Apis mellifera), the Eastern honey bee (Apis cerana) and bumble bee species (Bombus spp.). Bee antiviral defense mechanisms include RNA interference (RNAi), endocytosis, melanization, encapsulation, autophagy and conserved immune pathways including Jak/STAT , JNK , MAPK (mitogen-activated protein kinases) and the NF-κB mediated Toll and Imd (immune deficiency) pathways. Studies in Dipteran insects, including the model organism Drosophila melanogaster and pathogen-transmitting mosquitos, provide the framework for understanding bee antiviral defense. However, there are notable differences such as the more prominent role of a non-sequence specific, dsRNA-triggered, virus limiting response in honey bees and bumble bees. This virus-limiting response in bees is akin to pathways in a range of organisms including other invertebrates , as well as the mammalian interferon response. Current and future research aimed at elucidating bee antiviral defense mechanisms may lead to development of strategies that mitigate bee losses, while expanding our understanding of insect antiviral defense and the potential evolutionary relationship between sociality and immune function.Bees are important plant pollinators in both natural and agricultural ecosystems. Managed and wild bees have experienced high average annual colony losses, population declines, and local extinctions in many geographic regions. Multiple factors, including virus infections, impact bee health and longevity. The majority of bee-infecting viruses are positive-sense single-stranded RNA viruses. Bee-infecting viruses often cause asymptomatic infections but may also cause paralysis, deformity or death. The severity of infection is governed by bee host immune responses and influenced by additional biotic and abiotic factors. Herein, we highlight studies that have contributed to the current understanding of antiviral defense in bees, including the Western honey bee ( There are over 4000 bee species in the order Hymenoptera, including those that are social or solitary, native or introduced, managed or wild . Bees arApis mellifera) are eusocial, cavity nesting bees that are native to Europe, Africa and the Middle East; they were introduced into North America in the late 1600s [Apis cerana) is a related but distinct species endemic to Asia and detected in Australia in 2007 [Western honey bees . The ma in 2007 ,11. Sinc in 2007 ,15,16,17 in 2007 ,22,23,24Bombus terrestris and Bombus impatiens, are also important agricultural pollinators of crops such as tomatoes and peppers, as well as blueberries and other ecologically important plant species [B. terrestris) rear one generation per year [Nosema bombi) and trypanosomatid  infections [Bombus terrestris, Bombus cryptarum, and Bombus pascuorum) obtained from several locations in Belgium identified several known bee infecting viruses , Varroa destructor virus-1 VDV-1/DWV-B, DWV), including potentially different strains as well as numerous new bee-associated viruses including (+)ssRNA, (−)ssRNA, and dsDNA viruses [Bumble bees, including  species . Bumble  species . Unlike per year . This meper year ,29. Therper year ,35,36,37fections ,40,41,42fections ,46,47,48 viruses . Future  viruses . Virus i viruses ,53,54,55Megachile rotundata), blue orchard or mason bees (Osmia lignaria), and many other native and wild bee species are important plant pollinators. Some are generalist pollinators, whereas others are specialist pollinators that primarily interact with one or just a few plants  in Belgium resulted in strong support for bee macula-like 2 virus infection of A. haemorrhoa and detection of several new partial virus genomes including a nege-like virus and a toti-like virus in A. haemorrhoa and O. cornuta, respectively [Solitary bees including alfalfa leaf cutter bees (iewed in ). Intereiewed in ,58,59,60iewed in ). Therefiewed in , as welliewed in . For exaectively .A current focus of bee virus research is investigating intra- and inter- genera transmission of viruses. Though difficult to investigate, phylogenetic analyses of virus genome sequences obtained from co-foraging bee hosts have indicated that viruses are bidirectionally transmitted between managed and wild bee species ,63,64,65Apis mellifera) are the most investigated bee species and thus the majority of bee-infecting viruses were discovered in honey bee samples. Most bee viruses are positive-sense single-stranded RNA viruses with approximately 30 nm diameter icosahedral capsids. These include Dicistroviruses (black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV)), Iflaviruses (deformed wing virus (DWV), sacbrood virus (SBV), slow bee paralysis virus (SBPV)) and yet-to-be taxonomically classified viruses including chronic bee paralysis virus (CBPV) and the Lake Sinai virus (LSV) group . Recentiewed in ) includiiewed in . To dateiewed in . For a miewed in  and ChenVarroa destructor  [Bee viruses are transmitted vertically within species and horizontally, both within species and between different bee genera ,63,64,70PV, KBV) ,83,84,85PV, KBV) ) and at PV, KBV) ,88. PoorPV, KBV) ,91,92,93PV, KBV) ,93,94,95Virus infections in bees are primarily asymptomatic or they may result in deformity, paralysis, and/or death , and conserved pathways including Jak/STAT, JNK, MAPK, and the NF-κB mediated Toll and Imd pathways  Fi.Drosophila melanogaster [Drosophila melanogaster encodes three NF-κB transcription factors  which are involved in promoting the expression of specific antimicrobial peptides (AMPs) [dorsal and relish) involved in the Toll and Imd pathways, respectively. Honey bees lack dif but encode two dorsal homologues  and there are two isoforms of dorsal-1  [defensin-1, a Hymenoptera-specific AMP hymenoptaecin and both pathways govern expression of abaecin [Signal transduction cascades regulate the expression of genes, including those that are likely involved in limiting honey bee virus infections , thnogaster ,138,139 nogaster ,141. Fors (AMPs) ,142. Hon and 1B) . Toll an abaecin ,144,145. abaecin . The fun abaecin ,148.apidaecin, hymenoptaecin, abaecin, lysozyme and defensins), immune genes -S2, TEPs, prophenoloxidase, eater), transcription factors, components of the RNAi machinery  and differential expression of genes involved in metabolic pathways and cellular differentiation [protein lethal(2)essential for life-like, which encodes a protein in the small heat shock protein (Hsp20) family was differentially expressed in virus-infected bees [Drosophila melanogaster [Anopheles gambiae [There are several reports on the transcriptional level perturbations associated with virus infection and/or mite infestation of honey bees ,151,152.ntiation ,151,152.ted bees . The difnogaster ,154 and  gambiae .Apis mellifera) is the most thoroughly examined virus-bee host pair [Varroa destructor mite parasitization and mite-mediated virus transmission, as well as impairment of the antiviral NF-κB/Dorsal-1A pathway as a result of exposure to neonicotinoid insecticides  [Viruses by Nazzi and Pennacchio [dorsal-1A, both experimentally and naturally, favored virus proliferation [Amel\102, which is an immune gene likely involved in melanization and encapsulation; Amel\102 expression is reduced in DWV-infected bees [Deformed wing virus infection of honey bees  . The comnnacchio . Togetheferation . Furtherted bees ,150. Mitted bees ,150,159.ted bees ,160 and ted bees . Furtherted bees ,161.ATP channels, which respond to metabolic changes in the cell  play a role in limiting viral infection [ATP agonist prior to challenge with FHV survived longer and had reduced virus levels compared to untreated bees or bees fed a KATP antagonist prior to virus challenge [Drosophila melanogaster links metabolic function to antiviral immunity by suggesting that KATP channels control FHV infection in an RNAi-dependent manner [Nutritional status and metabolism also influence the outcome of virus infections in honey bees. Several transcriptome studies of virus-infected bees identified differential expression of genes involved in metabolic processes and bees fed higher quality diets had lower virus loads ,126,150.nfection . Specifihallenge . At least manner . Future t manner , covert t manner , and hont manner .During viral replication, most viruses produce long segments of dsRNA  that are recognized as PAMPs by host dsRNA-binding proteins. Host recognition of viral dsRNA may result in activation of sequence-specific RNAi and/or other antiviral immune pathways  Drosophila melanogaster infected with FHV, which is a positive-sense single stranded RNA (ssRNA) virus [ago-2 were shown to be hypersensitive to viral infection [ Drosophila studies have revealed that hemocytes and transposon-encoded reverse transcriptases are involved in amplification and systemic spread of siRNA-mediated antiviral defense [RNAi is a post-transcriptional sequence-specific gene silencing mechanism that is involved in regulating gene expression in most organisms ,167. TheA) virus . Later, nfection , furthernfection ,181. Rec defense ,183,184.Apis mellifera) antiviral defense was demonstrated in laboratory-based experiments in which bees and/or larvae that were fed virus-specific dsRNA harbored reduced levels of virus, as compared to controls [®) and IAPV determined that dsRNA-treated colonies in one of two locations had larger adult bee populations and produced a greater amount of honey, than colonies that were only fed IAPV, though virus abundance was not assessed [The role of RNAi in honey bee , as compared to mock-infected bees [Further support that RNAi plays an important antiviral role in honey bees is that the expression of ted bees ,126. Howted bees . In addited bees , larvae ted bees ,194, pupted bees ,195, andted bees ,198,199 hopscotch in the Jak/STAT pathway, toll-10 and tube in the Toll pathway, pirk and jra in the Imd pathway, antimicrobial peptides , numerous heat shock response genes, dicer and ago-2 and scavenger receptor class c, which is involved in dsRNA uptake in Drosophila; most DEGs were functionally uncharacterized [dicer, NCBI GeneID 552127) and a probable cyclin-dependent serine/threonine kinase (NCBI GenBank MF116383) was confirmed in individual bee gene knock-down experiments. Reduced expression of either dicer or MF116383 resulted in greater virus abundance [Drosophila melanogaster Dicer is involved in both specific and non-specific dsRNA responses including activating expression of the antiviral gene vago [Drosophila is not yet known. In mosquitos, Vago is a secreted protein that limits virus infection by linking the siRNA and Jak/STAT pathways through an unknown receptor [Vago expression is increased in DWV-infected honey bees [vago is not universally up-regulated in response to virus infection in honey bees  [While sequence-specific RNAi is an important honey bee antiviral defense mechanism and experimental gene silencing tool, dsRNA also triggers a general non-sequence-specific antiviral response . Incterized . The virbundance . In Drosene vago , though receptor ,201,202.ney bees , though P, IAPV) ,126.Recognition of non-self virally-produced dsRNA is the first step in activation of an antiviral state in many organisms. Cytosolically-located dsRNA recognition proteins include DExD (Asp-Glu-x-Asp) box helicases, such as Dicer, Retinoic acid-inducible gene I (RIG-I), and Melanoma differentiation-associated protein-5 (MDA-5), Protein kinase R, and endosomal Toll-like receptor 3 in diverse organisms including invertebrates and mammals ,205,206.B. terrestris) RNAi machinery  exhibits slightly increased expression in the context of virus infection  [B. terrestris [dicer-2 expression in B. terrestris did not result in increased abundance of IAPV or SBPV [Bumble bee and honey bee lineages diverged approximately 90 million years ago . Similarnd SBPV) ,189. Incrrestris ,208,209. or SBPV . IAPV, w or SBPV ,189, the or SBPV  and as e or SBPV  was not Bombus terrestris) were fed IAPV only, or IAPV and either virus-sequence specific dsRNA or non-sequence-specific dsRNA  for six consecutive days, beginning three days prior to virus-inoculation [Similar to honey bees, viral dsRNA triggers both sequence-specific RNAi and non-sequence specific virus limiting responses in bumble bees, though the non-sequence specific pathway may be even more important for bumble bees ,47,189. BtSVC-vago  vago) expression was reduced in IAPV-infected bees and not affected in SBPV-infected bees, unlike the observed up-regulation of this important antiviral gene in Drosophila melanogaster [BtSVC-vago expression did not impact IAPV or SBPV abundance in the abdomen [BtSVC-vago reduction resulted in greater IAPV abundance in the fat body [Dmvago by DmDicer-2, BtSVC-vago expression is governed by BtDicer-2 [BtSVC-vago expression did not impact Btdicer-2 or Bthop levels in fat bodies and thus to date a connection between BtSVC-vago expression and Jak/STAT activation has not been established [Bthop resulted in higher SBPV load 48 h post-infection, implicating the Jak/STAT pathway in bumble bee antiviral defense [BtSVC-vago regulates expression of four AMPs  remains to be elucidated [In addition to dsRNA-mediated antiviral responses, there are other immune pathways that likely play important roles in antiviral defense in bumble bees. Targeted analysis of canonical insect immune gene expression in virus-infected bumble bees unexpectedly indicated that nogaster . Experim abdomen , howeverfat body . SimilartDicer-2 . Reductiablished . However defense  (reviewe defense ) . The extent to which these pathways are involved varies for each co-evolved virus—host pair. Overall the role of RNAi is greater in plants, fruit-flies, and mosquitos, while the role of other immune pathways including Jak/STAT, JNK, Toll, and non-sequence specific dsRNA-triggered pathways are more important in bees, oysters, and mammals. Importantly, these generalities are only true for the specific species that have been studied including Arabidopsis thaliana, Xanthomonas oryzae, D. melanogaster, A. gambiae, A. mellifera, B. terrestris, C. gigas, Mus musculus, Homo sapiens, and others.RNA virus infection results in the production of dsRNA molecules  in host cells. This viral pathogen PAMP is recognized as non-self by PRRs across diverse taxa, including plants, invertebrates  and mammals ,219,220.The antiviral response(s) that are most important for any particular host-virus pair cannot be assumed based on broad organismal classification —each must be empirically determined. Since all host-virus interactions are inexorably complicated by the history of conflict and evolution shared (or not shared in the case of model viruses) by the host and a virus. These unique histories are reflected in the differential transcriptional responses and extent of parallel activation and/or cross-talk between host immune pathways, as well as identification of the virus-evolved counter defense mechanisms, including suppressors of RNAi and other immune pathways ,221.Drosophila melanogaster [Viruses focused on “Antiviral Defense in Invertebrates”, may help guide these studies.The parallels that exist between the antiviral responses, including the general, non-sequence specific dsRNA-triggered induction of an antiviral state, in organisms separated by large evolutionary distances including honey bees, bumble bees, sand flies, shrimp, oysters, and mammals, are very intriguing. Furthermore, it is interesting to hypothesize that in social bees this response may have evolved to rapidly respond to viruses and limit their transmission in the crowded hive environment and in the context of behaviors  that promote virus transmission between individuals within the super-organism. Investigation of antiviral defense in some of the thousands of under-explored bee species, will further our understanding of the general and specific mechanisms that bees have evolved to combat specific viruses. This short review highlights studies that have contributed to our current understanding of bee antiviral defense mechanisms. Under-explored, burgeoning research areas include elucidation of the roles of alternative splicing ,222, epinogaster ,183,184)


The inoculated chicken mortality rate in the A46 and Q29 groups was 1/5 and 5/5, respectively. Q29 virus replication was more efficient compared to the A46 virus in inoculated chickens. Infected chickens initiated viral shedding to naïve contact chickens through respiratory and digestive routes. Both viruses transmitted between chickens by naïve contact, but the Q29 virus had a higher pathogenicity in contact chickens than the A46 virus. Compared with early H7N9 LPAIVs, the pathogenicity and transmissibility of the emerging H7N9 HPAIV was stronger in chickens, indicating that H7N9 influenza virus may continue to threaten human and poultry health.There were five outbreaks of H7N9 influenza virus in humans in China since it emerged in 2013, infecting >1000 people. The H7N9 low pathogenic influenza virus was inserted into four amino acids in the HA protein cleavage site to mutate into the H7N9 highly pathogenic virus. This emerging virus caused 15 outbreaks in chickens from the end of 2016 to date. Two H7N9 avian influenza virus (AIV) strains, A/chicken/Guangdong/A46/2013 (LPAIV) and A/chicken/Guangdong/Q29/2017 (HPAIV), were selected to compare the pathogenicity and transmissibility between H7N9 LPAIVs and HPAIVs in chickens. We inoculated 3- to 4-week-old specific-pathogen-free (SPF) chickens with 6 log Orthomyxoviridae and genus Influenzavirus A [Avian influenza is an infectious disease that is caused by avian influenza virus (AIV) and it infects poultry and various species of wild birds. AIV is a single-stranded negative-sense RNA virus belonging to the family avirus A . The viravirus A . To dateavirus A ,3,4,5. AIn February 2013, a new H7N9 influenza virus first infected a man in Shanghai, China and was identified as a low pathogenic influenza virus ,7,8. As From November 2013 to the end of 2016, H7N9 had over three waves in China. In June 2016, four amino acids from H7N9 LPAIVs were inserted into the HA protein cleavage site to mutate it into the H7N9 HPAIVs . Since tTo better understand the differences of the pathogenicity and transmissibility between the H7N9 LPAIVs and HPAIVs, we purified and propagated two viruses, A/chicken/Guangdong/A46/2013(H7N9) (A46), which is representative of the H7N9 LPAIVs from 2013, and A/chicken/Guangdong/Q29/2017(H7N9) (Q29), which is representative of the H7N9 HPAIVs from 2017, to perform the infection experiments.The two H7N9 AIVs, A/chicken/Guangdong/A46/2013(H7N9) (A46) and A/chicken/Guangdong/Q29/2017(H7N9) (Q29), were isolated from swabs of chickens in live-bird markets in Guangdong during 2013 and 2017, respectively. These viruses were propagated using 9 to 10-day-old embryonated specific-pathogen-free (SPF) hen eggs through two-to-three rounds of the limiting dilution method ,19. We t10EID50 of the A46 virus in a volume of 100 μL via the ocular-nasal route, and this was the inoculated A46 group. Three chickens, including dead and euthanized ones, in the inoculated A46 group were dissected to collect the tissues at 1 and 3 days post-infection (dpi), respectively. The remaining five chickens were observed daily for clinical symptoms and mortality until 14 dpi or until all of them died of viral infection. Five chickens in the A46 group were inoculated with 100 μL phosphate buffered saline (PBS) via the ocular-nasal route and co-housed with the inoculated A46 group to assess the transmission efficiency between the inoculated chickens and contact chickens, and this was the contact A46 group. Sixteen chickens from the Q29 group were treated and grouped in the same way as above. In addition, we raised 11 3- to 4-week-old SPF chickens alone as the control group. All control chickens were inoculated with 100 μL PBS via the ocular–nasal route and were observed for clinical symptoms and mortality for 14 days.To investigate the pathogenicity and transmission of the H7N9 AIVs, A46 and Q29, in chickens, we designed our animal experiment according to previous studies ,21. We rThe chickens’ tissues including brain, kidneys, livers, lungs, spleen, trachea, ileum, bursa of Fabricius, and pancreas were collected from three chickens that were dissected randomly from the dead and euthanized chickens in the inoculated A46 and Q29 group at 1 and 3 dpi to assess the virus replication, respectively. Oropharyngeal and cloacal swabs were collected from all the chickens every other day to detect viral shedding until 13 days or until all the chickens were dead. All samples were homogenized in 1 mL of cold PBS and titrated for virus infectivity in 9- to 10-day-old SPF embryonated hen eggs. Virus titers were calculated using the Reed and Muench method .All experiments were carried out in ABSL-3 facilities in compliance with approved protocols (SCAUABSL2017-006) by the biosafety committee of South China Agriculture University. All animals were handled in accordance with the approved guidelines of the Experimental Animal Administration and Ethics Committee of South China Agriculture University .One inoculated chicken in the A46 group showed mild clinical symptoms such as depression and ruffled feathers, and one died of viral infection at 6 dpi; thus, the mortality of the inoculated A46 chickens was 1/5 . However10EID50 at 1 dpi. The virus was detected in all collected tissues, and the mean virus titers were 1.75–4.92 log10EID50 at 3 dpi . Consequently, Q29 virus replication was more efficient than that of the A46 virus in inoculated chickens.To assess the replication of H7N9 AIVs in the inoculated chickens, the brain, kidneys, livers, lungs, spleen, trachea, ileum, bursa of Fabricius, and pancreas were collected at 1 dpi and 3 dpi, respectively. In the inoculated A46 group, virus was detected in the kidneys, livers, lungs, trachea, and pancreas, with mean virus titers of 1.58–3.17 logat 3 dpi . In the 10EID50 at 1 dpi, 3 dpi, and 5 dpi, respectively. Virus titers in the cloacal swabs were 2.38, 3.75, and 2.13 log10EID50 at 1 dpi, 3 dpi, and 5 dpi, respectively. However, Q29 inoculated chickens’ swabs were collected at 1 and 3 dpi because all the chickens died within 3 days. The virus titers in the oropharyngeal swabs were 2.38 and 2.88 log10EID50 at 1 and 3 dpi, respectively. In addition, the virus titers in the cloacal swabs were 2.21 log10EID50 at 1 dpi and 3.13 log10EID50 at 3 dpi  were healthy without exhibiting any clinical signs and the virus was only detected in the trachea. Shedding of the virus was only detected in oral swab samples at 3 dpi, indicating that viral replication was limited to the upper respiratory tract . Zhang ehe virus . Jacob ehe virus . Jiao etnd 5 dpi . Here, wnd 5 dpi ,28, (2) 10EID50/0.1 mL of A/chicken/Guangdong/SD008/2017 (H7N9 HPAIV) died within 4 days. The virus replicated systemically in chickens and was detected in both pharyngeal and cloacal swabs [10EID50/0.1 mL of these H7N9 HPAIVs, causing all the infected chickens to die [Shi et al. found that all 6-week-old chickens intranasally inoculated with 6 logal swabs . Wang ets to die . Here, w6 TCID50 (50% tissue culture infective doses) of the A/Anhui/1/2013 (H7N9), the contact chickens could shed the virus through the respiratory route at 2 to 8 days postexposure, indicating successful transmission of the virus [10EID50 of the A/Anhui/1/2013 (H7N9) was used to intranasally inoculate chickens, Vidaña et al. found that no viral shedding was tested in the oropharyngeal and cloacal swabs of the contact chickens [10EID50 of the LPAIVs A/chicken/Guangdong/110/2013 and A/chicken/Guangdong/134/2013. They observed that no contact chickens showed obvious symptoms during the observation period, but both viruses were present in the oropharyngeal and cloacal swabs from the contact chickens [10EID50/g/0.1 mL, respectively [Numerous studies have shown that different H7N9 viruses exhibit different transmissibility. When 8-week-old chickens were co-housed with the chickens that were oculonasally inoculated with 10he virus . Howeverchickens . The abochickens . Jiao etchickens . In thisectively . Our resIn summary, both the early and emerging H7N9 AIVs could be detected in infected chickens, and the infected chickens initiated viral shedding to naïve contact chickens through respiratory and digestive routes. However, compared with the early H7N9 LPAIVs, the pathogenicity and transmissibility of the emerging H7N9 HPAIVs are stronger in chickens, suggesting that the H7N9 influenza virus may continue to threaten human and poultry health.


Culicoides spp. and susceptible hosts and cattle might provide the ideal host for reassortment to occur.Bluetongue is primarily a disease of sheep in South Africa, while cattle and goats are mostly subclinically infected. The viraemia of bluetongue virus in cattle lasts much longer than in sheep and the role of cattle in the epidemiology of bluetongue in South Africa is poorly understood. Bluetongue virus has a segmented double-stranded ribonucleic acid genome and reassortment of genomes is a common feature. The aim of the study was to investigate whether reassortment occurs between vaccine and field strains when simultaneously administered to cattle. Six cattle between the ages of 6 and 12 months were infected with five strains of modified live vaccine bluetongue virus and a virulent field isolate of bluetongue virus 4. Blood samples were subsequently collected daily from these animals from day 1 to day 39 post-inoculation. Viruses were directly isolated during viraemia from the buffy coat on Vero cells using the plaque forming unit method. Analysis of plaques indicated that no reassortants between virulent field and vaccine strains occurred and the virulent bluetongue virus 4 was identified as the predominant virus strain. However, a reassortant virus between two bluetongue virus vaccine strains was isolated from the buffy coat. Whole genome sequences from the vaccine viruses were compared to the suspected reassortant and it was found that segment 8 exchanged between the bluetongue virus 8 and bluetongue virus 9 vaccine strains. The use of the live-attenuated bluetongue virus multivalent vaccine in South Africa causes circulation of different vaccine serotypes in  Orbivirus that belongs to the family Reoviridae. The virus genome consists of 10 segments of double-stranded ribonucleic acid (dsRNA) that codes for seven structural viral proteins (VPs: VP 1 – VP 7) and five non-structural proteins (NS) as illustrated in Bluetongue (BT) is a non-contagious viral disease of domestic and wild ruminants with serious socio-economic impacts. Infection with bluetongue virus (BTV), the causative agent, results in clinical disease in cattle and sheep, including reproduction losses and consequently international movement restriction of ruminants  is the most abundant in the infected cells and forms tubular structures in the cell cytoplasm; the NS 1 is also a positive regulator of the virus’s expression that increases protein synthesis  and the occurrence of the disease depends on the presence and the abundance of competent vectors  Gerdes . BluetonIn SA, BT is primarily a disease of sheep and controlled by annual vaccination using a freeze-dried polyvalent modified live vaccine (MLV) (Reg. No. G 358 Act No 36/1947) produced by Onderstepoort Biological Products (Pty) Ltd. The vaccine consists of three bottles  containing five BTV strains each: Bottle A includes BTV serotypes 1, -4, -6, -12 and -14; Bottle B includes serotypes 3, -8, -9, -10 and -11; Bottle C includes serotypes 2, -5, -7, -13 and -19  from an SA BTV 2 MLV strain . The titres of the different virus serotypes in the vaccine are not indicated but according to Modumo and Venter , the min5.8 per mL TCID50 (1 mL). This virus was previously isolated from ethylenediaminetetraacetic acid-containing blood (EDTA) taken from a clinically sick ewe. All viruses were characterised by whole genome sequencing before infection  . Appetite and rectal temperatures of the animals were monitored daily.® method according to the method prescribed by the manufacturer . Single-stranded RNA was removed from the total RNA by precipitation with 2 M lithium chloride  were performed according to the methodology of Howell et al. . Total RWhole genome sequencing  was performed on control and 11 possible reassortant samples as identified by PAGE and using full-length amplification of complementary DNA as previously described by Potgieter et al. . SequencAnimal ethics clearance was obtained from the Ethics Committee of the University of Pretoria for the experimental work. Section 20 approval according to the Animal Disease Act, 1984, was obtained from the Department of Agriculture, Forestry and Fisheries. Animals were only released from the insect-free stables when virus could not be isolated from the collected blood as stipulated by the Department of Agriculture, Forestry and Fisheries.Daily monitoring revealed that two of the six cattle had slightly elevated temperatures of 39.1 °C and 39.4 °C on day 1 post-inoculation. Both the animals had no appetite on that specific day but returned to normal by day 2 post-inoculation. No further signs of distress were noted for any of the animals throughout the remainder of the trial. Blood samples were collected daily from the animals from day 1 to day 39 post-inoculation and viraemia was detected between day 2 and day 32 in four of the animals and in the two other animals viraemia could be detected until 39 days post-inoculation using virus isolation. Seroconversion was confirmed using a commercially available competitive ELISA ; all cattle tested negative on day 0 and seropositive on days 13, 15, 17, 19 and 21.Parental strains from the vaccine bottle and the BTV 4 field strain each demonstrated unique electrophoretic profiles and it was possible to distinguish between genome segments based on their PAGE migration profiles as illustrated in n = 11) with the vaccine strains and the BTV 4 wild-type revealed that nine of the 11 samples analysed clustered with the wild-type BTV 4 strain in which all 10 segments were identical to the 10 segments of BTV 4. Two of the viral isolates obtained from blood samples (samples 2b and 6b, Comparison of genomic sequences of each of the suspected reassortant isolates (Cattle in SA are normally not affected by BTV outbreaks. Clinical signs have been reported in only two outbreaks of BT in cattle: in 1996–1997 in the Delareyville District in the North-West Province and in Donkerhoek in Bronkhorstspruit District, Gauteng Province (Barnard, Gerdes & Meiswinkel During this trial, cattle showed no adverse effects of stress, with the exception of two of the cattle that were inoculated with the vaccine and the wild-type virus and developed a mild fever and loss of appetite for 1 day. Animals were viraemic from 2 to 39 days post-infection. No virus could be detected in four animals after day 33 post-infection using virus isolation, but two animals were viraemic up to 39 days post-infection. Only one of the animals with the prolonged viraemic period showed some mild signs as discussed earlier. This is in agreement with previous studies in which it has been reported that cattle can be viraemic for periods between 14 and 100 days post-infection (Sellers & Taylor When the isolated viruses were compared to each other and to the vaccine strains using PAGE, the majority of the samples were similar to BTV 4 the wild-type strain. This might be because BTV 4 was inoculated intravenously resulting in high titres of BTV 4 in the blood compared to the vaccine serotypes that were inoculated subcutaneously according to the guidelines of the manufacturer. Another reason might be that the fastest multiplying viruses showing plaques on cell cultures were preferentially selected; thus, the virulent field strain was isolated rather than the attenuated vaccine strains (Dungu et al. This study illustrated the generation of reassortant viruses between two vaccine strains of BTV in cattle. Segment 8 of MLV BTV 8 reassorted with MLV BTV 9. Segment 8 codes for the NS 2 which is mainly responsible for assembly of the virion (Kar et al. Whole genome sequences were performed on 116 BTV isolates from Europe, the Mediterranean region and African countries collected during 1958–2012. The isolates were compared to four MLV serotypes and compared to the 26 serotypes available on GenBank, which revealed reassortment between field isolates and vaccine strains as well as reassortment between field isolates (Nomikou et al. The impact of BTV reassortment on the epidemiology of the disease in SA has not been studied, but reported studies indicated that it is possible that new emerging viruses can infect novel species (Purse et al. It is not possible to predict to what extent the characteristics of new emerging viruses can change and what the limitations of such changes will be. The new virus can be more pathogenic especially if reassortment results in reversion to virulence, can cross the placenta increasing its overwintering capabilities, or susceptibility to the vector resulting in more efficient spread of the virus (reviewed by Wilson, Darpel & Mellor Culicoides midges creates the perfect opportunity for emergence of new viruses by means of reassortment (Veronesi et al. The live-attenuated vaccine serotypes introduced by annual vaccination of BTV and the presence of the 21 endemic BTV serotypes in SA increase the co-circulation of viruses and provide multiple opportunities for the establishment of BTV reassortants Gerdes . The pro


Viral diseases are a matter of great concern for poultry farmers in Pakistan. Multiple common viral respiratory diseases (CVRDs) cause huge economic losses in the poultry industry. The prevalence of CVRDs in many countries, including Pakistan, is not clearly understood.Incidences of 5 chicken respiratory viruses: avian influenza virus (AIV), Newcastle disease virus (NDV/AAVV-1), infectious bronchitis virus (IBV), avian metapneumovirus (aMPV) and infectious laryngotracheitis virus (ILTV) were assessed on commercial Pakistani farms with respiratory problems from 2014 through to 2016. While AIV and AAVV-1 were frequently detected (16 to 17% of farms), IBV and aMPV were rarely detected (in 3 to 5% of farms) and ILTV was not detected. We characterized H9 AIV of the G1 lineage, genotype VII AAVV-1, GI-13 IBV, and type B aMPV strains with very little genetic variability in the 2-year study period. Co-infections with AIV and AAVV-1 were common and wild type AAVV-1 was detected despite the use of vaccines. Control measures to limit the virus burden in chicken flocks are discussed.Our data shows that AIV (H9), AAVV-1, IBV and aMPV are prevalent in commercial poultry in Pakistan. Further studies are necessary to assess circulating strains, economic losses caused by infections and coinfections of these pathogens, and the costs and benefits of countermeasures. Furthermore, veterinarians and farmers should be informed of the pathogens circulating in the field and hence advised on the use of vaccines. Commercial poultry in Pakistan was established in the early 1960s, representing one of the largest agriculture-based segments of Pakistan’s economy with a significant contribution to national gross domestic product 1.3%) . In the % . In thThe aim of this study was to detect and characterize chicken respiratory viruses found in commercial Pakistan poultry, which is the first step of control measures implementation. Five major chicken respiratory viruses were investigated: AIV, AAVV-1, IBV, aMPV and ILTV.In Pakistan, samples were screened for viral respiratory pathogen nucleic acid within the commercial broiler and layer hen populations. Viral respiratory diseases are common even though vaccines are being used on a small scale in the country. In total, 161 pooled samples from 89 flocks were collected during 2014–2016 from different areas of Pakistan. Among these flocks, there were 15 (16.8%) positives for AIV (H9), 16 positives for AAVV-1 (17.9%), 4 positives for IBV (4.4%), and 2 positives for aMPV (2.2%). No ILTV positive sample was detected in our study , were indicative of high viral virulence. The phylogenetic tree clearly shows that AAVV-1/chicken/Pakistan/11/2014 clusters with sequences of genotype VII viruses   was phylogenetically compared with representatives of the 4 known aMPV genotypes (A to D). The Pakistani sequence clusters with aMPV type B viruses , GI-13 (793B) IBV and genotype B aMPV, with no difference from the summer of 2014 through to January 2016.Avian avulavirus 1 (AAVV-1) is a member of the genus Avulavirus of the family Paramyxoviridae. The AAVV-1 species include multiple strains of avian paramyxovirus 1 (APMV-1) virus. APMV-1 is of particular importance to poultry due to virulent strains which cause Newcastle disease, often referred to as Newcastle disease viruses (AAVV-1 s). At least two classes and 18 genotypes of AAVV-1 have been described. Despite the use of imported vaccines, AAVV-1 still remains the main poultry disease in both commercial and rural chickens flocks of Pakistan . RecentlDirect comparison of the IBV and aMPV findings with previous Pakistani ones is not possible due to the absence of published data. There has been so far no genomic characterization of IB viruses circulating in Pakistan, but there is serological evidence of the pathogen. Ahmed et al.  showed s. In addition, aMPV subtype A and subtype C have been reported in China [High seroprevalence of aMPV in broiler (48%) and breeder (93%) flocks has been reported in Iran . In a rein China –37. AgaiWhile most of the sampled farms did not vaccinate against any of the chicken viruses of interest, putative AAVV-1 vaccine failures were observed in farms 6, 13, 23, 62, 65, 66, 67, and 80 (Table The present study helps to understand the virus burden of Pakistani poultry production systems and highlights frequent AIV/AAVV-1 co-infections. This phenomenon has been observed previously in Bangladesh . This fiThe main limitations of this study are (i) the type of samples collected (no virus isolation is possible from FTA cards), (ii) the thus limited amount of viral nucleic acids, and (iii) the limited knowledge available on most avian viruses circulating in the country for comparison. Further studies are warranted to fully characterize the virus strains and evaluate vaccines efficacy to counter the different pathogens.Our data showed that AIV (H9), AAVV-1, IBV and aMPV were prevalent in Pakistani commercial poultry flocks. Further studies are necessary to assess circulating strains, economic losses due to infections and coinfections, and the costs and benefits of control measures. Furthermore, veterinarians and farmers should be informed of the pathogens circulation in the field and hence advised on the use of vaccines.From July 2014 through January 2016, 89 commercial poultry farms (broiler and layer farms) were sampled at different locations in Pakistan by participating veterinarians  from FTA cards were incubated at 4 °C for 24 h in 1 mL of phosphate- buffered saline (PBS).A comparative pilot experiment using both the QIAamp Viral RNA isolation and DNA isolation kits (Qiagen Germany) according to the instructions of the manufacturer with infectious bursal disease virus  and infectious laryngotracheitis virus  vaccines was performed. Real-time (RT-)PCRs were then carried out to compare the extracted material and the results were as follows: (i) extracting ILTV DNA with the QIAamp viral RNA isolation kit resulted in a loss of 1 Ct value , as compared to the DNA isolated with the DNA extraction kit; (ii) extracting IBDV RNA with the Qiagen DNA extraction kit resulted in a loss of 3 Ct values , as compared to the RNA isolated with the RNA extraction kit. We therefore considered that the loss of sensitivity in detecting the DNA virus was negligible and that we could use the QIAamp Viral RNA isolation kit to extract both RNA and DNA. Viral RNA and DNA from FTA cards were therefore extracted using the QIAamp® viral RNA isolation kit  following the manufacturer’s instructions. RNA/DNA was eluted in 50 μL elution buffer and stored at − 80 °C until further use.2O) controls were included in each reaction.Complementary DNA (cDNA) was generated for RNA viruses from 5 μL of extracted RNA using RevertAid first strand cDNA synthesis kit  following the manufacturer’s protocol. Briefly, 5 μL of total RNA was mixed with random hexamer primers (0.3 μg/μL) and incubated for 5 min at 65 °C. Then, 4 μL of 5x reaction buffer , 0.5 μL of RNase Out , 2 μL of 10 mmol/L deoxynucleoside triphosphate (dNTP) solution , and 1 μL of RevertAid reverse transcriptase  were added. Water was added to reach a final reaction volume of 20 μL. The RT reaction was completed following 10 min at 25 °C then 60 min at 42 °C, and finally 10 min at 70 °C. cDNA was used as the template for PCR amplification. The primers and PCR conditions used for the detection of AAVV-1, IBV, aMPV (types A and B), ILTV, and AIV are listed in Table PCR products were purified using the NucleoSpin®Gel and PCR Clean-up kit  following the manufacturer’s instructions. Purified products were quantified with a Qubit® 2.0 fluorometer . The PCR primers and 10 ng of DNA were used for sequencing in both directions using a Big Dye Terminator v.3.1 cycle sequencing kit (Applied Bio-systems) on a capillary sequencer . In case of nucleotide ambiguity, sequencing was repeated. Sequences generated in the present study were submitted to the EMBL/GenBank database under the accession numbers LT599493 to LT599497.Assembly and analysis of sequence data were conducted using the BioEdit Software version 5.0.9 . Additio


Cryptococcus gattii species complex. The diagnosis can be challenging due to its rarity, unspecific symptoms and long symptomless latency.Cryptococcosis in immunocompetent adults is a rare disease in Europe, mostly induced by members of the C. gattii sensu lato. Molecular genetic analysis identified the involved cryptococcal species as genotype AFLP6/VGII, recently described as Cryptococcus deuterogattii, which is known to be endemic to the west-coast of Canada and the USA. Additional heteroanamnestic information revealed that she had spent her holidays on Vancouver Island, Canada, two years before disease onset, indicating that infection during this stay seems to be plausible.A 49-year-old woman with a three weeks history of headache was admitted to the hospital due to discrete ataxia and impaired vision. Cranial magnetic resonance imaging (MRI) showed a contrast-enhancing mass in the cerebellum. Further investigations detected a slight leukocytosis and a single subpleural nodule in the right inferior lung lobe. The cerebral lesion was surgically removed, and a direct frozen section only showed an unspecific inflammation. In the course of her admission she developed non-treatable cerebral edema and died ten days after surgical intervention. Histopathological examination of the surgical specimen and postmortem evaluation of the lung and the cerebrum demonstrated fungal elements. Molecular identification of the fungal elements in formalin-fixed paraffin-embedded tissue lead to the diagnosis of cryptococcosis induced by C. deuterogattii is a rarely encountered fungal disease in Europe, not particularly associated with immunodeficiency, and infection is likely to be contracted in endemic areas. Due to its rarity, long symptomless latency, unspecific symptoms and misleading radiological features the diagnosis can be challenging. Physicians need to be aware of this differential diagnosis in immunocompetent patients, as early adequate therapy can be lifesaving.Cryptococcosis due to  Cryptococcus gattii infections have increased during the last two decades [C. gattii can cause severe systemic disease in immunocompromised and immunocompetent hosts [Cryptococcal yeasts usually enter the host via the respiratory tract [Cryptococcus neoformans and C. gattii species complexes show a high affinity for the central nervous system [C. gattii species complex can be differentiated into five distinct genotypes [C. neoformans/C. gattii species complexes have been recently thoroughly revised and the five C. gattii species complex genotypes were all raised to species as C. gattii sensu stricto (genotype AFLP4/VGI), Cryptococcus bacillisporus (genotype AFLP5/VGIII), Cryptococcus deuterogattii (genotype AFLP6/VGII), Cryptococcus tetragattii (genotype AFLP7/VGIV) and Cryptococcus decagattii (genotype AFLP10) [Cryptococcus gattii s.s. and C. deuterogattii are often involved in disease in apparently immunocompetent subjects, while the other three species are geographically restricted and have a predilection for immunocompromised hosts, mostly HIV-infected individuals [C. deuterogattii). The outbreak expanded to the Pacific Northwest of the USA, although different subgenotypes were involved [C. gattii s.l. infections. As molecular analysis identified that only half of the infections were acquired abroad [C. gattii s.l. in Germany is an important challenge to assess the associated risk of infection [C. deuterogattii infection that was likely to have been acquired on Vancouver Island two years before the onset of disease.In Western Europe  decades . C. gattnt hosts . Cryptocry tract . Pulmonary tract . Both Crs system . The meds system . By usinenotypes . Due to  AFLP10) . The gloividuals . An ongoinvolved  and has involved . In Germnfection . Here we−1). Cranial magnetic resonance imaging (MRI) revealed a contrast-enhancing lesion with surrounding edema in the left cerebellum hemisphere next to the cerebellar pedunculus . Natural reservoirs for C. gattii are found in Australia, Asia, Africa and some regions of America, including Vancouver Island which has been determined to be an endemic area [C. gattii has been previously isolated in Greece, southern Italy and Spain [C. deuterogattii, formerly known as C. gattii genotype AFLP6/VGII. By using phylogenetic analysis, it was determined that this strain clustered within the clade of Vancouver-Island-outbreak-related C. deuterogattii strains (A 49-year-old woman was hospitalized in the University Hospital Halle/Saale, Germany, with an acute presentation of headache, discrete ataxia and impaired vision. No episodes of pyrexia were reported. Serum inflammatory markers were inconspicuous with only a slight leukocytosis (14.53 Gpt ldunculus a, b. In dunculus . To ruledunculus c, d. Posdunculus a, b. Thedunculus c, d. Crynd Spain . When cond Spain . Subsequ strains , which sCryptococcus is a common yeast fungus with a natural reservoir on decaying wood and bird droppings [Cryptococcus included over 100 species, but after a thorough taxonomic revision only ten remained [C. gattii species complex, which is common in Sub-Saharan Africa, Asia, Australia, Europe, South and North America, can infect persons without immunodeficiency [C. gattii species complex [in vitro antifungal susceptibility varies [C gattii s.l. between 2004 and 2013 [C. gattii s.l. infections might also occur in Northern Europe, probably as a consequence of climate change [C. gattii s.l. within Europe have been described [C. gattii s.l. was detected in Italy, Greece, Portugal, and Spain [C. gattii s.s. without a travel history to endemic regions have been documented [roppings . Until rremained . Membersficiency . As a dificiency . Frequenficiency . Additio complex , even iny varies . Concerny varies . The hypy varies . Accordiand 2013  and survand 2013 . Howevere change . Therefoe change  and endeescribed  and earlnd Spain . Furthercumented . Nonethecumented . In summcumented .C. gattii, which has been formerly known to be a pathogen in tropical and subtropical regions [In Western Europe, increased international travel leads to a rising number of infections with rare pathogens . Additio regions , has bee regions . In cons regions . Early d regions .


The second one is a ZnO/γ-Al2O3 material with intercropped hexagons on its surface. To determine the antimicrobial efficiency of these materials in life-like conditions, a pilot device was constructed that allows the test of the materials in dynamic conditions and agar diffusion inhibitory tests were also conducted. The results of the tests showed that the MnO2/AlPO4/γ-Al2O3 material has a germicidal effect in static conditions whereas ZnO/γ-Al2O3 does not. In dynamic conditions, the oxidizing MnO2/AlPO4/γ-Al2O3 material is the most efficient when using low air speed whereas the ZnO/γ-Al2O3 one becomes more efficient than the other materials when increasing the air linear speed. This ZnO/γ-Al2O3 dark-operating germicidal material manifests the ability to proceed the mechanical destruction of bacterial cells. Actually, the antimicrobial efficiency of materials in dynamic conditions varies regarding the air speed through the materials and that static tests are not representative of the behavior of the material for air disinfection. Depending on the conditions, the best strategy to inactivate microorganisms changes and abrasive structures are a field that needs further exploration as they are in most of the conditions tested the best way to quickly decrease the number of microorganisms.As people spend more and more time inside, the quality of indoor air becomes crucial matter. This study explores the germicidal potential of two dark-operating germicidal composite materials designed to be applied for the indoor air disinfection under flow conditions. The first material, MnO In developed countries, people spend more than 85% of their time in enclosed areas , 2. In tet al. started to explore titanium dioxide TiO2 for bacteria inactivation  m3/h and [4.0–4.25] m3/h. As it was pointed out before, PVC tubes of different widths were also used: 4.5*10−2 m and 1.9*10−2 m of inner diameter. The air linear speeds (m/s) applied during the tests are shown in Several flow rates were applied: [1.0–1.25] mBacillus atrophaeus DSM 675 as the selected bacteria strain. 150μL of this strain were put in 2mL of a solution of modified chopped meat growth medium (American Type Culture Collection medium 1490). The resulting suspension was incubated under continuous stirring at 303 K and 183 rpm for 4 hours. After incubation, a suspension containing approximately 4*105 cells/mL has been prepared in 9g/L NaCl solution (Sigma-Aldrich).Static microbial tests have been conducted using the agar disc diffusion method, with 5 mL of the diluted bacterial suspension was used to seed Tryptic Soy Agar (TSA) Petri dishes. After three minutes, the supernatant was taken out. The tested samples were then placed on the Petri dishes . The Petri dishes were incubated for 22 hours at 304 K. After incubation, the mean inhibition radii were measured using six radius inhibition measures.To follow the evolution of the concentration of microorganisms in the pilot device, the BioTrak 9510-BD was used. Equipped with a laser diffraction detector, this device counts all the particles occurring in the gas phase in the range of diameters from 0.5 to 25 μm  thanks −2 m of inner diameter. During this test series 13 samples were collected by the Biotrak 9510-BD  with a 570 s break before each sampling operation. The whole procedure duration was 2 hours and 30 seconds (7230 s). This first protocol is not suitable for higher linear speeds of the gaseous phase as the decrease of the number of viable particles becomes so fast that it cannot be analyzed with the 2 hours and 30 seconds long protocol. The second protocol was used with the PVC sections having 1.9*10−2 m of inner diameter: 10 samples were collected by the Biotrak 9510-BD  with a 160 s break before each sampling operation. The whole procedure duration was 41 minutes (2460 s). For each of the six velocities applied, the tests were repeated three times to make sure the differences in the results are significant ones. The experiments have also been randomized.Two protocols were used; each one has been applied for different air flow rates.The first protocol was employed when using the polyvinyl chloride (PVC) sections having 4.5*102O3 beads which show a “glob”-like morphology  analyses carried out using back-scattered electrons (BSE) confirmed the presence of a zinc oxide ZnO coating. As shown in rphology . There a2O3 beads. The beads are in average made of 41.9 wt% of ZnO and 46.3 wt% of Al2O3.Together with the SEM analyses, an energy-dispersive X-ray spectroscopy (EDX) analysis was also carried out. 2/AlPO4/γ-Al2O3 beads was determined  adsorption method using the Tristar II PLUS (Micrometrics). The results are displayed in The images of the incubated Petri dishes are displayed in 2/AlPO4/γ-Al2O3 composite material. This implies that neither the non-modified γ-Al2O3 beads nor the ZnO/γ-Al2O3 beads manifest germicidal ability in the selected test conditions.As it can be seen on 2O3 beads are shown in The data resulting from experiments without and with non-modified γ-Al2O3 beads, ZnO/γ-Al2O3 beads and MnO2/AlPO4/γ-Al2O3 beads were tested using the first protocol at 0.2, 0.4 and 0.7 m/s linear air speed. The results are presented in Non-modified γ-Al2/AlPO4/γ-Al2O3 beads manifest higher performance than the two other materials. Indeed, it needs 40 minutes to clean out 95% of the initial quantity of bioaerosols. It is twice as efficient as with the non-coated alumina beads which need 1h20 to achieve the same result. In 30 minutes with a 0.2 m/s air linear speed the air goes through the system twice. In one hour, respectively, it goes through the system four times. When the ZnO/γ-Al2O3 beads are applied, during the first hour the microbial concentration decrease follows practically the same way as non-coated alumina beads. This fact may be due to a partial discharge of the microorganisms remaining alive from the surface of the sample. During the second part of the experiment, probably from the third entrance of the treated air into the circuit, an important decrease of the number of airborne microorganisms was observed.As shown in 2/AlPO4/γ-Al2O3 beads are still the most efficient as they eliminate 95% of the initial microbial population in 30 minutes with a slight discharge at 40 minutes approximately. With the γ-Al2O3 beads, the concentration of microorganisms drops to 5% of its initial value in 30 minutes too but the discharge phenomenon which happens thereafter is more important. ZnO/γ-Al2O3 beads do not manifest a discharge phenomenon and eliminate 95% of the initial population of microorganisms in 50 minutes, which is still less efficient than in the case of MnO2/AlPO4/γ-Al2O3 beads.As illustrated by 2O3 beads become the only material without a discharge phenomenon which inhibits 95% of the airborne microorganisms in 30 minutes. The MnO2/AlPO4/γ-Al2O3 beads make the microbial population decrease down to 5% of its original value in just 20 minutes. However, a discharge then occurs and stability under 5% of the initial population is reached at 1 hour. The γ-Al2O3 beads perform poorly as they reach the 5% threshold for good after 1h20.At 0.7 m/s, the ZnO/γ-Al2O3/AlPO4/MnO2 beads were already foreseen before the series of dynamic tests: in the case of photocatalytic air sanitation which also involves chemical surface reactions, the reactive oxygen species (ROS) generators require to be exposed to the treated media for relatively long periods of time. For instance, at the 0.2 m/s air linear velocity, the contact time between the tested ROS-generating material (MnO2/AlPO4/γ-Al2O3 beads) and the indoor air was 0.2 s. A contact time equal or superior to some tenths of seconds is absolutely necessary both in the case of photocatalytic and dark-operating oxidative antimicrobial air conditioning because the lysis of the bacteria and fungi is dominantly carried out in both cases by the adsorbed hydroxyl radicals HO∙ads.The results obtained with the γ-Al2O3 beads and the MnO2/AlPO4/γ-Al2O3 beads were tested with the second protocol, at 1.0–1.2, 2.0–2.2 and 3.9–4.2 m/s linear air speed. The obtained results are shown in The non-coated alumina beads, the ZnO/γ-Al2/AlPO4/γ-Al2O3 beads and the non-modified γ-Al2O3 beads have similar decrease paths, reaching 95% of viable cells removal at the end of the experiment, whereas the ZnO/γ-Al2O3 beads need 31 minutes and 30 seconds to do the same. The same pattern can be observed when applying a 2.0–2.2 m/s air flow speed. Indeed, when applying a 2.0–2.2 m/s air linear speed, the ZnO/γ-Al2O3 beads eliminate 95% of the initial quantity of bioaerosols in 26 minutes. The two other tested materials need 31 minutes and 30 seconds to do the same. When doubling the air linear speed and reaching 3.9 to 4.2 m/s, the ZnO/γ-Al2O3 beads keep being the most efficient material  and do not manifest any reportable discharge behavior. With the MnO2/AlPO4/γ-Al2O3 beads, the decrease of the microbial population is also 22.5 minutes long but the microbial concentration remains between 4 and 7% of its initial value. The non-modified γ-Al2O3 beads, however, do not achieve 95% of removal efficiency in these conditions.At 1.0–1.2 m/s, the MnOThe obtained results testify major differences in the behavior of the two types of developed dark-operating germicidal materials (DOGM).2-based interactive composites  can be attributed to a significant oxidative capacity of manganese dioxide.The germicidal activity of the MnO2/AlPO4/γ-Al2O3 samples , manganese dioxide crystallizes preferably in β-MnO2, a crystallographic analogue of a natural mineral pyrolusite. The β-MnO2 manifests the properties of n-semiconductor ext–[MnCB(D)–OVB(A)–Al]int and [H∙∙∙∙∙OVB(D)]ext–[MnCB(D)–OVB(A)–P]int cross-linking bonds at the surface. The external spheres of these clusters are constituted with Bronsted acid sites. The subscript “VB(D)” indicates the belonging of all external oxygen anions to the valence band of the donor component. In turn, the internal cluster spheres have mixed electronic structures: the manganese cations constituting the donor’s conduction band (“CB(D)”) are chemically linked to the oxygen anions playing the role of bridging atoms and creating the valence band of the acceptor support (“VB(A)”).Due to the applied method of synthesis, all components of the developed composite material remain chemically bound and form thus new donor-acceptor covalent clusters with [HMnO2) may be considered as free charge carriers (FCC): these electrons are able to move freely from the VBMnO2 to its conduction band (CBMnO2). According to the evaluation results obtained using the Eq ]ext–[MnCB(D)–OVB(A)–Al]int and [H∙∙∙∙∙OVB(D)]ext–[MnCB(D)–OVB(A)–P]int, the FCC part can be noticeably greater due to intensive electron pumping on the side of acceptor supports.Inside of the mentioned donor-acceptor clusters a part of the electrons placed in the manganese dioxide valence band  which are covalent donor-acceptor clusters, have to be energetically best positioned. Inside the FMS, the FCC cannot return to the VBMnO2 because the manganese dioxide HOMO  edge is situated at a considerably higher energy level than the ones of Al2O3 and AlPO4  due to the tunneling effect. Secondly, the appeared FCC are transferred within the internal spheres of the created covalent clusters [H∙∙∙∙∙OVB(D)]ext–[MnCB(D)–OVB(A)–Al]int and [H∙∙∙∙∙OVB(D)]ext–[MnCB(D)–OVB(A)–P]int from the CBMnO2 towards the valence bands of the acceptor components: their HOMO are positioned at lower energies than the HOMO of β-MnO2. The electron holes h+ created in the VBMnO2 by the transfer process mentioned above are significantly more powerful than the holes of pure β-MnO2  ensure therefore their ability to proceed with the elementary reactions (2–4) without any external excitation, only by means of continuous donor-acceptor interactions inside of the covalent clusters [H∙∙∙∙∙OVB(D)]ext–[MnCB(D)–OVB(A)–Al]int and [H∙∙∙∙∙OVB(D)]ext–[MnCB(D)–OVB(A)–P]int. This conclusion is objectively proven by the results of inhibition radius tests . Using the formula (1) on can evaluate a probability of the tunneling effect in ZnO: (U0–E) = 3.73 eV, where U0 = 3.73 eV (the bandgap energetic barrier) [d = 1.98 Å or 0.198 nm  would be carried out at the surfaces of the ZnO/γ-Albarrier) , d = 1.9d in ZnO ), D ≈ 0. samples . These c2O3 composite material are perfectly in keeping with typical bacterial cell dimensions . It is worthwhile to underline that the applied Mecha-DOGM had to operate under harsh dynamic conditions, at an extremely high volumetric flow velocity calculated as the quotient of the gas flow rate divided by the apparent volume of the used active material. This fact tends to prove a high productivity of the ZnO/γ-Al2O3 composite material when applied for dynamic antimicrobial conditioning of the indoor air.It may be seen that, at relatively moderated gas linear velocities and with only single indoor air run through the ZnO/γ-AlAs also follows from the data presented in 2O3 beads seem to have a mechanical abrasive effect on microorganisms which cannot be observed in static conditions. With the dynamic tests, however, which approached life-like use of the materials , the germicidal effect of this material was noticed. It is important to stress that the results of the tests were influenced by its operational conditions and especially by the air velocity. When employing an air velocity below 0.7 m/s, the MnO2/AlPO4/ γ-Al2O3 beads were the most efficient of the tested materials for the inactivation of the airborne microorganisms, which is consistent with the inhibition zone test results. Indeed, its structure allows the generation of hydroxyl radicals at room temperature without any energetic assistance thanks to tunneling effect and also to donor-acceptor interactions between MnO2 and AlPO4/γ-Al2O3. These hydroxyl radicals are a source of oxidative stress for microorganisms and are located on the surface of the beads, which explain that this material works best when using low speeds and thus high contact times. However, when applying higher velocities (between 0.7 and 4 m/s), the ZnO/γ-Al2O3 beads became the most effective material. This is concordant with an abrasive behavior. To conclude, testing the germicidal potential of materials in realistic conditions provided unique and key information. Therefore, it is to be hoped that the use of dynamic tests such as ours will be generalized in the near future for the research of materials for the disinfection of air.Static tests such as agar diffusion inhibitory test (ADT) are to this day the standard procedure to explore the germicidal potential of materials. However, as our study highlighted, the information these tests provided is simply not enough when searching for materials for the disinfection of indoor air. Indeed, some materials such as the ZnO/γ-AlS1 DataThis file contains the data obtained for the experiments analyzed via the BioTrak 9510-BD. The data set has been sorted out to facilitate reading.(XLSX)Click here for additional data file.


When compared with 51 BYMV genomes, it closely resembled the Western Australian isolate PN83A (Lupinus angustifolius), with 98.4% nucleotide identity.Using RNA strand-specific sequencing followed by  de novo assembly, a Bean yellow mosaic virus (BYMV) genome was obtained from a lentil sample (Aus14BY) collected in Victoria, Australia, in 2005. When compared with 51 BYMV genomes, it closely resembled the Western Australian isolate PN83A (Lupinus angustifolius), with 98.4% nucleotide identity.Using RNA strand-specific sequencing followed by  Bean yellow mosaic virus (BYMV)  is a single-stranded positive-sense RNA virus that occurs globally. It has a wide host range that includes monocots and dicots in both domesticated and wild plant species (Vicia faba), field peas (Pisum sativum), lentils (Lens culinaris), and lupins (Lupinus spp.). As part of a project that aims to apply high-throughput sequencing (HTS) to improve the efficiency of pulse seed diagnostic testing, one leaf sample (preserved with CaCl2) that had been collected from a lentil crop growing in Wimmera  and showing virus-like symptoms was subjected to HTS for virus detection, and a complete BYMV (14BY) genome was obtained. species , 2. It i species . BYMV ca species , 5. In ATotal RNA was extracted from the sample using a Zymo Research plant RNA miniprep kit, and quality control checks were performed as described previously . The RNAde novo assembly was performed using the metaSPAdes v3.13.0 genome assembler . This contig consisted of 9,868 nucleotides, with an average coverage depth of 2,307× and a GC content of 39.4%. The phylogenetic analysis revealed nine minor phylogroups (phylogroups I to IX)  , represeI to IX) . IsolateI to IX) , which iThis study forms part of a wider safer-seed certification project, which aims to apply HTS to increase the efficiency of current biosecurity procedures to safeguard the movement of pulse germplasm into and out of Australia. This new sequence from lentil also provides new phylogenetic insights into BYMV.LC500882. The raw reads were deposited in GenBank under SRA number SRR10420664, which is part of BioProject number PRJNA588304.The sequence described here was deposited in GenBank under accession number


Integrated nutrition and agricultural interventions have the potential to improve the efficiency and effectiveness of investments in food security and nutrition. This article aimed to estimate the costs of an integrated agriculture and health intervention (Mama SASHA) focused on the promotion of orange-fleshed sweet potato (OFSP) production and consumption in Western Kenya. Programme activities included nutrition education and distribution of vouchers for OFSP vines during antenatal care and postnatal care (PNC) visits. We used expenditures and activity-based costing to estimate the financial costs during programme implementation (2011–13). Cost data were collected from monthly expense reports and interviews with staff members from all implementing organizations. Financial costs totalled US$507 809 for the project period. Recruiting and retaining women over the duration of their pregnancy and postpartum period required significant resources. Mama SASHA reached 3281 pregnant women at a cost of US$155 per beneficiary. Including both pregnant women and infants who attended PNC services with their mothers, the cost was US$110 per beneficiary. Joint planning, co-ordination and training across sectors drove 27% of programme costs. This study found that the average cost per beneficiary to implement an integrated agriculture, health and nutrition programme was substantial. Planning and implementing less intensive integrated interventions may be possible, and economies of scale may reduce overall costs. Empirical estimates of costs by components are critical for future planning and scaling up of integrated programmes. This is one of the few studies to address the challenge of capturing the costs of interventions that work across sectors to improve health and nutrition.The cost per beneficiary for the integrated agriculture-nutrition-health intervention was US$155, when considering women only, and reduced to US$110, when also including infants who attended postnatal care services. These costs are comparable with other community-based interventions.The costliest component of the intervention was integration, which included monthly feedback meetings and other co-ordination and monitoring activities by all partners and implementing agents (27%), followed by administration and overhead (23%) and training (15%).Scenarios for lowering costs through economies of scale and integration into routine services should be further explored. Agricultural and nutrition-sensitive interventions can complement health service and nutrition-specific interventions to improve child outcomes  antenatal care (ANC) services, with the aim of maximizing the benefits of orange-fleshed sweet potato (OFSP) on the health status of mothers and children <2 years of age. A comprehensive evaluation strategy assessed impact, acceptability, feasibility and affordability of the integrated approach  to estimate the total implementation costs and cost per beneficiary of the Mama SASHA intervention and (2) to identify potential efficiencies for scaling up the intervention to other locations where OFSP holds promise.The Sweetpotato Action for Security and Health in Africa (SASHA) was a 5-year multi-partner project designed to improve the food security and livelihoods of poor families in sub-Saharan Africa by exploiting the untapped potential of sweet potato.The Mama SASHA intervention was conducted through a combination of health facility and community-based strategies targeting pregnant women. After an initial pilot in 2009, the implementation start-up activities (planning and training) began in late 2010 and recurrent implementation activities ran from March 2011 to December 2013. See Mama SASHA implementing partners  received training to support the pregnant women with nutrition counselling, accessing vines and offering OFSP production support. Co-ordination and monitoring activities were critical for integrating the Mama SASHA project into existing ANC and postnatal care (PNC) services and for ensuring a shared understanding and coherent implementation of activities. Integration activities included annual, quarterly and monthly feedback meetings, transportation and per diems for all meeting participants, and NGO labour to support integration.This cost analysis estimated incremental total financial and unit costs for start-up and recurrent activities related to delivering the Mama SASHA activities, described above, at health facilities already supported by the USAID APHIAplus project. We used a mix of top-down expenditure analysis and bottom-up activity-based costing to understand resource use and financial costs. The analysis followed existing methodological guidance for primary health care costing in low- and middle-income countries , or the costs of community-based activities by established cadres of CHWs already supported by APHIA II and APHIAplus. For example, we included the costs associated with new NGO activities like managing the receipt and redemption of vouchers, supporting PWC, or initiating and supervising agricultural activities for establishing vine multiplication of new varieties of OFSP. The current analysis adopted a provider prospective, where the provider was a combination of the Kenyan government and NGO services based in Kenya.We also classified costs based on input categories for personnel, agriculture supplies, agriculture equipment, other consumable supplies, other capital equipment, transportation, travel per diem, allowances, mixed inputs, vouchers and other costs . We estiStart-up costs included costs on activities for planning, education and communication (IEC) materials development (December 2009 to December 2010), training programme development, establishing secondary VM plots, and promotion and awareness-raising activities (September 2010 to February 2011). Start-up costs were considered a type of fixed or capital cost, where we estimated their useful life years as 5 years (i.e. the life of the project). Recurrent costs were for inputs associated with maintaining the supply and demand for vines, supervision and support for health facilities, and integration activities, including any refresher training, occurring from March 2011 through December 2013.All research-related costs were excluded from this analysis. In addition to implementing the Mama SASHA project described here, some implementing organizations had additional research costs to support the overarching SASHA project research objectives. Personnel, transportation and travel costs were proportionately allocated to research based on staff time spent between research and implementation activities. Only personnel time spent on Mama SASHA implementation was included.We transcribed the international and local NGO project expense reports into an excel template. All data were entered by organization and assigned activity and input codes based on the cost categories described above and in The cost analysis uses two main summary measures of overall intervention service provision: (1) the number of beneficiaries reached and (2) the total points of contacts with beneficiaries. We estimated both using monthly data collected as part of routine Mama SASHA monitoring  the total number of single-count women who received and redeemed vouchers; and (2) 1+ the number of infants who attended a PNC visit with their mother.We aggregated incremental financial costs by year and by NGO organization for the implementation phase (2011–13). Unit costs were estimated as incremental financial costs per service delivery output described above , administration and overhead (23%) and training (15%). An additional one-fifth (22%) of the total costs were used to support agricultural activities related to establishing a continuous supply of vines (9%), ensuring adequate and continuous supply of roots to households (7%) and improving knowledge and practices of OFSP among beneficiaries and the community (6%), while a relatively smaller share of financial costs went to supporting health facilities in vine voucher programme distribution (5%). The remaining budget (9%) was allocated to planning, development of training and ICT materials, awareness-raising and sensitization, and depreciation of capital investments .Total recurrent costs and output declined over the 3-year period . The proTo our knowledge, this is the first study in sub-Saharan Africa to empirically estimate the costs of an integrated agriculture and health intervention implemented at both the community and facility levels. We estimated that the financial costs of reaching nearly 6000 beneficiaries (and over 18 000 contacts) in a 3-year period were just over US$500 000 or approximately US$170 000 per year, including start-up costs. When considering mother–baby pairs, the average unit costs were US$110 per beneficiary and US$28 per contact. Vine vouchers were the project’s critical integrating element: over 7000 pairs of vouchers were distributed to over 4600 women and infants. Each voucher and each mother receiving one or more vouchers were tracked from the health facility to the field. The financial value of the vouchers was low (US$1 per voucher pair redeemed), so the total reimbursement costs of vouchers comprised only a fraction of the overall project expenditure (1%). However, when considering more comprehensive costs of establishing a continuous supply of vines and the requisite health implementation activities for distributing and tracking the vouchers, the cost share of the voucher component was more substantial (around 20%).This study estimated intervention costs in several ways in order to understand each sector’s separate and joint financial contributions to an integrated agriculture and health intervention. Inter-sector co-ordination and feedback mechanisms supported demand generation and ensured a sustainable supply of vines but significantly increased the cost per beneficiary. Early operational research evaluating the pilot component  highlighPrevious studies have presented costs for nutrition interventions with similar complexities in linking health and community-based activities, such as treatment of severely malnourished children, and introducing OFSP through agriculture extension with enhanced nutrition education  shares several similarities with the Mama SASHA project, though CMAM does not include an agricultural component. In Bangladesh, Other household food-based strategies have varied implementation costs. Unpublished reports by Helen Keller International provided cost estimates for a 1996 pilot of homestead food production programme promoting household gardening of vitamin A-rich foods in Bangladesh at an adjusted cost of 2013 US$68 per household garden . More reSeveral study limitations are worth noting. First, this analysis presented information on financial costs and did not estimate the opportunity cost of time for health workers, agriculture extension agents, community volunteers and beneficiaries. However, key informant data presented in Second, in parsing out non-project-related expenses, we tried to make consistent decisions on what to include and exclude but were challenged by different financial accounting methods across the four implementing organizations. NGOs coded or categorized inputs or activities differently for many expenses related to workshops, training, personnel, travel, transportation and indirect/overhead expenses. In particular, we had to use our judgement to allocate vehicles and transportation costs to research and implementation activities, since we did not have detailed information on how vehicles and transport supported project activities. We may, therefore, have underestimated implementation costs. Alternatively, we may have overestimated costs as vehicle and transport costs sometimes served both purposes.Third, international NGOs incurred indirect costs related to fringe benefits and housing allowances for expatriate staff beyond the scope of the Mama SASHA project, so we estimated and included only the portion of those indirect costs that were directly related to the Mama SASHA project implementation. Further, we did not include labour and travel costs of international technical collaborators that contributed to the design of the proof-of-project concept and to annual work planning meetings, as the majority of these costs were for research design and management not directly related to intervention service delivery. Our results from the sensitivity analysis indicated that our cost estimates are fairly robust to changes in transportation, salaries of personnel and indirect expenses.Fourth, we did not include the costs of implementing and evaluating the 2010 pilot programme, though it was instrumental in refining the programme, helping implementation to run more smoothly, and achieving higher uptake. Including the pilot would increase the overall project costs, though it would also increase the total number of beneficiaries reached by 2453 pregnant and lactating women. Finally, our cost per beneficiary does not capture the total number of infants and family members who consumed OFSP as an indirect result of Mama SASHA. Doing so would yield lower unit costs.We estimated the Mama SASHA project to cost in the range of US$110 to $155 per direct beneficiary. Whether or not Mama SASHA is considered affordable will depend upon: the priorities of ministries of health and agriculture and international donors; sectoral organizations willingness to fund activities which cross sectors; the level of effort required to sustain or increase uptake over time; and the effects of moving to scale. At scale, we might observe economies of scope, as integrating activities allowed healthcare workers to more effectively reach their target population, leading to secondary cost savings  as well Future work might consider innovative ways to harness market forces and the private sector. In Western Kenya, there was a strong demand for OFSP vines by NGOs and community-based projects to improve agriculture, health and nutrition . As a result of Mama SASHA, a dozen VMs established sustainable businesses to produce and sell surplus vines to other local NGOs. Previous studies have demonstrated the importance of early OFSP adopters and vine producers that can effectively lower the costs of extension services to neighbouring communities and encourage more rapid diffusion (This study estimated the incremental costs of implementing an integrated nutrition and agricultural intervention in rural Kenya. Results suggest that the financial cost per beneficiary and the share of those costs needed for integrating activities across sectors can be substantial. Budgeting explicitly for joint training, regular monthly, quarterly and annual meetings will help future projects ensure that each sector has sufficient resources. Planning and implementing less intensive multi-sectoral interventions may be possible, and economies of scale may help to reduce overall costs. Empirical estimates of costs and their breakdown across sectors are critical to inform the design, planning and implementation of multi-sectoral programmes. Knowing costs by components enables thoughtful examination of where cost savings can be made with the goal of achieving the same level of impact.All research protocols were approved by the institutional review boards at PATH and the Kenya Medical Research Institute.czz083_Supplementary_AppendixClick here for additional data file.


Poxviridae family continue to be the focus of an increasing number of studies. Among these studies, prominently stands vaccinia virus, an orthopoxvirus that is associated with bovine vaccinia outbreaks. Although more frequently associated with infections in cattle and humans, the host range of vaccinia virus is not restricted only to these hosts. There are several instances of molecular and serological evidence of circulation of vaccinia virus among wildlife species. In addition, viral isolation has confirmed a broad spectrum of vaccinia virus hosts. In this report, we provide a brief update on the host range of Brazilian vaccinia virus, and present a case description of an outbreak in domestic buffalo calves from Northeastern Brazil that corroborates previous serological and molecular studies. Furthermore, in the present study, vaccinia virus has been isolated for the first time in buffaloes, and referred to as vaccinia virus Pernambuco (VACV-PE). Phylogenetic reconstruction was based on A56R clustered VACV-PE with vaccinia virus isolates belonging to group 1 Brazilian vaccinia virus. Furthermore, the vaccinia virus genome was detected in the milk of a lactating cow, which thereby revealed a pathway for future studies on the possible impact of vaccinia virus on buffalo milk and milk products. Taken together, these results provide the first description of clinical disease caused by vaccinia virus in buffaloes in South America. They also raise new questions about the chain of transmission of this virus.Even nearly forty years after the eradication of smallpox, members of the  Orthopoxvirus (OPV) are still relevant and have a considerable impact on human and veterinary health  in the Southeast region of Brazil   . The watbubalis) . Studiesbubalis) . Introdububalis) .In the present study, we describe a VACV outbreak in domestic buffalo calves from Northeastern Brazil that corroborates previous serological and molecular studies. Furthermore, we describe the first isolation of Brazilian VACV from buffaloes.In September 2017, three buffalo calves on a farm in Ribeirão County, Pernambuco State, Brazil (08°30′11″S and 35°22′26″W) Figure , present2 atmosphere at 37°C, in Eagle’s minimum essential medium (MEM) , supplemented with 5% fetal bovine serum ; 2.5 μg/mL amphotericin B (Fungizone) ; 500 U/mL penicillin ; and 50 μg/mL streptomycin . Vero cells were used for viral isolation. The BSC-40 cells were used for the neutralization test. The vaccinia virus Western Reserve (VACV-WR) virus was kindly provided by Dr. C. Jungwirth , and was used in the plaque reduction neutralization test (PRNT). The VACV-WR was purified on a sucrose gradient as described by African green monkey kidney BSC-40 cells [American Type Culture Collection (ATCC) CRL-2761] and Vero cells (ATCC CCL-81) were maintained in a 5% COg for 5 min. The milk was diluted 10× in PBS and homogenized in a vortex apparatus. Vero cells were cultured in 25-cm2 flasks and infected with the specimen supernatants, to isolate the virus at 37°C, until a cytopathic effect was detected. The isolates were obtained after three additional rounds of plaque purification in Vero cells.Fragments of the tongue lesions on buffalo calves were macerated in phosphate-buffered saline (PBS), containing amphotericin B (20 μg/mL), penicillin (1000 U/mL), and streptomycin (500 μg/mL) in a ratio of 0.1 g sample/0.9 mL PBS. The macerated fragments were then homogenized using a Mini-BeadBeater-24 , and centrifuged at 3,000 × Tissue fragments excised from tongue lesions were collected and fixed in 10% buffered formalin. The fragments were routinely processed, sectioned at 5 μm, and then stained with hematoxylin and eosin (H&E).2), monolayers were washed twice with PBS, and overlain with solid medium, prepared by mixing equal proportions of 1% agarose and 2× Eagle’s MEM , supplemented with 2% FBS . After 48 h of incubation , cells were fixed with formaldehyde and stained with crystal violet for plaque size analysis. The method was developed using control viruses with large and small plaque phenotypes, as previously described .For the PRNT, serum samples were heat-inactivated at 56°C for 30 min, initially diluted in a 1:20 ratio in MEM, and incubated at 37°C for 15 h  with theC11R (F-5′CGCTACAACAGATATTCCAGCTATCAG3′-R-5′AGCGTGGATACAGTCACCGTGTAA3′) and viral hemagglutinin gene/ A56R (F-5′CATCATCTGGAATTGTCACTACTAAA3′- R-5′ACGGCCGACAATATAATTAATGC3′), as previously described by B5R (F-5′TTTTAGTGCTGCACAGTG3′-R-5′AGTAAAAATGCTCTAACG3′) was amplified (A56R and B5R fragments were directly sequenced in both orientations (GenBank B5R Accession Number MK210281 and A56R Accession Number MK210282), and in triplicate using an ABI3730 sequencer . The sequences were aligned with previously published OPV sequences from GenBank using the ClustalW method, and they were manually aligned using the MEGA software version X . The jModelTest 2.1.9 software was used to determine which model of evolution was most appropriate for our datasets , was submitted to three additional rounds of plaque purification followed by viral replication in Vero cells and subjected to the plaque phenotypic assay. Only the small plaque phenotype was evident Figure .Histopathology of the tongue lesions showed ulcerative superficial glossitis at multifocal areas, with superficial necrosis of the epithelium. This was associated with pustular formations characterized by the presence of eosinophilic debris, interspersed with cellular debris and degenerated neutrophils Figure . In the C23L gene showed a profile similar to that of Brazilian VACV GI, which corroborated the small plaque phenotype . Partial A56R gene nucleotide alignment showed that the VACV-PE sequence contained the signature deletion (18 nt) that was also present in other Brazilian sequences of VACV isolates in GI, but not in GII. Interestingly, this signature was also absent in the sequences of the buffalopox virus of India (Figure A56R (Figure B5R nucleotide sequences (Figure Amplified DNA from the two different reactions for the e Figure . The DNAa Figure . PhylogeR Figure  and B5R s Figure , also clThe present study reports the first description of clinical manifestation of VACV in buffaloes in the Western Hemisphere, and the first isolation of Brazilian VACV in a bubaline host. Here, we detected the presence of neutralizing antibodies in buffalo calves Figure . Only twA56R gene showed that the 18 nt deletion can be used as a molecular signature of Brazilian VACV GI (Figure A56R sequences of buffaloes from Ilha do Marajó also showed this deletion and were grouped in GI (A56R and B5R genes and others, such as C18L, E3L, K3L, and C7L, have demonstrated that BPXV isolates from India are grouped separately in a single cluster apart from the Brazilian isolates and vaccine isolates (Analysis of the VACV-PE I Figure . The A56ed in GI . A broadisolates . Our phyisolates .Another noteworthy issue was the detection of viral DNA in the milk of the cow that suckled the calves, as previous studies point to VACV as a possible foodborne pathogen . The VACGenerally, during BV outbreaks in lactating cows, calves comprise the most severely affected group among the herd showing clinical signs of the disease . SimilarIn support of these observations, we propose an update of the hypothetical model previously described , that hiBuffaloes exhibited clinical signs of viral infection that could be confused with those of BV. The possible etiological agents that produce similar clinical signs include foot-and-mouth disease virus, bluetongue virus, vesicular stomatitis viruses, bovine viral diarrhea virus types 1 and 2, bovine papular stomatitis virus, pseudocowpox virus, ovine herpesvirus-2, caprine herpesvirus-2, and bovine herpesviruses 1 and 2 . Based oML, JA, RS, CdM, AD, JSA, and EK conceived and designed the experiments. ML, GO, JA, and AD performed the experiments. ML, GO, JA, JSA, and EK analyzed the data. ML and EK wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arenaviridae is divided into three genera: Mammarenavirus, Reptarenavirus, and Hartmanivirus. The Mammarenaviruses contain viruses responsible for causing human hemorrhagic fever diseases including New World viruses Junin, Machupo, Guanarito, Sabia, and Chapare virus and Old World viruses Lassa, and Lujo virus. These two groups of arenaviruses share the same genome organization composed of two ambisense RNA segments. These segments contain four open reading frames that encode for four proteins: the nucleoprotein, glycoprotein precursor, L protein, and Z. Despite their genome similarities, these groups exhibit marked differences in their replication life cycles. This includes differences in attachment, entry, and immune evasion. By understanding the intricacy of replication in each of these viral species we can work to develop counter measures against human diseases. This includes the development of vaccines and antivirals for these emerging viral threats. Currently only the vaccine against Junin virus, Candid#1, is in use as well as Ribavirin for treatment of Lassa Fever. In addition, small molecule inhibitors can be developed to target various aspects of the virus life cycle. In these ways an understanding of the arenavirus replication cycle can be used to alleviate the mortality and morbidity of these infections worldwide.The family  Arenaviridae is a viral family encompassing three newly separated genera: Mammarenavirus, Reptarenavirus, and Hartmanivirus. As noted in the names, genus Mammarenavirus encompasses those viruses that infect mammalian hosts whereas the reptarenaviruses and hartmaniviruses infect reptilian hosts. The genus Mammarenavirus is further sub-divided into the Old World and New World groups. Currently, the International Committee for Taxonomy of Viruses (ICTV) recognizes 41 distinct viral species in the genus Mammarenavirus  and Lassa virus (LASV) as well as newly emerged Lujo virus. The Old World viruses are generally geographically confined to the African continent with the exception of LCMV that circulates globally in the Mus musculus host. LASV strains were previously subdivided into 4 distinct lineages based on genetic relationship , with the exception of TCRV, which is found in Artibeus bats . The human hemorrhagic pathogens Junin virus (JUNV), Machupo virus (MACV), Guanarito virus (GTOV), Sabia virus (SABV), and Chapare virus, cluster in clade B together with the prototypic Tacaribe virus (TCRV). JUNV, MACV, GTOV, and SABV are the causative agents of Argentine Hemorrhagic Fever (AHF), Bolivian Hemorrhagic Fever (BHF), Venezuelan Hemorrhagic Fever (VHF), and Brazilian Hemorrhagic Fever (BzHF), respectively. Pichinde virus (PICV) is a prototypic clade A virus that is not pathogenic to humans . To dateeus bats . Recentlod hosts . No furtRecent surveillance has uncovered the circulation of novel arenaviruses in Asia. Wenzhou Virus (WENV) was isolated in China and is phylogenetically related to the Old World viruses . FurtherBoth Old World and New World viruses cause significant disease burden in their endemic areas of circulation. According to the latest , 2015 reLassa virus and JUNV cause the prototypic human disease manifestations for the Old World and New World viruses, respectively. Lassa fever is a zoonotic disease usually resulting from contact with infected murine feces or urine, however, human-to-human transmission is possible. Lassa virus infections have an average incubation time of 10 days and are usually characterized by general flu-like symptoms: fever, malaise, and headache. Patients that progress to severe cases of LF can then develop hemorrhaging and/or neurologic involvement that may be fatal . In LF sTable 1 for summary of arenavirus entry). Following endocytosis and acidification of the endosome, GP2, a class I fusion protein, facilitates membrane fusion and the release of the viral genome and replication complexes into the cell  of the ESCRT pathway . RecentlDuring replication, viral evasion from the immune system is critical for productive replication and dissemination in the host. Arenaviruses have been shown to accomplish this in many ways. First, the NP protein has been shown to have strong anti-immune functions in the cell. Interferon regulatory factor 3 (IRF-3) is an important component of the type 1 interferon response. Studies have shown that NP inhibits IRF-3 activation of downstream genes resulting in a limited interferon response , 2008. ITable 2 for a summary of arenavirus immune evasion).In New World viruses, the Z protein has been shown to also play a role in immune evasion. The Z protein binds to the retinoic acid-inducible gene I (RIG-I) and inhibits interferon-beta production . RIG-I nex vivo model. When these agonist were used to treat in vitro cells chronically infected with LCMV, there was no significant difference in the TLR 7 and 9 response. Further studies are necessary to determine the exact contributions of each of these signaling pathways and the viral impact on their activation.Another way in which arenaviruses may activate the innate immune system and promote a Type I interferon response is through the activation of various Toll-like receptors (TLRs) . JUNV inFinally, Old World and New World viruses appear to exhibit differences in terms of immune protection against fatal infection. In New World JUNV, passive antibody transfer from immune sera has been shown to dramatically improve survival of infection in humans . Old WorArenaviridae encompasses a large variety of viral species found in a variety of animal hosts. Within the genus Mammarenavirus, great diversity is still seen in terms of cellular entry strategies, and host immune evasion. Old World and New World viruses demonstrate a marked divergence in these pathways that makes an understanding of the varied mechanisms crucial to the study of their replication and pathogenesis. Within these broader groups there is still an observed divergence in cellular receptor usage and natural host range adding additional complexity to their life cycles. As arenavirus research continues, similarities and differences between the varied species will further allow for the development of pathogenesis models and treatments that are encompassing for species that share those similarities, but not be mistakenly utilized on divergent members. This refinement of arenavirus biology is crucial for all future research and development utilizing these agents.The family SH compiled and wrote the manuscript. TK, JM, and SP were involved with reviewing and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zaire ebolavirus, can cause Ebola virus disease (EVD),formerly known as Ebola hemorrhagic fever, in humans. EVD is associated withcase-fatality rates as high as 90%, and there is currently no specific treatment orlicensed vaccine available against EVD. Understanding the molecular biology andpathogenesis of ebolaviruses is important for the development of antiviraltherapeutics. Ebolavirus encodes several forms of glycoproteins (GPs), which havesome interesting characteristics, including the transcriptional editing codingstrategy and extensive O-glycosylation modification, clustered in the mucin-likedomain of GP1, full-length GP , and shed GP. Inaddition to the canonical role of the spike protein, GP1,2,in viral entry, ebolavirus GPs appear to have multiple additional functions, likelycontributing to the complex pathogenesis of the virus. Here, we review the roles ofebolavirus GPs in viral pathogenesis. Ebolaviruses are highly dangerous pathogens exhibiting extreme virulence in humansand nonhuman primates. The majority of ebolavirus species, most notably