Rabies is known to be lethal in human. Treatment with passive immunity for the rabies is effective only when the patients have not shown the central nerve system (CNS) signs. The blood–brain barrier (BBB) is a complex functional barrier that may compromise the therapeutic development in neurological diseases. The goal of this study is to determine the change of BBB integrity and to assess the therapeutic possibility of enhancing BBB permeability combined with passive immunity in the late stage of rabies virus infection.The integrity of BBB permeability in rats was measured by quantitative ELISA for total IgG and albumin levels in the cerebrospinal fluid (CSF) and by exogenously applying Evans blue as a tracer. Western blotting of occludin and ZO-1, two tight junction proteins, was used to assess the molecular change of BBB structure.The breakdown of BBB with hypertonic arabinose, recombinant tumor necrosis factor-alpha (rTNF-γ), and focused ultrasound (FUS) were used to compare the extent of BBB disruption with rabies virus infection. Specific humoral immunity was analyzed by immunofluorescent assay and rapid fluorescent focus inhibition test. Virus-neutralizing monoclonal antibody (mAb) 8-10E was administered to rats with hypertonic breakdown of BBB as a passive immunotherapy to prevent the death from rabies.The BBB permeability was altered on day 7 post-infection. Increased BBB permeability induced by rabies virus infection was observed primarily in the cerebellum and spinal cord. Occludin was significantly decreased in both the cerebral cortex and cerebellum. The rabies virus-specific antibody was not strongly elicited even in the presence of clinical signs. Disruption of BBB had no direct association with the lethal outcome of rabies. Passive immunotherapy with virus-neutralizing mAb 8-10E with the hypertonic breakdown of BBB prolonged the survival of rabies virus-infected rats.We demonstrated that the BBB permeability was altered in a rat model with rabies virus inoculation. Delivery of neutralizing mAb to the infected site in brain combined with effective breakdown of BBB could be an aggressive but feasible therapeutic mode in rabies when the CNS infection has been established. Rabies is a highly lethal disease caused by a neurotrophic rabies virus. It has been estimated that about 55,000 persons died from rabies each year. In spite of the prevalence occurred primarily in Africa and Asia, the disease exists globally . VaccineRecently, it had been reported that the BBB was more permeable in mice infected with laboratory-attenuated CVS-F3 than mice infected with silver-haired bat rabies virus (SHBRV) ,10. The In the present study, we determined the BBB permeability and assessed the correlation between BBB integrity and fatality in a rat model infected with pathogenic rabies virus isolated from a dog-bite patient. Further, we used passive immunotherapy with virus-neutralizing mAb with BBB disruption to assess the practicability of this therapeutic strategy in the late stage of rabies.6 f.f.u. (focus-forming units) rabies virus  in 100 μl phosphate buffer saline (PBS); control animals received the same volume of PBS. The animals were monitored twice daily and euthanized by intraperitoneal injection of pentobarbital (150 mg/kg). In passive immunity studies, the rats were injected with rabies virus glycoprotein-specific, neutralizing mAb 8-10E  or with normal saline alone at the indicated time. All procedures were carried out according to the protocols approved by the Institutional Animal Care and Use Committee of Tzu-Chi University.Eight- to ten-week-old male LEW/SsNNarl (LEW) rats were purchased from National Laboratory Animal Center, Taipei, Taiwan. They were infected via gastrocnemius muscle inoculation with 2 × 10The surgery was performed according to pervious studies with minor modifications ,17. BrieImmunoglobulin G (IgG) and albumin in the CSF and serum were quantitated by sandwich ELISA using commercial kits . Total IgG and albumin concentrations were measured in duplicate.BBB permeability was quantitatively evaluated by measuring the amount of extravasated Evans blue . EB  was injected intravenously and allowed to circulate for 1 h . After wConventional Western blotting was used. Briefly, brain samples were homogenized in ice-cold radioimmunoprecipitation (RIPA) buffer, supplemented with protease inhibitor cocktail tablets . The homogenates were centrifuged and protein concentrations of the supernatants were determined using a Pierce protein assay kit . Equal amounts of protein (80 μg) were loaded onto 7.5% or 10% sodium dodecyl sulfate-polyacylamide gel, transferred onto polyvinylidene difluoride membranes . The membrane was then blocked with 5% nonfat milk and incubated with primary antibodies overnight at 4°C. The primary antibodies and concentrations used were as follows: rabbit polyclonal anti-occludin , rabbit polyclonal anti-ZO-1 , and mouse monoclonal anti-β-actin . Following washing and incubation with the secondary antibody  for 60 min at room temperature, the membranes were then probed with chemiluminescence reagents using a commercially available kit  to visualize the signals, followed by exposure to X-ray films . Intensities of the band were quantified with a densitometric analysis system , and calculated as the optical density x area of band.6 IU human recombinant TNF-α protein . FUS treatment was described previously [Three methods were use to disrupt the BBB permeability. Hyperosmotic solution and TNF-α were performed as described previously -21. Brieeviously ,23. SonoLevels of rabies virus-specific total IgG from rats infected with rabies virus as well as uninfected control groups were measured by indirect immunofluorescent assay (IFA). The titer was defined as the reciprocal of the highest dilution factor of test samples in which 50% or more of the fields examined contained specifically fluorescing cells. Virus-neutralizing antibody (VNA) titers were determined by the rapid fluorescent focus inhibition test (RFFIT) as previously described ,25. The Experiments were divided into two parts: (1) for the passive immunotherapy alone, the rabies virus-infected rats were intraperitoneally administrated 10 mg mAb 8-10E, and (2) for the passive immunotherapy combined with BBB opening (BBBO), mAb 8-10E was administered 6 hrs before BBB disruption. The studies were carried out on day 7 and day 9 post-infection (p.i.). The rats were continually observed to day 40 p.i. and then euthanized for analysis.Total RNA was extracted from the cerebral cortex, cerebellum, and spinal cord of rabies virus infected and control rats using the RNeasy Mini kit  according to the manufacturer’s instructions. The nucleoprotein (N) gene of rabies virus was amplified by one-step RT-PCR. The nucleotide sequences of the primers used for PCR are forward primer, 5'- CTACAATGGATGCCGAC-3' and reverse primer, 5'-TCATAACGGAGAGATCGCCAC-3'. RT-PCR was performed with 4 μl of RNA (0.2-0.4 μg) and the OneStep RT-PCR kit  according to the manufacture’s instructions. Each reaction mixture was incubated at 50°C for 30 min, followed by 40 cycles at 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, and 72°C for 10 min. Equal amounts of amplified PCR products were electrophoresed in 1.5% agarose gels containing ethidium bromide. The bands were visualized under UV light and photographed.t-test was used to determine the significance between groups. The survival rate was analyzed by the Kaplan–Meier method and log-rank statistics were used to test the difference between groups. The GraphPad Prism statistical package, version 5.0c  was used for comparison. Differences were considered statistically significant at p < 0.05.All data were expressed as mean ± SEM. One-way ANOVA was used to compare means between experimental groups. Student’s Clinical signs of rabies infection appeared on day 9–10 p.i.. Unsteady gait and hunched back were observed in the beginning, swimming movement, slow motion, and limbs paralysis in the late stage. Rats died from the infection within 2 to 4 days once the neural signs were observed. The viral genome was detected by IFA, RT-PCR and immunohistochemistry on day 5 p.i. in the brain and spread out within the next 2 days (data not shown).To determine the possible alteration of BBB permeability, the CSF were serially sampled and analyzed. The status of the BBB permeability was monitored post-infection every single day by quantifying the albumin and total IgG in the CSF using quantitative ELISA. The results showed that the albumin and total IgG levels were elevated significantly on day 7 and continually elevated to day 12 p.i.  was significant elevated following hypertonic arabinose infusion in rats. The enhancement of BBB permeability with hypertonic arabinose was performed on day 7 and 9 p.i.. The rats treated with or without hyperosmotic solution all died after rabies virus infection. Figure To estimate the humoral immunity of the rabies virus-infected rats, we measured the rabies virus-specific IgG and virus-neutralizing antibody (VNA) titer in the serum and CSF using IFA and RFFIT. The IFA results showed that the rabies virus-specific antibody was detected in the sera, but the mean of dilution factor was lower than 1:256 on day 11 p.i.. In the CSF, the antibody level was very low and the mean of dilution factor was less than 1:8  was administered to rabies virus-infected rats with or without BBBO Figure A. AdminiThe BBB is a functional and structural component of the CNS vasculature that serves important protective functions for the CNS. Many clinical symptoms, immune responses, pathological changes and disease outcome in neurodegeneration or infectious diseases have been associated with the BBB permeability . AlteratSerial sampling of CSF from rabies virus-infected rats presents a dynamic change of the BBB permeability. We found that serum proteins, albumin and total IgG, were elevated on day 7 p.i. and increased further afterwards. Our preliminary study showed that the rabies virus could retrogradely replicate in the brain tissues on day 5 p.i. and clinical symptoms were observed on day 9 p.i.. We postulate that the initiation of the BBB opening was behind the replication of rabies virus in the CNS but prior to the appearance of clinical signs. Further, we also demonstrated that the disruption of BBB could allow macromolecular markers, e.g., albumin and IgG, to leak into the CNS. This result was substantiated by the Evans blue staining. The greater permeability was seen in the spinal cord and cerebellum compared with the cerebral cortex on day 10 to 11 p.i.. This similar regional difference was reported also in the attenuated CVS-F3 study in mice . TogetheTight junction proteins, the essential components of the BBB, are the primary barrier to maintain the brain homeostasis . AlteratAccording to the results of quantitative ELISA and Evans blue study, the rabies virus infection induced BBB leakage of macromolecules was restricted to the cerebellum and spinal cord. We compared hypertonic arabinose, TNF-α, and FUS to find out which method could enhance greater BBB disruption and allow more macromolecules, such as IgG, to enter the brain. Intracarotid infusion of a hypertonic solution caused a transient and reversible shrinkage of the endothelial cells and opening of the endothelial tight junctions and increasing the delivery of antineoplastic agents, neutralizing antibodies, mAbs, nanoparticles and viral vectors into the CNS -40. IntrAlthough BBB permeability was changed after rabies virus infection, the mortality was not altered. This is mainly resulted from the insufficient titer of neutralization antibody for rabies virus, which was substantiated by the finding of lower levels of virus-specific and neutralizating antibody in the sera and the CSF. This result is similar to human naturally infected with rabies virus. They do not receive PEP and generally do not mount a strong immune response to rabies relatively late in the disease ,45. VNA It has also been reported that exogenous neutralizing antibodies accompanied with the enhancement of BBB permeability can raise the survival more than the administration of monoclonal antibodies alone ,47. In oUntil now, besides the success of the Wisconsin’s patient treated with the Milwaukee Protocol, several others have employed similar treatment regimens with minimal beneficial outcomes ,49-51. AThe present study revealed that BBB permeability was altered after pathogenic rabies virus infection in rats. Sufficient virus-neutralizing antibody plays a major role in determining the survival from rabies virus infection. Our study indicates that enhancing BBB opening combined with delivering sufficient virus-neutralizing mAb to the brain may provide an effective treatment when the CNS infection has been established.The authors declare that they have no competing interests.PHL performed the major experiments and wrote the manuscript. HHY and PTC were responsible for virus isolation and mAb production. PCC and HLL were for technical support of FUS study. MHW participated in data interpretation and manuscript improvement. LKC conceived the study, designed the experiments and analyzed the data. All authors read and approved the final manuscript.


Citrus tristeza virus  contains 12 open reading frames, with the 3′-terminal one corresponding to a protein of 209 amino acids (p23) that is expressed from an abundant subgenomic RNA. p23, an RNA-binding protein with a putative zinc-finger domain and some basic motifs, is unique to CTV because no homologs have been found in other closteroviruses, including the type species of the genus Beet yellows virus (despite both viruses having many homologous genes). Consequently, p23 might have evolved for the specific interaction of CTV with its citrus hosts. From a functional perspective p23 has been involved in many roles: (i) regulation of the asymmetrical accumulation of CTV RNA strands, (ii) induction of the seedling yellows syndrome in sour orange and grapefruit, (iii) intracellular suppression of RNA silencing, (iv) elicitation of CTV-like symptoms when expressed ectopically as a transgene in several Citrus spp., and (v) enhancement of systemic infection (and virus accumulation) in sour orange and CTV release from the phloem in p23-expressing transgenic sweet and sour orange. Moreover, transformation of Mexican lime with intron-hairpin constructs designed for the co-inactivation of p23 and the two other CTV silencing suppressors results in complete resistance against the homologous virus. From a cellular point of view, recent data indicate that p23 accumulates preferentially in the nucleolus, being the first closterovirus protein with such a subcellular localization, as well as in plasmodesmata. These major accumulation sites most likely determine some of the functional roles of p23.The large RNA genome of  Citrus tristeza virus (CTV) – a member of the genus Closterovirus, family Closteroviridae – features singular biological and physical properties. In natural infections it is confined to the phloem of most species of the genera Citrus and Fortunella within the subfamily Aurantioideae (Agrobacterium tumefaciens it may incite systemic infection and symptoms into the presumed non-host Nicotiana benthamiana (Citrus aurantium L.) rootstock, was the first syndrome observed in the 1930s’ decade, after this rootstock species became massively used worldwide to avoid root rot caused by oomycetes of the genus Phytophthora. In addition, some CTV isolates incite yellowing, stunting, and occasional growth arrest of sour orange, lemon (C. limon (L.) Burm. f.), and grapefruit (C. paradisi Macf.) seedlings [referred to as the seedling yellows (SY) syndrome], and/or stem pitting  on different citrus species irrespective of the rootstock used for their propagation, reducing the vigor, production, and fruit quality . The absence of homologs in other closteroviruses, including the type species of the genus Beet yellows virus (BYV) with which CTV has many homologous genes  (+) genome (19.3 kb), the largest reported for a plant virus, is organized in 12 open reading frames (ORFs) potentially encoding at least 17 proteins, confined between 5′ and 3′ untranslated regions , the C-terminal region has a net negative charge (the pI of the segment corresponding to the last 54 amino acids is 4.35). This asymmetrical charge distribution, and the putative zinc-finger domain, have been observed in the transactivating domains of some transcription factors , and increased earlier than other sgRNAs , was required for asymmetrical RNA accumulation. Further alanine substitution mutants mapped the conserved cysteines in the zinc-finger domain as critical for this regulatory activity of p23. Moreover, whereas non-functional p23 essentially resulted in a significant overaccumulation of minus strands, functional p23 induced a decrease in the accumulation of the minus-stranded sgRNA corresponding to the major coat protein (p25) but not in the accumulation of this protein , suggesting that the availability of the corresponding plus-stranded sgRNA as a messenger is blocked by the excess of its minus-stranded counterpart. In summary, by downregulating minus-stranded RNA accumulation (and increasing indirectly expression of the 3′ genes), p23 would control the unbalanced accumulation of CTV RNAs (The finding that CTV infection results in the production of more plus than minus strands (of both genomic and subgenomic length) is typical of ssRNA viruses. However, this asymmetrical proportion was perturbed in a CTV mutant replicon with all of the 3′ genes deleted, which replicated efficiently in  strands . BecauseCTV RNAs .Figure 1), which occasionally may be transitory with the plants resuming normal growth after some time. Although economically not relevant, this syndrome can be studied under greenhouse conditions more easily than decline and SP. The construction of an infectious CTV-cDNA clone , a sensitive CTV host, resulting in systemic infections. When tissues from these infected plants were graft-inoculated into sour orange and grapefruit seedlings, three of the T30/T36 recombinants incited SY symptoms identical to those of T36, whereas the other two – with T30 substitutions in the p23-3′UTR – did not. Moreover, the presence of a non-SY p23-3′UTR recombinant in sour orange seedlings resulted in their protection against challenge inoculation with the parental T36 virus, thus showing the potential for cross-protection of the engineered CTV constructs  Swing.], another particularly sensitive host, results in the high accumulation of virus-derived small RNAs (vsRNAs)  . The sRN(vsRNAs) . To cope(vsRNAs) . Since t(vsRNAs) , althoug(vsRNAs) .N. benthamiana (16c) expressing GFP constitutively  in a transgenic line of tutively . Analysitutively . The RSSC. sinensis (L.) Osb.; in which the virus titer is relatively high], but only amount to 3.5% in sour orange (with a considerably lower CTV titer), (ii) have a predominant size of 22- 21-nt, with an uneven balance in their 5′ nucleotide and a moderate overaccumulation of those of (+) polarity, and (iii) result from all the CTV genome, permitting its entire assembly from viral sRNA contigs, but display an asymmetrical profile characterized by a major hotspot corresponding to the 3′-terminal 2500 nt. These observations suggest that the genesis of the 22- and 21-nt CTV sRNAs is most likely catalyzed by the citrus homologs of DCL 2 and 4, respectively, and that these two ribonuclease isoenzymes operate on the gRNA as well as on the 3′ co-terminal sgRNAs, and, particularly, on their double-stranded forms. In the three mock-inoculated controls, the sRNA pattern was very similar and dominated by the 24-nt sRNAs. This pattern remained essentially unaffected by CTV infection in sour orange, while a clear reduction of the 24-nt sRNAs was detected in Mexican lime and sweet orange. In addition, CTV influences the accumulation of some miRNAs. For instance, miRNA 168 is upregulated by CTV in the three hosts , and bioinformatic approaches in young bark of three natural hosts infected by a severe CTV isolate, as well as in their corresponding mock-inoculated controls. The data obtained  show thaee hosts . This miee hosts  and vsRNee hosts . While Aee hosts .Cauliflower mosaic virus (CaMV) resulted in phenotypic aberrations – including intense vein clearing, general epinasty and chlorotic pinpoints in leaves, pitting and necrosis in the stems, and collapse – even more pronounced than those caused by CTV in non-transgenic plants , whereas Mexican limes expressing transgenically a truncated version of p23 were normal . Therefore, induction of CTV-like symptoms was associated with the expression of p23, and its accumulation level paralleled symptom intensity (Poncirus trifoliata (L.) Raf.], also incited CTV-like leaf symptoms , which again were not expressed in the controls transformed with a truncated p23 version . However, in contrast with the situation observed in Mexican lime, p23 was nearly undetectable in transgenic sour, sweet, and trifoliate orange, although symptom intensity correlated with levels of the p23 transcript. Because p23 also accumulates poorly in non-transgenic sweet and sour orange inoculated with CTV in comparison with Mexican lime, these results suggest that even minimal levels of p23 may induce toxic effects in the two former species. On the other hand, transgenic expression of p23 in N. benthamiana and N. tabacum resulted in the accumulation of p23 without visible phenotypic aberrations, indicating that p23 interference with plant development is citrus-specific . This was also the case with p23, the deduced amino acid sequence of which correlated in phylogenetic reconstructions with mild and severe strains . Interesntensity . An extespecific .Commelina yellow mottle virus  determines, at least in part, CTV pathogenesis in Mexican lime.Also in this context, examination of the phenotypic aberrations resulting from the transgenic expression of p23 under the control of a phloem-specific promoter should better mimic the symptoms incited by CTV, which is a phloem-confined virus. Indeed, it will be interesting to see whether restricting the expression of p23-derived transgenes to phloem-associated cells of Mexican lime by using a promoter from le virus  results le virus  and, (iiN. benthamiana with the transcript from an infectious CTV-cDNA clone (strain T36) expressing the GFP as an extra ORF  and sour orange expressing ectopically p23  were graAltogether these results show that, when expressed ectopically, p23 facilitates CTV phloem escaping and additionally enhances systemic infection of the less-susceptible sour orange host. Moreover, the distinct reaction observed in sweet and sour orange implies a multifaceted interaction between p23 and other virus- and host-encoded proteins for traversing diverse cell boundaries. In this context, the possibility that CTV exit from the vascular system in sweet and sour orange could be mediated by a p23 function unrelated to silencing suppression should be entertained. The finding that p23 accumulates preferentially in plasmodesmata, in a addition to the nucleolus (see below), is consistent with the involvement of this protein in CTV cell-to-cell and long-distance movement .p23 appeared normal and manifested the typical features of RNA silencing: multiple copies and methylation of the transgene, presence of p23-specific siRNAs, and low accumulation of the corresponding mRNA. CTV inoculation, by grafting or aphids, of propagations from these latter lines resulted in three responses: some were immune (they neither expressed symptoms nor accumulated the virus), others were partially resistant (with delayed and attenuated symptoms compared to controls transformed with the empty vector), and a third group was susceptible (with symptoms and virus titer similar to the controls). This erratic response of clonal propagations denoted that RNA-mediated resistance is also influenced by factors other than the transgenic background, like the developmental stage or growing conditions of each plant  was to test whether, under such conditions, this regulatory protein could interfere with CTV replication and provide resistance. Although most transgenic lines showed phenotypic aberrations similar to CTV symptoms (see above), certain lines of Mexican lime transformed with gene ch plant .p23 and the 3′UTR of the gRNA, a segment highly conserved (>90% identity) in different virus isolates, was used to transform Mexican lime in intron-hairpin, sense, and antisense formats. When graft-inoculated with CTV, all propagations from sense, antisense, and empty-vector transgenic lines were virus-susceptible, except one (out of seven) from a sense-line showing transgene-derived siRNAs, low accumulation of the transgene-derived transcript, and an intricate transgene integration profile. In contrast, 9 of 30 intron-hairpin lines were partly resistant to CTV: 9–56% of their propagations (depending on the line) remained uninfected, with the others being susceptible. Focusing on intron-hairpin lines with a single-transgene integration (to facilitate comparison between lines), the low accumulation of the transgene-derived transcript was a better predictor of CTV resistance than the high accumulation of the transgene-derived siRNAs, possibly because only a part of the latter are competent for RNA silencing  to silence concurrently the expression of the three RSS of CTV in infected cells. Graft-inoculation with an isolate of the same viral strain, in the scion or in the rootstock, revealed that three transgenic lines were completely resistant: all their propagations remained asymptomatic and virus-free, with the accumulation of transgene-derived siRNAs being necessary but insufficient for CTV resistance. However, resistance was only partial following inoculation with an isolate of a severe SP strain (T318A), with nucleotide identities with T36 of 91–92% for the three genes, thus showing the involvement of a sequence-dependent mechanism. Apart from representing a step ahead in the quest for developing full transgenic resistance to CTV, these results show that the simultaneous inactivation of the three viral RSS is crucial for this aim, although the participation of other concomitant RNA silencing mechanisms cannot be dismissed . These results, confirmed by coexpression experiments with proteins marking specifically the nucleolus (fibrillarin) and plasmodesmata (the movement protein of an ilarvirus), strongly suggested the presence in p23 of a nucleolar localization signal (NoLS) and of a plasmodesmatal localization signal (PLS). Because NoLS  are formed by short motifs rich in basic amino acids, the possibility that motifs of this class identified previously in p23 might form part of its NoLS was examined. Assay of seven truncated versions of p23 fused to GFP showed that regions 50–86 and 100–157 (excluding fragment 106–114), both with basic motifs and the first with a predicted zinc-finger domain , contain what appears a bipartite NoLS. Additional data obtained with 10 alanine substitution mutants confirmed and delimited this signal to three cysteines of the zinc-finger domain and to some basic amino acids. It is also worth noting that, even if a fine dissection of the PLS was not carried out , all deletion mutants  lost their PLS , the protein was additionally expressed as a sgRNA of Potato virus X (PVX) because different RSS act as pathogenicity determinants in this experimental context , as well as in plasmodesmata, is most likely instrumental for these effects. Considerable progress has been also achieved in dissecting the structural motifs of p23 associated with its functions. However, many facets of this singular protein await a deeper analysis, including its potential interactions with other proteins from the virus  and the host (including different AGOs involved in silencing), as well as with nucleic acids also of viral and host origin . These interactions should be ultimately mapped to specific p23 motifs/domains, as recently reported for other multifunctional virus proteins .Vitivirus, Carlavirus, and Benyvirus – encode in their 3′-proximal region proteins containing basic amino acid motifs and a putative zinc-finger domain (N. benthamiana when launched from PVX (Closterovirus (CTV) has followed this evolutionary pathway, remains an intriguing conundrum.As noted above, p23 is unique to CTV within closteroviruses. However, the gRNAs of some filamentous viruses – from genera r domain , 2013. Sfrom PVX . Why virThe 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.


In 2011, a new orthobunyavirus, named the Schmallenberg virus (SBV), was discovered in Europe. Like the related Shamonda virus, SBV is an arbovirus (arthropod-borne virus). After its discovery, the virus was detected in a wide area in north-western Europe, an unexpected finding in a territory where climatic conditions would not seem ideal for arbovirus transmission. This sudden expansion suggests the effect of 2011 drought as a key factor that may have triggered SBV circulation. The possible influence of drought, recorded in north-western Europe in early 2011, on virus circulation was evaluated.The locations of SBV detections in Europe until April 2012 were obtained, and area of virus circulation was evaluated by kernel density estimation. Precipitation data in SBV circulation area, summarized by the 3 month precipitation indexes of May, were compared with precipitation data outside that area, confirming driest conditions in that area.The onset of drought conditions recorded in the SBV detection area in early 2011 may have promoted the circulation of this virus. A correlation between circulation of some arboviruses and drought has been reported elsewhere. This was mainly explained by an effect of water deficit on the environment, which altered the relationships between vectors and reservoirs, but this correlation might be also the result of unknown effects of drought on the vectors. The effect of drought conditions on arbovirus circulation is most likely underestimated and should be considered, since it could promote expansion of arboviruses into new areas in a global warming scenario. The Schmallenberg virus (SBV) causes congenital malformations and stillbirths in cattle, sheep, and goats. Clinical signs of infection in adult animals are either absent or non-specific, but fever, loss of appetite, reduction in milk yield, and diarrhoea have been recorded in cattle ,2. ThereCulicoides spp.), which are biting midges of the Ceratopogonidae family [The expansion of SBV in north-western Europe was the second event of spread of an arbovirus in this area, after the unexpected arrival of Blue tongue virus 8 (BTV-8) in 2006 . While te family . The surCulex genus, and has its main reservoirs in wild birds [In Italy, an enhancing influence of drought was reported in mosquito circulation of the Usutu virus (USUV) , a flavild birds , althougld birds .The influence of drought conditions was previously reported for the circulation of other two flaviviruses that, similarly to Usutu, are maintained by a bird-mosquito transmission cycle: St. Louis encephalitis virus (SLEV) ,11 and WThe epidemiology of culicoid-borne viruses is not well known as the mosquito-borne viruses one, because of the gaps in knowledge of biting midge taxonomy and biology , neverthInterestingly, in 2011, an event of drought, characterized by the greatest deficit in accumulated rainfall in May, was recorded in north-western Europe, particularly in areas in which SBV was subsequently reported . The posA dataset of 3,309 SBV positive sites was obtained Figure a, with an 1,069) and the average outside of the contour was -0.2   inside the obtained 95% KDE volume contour was -2.7   . The aveThe 2011 total number of bovine animals, sheep and goats were obtained for European territorial units (NUTS) available in the Agriculture database of statistical office of the European Union (Eurostat) . LivestoThe locations of SBV detections were obtained from the EMPRES Global Animal Disease Information System . The SBVKDE was performed using ESRI ArcGIS 9.3 and the Spatial Analyst extension and was applied to this dataset to evaluate the area of virus circulation Figure b. The KDSBV: Schmallenberg virus; BTV: Blue tongue virus; USUV: Usutu virus; SLEV: St. Louis encephalitis virus; WNV: West Nile virus; CHIKV: Chikungunya virus; ANNA: Average nearest neighbor analysis; KDE: Kernel density estimation; SPI3: 3 month standardized precipitation indexThe authors declare that they have no competing interests.MC performed the analysis and drafted the manuscript. AA analysed data in geographic information system. Both authors read and approved the final manuscript.Livestock density and Schmallenberg virus (SBV) circulation area in 2011. The 2011 livestock densities , obtained for the different European territorial units, are shown with the overlaying of the 95% and 50% volume contours of KDE of SBV (yellow line and red line respectively).Click here for file


To the Editor: Crimean-Congo hemorrhagic fever virus (CCHFV) causes a disease in humans that is characterized by fever and hemorrhagic manifestations, with death rates up to 30%. Humans are infected through tick bites or contact with the viremic blood of patients or livestock. CCHFV belongs to the genus Nairovirus (family Bunyaviridae), which contains 7 serogroups: CCHFV, Dugbe virus, Hughes virus, Sakhalin virus, Dera Ghazi Khan virus, Qalyub virus, and Thiafora virus.Rhipicephalus bursa ticks collected in 1975 from goats in Vergina, a village in northern Greece .Serum samples were tested for CCHFV immunoglobulin (Ig) G by ELISA . The data were analyzed by using Stata Special Edition 9 . Multivariate logistic regression modeling was adopted to identify potential risk factors for acquisition of CCHFV infection. Odds ratios (ORs) with 95% confidence intervals (CIs) were obtained. p values <0.05 were considered significant.In total, 37 (3.14%) of 1,178 persons were positive for CCHFV by IgG. The mean ± SE age of seropositive and seronegative persons was 68.7 ± 2.54 years (range 0–87 years) and 55.6 ± 0.79 years (range 0–97 years). The female:male ratio was 1.6 among tested persons and 0.6 among seropositive persons. Seroprevalence differed among prefectures: Rodopi, where the fatal Crimean-Congo hemorrhagic fever case was observed, and Evros had the highest values (4.95% and 4.49%), Drama and Xanthi the lowest (1.34% and 1.09%), and no IgG-positive person was found in Kavala. The distribution of regions where IgG-positive persons were found is presented in the Figure. Seropositive persons lived in rural areas at an altitude of 10m to 270 m; however, this factor was not significant (p = 0.358).Crude analysis showed that age, sex, prefecture, occupation, contact with goats and sheep, slaughtering, and history of tick bite were significantly associated with seropositivity. Multivariate analysis showed that the following variables were significant risk factors for acquisition of CCHFV infection: age , residence in Rodopi prefecture (with Drama prefecture as reference category) , contact with goats , history of slaughtering , and history of tick bite .When we compared our results with those of Antoniadis et al. (Further studies are necessary to estimate the seroprevalence in the whole country and detect disease-endemic foci of the disease. In addition, surveys for CCHFV in Ixodid ticks are necessary to enable the construction of risk maps and risk assessment analysis.


Camelidae. They represent the smallest antigen-binding fragment of an antibody (human IgG = 150 kDA). VHH are currently being explored for a number of applications.VHH are polypeptides (15 kDa) derived from the variable domain of single heavy-chain antibodies of in vivo.Anti-rabies virus VHH were cloned from lymphocytes of vaccinated llamas and selected for their affinity with the viral spike glycoprotein G and neutralizing potency in a cellular infection assay. Linkage of two VHH allowed recognition of two identical or different epitopes and increased the neutralizing potency in cells more than a hundred-fold. Next, we examined the protection efficiency of these bimeric VHH Contrary to irrelevant control VHH, pre-incubation of the virus with anti-G VHH fully protected mice against disease and mortality upon inoculation of the virus-VHH mix in the nose, muscle or brain. Preventive administration of anti-G VHH in the nose, 24 hours prior to intranasal virus challenge, also almost completely prevented disease and lethal infection. This suggests that VHH remain sufficiently active for at least 24 hours at the site of administration in the nose to neutralize a significant part of the invading virus. Post exposure prophylaxis (PEP) by injection of anti-G VHH in the left quadriceps muscle 10 minutes after virus challenge in the right quadriceps muscle reduced mortality by 50%. Treatment 24 hours after virus challenge was however no longer effective, most likely because the virus had already reached the central nervous system and was no longer exposed to locally administered VHH.Our results show that anti-G VHH can neutralize rabies virus in an Fc-independent way. VHH probably hinder the recognition of cellular receptors or interfere with the fusion of viral and cellular membranes. The bimeric constructs proved protective in different challenge models, but that protection in the PEP model was weak. This might be due to the short half-life of the used VHH. Considering that prolongation of the half-life of VHH is feasible by different approaches, VHH technology may offer perspectives as an alternative to antibodies for PEP. VHH have a low production cost, not contaminated by blood-borne pathogenic agents and are less likely to evoke allergic or immunopathological reactions. They have good thermal stability, which is an advantage in developing countries, where the cold chain for distribution and preservation can not always be guaranteed.


Arctium species (Asteraceae) are distributed worldwide and are used as food and rich sources of secondary metabolites for the pharmaceutical industry, e.g., against avian influenza virus. RNA silencing is an antiviral defense mechanism that detects and destroys virus-derived double-stranded RNA, resulting in accumulation of virus-derived small RNAs (21–24 nucleotides) that can be used for generic detection of viruses by small-RNA deep sequencing (SRDS).Arctium tomentosum  displaying virus-like symptoms of vein yellowing and leaf mosaic in southern Finland. Assembly of the small-RNA reads resulted in contigs homologous to Alstroemeria virus X (AlsVX), a positive/single-stranded RNA virus of genus Potexvirus (family Alphaflexiviridae), or related to negative/single-stranded RNA viruses of the genus Emaravirus. The coat protein gene of AlsVX was 81% and 89% identical to the two AlsVX isolates from Japan and Norway, respectively. The deduced, partial nucleocapsid protein amino acid sequence of the emara-like virus was only 78% or less identical to reported emaraviruses and showed no variability among the virus isolates characterized. This virus—tentatively named as Woolly burdock yellow vein virus—was exclusively associated with yellow vein and leaf mosaic symptoms in woolly burdock, whereas AlsVX was detected in only one of the 52 plants tested.SRDS was used to detect viruses in the biennial wild plant species Acrtium species and reveal woolly burdock as the first natural host of AlsVX besides Alstroemeria (family Alstroemeriaceae). Emaravirus genus and demonstrated applicability of SRDS to detect negative-strand RNA viruses. SRDS potentiates virus surveys of wild plants, a research area underrepresented in plant virology, and helps reveal natural reservoirs of viruses that cause yield losses in cultivated plants.These results provide novel information about natural virus infections in  Ipomoea (family Convolvulaceae) occur in Uganda where they are infected with the same viruses and virus strains as the cultivated sweetpotato (Ipomoea batatas Lam.) believed to have originated in Latin America. Some of the viruses were most likely introduced to Uganda with sweetpotatoes because those viruses are globally distributed in the crop, unlike some other viruses that seem to be of East African origin Hardenbergia comptoniana (Andrews) Benth. (family Fabaceae) is a virus reservoir with demonstrated potential to act as a source of virus infection of the cultivated legume Lupinus angustifolius L. (family Fabaceae) Viruses occurring in communities of non-cultivated plants have gained limited attention as compared to those causing significant yield losses in cultivated plants etc.One reason for the limited number of studies addressing virus infections in wild plants is that such studies are particularly challenging. The serological and molecular methods routinely used for virus detection require at least some advance knowledge of the viruses expected in the samples so that appropriate antibodies, primers, or probes can be selected for use. When reasonable presumptions are lacking, virus detection becomes an arduous and time-consuming research process requiring many experiments and techniques, e.g., electron microscopy to observe virus particles, transmission of the virus to a range of indicator plants by mechanical or graft-inoculation to observe infectivity and symptoms, isolation of double-stranded RNA (dsRNA) for characterization of viral genome fragments, A recently developed, novel molecular method  provides an effective solution to the aforementioned challenges in virus detection. It is a generic method that allows detection of both RNA and DNA viruses, provided that their genomes contain regions 30–40% identical to previously described viruses RNA silencing is seemingly always activated in virus-infected plants, also in those that display disease symptoms. The reason why viruses can multiply and spread in plants despite RNA silencing is that virus genomes encode proteins that partially suppress the silencing Caulimoviridae and Geminiviridae in sweetpotato Sweet potato feathery mottle virus  and Sweet potato chlorotic stunt virus , and high numbers of vsiRNAs were detected Foveavirus (family Betaflexiviridae), Maculavirus, Marafivirus (family Tymoviridae), and Nepovirus (family Secoviridae) in grapevine plants  infected in the field Cereal yellow dwarf virus  in wild cocksfoot grass  in the United Kingdom The first study to utilize SRDS to detect unknown viruses identified three new ssDNA viruses belonging to the families Arctium spp., family Asteraceae) are rich sources of pharmaceutical compounds. Arctiin and its aglucone obtained from the tap roots of burdock can interfere with early stages of replication of the avian influenza virus and can also hamper progeny virus release in mammalian cells. Therefore, these compounds are studied as alternative antiviral medicines against the virus strains resistant to oseltamivir (Tamiflu) A. lappa L.) are consumed particularly in Asia Arctium tomentosum Mill.) is a wild species common throughout most parts of Europe, northern USA, Canada, and Asia etc.Burdock plants  in Finland in May (2010) and June (2009 and 2010). The plants had not started flowering or were at an early stage of flowering at the time of inspection. Symptomless plants and plants displaying vein yellowing and mosaic symptoms in leaves were sampled at random. The leaf samples were put into liquid nitrogen immediately after collection and stored at −80°C until use for RNA extraction. The six symptomatic plants that were subjected to SRDS were sampled in Pihlajamäki in June 2009; all other samples were collected from the aforementioned four places in June 2010. No specific permits were required for the described field studies. The locations were not privately owned, and field studies did not involve endangered species.Plants of matK) gene sequence was determined and used as a plant “barcode" for comparison with other species Arctium species share similar growth habitats and can cross-breed, which may hamper morphology-based species identification of the plants. Total DNA was extracted from leaves of two plants using the cetyltrimethylammonium bromide method 5′-ATCCATCTGGAAATCTTAGTTC-3′) and matK5r (5′- GTTCTAGCACAAGAAAGTCG-3′) as listed on Kew Royal Botanic Garden's DNA Barcoding website (http://www.kew.org/barcoding/protocols.html), was used to amplify the gene by polymerase chain reaction (PCR). PCR products of the expected size were purified from an agarose gel using E.Z.N.A. gel extraction kit D2500-02  according to the manufacturer's instructions. DNA sequencing was done at Haartman Institute (University of Helsinki). Sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI). The matK sequences of the two plants were identical and showed the closest (98%) nucleotide identity to matK of Arctium lappa L. (NCBI sequence accession no. HM989769). These results supported morphological identification of the plants to the species A. tomentosum whose matK sequence was not available prior to our study. The sequence was submitted to the NCBI sequence database under the accession no. JQ354896.The sampled plants were identified based on morphological characteristics Total RNA was extracted from leaf samples (0.4 g) using Trizol reagent . The concentration and purity of RNA samples were determined with a spectrophotometer. Agarose gel electrophoresis was used to check integrity of the high molecular weight RNA.A. tomentosum plants were pooled, and 10 µg from the RNA pool was sequenced by Fasteris SA . The main steps of SRDS included separation of RNA by polyacrylamide gel electrophoresis, isolation of small RNA (<30 nt) from the gel, and ligation with a single-stranded 3′-adapter and a bar-coded 5′-adapter. Ligated RNA was reverse transcribed and amplified by PCR to generate a DNA colony template library, which was purified and diluted to a concentration of 10 nM. An Illumina Genome Analyzer was used for high-throughput DNA sequencing.For SRDS, equal amounts of total RNA from six symptomatic de novo assembly of siRNA reads as in previous studies k nucleotides (k-mer) k-mer (number of nucleotides overlapping between reads), which was varied during the analysis because a larger k-mer brings out more specific contigs but decreases the read coverage whereas a small k-mer produces contigs with high coverage but with lower specificity. The error-correction functions of Velvet were utilized The program Velvet A. tomentosum. MultAlin and ClustalW2 were used to align multiple sequences and analyze coverage quality.When viral sequences had been identified among the contigs, complete sequences of the most similar viruses were retrieved from sequence databases and used as references for alignment of small-RNA reads with MAQ  as in previous studies Moloney murine leukemia virus (M-MLV) reverse transcriptase , following the manufacturer's instructions. cDNA was diluted 2-fold and stored at −20°C until use.The viruses detected by SRDS were characterized further by amplification of partial genome sequences using reverse transcription PCR (RT-PCR). cDNA was synthesized using an Oligo(T)23 primer or random hexamer primers . The RT reaction containing 0.1–02 µg of total RNA and http://simgene.com/Primer3) to amplify a 3′-proximal region, including the coat protein (CP) gene of Alstroemeria virus X (AlsVX) and the putative nucleocapsid protein (NP) gene of a Fig mosaic-like virus detected in this study . Nested PCR was used to amplify AlsVX. For the first PCR, the forward primer  was designed according to the isolate AlsVX-Jap described in Japan ACAACATTCTCT-3′5′-GGCGACCA  and 5′-CAAACTGTAGAAAAACACCTCC-3′ . The PCR program differed from the first PCR, as follows: denaturation 3 min at 94°C, 35 cycles of 30 sec at 94°C, 30 sec at 56°C, and 75 sec at 72°C, followed by final extension for 10 min at 72°C. The expected size of the PCR product was 1120 nt.Primers were designed with the program Primer3 (5′-TCAACAAACGACTTCCTCACTG-3′) and reverse (5′-AGGGTGCTGATCTGTCTGCT -3′) primers were designed according to the contigs built by Velvet and, based on comparison to FMV sequences, were expected to amplify a 604-nt region of the NP gene. PCR products were purified and sequenced at Haartman Institute as described above. Sequences were analyzed with ClustalW2 and BLAST. All new sequence data were deposited in the NCBI sequence database.To detect WBYVV, the PCR reactions and cycling were similar to those used in the first PCR for AlsVX amplification, except that the 30 cycles of PCR were as follows: 30 sec at 94°C, 30 sec at 53°C, and 75 sec at 72°C. The forward , and European mountain ash ringspot-associated virus (EMARAV) representing multipartite, negative-strand ssRNA [(−)ssRNA] viruses that belong to the genus Emaravirus (not assigned to any family) Potexvirus (family Alphaflexiviridae) Velvet could assemble 97 and 75 contigs in the size range of 100–299 nt using the A similar number of vsiRNA reads (21–24 nt) were mapped to the viral genomes  using MAIn general, 45.6% and 64.9% of all the small-RNA reads that mapped to AlsVX and FMAV genomes, respectively, belonged to the 21-nt class. The 22-nt vsiRNAs were the next most abundant group , followed by 24-nt (13.3% and 11.8%) and 23-nt (6.6% and 5.7%) vsiRNAs .Asparagus virus 3, Lettuce virus X, Scallion virus X, Narcissus mosaic virus, Chenopodium mosaic virus X , and Pepino mosaic virus belonging to the genus Potexvirus, and EMARAV (genus Emaravirus)  . These mPrimers were designed to AlsVX genome regions flanking the CP gene that were covered by high numbers of vsiRNA reads . Subsequently, the region corresponding to nt 5880–6959 in AlsVX-Jap (genome length 7009 nt) A. tomentosum included in the pool of RNA subjected to SRDS were all PCR-positive, and the sequences obtained were identical (NCBI accession no. JQ354894). The virus was designated tentatively as WBYVV, because the partial NP sequence showed only 75–78% nucleotide and amino acid sequence identity with previously described FMV isolates (Gr10 and Can01) and FMAV. The corresponding regions in FMV-Gr10, FMV-Can01, and FMAV were 94–98% and 99–100% identical at the nucleotide and amino acids levels, respectively (Rose rosette virus (RRV) were 69% and 74% identical, respectively, to WBYVV  Phylogenetic analysis of the partial NP gene sequences indicated that WBYVV is only distantly related to the FMV strains from Canada (FMV-Can01) and Italy (FMV-Gr10) A. tomentosum  tested by SRDS in 2009 grew within a distance of 50 m from each other at the edge of a pedestrian pathway. The same and three additional locations 1–10 km apart from each other were surveyed for virus-like symptoms in A. tomentosum in the next year. In all locations, most A. tomentosum plants displayed virus-like symptoms, such as vein yellowing, mild mosaic and epinasty in leaves  in Japan where it co-infects this ornamental plant with other viruses Alstroemeria forms rhizomes and storage roots, which are used in vegetative propagation of plants. The infected propagules disseminate AlsVX and other viruses to new plants of AlstroemeriaAlstroemeria because the virus is symptomless in this host AlsVX was originally detected in Arctium as a new natural host for AlsVX. Previously, AlsVX was known to naturally infect only Alstroemeria plants in Japan and has been recently reported in Norway Helichrysum bracteatum (Vent.) Andrews, synonymous to Xerochrysum bracteatum (Vent.) Tzvelev] that also belongs to family Asteraceae Alstroemeria originated in South America and is a popular ornamental plant grown in most parts of the world. Whether AlsVX evolved in Alstroemeria or in other species remains to be studied. Similarly, the origin of AlsVX found in a wild plant of A. tomentosum in this study remains enigmatic. These issues could be addressed in future studies by using SRDS to detect and characterize more isolates of AlsVX in other plants and countries.Our study reveals Arctium species, but common (lesser) burdock [Arctium minus (Hill.) Bernh.] is a natural host of Iris yellow spot virus  in the state of New York Allium cepa L.; Amaryllidaceae) in the western USA and occurs in many annual and perennial weeds and crop plants in the field in various other parts of the USA. Common burdock is also a host for onion thrips (Thrips tabaci Lindeman), the vector of IYSV Biennial growth BunyaviridaePigeon pea sterility mosaic virusEmaravirus). Our results show that (−)ssRNA viruses can be readily detected by SRDS.The majority of the woolly burdock plants we tested were naturally infected with WBYVV. The (−)ssRNA emaraviruses have not been assigned to any viral family, and in contrast to IYSV belonging to family Sorbus aucuparia L.; Rosaceae) distributed over a much larger area of Finland and Russia (500×500 km). The genes were 97–99% identical and found to be under strong purifying selection The partial NP gene sequences determined from eight isolates of WBYVV were identical, although the isolates were collected from different locations up to 10 km apart. Similarly, only limited genetic variability was observed among the NP gene sequences of 20 isolates of EMARAV characterized from mountain ash trees (Plants encode four DCLs (DCL1 to DCL4) with partially complementary or redundant activities Tomato spotted wilt virus, a (−)ssRNA virus from genus Tospovirus (Bunyaviridae) Human immunodeficiency virus 1 in a mammalian cell line Our study provides further evidence that SRDS is a powerful method for plant virus diagnostics, and also suitable for screening plants for known or new (−)ssRNA viruses. In a previous study SRDS was used to analyze vsiRNA profiles in plants experimentally inoculated with matK gene of A. tomentosum), JQ354895 (CP gene sequence of AlsVX-Hel), JQ354894 .JQ354896 (


Crimean-Congo hemorrhagic fever (CCHF) virus is a serious human pathogen causing severe hemorrhagic disease with a fatality rate of up to approximately 30%. We have determined the viral genomic sequence from an isolate that caused a fatal case of imported CCHF in the United Kingdom in October 2012. Nairovirus genus in the Bunyaviridae family, can be found across much of Asia, from China and the Indian subcontinent, to Eastern Europe, as well as much of northern, eastern, and central Africa. It has a genome consisting of three negative-sense single-stranded linear RNA segments: small (S) (1.7 kb), medium (M) (5.4 kb), and large (L) (12.2 kb).Crimean-Congo hemorrhagic fever virus (CCHFv) causes severe hemorrhagic fever with fatal outcome in approximately 30% of cases ; it is aHyalomma species tick vector or from contact with infected animal body fluids. The virus can also spread nosocomially, particularly to health care workers from hospitalized patients   from AfgViral RNA was extracted from the patient serum sample using an EZ1 DSP virus kit (Qiagen) as part of the Health Protection Agency (HPA) initial emergency diagnostic response. Having been confirmed positive by quantitative reverse transcription PCR (qRT-PCR) targeted at the S segment , the comAll sequencing was performed using standard Sanger dideoxy sequencing technologies and a 3130xl sequencer (Life Technologies), and contiguous sequences were assembled using DNASTAR SeqMan . In all reactions, viral RNA from the patient serum sample was used as the template and reactions were initiated using the Virology and Pathogenesis Group’s (HPA) collection of CCHFv-specific primers, which were validated against our unique collection of CCHF virus isolates and were designed to provide overlapping reads of the entire segments; this facilitated rapid sequencing to commence immediately following the initial diagnosis.Since CCHF viruses are highly diverse, this sequence information will ensure that assays remain current and are able to detect circulating strains of the virus, in addition to allowing current phylogenetic and epidemiological studies. This rapid response was, and continues to be, underpinned by several ongoing assay development and surveillance-based projects that ensure the capacity to respond urgently to such cases, as well as to carry out important surveillance programs in endemic areas –10.JX908640, for M-Segment, KC344856, and for L-Segment, KC344855.Sequence information for each of the three segments was submitted to GenBank as each was completed, and they have the following accession numbers: for S-Segment,


West Nile virus (WNV) infection and resistance in a primary WNV vector, and to assess the extent to which virus-vector relationships are species-specific, we performed fitness studies with and without WNV exposure using a highly susceptible Culex pipiens mosquito colony. Specifically, we measured and compared survival, fecundity, and feeding rates in bloodfed mosquitoes that were (i) infected following WNV exposure (susceptible), (ii) uninfected following WNV exposure (resistant), or (iii) unexposed.Understanding the phenotypic consequences of interactions between arthropod-borne viruses (arboviruses) and their mosquito hosts has direct implications for predicting the evolution of these relationships and the potential for changes in epidemiological patterns. Although arboviruses are generally not highly pathogenic to mosquitoes, pathology has at times been noted. Here, in order to evaluate the potential costs of Cx. tarsalis colony, WNV infection did not alter fecundity or blood-feeding behaviour of Cx. pipiens, yet results do indicate that resistance to infection is associated with a fitness cost in terms of mosquito survival.In contrast to our previous findings with a relatively resistant The identification of species-specific differences provides an evolutionary explanation for variability in vector susceptibility to arboviruses and suggests that understanding the costs of infection and resistance are important factors in determining the potential competence of vector populations for arboviruses. West Nile virus  is vital to describing the ways in which selective pressures might drive the co-evolution of vector and virus. With the ultimate evolutionary goal of pathogens being dispersal and transmission, and the probabilities of these generally increasing with increased vector fitness, evolution should tend to favor an innocuous relationship between arboviruses and their mosquito hosts . Indeed,rus  may parOn the host side, evolution should independently favor resistance and/or tolerance to pathogenic viruses, yet these immune defences are often associated with their own fitness costs, so predicting the way in which host-pathogen interactions will affect the evolution of host response requires a detailed understanding of the balance between the costs of infection and immunity in individual species.West Nile virus is the most widespread arbovirus in the United States and is primarily vectored by Culex mosquitoes including Cx. pipiens in the northeast and north central, Cx. tarsalis in the west, and Cx. quinquefasciatus in the south and southwest USA  and between replicates were done with a log-rank test, which is equivalent to the Mantel-Haenszel test. Resistance in this study is defined only as an inability to become infected under these experimental conditions and therefore does not necessarily imply resistance at higher WNV doses. Both survival and reproductive data were used to construct life history tables for each group in separate replicates. Specific calculations included survival (lln R0/T) ,38 were All authors have read and approved the final manuscript. ATC analyzed and interpreted the data and wrote the manuscript. LMS designed and carried out the experiments. MAM carried out the experiments. LDK conceived and coordinated the experiments.


Rabies remains a major public health threat in many parts of the world and is responsible for an estimated 55,000 human deaths annually. The burden of rabies is estimated to be around US$20 million in Africa, with the highest financial expenditure being the cost of post-exposure prophylaxis (PEP). However, these calculations may be substantial underestimates because the costs to households of coping with endemic rabies have not been investigated. We therefore aimed to estimate the household costs, health-seeking behaviour, coping strategies, and outcomes of exposure to rabies in rural and urban communities in Tanzania.Extensive investigative interviews were used to estimate the incidence of human deaths and bite exposures. Questionnaires with bite victims and their families were used to investigate health-seeking behaviour and costs  associated with exposure to rabies. We calculated that an average patient in rural Tanzania, where most people live on less than US$1 per day, would need to spend over US$100 to complete WHO recommended PEP schedules. High costs and frequent shortages of PEP led to poor compliance with PEP regimens, delays in presentation to health facilities, and increased risk of death.The true costs of obtaining PEP were twice as high as those previously reported from Africa and should be considered in re-evaluations of the burden of rabies. Rabies remains a major public health problem, although the means to control and prevent this disease are available through mass dog vaccination and provision of post-exposure prophylaxis (PEP) to people exposed to bites by rabid or suspect rabid animals. Despite its necessity as a life-saving measure to prevent the fatal onset of rabies, access to PEP is a major problem in developing countries. We used extensive investigative interviews to estimate rabies incidence (deaths and exposures) and questionnaires to bite victims and their families to investigate health-seeking behaviour and costs associated with receiving PEP, in four districts covering both rural and urban Tanzania. Frequent shortages at health centres limited prompt access to PEP. Suspect bite victims often had to travel long distances to major hospitals to receive costly PEP, causing delays and increasing the risk of developing rabies. We calculated that an average patient in rural Tanzania would need to spend over $100 to complete the WHO recommended PEP schedules, unaffordable for many Tanzanians, who survive under the poverty line. Our data shows that rabies imposes a disproportionate financial hardship and high risk of dying of rabies to rural poor families and highlights the need to re-evaluate the burden of rabies in Africa. SerengeWe used the criteria of the Tanzanian National Bureau of Statistics (NBS) in differentiating rural and urban areas Two methodologies were used in this study: 1) Extensive investigative interviews to determine rabies exposure incidence in the study areas, conducted in the three districts of Serengeti, Ulanga and Kilombero; and 2) questionnaires administered in all 4 study districts to determine health seeking behaviour, associated costs and coping strategies.We compiled animal bite records and case reports from veterinary and livestock offices and details of patients reporting with animal-bite injuries from hospitals, clinics and dispensaries within the focal districts of Kilombero, Ulanga and Serengeti. These records were used to initiate extensive investigative interviews to determine networks of transmission; we actively searched and traced all identified sources of infection  and subsequent cases of onward transmission/bite exposure, as previously described The aforementioned clinical signs were used to evaluate whether the attacking animal could be clinically categorized as rabid, based on the criteria of the ‘six-step’ method A structured open-ended questionnaire was also administered to a subset of rabid animal bite victims and the families of rabies victims  bitten between January 2006 to December 2008. We collected information on demographic characteristics of the victims, costs related to the bite and seeking medical attention and coping strategies of bitten individuals and their families. We recorded the number of health facilities visited by the victim whilst seeking PEP, the number of days spent at each health facility, the amount of money paid for medical services, travel and other costs and how these funds were raised. When patients were escorted by an adult family member/guardian during the PEP seeking process, related costs were also captured. In order to validate costs incurred, heads of households were asked to produce receipts and in the absence of receipts, market prices or local fares were used to estimate the costs. Respondents were also asked to mention how much time both the patient and carer spent in seeking PEP. However, the questionnaire did not capture losses due to the premature death of a household member due to rabies or of being absent from school for school children.Human demographic data for the study were drawn from the Tanzanian national population and housing census of 2002 For this analysis, the direct medical costs included the costs of biologicals  and the costs associated with wound care, such as antibiotics, tetanus immunizations and disinfection. The price year was 2010. The indirect costs included out-of-pocket expenses for patients, such as transport costs to and from health centres and hospitals, accommodation and other costs for communication and subsistence while seeking PEP. Non-medical costs included productivity losses due to time spent seeking PEP. Time lost for both patients and escorts was valued in monetary terms according to projections of per capita daily income from the Tanzanian Household Budget Survey (HBS) of 2007 We calculated (i) average costs per suspect bite victim and (ii) average costs per dose. The average cost per suspect bite was defined as the average amount of cash spent by victims and caretakers following a bite, including costs of receiving PEP. Therefore suspect rabies bite victims including those who did not seek medical attention were included in this calculation. Average cost per dose was defined as the average amount of cash spent by patients and their carer(s) in receiving a single PEP dose. Therefore only patients who sought and successfully obtained at least one dose of PEP are included in this calculation. This was estimated by summing all cash costs spent on obtaining PEP and dividing by the total number of doses delivered. We also calculated the predicted average costs of PEP according to different regimens including intramuscular (IM) and intradermal (ID) administration and methods of subsidization that could be used in the future. These were calculated as the mean cost per dose plus annual income lost per dose, all multiplied by the number of clinic visits in the PEP regimen. These losses were converted into the percentage of annual income lost and equivalent days wages lost.Chi-square tests were used to examine differences in sources of funds used to pay for PEP between victims from rural and urban areas and the proportion of victims that were escorted while seeking PEP. The Welch Two Sample t-test was used to determine the differences in average costs per suspect bite, numbers of hospitals visited before receiving PEP and delays in receiving PEP, between patients from rural and urban areas. Regressions were applied to assess factors affecting the costs of acquiring PEP, delays to obtaining PEP, probability of completing PEP (logistic regression) and probability of death following exposure to rabies (logistic regression). Explanatory variables investigated included direct medical costs, travel costs, accommodation costs, other costs, and loss of productivity costs, gender, residence , district and distance to PEP delivering hospitals. Every explanatory variable was tested to assess significance (at P<0.05) then a final multivariate regression model was developed using backward stepwise variable selection. All statistical analyses were implemented within the R statistical programming environment The study protocol was approved by the Medical Research Coordinating Committee of the National Institute for Medical Research of Tanzania, with approval number NIMR/HQ/R.8a/vol.IX/994 and the Institutional Review Board of the Ifakara Health Institute, including the use of oral consent for the collection of interview data. Written consent was not obtained as the study followed established procedures for collecting interview data in Tanzania without the collection of biological samples from humans. The study was cleared by the District Executive Director in every study district and Village Executive Officers were asked for permission prior to starting work in each village. Before administering questionnaires, participants were orally informed about the purpose of the study, the data to be collected and the freedom of their participation and their right to withdraw from the study at any time during the interview. Participants were then asked if they would wish to be interviewed. All respondents agreed verbally and the interviews were conducted.Animal bite injuries were traced and investigated across three districts , from January 2006 to December 2009. Active searching revealed 599 animal bite injuries that met the case definition of being caused by suspect rabid animals as per criteria of the ‘six-step’ method: 136 in Kilombero district, 248 in Ulanga district and 215 in Serengeti district. Estimates of the annual incidence of bites from suspected rabid dogs per 100,000 persons, and annual incidence of human rabies deaths are summarised in Structured open-ended questionnaires were administered to 415 bite victims bitten by suspect rabid animals between January 2006 and December 2008; 97 in Kilombero, 141 in Ulanga, 152 in Serengeti and 25 in Musoma Urban that were identified from hospital records. Questionnaires were not administered to all cases traced through active searching because of time constraints during fieldwork. Only 12% of these bite victims received PEP free of charge on a first-come first-served basis, the reminder had to pay for PEP, with rural bite victims paying around $12 per PEP dose and urban bite victims paying $8 per dose . Direct 2 = 10.91, df = 3, p = 0.01). Of 272 bite victims who lived far from district hospitals (more than 10 kilometres), only 39% received PEP within 7 days after exposure whereas, 64% of those who lived close to district hospitals received PEP within 7 days after exposure . Bite victims from urban areas where the district hospital was located in their home town did not incur many indirect costs compared with patients from rural areas who had to travel further to these hospitals. If PEP was not in stock at district hospitals, victims had to either wait for hospitals to procure PEP or travel elsewhere to receive PEP. We found that 40% (156/391) of patients attended multiple heath facilities (maximum of five heath facilities) for PEP. Of these 85% (132/156) were patients from rural areas .Ninety-four percent (391/415) of these suspect bite victims reported to health facilities for PEP. About half of these bite victims reported, immediately after being bitten by animals but there was considerable variance in delays before receiving the first PEP dose between rural and urban bite victims. Bite victims in rural areas took longer, on average, to receive PEP than those in urban areas . Families adopted various coping strategies to meet the costs of obtaining PEP. Families that had no savings had to transform their assets into cash. Poor rural families with little or no assets often were unable to afford PEP and experienced financial hardship while raising funds for PEP. We found that residence  had a significant impact on the source from which households obtained funds to pay for PEP . Patients from urban areas were more likely to use money from their salaries or sell their assets whereas patients from rural areas either obtained funds from selling crops or selling livestock (The majority (73%) of suspect rabies bite victims were escorted by an adult family member to hospital. There was no significant difference in whether bite victims from rural versus urban areas were escorted to hospital  of suspect bite victims received at least one dose of PEP. Of those that received their first dose, only 67% returned for a second dose, i.e. 5% of patients dropped out after a single dose. There was a further drop out of 16% after the second dose and very few patients received the last two doses . PatientCosts were a significant obstacle for bite victims to both obtain and complete PEP (p<0.05). We therefore calculated the predicted costs of completing PEP under different regimens that are recommended by WHO, assuming PEP is provided free-of-charge, as well as being made available at local health centres and not just at district hospitals therefore minimizing travel costs. The costs of completing PEP under these scenarios are presented in We found that 28% (117/415) of bite victims did not receive PEP of any kind, of them 18/117 (15%) developed rabies, whereas none of those who received a single dose of PEP developed rabies. Most of the deaths occurred in households dependent on subsistence farming (89%), involved children less than 16 years of age (78%) and occurred in areas far from hospital . In a multivariate analysis, distance to the district hospital and direct medical costs were the only significant predictors of human rabies deaths (p<0.05).The present study confirms that rabies remains a serious public health and economic problem in developing countries where canine rabies is endemic. Bites from suspect rabid animals impose a substantial financial burden on affected households, especially for poor rural bite victims who suffer excessively high costs in obtaining PEP compared to those from urban areas, and are less likely to receive vaccine. PEP is vital for human rabies prevention and requires repeat visits to hospital to complete a full course. Rural bite victims often live far from major health facilities in urban areas, where PEP can be obtained; however, even in these areas the availability of PEP is not guaranteed. In remote areas, travel to a regional hospital where PEP shortages are less frequent, can take more than 18 hours and is expensive. Moreover, costs were almost double for patients that needed to be escorted by a family member/adult, with rural bite victims incurring disproportionately higher indirect costs and a higher risk of developing rabies and dying from the disease. Delays to receiving PEP, due to hospital shortages and time spent raising money or waiting for district hospitals to procure PEP increased the risk of developing this fatal disease. The need to repeatedly visit hospital to complete PEP may deter poor people living in remote rural areas from obtaining and completing PEP, which may explain why the risk of rabies was higher for those living further from major hospitals. More generally, the distance to major hospitals seems to be an obstacle for people living in rural areas in Africa. A study in Ivory Coast reported that over 75% of patients who discontinued of PEP were from outside the capital city, Abidjan Adoption of a reduced 4-dose regimen such as the Essen 4-dose or the Zagreb regimen could reduce the number of hospital visits that patients need to make to complete their PEP course, in comparison to the 5-dose Essen regimen currently recommended by WHO. In several Asian countries ID regimens are being used that can reduce the total volume of vaccine required by up to 80%, and also only require 4 hospital visits In Tanzania, although the government aims to provide PEP free-of-charge, budgets allocated for this are often insufficient, resulting in shortages. We found that PEP was provided free-of-charge to less than 20% of bite victims, whilst the reminder had to pay, incurring costs that were equivalent to around two months income. If patients fully adhered to recommended regimens these costs would be considerably higher. We show that if PEP was fully subsidized and reduced dose regimens were instead adopted, costs to patients would be substantially reduced. Therefore, it is important for governments to consider strategies that increase accessibility and affordability of PEP to the rural poor who are at the most risk from the disease. This would assist poor families with few assets or little means to pay, especially poor farmers who depend on selling farm outputs to obtain money for PEP.In comparison to the literature, our study suggests that costs of seeking PEP are substantially underestimated. A previous study estimated that a full course of PEP  to be US$ 39.57 in Africa including 1% of patients receiving RIG One of the most effective methods to reduce expenditure on PEP is for veterinary services to invest in mass dog vaccination. Studies in northwest Tanzania have demonstrated the impact of mass dog vaccination on reducing animal-bite injuries and demand for PEP Recently we have seen an increased effort by the international community to improve the health of the world's poor. Attention has been focused on the relationship between health and poverty, particularly in relation to the Millennium Development Goals (MDGs)


Salmonella and extended-spectrum cephalosporin-resistantEscherichia coli (ESCREC). Stool cultures were performedfor 22 symptomatic campers, three asymptomatic food handlers, and 10 healthyhousehold members. Of the 22 campers, 19 had Salmonellaenterica, 9 had an ESCREC strain carrying an extended-spectrumβ-lactamase, and 2 had a second ESCREC strain carrying a plasmidiccephamycinase. Related ESCREC were detected in two asymptomatic food handlers and in none of the healthy household members. FecalESCREC and its β-lactamases and plasmids were extensivelycharacterized. Three of the five ESCREC clones were recovered from multiplehosts. The apparent dissemination of ESCREC suggests a food or water vehicle.The observed distribution of resistance plasmids and β-lactamasegenes in several clones indicates a high degree of horizontal transfer.Heightened vigilance and increased efforts must be made to discover thereservoirs and vehicles for community dissemination of ESCREC.Investigation of an acute gastroenteritis outbreak involving >100 personsat a summer camp in Girona, Spain, in June 2002 led to the detection of Escherichia coli that produce enzymes capable of degradingextended- spectrum cephalosporins (ESCs), i.e., extended-spectrumβ-lactamases (ESBLs), or these drugs plus cephamycins, i.e., plasmidic orhyperproduction of chromosomal cephamycinases have recently emerged as importantnosocomial pathogens  have involved hospitalizedpatients or nursing home residents . Byusing this media, we have isolated several resistant enterobacteriaceae strains frompatients with sporadic cases of gastroenteritis (unpub. data). During an investigationof a summer camp-associated salmonellosis outbreak, we observed that stool cultures fromnine campers unexpectedly yielded, on cefoperazone-containing medium, coloniesresembling enterobacteriaceae, with a uniform mucoid appearance. This result suggestedthe possibility that the same, probably ESC-resistant, enterobacterial strain waspresent in all these persons, findings consistent with possible foodborne spread.Consequently, all samples were reevaluated on media containing cefotaxime (see Methods)to increase sensitivity for detection of ESC-resistant organisms. To gain more knowledgeof foodborne spread as a potential mechanism of dissemination of resistance genes, weundertook an extensive molecular epidemiologic analysis of these isolates.The cefoperazone-containing medium routinely used in our laboratory for the isolation ofTwo hundred twenty-five elementary and secondary school students and 11 teacherswere spending a week  at a summer camp inPalafrugell, Girona (Spain), when an outbreak of gastroenteritis began on June14. An epidemiologic investigation involving 200 campers and staff failed toidentify the source of the outbreak. Clinical and epidemiologic studies wereinitiated at the onset of the outbreak, but our participation as referencelaboratory began later. Consequently, a limited number of stools fromsymptomatic patients and related persons were available to us for analysis.From June 16 to 19, stool samples from 22 ill campers were collected foranalysis. On July 6 (18–19 days later), additional stool samples werecollected from four ill campers and from 10 asymptomatic household members ofthe four ill campers. Stool samples were also collected from three asymptomaticfood handlers, on June 19, 22, and 28.Stool samples were processed according to conventional protocols forisolation of enteropathogenic bacteria, in addition to special selection forresistant organisms (as described below). Isolates were identified byconventional methods , which after 18 h of incubation at 35°C, wasadded to similarly supplemented MacConkey broth (MacC-broth-CTX). After an 18-hincubation, a loopful of this broth was spread on similarly supplementedMacConkey agar. A colony of each distinct morphotype was analyzed further.Salmonella were analyzed further.Stool samples were processed as for isolation of ESCREC, but after the initialgrowth the MacC-broth-CTX was added to selenite broth. After 8 h of incubationat 35°C, this broth was spread on cefotaxime-supplementedxylose-lysine-deoxycholate agar. Colonies suspected of representingSalmonella and ESCREC as described for stool samples,except that the initial TSB-CTX was replaced by peptone yeast extract broth.Eight unprepared food items from the camp kitchen were analyzed forDisk diffusion susceptibility testing to 28 antibiotics  was perfE. coli HB101  for which transconjugants wereselected, according to described methods . The serogroup of the ESCREC was determined at the Laboratoriode Referencia, Lugo, Universidad de Santiago de Compostela (J. Blanco).The biotype, as determined for 12 metabolic reactions, was expressed as a 4-digitcode  . For southern hybridization(CTX-M-9bla (850 bp) andCMY-2bla , as labeled and detectedusing the ECLTM Direct Nucleic Acid Labeling and Detection System(Amersham Biosciences).Plasmid DNA was isolated by using a commercial kit and subjected to 0.8% agarose gel electrophoresis both without digestions andafter cleavage with Comparisons of proportions were tested by using the Fisher exact test(two-tailed).The 225 student campers, 10–16 years of age, and 11 teachers were fromthree schools in three cities, Tarragona (T), El Prat (EP), and Sant Boi (SB).Of the 200 campers and staff interviewed, 109 (54.5%), including 3 teachers, hadsymptoms of gastroenteritis, with no significant differences between the threeschools . The most frequent symptoms wereabdominal pain (80%), diarrhea (79%), and headache (64%). Two students wereadmitted to hospital, but after supportive therapy were discharged within 48hours. No person received antibiotic therapy.S. enterica serovar Enteritidis phagetype 4, which was susceptible to all tested antimicrobial agents. No fecalpathogens were detected in the remaining three ill campers.Of the 109 ill campers, 22 provided stool samples that were available formicrobiologic study in our laboratory. Nineteen (86%) of the acute-phasestool samples yielded R-cefoxitin FOXS-ESCREC), consistentwith production of an ESBL. Two of these samples also yielded a secondESCREC type that was resistant to penicillins and cephalosporins, includingESCs and cephamycins, but susceptible to carbapenems(CTXR-FOXR-ESCREC), consistent withhyperproduction of E. coli AmpC chromosomalβ-lactamase or presence of a plasmidic cephamycinase. All ESCRECisolates exhibited multiple additional resistance markers of the 22 campers, including 8 (42%) of 19 with salmonellae and 1 with nodetectable enteric pathogen .All nin markers .Follow-up stool samples (collected 18–19 days later) wereavailable for four campers. Neither salmonellae nor ESCREC was recovered,except in one camper, from whom the initial ESCREC strain was isolated.R-FOXS-ESCREC at the second sampling, whereas theother had CTXR-FOXR-ESCREC at both the second and thirdsamplings.Although stool samples collected from the three (asymptomatic) food handlers werenegative for enteropathogens, two of these persons were carriers of ESCREC. Onehad CTXIn contrast, neither enteropathogens nor ESCREC were detected in stool samplesfrom 10 healthy household members of four ill campers who had both ESCREC andsalmonellae in their acute-phase stool sample .S. entericaserovar Enteritidis nor ESCREC.The eight cultured camp food items yielded neither E. coli isolates phenotypically suspected of ESBLproduction, a β-lactamase with an isoelectric point of 8.0 wasdetected. PCR with blaCTX-M-9-specific primers andsequence analysis confirmed the presence of the CTX-M-9 β-lactamase(not shown).In the 11 blaTEM-specific primers confirmed the presenceof a TEM-1-like β-lactamase .All four isolates phenotypically suspected of AmpC β-lactamaseproduction were PCR-positive with Collectively, the 15 ESCREC isolates represented five distinct biotypes . OverallClone A (CTX-M-9), the most prevalent, was recovered from students from all threecities, EP, SB, and T . Clone BCTX-M-9bla andCMY-2bla hybridized to a single large(>150 kb) plasmid  exhibited minor diversity within and greaterdiversity among clones  were indistinguishable in both the uncut plasmid blot .In contrast, the mid blot  and the E. coli HB101 but not to a S. entericaisolate from a patient. In contrast, a CMY-2-positive isolate was successfullyconjugated with this S. enterica isolate but not withE. coli HB101 .One CTX-M-9-positive isolate transferred its resistance by in vitro conjugationto Salmonellagastroenteritis at a summer camp uncovered the unsuspected dissemination amongcampers and camp staff of multiple clones of ESCREC containing diverse conjugallytransferable β-lactamases. Several lines of evidence indicated thatdissemination of ESCREC occurred within the summer camp. Sharing of ESCREC cloneswas observed among multiple hosts who had no contact with one another before camp,yet at camp lived together and shared a common food and water supply. Isolation ofESCREC was limited to camp attendees, to the exclusion of members of thecampers’ households who did not attend camp. Finally, the high observedprevalence of fecal ESCREC among camp attendees  contrasts strikinglywith the low prevalence of ESCREC detected by using similar methods in referencefecal samples from 707 outpatients without an infectious disease diagnosis whoattended Sant Pau Hospital from February through May, 2001  and among E. coli isolates from our hospital clinicalmicrobiology laboratory   might have served as a vehicle for ESCRECwithin the camp. Indeed, ESCREC clones A and B were confined to hosts who also hadsalmonellae. Since only eight food items were cultured, the failure to recoverESCREC from camp foods provides little evidence to rule out foodborne transmission.Although food handlers have been implicated in many foodborne outbreaks of intestinaldisease  and an outbreak S. enterica isolate (CMY-2). Thesefindings, which are consistent with our previous work and that of others , short longitudinal follow-up, unusual circumstances , and absence of data regarding priorantibiotic use and hospital contact. Future studies should seek ESCREC in a similarsetting, and in the general community, during a period without a known infectiousdisease outbreak.Salmonella gastroenteritis at a summer camp showed the unsuspecteddissemination among campers and staff of multiple clones of ESCREC that containeddiverse, conjugally transferable β-lactamases. Dissemination of ESCRECwithin the summer camp, possibly through food or water, was suggested by severallines of evidence. Confirmation of community-based transmission of ESCREC in othercontexts and locales would indicate a need for heightened vigilance and efforts todiscover the reservoirs and vehicles for dissemination of ESCREC within thecommunity.In summary, our microbiologic evaluation of an outbreak of


Interest in peste des petits ruminants virus (PPRV) has been stimulated by recent changes in its host and geographic distribution. For this study, biological specimens were collected from camels, sheep, and goats clinically suspected of having PPRV infection in Sudan during 2000–2009 and from sheep soon after the first reported outbreaks in Morocco in 2008. Reverse transcription PCR analysis confirmed the wide distribution of PPRV throughout Sudan and spread of the virus in Morocco. Molecular typing of 32 samples positive for PPRV provided strong evidence of the introduction and broad spread of Asian lineage IV. This lineage was defined further by 2 subclusters; one consisted of camel and goat isolates and some of the sheep isolates, while the other contained only sheep isolates, a finding with suggests a genetic bias according to the host. This study provides evidence of the recent spread of PPRV lineage IV in Africa. Peste des petits ruminants virus (PPRV) belongs to the genus Morbillivirus, in the family Paramyxoviridae. Like other members of the same genus, such as rinderpest virus, Canine distemper virus, Measles virus, and marine mammal viruses, PPRV is highly pathogenic for its natural hosts . A second aliquot was used for viral RNA extraction and virus isolation in tissue culture. Reverse transcription PCR (RT-PCR) amplified the N protein gene directly from tissue samples by using a set of pan-morbillivirus primers as described by Kwiatek et al. . Phylogenetic analysis was carried out by using the criterion of neighborhood based on the principle of parsimony , 5 goats (100%), and 38 camels (77.6%) drawn from all of the regions studied: Khartoum, Blue Nile, Northern Sudan, Kassala, Kordofan, and Darfur . Of the Of the tissue samples tested, lung and lymph nodes were the most suitable for sequencing because they had the highest viral load, thereby yielding a sufficient amount of PCR product. Sequence also was obtained from several virus isolates, including the historical Gedarif isolate from 1971. The partial N gene sequences were obtained for 26 of the 64 samples from Sudan and 6 of the 16 samples from Morocco. Sequences were aligned with an extended set of PPRV isolate sequences that either were in the database or were retrieved from GenBank where they were described by Kwiatek et al. ; only a few remained in lineage III (2). Molecular typing also showed for the first time the presence of PPRV lineage IV in Morocco .Lineage IV isolates from Sudan could be further distinguished into 2 clusters. The grouping of the sequences within the clusters was supported by bootstrap percentages >70%. In the first group, 17 sequences from all camel and goat isolates and some sheep isolates  showed 100% identity and matched with Saudi Arabia_1999_7 strain. Sheep Sudan Ov_41 differed from the previous sequences by 2 nt at position 68 (C to T) and 142 (A to G). The divergence of this cluster with consensus IV ranged from 1.2% (Cam_318) to 2% (Ov_41). Furthermore, the Saudi Arabia cluster was related closely to the 6 strains from Morocco collected from the 2008 outbreak, the closest differing by 4 nt.A separate cluster containing only sheep samples collected during 2000–2008  matched with 2 strains from central Africa  (Only 2 isolates collected at the start of the study (in mid-2000) from sheep in western and eastern Sudan fell into lineage III. Sequences of these 2 field isolates remained similar to the historical strains of Sinnar 72 that was isolated 40 years earlier from the Blue Nile region . For the measles virus, it appears that the lower the level of circulation, the higher the sequence conservation (Camels were not regarded as possible hosts for PPRV until 1992, when a number of authors reported PPRV seroconversion in these animals (The reason that isolates of lineage IV became predominant in Sudan, progressively replacing lineage III viruses, during the last decade remains unclear. Lineage IV has been present in Asia and in part of the Middle East for a long time, probably as low virulent strains (