wilks’ Lambda = 0.0017, F16,205.33 = 89.08, P ≤ 0.0001; Fig. R. padi carrying BYDV-PAV, R. padi fecundity increased by 150% and by 125% in R. maidis relative to controls (Fig. R. padi decreased 25% in plants pre-inhabited by R. maidis (Fig. R. maidis and infected with BYDV-RMV strain. The same trend was also observed in the field (Fig. Populations of both dis Fig. . Howevereld Fig. .Figure 2The presence of viruses increased the nutritional content of wheat leaves, specifically the free carbohydrates, sterols, and amino acids Fig. . The conOur experiments demonstrate that pre-inhabitation of a host plant by an aphid species can produce both intra and interspecific facilitation. Results also show that plant viruses magnify the facilitative effects that plant pre-inhabitation has on late-coming species.R. padi – the typical pioneer species – significantly increased the foraging capacity and fecundity of late coming R. maidis aphids. Furthermore, plant virus had a strong effect on the symmetry of the interspecific interactions. For example, without the virus, interspecific facilitation is asymmetric because pre-inhabitation by R. maidis hampers the foraging performance of R. padi. However, R. padi had no problem inhabiting areas previously occupied by R. maidis when the BYDV-RMV strain – exclusively transmitted by R. maidis – was present in plants. Although facilitation is a well-recognized process influencing the assembly of bacteria12, protozoan30, fungal32, plant32, animal33 and mutualistic communities34, this is the first time that it is shown to be mediated by a virus in a community of putative insect competitors.Particularly, the virus strain transmitted by R. padi (Fig. 36 possibly shortening the distance between the vascular bundles and the surface of the leaf that serve as a foraging cue, facilitating foraging for later arriving aphids37.Virus strains changed host plants in ways that increased the fitness of both aphid species. Aphids fed for longer periods and boosted their fecundity in the presence of virus strains Fig. . But viradi Fig. . For exa39. Higher concentrations of sterols are also correlated to enhanced fitness in aphids40. In addition to sterols and sugars, we found significant increases in the concentration of amino acids in our virus-infected plants (Fig. 41 However, the concentrations reported in the present study are lower than the previously reported, possibly due to differences in growth conditions, strain virulence, or method of detection. Still, our analysis of the nutritional content of plant leaf tissues are only a proxy for the potential effects of viruses on plant physiology as it relates to phloem-feeding insects since ideally the phloem sap should be collected for plant nutritional content analysis43. Thus, since aphids are specialist phloem-feeders, additional studies are needed to study in more detail how changes in sap nutritional content affects aphid interactions and population dynamics.Underscored are the significant increases of nutrients like plant sterols in virus-infected plants Fig. . SterolsR. maidis had a negative impact on the foraging behavior and fecundity of a late-coming R. padi (Figs R. padi to the same levels reached on control plants in the field, or attaining higher fecundity than controls in laboratory conditions (Fig. R. padi had consistently larger positive effects on the fecundity of any succeeding aphid species compared with the virus transmitted by R. maidis.For both aphid species, viruses increased the magnitude and symmetry of interspecific facilitative effects of prior foraging, although some outcomes were species-specific. For example, in the absence of virus, prior foraging by adi Figs  and 2. B45. Thus, our results add to the mounting evidence that viruses and other pathogens modify the behavior of animal vectors in ways that are conducive to fitness gains for the pathogen. More research is necessary to further uncover the implications of this type of animal-plant-pathogen interactions on the stability and coexistence of insect populations, and the dynamics of diseases given the improbable situation where, for competing insects – “the enemies of our food made us friends”.Our results suggest that viruses can play an important role in changing the nature of intra and interspecific interactions of competing species by promoting trophic facilitation, and affecting processes of community assembly. The longer feeding times aphids exhibit when exposed to a pre-inhabited virus-infected host should increase probabilities of virus acquisition and transmissionSupplementary information 1: MethodsSupplementary information 2 Tables


OBJECTIVES/SPECIFIC AIMS: We aimed to assess trends in incidence of genital warts across human papillomavirus (HPV) vaccine-eligible and nonvaccine-eligible age groups to determine the impact of the HPV vaccine among Medicaid enrollees in the state of Tennessee. METHODS/STUDY POPULATION: We analyzed 2006–2014 medical and pharmaceutical claims data from TennCare (Tennessee’s Medicaid program) enrollees aged 15–64 years. Incident cases of genital warts were defined as persons 12 months disease free and: (1) a diagnosis of condyloma acuminatum, or (2) a diagnosis of viral warts and genital-specific procedure, or (3) a prescription for genital warts medication and genital-specific procedure. Mann-Kendall trend tests were performed to assess for significant trends in incidence of genital warts by sex and age group; average annual percent changes were calculated to quantify these trends. RESULTS/ANTICIPATED RESULTS: Our analysis is in progress. We hypothesize that we will observe declines in genital warts among younger, vaccine-eligible age groups and no changes in older, nonvaccine-eligible age groups, with largest declines among females aged 15–19 years from 2006 to 2014. We also expect to see declines among younger males due to herd protection, with greater declines after 2011, when the vaccine was approved for males. DISCUSSION/SIGNIFICANCE OF IMPACT: Significant declines among younger compared with older age groups would suggest HPV vaccine effectiveness for preventing genital warts.


Current and reoccurring viral epidemic outbreaks such as those caused by the Zika virus illustrate the need for rapid development of antivirals. Such development would be facilitated by computational approaches that can provide experimentally testable predictions for possible antiviral strategies. To this end, we focus here on the fact that viruses are directly dependent on their host metabolism for reproduction. We develop a stoichiometric, genome-scale metabolic model that integrates human macrophage cell metabolism with the biochemical demands arising from virus production and use it to determine the virus impact on host metabolism and vice versa. While this approach applies to any host–virus pair, we first apply it to currently epidemic viruses Chikungunya, Dengue and Zika in this study. We find that each of these viruses causes specific alterations in the host metabolic flux towards fulfilling their biochemical demands as predicted by their genome and capsid structure. Subsequent analysis of this integrated model allows us to predict a set of host reactions, which, when constrained, inhibit virus production. We show that this prediction recovers known targets of existing antiviral drugs, specifically those targeting nucleotide production, while highlighting a set of hitherto unexplored reactions involving both amino acid and nucleotide metabolic pathways, with either broad or virus-specific antiviral potential. Thus, this computational approach allows rapid generation of experimentally testable hypotheses for novel antiviral targets within a host. GivenAlpha- and Flavi-viruses. The rather simple physical and genomic structure of these viruses  of the original value for at least one virus . Interestingly, many of these 29 reactions are interconnected and are involved in the de novo synthesis of RNA nucleotides (both purine and pyrimidine pathways) and amino acid interconversions  [DHORD9) [These identified reactions are potential antiviral targets, in the sense that altering their fluxes can limit virus production within the host. Thus, we explored if these reactions match with known antiviral drug targets. Performing a literature analysis, we found that there are currently 10 antivirals, specific to RNA viruses, and that these target only five unique metabolic enzymes . Of these five drug targets (and the associated drugs), one has been experimentally verified to be effective against CHIKV  ; and ano[DHORD9) . While t[DHORD9) , they arIMPD [DHORD9 [OMPDC) [AHC) [AHC reaction flux to zero abolishes virus growth for all three viruses . Finally, CTP synthase, which has been indicated to exhibit an effect on several RNA viruses [CTPS1) or glutamine (mediated by CTPS2) as a nitrogen source [We found that out of these five known antiviral targets, all are implicated in our analysis. The three known antiviral target reactions involving the genes IMPD ; DHORD9  [DHORD9 ; and oro [OMPDC)  are founC) [AHC)  is predi viruses , is incln source  and ther2.6.Considering the computationally predicted potential of the additional reactions identified as antiviral targets, we have searched for these reactions in a database of known inhibitor-like compounds . We foun3.We present a computational approach that combines application of FBA and FVA with the development of integrated host–virus metabolic models. We show that this approach recovers the known metabolic antiviral targets within a human macrophage cell and predicts new potential targets. These predicted reactions fall primarily onto pathways involving nucleotides and amino acids that are differentially used by the host and virus. The results of this study are in line with an integrated perspective that views the virus as an additional metabolic burden on the host cells that could be met or avoided by tinkering of host metabolic fluxes. The observed overlap between predicted reactions and known antiviral drugs gives confidence to this integrated modelling approach and highlights its potential as a rapid prediction tool to guide experimental design. This can be especially useful in the case of new and emerging viruses for which limited clinical and experimental data may be available to inform drug target identification.The integrated stoichiometric metabolic modelling approach focuses on metabolic changes as a driver of virus production and does not consider factors associated with virus–host cell recognition, viral entry, lipid envelope production and release . In addiFuture efforts to improving model curation and standardization  would op4.4.1.S is the stoichiometric matrix, defining the stoichiometry of metabolites in different reactions, v is a vector of metabolic reaction fluxes, and c is a vector of the same size as v and encoding the objective function Z (e.g. c might be a binary vector with 1 at the index of the reaction(s) composing the desired objective, and 0 elsewhere). Additional constraints on each reaction flux iv, is defined through the minimal (lb) and maximal (ub) flux bounds.FBA is a mathematical, constraint-based modelling method used to simulate reconstructions of cellular metabolic networks . FBA assHere, we implement FBA and its close variant FVA for specific analyses of an integrated host–virus metabolic model as described in further detail below. FVA provides an additional layer of information to FBA by predicting the permissible flux values for each reaction. Thus, FVA yields two flux distribution vectors, which predict the minimum and maximum flux value that a reaction can have (defined as its flux range) given the objective function.4.2.To implement the FBA approach to studying virus infections from a metabolic stance, we define a pseudoreaction accounting for the production of virus particles from its constituents. We call this reaction a virus biomass objective function (VBOF). To account for metabolic fluxes associated with the virus production, the VBOF needs to capture the stoichiometry of nucleotide, amino acid and associated energy metabolites relating to virus production, similar to biomass production function used for microbial metabolic models . We deri4.2.1.http://osslab.lifesci.warwick.ac.uk/?pid=resources). Viruses can be classified by their replication methods, known as the Baltimore Classification System [The genome sequences used in the present study are obtained from the NCBI genomic database  using thn System , and depn System . The ratn System , and 1 :n System . More br4.2.2.Cg):−1) of the nucleotides vM; see equation (4.15) below). Finally, the stoichiometric coefficients of each nucleotide in the VBOF are expressed as millimoles per gram of virus :Cnp is 1 for all viruses studied here, while Csp is 240 for CHIKV [−1) of each amino acid (XM):The total moles of each amino acid per mole of virus particle or CHIKV , and 180or CHIKV . The mol4.2.4.kATP (=4) The overall moles of ATP (ATOT) required to form the structural (ASP) and non-structural (ANP) polyproteins are calculated from the respective amino acid counts:ATOT, we calculate the stoichiometry of ATP in the VBOF as millimoles per gram of virus (SATP):The polymerization of amino acid monomers requires approximately four ATP molecules per peptide bond , definedi and H+) are also accounted for in the VBOF (see equation (4.16)).As ATP is hydrolysed in this process, the water requirement for polymerization 4.2.5.i molecule [kPPi (=1). The overall moles of PPi (PTOT) required to form the viral genome (GP) and replication intermediates (RP) are calculated from the respective nucleotide counts:The polymerization of nucleotide monomers to form the RNA viral genome liberates a PPmolecule , definedi stoichiometry in the VBOF as millimoles per gram of virus (SPPi), we again use the overall molar mass (g mol−1) of one mole of virus:To convert this into the PP4.2.6.vM is calculated from the total mass of the genome and proteome components asThe total molar mass of the virus 4.2.7.The left- and right-hand side terms of VBOF are based on the above calculations of stoichiometric coefficients. The final stoichiometry for the VBOF (pseudoreaction) isThis pseudoreaction accounts for the virus' biomass, and the energy requirements associated with its production, and can be incorporated into stoichiometric metabolic models of the host to represent the presence of a virus in that system.4.3.The human macrophage metabolic model, iAB-AMØ-1410, was constructed in a previous study . This mo4.4.The VBOFs for the three viruses  were integrated into three separate instances of the ‘host’ macrophage model (iAB-AMØ-1410) . In each case, the respective VBOF was appended into the existing macrophage model, with a lower flux bound of zero and an upper bound of infinity, reflecting no upper constraints on this flux . No othe4.5.i is over nucleotides (or amino acids) and k is over all biomass precursors, and the subscripts H and V indicate the use of the host and virus biomass functions, respectively. A positive value indicates a higher usage of nucleotide (or amino acid) i by the virus than the host, while a negative value indicates a lower usage.For both the host (iAB-AMØ-1410) and viruses  we have pseudoreactions that capture the metabolic requirements for the maintenance/production of their respective biomass. By comparing these pseudoreaction stoichiometries, we can quantify the differences in amino acid and nucleotide requirements to fulfil the host or virus objectives. To do so, we calculate the fold change in nucleotide and amino acid usage by normalizing their individual stoichiometric coefficients against the sum of stoichiometries of all metabolites present in the objective function (other than ATP):4.6.For all analyses, the generated host–virus integrated models were optimized, and reaction fluxes predicted using the linear optimization approach known as flux balance analysis (FBA) . Linear iA) and then collating the mean flux values for reactions associated with given subprocesses (aggregated subsystems) as a percentage of total flux through that process. More formally, we define the differential distribution of reaction flux for each subprocess (i) between the host- and virus-optimized models in terms of a fold change:k is over all reactions of the model, while the indexation m is over reactions that belong to subprocess i. The superscript indicates the use of flux values from the host (H)- and virus (V)-optimized models, respectively. A positive value indicates a higher mean flux for subprocess i in the virus- versus host-optimized model, while a negative value indicates a lower mean flux.To simulate a virus-optimal state, the models are optimized using the respective VBOFs of CHIKV, DENV and ZIKV viruses as the objective function, while to simulate a host-optimal state, the models are optimized using the existing biomass maintenance reaction for the human macrophage as presented in . Besides4.7..Knockout analysis Knockout analysis considers the effect of systematically setting individual reaction fluxes to zero, and then attempting to maximize for VBOF. The knockout optima for the virus production reaction flux Host-derived enforcement. Host-derived enforcement considers the effect of maintaining a metabolic system in a host-optimized state while attempting to optimize the model for VBOF. For this approach, we systematically set individual lower and upper flux bounds of individual reactions to a specific flux range. For each reaction, this range (rɛ) is derived from the corresponding minimum (−F) and maximum (+F) flux values for that reaction obtained from the FVA using the host (H) and virus (V) optimization (as described above). The range (rɛ) is bounded by minimum (−ɛ) and maximum (+ɛ) flux values, which are given by the following conditional arguments:To find reactions that can preferentially alter virus-optimized state of the model, we considered the effect of systematically constraining individual reactions. These calculated flux ranges for each individual reaction are then used to constrain the model, and the model is optimized for the VBOF. The resulting optima for the virus production reaction flux,


In 2016, during a major Zika virus (ZIKV) outbreak in Maracaibo, Venezuela, a 49-year-old woman and an unrelated 4-year-old boy developed bilateral optic neuritis 2–3 weeks after presenting an acute febrile illness characterized by low-grade fever, rash and myalgia [Which of the following statements is accurate about non-congenital severe ocular complications of Zika virus (ZIKV) infection?1. They are unique to ZIKV infection and readily distinguishable from complications caused by other flaviviruses.2. Serious ocular complications are related to the severity of the acute exanthematous illness.3. The diagnosis can be conclusively established by detecting anti-Zika IgM and/or IgG in the patient’s serum.4. These complications can lead to permanent visual impairment.5. There is specific treatment for ocular manifestations caused by ZIKV infection.Correct Answer: 4. These complications can lead to permanent visual impairment.Flavivirus of the family Flaviviridae [ZIKV is a mosquito-borne RNA virus belonging to the genus iviridae . The claiviridae , chikungiviridae , West Niiviridae  and Riftiviridae  (Table 1iviridae .Non-congenital ocular complications are infrequent, but serious, consequences of ZIKV and other arboviral infections. The complications may appear at the end of the acute febrile illness, but more commonly occur within 2 weeks to 1 month after the onset of symptoms. There is no evidence to suggest that serious ocular complications correlate with the severity of the acute febrile illness. One study, however, found that the white cell count and serum albumin are significant predictors of ocular complications of DENV .Serological testing for arboviral diseases should be performed in all patients with ocular complications and a recent history of acute febrile exanthematous infection, who live, or have travelled to, endemic regions. The presence of IgM to ZIKV strongly suggests that the ocular manifestation is associated with this virus. A causative aetiology, however, can only be established by documenting the presence of the virus in body fluids, either by cell culture or by PCR. It should be noted that other viruses, such as herpes simplex virus and human immunodeficiency virus, can also cause retinal damage and optic neuritis. Furthermore, as in the cases presented here, the diagnosis is complicated by cross-reactivity among flaviviruses, and by the co-circulation of arboviruses.Most patients with ocular complications of arboviral infections recover completely. Nevertheless, physicians should be aware that a small percentage of patients have permanent damage with long-life visual impairment.There is no specific or established treatment for optic neuritis caused by any arboviral infection. Systemic steroids may be used to reduce inflammation and resulting ischaemia. Corticosteroids have been used in combination with acyclovir to treat chikungunya-associated optic neuritis, but efficacy has not been proven .


Monkeypox virus is a zoonotic disease endemic to Africa. In 2017, we confirmed a case of human monkeypox virus in Sierra Leone by molecular and serologic methods. Sequencing analysis indicated the virus belongs to the West African clade and data suggest it was likely transmitted by wild animals. Orthopoxvirus, was identified in captive cynomolgus monkeys in Copenhagen in 1958 (Monkeypox virus (MPXV), of the genus Ecologic niche modeling shows that Sierra Leone is in a geographic region suitable for transmission of MPXV  and on May 10 (day 55 after the patient sought treatment) to the Sierra Leone–China Friendship Biologic Safety Laboratory in Freetown, Sierra Leone. We conducted MPXV-specific real-time PCR, as previously described  and aligned these to Sierra Leone 2017 (https://www.lynnon.com/dnaman.html). We found that Sierra Leone 2017 was closely related to UTC_1964. In addition, a 6-nucleotide deletion  was more evident in the dUTPase region of Sierra Leone 2017 compared with other MPXVs from Africa. Also, a 25-nucleotide insertion (5× repeat of TCCAT unit) in the kelch-like protein-encoding region was more evident in the USA_2003_039 genome compared with most MPXV isolates from Africa. This insertion leads the N-terminal amino acids of the protein sequence that changed from EWNGMEWNGK in USA_2003_039 to VNNFEIK in Sierra Leone 2017.For further comparison, we selected previous MPXV sequences from NCBI  Sierra Leone 2017 and other collected MPXV isolates.


Several ruminant species have been shown to be susceptible to Schmallenberg virus (SBV), but adult animals usually recover after showing mild or no clinical signs. However, transplacental infection can occur and lead to abortion, malformations and stillborn lambs, calves and goat kids. During November and December 2014, malformations were observed in 11 stillborn calves from two farms in the north-eastern region of Namibia. Blood samples were collected from 9 of the 11 cows that delivered stillborn and malformed calves. All these animals tested negative for Rift Valley fever, bovine viral diarrhoea and infectious bovine rhinotracheitis and were serologically positive for bluetongue virus, SBV and epizootic haemorrhagic disease virus. Clinical findings and serological results suggested that SBV may be circulating in Namibia. Orthobunyavirus within the Bunyaviridae family, is a member of the Simbu serogroup and is closely related to Akabane and Shamonda viruses , a recently discovered ® Schmallenberg virus Competition Multi-species ELISA  according to manufacturer’s instructions, whereas the serum neutralisation test was performed using the BH80/11–4 isolate . Antibody detection for bluetongue virus (BTV) was performed using the competitive ELISA described by Lelli et al. (2013). A commercially available competitive ELISA kit  was chosen to test for antibodies against epizootic haemorrhagic disease virus (EHDV) according to the manufacturer’s instructions, while the serum neutralisation test for BTV and EHDV was performed using the method described by Gard and Kirkland , and because calves were conceived between February and June, this is the only window (between 30 and 150 days after conception) that may have led to porencephaly or hydranencephaly-micromyelia-arthrogryposis syndrome (Bayrou et al.


Avian orthoavulavirus 13 (AOAV-13), also named avian paramyxovirus 13 (APMV-13), has been found sporadically in wild birds around the world ever since the discovery of AOAV-13 (AOAV-13/wild goose/Shimane/67/2000) in a wild goose from Japan in 2000. However, there are no reports of AOAV-13 in China. In the present study, a novel AOAV-13 virus (AOAV-13/wild goose/China/Hubei/V93-1/2015), isolated from a wild migratory waterfowl in a wetland of Hubei province of China, during active surveillance from 2013 to 2018, was biologically and genetically characterized. Phylogenetic analyses demonstrated a very close genetic relationship among all AOAV-13 strains, as revealed by very few genetic variations. Moreover, pathogenicity tests indicated that the V93-1 strain is a low virulent virus for chickens. However, the genome of the V93-1 virus was found to be 16,158 nucleotides (nt) in length, which is 12 nt or 162 nt longer than the other AOAV-13 strains that have been reported to date. The length difference of 12 nt in strain V93-1 is due to the existence of three repeats of the conserved sequence, “AAAAAT”, in the 5′-end trailer of the genome. Moreover, the HN gene of the V93-1 virus is 2070 nt in size, encoding 610 aa, which is the same size as the AOAV-13 strain from Japan, whereas that of two strains from Ukraine and Kazakhstan are 2080 nt in length, encoding 579 aa. We describe a novel AOAV-13 in migratory waterfowl in China, which suggests that diversified trailer region sequences and HN gene lengths exist within serotype AOAV-13, and highlight the need for its constant surveillance in poultry from live animal markets, and especially migratory birds. Paramyxoviridae circulated in various vertebrates, including mammals, birds, reptiles and fish, have been newly identified [Avulavirinae, previously designated avian avulaviruses (AAvV), are enveloped, non-segmented negative-sense single stranded RNA viruses, which are in the range of 14.9 to 17.4 kb in length, encode up to nine different proteins [Metaavulavirus, Orthoavulavirus and Paraavulavirus) within Avulavirinae, including 20 described serotypes (-1 to -20), based on haemagglutination inhibition (HI) and neuraminidase inhibition (NI) assays and genetic sequencing [Metaavulavirus contains ten species of avian metaavulavirus (AMAV), including serotypes -2, -5, -6, -7, -8, -10, -11, -14, -15 and -20, Orthoavulavirus contains eight species of avian orthoavulavirus (AOAV), including serotypes -1, -9, -12, -13, -16, -17, -18 and -19, while only two serotypes (-3 and -4) of avian paraavulavirus (APAV) are grouped into the genus of Paraavulavirus [Viruses of the family entified ,2. Virusproteins  and havequencing ,7,8,9,10Avulavirinae [Avulavirinae.While little is known about the biological and genetic information of serotypes 2 to 20, extensive studies have been conducted on AOAV-1  because of its high mortality and heavy losses on the economy in relation to the poultry industry worldwide ,13. AOAVavirinae . Meanwhiavirinae . HoweverAll animal experimental protocols used in this study was approved by the Institutional Animal Care and Use Committee of Jilin University, China .n = 3974) and fresh fecal samples from migratory birds in wetlands  were collected from six provinces of China, including two provinces in Central China (Hubei and Hunan), one in East China (Anhui), one in Northeast China (Jilin), one in Northwest China (Qinghai) and one in North China (Neimenggu), as part of both the avian influenza virus (AIV) and NDV surveillance program [During the years from 2013 to 2018, both cloacal and oropharyngeal swab samples from clinically healthy domestic poultry from the live bird market  embryonated chicken eggs using standard techniques, as previously described for ND . The preRNA extraction, cNDA synthesis, and seminested PCR and sanger sequencing for L gene of paramyxoviruses were performed according to the methods described in our previous study . To explThe pathogenicity test of the V93-1 virus, including the mean death time (MDT), the intra-cerebral pathogenicity index (ICPI), and intravenous pathogenicity index (IVPI), were determined according to the protocols described in the Office International des Epizooties (OIE) manual for ND . Virus iTo further determine the biological characteristic of this V93-1 virus, eight 21-day-old SPF chickens were inoculated with a single dose of 100 µL allantoic fluid (HA titer of 1024 per 25 µL) via intraocular-nasal dropping. Then all chickens were monitored for clinical signals for 10 days. Cloacal and oropharyngeal swab samples were harvested at 2, 4, 7, and 10 dpi (days post infection) for the detection of virus shedding. In addition, at 2, 4, 7, and 10 dpi, two chickens were randomly sacrificed and tissue samples  and blood were harvested for the detection of virus load in vivo. For detection of virus shedding and virus load in infected chickens, seminested PCR for L gene of V93-1 virus was applied .To understand genetic relationships of the V93-1 virus, phylogenetic trees were constructed based on the complete nt sequences of the genome and F gene of the AOAV-13 isolates, including V93-1 and other viruses of serotypes, using the maximum likelihood method based on the Tamura–Nei model . The perIn this study, for the first time, we obtained a novel AOAV-13 virus  from migratory waterfowl at the Chenhu Lake Wetland of Hubei province , China, in 2015, and compared it to other available AOAV-13 isolates in GenBank to assess the biological characteristics and epidemiological genetic relationships of the V93-1 virus. In line with the reported studies ,15,16, nThe V93-1 isolate was successfully propagated in 9- to 10-day-old SPF chicken embryos, and the harvested infective allantoic fluid had a hemagglutination (HA) titer of 512–1024 per 25 µL. Meanwhile, the V93-1 isolate had a high HI titer (1:512) for homologous V93-1 specific chicken antibodies, but low (less than 1:8) or no cross-reactivity with other serotypes, including AOAV-1, APAV-4 and AMAV-6. In addition, the mean death time (MDT) score in 9- to 10-day-old embryonated SPF chicken embryos was more than 168 h, with no mortality after 7 days, and both the intra-cerebral pathogenicity index (ICPI) in 1-day-old SPF chickens and the intravenous pathogenicity index (IVPI) in 6-week-old chickens were 0, suggesting that this V93-1 isolate is a low virulent virus for chickens.To further determine the biological characteristic of this V93-1 virus, eight 21-day-old SPF chickens were inoculated with a single dose of 100 µL allantoic fluid (HA titer of 1024 per 25 µL) via intraocular-nasal dropping. However, no clinical signals were observed in virus-infected chickens during 2–10 days of post inoculation. Meanwhile, the viral load could not be detected in the organs  and blood of the experimentally infected chickens, and cloacal, oropharyngeal swab samples from the infected chickens were found to be negative by PCR. In addition, the virus did not cause a cytopathic effect without exogenous protease in tested MDCK, HD 11 and DF-1 cell lines. However, the growth and other biological characteristics need to be detected with other cell lines.The genome of the V93-1 virus was found to be 16,158 nucleotides (nt) in length, with a GC content of 42.6%, which is 12 nt longer than those of strains from Japan (GenBank: LC041132) and Ukraine (KX119151) and 162 nt longer than the strain from Kazakhstan (KU646513) A. The geTo further determine the genetic relationships of the V93-1 virus, phylogenetic trees were constructed, based on the complete nt sequences of the genome and F gene of the AOAV-13 isolates, including V93-1 and other viruses of serotypes . PhylogeTo our knowledge, our study is the first to describe an AOAV-13 isolate in wild migratory waterfowl from China, demonstrating that not only does the V93-1 virus harbor the longest genome size  and three repeats of the uniquely conserved sequence (AAAAAT) within the 5′ trailer, as compared to the three previously reported completely sequenced isolates, but also at least two different HN proteins, which exist within the serotype AOAV-13. Furthermore, the emergence of the V93-1 virus in China is epidemiologically connected to the AOAV-13 from other geographical regions, such as Kazakhstan. Therefore, its presence signified a potential risk of AOAV-13 being introduced into the territory and country. It also necessitates the demand for constant epidemiological surveillance, including geographic, ecological and/or host-species factors for AOAV-13 isolates among wild birds and domestic poultry in China, to prevent and control the potential transmission of novel variants from other regions.


In the re-emergence of Highly Pathogenic Avian Influenza (HPAI), live bird markets have been identified to play a critical role. In this repeated cross-sectional study, we combined surveillance data collected monthly on Jakarta’s live bird markets over a five-year period, with risk factors related to the structure and management of live bird markets, the trading and slaughtering of birds at these markets, and environmental and demographic conditions in the areas where the markets were located. Over the study period 36.7%  of samples (N = 1315) tested HPAI H5 virus positive. Using General Estimation Equation approaches to account for repeated observations over time, we explored the association between HPAI H5 virus prevalence and potential risk factors. Markets where only live birds and carcasses were sold, but no slaughtering was conducted at or at the vicinity of the markets, had a significantly reduced chance of being positive for H5 virus . Also, markets, that used display tables for poultry carcasses made from wood, had reduced odds of being H5 virus positive , while having at least one duck sample included in the pool of samples collected at the market increased the chance of being H5 virus positive . Markets where parent stock was traded, were more at risk of being H5 virus positive compared to markets where broilers were traded. Finally, the human population density in the district, the average distance between markets and origins of poultry sold at markets and the total rainfall per month were all positively associated with higher H5 virus prevalence. In summary, our results highlight that a combination of factors related to trading and marketing processes and environmental pressures need to be considered to reduce H5 virus infection risk for customers at urban live bird markets. In particular, the relocation of slaughter areas to well-managed separate locations should be considered. Since the first wave of highly pathogenic H5N1 (HPAI H5N1) occurred in 2003/04 in South East Asia, the virus has managed to maintain itself in several countries, resulting in an endemic state and regular disease outbreaks. Especially in the early years of the incursion of HPAI H5N1, many human cases were reported, arousing fears of an emerging human pandemic. By far, Indonesia reported the highest number of human cases in the region. Interestingly, many human cases were not reported from areas of high poultry density, but rather from urban areas in which hardly any poultry is produced. Investigations revealed that most human cases of not only HPAI H5N1, but also of the more recently discovered strains H7N9 and H10N8, have been linked to visits of live bird markets (LBM) . ResultsBackyard poultry is usually purchased by ‘middlemen’ or small-volume poultry traders who visit several villages on motorbikes to trade with farmers or they purchase birds from small village markets. They then sell these birds at LBMs in rural areas, from where birds are transported to smaller poultry wholesale markets. There, birds are re-assorted and mixed according to size, before being transported to larger wholesale poultry markets and further distributed to LBMs in urban centres . Thus, tTherefore, the high HPAI virus prevalence observed at LBM is probably related to the duration poultry remains in the trading chain before being sold at LBM and is influenced by the number and frequency of contacts of susceptible birds with infected poultry or with HPAI virus contaminated surfaces. Since it is difficult to acquire data explaining these associations, conclusion on the impact of trading on HPAI virus spread are mainly based on infectious disease modelling , 9 suppoHowever, most of these studies focussed only on one group of risk factors, either market characteristics or environmental and infrastructural conditions that might be associated with HPAI virus prevalence while using cross-sectional diagnostic data collected only at a single time point.In our study, we combined regular surveillance results collected repeatedly from LBMs in the Greater Jakarta region, Indonesia, over a period of five years with information on market characteristics, trading parameters, market density, environmental conditions and sampling characteristics to provide holistic insights into the HPAI H5 infection dynamics at LBMs.The data collection was conducted in the 13 districts of the Greater Jakarta region, which included the following: Bekasi, Bogor, Jakarta Barat, Jakarta Pusat, Jakarta Selatan, Jakarta Timur, Jakarta Utara, Kota Bekasi, Koa Bogor, Kota Depok, Kota Tangerang, Tangerang and Tangerang Selatan. The Government of Indonesia, in collaboration with the Food and Agriculture Organisation (FAO), has been conducting environmental LBM surveillance since 2009. It involves the collection of environmental samples from individual vendors. A vendor represents a single retailer who sells live birds or poultry carcasses to customers and who may or may not slaughter birds on site. The number of vendors in a market can vary greatly, between one and more than 50 . Initially, surveillance of LBMs in Jakarta included all of the 260 identified LBMs, but to reduce the costs associated with these activities, surveillance was reduced to 86 LBMs in 2012. A risk-based approach was used to select LBMs that should undergo further surveillance: LBMs were ranked according to their completeness of surveillance data and frequency of HPAI H5 positive results, and by ensuring that a good geographic coverage of all districts in the Greater Jakarta region is maintained .LBM’s were visited approximately monthly by a government official and a pooled environmental sample was collected from six vendors, with one environmental swab obtained per vendor. Monthly collection of pooled samples commenced in March 2009, but in May 2012, sample collection was changed from a monthly to a quarterly protocol, and returned to monthly sampling in February 2013.The sample sites for the pooled samples were selected based on research by Indriani et al. 2010, who tested a multitude of environmental sampling sites at markets and found six locations to be the most sensitive for the detection of H5N1 . The envR Viral RNA Mini Kit (Qiagen inc.). The RNA template was used for Influenza A virus detection (M gene) with the Influenza Virus Type A & Avian Influenza Virus TaqMan PCR assay [RNA extraction was carried out using QiampCR assay . A H5 suCR assay .Risk factor information on LBM trading, poultry processing and selling practices was collected through questionnaire surveys conducted between 2008 and 2014 on LBMs in the Greater Jakarta region. Questionnaires used in the surveys were pilot-tested with at least five interviewees and modified if required. The government officials conducting the surveys were trained in interviewing techniques for each survey during one to two-days workshops. The following surveys were conducted:A survey of poultry markets  was carried out in 2008 to describe the trading volume and practices . The geographical locations of these poultry markets were used to calculate their density (number of markets/square km) for each of the districts in the Greater Jakarta region. The information was also used to calculate the average distance between the centroid of districts supplying the poultry and the district centroid of the market (representing the average distance for transportation of poultry to the LBM).A survey of all the 86 LBM’s under surveillance was carried out in March 2013 by government officials, as part of the routine monthly surveillance activities. The survey was designed by FAO Indonesia and explored general trading information, data on volumes and species of poultry traded and data on slaughtering facilities .A follow-up survey was conducted in February 2014 by local government officers during the monthly surveillance activities. Data were collected on the market layouts, positioning of live bird holdings, slaughtering facilities and slaughtering and cleaning practices, as well as implementation of control and biosecurity practices. Subsequently, the physical structure of LBMs was categorized. Since LBMs differ significantly with regard to their layout of slaughtering and live bird storage facilities, the eight most common designs were captured in reference sketches in a participatory approach with government officials involved in the surveillance activities. Markets were then categorized as per the layout sketch that most resembled their structure.https://www.wunderground.com). Furthermore, data on the specific characteristics of each pooled sample were recorded: if samples were obtained from broilers only, if at least one sample of the pool was obtained from a Kampung chicken, if at least one sample of the pool was obtained from a duck, if samples were obtained from the slaughter or from the display area only. Thus a total of 29 risk factors were compiled.A total of 22 risk factors potentially influencing HPAI H5 virus prevalence were compiled from the survey data. This included data on market characteristics , data on the poultry management at the markets , density of poultry retail and wholesale markets and average distance between market and location from where poultry was supplied to the market (kilometre). In addition, total monthly rainfall (mm) data for each month in the study period for Jakarta was sourced from “Weather Underground”  were used to account for this within-subject correlation. GEE models are a suitable solution for longitudinal panel data where multiple results are recorded for one subject and are therefore likely to be correlated, like the HPAI H5 virus status of sampled LBMs over time. The correlation matrix in GEE models represents the within-subject dependencies and parameter estimation is conducted using an iterative quasi-likelihood estimation. We used an exchangeable correlation structure in our model as it produced the best fit, although GEE models are very robust to correlation misspecification . A weighWe started the analysis with a univariate analysis. Variables with p<0.1 in the univariate analysis were considered for the multivariable analysis. We also evaluated correlations between variables using the Pearson correlation coefficient for continuous variables and tetrachoric and polychoric correlations for dichotomous and categorical variables. If two predictors were highly correlated (>0.7), only one of the variables was used in the multivariable model. We used a backward selection processes to develop the final model with predictors significant at p<0.05. Generalised joint Wald tests were used to test the significance of each fitted categorical variable with more than two levels. All reported p-values are two sided. Stata 14.0  was used for the statistical analyses. Categorized period prevalence values were plotted for each market location using ArcGIS 10.4 (Esri Inc).A total of 4,213 pooled LBM samples were collected from 86 LBMs between March 2009 and July 2014. Although all the 86 LBMs should have been sampled every month, in practice not all were sampled monthly. Over the study period of 65 months, a single LBM was sampled on average 51 times ranging from 17 to 59 times. Complete risk factor information and diagnostic data was available for 79 LBMs, representing 3,579 pooled LBM samples, which were used for the analysis. LBMs were trading on average 4,739 birds per day ranging from 65 to 102,900. The average number of vendors per market was 17, ranging from 1 to 64.nd half of the year, peaking between October-February in 2009, 2010, 2011 and also in 2013. The seasonal peak of the October-February period in 2011 was lower than in the previous years (by only about 20%). As LBM sampling was infrequently conducted in the 2nd part of 2012, clear seasonal patterns could not be observed. During the high risk period of 2013, LBM H5 virus prevalence again reached the 2009 values of about 50%.Over the whole study period 36.7%   LBM samples tested HPAI H5 virus positive. The number of LBMs tested and the monthly HPAI H5 virus prevalence  are displayed in There was a considerable variation between districts  with JakThe proportion of HPAI H5 virus positive and negative samples, the odds ratios for being H5 virus positive  and the p-values for all risk factors evaluated are displayed in Samples from slaughter area only (correlated with Slaughter at the market) and Market location (correlated with Density of poultry retail markets in the district and Average distance between market and origin of poultry sold at market) and Human population density) and Density of poultry wholesale markets in the district (correlated with Human population density).After exploring bivariate correlations between potential risk factors, we excluded one of the two risk factors with a correlation of larger than 0.7. These included The final multivariable model included risk factors related to the market characteristics, the poultry management on the market, the sampling conducted at the market and some environmental factors .Markets where only carcasses were sold, but no slaughtering was conducted at or near these markets (market layout: SALE 2 only), had a significantly reduced chance of being positive for H5 virus (OR = 0.2 (95% CI 0.1–0.5), p = 0.002). Also, markets where live bird sales were conducted outside the LBM, and slaughter and carcass sales were conducted in separated areas inside the LBM (market layout: F) had marginally lower odds of being infected with HPAI H5 virus (OR = 0.4 (95% CI 0.2–1.1), p = 0.071). Interestingly, markets where parent stock was traded or slaughtered , were more at risk of being H5 virus positive compared to markets where broilers were traded (OR = 5.7 (95% CI 3.6–9.2), p<0.001). Markets, that used display tables for poultry carcasses made from wood, had reduced odds of being H5 virus positive (OR = 0.7 (95% CI 0.5–1.0), p = 0.047), while having at least one duck sample included in the pool of samples collected at a market increased the chance of the sample being H5 virus positive (OR = 5.7 (95%CI 3.6–9.2), p<0.001). Finally, the human population density in the district, the average distance between market and the origin of the poultry sold at market and total rainfall per month were all positively associated with higher H5 virus prevalence.Our study aimed to identify risk factors for HPAI H5 virus prevalence, by evaluating long-term surveillance data collected at urban live bird markets in Indonesia. Our results highlight the role of the poultry trading chain and its impact on the infections status of markets.There is a strong relationship between human population density and the risk of markets being virus positive, also highlighted by the fact that district Jakarta Timur, with the highest population density in the Greater Jakarta region, had also the highest H5 virus prevalence over the study period. Human population density has been previously described as a risk factor when identifying HPAI in backyard village chickens through participatory disease surveillance in Indonesia , but detIn our study, we identified a strong correlation between human density and wholesale market density and therefore only included human population density in our multivariable model. However, the density of wholesale markets is likely to be an indicator for density of settlements and people living in an area as well as an indicator for connectivity between traders. The closer the wholesale markets are to each other, the shorter the transport time for poultry being traded between markets. It is also likely that the same traders will visit multiple wholesale markets to sell birds. Due to the large number of birds being traded at wholesale markets, each market will ‘provide’ a substantial source of new susceptible birds that can be infected by HPAI H5 virus. This association was recently highlighted by Gilbert et al. 2014, who found that market density was the most important predictor for market H7N9 prevalence [We also identified that the distance between the area where poultry originated and the LBM increased the chance of a LBM being infected. This observation supports the importance of disease spread during trading, heightened through many players being involved in the trade, as is the case with backyard chickens. The longer the distances and the more frequent the re-assortment of poultry, the more time the virus has to find new susceptible hosts.Higher monthly rainfall also contributed to an increased chance of markets testing H5 virus positive. In wet conditions, the virus can survive for several days and this has been previously demonstrated to be of importance for virus persistence in LBMs . This suOverall, market layouts that did not have slaughtering conducted in the direct vicinity of the market had lower odds of being H5 virus positive (SALE 2 type of markets). During the slaughter process, body fluids are released and contaminate surface areas, floors and equipment providing a source for cross-contamination of H5 virus. Confining the slaughter to a separate location at the LBM, or even better far away from the LBM, where neither live poultry or displayed carcasses are present, is likely to reduce the risk of H5 spread and the infection risk for consumers.Interestingly, there was a lower chance of samples being positive when display areas were made of wood. Although a previous study identified wooden tables as a risk factor for H5 virus market positivity , it did Finally, our results demonstrated that if pooled samples collected contained at least one duck sample, then the chance of a market being H5 virus positive was also increased. The role of ducks with unapparent clinical signs of HPAI infection in the maintenance of HPAI H5 virus infection has being highlighted previously , 28 and Our study used long term surveillance data in combination with data collected during cross-sectional studies. This implies that some risk factor variables were not regularly evaluated. It is possible that some biosecurity practices might have varied throughout the study period, but most risk factors we evaluated would have stayed the same . However, other risk factors such as rainfall and type of samples were actually measured repeatedly throughout the study period . We did not have suitable data available on the dedicated cleaning and disinfection programmes conducted at these markets. Cleaning and disinfection may reduce the virus survival in the market somewhat, but is unlikely to counter the effect of constant virus influx. Similar observations were made on LBMs in Bangladesh and New York respectively , 29. IndWhile our study design was not that of a conventional case-control study, it showed that surveillance conducted over a long time period can provide valuable insights into the risks associated with HPAI H5 infection at LBMs. Furthermore, our analysis included 79 out of the total of 260 LBMs in the Greater Jakarta area, which is approximately a third of all LBMs. This large geographical coverage strengthens the value of our findings, especially considering that markets had been tested on an average 51 times during the study period.Overall, our diagnostic results mirror findings from other studies on LBMs in Asia and all significant risk factors identified are epidemiologically plausible. We therefore believe that our results can be extrapolated to countries with similar poultry trading systems.In summary, our results indicate the reason that LBMs posing a high risk of H5 infection is a combination of virus accumulation at the markets, spread of virus during the trading process and environmental pressures. For effective risk reduction of H5 virus infection at urban LBMs, all three aspects need to be considered. Possible ameliorative measures could include the regulation of poultry trading processes, the relocation of slaughter areas at LMMs to well-managed separate locations and the relocation of wholesale poultry markets outside urban areas.Data collection for this study was carried out according to the guidelines of the Laboratory of Animal and Fish Health Center (BKHI) Jakarta, Indonesia and was approved by Directorate General of Livestock Services, Indonesia. Market vendors were informed that their participation was strictly voluntarily and verbal consent for the interviews was obtained. The survey responses used in this study were anonymized by the authors. Since all activities were carried out during routine market surveillance no live animals were sampled and no additional animal or human ethics approval was required.S1 Table(DOCX)Click here for additional data file.